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Immunopharmacology and Immunotoxicology, 30:135–151, 2008Copyright © Informa Healthcare USA, Inc.ISSN: 0892-3973 print / 1532-2513 onlineDOI: 10.1080/08923970701812704
LIPI0892-39731532-2513Immunopharmacology and Immunotoxicology, Vol. 30, No. 1, January 2008: pp. 1–29Immunopharmacology and ImmunotoxicologyAnti-inflammatory Mechanism of Rungia pectinata (Linn.) NeesAnti-inflammation of Rungia pectinata (Linn.) NeesL. Zhao et al.
Lei Zhao,1 Jun-Yan Tao,2 Shu-Ling Zhang,1 Feng Jin,3 Ran Pang,1 Ji-Hua Dong,4 Yuan-Jin Guo,5 and Pian Ye1
1Department of Hepatology & Infectious Disease, Union Hospital, Tongji MedicalCollege, Huazhong University of Science and Technology, Wuhan, P.R. China2College of Life Science and Technology, Huazhong University of Science andTechnology, Wuhan, P.R. China3Department of Neurosurgery, Union Hospital, Tongji Medical College, HuazhongUniversity of Science and Technology, Wuhan, P.R. China4Central Lab, Union Hospital, Tongji Medical College, Huazhong University of Scienceand Technology, Wuhan, P.R. China5Department of Neurology, Union Hospital, Tongji Medical College, HuazhongUniversity of Science and Technology, Wuhan, P.R. China
This study is to explore the inner anti-inflammatory mechanism of the ethanol extractof Rungia pectinata (Linn.) Nees. As a result, the ethanol extract of Rungia pectinata(Linn.) Nees could not only strongly reduce production of pro-inflammatory cytokinesand mediators via blocking NF-κB activation but slightly promote release of anti-inflammatory mediator HO-1 and suppress IL-10 secretion. In conclusion, compared toDexamethasone, Rungia pectinata (Linn.) Nees has not only similar effects on anta-gonizing pro-inflammatory mediators and cytokines but also mild effects on promotingproduction of anti-inflammatory mediators.
Keywords Rungia pectinata (Linn.) Nees, RAW 264.7 cell line, NF-κB, HO-1.
INTRODUCTION
Rungia pectinata (Linn.) Nees, a member of the Acanthaceae family distri-buted in Far East and Southeast Asia region, is used as folk plant medicine.In south China, Rungia pectinata (Linn.) Nees is used for treating hepatitis,acute conjunctivitis, children’s dyspepsia, dysentery, lymphoid tuberculosis
Authors Zhao and Tao have contributed equally to this work.Address correspondence to Prof. Shu-Ling Zhang, Department of Hepatology & Infec-tious Disease, Union Hospital, Tongji Medical College, Huazhong University of Scienceand Technology, Wuhan 430022, P.R. China; E-mail: [email protected]
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etc.(1) However, the anti-inflammatory mechanism of that folk herbal medi-cine has not been investigated clearly.
Generally considering, macrophages play an important role in inflam-matory diseases by producing cytokines, such as interleukin-1 beta (IL-1β),(2) interleukin-6 (IL-6)(3) and tumor necrosis factor-alpha (TNF-α),(4) andother inflammatory mediators, such as nitric oxide (NO)(5) and prostaglan-dins.(6) These cytokines and mediators from macrophages have been foundin many inflammatory cells and tissues, along with increased expression oftheir mRNA, following exposure to immune stimulants, including bacterialendotoxin lipopolysaccharide (LPS). Those pro-inflammatory cytokines andmediators are regulated by nuclear factors, such as nuclear factor-kappaB(NF-κB).(7) Meanwhile, following pro-inflammatory mediators are released,(8)
anti-inflammatory cytokines and enzymes,(9) such as interleukin-10 (IL-10)(10) and heme oxygenase-1 (HO-1)(11) are activated and secreted. Activa-tion of pro-inflammatory and anti-inflammatory cytokines and mediators isthe key procedure of inflammatory reaction and leads consequent inflam-matory impairment and restoration.(12) In experimental practice, RAW264.7 mouse macrophage cell line stimulated by LPS is widely used as theinflammatory cellular model to study the effect of anti-inflammatory drugsand herbs.(13–15)
In this study, we chose the inflammation cellular model to explore theanti-inflammatory effect of the ethanol extract of Rungia pectinata (Linn.)Nees and the inner anti-inflammatory mechanisms of that medicinal plant.
MATERIALS AND METHODS
Plant MaterialsRungia pectinata (Linn.) Nees was provided and identified by Prof. Ke-Li
Chen (School of Pharmacy, Hubei College of Traditional Chinese Medicine).The plant materials were stored in the plant specimen department, school ofpharmacy, Hubei College of Traditional Chinese Medicine.
Preparation of Ethanol Extract from Rungia pectinata (Linn.) NeesFirst, 50 g air-dried whole grass of Rungia pectinata (Linn.) Nees was
powdered and extracted with 70% ethanol at 85°C (2 × 500 mL, 1.5 h each).Then the extracting liquid was filtered, combined and concentrated in vac-uum. Subsequently the concentrated liquid was diluted by deionizer water toa concentration of 1 g herb weight per 1 mL water. The solution was diluted by1640 medium to the concentration of 10 μg/mL, 5 μg/mL and 1 μg/mL for inter-fering inflammation cellular model.
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Anti-inflammation of Rungia pectinata (Linn.) Nees 137
Chemicals and ReagentsRPMI1640 was from Gibico (Grand Island, NY). Lipopolysaccharide (LPS)
(Escherichia coli O111:B4) and methyl thiazolyl tetrazolium (MTT) were obtainedfrom Sigma (St. Louis, MO). Affinity-purified goat anti-mouse HO-1 antibody wasobtained from R&D Systems (Minneapolis, MN). Affinity-purified goat anti-mouseCOX-2 antibody and rabbit anti-mouse NF-κB p65 IgG antibody were gotten fromSanta Cruz Biotechnology (Santa Cruz, CA). Mouse TNF-α, IL-1β and IL-6 ELISAkits were purchased from Quantikine, R&D Systems (Minneapolis, MN). Griessreagent nitric oxide assay kit was from Beyotime Biotech (Jiangsu, P.R. China).Mouse IL-10 ELISA kit was obtained from Bender Medsystem (Vienna, Austria).Trizolwas purchased from Gibico (Grand Island, NY). M-MLV Reverse Tran-scriptase was afforded from Promega (Madison, WI). SYBR Green I was takenfrom Biotium (Hayward, CA). The Oligo(dT18) and primers was synthesized byShanghai Invitrogen (Shanghai, China). The dNTP was obtained from Promega(Madison, WI). All the material under study is endotoxin free.
Cell CultureMurine macrophage cell line RAW264.7 was from China Center for Typi-
cal Culture Collection (CCTCC) (Wuhan, China). The cells were maintained inRPMI1640 medium supplemented with 100 U/mL of penicillin, 100 μg/mL ofstreptomycin and 10% fetal bovine serum and was cultured at 37°C and 5%CO2 in humidified air.
Cellular Model Establishment and InterventionFirst, 24 hours prior to LPS treatment, the cells were inoculated into 6, 24
or 96 micro-well plates. 24 hours later, when the cells were observed foradherence at the bottom of the well, cell supernatants were disposed and10 ng/mL LPS with prepared exact solution were added into the well. Thestimulation and intervention lasted for different hours and the supernatantsand the cells were harvested for ELISA, real-time PCR, and western-blot aswell as immunocytochemical test.
Control EstablishmentDexamethasone, which is known as a classic glucocorticosteroid drug
widely used in clinical practice, was chosen as positive control with concentra-tion of 0.5 μg/mL. Astragalus polysaccharides (APS) is an immunomodulatorto reinforce immune response on TNF-α, IL-1β and IL-6.(16) According to theliterature,(16) APS with concentration of 100 μg/mL was selected as negativecontrol for monitoring the procedure. Cells stimulated by LPS without anyintervention were observed as blank control, whereas cells incubated by 1640medium were as normal control.
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MTT Assay for Measuring Cell ProliferationBased on the instruction from the American Type Culture Collection
(ATCC), cytotoxic effect of the extract was evaluated by MTT assay. Fourhours prior to the culture termination, 20 μL MTT solution (5 mg/mL in aphosphate-buffered saline, pH 7.4) was added. When cell culture was termi-nated, 150 μL dimethyl sulfoxide (DMSO) was added into each well for solubi-lization. The optical density (OD) at 490 nm was measured by a Spectramax250 microplate reader.
Cytopathic Effect Test for Cell Morphous ObservationAfter cell model was interfered by the extract for 24 hours, cytopathic
effect test was performed for observing cell morphous.
Detection of TNF-a, IL-1b and IL-6 in SupernatantInhibitory effects of 10 μg/mL, 5 μg/mL and 1 μg/mL the extract on the
cytokine TNF-α, IL-1β and IL-6 production from LPS-treated RAW264.7 cellswere determined by sandwich ELISA. After stimulation and intervention onRAW264.7 cells for 24 hours, supernatants were harvested and assayed forTNF-α, IL-1β and IL-6 by respective ELISA kits. The procedure obeyed toinstructions from related kits. Results of 3 independent experiments wereused for statistical analysis.
Analysis of Nitric OxideLevels of the NO derivative nitrite were determined with the Griess reac-
tion. The nitrite detection kit was used according to instructions provided bythe manufacturer. The samples were assayed in triplicate, and a standardcurve using NaNO2 was generated for each experiment for quantification.Briefly, 100 μL of medium or standard NaNO2 was mixed with 100 μL ofGriess reagent in a 96-well plate. After 15 min, optical density was read in amicroplate reader at 540 nm.
Real-time PCR for Detecting mRNA of TNF-a, COX-2, iNOS and HO-1The total RNA from stimulated and interfered cells was prepared by adding
TRIzol Reagent according to manufacturer’s protocol. The RNA on TNF-α, induc-ible nitric oxide synthase (iNOS) and cycloxygenase-2 (COX-2) was extracted 4hours after stimulation and intervention and the RNA on HO-1 was obtained 18hours after stimulation and intervention. Quantitative PCR was performed inABI-7700 Sequence Detector (Applied Biosystems, Foster City, CA). The reversetranscription was performed with M-MLV Reverse Transcriptase.
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Anti-inflammation of Rungia pectinata (Linn.) Nees 139
The Reverse Transcription reaction system included: 5.5 μL H2O, 1.0 μLOligo (dT18) (50 μg/ml), Total RNA 6.0 μL, 70°C 5 min to ice for unfolding sec-ondary structure of mRNA; 0.5 μL RNasin (40 U/μL), 4.0 μL 5 × buffer, 2.0 μLdNTP (10 mM), 1.0 μL RTase (200 U/μL), 42°C, 60 min to 95°C, 5 min to 4°C.Real-time PCR reaction was performed with SYBR Green I fluorochrome. Thestandard curve of each sample was obtained and cycle threshold (Ct) valuewas calculated. Each 50 μL PCR system contained 1/50 of the original cDNAsynthesis reaction, 7 μL (25mM) MgCl2, 0.8 μL (20 pmol/μL) of each primer,1 μL (10 mM) dNTP, 1 μL SYBR Green I, 0.5 μL (5 U/μL) Taq and 5 μL 10 ×Buffer. Fifty cycles of amplification were performed: after 94°C, 3 min, reac-tion cycle with 94°C, 30s, to 57°C, 30s, then to 72°C, 30s was carried out for 50times. The fluorescence signal was detected at the end of each cycle. Meltingcurve analysis was used to confirm the specificity of the products. The 2-ΔΔCT
method was performed to analyze the results.(17) The primer was as follows:
Mus-COX-2:Forward: 5’-GAAGTCTTTGGTCTGGTGCCTG-3’,Reverse: 5’-GTCTGCTGGTTTGGAATAGTTGC-3’;
Mus-iNos:Forward: 5’-GGAGCGAGTTGTGGATTGTC-3’,Reverse:5’-GTGAGGGCTTGGCTGAGTGAG-3’;
Mus-TNF-α:Forward: 5’-GTGGAACTGGCAGAAGAGGC-3’,Reverse: 5’-AGACAGAAGAGCGTGGTGGC-3’;
Mus-HO-1:Forward: 5’-CACAGATGGCGTCACTTCGTC-3’,Reverse: 5’-GTGAGGACCCACTGGAGGAG-3’.
Mus-β-actin:Forward: 5’- GCTACAGCTTCACCACCACAG-3’,Reverse: 5’- GGTCTTTACGGATGTCAACGTC-3’.
Western Blot Analysis of COX-2 and HO-1The treated cells were harvested and incubated on ice for 15 minutes in a
lysis buffer of 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 100 mg/mL phenyl meth-ylsulfonyl fluoride, 1 mg/mL aprotinin, and 1% Triton X-100. Cell debris wasremoved by centrifugation at 10,000 rpm and 4°C for 10 minutes. The proteinconcentration of each cell lysate was determined together with a Bio-Rad(Hercules, CA, USA) protein assay kit. To each tube, an equivalent volume of2 × sodium dodecyl sulfate (SDS) loading buffer (100 mM Tris-HCl, pH 6.8, 4%SDS, 20% glycerine, 10% b-mercaptoethanol, and 0.2% bromophenol blue) was
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added and mixed again. The mixtures were then denatured at 95°C for 10 min-utes, and about 10 mg of the protein mixture was loaded and separated in eachwell on 10% SDS-polyacrylamide electrophoresis gels. After separation for about80 minutes, the proteins were transblotted onto nitrocellulose membranes(Bio-Rad), and the membranes were saturated and blocked with 5% fat-free milkat 37°C for 1 hour.
Membranes were probed with goat polyclonal anti-COX-2, anti-HO-1(1:6000) and then with horseradish peroxidase-conjugated secondary immuno-globulin G (IgG, Kangcheng, Shanghai, P.R. China). The membranes werethen treated with an enhanced chemiluminescence reagent (Amersham,Piscataway, NJ), and the signals were detected by exposure of the membranesto X-ray films (Kodak, Rochester, NY, USA). The relative signal intensity wasquantified by densitometry with Gel pro3.0 image software (Media Cybernetics,Silverspring, MD, USA) on an IBM-compatible personal computer. All experi-ments were independently performed 3 times.
Immunocytochemistry Assay on NF-kBSP immunocytochemical assay was employed to detect expression of the
nuclear translocation of NF-κB. Coverslips were soaked in polylysine for awhole night. After cell crawling to the coverlslips and subsequent LPS stimu-lation and the extract intervention, the cells were fixed by acetone. Then theslides with cells were soaked in 3% H2O2-methanol solution for 20 min inorder to block endogenous peroxydase. Next 1%Triton X-100 was added at37°C for 5min, followed by PBS washing. After incubated with normal goatserum at room temperature for 20 min, rabbit anti-mouse NF-κB p65 IgGantibody was dropwised and the slides were stored at 4°C for a whole night.
The next day, following PBS washing, biotin-conjugated goat anti rabbitIgG was dropwised and the incubation lasted 30 min at 37°C. Then with aPBS wash again, streptavidin-HRP was added and incubated with cells for 30min at 37°C. Subsequently with PBS washing for 5 min, 3 times, DAB colora-tion was performed. Following normal dehydration, lucidification and mount-ing, the slides were observed and pictures taken.
RESULTS
In Vitro Cytotoxicity of the Extract of Rungia pectinata (Linn.) NeesBased on the MTT assay, it showed that pretreatment on unstimulated
RAW264.7 cells lines with prepared solution of the extract of Rungia pectinata(Linn.) Nees (concentration mentioned above) for 24 hours did not signifi-cantly affect cell viability (data not shown). The Cytopathic effect (CPE) testpresented the same result (Fig. 1).
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Anti-inflammation of Rungia pectinata (Linn.) Nees 141
Inflammatory Model Establishment and Procedure MonitoringAs shown from figures on pro-inflammatory cytokines and mediators, the
levels of those factors from cells stimulated by LPS were significantly higherthan those from normal cells (p < 0.01), which implied the successful estab-lishment on model of inflammation. Meanwhile, the levels of those factorsfrom cells by Dexamethasone intervention was significantly lower than thosefrom single LPS stimulation (p < 0.01). The levels of those factors from cells byAPS intervention, an immunomodelator for enhancing immune response onTNF-α, IL-1β and IL-6, was significantly higher than those from single LPSstimulation (p < 0.01). The effects of Dexamethasone and APS interventiondemonstrated experimental procedure was proper.
Effect of the Extract of Rungia pectinata (Linn.) Nees on Pro-inflammatory CytokinesAs shown in Figures 2A–2C, after LPS stimulation with the extract of
Rungia pectinata (Linn.) Nees intervention for 24 h, secretion of TNF-α, IL-1βand IL-6 were significantly decreased than that in single LPS stimulation (p <0.01). Furthermore, when the dosage of the extract of Rungia pectinata (Linn.)Nees increased, the effects of antagonizing pro-inflammatory cytokines weresignificantly elevated (p < 0.05 or 0.01), which showed dose-dependent rela-tion between anti-inflammatory effect and the extract of Rungia pectinata(Linn.) Nees.
Figure 1: A. The RAW 264.7 cells before intervention by the extract of Rungia pectinata (Linn.)Nees; B. The cells after intervention by 10 μg/mL the extract of Rungia pectinata (Linn.) Nees.
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Figure 2: Effects of the extract of Rungia pectinata (Linn.) Nees on pro-inflammatory cytokineand mediator production. RAW264.7 cells were treated with LPS (10 ng/ml) in the presence ofdifferent concentrations of the extract of Rungia pectinata (Linn.) Nees for 24 h. Cytokine ormediator levels were measured by ELISA or Griess method. Data were shown as mean ± SD(n = 3). *p < 0.05 compared to LPS alone; **p < 0.01 compared to LPS alone; §p < 0.05 com-pared to normal cell; §§p < 0.01 compared to normal cell. A: effect on TNF-α production; B:effect on IL-1β production; C: effect on IL-6 production; D: effect on NO production.
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Anti-inflammation of Rungia pectinata (Linn.) Nees 143
Effect of the Extract of Rungia pectinata (Linn.) Nees on Protein of COX-2The extract of Rungia pectinata (Linn.) Nees displayed strikingly
decreased level of COX-2 protein as shown in Figure 5 by the method ofWestern blot analysis (p < 0.01). It suggested that the extract of Rungiapectinata (Linn.) Nees could control pro-inflammatory mediator production atprotein levels.
Effect of the Extract of Rungia pectinata (Linn.) Nees on mRNA of Pro-inflammatory Cytokines and MediatorsThe extract of Rungia pectinata (Linn.) Nees displayed strikingly decreased
level of TNF-α and COX-2 mRNA as shown in Figures 3A–3B (p < 0.01). It issuggested that the extract of Rungia pectinata (Linn.) Nees may control pro-inflammatory cytokine and mediator production at gene levels. Furthermore,when the dosage of the extract of Rungia pectinata (Linn.) Nees increased, theeffects of antagonizing pro-inflammatory gene expression were significantlyelevated (p < 0.05 or 0.01), which showed dose-dependent relation betweencontrolling effect on inflammatory gene and the extract of Rungia pectinata(Linn.) Nees.
Effect of the Extract of Rungia pectinata (Linn.) Nees on NO and iNOSAs shown in Figure 2D, after LPS stimulation with the extract of
Rungia pectinata (Linn.) Nees intervention, secretion of nitric oxide (NO)was significantly decreased than that by single LPS stimulation (p <0.01). Furthermore, when the dosage of the extract of Rungia pectinata(Linn.) Nees increased, the effects of antagonizing NO releasing signifi-cantly elevated (p < 0.05), which showed dose-dependent relationbetween controlling effect on NO and the extract of Rungia pectinata(Linn.) Nees. As shown from Figure 3C, effect of the extract of Rungiapectinata (Linn.) Nees on mRNA expression of iNOS was coincident withthat on NO (p < 0.01).
Effect of the Extract of Rungia pectinata (Linn.) Nees on IL-10As shown in Figure 4A the level of IL-10 was the most decreased under
APS condition, which illustrated the effect of APS on reinforcing cellularimmunity and inhibiting humoral immunity. Increase of IL-10 on single LPSstimulation displayed the regulatory action on cells after inflammatory reac-tion. The levels of IL-10 on the extract of Rungia pectinata (Linn.) Nees inter-vention were similar to that on Dexamethasone intervention.
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Figure 3: Effects of the extract of Rungia pectinata (Linn.) Nees on mRNA expression of pro-inflammatory cytokine and mediator. RAW264.7 cells were treated with LPS (10 ng/ml) in thepresence of different concentrations of the extract of Rungia pectinata (Linn.) Nees for 4 h.The mRNA levels were measured by Real-time PCR. Data were shown as mean ± SD (n = 3).*p < 0.05 compared to LPS alone; **p < 0.01 compared to LPS alone; §p < 0.05 compared tonormal cell; §§p < 0.01 compared to normal cell. A: effect on TNF-α mRNA expression;B: effect on COX-2 mRNA expression; C: effect on iNOS mRNA expression.
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Effect of the Extract of Rungia pectinata (Linn.) Nees on HO-1 mRNA and Protein ExpressionAs shown in Figure 4B and Figure 6, the levels of HO-1 mRNA and pro-
tein in single LPS-stimulated cells and Dexamethasone-interfered cells werenot different to the level of normal incubated cells. The levels of HO-1 mRNAand protein on APS intervention were significantly higher than those on sin-gle LPS stimulation and normal incubation (p < 0.01), which suggested thatAPS could promote regression of inflammatory. Although the levels of HO-1mRNA and protein on the extract of Rungia pectinata (Linn.) Nees intervention
Figure 4: Effects of the extract of Rungia pectinata (Linn.) Nees on anti-inflammatory cytokineand mediator expression. RAW264.7 cells were treated with LPS (10 ng/ml) in the presence ofdifferent concentrations of the extract of Rungia pectinata (Linn.) Nees for 24 h (IL-10) or 18 h(HO-1 mRNA). The IL-10 levels were measured by ELISA and HO-1 mRNA levels were mea-sured by Real-time PCR. Data were shown as mean ± SD (n = 3). *p < 0.05 compared to LPSalone; **p < 0.01 compared to LPS alone; §p < 0.05 compared to normal cell; §§p < 0.01 com-pared to normal cell. A: effect on IL-10 production; B: effect on HO-1 mRNA expression.
Figure 5: Effects of the extract of Rungia pectinata (Linn.) Nees on COX-2 protein expressionassayed by western blot analysis. RAW264.7 cells were treated with LPS (10 ng/ml) in the pres-ence of different concentrations of the extract of Rungia pectinata (Linn.) Nees for 24 h.
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were higher than those on single LPS stimulation and normal incubation (p < 0.01or 0.05), the effects of the extract on HO-1 were significantly inferior to that ofAPS (p < 0.01), which implied a slight promotion of Rungia pectinata (Linn.)Nees on anti-inflammatory factor, HO-1.
Inhibition Effect of the Extract of Rungia pectinata (Linn.) Nees on NF-kBThe results of immunocytochemistry in Figure 7 showed NF-κB activation
was significantly blocked by the extract of Rungia pectinata (Linn.) Nees (p <0.01). Those results suggested that suppression of IL-1β, TNF-α, iNOS, andCOX-2 expression by the extract of Rungia pectinata (Linn.) Nees might bedue to the attenuation of NF-κB activation.
DISCUSSION
Ethanol extraction is the most common method to study plant ingredientsbecause of ethanol’s both hydrophilic and lipophilic properties.(18) Therebyin this research we chose ethanol to extract the active components in Rungiapectinata (Linn.) Nees.
Inflammation is the complex biological response of tissues to harmfulstimuli. However, inflammation might be self-damaging and therefore thetreatment on controlling inflammation is widely used in clinical practice.(19) Ininflammation progress, a series of cytokines and mediators contribute to evok-ing and regression of inflammation.(20) Thereby, in this study we examined theinfluence of the extract from Rungia pectinata (Linn.) Nees on inflammation-related cytokines and mediators.
TNF-α is a cytokine involved in systemic inflammation and is a member ofa group of cytokines that all stimulate the acute phase reaction.(21,22) IL-1βforms an important part of the inflammatory response of the body againstinfection by increasing the expression of adhesion factors on endothelial cellsto enable transmigration of leukocytes and re-setting the hypothalamus ther-moregulatory center.(23) IL-6 is one of the most important mediators of fever
Figure 6: Effects of the extract of Rungia pectinata (Linn.) Nees on HO-1 protein expressionassayed by Western blot analysis. RAW264.7 cells were treated with LPS (10 ng/ml) in the pres-ence of different concentrations of the extract of Rungia pectinata (Linn.) Nees for 24 h.
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Figure 7: Effects of the extract of Rungia pectinata (Linn.) Nees on NF-κB translocation.RAW264.7 cells were treated with LPS (10 ng/ml) in the presence of different concentrationsof the extract for 2 h. Cell nucleus stained to brown presented NF-κB activation and cyto-plasm stained to brown presented NF-κB inactivation. 7A: 10 μg/mL the extract plus LPS; 7B: 5μg/mL the extract plus LPS; 7C: 1 μg/mL the extract plus LPS; 7D: Dexamethasone plus LPS; 7E:APS plus LPS; 7F: simple LPS; 7G: normal medium.
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and the acute phase response.(24) Thereby, we chose TNF-α, IL-1β and IL-6 asa parameter to investigate the anti-inflammatory effect of the extract. In thisresearch, the extract showed the significantly anti-inflammatory efficacy thathad a dose-effect relationship with the extract, which presented the potentialactivity of Rungia pectinata (Linn.) Nees to treat typical inflammation-relateddisorders.
Cyclooxygenease-2 (COX-2) is the key enzyme regulating the productionof prostaglandins, the central mediators of inflammation. Each step ofcyclooxygenease-2 regulation can be used as potential therapeutic target.(25)
Thereby, we detected mRNA and protein expression of COX-2 to evaluate theinner anti-inflammatory mechanism of the extract. It could be observed thatthe extract could suppress the COX-2 expression at both gene and proteinlevel, which showed strong evidence that Rungia pectinata (Linn.) Nees hadan activity to inhibit inflammation.
Nitric oxide (NO) is recognized as a mediator and regulator of inflamma-tory responses. NO is produced in high amounts by inducible nitric oxide syn-thase (iNOS) by activated inflammatory cells.(26) Cyclooxygenease-2 also canbe affected directly at its enzymatic activity by nitric oxide and induciblenitric oxide synthase (iNOS) (25). Therefore in this exploration we examinedthe expression of NO and iNOS before and after the extract intervention. Theresults showed Rungia pectinata (Linn.) Nees had a significant effect on NOand iNOS. Furthermore, as nitric oxide can contribute to reperfusion injurywhen excessive amount produced during reperfusion (following a period ofischemia) reaction with superoxide to produce the damaging free radical per-oxynitrite,(27) it deserves an investigation whether Rungia pectinata (Linn.)Nees can relieve reperfusion injury after ischemia.
NF-κB is one of the most important mediators that play a key role in theregulated expression of a large number of pro-inflammatory cytokines andmediators that are known to lead to organ destruction in some inflammatoryand autoimmune diseases.(28,29) During the observation, we found NF-κBactivity was potently inhibited by the medicinal plant extract, which impliedthe effect of Rungia pectinata (Linn.) Nees on intronuclear anti-inflammatorypathway.
Apart from the effect on pro-inflammatory cytokines and mediators, pro-motional effects on anti-inflammatory factors were also investigated. IL-10has been attached much attention because of its anti-inflammatory proper-ties.(30,31) In literatures, Dexamethasone can inhibit the release of IL-10 athigh dosage and promote the release at low dosage.(32) In our research, wechose high dosage Dexamethasone as the control and found the extract had asimilar effect to Dexamethasone. As Dexamethasone at high dosage caninduce some immune cells into apoptosis, it deserves further investigation asto whether Rungia pectinata (Linn.) Nees can perform anti-inflammation onsome conditions via inducing apoptosis on some immune cells.
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Heme oxygenase-1 (HO-1) or HSP32 is the inducible isoform of heme oxy-genase.(33) HO-1 is an important anti-inflammatory enzyme featured by itsantioxidant activity.(34) Experimental models of various diseases, includingacute inflammation, have demonstrated that the induction of HO-1 can pre-vent or mitigate the symptoms associated with these ailments.(35) It can beinferred from result of our research the extract had slight anti-inflammatoryactivity via anti-oxidation by inducing HO-1.
Recently, herbal pharmacological researches were focused increasingly.(36,37)
However, so far the effect of Rungia pectinata (Linn.) Nees on pro-inflammatorycytokines and mediators, on blocking translocation of NF-kappaB, on promot-ing anti-inflammatory mediators and on comparing to Dexamethesone havenot been well explored. In this research, it can be disclosed that Rungia pecti-nata (Linn.) Nees has a strong efficacy on blocking release of pro-inflammatoryfactors, but also a slight effect on promoting anti-inflammatory factors. Basedon those activities, more pharmacological efficacies of Rungia pectinata(Linn.) Nees will be discovered in the future. When acute inflammatory andsuper-coagulative response occurs, when hepatitis and jaundice takes place,when a viral infection happens,(38) a strong anti-inflammatory agent is neces-sary. Our next-step exploration is to investigate the effects of Rungia pectinata(Linn.) Nees on these disorders and the related molecular biological mechanisms.
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