Research ArticleAstragalus Polysaccharide Protects Astrocytes from BeingInfected by HSV-1 through TLR3NF-120581B Signaling Pathway

Lihong Shi1 Fengling Yin2 Xiangui Xin3 Shumei Mao1 Pingping Hu1

Chunzhen Zhao1 and Xiuning Sun4

1 Department of Pharmacology Weifang Medical University 7166 Baotong West Street Weifang 261053 China2Hospital of Weifang Medical University 7166 Baotong West Street Weifang 261053 China3The Affiliated Hospital of Shandong Traditional Chinese Medicine Junior College 508 Binhai East Road Yantai 264199 China4Department of Parasitology Weifang Medical University 7166 Baotong West Street Weifang 261053 China

Correspondence should be addressed to Xiuning Sun sxn321163com

Received 17 April 2014 Revised 10 June 2014 Accepted 12 June 2014 Published 26 June 2014

Academic Editor Shi-Biao Wu

Copyright copy 2014 Lihong Shi et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Astragalus polysaccharide (APS) is themost immunoreactive substance in Astragalus APS can regulate the bodyrsquos immunity and iswidely used inmany immune related diseasesHowever till now there is little information about its contribution to the protection ofastrocytes infected by virus Toll-like receptor 3 (TLR3) is a key component of the innate immune system and has the ability to detectvirus infection and trigger host defence responses This study was undertaken to elucidate the protective effect of APS on herpessimplex virus (HSV-1) infected astrocytes and the underlying mechanisms The results showed that APS protected the astrocytesfromHSV-1 induced proliferation inhibition along with increasing expression of tumor necrosis factor-120572 (TNF-120572) and interleukin-6 (IL-6) markedly Moreover APS significantly promoted the expression of Toll-like receptor 3 (TLR3) and the activation of nuclearfactor-120581B (NF-120581B) in astrocytes In addition while astrocytes were pretreated with TLR3 antibody before adding HSV-1 and APSthe expression of TLR3 TNF-120572 and IL-6 and the activation of NF-120581B decreased sharplyThese results indicate that APS can protectastrocytes by promoting immunological function provoked by HSV-1 through TLR3NF-120581B pathway

1 Introduction

Herpes simplex encephalitis (HSE) is the most commonsporadic and nonepidemic viral encephalitis in both childrenand adults Untreated HSE can result in prolonged neu-roinflammation and compromised brain function or death[1ndash4] Furthermore the majority of treated patients wouldsuffer various degrees of sequela despite the improvementsin diagnosis and therapy [5] HSV-1 infection of the brainresults in devastating necrotizing encephalitis Murinemodelof herpes simplex encephalitis showed that HSV-1 infectiontriggered a robust immune response including infiltration ofleukocytes production of proinflammatory mediators acti-vation of resident microglial cells and focal tissue damage

Toll-like receptor is an important class of immune recog-nition receptors that can recognize pathogens moleculesactivate the innate immune response and invoke the releasingof cytokines and initiate adaptive immunity [6 7] As

an important member of the Toll-like receptor family TLR3can recognize double-stranded RNA (dsRNA) of viruses andinduce related signal pathway to play important role in hostdefense against viruses [8 9] It is a remarkable fact that TLR3is vital for natural immunity to HSV-1 in the central nervoussystem [10 11]

Astrocytes are the most abundant cells in the centralnervous system (CNS) The main function of astrocytes isto provide nutrition and support In addition astrocytes cansecrete cytokines and neurotrophic factors to regulate theimmune function [12 13] Whenmice embryos were infectedby HSV-1 neuronal injury and necrosis accompanied bypathological changes of astrocytes would occur [14] RecentlyFurr et al reported that astrocytes increased expression ofDNA-dependent activator of interferon-regulatory factors(DAI) as an important innate immune mechanism underly-ing the rapid and potentially lethal inflammation associatedwith HSV-1 infection [15] Our previous studies revealed

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2014 Article ID 285356 6 pageshttpdxdoiorg1011552014285356

2 Evidence-Based Complementary and Alternative Medicine

that when astrocytes were infected by HSV-1 NF-120581B wasactivated through Toll-like receptor 3 (TLR3) and then theactivated NF-120581B would translocate from the cytoplasm tothe nucleus so as to promote the production of TNF-120572 andIL-6 to play antiviral roles [16] These results demonstratethat astrocytes play significant roles in the inflammatoryresponses of resident CNS cells to HSV-1 challenge

Astragalus is a traditional Chinese medicine which con-tains polysaccharides saponins flavonoids amino acidslinoleic acid alkaloids and so forth Astragalus polysac-charide (APS) is the most immunoreactive substance inAstragalus which can regulate the body immunity APShas been identified as a class of macromolecules that canprofoundly affect the immune system and is widely used asan immune adjuvant inChina APS can induce the expressionof surface antigens on lymphocytes promote the productionof antibodies affect the secretion of cytokines and evenstimulate cell proliferation [17 18] Previous studies provedthe effective immunostimulatory roles of APS against variousviruses [17 19 20]

In this paper based on our previous research the antiviraleffect of APS on the HSV-1 infected astrocytes was inves-tigated Furthermore the immunoregulatory effect and thepossible immunization mechanisms of APS were evaluated

2 Materials and Methods

21 Laboratory Animals Cells and Virus The BALBc micewere purchased from Medicine Animal Center of ShandongUniversity HSV-1 SM44 strain was kept in Central Labo-ratory of Weifang Medical University at minus80∘C The rabbitanti-mouse antibody TLR3 NF-120581B and 120573-actin were fromInvitrogen Nuclear and Cytoplasmic Protein Extraction Kitwere from Beyotime Institute of Biotechnology MTT kit waspurchased from Sigma 96T enzyme-linked immunosorbentassay (ELISA) kit was purchased from ADL APS was pur-chased from Tianjin Sino Pharmaceutical Company batchnumber is 120102

This research was conducted in strict accordance withthe recommendations in the Guide for the Care and Useof Laboratory Animals of the National Institutes of HealthSurgery was performed under sodium pentobarbital anesthe-sia all efforts were made to minimize suffering The protocolwas approved by the Committee on the Ethics of AnimalExperiments of Weifang Medical University (number 2012-056)

22 HSV-1 Multiplication and Tittering The HSV-1 strainSM44 propagated in Vero cells the supernatant was collectedwhen cytopathic effect (CPE) was in confluence to 75By freezing in minus80∘C and thawing for three times HSV-1 particles were released By centrifuging for 8min with1200 rpm the supernatant was collected as inoculum andstocked in freezer All of the HSV-1 samples used in thisstudy were of the same batch In following research finalconcentration of HSV-1 TCID

50was 10minus6mLminus1

23 Astrocytes Culture and Purification Newborn BALBcmice in day 1 to day 3 were taken and cells culture and purifi-cation were performed according to McNaughtrsquos protocol[21] Take the 3rd generation of astrocytes for experimentalresearch according to our teamrsquos protocol [16]

24 MTT Cell Proliferation Assay Calculating the MedianEffective Concentration (EC

50) of APS Astrocytes (1 times

106mLminus1) were cultured inDMEMF12media in the presenceof HSV-1 (final TCID

50 10minus6mLminus1) with APS (0 50 100

200 and 300120583gmLminus1) 5 repeating groups for each con-centration 20120583L MTT solution (5mg mLminus1) was added tocell media at various time points (6 12 18 and 24 h) afterthe addition of HSV-1 and APS incubated for 4 h and thensupernatants were gently removed and 200120583L dimethyl sul-foxide was added into each pore After 10 minutesrsquo vibrationthe absorbance (OD value) was measured at a wavelengthof 492 nm by enzyme-linked immunosorbent detector ODvalue is proportional to the proliferation of living cells and isan indicator of cell proliferation

The mean and standard deviation of OD values foreach concentration of APS at every action time point werecalculated The results showed that cell proliferation is themost exuberant at 12 h time point for every concentration ofAPS so 12 h is the detection timepoint in the following assaysEC50ofAPSwas 120120583gmLminus1 so in the following research the

final concentration of APS was 120120583gmLminus1 The formula tocalculated EC

50is

effect =ODdrug minusODmodel

ODcontrol minusODmodeltimes 100 (1)

25 Grouping and Detecting the Effect of APS on AstrocytesProliferation by MTT Assay Astrocytes were seeded at den-sity of 1 times 106mLminus1 into 24-well plates four groups were setup according to the different intervention conditions HSV-1group HSV-1 + APS group TLR3 antibody + HSV-1 + APSgroup and blank control group In HSV-1 groups all flaskcells were inoculated with viral suspension (final TCID

50

10minus6mLminus1) in HSV-1 + APS group all flask cells were inoc-ulated with viral suspension (final TCID

50 10minus6mLminus1) and

APS (final concentration 120120583gmLminus1) in TLR3 antibody+ HSV-1 + APS group TLR3 antibody (final concentration10 120583gmLminus1) was used to pretreat cells for 30min and thenviral suspension (final TCID

50 10minus6mLminus1) and APS (final

concentration 120120583gmLminus1) were added to cells in blankcontrol group the same volume of cell culture medium asthe aforementioned groups was added into each flask cellsFor each group 3 repeating experiments were performedTheproliferation rate of cells was assayed by MTT after culturingfor 12 h

26 Determination of the Levels of TNF-120572 and IL-6 by ELISAAstrocytes were seeded at density of 1 times 106mLminus1 into 12flasks (25 cm2) Grouping and treatment were performedas previously mentioned Supernatants were collected andfiltered TNF-120572 and IL-6 in the supernatant were measured

Evidence-Based Complementary and Alternative Medicine 3

20120583m

(a)

20120583m

(b)

20120583m

(c)

20120583m

(d)

Figure 1 Effect of APS on the growth and proliferation of astrocytes (a) Blank control group the astrocytes grew in good condition withactive proliferation (b)HSV-1 group 12 h after infectionwithHSV-1 the proliferation of astrocytes was significantly inhibited and the infectedastrocytesrsquo bodies were gradually swollen into round and giant appearance (c)HSV-1 +APS group the proliferation inhibitory effect ofHSV-1could be suppressed by APS and most astrocytes grew in good conditiosn (d) TLR3 antibody + HSV-1 + APS group when astrocytes werepretreatedwith TLR3 antibody the proliferation of astrocytes reducedmarkedly and some infected astrocytesrsquo bodies were swollen into roundappearance Scale bar 20 120583m

by ELISAThe absorbance (OD value) was determined usinga microplate reader at a wavelength of 450 nm For eachsample the measurement was repeated 3 times and theaverage concentration of TNF-120572 and IL-6 was set as the finalresult

27 Detection of TLR3 Protein in Cells and NF-120581B Proteinin Cell Nuclei by Western Blot In brief total protein andnuclear protein were extracted following the reagent com-panyrsquos instruction Protein samples were separated by SDS-PAGE transferred to polyvinylidene difluoride membranesimmunoblotted using the appropriate primary and the HRPconjugated secondary antibodies and visualized by usingenhanced chemiluminescence reagents ECL Anti-120573-actin orLMNB1 monoclonal antibody was used as loading controlThe intensities of bands in Western blots were quantifiedby densitometry analysis using AlphaImager HP (AlphaInnotech USA) and NIH Image J software (Rockville MDUSA) Western blot data shown in the paper are representa-tives of three independent experiments

28 Statistical Analysis All data were analyzed using theSPSS 130 statistical software Values were expressed as meanplusmn standard deviation (119883 plusmn 119878) The significance of differences

between groups was determined using the one-way ANOVAStatistical significance was accepted for 119875 values lt005

3 Result

APS promotes the growth and proliferation of astrocytesinfected by HSV-1 Observation under microscope showedthat in the blank control group the uninfected astrocyteswere in thin and flat appearance with good refraction andgrew in good conditionwith active proliferation (Figure 1(a))in the HSV-1 group the proliferation of astrocytes wassignificantly inhibited and the infected astrocytesrsquo bodieswere gradually swollen into round and giant appearance(Figure 1(b)) the inhibited proliferation of astrocytes infectedbyHSV-1 could be rescued byAPS apparently inHSV-1 +APSgroup (Figure 1(c)) when astrocytes were pretreated withTLR3 antibody and then exposed to HSV-1 and APS con-currently the proliferation of astrocytes reduced markedlycompared with the HSV-1 + APS group (Figure 1(d))

MTT analysis (Figure 2) showed that when astrocyteswere exposed to HSV-1 the proliferation of astrocytes wassignificantly inhibited compared to the blank control groupThe inhibited proliferation of astrocytes infected by HSV-1 could be rescued by APS apparently in the HSV-1 + APS

4 Evidence-Based Complementary and Alternative Medicine

Control HSV-1 HSV-1 + APS HSV-1+ TLR3 + APS

lowast

08

07

06

05

04

03

02

01

0

OD

998779

Figure 2 Astrocytes proliferation detected by MTT lowast119875 lt 001versus the HSV-1 group Δ119875 lt 005 versus the TLR3 antibody +HSV-1 + APS group 119899 = 3

group In the presence of APS the proliferation of astrocytesincreased to some extent and the OD value of HSV-1 +APS group was greater than that of the HSV-1 group (119875 lt001) which suggests that APS can protect astrocytes fromHSV-1 induced proliferation inhibition Interestingly whenastrocytes were pretreated with TLR3 antibody before addingHSV-1 and APS the proliferation of astrocytes decreasedmarkedly when compared to the HSV-1 + APS group (119875 lt005) This result indicates that the protective effect ofAPS against HSV-1 infection may be associated with TLR3pathway

Secretion levels of TNF-120572 and IL-6 in culture supernatantwere detected by ELISA (Figure 3) The concentrations ofTNF-120572 and IL-6 were very low in culture supernatant of theblank control group whereas in culture supernatant of theHSV-1 group the concentrations of both TNF-120572 and IL-6increased obviously (119875 lt 001) In the presence of APS HSV-1 infected astrocytes expressed higher amount of TNF-120572 andIL-6 than that of the HSV-1 group Remarkably pretreatmentof astrocytes with TLR3 antibody decreased the expression ofTNF-120572 and IL-6 in the TLR3 antibody + HSV-1 + APS group(119875 lt 005)

Western blot analysis showed that the expression level ofTLR3 and the activation of NF-120581B were low in the controlgroup (Figure 4) In the HSV-1 group after being exposedto HSV-1 for 12 h astrocytes expressed higher level of TLR3than the control group At the same time HSV-1 infectioninduced the activation of NF-120581B significantly Moreover inthe presence of APS the expression level of TLR3 and theactivation level of NF-120581B were obviously higher than those ofthe HSV-1 group However when astrocytes were pretreatedwith TLR3 antibody before addition of HSV-1 and APSastrocytes reduced the expression of TLR3 and the activationof NF-120581B significantly Considering the different secretionlevels of TNF-120572 and IL-6 in different groups these resultsindicate that APS may activate NF-120581B which leads to theproduction of TNF-120572 and IL-6 through TLR3 pathway (119875 lt005)

4 Discussion

Recent studies have confirmed thatmany herbs have effects ofmodulating immunity and inhibiting and killing pathogenic

05

10152025303540

Control HSV-1 HSV-1 + APS HSV-1+ TLR3 + APS

lowast

lowast

TNF-120572IL-6

TNF-120572

and

IL-6

(ng

mL)

998779

998779

Figure 3 Concentration of TNF-120572 and IL-6 (ngmLminus1) in cellculture medium detected by ELISA lowast119875 lt 001 versus the HSV-1group Δ119875 lt 005 versus the TLR3 antibody + HSV-1 + APS group119899 = 3

microorganisms [22 23] As one of the bioactive ingredientsfrom the natural traditional Chinese medicinal herb Astra-galus membranaceus APS is used widely as an immunomod-ulator in China Evidences indicate that APS can enhancelymphocyte blastogenesis and stimulate macrophage activa-tion without cytotoxic effects [7 8] APS cannot inhibit virusdirectly but it can activate the immune system and inducethe production of cytokines to initiate an antiviral response[24 25] Consistent with these previous reports the currentstudy showed that HSV-1 infection induced the secretionof TNF-120572 and IL-6 in astrocytes and the secretion notablyincreased in response to APSThese results indicate that APScan promote immunomodulatory effects of astrocytes

In addition we also found that HSV-1 infection induceda higher expression of TLR3 and decreased the proliferationof astrocytes these results are in accordance with previousreports which showed that high expression of TLR3 impairedcell proliferation and inhibited cell cycle progress [26 27]Furthermore we found that APS was capable of promotingthe proliferation of astrocytes infected by HSV-1 This resultsquares with what we already know about the role of APSon cell proliferation [18 28 29] Although the potentialmechanism is still to be clarified regarding the currentunderstanding of TLR3 signaling function we infer thatthe effect of APS on proliferation of astrocytes may not beassociated with its effect on TLR3

Antiviral innate immunity depends on different sensorsystems that recognize viral-pathogen-associated molecularpatterns (PAMPs) and affect specific signaling pathwaysincluding those leading to the activation of NF-120581B Recentstudies demonstrate that TLR3 is also present in cells as asensor to recognize the structure of the pathogens and par-ticipate in the adaptive immune response directly [30] TLR3can recognize the dsRNA of viruses initiates intracellularsignal transduction pathway and then induces the activationof NF-120581B [30 31] Activated NF-120581B translocates from cyto-plasm to the nucleus combines with target genes to triggergene transcriptions and increases the production of certain

Evidence-Based Complementary and Alternative Medicine 5

TotalTLR3

120573-Actin

1 2 3 4

(a)

0005

01015

02025

03035

04

Control HSV-1 HSV-1 + APS HSV-1+ TLR3 + APS

Relat

ive i

nten

sity

(TLR

3120573

-act

in)

lowast

998779

(b)

1 2 3 4

NuclearNF-120581B

LMNB1

(c)

0010203040506070809

1

Control HSV-1 HSV-1 + APS HSV-1+ TLR3 + APS

Relat

ive i

nten

sity

(NF-120581

BLM

NB1

)

lowast

998779

(d)

Figure 4 Western blot analysis of expression levels of total TLR3 and nuclear NF-120581B (activated NF-120581B) (a) Expression levels of total TLR3protein in blank control group HSV-1 group HSV-1 + APS group and TLR3 antibody + HSV-1 + APS group 120573-Actin was used as a loadingcontrol (b)The relative intensity of total TLR3 protein as analyzed byWestern blot (c) Expression levels of nuclear NF-120581B protein (activatedNF-120581B) in blank control group HSV-1 group HSV-1 + APS group and TLR3 antibody + HSV-1 + APS group LMNB1 was used as a loadingcontrol (d)The relative intensity of nuclear NF-120581B protein (activated NF-120581B) as analyzed byWestern blot lowast119875 lt 001 versus the HSV-1 groupΔ

119875 lt 005 versus the TLR3 antibody + HSV-1 + APS group The results of Western blot were from a representative of at least three repeatedexperiments 119899 = 3

antiapoptotic proteins and proinflammatory cytokines [26ndash28] Our previous study revealed that when astrocytes wereinfected by HSV-1 the NF-120581B was activated through TLR3and the generation of TNF-120572 and IL-6 increased obviously[5] In this paper we further confirmed that TLR3 and NF-120581B positively contribute to the immune response to HSV-1 infection Moreover we showed that for HSV-1 infectedastrocytes APS could promote the expression of TLR3and the activation of NF-120581B which elicits the secretion ofinflammatory cytokines TNF-120572 and IL-6 indicating that APScan protect astrocytes by promoting immunological functionprovoked by HSV-1 through TLR3NF-120581B pathway

5 Conclusion

In conclusion APS can protect the astrocytes against HSV-1induced proliferation inhibition and enhance the immuno-logical function of astrocytes by upregulating the TLR3NF-120581B signaling pathway along with increasing expression ofTNF-120572 and IL-6The study suggests that APS has potential inthe treatment of HSV-1 infectious diseases in central nervoussystem

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by a Project of Shandong ProvinceHigh Educational Science and Technology Program (noJ12LK04) Traditional Chinese Medicine Science and Tech-nology Research Projects of Shandong Province (nos 2013-140 2013-236 and 2013-240) the Natural Science Foun-dation of Shandong Province (nos ZR2011HL047 andZR2011HL066) and Natural Science Foundation of China(81341137)

References

[1] R N Aravalli S Hu T N Rowen J M Palmquist and J RLokensgard ldquoCutting edge TLR2-mediated proinflammatorycytokine and chemokine production by microglial cells inresponse to herpes simplex virusrdquo Journal of Immunology vol175 no 7 pp 4189ndash4193 2005

6 Evidence-Based Complementary and Alternative Medicine

[2] A G Armien S Hu M R Little et al ldquoChronic corticaland subcortical pathology with associated neurological deficitsensuing experimental herpes encephalitisrdquoBrain Pathology vol20 no 4 pp 738ndash750 2010

[3] C P Marques M-C Cheeran J M Palmquist S Hu SL Urban and J R Lokensgard ldquoProlonged microglial cellactivation and lymphocyte infiltration following experimentalherpes encephalitisrdquoThe Journal of Immunology vol 181 no 9pp 6417ndash6426 2008

[4] J Kavouras E Prandovszky K Valyi-Nagy et al ldquoHerpessimplex virus type 1 infection induces oxidative stress and therelease of bioactive lipid peroxidation by-products in mouseP19N neural cell culturesrdquo Journal of NeuroVirology vol 13 no5 pp 416ndash425 2007

[5] R J Whitley and J W Gnann ldquoViral encephalitis familiarinfections and emerging pathogensrdquo The Lancet vol 359 no9305 pp 507ndash513 2002

[6] J M Thompson and A Iwasaki ldquoToll-like receptors regulationof viral infection and diseaserdquoAdvanced Drug Delivery Reviewsvol 60 no 7 pp 786ndash794 2008

[7] K Tabeta P Georgel E Janssen et al ldquoToll-like receptors 9 and3 as essential complonents of innate immune defense againstmouse cytomegalovirus infectionrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 101 no10 pp 3516ndash3521 2004

[8] K Fukuda T Tsujita M Matsumoto et al ldquoAnalysis of theinteraction between human TLR3 ectodomain and nucleicacidsrdquo Nucleic Acids Symposium Series no 50 pp 249ndash2502006

[9] Z-Y Lu S P Yu J-F Wei and L Wei ldquoAge-related neuraldegeneration in nuclear-factor 120581B p50 knockout micerdquo Neuro-science vol 139 no 3 pp 965ndash978 2006

[10] S Zhang E Jouanguy S Ugolini et al ldquoTLR3 deficiency inpatients with herpes simplex encephalitisrdquo Science vol 317 no5844 pp 1522ndash1527 2007

[11] J Tabiasco E Devevre N Rufer et al ldquoHuman effector CD8+T lymphocytes express TLR3 as a functional coreceptorrdquo TheJournal of Immunology vol 177 no 12 pp 8708ndash8713 2006

[12] C S Jack N Arbour J Manusow et al ldquoTLR signaling tailorsinnate immune responses in human microglia and astrocytesrdquoThe Journal of Immunology vol 175 no 7 pp 4320ndash4330 2005

[13] K R Jessen ldquoGlial cellsrdquo International Journal of Biochemistryand Cell Biology vol 36 no 10 pp 1861ndash1867 2004

[14] W Schabitz C Berger R Kollmar et al ldquoEffect of brain-derivedneurotrophic factor treatment and forced armuse on functionalmotor recovery after small cortical ischemiardquo Stroke vol 35 no4 pp 992ndash997 2004

[15] S R Furr V S Chauhan M J Moerdyk-Schauwecker and IMarriott ldquoA role for DNA-dependent activator of interferonregulatory factor in the recognition of herpes simplex virus type1 by glial cellsrdquo Journal of Neuroinflammation vol 8 article no99 2011

[16] Z Liu Y Guan X Sun et al ldquoHSV-1 activates NF-kappaB inmouse astrocytes and increases TNF-alpha and IL-6 expressionvia Toll-like receptor 3rdquoNeurological Research vol 35 no 7 pp755ndash762 2013

[17] J Li Y Zhong H Li et al ldquoEnhancement of Astragalus polysac-charide on the immune responses in pigs inoculated withfoot-and-mouth disease virus vaccinerdquo International Journal ofBiological Macromolecules vol 49 no 3 pp 362ndash368 2011

[18] X Kong Y Hu R Rui D Wang and X Li ldquoEffects of Chineseherbal medicinal ingredients on peripheral lymphocyte prolif-eration and serum antibody titer after vaccination in chickenrdquoInternational Immunopharmacology vol 4 no 7 pp 975ndash9822004

[19] K Lim S D Jazayeri S K Yeap et al ldquoCo-administration ofavian influenza virus H5 plasmid DNA with chicken IL-15 andIL-18 enhanced chickens immune responsesrdquo BMC VeterinaryResearch vol 8 article 132 2012

[20] H Chen Y Shang H Yao et al ldquoImmune responses of chickensinoculated with a recombinant fowlpox vaccine coexpressingHA of H9N2 avain influenza virus and chicken IL-18rdquo AntiviralResearch vol 91 no 1 pp 50ndash56 2011

[21] K S McNaught and P Jenner ldquoExtracellular accumulation ofnitric oxide hydrogen peroxide and glutamate in astrocyticcultures following glutathione depletion complex I inhibi-tion andor lipopolysaccharide-induced activationrdquo Biochem-ical Pharmacology vol 60 no 7 pp 979ndash988 2000

[22] S E Dorman and S M Holland ldquoInterferon-120574 and interleukin-12 pathway defects and human diseaserdquo Cytokine and GrowthFactor Reviews vol 11 no 4 pp 321ndash333 2000

[23] Z Zhuge Y Zhu P Liu et al ldquoEffects of astragalus polysaccha-ride on immune responses of porcine PBMC stimulated withPRRSV or CSFVrdquo PLoS ONE vol 7 no 1 Article ID e293202012

[24] X Du X Chen B Zhao et al ldquoAstragalus polysaccharidesenhance the humoral and cellular immune responses of hep-atitis B surface antigen vaccination through inhibiting theexpression of transforming growth factor 120573 and the frequencyof regulatory T cellsrdquo FEMS Immunology and Medical Microbi-ology vol 63 no 2 pp 228ndash235 2011

[25] S Makino S Ikegami H Kano et al ldquoImmunomodulatoryeffects of polysaccharides produced by Lactobacillus delbrueckiissp bulgaricus OLL1073R-1rdquo Journal of Dairy Science vol 89no 8 pp 2873ndash2881 2006

[26] M Yang Z Xiao Q Lv et al ldquoThe functional expressionof TLR3 in EPCs impairs cell proliferation by induction ofcell apoptosis and cell cycle progress inhibitionrdquo InternationalImmunopharmacology vol 11 no 12 pp 2118ndash2124 2011

[27] C Meyer R Pries and B Wollenberg ldquoEstablished and novelNF-120581B inhibitors lead to downregulation of TLR3 and theproliferation and cytokine secretion inHNSCCrdquoOralOncologyvol 47 no 9 pp 818ndash826 2011

[28] C J Xu X C Jian F Guo Q P Gao J Y Peng and X PXu ldquoEffect of astragalus polysaccharides on the proliferationand ultrastructure of dog bone marrow stem cells induced intoosteoblasts in vitrordquo Hua Xi Kou Qiang Yi Xue Za Zhi vol 25no 5 pp 432ndash436 2007

[29] Y LiuW JWangW H Chen and J Yin ldquoEffects of Astragaluspolysaccharides on proliferation and differentiation of 3T3-L1preadipocytesrdquo Zhong Xi Yi Jie He Xue Bao vol 5 no 4 pp421ndash426 2007

[30] K A Fitzgerald S M McWhirter K L Faia et al ldquoIKKE andTBKI are essential components of the IRF3 signalling pathwayrdquoNature Immunology vol 4 no 5 pp 491ndash496 2003

[31] Y Y Wang L Li K J Han Z Zhai and H B Shu ldquoA20 is apotent inhibitor of TLR3- and Sendai virus-induced activationof NF-120581B and ISRE and IFN-120573 promoterrdquo FEBS Letters vol 576no 1-2 pp 86ndash90 2004

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Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

2 Evidence-Based Complementary and Alternative Medicine

that when astrocytes were infected by HSV-1 NF-120581B wasactivated through Toll-like receptor 3 (TLR3) and then theactivated NF-120581B would translocate from the cytoplasm tothe nucleus so as to promote the production of TNF-120572 andIL-6 to play antiviral roles [16] These results demonstratethat astrocytes play significant roles in the inflammatoryresponses of resident CNS cells to HSV-1 challenge

Astragalus is a traditional Chinese medicine which con-tains polysaccharides saponins flavonoids amino acidslinoleic acid alkaloids and so forth Astragalus polysac-charide (APS) is the most immunoreactive substance inAstragalus which can regulate the body immunity APShas been identified as a class of macromolecules that canprofoundly affect the immune system and is widely used asan immune adjuvant inChina APS can induce the expressionof surface antigens on lymphocytes promote the productionof antibodies affect the secretion of cytokines and evenstimulate cell proliferation [17 18] Previous studies provedthe effective immunostimulatory roles of APS against variousviruses [17 19 20]

In this paper based on our previous research the antiviraleffect of APS on the HSV-1 infected astrocytes was inves-tigated Furthermore the immunoregulatory effect and thepossible immunization mechanisms of APS were evaluated

2 Materials and Methods

21 Laboratory Animals Cells and Virus The BALBc micewere purchased from Medicine Animal Center of ShandongUniversity HSV-1 SM44 strain was kept in Central Labo-ratory of Weifang Medical University at minus80∘C The rabbitanti-mouse antibody TLR3 NF-120581B and 120573-actin were fromInvitrogen Nuclear and Cytoplasmic Protein Extraction Kitwere from Beyotime Institute of Biotechnology MTT kit waspurchased from Sigma 96T enzyme-linked immunosorbentassay (ELISA) kit was purchased from ADL APS was pur-chased from Tianjin Sino Pharmaceutical Company batchnumber is 120102

This research was conducted in strict accordance withthe recommendations in the Guide for the Care and Useof Laboratory Animals of the National Institutes of HealthSurgery was performed under sodium pentobarbital anesthe-sia all efforts were made to minimize suffering The protocolwas approved by the Committee on the Ethics of AnimalExperiments of Weifang Medical University (number 2012-056)

22 HSV-1 Multiplication and Tittering The HSV-1 strainSM44 propagated in Vero cells the supernatant was collectedwhen cytopathic effect (CPE) was in confluence to 75By freezing in minus80∘C and thawing for three times HSV-1 particles were released By centrifuging for 8min with1200 rpm the supernatant was collected as inoculum andstocked in freezer All of the HSV-1 samples used in thisstudy were of the same batch In following research finalconcentration of HSV-1 TCID

50was 10minus6mLminus1

23 Astrocytes Culture and Purification Newborn BALBcmice in day 1 to day 3 were taken and cells culture and purifi-cation were performed according to McNaughtrsquos protocol[21] Take the 3rd generation of astrocytes for experimentalresearch according to our teamrsquos protocol [16]

24 MTT Cell Proliferation Assay Calculating the MedianEffective Concentration (EC

50) of APS Astrocytes (1 times

106mLminus1) were cultured inDMEMF12media in the presenceof HSV-1 (final TCID

50 10minus6mLminus1) with APS (0 50 100

200 and 300120583gmLminus1) 5 repeating groups for each con-centration 20120583L MTT solution (5mg mLminus1) was added tocell media at various time points (6 12 18 and 24 h) afterthe addition of HSV-1 and APS incubated for 4 h and thensupernatants were gently removed and 200120583L dimethyl sul-foxide was added into each pore After 10 minutesrsquo vibrationthe absorbance (OD value) was measured at a wavelengthof 492 nm by enzyme-linked immunosorbent detector ODvalue is proportional to the proliferation of living cells and isan indicator of cell proliferation

The mean and standard deviation of OD values foreach concentration of APS at every action time point werecalculated The results showed that cell proliferation is themost exuberant at 12 h time point for every concentration ofAPS so 12 h is the detection timepoint in the following assaysEC50ofAPSwas 120120583gmLminus1 so in the following research the

final concentration of APS was 120120583gmLminus1 The formula tocalculated EC

50is

effect =ODdrug minusODmodel

ODcontrol minusODmodeltimes 100 (1)

25 Grouping and Detecting the Effect of APS on AstrocytesProliferation by MTT Assay Astrocytes were seeded at den-sity of 1 times 106mLminus1 into 24-well plates four groups were setup according to the different intervention conditions HSV-1group HSV-1 + APS group TLR3 antibody + HSV-1 + APSgroup and blank control group In HSV-1 groups all flaskcells were inoculated with viral suspension (final TCID

50

10minus6mLminus1) in HSV-1 + APS group all flask cells were inoc-ulated with viral suspension (final TCID

50 10minus6mLminus1) and

APS (final concentration 120120583gmLminus1) in TLR3 antibody+ HSV-1 + APS group TLR3 antibody (final concentration10 120583gmLminus1) was used to pretreat cells for 30min and thenviral suspension (final TCID

50 10minus6mLminus1) and APS (final

concentration 120120583gmLminus1) were added to cells in blankcontrol group the same volume of cell culture medium asthe aforementioned groups was added into each flask cellsFor each group 3 repeating experiments were performedTheproliferation rate of cells was assayed by MTT after culturingfor 12 h

26 Determination of the Levels of TNF-120572 and IL-6 by ELISAAstrocytes were seeded at density of 1 times 106mLminus1 into 12flasks (25 cm2) Grouping and treatment were performedas previously mentioned Supernatants were collected andfiltered TNF-120572 and IL-6 in the supernatant were measured

Evidence-Based Complementary and Alternative Medicine 3

20120583m

(a)

20120583m

(b)

20120583m

(c)

20120583m

(d)

Figure 1 Effect of APS on the growth and proliferation of astrocytes (a) Blank control group the astrocytes grew in good condition withactive proliferation (b)HSV-1 group 12 h after infectionwithHSV-1 the proliferation of astrocytes was significantly inhibited and the infectedastrocytesrsquo bodies were gradually swollen into round and giant appearance (c)HSV-1 +APS group the proliferation inhibitory effect ofHSV-1could be suppressed by APS and most astrocytes grew in good conditiosn (d) TLR3 antibody + HSV-1 + APS group when astrocytes werepretreatedwith TLR3 antibody the proliferation of astrocytes reducedmarkedly and some infected astrocytesrsquo bodies were swollen into roundappearance Scale bar 20 120583m

by ELISAThe absorbance (OD value) was determined usinga microplate reader at a wavelength of 450 nm For eachsample the measurement was repeated 3 times and theaverage concentration of TNF-120572 and IL-6 was set as the finalresult

27 Detection of TLR3 Protein in Cells and NF-120581B Proteinin Cell Nuclei by Western Blot In brief total protein andnuclear protein were extracted following the reagent com-panyrsquos instruction Protein samples were separated by SDS-PAGE transferred to polyvinylidene difluoride membranesimmunoblotted using the appropriate primary and the HRPconjugated secondary antibodies and visualized by usingenhanced chemiluminescence reagents ECL Anti-120573-actin orLMNB1 monoclonal antibody was used as loading controlThe intensities of bands in Western blots were quantifiedby densitometry analysis using AlphaImager HP (AlphaInnotech USA) and NIH Image J software (Rockville MDUSA) Western blot data shown in the paper are representa-tives of three independent experiments

28 Statistical Analysis All data were analyzed using theSPSS 130 statistical software Values were expressed as meanplusmn standard deviation (119883 plusmn 119878) The significance of differences

between groups was determined using the one-way ANOVAStatistical significance was accepted for 119875 values lt005

3 Result

APS promotes the growth and proliferation of astrocytesinfected by HSV-1 Observation under microscope showedthat in the blank control group the uninfected astrocyteswere in thin and flat appearance with good refraction andgrew in good conditionwith active proliferation (Figure 1(a))in the HSV-1 group the proliferation of astrocytes wassignificantly inhibited and the infected astrocytesrsquo bodieswere gradually swollen into round and giant appearance(Figure 1(b)) the inhibited proliferation of astrocytes infectedbyHSV-1 could be rescued byAPS apparently inHSV-1 +APSgroup (Figure 1(c)) when astrocytes were pretreated withTLR3 antibody and then exposed to HSV-1 and APS con-currently the proliferation of astrocytes reduced markedlycompared with the HSV-1 + APS group (Figure 1(d))

MTT analysis (Figure 2) showed that when astrocyteswere exposed to HSV-1 the proliferation of astrocytes wassignificantly inhibited compared to the blank control groupThe inhibited proliferation of astrocytes infected by HSV-1 could be rescued by APS apparently in the HSV-1 + APS

4 Evidence-Based Complementary and Alternative Medicine

Control HSV-1 HSV-1 + APS HSV-1+ TLR3 + APS

lowast

08

07

06

05

04

03

02

01

0

OD

998779

Figure 2 Astrocytes proliferation detected by MTT lowast119875 lt 001versus the HSV-1 group Δ119875 lt 005 versus the TLR3 antibody +HSV-1 + APS group 119899 = 3

group In the presence of APS the proliferation of astrocytesincreased to some extent and the OD value of HSV-1 +APS group was greater than that of the HSV-1 group (119875 lt001) which suggests that APS can protect astrocytes fromHSV-1 induced proliferation inhibition Interestingly whenastrocytes were pretreated with TLR3 antibody before addingHSV-1 and APS the proliferation of astrocytes decreasedmarkedly when compared to the HSV-1 + APS group (119875 lt005) This result indicates that the protective effect ofAPS against HSV-1 infection may be associated with TLR3pathway

Secretion levels of TNF-120572 and IL-6 in culture supernatantwere detected by ELISA (Figure 3) The concentrations ofTNF-120572 and IL-6 were very low in culture supernatant of theblank control group whereas in culture supernatant of theHSV-1 group the concentrations of both TNF-120572 and IL-6increased obviously (119875 lt 001) In the presence of APS HSV-1 infected astrocytes expressed higher amount of TNF-120572 andIL-6 than that of the HSV-1 group Remarkably pretreatmentof astrocytes with TLR3 antibody decreased the expression ofTNF-120572 and IL-6 in the TLR3 antibody + HSV-1 + APS group(119875 lt 005)

Western blot analysis showed that the expression level ofTLR3 and the activation of NF-120581B were low in the controlgroup (Figure 4) In the HSV-1 group after being exposedto HSV-1 for 12 h astrocytes expressed higher level of TLR3than the control group At the same time HSV-1 infectioninduced the activation of NF-120581B significantly Moreover inthe presence of APS the expression level of TLR3 and theactivation level of NF-120581B were obviously higher than those ofthe HSV-1 group However when astrocytes were pretreatedwith TLR3 antibody before addition of HSV-1 and APSastrocytes reduced the expression of TLR3 and the activationof NF-120581B significantly Considering the different secretionlevels of TNF-120572 and IL-6 in different groups these resultsindicate that APS may activate NF-120581B which leads to theproduction of TNF-120572 and IL-6 through TLR3 pathway (119875 lt005)

4 Discussion

Recent studies have confirmed thatmany herbs have effects ofmodulating immunity and inhibiting and killing pathogenic

05

10152025303540

Control HSV-1 HSV-1 + APS HSV-1+ TLR3 + APS

lowast

lowast

TNF-120572IL-6

TNF-120572

and

IL-6

(ng

mL)

998779

998779

Figure 3 Concentration of TNF-120572 and IL-6 (ngmLminus1) in cellculture medium detected by ELISA lowast119875 lt 001 versus the HSV-1group Δ119875 lt 005 versus the TLR3 antibody + HSV-1 + APS group119899 = 3

microorganisms [22 23] As one of the bioactive ingredientsfrom the natural traditional Chinese medicinal herb Astra-galus membranaceus APS is used widely as an immunomod-ulator in China Evidences indicate that APS can enhancelymphocyte blastogenesis and stimulate macrophage activa-tion without cytotoxic effects [7 8] APS cannot inhibit virusdirectly but it can activate the immune system and inducethe production of cytokines to initiate an antiviral response[24 25] Consistent with these previous reports the currentstudy showed that HSV-1 infection induced the secretionof TNF-120572 and IL-6 in astrocytes and the secretion notablyincreased in response to APSThese results indicate that APScan promote immunomodulatory effects of astrocytes

In addition we also found that HSV-1 infection induceda higher expression of TLR3 and decreased the proliferationof astrocytes these results are in accordance with previousreports which showed that high expression of TLR3 impairedcell proliferation and inhibited cell cycle progress [26 27]Furthermore we found that APS was capable of promotingthe proliferation of astrocytes infected by HSV-1 This resultsquares with what we already know about the role of APSon cell proliferation [18 28 29] Although the potentialmechanism is still to be clarified regarding the currentunderstanding of TLR3 signaling function we infer thatthe effect of APS on proliferation of astrocytes may not beassociated with its effect on TLR3

Antiviral innate immunity depends on different sensorsystems that recognize viral-pathogen-associated molecularpatterns (PAMPs) and affect specific signaling pathwaysincluding those leading to the activation of NF-120581B Recentstudies demonstrate that TLR3 is also present in cells as asensor to recognize the structure of the pathogens and par-ticipate in the adaptive immune response directly [30] TLR3can recognize the dsRNA of viruses initiates intracellularsignal transduction pathway and then induces the activationof NF-120581B [30 31] Activated NF-120581B translocates from cyto-plasm to the nucleus combines with target genes to triggergene transcriptions and increases the production of certain

Evidence-Based Complementary and Alternative Medicine 5

TotalTLR3

120573-Actin

1 2 3 4

(a)

0005

01015

02025

03035

04

Control HSV-1 HSV-1 + APS HSV-1+ TLR3 + APS

Relat

ive i

nten

sity

(TLR

3120573

-act

in)

lowast

998779

(b)

1 2 3 4

NuclearNF-120581B

LMNB1

(c)

0010203040506070809

1

Control HSV-1 HSV-1 + APS HSV-1+ TLR3 + APS

Relat

ive i

nten

sity

(NF-120581

BLM

NB1

)

lowast

998779

(d)

Figure 4 Western blot analysis of expression levels of total TLR3 and nuclear NF-120581B (activated NF-120581B) (a) Expression levels of total TLR3protein in blank control group HSV-1 group HSV-1 + APS group and TLR3 antibody + HSV-1 + APS group 120573-Actin was used as a loadingcontrol (b)The relative intensity of total TLR3 protein as analyzed byWestern blot (c) Expression levels of nuclear NF-120581B protein (activatedNF-120581B) in blank control group HSV-1 group HSV-1 + APS group and TLR3 antibody + HSV-1 + APS group LMNB1 was used as a loadingcontrol (d)The relative intensity of nuclear NF-120581B protein (activated NF-120581B) as analyzed byWestern blot lowast119875 lt 001 versus the HSV-1 groupΔ

119875 lt 005 versus the TLR3 antibody + HSV-1 + APS group The results of Western blot were from a representative of at least three repeatedexperiments 119899 = 3

antiapoptotic proteins and proinflammatory cytokines [26ndash28] Our previous study revealed that when astrocytes wereinfected by HSV-1 the NF-120581B was activated through TLR3and the generation of TNF-120572 and IL-6 increased obviously[5] In this paper we further confirmed that TLR3 and NF-120581B positively contribute to the immune response to HSV-1 infection Moreover we showed that for HSV-1 infectedastrocytes APS could promote the expression of TLR3and the activation of NF-120581B which elicits the secretion ofinflammatory cytokines TNF-120572 and IL-6 indicating that APScan protect astrocytes by promoting immunological functionprovoked by HSV-1 through TLR3NF-120581B pathway

5 Conclusion

In conclusion APS can protect the astrocytes against HSV-1induced proliferation inhibition and enhance the immuno-logical function of astrocytes by upregulating the TLR3NF-120581B signaling pathway along with increasing expression ofTNF-120572 and IL-6The study suggests that APS has potential inthe treatment of HSV-1 infectious diseases in central nervoussystem

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by a Project of Shandong ProvinceHigh Educational Science and Technology Program (noJ12LK04) Traditional Chinese Medicine Science and Tech-nology Research Projects of Shandong Province (nos 2013-140 2013-236 and 2013-240) the Natural Science Foun-dation of Shandong Province (nos ZR2011HL047 andZR2011HL066) and Natural Science Foundation of China(81341137)

References

[1] R N Aravalli S Hu T N Rowen J M Palmquist and J RLokensgard ldquoCutting edge TLR2-mediated proinflammatorycytokine and chemokine production by microglial cells inresponse to herpes simplex virusrdquo Journal of Immunology vol175 no 7 pp 4189ndash4193 2005

6 Evidence-Based Complementary and Alternative Medicine

[2] A G Armien S Hu M R Little et al ldquoChronic corticaland subcortical pathology with associated neurological deficitsensuing experimental herpes encephalitisrdquoBrain Pathology vol20 no 4 pp 738ndash750 2010

[3] C P Marques M-C Cheeran J M Palmquist S Hu SL Urban and J R Lokensgard ldquoProlonged microglial cellactivation and lymphocyte infiltration following experimentalherpes encephalitisrdquoThe Journal of Immunology vol 181 no 9pp 6417ndash6426 2008

[4] J Kavouras E Prandovszky K Valyi-Nagy et al ldquoHerpessimplex virus type 1 infection induces oxidative stress and therelease of bioactive lipid peroxidation by-products in mouseP19N neural cell culturesrdquo Journal of NeuroVirology vol 13 no5 pp 416ndash425 2007

[5] R J Whitley and J W Gnann ldquoViral encephalitis familiarinfections and emerging pathogensrdquo The Lancet vol 359 no9305 pp 507ndash513 2002

[6] J M Thompson and A Iwasaki ldquoToll-like receptors regulationof viral infection and diseaserdquoAdvanced Drug Delivery Reviewsvol 60 no 7 pp 786ndash794 2008

[7] K Tabeta P Georgel E Janssen et al ldquoToll-like receptors 9 and3 as essential complonents of innate immune defense againstmouse cytomegalovirus infectionrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 101 no10 pp 3516ndash3521 2004

[8] K Fukuda T Tsujita M Matsumoto et al ldquoAnalysis of theinteraction between human TLR3 ectodomain and nucleicacidsrdquo Nucleic Acids Symposium Series no 50 pp 249ndash2502006

[9] Z-Y Lu S P Yu J-F Wei and L Wei ldquoAge-related neuraldegeneration in nuclear-factor 120581B p50 knockout micerdquo Neuro-science vol 139 no 3 pp 965ndash978 2006

[10] S Zhang E Jouanguy S Ugolini et al ldquoTLR3 deficiency inpatients with herpes simplex encephalitisrdquo Science vol 317 no5844 pp 1522ndash1527 2007

[11] J Tabiasco E Devevre N Rufer et al ldquoHuman effector CD8+T lymphocytes express TLR3 as a functional coreceptorrdquo TheJournal of Immunology vol 177 no 12 pp 8708ndash8713 2006

[12] C S Jack N Arbour J Manusow et al ldquoTLR signaling tailorsinnate immune responses in human microglia and astrocytesrdquoThe Journal of Immunology vol 175 no 7 pp 4320ndash4330 2005

[13] K R Jessen ldquoGlial cellsrdquo International Journal of Biochemistryand Cell Biology vol 36 no 10 pp 1861ndash1867 2004

[14] W Schabitz C Berger R Kollmar et al ldquoEffect of brain-derivedneurotrophic factor treatment and forced armuse on functionalmotor recovery after small cortical ischemiardquo Stroke vol 35 no4 pp 992ndash997 2004

[15] S R Furr V S Chauhan M J Moerdyk-Schauwecker and IMarriott ldquoA role for DNA-dependent activator of interferonregulatory factor in the recognition of herpes simplex virus type1 by glial cellsrdquo Journal of Neuroinflammation vol 8 article no99 2011

[16] Z Liu Y Guan X Sun et al ldquoHSV-1 activates NF-kappaB inmouse astrocytes and increases TNF-alpha and IL-6 expressionvia Toll-like receptor 3rdquoNeurological Research vol 35 no 7 pp755ndash762 2013

[17] J Li Y Zhong H Li et al ldquoEnhancement of Astragalus polysac-charide on the immune responses in pigs inoculated withfoot-and-mouth disease virus vaccinerdquo International Journal ofBiological Macromolecules vol 49 no 3 pp 362ndash368 2011

[18] X Kong Y Hu R Rui D Wang and X Li ldquoEffects of Chineseherbal medicinal ingredients on peripheral lymphocyte prolif-eration and serum antibody titer after vaccination in chickenrdquoInternational Immunopharmacology vol 4 no 7 pp 975ndash9822004

[19] K Lim S D Jazayeri S K Yeap et al ldquoCo-administration ofavian influenza virus H5 plasmid DNA with chicken IL-15 andIL-18 enhanced chickens immune responsesrdquo BMC VeterinaryResearch vol 8 article 132 2012

[20] H Chen Y Shang H Yao et al ldquoImmune responses of chickensinoculated with a recombinant fowlpox vaccine coexpressingHA of H9N2 avain influenza virus and chicken IL-18rdquo AntiviralResearch vol 91 no 1 pp 50ndash56 2011

[21] K S McNaught and P Jenner ldquoExtracellular accumulation ofnitric oxide hydrogen peroxide and glutamate in astrocyticcultures following glutathione depletion complex I inhibi-tion andor lipopolysaccharide-induced activationrdquo Biochem-ical Pharmacology vol 60 no 7 pp 979ndash988 2000

[22] S E Dorman and S M Holland ldquoInterferon-120574 and interleukin-12 pathway defects and human diseaserdquo Cytokine and GrowthFactor Reviews vol 11 no 4 pp 321ndash333 2000

[23] Z Zhuge Y Zhu P Liu et al ldquoEffects of astragalus polysaccha-ride on immune responses of porcine PBMC stimulated withPRRSV or CSFVrdquo PLoS ONE vol 7 no 1 Article ID e293202012

[24] X Du X Chen B Zhao et al ldquoAstragalus polysaccharidesenhance the humoral and cellular immune responses of hep-atitis B surface antigen vaccination through inhibiting theexpression of transforming growth factor 120573 and the frequencyof regulatory T cellsrdquo FEMS Immunology and Medical Microbi-ology vol 63 no 2 pp 228ndash235 2011

[25] S Makino S Ikegami H Kano et al ldquoImmunomodulatoryeffects of polysaccharides produced by Lactobacillus delbrueckiissp bulgaricus OLL1073R-1rdquo Journal of Dairy Science vol 89no 8 pp 2873ndash2881 2006

[26] M Yang Z Xiao Q Lv et al ldquoThe functional expressionof TLR3 in EPCs impairs cell proliferation by induction ofcell apoptosis and cell cycle progress inhibitionrdquo InternationalImmunopharmacology vol 11 no 12 pp 2118ndash2124 2011

[27] C Meyer R Pries and B Wollenberg ldquoEstablished and novelNF-120581B inhibitors lead to downregulation of TLR3 and theproliferation and cytokine secretion inHNSCCrdquoOralOncologyvol 47 no 9 pp 818ndash826 2011

[28] C J Xu X C Jian F Guo Q P Gao J Y Peng and X PXu ldquoEffect of astragalus polysaccharides on the proliferationand ultrastructure of dog bone marrow stem cells induced intoosteoblasts in vitrordquo Hua Xi Kou Qiang Yi Xue Za Zhi vol 25no 5 pp 432ndash436 2007

[29] Y LiuW JWangW H Chen and J Yin ldquoEffects of Astragaluspolysaccharides on proliferation and differentiation of 3T3-L1preadipocytesrdquo Zhong Xi Yi Jie He Xue Bao vol 5 no 4 pp421ndash426 2007

[30] K A Fitzgerald S M McWhirter K L Faia et al ldquoIKKE andTBKI are essential components of the IRF3 signalling pathwayrdquoNature Immunology vol 4 no 5 pp 491ndash496 2003

[31] Y Y Wang L Li K J Han Z Zhai and H B Shu ldquoA20 is apotent inhibitor of TLR3- and Sendai virus-induced activationof NF-120581B and ISRE and IFN-120573 promoterrdquo FEBS Letters vol 576no 1-2 pp 86ndash90 2004

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 3

20120583m

(a)

20120583m

(b)

20120583m

(c)

20120583m

(d)

Figure 1 Effect of APS on the growth and proliferation of astrocytes (a) Blank control group the astrocytes grew in good condition withactive proliferation (b)HSV-1 group 12 h after infectionwithHSV-1 the proliferation of astrocytes was significantly inhibited and the infectedastrocytesrsquo bodies were gradually swollen into round and giant appearance (c)HSV-1 +APS group the proliferation inhibitory effect ofHSV-1could be suppressed by APS and most astrocytes grew in good conditiosn (d) TLR3 antibody + HSV-1 + APS group when astrocytes werepretreatedwith TLR3 antibody the proliferation of astrocytes reducedmarkedly and some infected astrocytesrsquo bodies were swollen into roundappearance Scale bar 20 120583m

by ELISAThe absorbance (OD value) was determined usinga microplate reader at a wavelength of 450 nm For eachsample the measurement was repeated 3 times and theaverage concentration of TNF-120572 and IL-6 was set as the finalresult

27 Detection of TLR3 Protein in Cells and NF-120581B Proteinin Cell Nuclei by Western Blot In brief total protein andnuclear protein were extracted following the reagent com-panyrsquos instruction Protein samples were separated by SDS-PAGE transferred to polyvinylidene difluoride membranesimmunoblotted using the appropriate primary and the HRPconjugated secondary antibodies and visualized by usingenhanced chemiluminescence reagents ECL Anti-120573-actin orLMNB1 monoclonal antibody was used as loading controlThe intensities of bands in Western blots were quantifiedby densitometry analysis using AlphaImager HP (AlphaInnotech USA) and NIH Image J software (Rockville MDUSA) Western blot data shown in the paper are representa-tives of three independent experiments

28 Statistical Analysis All data were analyzed using theSPSS 130 statistical software Values were expressed as meanplusmn standard deviation (119883 plusmn 119878) The significance of differences

between groups was determined using the one-way ANOVAStatistical significance was accepted for 119875 values lt005

3 Result

APS promotes the growth and proliferation of astrocytesinfected by HSV-1 Observation under microscope showedthat in the blank control group the uninfected astrocyteswere in thin and flat appearance with good refraction andgrew in good conditionwith active proliferation (Figure 1(a))in the HSV-1 group the proliferation of astrocytes wassignificantly inhibited and the infected astrocytesrsquo bodieswere gradually swollen into round and giant appearance(Figure 1(b)) the inhibited proliferation of astrocytes infectedbyHSV-1 could be rescued byAPS apparently inHSV-1 +APSgroup (Figure 1(c)) when astrocytes were pretreated withTLR3 antibody and then exposed to HSV-1 and APS con-currently the proliferation of astrocytes reduced markedlycompared with the HSV-1 + APS group (Figure 1(d))

MTT analysis (Figure 2) showed that when astrocyteswere exposed to HSV-1 the proliferation of astrocytes wassignificantly inhibited compared to the blank control groupThe inhibited proliferation of astrocytes infected by HSV-1 could be rescued by APS apparently in the HSV-1 + APS

4 Evidence-Based Complementary and Alternative Medicine

Control HSV-1 HSV-1 + APS HSV-1+ TLR3 + APS

lowast

08

07

06

05

04

03

02

01

0

OD

998779

Figure 2 Astrocytes proliferation detected by MTT lowast119875 lt 001versus the HSV-1 group Δ119875 lt 005 versus the TLR3 antibody +HSV-1 + APS group 119899 = 3

group In the presence of APS the proliferation of astrocytesincreased to some extent and the OD value of HSV-1 +APS group was greater than that of the HSV-1 group (119875 lt001) which suggests that APS can protect astrocytes fromHSV-1 induced proliferation inhibition Interestingly whenastrocytes were pretreated with TLR3 antibody before addingHSV-1 and APS the proliferation of astrocytes decreasedmarkedly when compared to the HSV-1 + APS group (119875 lt005) This result indicates that the protective effect ofAPS against HSV-1 infection may be associated with TLR3pathway

Secretion levels of TNF-120572 and IL-6 in culture supernatantwere detected by ELISA (Figure 3) The concentrations ofTNF-120572 and IL-6 were very low in culture supernatant of theblank control group whereas in culture supernatant of theHSV-1 group the concentrations of both TNF-120572 and IL-6increased obviously (119875 lt 001) In the presence of APS HSV-1 infected astrocytes expressed higher amount of TNF-120572 andIL-6 than that of the HSV-1 group Remarkably pretreatmentof astrocytes with TLR3 antibody decreased the expression ofTNF-120572 and IL-6 in the TLR3 antibody + HSV-1 + APS group(119875 lt 005)

Western blot analysis showed that the expression level ofTLR3 and the activation of NF-120581B were low in the controlgroup (Figure 4) In the HSV-1 group after being exposedto HSV-1 for 12 h astrocytes expressed higher level of TLR3than the control group At the same time HSV-1 infectioninduced the activation of NF-120581B significantly Moreover inthe presence of APS the expression level of TLR3 and theactivation level of NF-120581B were obviously higher than those ofthe HSV-1 group However when astrocytes were pretreatedwith TLR3 antibody before addition of HSV-1 and APSastrocytes reduced the expression of TLR3 and the activationof NF-120581B significantly Considering the different secretionlevels of TNF-120572 and IL-6 in different groups these resultsindicate that APS may activate NF-120581B which leads to theproduction of TNF-120572 and IL-6 through TLR3 pathway (119875 lt005)

4 Discussion

Recent studies have confirmed thatmany herbs have effects ofmodulating immunity and inhibiting and killing pathogenic

05

10152025303540

Control HSV-1 HSV-1 + APS HSV-1+ TLR3 + APS

lowast

lowast

TNF-120572IL-6

TNF-120572

and

IL-6

(ng

mL)

998779

998779

Figure 3 Concentration of TNF-120572 and IL-6 (ngmLminus1) in cellculture medium detected by ELISA lowast119875 lt 001 versus the HSV-1group Δ119875 lt 005 versus the TLR3 antibody + HSV-1 + APS group119899 = 3

microorganisms [22 23] As one of the bioactive ingredientsfrom the natural traditional Chinese medicinal herb Astra-galus membranaceus APS is used widely as an immunomod-ulator in China Evidences indicate that APS can enhancelymphocyte blastogenesis and stimulate macrophage activa-tion without cytotoxic effects [7 8] APS cannot inhibit virusdirectly but it can activate the immune system and inducethe production of cytokines to initiate an antiviral response[24 25] Consistent with these previous reports the currentstudy showed that HSV-1 infection induced the secretionof TNF-120572 and IL-6 in astrocytes and the secretion notablyincreased in response to APSThese results indicate that APScan promote immunomodulatory effects of astrocytes

In addition we also found that HSV-1 infection induceda higher expression of TLR3 and decreased the proliferationof astrocytes these results are in accordance with previousreports which showed that high expression of TLR3 impairedcell proliferation and inhibited cell cycle progress [26 27]Furthermore we found that APS was capable of promotingthe proliferation of astrocytes infected by HSV-1 This resultsquares with what we already know about the role of APSon cell proliferation [18 28 29] Although the potentialmechanism is still to be clarified regarding the currentunderstanding of TLR3 signaling function we infer thatthe effect of APS on proliferation of astrocytes may not beassociated with its effect on TLR3

Antiviral innate immunity depends on different sensorsystems that recognize viral-pathogen-associated molecularpatterns (PAMPs) and affect specific signaling pathwaysincluding those leading to the activation of NF-120581B Recentstudies demonstrate that TLR3 is also present in cells as asensor to recognize the structure of the pathogens and par-ticipate in the adaptive immune response directly [30] TLR3can recognize the dsRNA of viruses initiates intracellularsignal transduction pathway and then induces the activationof NF-120581B [30 31] Activated NF-120581B translocates from cyto-plasm to the nucleus combines with target genes to triggergene transcriptions and increases the production of certain

Evidence-Based Complementary and Alternative Medicine 5

TotalTLR3

120573-Actin

1 2 3 4

(a)

0005

01015

02025

03035

04

Control HSV-1 HSV-1 + APS HSV-1+ TLR3 + APS

Relat

ive i

nten

sity

(TLR

3120573

-act

in)

lowast

998779

(b)

1 2 3 4

NuclearNF-120581B

LMNB1

(c)

0010203040506070809

1

Control HSV-1 HSV-1 + APS HSV-1+ TLR3 + APS

Relat

ive i

nten

sity

(NF-120581

BLM

NB1

)

lowast

998779

(d)

Figure 4 Western blot analysis of expression levels of total TLR3 and nuclear NF-120581B (activated NF-120581B) (a) Expression levels of total TLR3protein in blank control group HSV-1 group HSV-1 + APS group and TLR3 antibody + HSV-1 + APS group 120573-Actin was used as a loadingcontrol (b)The relative intensity of total TLR3 protein as analyzed byWestern blot (c) Expression levels of nuclear NF-120581B protein (activatedNF-120581B) in blank control group HSV-1 group HSV-1 + APS group and TLR3 antibody + HSV-1 + APS group LMNB1 was used as a loadingcontrol (d)The relative intensity of nuclear NF-120581B protein (activated NF-120581B) as analyzed byWestern blot lowast119875 lt 001 versus the HSV-1 groupΔ

119875 lt 005 versus the TLR3 antibody + HSV-1 + APS group The results of Western blot were from a representative of at least three repeatedexperiments 119899 = 3

antiapoptotic proteins and proinflammatory cytokines [26ndash28] Our previous study revealed that when astrocytes wereinfected by HSV-1 the NF-120581B was activated through TLR3and the generation of TNF-120572 and IL-6 increased obviously[5] In this paper we further confirmed that TLR3 and NF-120581B positively contribute to the immune response to HSV-1 infection Moreover we showed that for HSV-1 infectedastrocytes APS could promote the expression of TLR3and the activation of NF-120581B which elicits the secretion ofinflammatory cytokines TNF-120572 and IL-6 indicating that APScan protect astrocytes by promoting immunological functionprovoked by HSV-1 through TLR3NF-120581B pathway

5 Conclusion

In conclusion APS can protect the astrocytes against HSV-1induced proliferation inhibition and enhance the immuno-logical function of astrocytes by upregulating the TLR3NF-120581B signaling pathway along with increasing expression ofTNF-120572 and IL-6The study suggests that APS has potential inthe treatment of HSV-1 infectious diseases in central nervoussystem

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by a Project of Shandong ProvinceHigh Educational Science and Technology Program (noJ12LK04) Traditional Chinese Medicine Science and Tech-nology Research Projects of Shandong Province (nos 2013-140 2013-236 and 2013-240) the Natural Science Foun-dation of Shandong Province (nos ZR2011HL047 andZR2011HL066) and Natural Science Foundation of China(81341137)

References

[1] R N Aravalli S Hu T N Rowen J M Palmquist and J RLokensgard ldquoCutting edge TLR2-mediated proinflammatorycytokine and chemokine production by microglial cells inresponse to herpes simplex virusrdquo Journal of Immunology vol175 no 7 pp 4189ndash4193 2005

6 Evidence-Based Complementary and Alternative Medicine

[2] A G Armien S Hu M R Little et al ldquoChronic corticaland subcortical pathology with associated neurological deficitsensuing experimental herpes encephalitisrdquoBrain Pathology vol20 no 4 pp 738ndash750 2010

[3] C P Marques M-C Cheeran J M Palmquist S Hu SL Urban and J R Lokensgard ldquoProlonged microglial cellactivation and lymphocyte infiltration following experimentalherpes encephalitisrdquoThe Journal of Immunology vol 181 no 9pp 6417ndash6426 2008

[4] J Kavouras E Prandovszky K Valyi-Nagy et al ldquoHerpessimplex virus type 1 infection induces oxidative stress and therelease of bioactive lipid peroxidation by-products in mouseP19N neural cell culturesrdquo Journal of NeuroVirology vol 13 no5 pp 416ndash425 2007

[5] R J Whitley and J W Gnann ldquoViral encephalitis familiarinfections and emerging pathogensrdquo The Lancet vol 359 no9305 pp 507ndash513 2002

[6] J M Thompson and A Iwasaki ldquoToll-like receptors regulationof viral infection and diseaserdquoAdvanced Drug Delivery Reviewsvol 60 no 7 pp 786ndash794 2008

[7] K Tabeta P Georgel E Janssen et al ldquoToll-like receptors 9 and3 as essential complonents of innate immune defense againstmouse cytomegalovirus infectionrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 101 no10 pp 3516ndash3521 2004

[8] K Fukuda T Tsujita M Matsumoto et al ldquoAnalysis of theinteraction between human TLR3 ectodomain and nucleicacidsrdquo Nucleic Acids Symposium Series no 50 pp 249ndash2502006

[9] Z-Y Lu S P Yu J-F Wei and L Wei ldquoAge-related neuraldegeneration in nuclear-factor 120581B p50 knockout micerdquo Neuro-science vol 139 no 3 pp 965ndash978 2006

[10] S Zhang E Jouanguy S Ugolini et al ldquoTLR3 deficiency inpatients with herpes simplex encephalitisrdquo Science vol 317 no5844 pp 1522ndash1527 2007

[11] J Tabiasco E Devevre N Rufer et al ldquoHuman effector CD8+T lymphocytes express TLR3 as a functional coreceptorrdquo TheJournal of Immunology vol 177 no 12 pp 8708ndash8713 2006

[12] C S Jack N Arbour J Manusow et al ldquoTLR signaling tailorsinnate immune responses in human microglia and astrocytesrdquoThe Journal of Immunology vol 175 no 7 pp 4320ndash4330 2005

[13] K R Jessen ldquoGlial cellsrdquo International Journal of Biochemistryand Cell Biology vol 36 no 10 pp 1861ndash1867 2004

[14] W Schabitz C Berger R Kollmar et al ldquoEffect of brain-derivedneurotrophic factor treatment and forced armuse on functionalmotor recovery after small cortical ischemiardquo Stroke vol 35 no4 pp 992ndash997 2004

[15] S R Furr V S Chauhan M J Moerdyk-Schauwecker and IMarriott ldquoA role for DNA-dependent activator of interferonregulatory factor in the recognition of herpes simplex virus type1 by glial cellsrdquo Journal of Neuroinflammation vol 8 article no99 2011

[16] Z Liu Y Guan X Sun et al ldquoHSV-1 activates NF-kappaB inmouse astrocytes and increases TNF-alpha and IL-6 expressionvia Toll-like receptor 3rdquoNeurological Research vol 35 no 7 pp755ndash762 2013

[17] J Li Y Zhong H Li et al ldquoEnhancement of Astragalus polysac-charide on the immune responses in pigs inoculated withfoot-and-mouth disease virus vaccinerdquo International Journal ofBiological Macromolecules vol 49 no 3 pp 362ndash368 2011

[18] X Kong Y Hu R Rui D Wang and X Li ldquoEffects of Chineseherbal medicinal ingredients on peripheral lymphocyte prolif-eration and serum antibody titer after vaccination in chickenrdquoInternational Immunopharmacology vol 4 no 7 pp 975ndash9822004

[19] K Lim S D Jazayeri S K Yeap et al ldquoCo-administration ofavian influenza virus H5 plasmid DNA with chicken IL-15 andIL-18 enhanced chickens immune responsesrdquo BMC VeterinaryResearch vol 8 article 132 2012

[20] H Chen Y Shang H Yao et al ldquoImmune responses of chickensinoculated with a recombinant fowlpox vaccine coexpressingHA of H9N2 avain influenza virus and chicken IL-18rdquo AntiviralResearch vol 91 no 1 pp 50ndash56 2011

[21] K S McNaught and P Jenner ldquoExtracellular accumulation ofnitric oxide hydrogen peroxide and glutamate in astrocyticcultures following glutathione depletion complex I inhibi-tion andor lipopolysaccharide-induced activationrdquo Biochem-ical Pharmacology vol 60 no 7 pp 979ndash988 2000

[22] S E Dorman and S M Holland ldquoInterferon-120574 and interleukin-12 pathway defects and human diseaserdquo Cytokine and GrowthFactor Reviews vol 11 no 4 pp 321ndash333 2000

[23] Z Zhuge Y Zhu P Liu et al ldquoEffects of astragalus polysaccha-ride on immune responses of porcine PBMC stimulated withPRRSV or CSFVrdquo PLoS ONE vol 7 no 1 Article ID e293202012

[24] X Du X Chen B Zhao et al ldquoAstragalus polysaccharidesenhance the humoral and cellular immune responses of hep-atitis B surface antigen vaccination through inhibiting theexpression of transforming growth factor 120573 and the frequencyof regulatory T cellsrdquo FEMS Immunology and Medical Microbi-ology vol 63 no 2 pp 228ndash235 2011

[25] S Makino S Ikegami H Kano et al ldquoImmunomodulatoryeffects of polysaccharides produced by Lactobacillus delbrueckiissp bulgaricus OLL1073R-1rdquo Journal of Dairy Science vol 89no 8 pp 2873ndash2881 2006

[26] M Yang Z Xiao Q Lv et al ldquoThe functional expressionof TLR3 in EPCs impairs cell proliferation by induction ofcell apoptosis and cell cycle progress inhibitionrdquo InternationalImmunopharmacology vol 11 no 12 pp 2118ndash2124 2011

[27] C Meyer R Pries and B Wollenberg ldquoEstablished and novelNF-120581B inhibitors lead to downregulation of TLR3 and theproliferation and cytokine secretion inHNSCCrdquoOralOncologyvol 47 no 9 pp 818ndash826 2011

[28] C J Xu X C Jian F Guo Q P Gao J Y Peng and X PXu ldquoEffect of astragalus polysaccharides on the proliferationand ultrastructure of dog bone marrow stem cells induced intoosteoblasts in vitrordquo Hua Xi Kou Qiang Yi Xue Za Zhi vol 25no 5 pp 432ndash436 2007

[29] Y LiuW JWangW H Chen and J Yin ldquoEffects of Astragaluspolysaccharides on proliferation and differentiation of 3T3-L1preadipocytesrdquo Zhong Xi Yi Jie He Xue Bao vol 5 no 4 pp421ndash426 2007

[30] K A Fitzgerald S M McWhirter K L Faia et al ldquoIKKE andTBKI are essential components of the IRF3 signalling pathwayrdquoNature Immunology vol 4 no 5 pp 491ndash496 2003

[31] Y Y Wang L Li K J Han Z Zhai and H B Shu ldquoA20 is apotent inhibitor of TLR3- and Sendai virus-induced activationof NF-120581B and ISRE and IFN-120573 promoterrdquo FEBS Letters vol 576no 1-2 pp 86ndash90 2004

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

4 Evidence-Based Complementary and Alternative Medicine

Control HSV-1 HSV-1 + APS HSV-1+ TLR3 + APS

lowast

08

07

06

05

04

03

02

01

0

OD

998779

Figure 2 Astrocytes proliferation detected by MTT lowast119875 lt 001versus the HSV-1 group Δ119875 lt 005 versus the TLR3 antibody +HSV-1 + APS group 119899 = 3

group In the presence of APS the proliferation of astrocytesincreased to some extent and the OD value of HSV-1 +APS group was greater than that of the HSV-1 group (119875 lt001) which suggests that APS can protect astrocytes fromHSV-1 induced proliferation inhibition Interestingly whenastrocytes were pretreated with TLR3 antibody before addingHSV-1 and APS the proliferation of astrocytes decreasedmarkedly when compared to the HSV-1 + APS group (119875 lt005) This result indicates that the protective effect ofAPS against HSV-1 infection may be associated with TLR3pathway

Secretion levels of TNF-120572 and IL-6 in culture supernatantwere detected by ELISA (Figure 3) The concentrations ofTNF-120572 and IL-6 were very low in culture supernatant of theblank control group whereas in culture supernatant of theHSV-1 group the concentrations of both TNF-120572 and IL-6increased obviously (119875 lt 001) In the presence of APS HSV-1 infected astrocytes expressed higher amount of TNF-120572 andIL-6 than that of the HSV-1 group Remarkably pretreatmentof astrocytes with TLR3 antibody decreased the expression ofTNF-120572 and IL-6 in the TLR3 antibody + HSV-1 + APS group(119875 lt 005)

Western blot analysis showed that the expression level ofTLR3 and the activation of NF-120581B were low in the controlgroup (Figure 4) In the HSV-1 group after being exposedto HSV-1 for 12 h astrocytes expressed higher level of TLR3than the control group At the same time HSV-1 infectioninduced the activation of NF-120581B significantly Moreover inthe presence of APS the expression level of TLR3 and theactivation level of NF-120581B were obviously higher than those ofthe HSV-1 group However when astrocytes were pretreatedwith TLR3 antibody before addition of HSV-1 and APSastrocytes reduced the expression of TLR3 and the activationof NF-120581B significantly Considering the different secretionlevels of TNF-120572 and IL-6 in different groups these resultsindicate that APS may activate NF-120581B which leads to theproduction of TNF-120572 and IL-6 through TLR3 pathway (119875 lt005)

4 Discussion

Recent studies have confirmed thatmany herbs have effects ofmodulating immunity and inhibiting and killing pathogenic

05

10152025303540

Control HSV-1 HSV-1 + APS HSV-1+ TLR3 + APS

lowast

lowast

TNF-120572IL-6

TNF-120572

and

IL-6

(ng

mL)

998779

998779

Figure 3 Concentration of TNF-120572 and IL-6 (ngmLminus1) in cellculture medium detected by ELISA lowast119875 lt 001 versus the HSV-1group Δ119875 lt 005 versus the TLR3 antibody + HSV-1 + APS group119899 = 3

microorganisms [22 23] As one of the bioactive ingredientsfrom the natural traditional Chinese medicinal herb Astra-galus membranaceus APS is used widely as an immunomod-ulator in China Evidences indicate that APS can enhancelymphocyte blastogenesis and stimulate macrophage activa-tion without cytotoxic effects [7 8] APS cannot inhibit virusdirectly but it can activate the immune system and inducethe production of cytokines to initiate an antiviral response[24 25] Consistent with these previous reports the currentstudy showed that HSV-1 infection induced the secretionof TNF-120572 and IL-6 in astrocytes and the secretion notablyincreased in response to APSThese results indicate that APScan promote immunomodulatory effects of astrocytes

In addition we also found that HSV-1 infection induceda higher expression of TLR3 and decreased the proliferationof astrocytes these results are in accordance with previousreports which showed that high expression of TLR3 impairedcell proliferation and inhibited cell cycle progress [26 27]Furthermore we found that APS was capable of promotingthe proliferation of astrocytes infected by HSV-1 This resultsquares with what we already know about the role of APSon cell proliferation [18 28 29] Although the potentialmechanism is still to be clarified regarding the currentunderstanding of TLR3 signaling function we infer thatthe effect of APS on proliferation of astrocytes may not beassociated with its effect on TLR3

Antiviral innate immunity depends on different sensorsystems that recognize viral-pathogen-associated molecularpatterns (PAMPs) and affect specific signaling pathwaysincluding those leading to the activation of NF-120581B Recentstudies demonstrate that TLR3 is also present in cells as asensor to recognize the structure of the pathogens and par-ticipate in the adaptive immune response directly [30] TLR3can recognize the dsRNA of viruses initiates intracellularsignal transduction pathway and then induces the activationof NF-120581B [30 31] Activated NF-120581B translocates from cyto-plasm to the nucleus combines with target genes to triggergene transcriptions and increases the production of certain

Evidence-Based Complementary and Alternative Medicine 5

TotalTLR3

120573-Actin

1 2 3 4

(a)

0005

01015

02025

03035

04

Control HSV-1 HSV-1 + APS HSV-1+ TLR3 + APS

Relat

ive i

nten

sity

(TLR

3120573

-act

in)

lowast

998779

(b)

1 2 3 4

NuclearNF-120581B

LMNB1

(c)

0010203040506070809

1

Control HSV-1 HSV-1 + APS HSV-1+ TLR3 + APS

Relat

ive i

nten

sity

(NF-120581

BLM

NB1

)

lowast

998779

(d)

Figure 4 Western blot analysis of expression levels of total TLR3 and nuclear NF-120581B (activated NF-120581B) (a) Expression levels of total TLR3protein in blank control group HSV-1 group HSV-1 + APS group and TLR3 antibody + HSV-1 + APS group 120573-Actin was used as a loadingcontrol (b)The relative intensity of total TLR3 protein as analyzed byWestern blot (c) Expression levels of nuclear NF-120581B protein (activatedNF-120581B) in blank control group HSV-1 group HSV-1 + APS group and TLR3 antibody + HSV-1 + APS group LMNB1 was used as a loadingcontrol (d)The relative intensity of nuclear NF-120581B protein (activated NF-120581B) as analyzed byWestern blot lowast119875 lt 001 versus the HSV-1 groupΔ

119875 lt 005 versus the TLR3 antibody + HSV-1 + APS group The results of Western blot were from a representative of at least three repeatedexperiments 119899 = 3

antiapoptotic proteins and proinflammatory cytokines [26ndash28] Our previous study revealed that when astrocytes wereinfected by HSV-1 the NF-120581B was activated through TLR3and the generation of TNF-120572 and IL-6 increased obviously[5] In this paper we further confirmed that TLR3 and NF-120581B positively contribute to the immune response to HSV-1 infection Moreover we showed that for HSV-1 infectedastrocytes APS could promote the expression of TLR3and the activation of NF-120581B which elicits the secretion ofinflammatory cytokines TNF-120572 and IL-6 indicating that APScan protect astrocytes by promoting immunological functionprovoked by HSV-1 through TLR3NF-120581B pathway

5 Conclusion

In conclusion APS can protect the astrocytes against HSV-1induced proliferation inhibition and enhance the immuno-logical function of astrocytes by upregulating the TLR3NF-120581B signaling pathway along with increasing expression ofTNF-120572 and IL-6The study suggests that APS has potential inthe treatment of HSV-1 infectious diseases in central nervoussystem

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by a Project of Shandong ProvinceHigh Educational Science and Technology Program (noJ12LK04) Traditional Chinese Medicine Science and Tech-nology Research Projects of Shandong Province (nos 2013-140 2013-236 and 2013-240) the Natural Science Foun-dation of Shandong Province (nos ZR2011HL047 andZR2011HL066) and Natural Science Foundation of China(81341137)

References

[1] R N Aravalli S Hu T N Rowen J M Palmquist and J RLokensgard ldquoCutting edge TLR2-mediated proinflammatorycytokine and chemokine production by microglial cells inresponse to herpes simplex virusrdquo Journal of Immunology vol175 no 7 pp 4189ndash4193 2005

6 Evidence-Based Complementary and Alternative Medicine

[2] A G Armien S Hu M R Little et al ldquoChronic corticaland subcortical pathology with associated neurological deficitsensuing experimental herpes encephalitisrdquoBrain Pathology vol20 no 4 pp 738ndash750 2010

[3] C P Marques M-C Cheeran J M Palmquist S Hu SL Urban and J R Lokensgard ldquoProlonged microglial cellactivation and lymphocyte infiltration following experimentalherpes encephalitisrdquoThe Journal of Immunology vol 181 no 9pp 6417ndash6426 2008

[4] J Kavouras E Prandovszky K Valyi-Nagy et al ldquoHerpessimplex virus type 1 infection induces oxidative stress and therelease of bioactive lipid peroxidation by-products in mouseP19N neural cell culturesrdquo Journal of NeuroVirology vol 13 no5 pp 416ndash425 2007

[5] R J Whitley and J W Gnann ldquoViral encephalitis familiarinfections and emerging pathogensrdquo The Lancet vol 359 no9305 pp 507ndash513 2002

[6] J M Thompson and A Iwasaki ldquoToll-like receptors regulationof viral infection and diseaserdquoAdvanced Drug Delivery Reviewsvol 60 no 7 pp 786ndash794 2008

[7] K Tabeta P Georgel E Janssen et al ldquoToll-like receptors 9 and3 as essential complonents of innate immune defense againstmouse cytomegalovirus infectionrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 101 no10 pp 3516ndash3521 2004

[8] K Fukuda T Tsujita M Matsumoto et al ldquoAnalysis of theinteraction between human TLR3 ectodomain and nucleicacidsrdquo Nucleic Acids Symposium Series no 50 pp 249ndash2502006

[9] Z-Y Lu S P Yu J-F Wei and L Wei ldquoAge-related neuraldegeneration in nuclear-factor 120581B p50 knockout micerdquo Neuro-science vol 139 no 3 pp 965ndash978 2006

[10] S Zhang E Jouanguy S Ugolini et al ldquoTLR3 deficiency inpatients with herpes simplex encephalitisrdquo Science vol 317 no5844 pp 1522ndash1527 2007

[11] J Tabiasco E Devevre N Rufer et al ldquoHuman effector CD8+T lymphocytes express TLR3 as a functional coreceptorrdquo TheJournal of Immunology vol 177 no 12 pp 8708ndash8713 2006

[12] C S Jack N Arbour J Manusow et al ldquoTLR signaling tailorsinnate immune responses in human microglia and astrocytesrdquoThe Journal of Immunology vol 175 no 7 pp 4320ndash4330 2005

[13] K R Jessen ldquoGlial cellsrdquo International Journal of Biochemistryand Cell Biology vol 36 no 10 pp 1861ndash1867 2004

[14] W Schabitz C Berger R Kollmar et al ldquoEffect of brain-derivedneurotrophic factor treatment and forced armuse on functionalmotor recovery after small cortical ischemiardquo Stroke vol 35 no4 pp 992ndash997 2004

[15] S R Furr V S Chauhan M J Moerdyk-Schauwecker and IMarriott ldquoA role for DNA-dependent activator of interferonregulatory factor in the recognition of herpes simplex virus type1 by glial cellsrdquo Journal of Neuroinflammation vol 8 article no99 2011

[16] Z Liu Y Guan X Sun et al ldquoHSV-1 activates NF-kappaB inmouse astrocytes and increases TNF-alpha and IL-6 expressionvia Toll-like receptor 3rdquoNeurological Research vol 35 no 7 pp755ndash762 2013

[17] J Li Y Zhong H Li et al ldquoEnhancement of Astragalus polysac-charide on the immune responses in pigs inoculated withfoot-and-mouth disease virus vaccinerdquo International Journal ofBiological Macromolecules vol 49 no 3 pp 362ndash368 2011

[18] X Kong Y Hu R Rui D Wang and X Li ldquoEffects of Chineseherbal medicinal ingredients on peripheral lymphocyte prolif-eration and serum antibody titer after vaccination in chickenrdquoInternational Immunopharmacology vol 4 no 7 pp 975ndash9822004

[19] K Lim S D Jazayeri S K Yeap et al ldquoCo-administration ofavian influenza virus H5 plasmid DNA with chicken IL-15 andIL-18 enhanced chickens immune responsesrdquo BMC VeterinaryResearch vol 8 article 132 2012

[20] H Chen Y Shang H Yao et al ldquoImmune responses of chickensinoculated with a recombinant fowlpox vaccine coexpressingHA of H9N2 avain influenza virus and chicken IL-18rdquo AntiviralResearch vol 91 no 1 pp 50ndash56 2011

[21] K S McNaught and P Jenner ldquoExtracellular accumulation ofnitric oxide hydrogen peroxide and glutamate in astrocyticcultures following glutathione depletion complex I inhibi-tion andor lipopolysaccharide-induced activationrdquo Biochem-ical Pharmacology vol 60 no 7 pp 979ndash988 2000

[22] S E Dorman and S M Holland ldquoInterferon-120574 and interleukin-12 pathway defects and human diseaserdquo Cytokine and GrowthFactor Reviews vol 11 no 4 pp 321ndash333 2000

[23] Z Zhuge Y Zhu P Liu et al ldquoEffects of astragalus polysaccha-ride on immune responses of porcine PBMC stimulated withPRRSV or CSFVrdquo PLoS ONE vol 7 no 1 Article ID e293202012

[24] X Du X Chen B Zhao et al ldquoAstragalus polysaccharidesenhance the humoral and cellular immune responses of hep-atitis B surface antigen vaccination through inhibiting theexpression of transforming growth factor 120573 and the frequencyof regulatory T cellsrdquo FEMS Immunology and Medical Microbi-ology vol 63 no 2 pp 228ndash235 2011

[25] S Makino S Ikegami H Kano et al ldquoImmunomodulatoryeffects of polysaccharides produced by Lactobacillus delbrueckiissp bulgaricus OLL1073R-1rdquo Journal of Dairy Science vol 89no 8 pp 2873ndash2881 2006

[26] M Yang Z Xiao Q Lv et al ldquoThe functional expressionof TLR3 in EPCs impairs cell proliferation by induction ofcell apoptosis and cell cycle progress inhibitionrdquo InternationalImmunopharmacology vol 11 no 12 pp 2118ndash2124 2011

[27] C Meyer R Pries and B Wollenberg ldquoEstablished and novelNF-120581B inhibitors lead to downregulation of TLR3 and theproliferation and cytokine secretion inHNSCCrdquoOralOncologyvol 47 no 9 pp 818ndash826 2011

[28] C J Xu X C Jian F Guo Q P Gao J Y Peng and X PXu ldquoEffect of astragalus polysaccharides on the proliferationand ultrastructure of dog bone marrow stem cells induced intoosteoblasts in vitrordquo Hua Xi Kou Qiang Yi Xue Za Zhi vol 25no 5 pp 432ndash436 2007

[29] Y LiuW JWangW H Chen and J Yin ldquoEffects of Astragaluspolysaccharides on proliferation and differentiation of 3T3-L1preadipocytesrdquo Zhong Xi Yi Jie He Xue Bao vol 5 no 4 pp421ndash426 2007

[30] K A Fitzgerald S M McWhirter K L Faia et al ldquoIKKE andTBKI are essential components of the IRF3 signalling pathwayrdquoNature Immunology vol 4 no 5 pp 491ndash496 2003

[31] Y Y Wang L Li K J Han Z Zhai and H B Shu ldquoA20 is apotent inhibitor of TLR3- and Sendai virus-induced activationof NF-120581B and ISRE and IFN-120573 promoterrdquo FEBS Letters vol 576no 1-2 pp 86ndash90 2004

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 5

TotalTLR3

120573-Actin

1 2 3 4

(a)

0005

01015

02025

03035

04

Control HSV-1 HSV-1 + APS HSV-1+ TLR3 + APS

Relat

ive i

nten

sity

(TLR

3120573

-act

in)

lowast

998779

(b)

1 2 3 4

NuclearNF-120581B

LMNB1

(c)

0010203040506070809

1

Control HSV-1 HSV-1 + APS HSV-1+ TLR3 + APS

Relat

ive i

nten

sity

(NF-120581

BLM

NB1

)

lowast

998779

(d)

Figure 4 Western blot analysis of expression levels of total TLR3 and nuclear NF-120581B (activated NF-120581B) (a) Expression levels of total TLR3protein in blank control group HSV-1 group HSV-1 + APS group and TLR3 antibody + HSV-1 + APS group 120573-Actin was used as a loadingcontrol (b)The relative intensity of total TLR3 protein as analyzed byWestern blot (c) Expression levels of nuclear NF-120581B protein (activatedNF-120581B) in blank control group HSV-1 group HSV-1 + APS group and TLR3 antibody + HSV-1 + APS group LMNB1 was used as a loadingcontrol (d)The relative intensity of nuclear NF-120581B protein (activated NF-120581B) as analyzed byWestern blot lowast119875 lt 001 versus the HSV-1 groupΔ

119875 lt 005 versus the TLR3 antibody + HSV-1 + APS group The results of Western blot were from a representative of at least three repeatedexperiments 119899 = 3

antiapoptotic proteins and proinflammatory cytokines [26ndash28] Our previous study revealed that when astrocytes wereinfected by HSV-1 the NF-120581B was activated through TLR3and the generation of TNF-120572 and IL-6 increased obviously[5] In this paper we further confirmed that TLR3 and NF-120581B positively contribute to the immune response to HSV-1 infection Moreover we showed that for HSV-1 infectedastrocytes APS could promote the expression of TLR3and the activation of NF-120581B which elicits the secretion ofinflammatory cytokines TNF-120572 and IL-6 indicating that APScan protect astrocytes by promoting immunological functionprovoked by HSV-1 through TLR3NF-120581B pathway

5 Conclusion

In conclusion APS can protect the astrocytes against HSV-1induced proliferation inhibition and enhance the immuno-logical function of astrocytes by upregulating the TLR3NF-120581B signaling pathway along with increasing expression ofTNF-120572 and IL-6The study suggests that APS has potential inthe treatment of HSV-1 infectious diseases in central nervoussystem

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by a Project of Shandong ProvinceHigh Educational Science and Technology Program (noJ12LK04) Traditional Chinese Medicine Science and Tech-nology Research Projects of Shandong Province (nos 2013-140 2013-236 and 2013-240) the Natural Science Foun-dation of Shandong Province (nos ZR2011HL047 andZR2011HL066) and Natural Science Foundation of China(81341137)

References

[1] R N Aravalli S Hu T N Rowen J M Palmquist and J RLokensgard ldquoCutting edge TLR2-mediated proinflammatorycytokine and chemokine production by microglial cells inresponse to herpes simplex virusrdquo Journal of Immunology vol175 no 7 pp 4189ndash4193 2005

6 Evidence-Based Complementary and Alternative Medicine

[2] A G Armien S Hu M R Little et al ldquoChronic corticaland subcortical pathology with associated neurological deficitsensuing experimental herpes encephalitisrdquoBrain Pathology vol20 no 4 pp 738ndash750 2010

[3] C P Marques M-C Cheeran J M Palmquist S Hu SL Urban and J R Lokensgard ldquoProlonged microglial cellactivation and lymphocyte infiltration following experimentalherpes encephalitisrdquoThe Journal of Immunology vol 181 no 9pp 6417ndash6426 2008

[4] J Kavouras E Prandovszky K Valyi-Nagy et al ldquoHerpessimplex virus type 1 infection induces oxidative stress and therelease of bioactive lipid peroxidation by-products in mouseP19N neural cell culturesrdquo Journal of NeuroVirology vol 13 no5 pp 416ndash425 2007

[5] R J Whitley and J W Gnann ldquoViral encephalitis familiarinfections and emerging pathogensrdquo The Lancet vol 359 no9305 pp 507ndash513 2002

[6] J M Thompson and A Iwasaki ldquoToll-like receptors regulationof viral infection and diseaserdquoAdvanced Drug Delivery Reviewsvol 60 no 7 pp 786ndash794 2008

[7] K Tabeta P Georgel E Janssen et al ldquoToll-like receptors 9 and3 as essential complonents of innate immune defense againstmouse cytomegalovirus infectionrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 101 no10 pp 3516ndash3521 2004

[8] K Fukuda T Tsujita M Matsumoto et al ldquoAnalysis of theinteraction between human TLR3 ectodomain and nucleicacidsrdquo Nucleic Acids Symposium Series no 50 pp 249ndash2502006

[9] Z-Y Lu S P Yu J-F Wei and L Wei ldquoAge-related neuraldegeneration in nuclear-factor 120581B p50 knockout micerdquo Neuro-science vol 139 no 3 pp 965ndash978 2006

[10] S Zhang E Jouanguy S Ugolini et al ldquoTLR3 deficiency inpatients with herpes simplex encephalitisrdquo Science vol 317 no5844 pp 1522ndash1527 2007

[11] J Tabiasco E Devevre N Rufer et al ldquoHuman effector CD8+T lymphocytes express TLR3 as a functional coreceptorrdquo TheJournal of Immunology vol 177 no 12 pp 8708ndash8713 2006

[12] C S Jack N Arbour J Manusow et al ldquoTLR signaling tailorsinnate immune responses in human microglia and astrocytesrdquoThe Journal of Immunology vol 175 no 7 pp 4320ndash4330 2005

[13] K R Jessen ldquoGlial cellsrdquo International Journal of Biochemistryand Cell Biology vol 36 no 10 pp 1861ndash1867 2004

[14] W Schabitz C Berger R Kollmar et al ldquoEffect of brain-derivedneurotrophic factor treatment and forced armuse on functionalmotor recovery after small cortical ischemiardquo Stroke vol 35 no4 pp 992ndash997 2004

[15] S R Furr V S Chauhan M J Moerdyk-Schauwecker and IMarriott ldquoA role for DNA-dependent activator of interferonregulatory factor in the recognition of herpes simplex virus type1 by glial cellsrdquo Journal of Neuroinflammation vol 8 article no99 2011

[16] Z Liu Y Guan X Sun et al ldquoHSV-1 activates NF-kappaB inmouse astrocytes and increases TNF-alpha and IL-6 expressionvia Toll-like receptor 3rdquoNeurological Research vol 35 no 7 pp755ndash762 2013

[17] J Li Y Zhong H Li et al ldquoEnhancement of Astragalus polysac-charide on the immune responses in pigs inoculated withfoot-and-mouth disease virus vaccinerdquo International Journal ofBiological Macromolecules vol 49 no 3 pp 362ndash368 2011

[18] X Kong Y Hu R Rui D Wang and X Li ldquoEffects of Chineseherbal medicinal ingredients on peripheral lymphocyte prolif-eration and serum antibody titer after vaccination in chickenrdquoInternational Immunopharmacology vol 4 no 7 pp 975ndash9822004

[19] K Lim S D Jazayeri S K Yeap et al ldquoCo-administration ofavian influenza virus H5 plasmid DNA with chicken IL-15 andIL-18 enhanced chickens immune responsesrdquo BMC VeterinaryResearch vol 8 article 132 2012

[20] H Chen Y Shang H Yao et al ldquoImmune responses of chickensinoculated with a recombinant fowlpox vaccine coexpressingHA of H9N2 avain influenza virus and chicken IL-18rdquo AntiviralResearch vol 91 no 1 pp 50ndash56 2011

[21] K S McNaught and P Jenner ldquoExtracellular accumulation ofnitric oxide hydrogen peroxide and glutamate in astrocyticcultures following glutathione depletion complex I inhibi-tion andor lipopolysaccharide-induced activationrdquo Biochem-ical Pharmacology vol 60 no 7 pp 979ndash988 2000

[22] S E Dorman and S M Holland ldquoInterferon-120574 and interleukin-12 pathway defects and human diseaserdquo Cytokine and GrowthFactor Reviews vol 11 no 4 pp 321ndash333 2000

[23] Z Zhuge Y Zhu P Liu et al ldquoEffects of astragalus polysaccha-ride on immune responses of porcine PBMC stimulated withPRRSV or CSFVrdquo PLoS ONE vol 7 no 1 Article ID e293202012

[24] X Du X Chen B Zhao et al ldquoAstragalus polysaccharidesenhance the humoral and cellular immune responses of hep-atitis B surface antigen vaccination through inhibiting theexpression of transforming growth factor 120573 and the frequencyof regulatory T cellsrdquo FEMS Immunology and Medical Microbi-ology vol 63 no 2 pp 228ndash235 2011

[25] S Makino S Ikegami H Kano et al ldquoImmunomodulatoryeffects of polysaccharides produced by Lactobacillus delbrueckiissp bulgaricus OLL1073R-1rdquo Journal of Dairy Science vol 89no 8 pp 2873ndash2881 2006

[26] M Yang Z Xiao Q Lv et al ldquoThe functional expressionof TLR3 in EPCs impairs cell proliferation by induction ofcell apoptosis and cell cycle progress inhibitionrdquo InternationalImmunopharmacology vol 11 no 12 pp 2118ndash2124 2011

[27] C Meyer R Pries and B Wollenberg ldquoEstablished and novelNF-120581B inhibitors lead to downregulation of TLR3 and theproliferation and cytokine secretion inHNSCCrdquoOralOncologyvol 47 no 9 pp 818ndash826 2011

[28] C J Xu X C Jian F Guo Q P Gao J Y Peng and X PXu ldquoEffect of astragalus polysaccharides on the proliferationand ultrastructure of dog bone marrow stem cells induced intoosteoblasts in vitrordquo Hua Xi Kou Qiang Yi Xue Za Zhi vol 25no 5 pp 432ndash436 2007

[29] Y LiuW JWangW H Chen and J Yin ldquoEffects of Astragaluspolysaccharides on proliferation and differentiation of 3T3-L1preadipocytesrdquo Zhong Xi Yi Jie He Xue Bao vol 5 no 4 pp421ndash426 2007

[30] K A Fitzgerald S M McWhirter K L Faia et al ldquoIKKE andTBKI are essential components of the IRF3 signalling pathwayrdquoNature Immunology vol 4 no 5 pp 491ndash496 2003

[31] Y Y Wang L Li K J Han Z Zhai and H B Shu ldquoA20 is apotent inhibitor of TLR3- and Sendai virus-induced activationof NF-120581B and ISRE and IFN-120573 promoterrdquo FEBS Letters vol 576no 1-2 pp 86ndash90 2004

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

6 Evidence-Based Complementary and Alternative Medicine

[2] A G Armien S Hu M R Little et al ldquoChronic corticaland subcortical pathology with associated neurological deficitsensuing experimental herpes encephalitisrdquoBrain Pathology vol20 no 4 pp 738ndash750 2010

[3] C P Marques M-C Cheeran J M Palmquist S Hu SL Urban and J R Lokensgard ldquoProlonged microglial cellactivation and lymphocyte infiltration following experimentalherpes encephalitisrdquoThe Journal of Immunology vol 181 no 9pp 6417ndash6426 2008

[4] J Kavouras E Prandovszky K Valyi-Nagy et al ldquoHerpessimplex virus type 1 infection induces oxidative stress and therelease of bioactive lipid peroxidation by-products in mouseP19N neural cell culturesrdquo Journal of NeuroVirology vol 13 no5 pp 416ndash425 2007

[5] R J Whitley and J W Gnann ldquoViral encephalitis familiarinfections and emerging pathogensrdquo The Lancet vol 359 no9305 pp 507ndash513 2002

[6] J M Thompson and A Iwasaki ldquoToll-like receptors regulationof viral infection and diseaserdquoAdvanced Drug Delivery Reviewsvol 60 no 7 pp 786ndash794 2008

[7] K Tabeta P Georgel E Janssen et al ldquoToll-like receptors 9 and3 as essential complonents of innate immune defense againstmouse cytomegalovirus infectionrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 101 no10 pp 3516ndash3521 2004

[8] K Fukuda T Tsujita M Matsumoto et al ldquoAnalysis of theinteraction between human TLR3 ectodomain and nucleicacidsrdquo Nucleic Acids Symposium Series no 50 pp 249ndash2502006

[9] Z-Y Lu S P Yu J-F Wei and L Wei ldquoAge-related neuraldegeneration in nuclear-factor 120581B p50 knockout micerdquo Neuro-science vol 139 no 3 pp 965ndash978 2006

[10] S Zhang E Jouanguy S Ugolini et al ldquoTLR3 deficiency inpatients with herpes simplex encephalitisrdquo Science vol 317 no5844 pp 1522ndash1527 2007

[11] J Tabiasco E Devevre N Rufer et al ldquoHuman effector CD8+T lymphocytes express TLR3 as a functional coreceptorrdquo TheJournal of Immunology vol 177 no 12 pp 8708ndash8713 2006

[12] C S Jack N Arbour J Manusow et al ldquoTLR signaling tailorsinnate immune responses in human microglia and astrocytesrdquoThe Journal of Immunology vol 175 no 7 pp 4320ndash4330 2005

[13] K R Jessen ldquoGlial cellsrdquo International Journal of Biochemistryand Cell Biology vol 36 no 10 pp 1861ndash1867 2004

[14] W Schabitz C Berger R Kollmar et al ldquoEffect of brain-derivedneurotrophic factor treatment and forced armuse on functionalmotor recovery after small cortical ischemiardquo Stroke vol 35 no4 pp 992ndash997 2004

[15] S R Furr V S Chauhan M J Moerdyk-Schauwecker and IMarriott ldquoA role for DNA-dependent activator of interferonregulatory factor in the recognition of herpes simplex virus type1 by glial cellsrdquo Journal of Neuroinflammation vol 8 article no99 2011

[16] Z Liu Y Guan X Sun et al ldquoHSV-1 activates NF-kappaB inmouse astrocytes and increases TNF-alpha and IL-6 expressionvia Toll-like receptor 3rdquoNeurological Research vol 35 no 7 pp755ndash762 2013

[17] J Li Y Zhong H Li et al ldquoEnhancement of Astragalus polysac-charide on the immune responses in pigs inoculated withfoot-and-mouth disease virus vaccinerdquo International Journal ofBiological Macromolecules vol 49 no 3 pp 362ndash368 2011

[18] X Kong Y Hu R Rui D Wang and X Li ldquoEffects of Chineseherbal medicinal ingredients on peripheral lymphocyte prolif-eration and serum antibody titer after vaccination in chickenrdquoInternational Immunopharmacology vol 4 no 7 pp 975ndash9822004

[19] K Lim S D Jazayeri S K Yeap et al ldquoCo-administration ofavian influenza virus H5 plasmid DNA with chicken IL-15 andIL-18 enhanced chickens immune responsesrdquo BMC VeterinaryResearch vol 8 article 132 2012

[20] H Chen Y Shang H Yao et al ldquoImmune responses of chickensinoculated with a recombinant fowlpox vaccine coexpressingHA of H9N2 avain influenza virus and chicken IL-18rdquo AntiviralResearch vol 91 no 1 pp 50ndash56 2011

[21] K S McNaught and P Jenner ldquoExtracellular accumulation ofnitric oxide hydrogen peroxide and glutamate in astrocyticcultures following glutathione depletion complex I inhibi-tion andor lipopolysaccharide-induced activationrdquo Biochem-ical Pharmacology vol 60 no 7 pp 979ndash988 2000

[22] S E Dorman and S M Holland ldquoInterferon-120574 and interleukin-12 pathway defects and human diseaserdquo Cytokine and GrowthFactor Reviews vol 11 no 4 pp 321ndash333 2000

[23] Z Zhuge Y Zhu P Liu et al ldquoEffects of astragalus polysaccha-ride on immune responses of porcine PBMC stimulated withPRRSV or CSFVrdquo PLoS ONE vol 7 no 1 Article ID e293202012

[24] X Du X Chen B Zhao et al ldquoAstragalus polysaccharidesenhance the humoral and cellular immune responses of hep-atitis B surface antigen vaccination through inhibiting theexpression of transforming growth factor 120573 and the frequencyof regulatory T cellsrdquo FEMS Immunology and Medical Microbi-ology vol 63 no 2 pp 228ndash235 2011

[25] S Makino S Ikegami H Kano et al ldquoImmunomodulatoryeffects of polysaccharides produced by Lactobacillus delbrueckiissp bulgaricus OLL1073R-1rdquo Journal of Dairy Science vol 89no 8 pp 2873ndash2881 2006

[26] M Yang Z Xiao Q Lv et al ldquoThe functional expressionof TLR3 in EPCs impairs cell proliferation by induction ofcell apoptosis and cell cycle progress inhibitionrdquo InternationalImmunopharmacology vol 11 no 12 pp 2118ndash2124 2011

[27] C Meyer R Pries and B Wollenberg ldquoEstablished and novelNF-120581B inhibitors lead to downregulation of TLR3 and theproliferation and cytokine secretion inHNSCCrdquoOralOncologyvol 47 no 9 pp 818ndash826 2011

[28] C J Xu X C Jian F Guo Q P Gao J Y Peng and X PXu ldquoEffect of astragalus polysaccharides on the proliferationand ultrastructure of dog bone marrow stem cells induced intoosteoblasts in vitrordquo Hua Xi Kou Qiang Yi Xue Za Zhi vol 25no 5 pp 432ndash436 2007

[29] Y LiuW JWangW H Chen and J Yin ldquoEffects of Astragaluspolysaccharides on proliferation and differentiation of 3T3-L1preadipocytesrdquo Zhong Xi Yi Jie He Xue Bao vol 5 no 4 pp421ndash426 2007

[30] K A Fitzgerald S M McWhirter K L Faia et al ldquoIKKE andTBKI are essential components of the IRF3 signalling pathwayrdquoNature Immunology vol 4 no 5 pp 491ndash496 2003

[31] Y Y Wang L Li K J Han Z Zhai and H B Shu ldquoA20 is apotent inhibitor of TLR3- and Sendai virus-induced activationof NF-120581B and ISRE and IFN-120573 promoterrdquo FEBS Letters vol 576no 1-2 pp 86ndash90 2004

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom