Research Article Cordyceps sinensis Increases Hypoxia …downloads.hindawi.com/journals/bmri/2013/569206.pdf · 2019-07-31 · Cordyceps sinensis claiming that it gives them energy

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 569206 13 pageshttpdxdoiorg1011552013569206

Research ArticleCordyceps sinensis Increases Hypoxia Tolerance by InducingHeme Oxygenase-1 and Metallothionein via Nrf2 Activation inHuman Lung Epithelial Cells

Mrinalini Singh Rajkumar Tulsawani Praveen Koganti Amitabh ChauhanManimaran Manickam and Kshipra Misra

Defence Institute of Physiology and Allied Science Lucknow Road Timarpur Delhi 110 054 India

Correspondence should be addressed to Rajkumar Tulsawani rktulsawaniyahoocom

Received 18 April 2013 Revised 11 July 2013 Accepted 11 July 2013

Academic Editor Dobromir Dobrev

Copyright copy 2013 Mrinalini Singh et alThis is an open access article distributed under the Creative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Cordyceps sinensis an edible mushroom growing in Himalayan regions is widely recognized in traditional system of medicineIn the present study we report the efficacy of Cordyceps sinensis in facilitating tolerance to hypoxia using A549 cell line as amodel system Treatment with aqueous extract of Cordyceps sinensis appreciably attenuated hypoxia induced ROS generationoxidation of lipids and proteins andmaintained antioxidant status similar to that of controls via induction of antioxidant gene HO1(heme oxygenase-1) MT (metallothionein) and Nrf2 (nuclear factor erythroid-derived 2-like 2) In contrast lower level of NF120581B(nuclear factor kappaB) and tumor necrosis factor-120572 observed which might be due to higher levels of HO1 MT and transforminggrowth factor-120573 Further increase in HIF1 (hypoxia inducible factor-1) and its regulated genes erythropoietin vascular endothelialgrowth factor and glucose transporter-1 was observed Interestingly Cordyceps sinensis treatment under normoxia did not regulatethe expression HIF1 NF120581B and their regulated genes evidencing that Cordyceps sinensis per se did not have an effect on thesetranscription factorsOverallCordyceps sinensis treatment inhibited hypoxia induced oxidative stress bymaintaining higher cellularNrf2 HIF1 and lowering NF120581B levels These findings provide a basis for possible use of Cordyceps sinensis in tolerating hypoxia

1 Introduction

Acclimatization is a major problem for people travellingto high altitudes for the first time Adverse environmentalconditions such as extreme cold hypoxia low humidityhigh wind velocity and high intensity of solar radiation[1ndash3] result in the risk of altitude sickness worldwide Thecommon problems are acute mountain sickness (AMS)insomnia lack of appetite tiredness lethargy upset stomachdisinclination to work bone and muscle degradation high-altitude pulmonary edema (HAPE) high-altitude cerebraledema (HACE) and all resulted in decrease in physical andmental performance in unacclimatized individuals [4ndash8]These problems may escalate rapidly and the results may alsobe lethal sometimes

For an individual or cells to adapt to hypoxic conditionsthey must be able to sense changes in oxygen tension andrespond accordingly The initiation of these responses can be

rapid involving biochemical homeostasis reprogramming oftranscription factors and gene expression and these changeslead to the production of proteins that exert a protectiveeffect on the cellMajor oxygen and redox-sensitive transcrip-tional factors (TFs) are nuclear factor- (erythroid-derived2-) like 2 (Nrf2) hypoxia-inducible factor 1 (HIF1) andnuclear factor kappa-B (NF120581B) Expression of antioxidant-responsive-element- (ARE-) driven genes and enzymes isdirected by Nrf2 caprsquonrsquocollar bZIP transcription factorsThese include glutathione peroxidase (GPx) glutathione-S-transferase (GST) heme oxygenase (HO) superoxide dis-mutase (SOD) and ferritin HIF1 is selectively stabilized inhypoxia and this in turn leads to activation of several genessuch as erythropoietin (EPO) that promotes erythropoiesisnitric oxide synthase (NOS) that modulates vascular tonevascular endothelial growth factor (VEGF) that promotesangiogenesis and glucose transporter-1 (GLUT1) that regu-lates energy metabolism Conversely NF120581B activates genes

2 BioMed Research International

particularly involved in the inflammatory response as wellas in modulating the cellular response to oxidative injuryNF120581B plays an important role in the innate and adaptiveimmunity and cellular survival through the induction ofgenetic networks [9 10] All these events are essential for thesurvival of cell to hypoxic environment

Herbs have been used throughout history to enhancephysical performance but scientific scrutiny with controlledclinical trials has only recently been used to study sucheffects Several herbs are currently used to enhance physicalperformance regardless of scientific evidence of effect onepotential supplement not yet well-characterized is Cordy-ceps sinensis (Berk) Sacc a fungus endemic to Himalayanalpine habitats (at an elevation of 3600ndash5000m) Purportedeffects of the fungus suggested a wide range of biologicalfunctions such as use as an aphrodisiac [11] analgesic [12]and immune modulator [13] and free radical scavenger [14]Human clinical trials have demonstrated the effectiveness ofCordyceps sinensis fermentedmycelia in combating decreasedlibido and virility [15 16] In a clinical study of elderlypatients with chronic fatigue results indicated that most ofthe subjects treated with Cordyceps sinensis pure myceliumreported a significant clinical improvement in the areasof fatigue cold intolerance dizziness frequent nocturiatinnitus hyposexuality and amnesia while no improvementwas reported in the placebo group [17ndash19] Inhabitants in thehigh altitude mountain regions of Tibet and Nepal consumeCordyceps sinensis claiming that it gives them energy andoffsets the symptoms of high altitude sickness while in Westit is consumed by both athletes and elderly people In recentyears Cordyceps sinensis has been investigated in animaland in vitro studies for antiaging effects activity on sexualfunction and immune modulation among other potentialuses [20 21]

Our study on the protective effect of Cordyceps sinensissupplementation on hypoxia-induced oxidative stress in lungepithelium cells (A549) is the first of its kind whereinthe effectiveness of Cordyceps sinensis in ameliorating theoxidative stress to hypoxia was studied at the molecular levelFurther evaluation of Cordyceps sinensis in acclimatizationprocess in high altitude ailments could also enhance the pos-sibility use Cordyceps sinensis as a potent naturaltherapeuticagent In view of above the present study was designed toevaluate the molecular mechanisms of action of Cordycepssinensis in the process of hypoxia tolerance

2 Material and Methods

21 Apparatus HPLC Waters ASE 350 Dionex Corporation(Sunnyvale CA USA) Spectrophotometer Bio-Rad ELISAreader (Molecular Devices USA) Spectrofluorimeter (Var-ian USA)

22 Reagents 111015840-Diphenyl-2-picrylhydrazly [DPPH] 345-trihydroxybenzoic acid [Gallic Acid] TPTZ [246-tripyridy-s-triazine] 6 Hydroxy-2578-Tetramethylchroman-2-Car-boxylic acid [Trolox] Rutin Folin-Ciocalteu reagent andall other chemicals were from Sigma Aldrich ChemicalsUSA Specific antibodies against all HIF1 NF120581B Nrf2 TNF120572

TGF120573 VEGF EPO GLUT1 HO1 MT1 and HRP conju-gated secondary antibodies were purchased from Santa CruzBiotechnology Inc California USA

23 Collection of Plant Material Cordyceps sinensis mycelia(Voucher specimen DIP-CS2011) were obtained fromDefence Institute of Bio Energy Research HaldwaniCordyceps sinensis was collected during the rainy season(May-June) from wood logs and tree stumps from the hillyregions (at an altitude of over 4000m) of the NorthwestHimalayas at different locations in Pithoragarh UttarakhandIndia where the plant growswidely under natural conditionsOnly themature fruiting bodies (seen as reddish-brown opencaps) were selected removed and washed with nanopurewater dried under shade in a clean dust-free environmentand milled into powder using pestle and mortar

24 Extraction Procedure Water extract ofCordyceps sinensiswas prepared using Accelerated Solvent Extraction system(ASE 350) equipped with a solvent controller unit (DionexCorporation Sunnyvale CA USA) The extraction was car-ried out in triplicate using water as a solvent The extractionprocedurewas as follows (i) the powdered samplewas loadedinto cell (ii) cell was filled with highly pure grade water up toa pressure of 1500 psi (iii) static extractionwas for 15min (iv)cell was rinsed with extraction solvent (60 of cell volume)and the solvent is purged from cell with N2 gas (viii) andfinally depressurization takes place Between extractions arinse of the complete system was made in order to overcomeany extract carryover The extract was lyophilized (AlliedFrost India) and stored at 4∘C

25 HPLC Fingerprinting HPLC analysis of the water extractof Cordyceps sinensis was performed using Waters HPLCsystem (Waters Corporation USA) equipped with Waters515HPLC pump Waters 717 plus autosampler and Waters2487UV detector Separation was performed in a symmetryC18 250mm times 47mm ID 5120583m column (Waters USA) bymaintaining the isocratic flow rate (1mLmin) of the mobilephase (001 M KH

2PO4pH 37 methanol 90 10) and peaks

were detected at 260 nm absorbance

26 Determination of Total Phenol Content The total phe-nolic content in extract was estimated using Folin-Ciocalteureagent (FCR) based assay using 20 120583L of stock solution(1mgmL) of the extract 80 120583l of water and 500120583L of FCR[22] After 5min incubation in dark at room temperature400 120583L of 75 sodium carbonate solution was added Themixture was further incubated in dark for 30min at roomtemperature and absorbance was read at 765 nm Totalphenols (mgg) in the extract were expressed as gallic acidequivalent (GAE) using standard curve prepared from gallicacid (01mgmL) solution

27 Determination of Total Flavonoid Content Total flavon-oid content was estimated by aluminum chloride (AlCl

3)

colorimetric assay as described elsewhere [23] using rutinas a standard 1mL of extract was added to 4mL distilled

BioMed Research International 3

water and subsequently 03mL of 5 NaNO2solution was

added After 5min 03mL of 10 AlCl3solution was added

and allowed to stand for 5min and then 02mL of 4NaOH solutionwas added to themixture and the volumewasadjusted up to 10mL with distilled water Absorbance of themixture was read at 510 nm Total flavonoid content (mgg)in the extract was expressed as rutin equivalent

28 FRAP Assay The FRAP assay was estimated usingmethod described elsewhere [24] The fresh FRAP workingsolution was prepared by mixing 25mL of 300mM acetatebuffer (31 g C

2H3NaO23H2O and 16mL C

2H4O2 pH 36)

25mL of 10mM TPTZ (246-tripyridyl-s-triazine in 40mMHCl) and 25mL of 20mM FeCl

3sdot6H2O solution which was

warmed at 37∘C prior to use 150120583L of extract (1mgmL) wasallowed to react with 2850120583L of the FRAP solution for 30minin dark and the absorbance of the color developed was read at593 nm Results were expressed as mg Trolox equivalentg ofextract using standard curve prepared from Trolox solution

29 DPPH Assay The DPPH assay was estimated usingmethod described elsewhere [25] DPPH assay stock solu-tion was prepared by dissolving 24mg DPPH with 100mLmethanol and then stored atminus20∘Cuntil neededTheworkingsolution was obtained by mixing 10mL stock solution with45mL methanol to obtain an absorbance of 110 plusmn 002units at 515 nm using the spectrophotometer 150 120583L of leafextract solution was allowed to react with 2850120583L of theDPPH solution for 2 h in the dark Then the absorbance wasmeasured at 515 nm The standard curve was linear between25 and 200 ppm Trolox Results are expressed in mg of TEgof extract

210 21015840-Azino-bis(3-ethylbenzothiazoline-6-sulfonic Acid) Di-ammonium Salt (ABTS) Assay The ABTS assay was per-formed using method described elsewhere [26] The stockABTS solution (7mM) and potassiumpersulphate (245mM)was mixed in 1 1 ratio and incubated overnight in dark atroom temperatureThe assay was performed by incubation of250120583L of chemically generated ABTS∙+ radical in potassiumpersulfate with 10 120583L of extracts or ascorbic acid standardAbsorbance was measured spectrophotometrically at 734 nmfor intervals of 1 2 5 10 and 15min after addition of extractor standard The extracts without ABTS∙+ were used as assaycontrol

211 Cells and Culture Conditions A549 cells (1 times 105cellsmL) were plated in tissue culture flasks and incubatedin normoxia (21 O

2 74 N

2 and 5 CO

2) at 37∘C in

DMEMmedium (Sigma St Louis USA) supplemented with10 heat-inactivated fetal bovine serum (Sigma St LouisUSA) and penicillin-streptomycin (50 120583gmL Invitrogen)The hypoxic conditions were achieved by culturing cells inan incubator (Jouan Saint-Nazaire France) with a 05 O

2

5 CO2 and 94 N

2atmosphere Cells were treated with

Cordyceps sinensis extract (25mgmL stock) dissolved inculture medium and after an hour of treatment cells wereexposed to hypoxia

212 MTT (Methylthiazole Tetrazolium) Cytotoxicity AssayThe MTT assay which is based on conversion of yellowtetrazolium salt to purple-formazan crystals by metabolicallyactive cells provides a quantitative determination of viablecells Cells seeded at the density of 10000 per well in 96well tissue culture plates were allowed to adhere for 24 h at37∘C Cells were then treated with 25 5 10 25 50 100 250500 and 1000 120583gmL concentrations of Cordyceps sinensisdissolved in DMEM media Cells were exposed to normoxiaand hypoxia for 24 h or 48 h and cytotoxicity was assessed byMTT assay Briefly 50 120583L of MTT (1mgmL) was added toeach well and incubated for 4 h at 37∘C Formazan crystalswere solubilized in 100 120583L of DMSO by incubating in shakingcondition at room temperature for 5min Absorbance wastaken at 570 nm with 630 nm as reference filter Absorbancegiven by untreated cells was taken as 100 cell survival

213 Biochemical Analysis After normoxic and hypoxicexposure the cells were harvested by trypsinization andwashed with sterile phosphate buffered saline (PBS pH74) The cells were then sonicated for 10 sec in PBS andcentrifuged (Sigma Co Munich Germany) at 1500 g for10min at 4∘C The pellet containing tissuecell debris wasdiscarded and the supernatant was used to determine ROSGSH lipid peroxidation the protein oxidation GR GPx andSOD activities The protein content in the homogenate wasdetermined by the method of Lowry et al [27]

214 Assay for Intracellular Redox State Intracellular redoxstate levels were measured using the fluorescent dye 27-dichlorofluorescein diacetate (H2-DCFH-DA) Briefly cellswere washed once with HBSS and incubated in the samebuffer containing 5ndash10120583g of DCFH-DA for 30min at 37∘CIntracellular fluorescence was detected with excitation at485 nm and emission at 530 nm using Spectra Max GeminiEM (Molecular Devices Sunnyvale CA)

215 Lipid Peroxidation Lipid peroxidation was assessedby measuring malondialdehyde (MDA) formed by thio-barbiturate (TBA) reaction as thiobarbituric acid reactivesubstances (TBARS) using the method described elsewhere[28] Thiobarbiturate was used as the standard and the levelof lipid peroxides was expressed as nmol MDAmg protein

216 Protein Oxidation Protein oxidation was measured bydetermining the carbonyl groups after derivatization with24-dinitrophenyl hydrazine (DNPH) [29] Carbonyl con-tent was calculated from its molar absorption coefficientas 22000mminus1 cmminus1 and results were expressed as nmolprotein carbonyl per mg protein Briefly equal volumes ofsupernatant and 10mM DNPH2M HCl were incubatedfor 60min at 50∘C Protein was then precipitated with20 trichloroacetic acid (TCA) and unreacted DNPH wasremoved by centrifugation at 1400 g for 10min The precipi-tate was washed three times with a cold ethanolethyl acetate(1 1) mixture and finally the precipitate was dissolved in1M NaOHThe absorbance was measured at 450 nm and thecarbonyl content was obtained as nmolmg protein


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217 Enzymatic and Nonenzymatic Antioxidants Reducedglutathione (GSH) levels were measured fluorometricallyby the method described elsewhere [30] The activities ofglutathione peroxidases (GPx) superoxide dismutase (SOD)and glutathione reductase (GR) were determined using com-mercial kits (Randox) as per the manufacturerrsquos instructions

218 Cellular Protein Preparation and Western Blotting Fol-lowing indicated treatments cells were washed thrice withice-cold phosphate buffered saline (PBS) and lysed in ice-cold lysis buffer (50mMTris-HCl pH 75 with 120mMNaCl10mM sodium fluoride 10mM sodium pyrophosphate2mM EDTA 1mM sodium orthovanadate 1mM phenyl-methylsulfonyl fluoride 1 NP-40 and protease inhibitorcocktail) Cellular lysates were clarified by centrifugation at12000 rpm for 30 minutes 40 120583g of protein was resolvedon 10ndash12 SDS-polyacrylamide gel which was subsequentlytransferred onto a nitrocellulose membrane (045 120583) Themembranes were probed with respective primary antibod-ies (Nrf2 HO1 MT NF120581B TNF120572 TGF120573 EPO GLUT1VEGF) followed by HRP conjugated secondary antibodiesImmunoblots were detected by chemiluminescence reagent

(Sigma USA) and the bands were developed using X-ray films (Kodak Rochester NY USA) Quantification wasperformed by densitometry using Labworks software (UVPBioImaging Systems)

219 Data Analysis All the experiments were performed onthree different occasions and data are presented as mean plusmnSE The data was analyzed by one-way ANOVA followedby Dunnetrsquos test for comparing control and the variousgroups using GraphPad software Statistical significance wasestimated at the 5 level

3 Results

31 Chemical Standardization of Water Extract of CordycepsSinensis by Its Total Phenolics Flavonoid Content AntioxidantActivity and Fingerprinting Figure 1 depicts yield totalphenolics flavonoid content and antioxidant activity ofwaterextract of Cordyceps sinensis The extract showed 159 plusmn008mgg of total phenols and 106 plusmn 005mgg of flavonoidcontent (Figure 1(a)) The antioxidant activity of extract wasfound to be 575 plusmn 038 302 plusmn 154 162 plusmn 102120583M TEg ofextract as evident by DPPH ABTS and FRAP assays respec-tively (Figure 1(b)) Figure 2 shows HPLC chromatograms at260 nm The presence of adenine the marker compound ofCordyceps sinensis was evident in the extract with a retentiontime of 643 Adenine was found to be 071ww

32 Effect of Cordyceps sinensis on Cell Viability and ROSProduction in Hypoxia Exposed A549 Cells We determinedthe effect of hypoxia alone using different percentages ofoxygen (01 05 1 and 5 O

2) or in combination

with various concentrations of Cordyceps sinensis extract(5ndash250120583gmL) on cell viability and generation of reactive


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Figure 3 Cytoprotective effects of aqueous extract of Cordyceps sinensis in A549 cells exposed to different oxygen percentages (01 05 1or 5 O

2 (a)ndash(d) for 48 h Cells exposed to hypoxia showed cell death (dark gray bar) in comparison to unexposed cells (black bar) which

was dependent on oxygen percentage Treatment of cells with aqueous extract of Cordyceps sinensis increased cell survival following hypoxiaexposure and the efficacy of extract was dose dependent (light gray bars) lowast119875 lt 005

oxygen species Cells exposed to varying percentage ofoxygen showed increased reactive oxygen species and celldeath The cytotoxic effect of hypoxia was dependent onthe concentration of oxygen (Figures 3(a)ndash3(d) and 4(a)ndash4(d) dark gray bars) The treatment of cells with variousconcentrations of extract prevented cell death and generationof reactive oxygen species following hypoxia exposure andthe protective effects were dose dependent (Figures 3(a)ndash3(d)and 4(a)ndash4(d) light gray bars) Because 05 O

2showed

approximately 51 cell death and 250120583gmL of Cordycepssinensis extract showed maximal effects on cell viability andROS generation all subsequent experiments were conductedusing the above-mentioned percentage of oxygen and con-centration of extract

33 Lipid Peroxidation Since hypoxia exposure led toincreased oxidative stress we determined the levels of lipidperoxidation Exposure to hypoxia caused a marked increase

in lipid peroxidation as observed by an increase in MDAlevels However Cordyceps sinensis treatment significantlyinhibited the hypoxia-induced lipid peroxidation as com-pared with the respective hypoxia exposed cells (Figure 5(a)119875 lt 005) No significant difference was observed in thenormoxic cells and Cordyceps sinensis treated cells kept innormoxic conditions

34 Protein Oxidation The effect of hypoxia on oxidationof proteins was measured by determining protein carbonylcontents in A549 cells after derivatization with DNPH Theresults showed a considerable increase in protein oxidation incells exposed to hypoxia as compared to the control Cordy-ceps sinensis treatment appreciably inhibited the formationof protein carbonyls levels (Figure 5(b) 119875 lt 005) afterexposure to hypoxia No significant change was observed inprotein oxidation in cells treated with Cordyceps sinensis keptin normoxic conditions relative to normoxic control cells


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Figure 4 Antioxidative effects of aqueous extract of Cordyceps sinensis in A549 cells exposed to different oxygen percentages (01 051 or 5 O

2 (a)ndash(d)) for 48 h Cells exposed to hypoxia showed elevated levels of reactive oxygen species (dark gray bars) compared to

unexposed cells (black bars) which was dependent on oxygen percentage Cordyceps sinensis decreased reactive oxygen species followinghypoxia exposure and the efficacy of extract was dose dependent (light gray bars) lowast119875 lt 005

35 Glutathione Status and Antioxidant Enzyme Activity Toassess the level of endogenous nonenzymatic antioxidantGSH was determined Exposure of A549 cells to hypobarichypoxia resulted in a significant decrease in GSH levels ascompared with normoxic control cells (Table 1 119875 lt 005)Cordyceps sinensis treatment significantly increased GSHlevels after exposure to hypoxia though no difference wasobserved between Cordyceps sinensis treatment normoxiccells and control normoxic cells (Table 1)

As shown in Table 1 exposure to hypoxia resulted in asignificant increase in SODactivity and a decrease inGPx andGR activities relative to normoxic cells (119875 lt 005) Cordycepssinensis treatment maintained these enzymes levels similar tothe control values indicating reduced oxidative stress

36 Heme Oxygenase-1 and Metallothionein Levels Con-sidering that Cordyceps sinensis treatment has significantlyinhibited ROS and maintained antioxidant status of hypoxiaexposed cells we sought whether this ability to preventoxidative stress is mediated via Nrf2 HO1 andMTTherefore

Table 1 Protective effects of aqueous extract of Cordyceps sinensison antioxidant status of cells exposed to hypoxia

Treatment GPx (UL) SOD (UmL) GR (UL) GSH (mM)Control 4007 plusmn 388 244 plusmn 174 423 plusmn 223 901 plusmn 032Hypoxia 3280 plusmn 222lowast 484 plusmn 346lowast 149 plusmn 082lowast 334 plusmn 026lowast

Control +extract 4009 plusmn 189 266 plusmn 202 438 plusmn 284 883 plusmn 033

Hypoxia +extract 3869 plusmn 204 327 plusmn 246lowast 447 plusmn 262 786 plusmn 038

A549 cells were exposed to hypoxia (05 O2) for 48 h in the absence orpresence of aqueous extract of Cordyceps sinensis (250120583gmL) A significantdecrease in GSH GPx and GR levels observed after hypoxia was restoredin Cordyceps sinensis treated cells Further an increase in SOD activity wasobserved in cells exposed to hypoxia and the effect was nearly neutralizedby Cordyceps sinensis treatment Results are means plusmn SE of three individualexperiments lowast119875 lt 005

the relative levels of Nrf2 HO1 and MT were determinedby immunoblotting Exposure of cells to hypoxia resulted


(nm

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Figure 5 Lipid peroxidation (TBARS thiobarbituric acid reactive substance) and protein oxidation (carbonyl groups after derivatizationof proteins with dinitrophenylhydrazine) in A549 cells were determined after (05 O

2) hypoxia exposure for 48 h by spectrophotometric

measurement A significant increase in both lipid peroxidation and protein oxidation was observed in cells exposed to hypoxia and suchchanges were attenuated in hypoxic cells treated with Cordyceps sinensis extract Experiments were done in triplicate Values are mean plusmn SEof three individual experiments lowast119875 lt 005

in a significant increase in Nrf2 HO1 and MT relative tonormoxic control cells (Figure 6) Cordyceps sinensis treat-ment and hypoxia exposure further increased expression ofNrf2 HO1 and MT levels compared to normoxic cells

37 Role of NF120581B in Cellular Protection To investigate therole of NF120581B-mediated mechanisms in the induction ofprotection the effect ofCordyceps sinensis treatment onNF120581Band its target genes were examined It is a key transcriptionalfactor that regulates inflammatorymediatorsWe determinedthe relative levels of active NF120581B by immunoblotting Expo-sure of cells to hypoxia resulted in amarked increase in NF120581Blevels however Cordyceps sinensis treatment significantlyattenuated NF120581B expression (Figure 7)

Since NF120581B regulates inflammatory mediators we deter-mined pro- and anti-inflammatory molecules like TNF120572 andTGF120573 There was a considerable increase in TNF120572 levelsupon hypoxic exposure relative to control cells However nochange in TGF120573 levels was noticed in the cells exposed tohypoxiaCordyceps sinensis treatment significantly attenuatedhypoxia induced increase in TNF120572 level Interestingly thelevels of TGF120573 increased upon Cordyceps sinensis treatment(Figure 7)

38 Effect of Cordyceps sinensis on HIF1 Expression and ItsRegulated Genes HIF1 level was measured in A549 cells byimmunoblotting The results showed a marked increase inHIF1 expression during exposure to hypoxia InterestinglyCordyceps sinensis treated cells under hypoxic conditionshowed further increase in HIF1 protein (Figure 8) To knowwhether increased HIF1 levels in hypoxia and Cordycepssinensis treated cells resulted in increased expression of its

downregulated genes western blotting was performed forEPO GLUT1 and VEGF which promote erythropoiesis glu-cose transport and angiogenesis respectively A significantincrease in VEGF and GLUT1 protein levels was observedin hypoxia and Cordyceps sinensis treated cells EPO proteinincreased slightly following hypoxia and Cordyceps sinensistreatment (Figure 8)

4 Discussion

In the preliminary experiments A549 cells were exposed todifferent percentages of oxygen 01 05 1 and 5 for24ndash48 h duration and the results showed 70ndash77 42ndash5132ndash44 and 15ndash27 cell death with an increase of 66ndash7453ndash67 43ndash51 and 35ndash43 fold in reactive oxygen speciesrespectively The findings suggested that the cell death wasdue to increased oxidative stress governed by percentage ofoxygen and duration of hypoxia exposure Based on prelimi-nary data optimal percentage of oxygen was found to be 05O2as 01 O

2percentage of oxygen reduced cell survival to

23ndash30while 1 and 5O2resulted inminimal cell death In

a separate study cells were exposed to various concentrationsof Cordyceps sinensis extract (25ndash1000 120583gmL) for 24 h and48 h and A549 cells did not show any toxicity per se Theconcentration of Cordyceps sinensis extract below 5120583gmLwas not effective and cells treated above 250120583gmL could notprovide additional improvement in efficacy of extract hencedata of effective dose range 5ndash250120583gmL is only presentedin Figures 3(a)ndash3(d) and 4(a)ndash4(d) The effective dose rangefor aqueous extract of Cordyceps sinensis under hypoxiaexposure of 48 h was found to be 5ndash250120583gmL howeverthe best results were obtained with 250120583gmL against all


ROD

0

50

100

150

200

Nrf2

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

lowastlowast

lowast

(a)

ROD

0

50

100

150

200

250

HO1

lowastlowast

lowast

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(b)

ROD

0

50

100

150

200

250

MT

lowast

lowast

lowast

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(c)

ROD

0

50

100

150

200

120573-Actin

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(d)

Figure 6 Effect of Cordyceps sinensis treatment on the expression of antioxidant transcription factor (Nrf2 (a)) and its regulated genes hemeoxygenase-1 (HO1 (b)) and metallothionein 1 (MT (c)) in A549 cells (05 O

2) hypoxia exposure for 48 h There was a significant increase

in Nrf2 HO1 and MT levels following hypoxia exposure and also in the presence of Cordyceps sinensis extract The figures are representativeof three independent experiments These were normalized with actin to observe any change in expression

the percentage of oxygen Therefore we have used 05 O2

and 250 120583gmL of Cordyceps sinensis extract in all our studiesexcept MTT and ROS

The present study shows that treatment of Cordy-ceps sinensis significantly improves tolerance to hypoxia asrevealed by increased Nrf2 and HIF1 and decrease in NF120581Btranscription factors This in turn led to increased antioxi-dant genes antioxidant enzymes EPO VEGF and GLUT1levels facilitating better oxygenation oxygen delivery andglucose transport At the same time decrease in NF120581B levelsresulted in lower expression of proinflammatory cytokineslike TNF120572 and increased expression of anti-inflammatory

cytokine TGF120573 All these changes contributed to the increasein hypoxic tolerance ofCordyceps sinensis treated cells To thebest of our knowledge we have demonstrated for the firsttime the potency of Cordyceps sinensis treatment in inducingtolerance to hypoxia

During hypoxia less oxygen is available to be reduced toH2O at cytochrome oxidase causing accumulation of highly

reactive oxygen intermediates (ROIs) Overproduction ofROIs can readily cause oxidative damage to various biologicalmacromolecules resulting in DNA damage oxidation of keyamino acid side chains formation of protein-protein cross-links oxidation of the polypeptide backbone resulting in


ROD

0

50

100

150

200NF120581B

lowast

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(a)

ROD

0

50

100

150

200

TNF120572

lowast

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(b)

ROD

0

50

100

150

200

250

lowast

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

TGF120573

(c)

ROD

0

50

100

150

200

120573-Actin

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(d)

Figure 7 Western blot analyses showed significant increase in NF120581B (a) and pro-inflammatory cytokines TNF120572 (b) while no change wasobserved in levels of anti-inflammatory cytokines TGF120573 (c) inA549 cells following hypoxia (05O

2) exposure for 48 hThe treatment of cells

with extract prevented increase in NF120581B and TNF120572 levels following hypoxia exposure Further an increase in TGF120573 levels was also evidentin Cordyceps sinensis treated cells The figures are representative of three independent experiments These were normalized with 120573-actin (d)to observe any change in expression

protein fragmentation and lipid peroxidation The presentstudy also reports that exposure of A549 cells to hypoxiaresulted in an appreciable increase in ROS levels whichin turn could be responsible for the observed increasein oxidation of cellular protein and lipids For hypoxia-associated free radicals antioxidant can safely interact withfree radicals and terminate the chain reaction before vitalbiological macromolecules are damaged Cellular antioxi-dants defense network comprising the well-known endoge-nous enzymes superoxide dismutase (SOD) catalase (CAT)and glutathione peroxidase (GPx) as well as nonenzymatic

antioxidants reduced glutathione (GSH) These participatein maintaining the proper balance of free radicals andantioxidants in the healthy cellsThe cells exposed to hypoxiashowed a decrease in GSH levels and to cope up with thishypoxia-induced stress a marked increase in activities ofcellular antioxidant enzymes such as SOD GPx and GRwere observed HoweverCordyceps sinensis supplementationmaintained the antioxidant enzymes levels similar to thoseof control values and restores the GSH level The findingssuggested that Cordyceps sinensis exerted its robust antioxi-dant defense abilities by activating endogenous antioxidant


ROD

0

50

100

150

200

250HIF1

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

lowast

lowast

(a)RO

D

0

50

100

150

200

250EPO

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

lowast lowast

(b)

ROD

0

50

100

150

200GLUT1

lowastlowast

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(c)

ROD

0

50

100

150

200VEGF

lowast

lowast

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(d)

ROD

0

50

100

150

200

250120573-Actin

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(e)

Figure 8 Immunoblot analysis of HIF1 (a) and HIF1 regulated genes namely EPO (b) GLUT1 (c) and VEGF (d) in A549 cells followinghypoxia (05O

2) exposure for 48 hThe treatment of cells withCordyceps sinensis increased levels ofHIF1 EPOGLUT1 andVEGF following

hypoxia exposure The figures are representative of three independent experiments These were normalized with 120573-actin (e) to observe anychange in expression

enzymes as well as inhibiting protein and lipid peroxidationdue to presence of higher amount of phenols flavonoidsnucleosides (adenosine) nucleobases (adenine and uracil)and polysaccharides these compounds absorb and neutralizefree radicals quench singlet and triplet oxygen or decomposeperoxides [31] This result is in agreement with other reportsthat aqueous extracts of natural and cultured Cordycepsspecies are more effective in free radical scavenging than inhydrophobic systems [32] Yaorsquos group [33] has reported thatthe water extracts of Cordyceps sinensis mycelia have directand moderate-to-potent antioxidant activities involving thescavenging of superoxide anion radical hydroxyl radical andinhibition of lipid peroxidation They also proposed that theantioxidant activities of the water extracts may be caused bya combined effect of proteins polysaccharides and mannitolor some other compounds in Cordyceps sinensis myceliaHowever Li et al [34] have observed that the water extractof Cordyceps sinensis mycelia possessed a strong antioxidantactivity and that partially purified polysaccharides from thewater extract showed greater antioxidant activities (10-fold to30-fold) than before purification Therefore polysaccharides

may be regarded as the key components of the antioxidantactivity of Cordyceps sinensismycelia

Growing evidence has indicated that cellular redox playsan essential role not only in cell survival but also in cellularsignaling pathways such as NF120581B Several laboratories havedemonstrated that treatment of cells with H

2O2(ROIs)

can activate the NF120581B pathway as reviewed by Bauerleand Henkel [35] Shih et al [36] demonstrated that thehydroxyl radical was primarily responsible for activationof NF120581B in Jurkat cells and macrophages During hypoxiaelevated levels of ROIs prompted speculation that ROIs mayfunction as commonmediators ofNF120581B activation also SinceNF120581B is involved in inflammation we therefore analyzed theexpression of NF120581B and its proinflammatorymediator TNF120572in cells exposed to hypoxia A marked increase in NF120581B andTNF120572 levels was seen during hypoxia however Cordycepssinensis treatment + hypoxia markedly inhibited NF120581B andTNF120572 expression possibly due to reduction in hypoxia-induced oxidative stress indicating towards cells defenseagainst the hypoxic stress This hypothesis is in agreementwith an earlier report in which evidence showing that nearly


Proinflammatorycytokine

Inflammation

Nrf2

HO1 MT SOD GPx GR

GLUT1 VEGF EPO

Anti-inflammatorycytokine

Tolerance to hypoxia

Oxidative stressROS GSH

Antioxidant genes

Antioxidant enzymes

HIF1

Hypoxia ( Cordyceps sinensis ( )

NF120581B

TNF120572 TGF120573

)Hypoxia +

Figure 9 Schematic representations of effects of hypoxia and aqueous extract of Cordyceps sinensis in A549 cells Putative corelationamong various transcription factors and mechanism of tolerance to hypoxia upregulationdownregulation after hypoxia (solid arrow) andupregulationdownregulation after Cordyceps sinensis treatment (dotted arrow)

all pathways leading to NF120581B activation could be blockedby a variety of antioxidants including pyrrolidine dithio-carbamate (PDTC) N-acetyl-L-cysteine (NAC) glutathione(GSH) or thioredoxin (Txn) or by overexpression of antiox-idant enzymes including superoxide dismutase glutathioneperoxidase or thioredoxin peroxidase As aforementionedCordyceps sinensis possesses robust antioxidant properties

Based on the results mentioned above and reported factswe hypothesized that Cordyceps sinensis treatment inhibitedoxidative stress and inflammation in the hypoxia exposedcells Thus we explored the probability of alterations in theexpression of Nrf2 transcription factor a master regulatorof endogenous antioxidative systems [36ndash38] being involvedin cell survival We found that Cordyceps sinensis + hypoxiatreatment resulted in a significant increase in Nrf2 levelsHaving confirmed the expression of Nrf2 we investigatedthe levels of HO1 which is known to possess antioxidantand anti-inflammatory activity and regulated by Nrf2 [39]Our results demonstrated that Cordyceps sinensis + hypoxiaresulted in higher levels of HO1 We also measured otheranti-inflammatory mediators such as TGF120573 and MT levelsin A549 cells The study revealed significant upregulation ofTGF120573 and MT after Cordyceps sinensis + hypoxia treatmentand all these genes might be responsible for the observed fallin proinflammatory cytokines after exposure to hypoxia Ourresults are in accordance with previous report [40] statingthat activation of Nrf2 effectively reduces oxidative stress andinflammation in rats Similarly overexpression of TGF120573 HO1and MT facilitates acclimatization and maintains the redoxbalance in the cells [41 42]The capacity ofCordyceps sinensisin limiting the oxidative stress is partly mediated by Nrf2pathway

Hypoxia has been shown to activateHIF1 thus an attemptwas made to investigate changes in the expression andto define a functional involvement of HIF1 in responseto Cordyceps sinensis treatment We found that Cordycepssinensis treatment 1 h before hypoxia exposure resulted in asignificant increase in HIF1 levels this suggests that HIF1-mediated gene expression may be involved in hypoxia-induced tolerance Therefore we determined the relativelevels of HIF1 regulated genes EPO GLUT1 and VEGF byimmunoblotting in control and Cordyceps sinensis treatedcells Cordyceps sinensis treatment under hypoxia resultedin higher EPO levels increased EPO levels and increasedO2

carrying capacity Further the protein levels of GLUT1mediating the transport of glucose were increased in hypoxiaand Cordyceps sinensis + hypoxia cells compared to the con-trol cells indicating enhanced glucose uptake for continuedenergy generation in hypoxic environments Our results arein accordance with previous studies suggest that CordyMaxCS-4 supplementation effectively lowers blood glucose andplasma insulin and improves glucose metabolism by enhanc-ing insulin sensitivity [43] It is presumed that when cellsin the tissue are deprived of oxygen they release angiogenicfactor that induce new capillary growth and hence better oxy-gen transport [6] Of the known angiogenic factors FGF andVEGF aremost commonly expressed [8] In our study too wefound a significant increase inVEGFprotein levels in hypoxiccells and Cordyceps sinensis + hypoxia exposed cells relativeto control cells These results indicate that the induction ofspecific HIF1 and its regulated genes in Cordyceps sinensistreated cells following hypoxia is an attempt by cells to initiatethe process of acclimatization to escape death Putative core-lation among various transcription factors andmechanism of


tolerance offered by aqueous extract of Cordyceps sinensis tohypoxia is schematically represented in Figure 9

To conclude exposure of cells to hypoxia resulted in asignificant increase in oxidative stress further there wasa marked increase in NF120581B activity and inflammatorymediators TNF120572 Treatment of cells by Cordyceps sinensis(250120583gmL) significantly attenuated the oxidative stress inhypoxia This was attributed to activation of Nrf2-ARE path-way reducedNF120581B activity and increased oxygen availabilityvia HIF1 signaling mechanisms Further higher levels ofantioxidant anti-inflammatory mediators namely TGF120573HO1 and MT levels in Cordyceps sinensis treated cellsmight also be responsible for tolerance of cells to hypoxiaThe findings of the study will help in the development ofnovel therapeutic strategies to use Cordyceps sinensis as anutraceutical for promoting tolerance to high altitude

Conflict of Interests

The authors declare that there is no conflict of interests

Acknowledgments

The authors are thankful to the Director of DIPAS Delhifor her constant support and constructive suggestions andthe Director of DIBER Haldwani India for providing studymaterial We acknowledge Dr Sayeed Ahmed Jamia Ham-dard University Delhi for carrying out HPLC analysis ofextract

References

[1] MWardMountainMedicine A Clinical Study of Cold and HighAltitude Crosby Lockwood staples London UK 1975

[2] MMoehrle B Dennenmoser andCGarbe ldquoContinuous long-term monitoring of UV radiation in professional mountainguides reveals extremely high exposurerdquo International Journalof Cancer vol 103 no 6 pp 775ndash778 2003

[3] L G Pugh ldquoTolerance to extreme cold at altitude in a NepalesePilgrimrdquo Journal of Applied Physiology vol 18 pp 1234ndash12381963

[4] B Shukitt-Hale L E Banderet and H R Lieberman ldquoEle-vation-dependent symptom mood and performance changesproduced by exposure to hypobaric hypoxiardquo InternationalJournal of Aviation Psychology vol 8 no 4 pp 319ndash334 1998

[5] A R Frisancho ldquoFunctional adaptation to high altitude hyp-oxiardquo Science vol 187 no 4174 pp 313ndash319 1975

[6] A J Peacock ldquoMedical problems of high altituderdquo Journal of theRoyal College of Physicians of Edinburgh vol 38 no 2 pp 126ndash128 2008

[7] I Singh P K KhannaM C SrivastavaM Lal S B Roy and CS Subramanyam ldquoAcute mountain sicknessrdquoThe New EnglandJournal of Medicine vol 280 no 4 pp 175ndash184 1969

[8] ldquoThe Lake Louise Consensus on the definition and quantifi-cation of altitude illnessrdquo in Hypoxia and Mountain MedicineProceedings of the 7th International Hypoxia Symposium Held atLake Louise Canada February 1991 J R Sutton G Coates andHHouston Eds vol 84 ofAdvances in the Biosciences pp 327ndash330 Pergamon Oxford UK 1992

[9] P J Barnes and M Karin ldquoNuclear factor-120581Bmdasha pivotaltranscription factor in chronic inflammatory diseasesrdquoTheNewEngland Journal ofMedicine vol 336 no 15 pp 1066ndash1071 1997

[10] M Karin ldquoThe beginning of the end I120581B kinase (IKK) and NF-120581B activationrdquo Journal of Biological Chemistry vol 274 no 39pp 27339ndash27342 1999

[11] N K Bhattarai ldquoFolk herbal medicines of Dolakha districtNepalrdquo Fitoterapia vol 64 no 5 pp 387ndash395 1993

[12] K Koyama T Imaizumi M Akiba et al ldquoAntinociceptivecomponents of Ganoderma lucidumrdquo Planta Medica vol 63no 3 pp 224ndash227 1997

[13] X J Gong H Ji S G Lu et al ldquoEffects of polysaccharides fromcultured Cordyceps sinensis on murine immunologic functionrdquoZhongguo Yaoke Daxue Xuebao vol 31 pp 53ndash55 2000

[14] Y Yamaguchi S Kagota K Nakamura K Shinozuka and MKunitomo ldquoAntioxidant activity of the extracts from fruitingbodies of cultured Cordyceps sinensisrdquo Phytotherapy Researchvol 14 no 8 pp 647ndash649 2000

[15] Y Z Guo ldquoMedicinal chemistry pharmacology and clinicalapplications of fermented mycelia of Cordyceps sinensis andJinShuBao capsulerdquo Journal of Modern Diagnostics and Thera-peutics vol 1 pp 60ndash65 1986

[16] J S Zhu and J M Rippe ldquoCordyMax enhances aerobic ca-pability endurance performance and exercise metabolism inhealthy mid-age to elderly sedentary humansrdquo The FASEBJournal vol 18 no 4 p A931 2004

[17] T Mizuno ldquoMedicinal effects and utilization of Cordyceps(Fr) Link (Ascomycetes) and Isaria Fr (Mitosporic fungi)Chinese caterpillar fungi lsquoTochukasorsquordquo International Journal ofMedicinal Mushrooms vol 1 no 3 pp 251ndash262 1999

[18] Z D Bao Z G Wu and F Zheng ldquoAmelioration of amino-glycoside nephrotoxicity by Cordyceps sinensis in old patientsrdquoChinese Journal of Integrated Traditional and Western Medicinevol 14 no 5 pp 259ndash271 1994

[19] D G Chen ldquoEffects of JinShuiBao capsule on the quality oflife of patients with heart failurerdquo Journal of Administration ofTraditional Chinese Medicine vol 5 pp 40ndash43 1995

[20] D B Ji J Ye and C L Li ldquoAntiaging effect of Cordyceps sinensisextractrdquo Phytotherapy Research vol 23 no 1 pp 116ndash122 2009

[21] D K Park W S Choi P-J Park et al ldquoImmunoglobulin andcytokine production frommesenteric lymph node lymphocytesis regulated by extracts ofCordyceps sinensis in C57Bl6NmicerdquoJournal of Medicinal Food vol 11 no 4 pp 784ndash788 2008

[22] V L Singleton and J A Rossi ldquoCalorimetry of total phenolicswith phosphomolybdic phosphotungstic acid reagentsrdquo Ameri-can Journal of Enology and Viticulture vol 16 pp 144ndash158 1965

[23] J Zhishen TMengcheng andW Jianming ldquoThedeterminationof flavonoid contents in mulberry and their scavenging effectson superoxide radicalsrdquo Food Chemistry vol 64 no 4 pp 555ndash559 1999

[24] I F F Benzie and J J Strain ldquoThe ferric reducing ability ofplasma (FRAP) as a measure of ldquoantioxidant powerrdquo the FRAPassayrdquo Analytical Biochemistry vol 239 no 1 pp 70ndash76 1996

[25] M Burits and F Bucar ldquoAntioxidant activity of Nigella sativaessential oilrdquo Phytotherapy Research vol 14 pp 323ndash328 2000

[26] R Re N Pellegrini A Proteggente A PannalaM Yang andCRice-Evans ldquoAntioxidant activity applying an improved ABTSradical cation decolorization assayrdquo Free Radical Biology andMedicine vol 26 no 9-10 pp 1231ndash1237 1999

[27] O H Lowry N J Rosebrough A L Faro and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951


[28] H Ohkawa N Ohishi and K Yagi ldquoAssay for lipid peroxidesin animal tissues by thiobarbituric acid reactionrdquo AnalyticalBiochemistry vol 95 no 2 pp 351ndash358 1979

[29] R L Levine D Garland C N Oliver et al ldquoDetermination ofcarbonyl content in oxidatively modified proteinsrdquo Methods inEnzymology vol 186 pp 464ndash478 1990

[30] P J Hissin and R Hilf ldquoA fluorometric method for determina-tion of oxidized and reduced glutathione in tissuesrdquo AnalyticalBiochemistry vol 74 no 1 pp 214ndash226 1976

[31] L Costantino A Albasini G Rastelli and S Benvenuti ldquoActiv-ity of polyphenolic crude extracts as scavengers of superoxideradicals and inhibitors of xanthine oxidaserdquo Planta Medica vol58 no 4 pp 342ndash344 1992

[32] M Y Hui B-S Wang S C Huang and P-D Duh ldquoCompar-ison of protective effects between cultured Cordyceps militarisand natural Cordyceps sinensis against oxidative damagerdquo Jour-nal of Agricultural and Food Chemistry vol 54 no 8 pp 3132ndash3138 2006

[33] C-H Dong and Y-J Yao ldquoIn vitro evaluation of antioxidantactivities of aqueous extracts from natural and culturedmyceliaof Cordyceps sinensisrdquo Food Science and Technology vol 41 no4 pp 669ndash677 2008

[34] S P Li P Li T T X Dong and K W K Tsim ldquoAnti-oxidationactivity of different types of natural Cordyceps sinensis andcultured Cordyceps myceliardquo Phytomedicine vol 8 no 3 pp207ndash212 2001

[35] P A Bauerle and T Henkel ldquoFunction and activation of NK-120581Bin the immune systemrdquo Annual Review of Immunology vol 12pp 141ndash179 1994

[36] A Y Shih P Li and T HMurphy ldquoA small-molecule-inducibleNrf2-mediated antioxidant response provides effective prophy-laxis against cerebral ischemia in vivordquo Journal of Neurosciencevol 25 no 44 pp 10321ndash10335 2005

[37] J Y Chan and M Kwong ldquoImpaired expression of glutathionesynthetic enzyme genes in mice with targeted deletion of theNrf2 basic-leucine zipper proteinrdquo Biochimica et BiophysicaActa vol 1517 no 1 pp 19ndash26 2000

[38] T Ishii K Itoh S Takahashi et al ldquoTranscription factor Nrf2coordinately regulates a group of oxidative stress-induciblegenes inmacrophagesrdquo Journal of Biological Chemistry vol 275no 21 pp 16023ndash16029 2000

[39] L E Otterbein M P Soares K Yamashita and F H BachldquoHeme oxygenase-1 unleashing the protective properties ofhemerdquo Trends in Immunology vol 24 no 8 pp 449ndash455 2003

[40] M Asghar L George and M F Lokhandwala ldquoExercise de-creases oxidative stress and inflammation and restores renaldopamine D1 receptor function in old ratsrdquo American Journalof PhysiologymdashRenal Physiology vol 293 no 3 pp F914ndashF9192007

[41] J Hollander R Fiebig M Gore et al ldquoSuperoxide dismu-tase gene expression in skeletal muscle fiber-specific adapta-tion to endurance trainingrdquo American Journal of PhysiologymdashRegulatory Integrative and Comparative Physiology vol 277 no3 pp R856ndashR862 1999

[42] S K Powers and M J Jackson ldquoExercise-induced oxidativestress cellularmechanisms and impact onmuscle force produc-tionrdquo Physiological Reviews vol 88 no 4 pp 1243ndash1276 2008

[43] C-S Zhao W-T Yin J-Y Wang et al ldquoCordyMaxŮ Cs-4improves glucose metabolism and increases insulin sensitivityin normal ratsrdquo Journal of Alternative and ComplementaryMedicine vol 8 no 3 pp 309ndash314 2002

Submit your manuscripts athttpwwwhindawicom

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Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Autoimmune Diseases


Anesthesiology Research and Practice

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Journal of


Pharmaceutics


MEDIATORSINFLAMMATION

of


particularly involved in the inflammatory response as wellas in modulating the cellular response to oxidative injuryNF120581B plays an important role in the innate and adaptiveimmunity and cellular survival through the induction ofgenetic networks [9 10] All these events are essential for thesurvival of cell to hypoxic environment

Herbs have been used throughout history to enhancephysical performance but scientific scrutiny with controlledclinical trials has only recently been used to study sucheffects Several herbs are currently used to enhance physicalperformance regardless of scientific evidence of effect onepotential supplement not yet well-characterized is Cordy-ceps sinensis (Berk) Sacc a fungus endemic to Himalayanalpine habitats (at an elevation of 3600ndash5000m) Purportedeffects of the fungus suggested a wide range of biologicalfunctions such as use as an aphrodisiac [11] analgesic [12]and immune modulator [13] and free radical scavenger [14]Human clinical trials have demonstrated the effectiveness ofCordyceps sinensis fermentedmycelia in combating decreasedlibido and virility [15 16] In a clinical study of elderlypatients with chronic fatigue results indicated that most ofthe subjects treated with Cordyceps sinensis pure myceliumreported a significant clinical improvement in the areasof fatigue cold intolerance dizziness frequent nocturiatinnitus hyposexuality and amnesia while no improvementwas reported in the placebo group [17ndash19] Inhabitants in thehigh altitude mountain regions of Tibet and Nepal consumeCordyceps sinensis claiming that it gives them energy andoffsets the symptoms of high altitude sickness while in Westit is consumed by both athletes and elderly people In recentyears Cordyceps sinensis has been investigated in animaland in vitro studies for antiaging effects activity on sexualfunction and immune modulation among other potentialuses [20 21]

Our study on the protective effect of Cordyceps sinensissupplementation on hypoxia-induced oxidative stress in lungepithelium cells (A549) is the first of its kind whereinthe effectiveness of Cordyceps sinensis in ameliorating theoxidative stress to hypoxia was studied at the molecular levelFurther evaluation of Cordyceps sinensis in acclimatizationprocess in high altitude ailments could also enhance the pos-sibility use Cordyceps sinensis as a potent naturaltherapeuticagent In view of above the present study was designed toevaluate the molecular mechanisms of action of Cordycepssinensis in the process of hypoxia tolerance

2 Material and Methods

21 Apparatus HPLC Waters ASE 350 Dionex Corporation(Sunnyvale CA USA) Spectrophotometer Bio-Rad ELISAreader (Molecular Devices USA) Spectrofluorimeter (Var-ian USA)

22 Reagents 111015840-Diphenyl-2-picrylhydrazly [DPPH] 345-trihydroxybenzoic acid [Gallic Acid] TPTZ [246-tripyridy-s-triazine] 6 Hydroxy-2578-Tetramethylchroman-2-Car-boxylic acid [Trolox] Rutin Folin-Ciocalteu reagent andall other chemicals were from Sigma Aldrich ChemicalsUSA Specific antibodies against all HIF1 NF120581B Nrf2 TNF120572

TGF120573 VEGF EPO GLUT1 HO1 MT1 and HRP conju-gated secondary antibodies were purchased from Santa CruzBiotechnology Inc California USA

23 Collection of Plant Material Cordyceps sinensis mycelia(Voucher specimen DIP-CS2011) were obtained fromDefence Institute of Bio Energy Research HaldwaniCordyceps sinensis was collected during the rainy season(May-June) from wood logs and tree stumps from the hillyregions (at an altitude of over 4000m) of the NorthwestHimalayas at different locations in Pithoragarh UttarakhandIndia where the plant growswidely under natural conditionsOnly themature fruiting bodies (seen as reddish-brown opencaps) were selected removed and washed with nanopurewater dried under shade in a clean dust-free environmentand milled into powder using pestle and mortar

24 Extraction Procedure Water extract ofCordyceps sinensiswas prepared using Accelerated Solvent Extraction system(ASE 350) equipped with a solvent controller unit (DionexCorporation Sunnyvale CA USA) The extraction was car-ried out in triplicate using water as a solvent The extractionprocedurewas as follows (i) the powdered samplewas loadedinto cell (ii) cell was filled with highly pure grade water up toa pressure of 1500 psi (iii) static extractionwas for 15min (iv)cell was rinsed with extraction solvent (60 of cell volume)and the solvent is purged from cell with N2 gas (viii) andfinally depressurization takes place Between extractions arinse of the complete system was made in order to overcomeany extract carryover The extract was lyophilized (AlliedFrost India) and stored at 4∘C

25 HPLC Fingerprinting HPLC analysis of the water extractof Cordyceps sinensis was performed using Waters HPLCsystem (Waters Corporation USA) equipped with Waters515HPLC pump Waters 717 plus autosampler and Waters2487UV detector Separation was performed in a symmetryC18 250mm times 47mm ID 5120583m column (Waters USA) bymaintaining the isocratic flow rate (1mLmin) of the mobilephase (001 M KH

2PO4pH 37 methanol 90 10) and peaks

were detected at 260 nm absorbance

26 Determination of Total Phenol Content The total phe-nolic content in extract was estimated using Folin-Ciocalteureagent (FCR) based assay using 20 120583L of stock solution(1mgmL) of the extract 80 120583l of water and 500120583L of FCR[22] After 5min incubation in dark at room temperature400 120583L of 75 sodium carbonate solution was added Themixture was further incubated in dark for 30min at roomtemperature and absorbance was read at 765 nm Totalphenols (mgg) in the extract were expressed as gallic acidequivalent (GAE) using standard curve prepared from gallicacid (01mgmL) solution

27 Determination of Total Flavonoid Content Total flavon-oid content was estimated by aluminum chloride (AlCl

3)

colorimetric assay as described elsewhere [23] using rutinas a standard 1mL of extract was added to 4mL distilled







2H4O2 pH 36)







2 74 N

2 and 5 CO

2) at 37∘C in


2

5 CO2 and 94 N









0

5

10

15

20

mg

g of

extr

act

Total phenols

Total flavonoids

120583M

trol

ox eq

uiva

lent

g o

f ext

ract


0

5

10

15

20

25

30

35

DPPH

ABTS

FRAP



0 5 0

10 15 20 25 30

100200300400500

Time (min)

Adenine

Volta

ge (m

V)






3 Results






00

02

04

06

08

10

Con

trol


MTT

assa

y (Δ

OD490)

lowastlowast

lowastlowast

lowast

lowastlowast

+5120583

gm

L

+10

120583g

mL

+25

120583g

mL

+50120583

gm

L

+100120583

gm

L

+250120583

gm

L

01

O

2

(a)

Con

trol


02

04

06

08

10

MTT

assa

y (Δ

OD490)

lowast

lowast

+5120583

gm

L

lowast

+10

120583g

mL

lowast

+25

120583g

mL

+50120583

gm

L

+100120583

gm

L

+250120583

gm

L

05

O

2

(b)

Con

trol


02

04

06

08

10

MTT

assa

y (Δ

OD490)

lowast

lowastlowast

lowast

+5120583

gm

L

+10

120583g

mL

+25

120583g

mL

+50120583

gm

L

+100120583

gm

L

+250120583

gm

L

1

O2

(c)

Con

trol


02

04

06

08

10M

TT as

say

(ΔO

D490)

lowastlowast

lowast+5120583

gm

L

+10

120583g

mL

+25

120583g

mL

+50120583

gm

L

+100120583

gm

L

+250120583

gm

L

5

O2

(d)





2showed






0

50

100

150

200

250

300

Relat

ive fl

uore

scen

ce u

nits

Con

trol

lowast

lowast

lowast

lowast

lowast

lowastlowast

+5120583

gm

L

+10

120583g

mL

+25

120583g

mL

+50120583

gm

L

+100120583

gm

L

+250120583

gm

L

01

O

2


(a)

0

50

100

150

200

250

Relat

ive fl

uore

scen

ce u

nits

Con

trol


lowast

lowast

+5120583

gm

L

lowast

+10

120583g

mL

lowast

lowast

+25

120583g

mL

+50120583

gm

L

+100120583

gm

L

+250120583

gm

L

05

O

2

(b)

0

50

100

150

200

250

Relat

ive fl

uore

scen

ce u

nits

Con

trol


lowast

lowast

lowast

lowast

+5120583

gm

L

+10

120583g

mL

+25

120583g

mL

+50120583

gm

L

+100120583

gm

L

+250120583

gm

L

1

O2

(c)

0

50

100

150

200

250

Relat

ive fl

uore

scen

ce u

nits

Con

trol


lowastlowast

lowast

+5120583

gm

L

+10

120583g

mL

+25

120583g

mL

+50120583

gm

L

+100120583

gm

L

+250120583

gm

L

5

O2

(d)














(nm

olm

g pr

otei

n)

00

05

10

15

20

25

Con

trol

Lipid per oxidation

CP 2

50 120583

gm

L al

one

lowast

05

O2

(hyp

oxia

)

05

O2

+ CP

250

120583g

mL

(a)

(nm

olm

g pr

otei

n)

0

10

20

30

40

50

60

Con

trol

Protein oxidation

CP 2

50 120583

gm

L al

one

lowast

05

O2

(hyp

oxia

)

05

O2

+ CP

250

120583g

mL

(b)









4 Discussion






ROD

0

50

100

150

200

Nrf2

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

lowastlowast

lowast

(a)

ROD

0

50

100

150

200

250

HO1

lowastlowast

lowast

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(b)

ROD

0

50

100

150

200

250

MT

lowast

lowast

lowast

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(c)

ROD

0

50

100

150

200

120573-Actin

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(d)











ROD

0

50

100

150

200NF120581B

lowast

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(a)

ROD

0

50

100

150

200

TNF120572

lowast

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(b)

ROD

0

50

100

150

200

250

lowast

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

TGF120573

(c)

ROD

0

50

100

150

200

120573-Actin

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(d)







ROD

0

50

100

150

200

250HIF1

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

lowast

lowast

(a)RO

D

0

50

100

150

200

250EPO

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

lowast lowast

(b)

ROD

0

50

100

150

200GLUT1

lowastlowast

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(c)

ROD

0

50

100

150

200VEGF

lowast

lowast

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(d)

ROD

0

50

100

150

200

250120573-Actin

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(e)







2O2(ROIs)




Inflammation

Nrf2

HO1 MT SOD GPx GR

GLUT1 VEGF EPO




Antioxidant genes

Antioxidant enzymes

HIF1


NF120581B

TNF120572 TGF120573

)Hypoxia +











Acknowledgments


References

















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of







2H4O2 pH 36)







2 74 N

2 and 5 CO

2) at 37∘C in


2

5 CO2 and 94 N









0

5

10

15

20

mg

g of

extr

act

Total phenols

Total flavonoids

120583M

trol

ox eq

uiva

lent

g o

f ext

ract


0

5

10

15

20

25

30

35

DPPH

ABTS

FRAP



0 5 0

10 15 20 25 30

100200300400500

Time (min)

Adenine

Volta

ge (m

V)






3 Results






00

02

04

06

08

10

Con

trol


MTT

assa

y (Δ

OD490)

lowastlowast

lowastlowast

lowast

lowastlowast

+5120583

gm

L

+10

120583g

mL

+25

120583g

mL

+50120583

gm

L

+100120583

gm

L

+250120583

gm

L

01

O

2

(a)

Con

trol


02

04

06

08

10

MTT

assa

y (Δ

OD490)

lowast

lowast

+5120583

gm

L

lowast

+10

120583g

mL

lowast

+25

120583g

mL

+50120583

gm

L

+100120583

gm

L

+250120583

gm

L

05

O

2

(b)

Con

trol


02

04

06

08

10

MTT

assa

y (Δ

OD490)

lowast

lowastlowast

lowast

+5120583

gm

L

+10

120583g

mL

+25

120583g

mL

+50120583

gm

L

+100120583

gm

L

+250120583

gm

L

1

O2

(c)

Con

trol


02

04

06

08

10M

TT as

say

(ΔO

D490)

lowastlowast

lowast+5120583

gm

L

+10

120583g

mL

+25

120583g

mL

+50120583

gm

L

+100120583

gm

L

+250120583

gm

L

5

O2

(d)





2showed






0

50

100

150

200

250

300

Relat

ive fl

uore

scen

ce u

nits

Con

trol

lowast

lowast

lowast

lowast

lowast

lowastlowast

+5120583

gm

L

+10

120583g

mL

+25

120583g

mL

+50120583

gm

L

+100120583

gm

L

+250120583

gm

L

01

O

2


(a)

0

50

100

150

200

250

Relat

ive fl

uore

scen

ce u

nits

Con

trol


lowast

lowast

+5120583

gm

L

lowast

+10

120583g

mL

lowast

lowast

+25

120583g

mL

+50120583

gm

L

+100120583

gm

L

+250120583

gm

L

05

O

2

(b)

0

50

100

150

200

250

Relat

ive fl

uore

scen

ce u

nits

Con

trol


lowast

lowast

lowast

lowast

+5120583

gm

L

+10

120583g

mL

+25

120583g

mL

+50120583

gm

L

+100120583

gm

L

+250120583

gm

L

1

O2

(c)

0

50

100

150

200

250

Relat

ive fl

uore

scen

ce u

nits

Con

trol


lowastlowast

lowast

+5120583

gm

L

+10

120583g

mL

+25

120583g

mL

+50120583

gm

L

+100120583

gm

L

+250120583

gm

L

5

O2

(d)














(nm

olm

g pr

otei

n)

00

05

10

15

20

25

Con

trol

Lipid per oxidation

CP 2

50 120583

gm

L al

one

lowast

05

O2

(hyp

oxia

)

05

O2

+ CP

250

120583g

mL

(a)

(nm

olm

g pr

otei

n)

0

10

20

30

40

50

60

Con

trol

Protein oxidation

CP 2

50 120583

gm

L al

one

lowast

05

O2

(hyp

oxia

)

05

O2

+ CP

250

120583g

mL

(b)









4 Discussion






ROD

0

50

100

150

200

Nrf2

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

lowastlowast

lowast

(a)

ROD

0

50

100

150

200

250

HO1

lowastlowast

lowast

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(b)

ROD

0

50

100

150

200

250

MT

lowast

lowast

lowast

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(c)

ROD

0

50

100

150

200

120573-Actin

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(d)











ROD

0

50

100

150

200NF120581B

lowast

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(a)

ROD

0

50

100

150

200

TNF120572

lowast

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(b)

ROD

0

50

100

150

200

250

lowast

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

TGF120573

(c)

ROD

0

50

100

150

200

120573-Actin

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(d)







ROD

0

50

100

150

200

250HIF1

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

lowast

lowast

(a)RO

D

0

50

100

150

200

250EPO

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

lowast lowast

(b)

ROD

0

50

100

150

200GLUT1

lowastlowast

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(c)

ROD

0

50

100

150

200VEGF

lowast

lowast

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(d)

ROD

0

50

100

150

200

250120573-Actin

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(e)







2O2(ROIs)




Inflammation

Nrf2

HO1 MT SOD GPx GR

GLUT1 VEGF EPO




Antioxidant genes

Antioxidant enzymes

HIF1


NF120581B

TNF120572 TGF120573

)Hypoxia +











Acknowledgments


References

















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


0

5

10

15

20

mg

g of

extr

act

Total phenols

Total flavonoids

120583M

trol

ox eq

uiva

lent

g o

f ext

ract


0

5

10

15

20

25

30

35

DPPH

ABTS

FRAP



0 5 0

10 15 20 25 30

100200300400500

Time (min)

Adenine

Volta

ge (m

V)






3 Results






00

02

04

06

08

10

Con

trol


MTT

assa

y (Δ

OD490)

lowastlowast

lowastlowast

lowast

lowastlowast

+5120583

gm

L

+10

120583g

mL

+25

120583g

mL

+50120583

gm

L

+100120583

gm

L

+250120583

gm

L

01

O

2

(a)

Con

trol


02

04

06

08

10

MTT

assa

y (Δ

OD490)

lowast

lowast

+5120583

gm

L

lowast

+10

120583g

mL

lowast

+25

120583g

mL

+50120583

gm

L

+100120583

gm

L

+250120583

gm

L

05

O

2

(b)

Con

trol


02

04

06

08

10

MTT

assa

y (Δ

OD490)

lowast

lowastlowast

lowast

+5120583

gm

L

+10

120583g

mL

+25

120583g

mL

+50120583

gm

L

+100120583

gm

L

+250120583

gm

L

1

O2

(c)

Con

trol


02

04

06

08

10M

TT as

say

(ΔO

D490)

lowastlowast

lowast+5120583

gm

L

+10

120583g

mL

+25

120583g

mL

+50120583

gm

L

+100120583

gm

L

+250120583

gm

L

5

O2

(d)





2showed






0

50

100

150

200

250

300

Relat

ive fl

uore

scen

ce u

nits

Con

trol

lowast

lowast

lowast

lowast

lowast

lowastlowast

+5120583

gm

L

+10

120583g

mL

+25

120583g

mL

+50120583

gm

L

+100120583

gm

L

+250120583

gm

L

01

O

2


(a)

0

50

100

150

200

250

Relat

ive fl

uore

scen

ce u

nits

Con

trol


lowast

lowast

+5120583

gm

L

lowast

+10

120583g

mL

lowast

lowast

+25

120583g

mL

+50120583

gm

L

+100120583

gm

L

+250120583

gm

L

05

O

2

(b)

0

50

100

150

200

250

Relat

ive fl

uore

scen

ce u

nits

Con

trol


lowast

lowast

lowast

lowast

+5120583

gm

L

+10

120583g

mL

+25

120583g

mL

+50120583

gm

L

+100120583

gm

L

+250120583

gm

L

1

O2

(c)

0

50

100

150

200

250

Relat

ive fl

uore

scen

ce u

nits

Con

trol


lowastlowast

lowast

+5120583

gm

L

+10

120583g

mL

+25

120583g

mL

+50120583

gm

L

+100120583

gm

L

+250120583

gm

L

5

O2

(d)














(nm

olm

g pr

otei

n)

00

05

10

15

20

25

Con

trol

Lipid per oxidation

CP 2

50 120583

gm

L al

one

lowast

05

O2

(hyp

oxia

)

05

O2

+ CP

250

120583g

mL

(a)

(nm

olm

g pr

otei

n)

0

10

20

30

40

50

60

Con

trol

Protein oxidation

CP 2

50 120583

gm

L al

one

lowast

05

O2

(hyp

oxia

)

05

O2

+ CP

250

120583g

mL

(b)









4 Discussion






ROD

0

50

100

150

200

Nrf2

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

lowastlowast

lowast

(a)

ROD

0

50

100

150

200

250

HO1

lowastlowast

lowast

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(b)

ROD

0

50

100

150

200

250

MT

lowast

lowast

lowast

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(c)

ROD

0

50

100

150

200

120573-Actin

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(d)











ROD

0

50

100

150

200NF120581B

lowast

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(a)

ROD

0

50

100

150

200

TNF120572

lowast

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(b)

ROD

0

50

100

150

200

250

lowast

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

TGF120573

(c)

ROD

0

50

100

150

200

120573-Actin

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(d)







ROD

0

50

100

150

200

250HIF1

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

lowast

lowast

(a)RO

D

0

50

100

150

200

250EPO

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

lowast lowast

(b)

ROD

0

50

100

150

200GLUT1

lowastlowast

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(c)

ROD

0

50

100

150

200VEGF

lowast

lowast

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(d)

ROD

0

50

100

150

200

250120573-Actin

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(e)







2O2(ROIs)




Inflammation

Nrf2

HO1 MT SOD GPx GR

GLUT1 VEGF EPO




Antioxidant genes

Antioxidant enzymes

HIF1


NF120581B

TNF120572 TGF120573

)Hypoxia +











Acknowledgments


References

















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


00

02

04

06

08

10

Con

trol


MTT

assa

y (Δ

OD490)

lowastlowast

lowastlowast

lowast

lowastlowast

+5120583

gm

L

+10

120583g

mL

+25

120583g

mL

+50120583

gm

L

+100120583

gm

L

+250120583

gm

L

01

O

2

(a)

Con

trol


02

04

06

08

10

MTT

assa

y (Δ

OD490)

lowast

lowast

+5120583

gm

L

lowast

+10

120583g

mL

lowast

+25

120583g

mL

+50120583

gm

L

+100120583

gm

L

+250120583

gm

L

05

O

2

(b)

Con

trol


02

04

06

08

10

MTT

assa

y (Δ

OD490)

lowast

lowastlowast

lowast

+5120583

gm

L

+10

120583g

mL

+25

120583g

mL

+50120583

gm

L

+100120583

gm

L

+250120583

gm

L

1

O2

(c)

Con

trol


02

04

06

08

10M

TT as

say

(ΔO

D490)

lowastlowast

lowast+5120583

gm

L

+10

120583g

mL

+25

120583g

mL

+50120583

gm

L

+100120583

gm

L

+250120583

gm

L

5

O2

(d)





2showed






0

50

100

150

200

250

300

Relat

ive fl

uore

scen

ce u

nits

Con

trol

lowast

lowast

lowast

lowast

lowast

lowastlowast

+5120583

gm

L

+10

120583g

mL

+25

120583g

mL

+50120583

gm

L

+100120583

gm

L

+250120583

gm

L

01

O

2


(a)

0

50

100

150

200

250

Relat

ive fl

uore

scen

ce u

nits

Con

trol


lowast

lowast

+5120583

gm

L

lowast

+10

120583g

mL

lowast

lowast

+25

120583g

mL

+50120583

gm

L

+100120583

gm

L

+250120583

gm

L

05

O

2

(b)

0

50

100

150

200

250

Relat

ive fl

uore

scen

ce u

nits

Con

trol


lowast

lowast

lowast

lowast

+5120583

gm

L

+10

120583g

mL

+25

120583g

mL

+50120583

gm

L

+100120583

gm

L

+250120583

gm

L

1

O2

(c)

0

50

100

150

200

250

Relat

ive fl

uore

scen

ce u

nits

Con

trol


lowastlowast

lowast

+5120583

gm

L

+10

120583g

mL

+25

120583g

mL

+50120583

gm

L

+100120583

gm

L

+250120583

gm

L

5

O2

(d)














(nm

olm

g pr

otei

n)

00

05

10

15

20

25

Con

trol

Lipid per oxidation

CP 2

50 120583

gm

L al

one

lowast

05

O2

(hyp

oxia

)

05

O2

+ CP

250

120583g

mL

(a)

(nm

olm

g pr

otei

n)

0

10

20

30

40

50

60

Con

trol

Protein oxidation

CP 2

50 120583

gm

L al

one

lowast

05

O2

(hyp

oxia

)

05

O2

+ CP

250

120583g

mL

(b)









4 Discussion






ROD

0

50

100

150

200

Nrf2

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

lowastlowast

lowast

(a)

ROD

0

50

100

150

200

250

HO1

lowastlowast

lowast

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(b)

ROD

0

50

100

150

200

250

MT

lowast

lowast

lowast

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(c)

ROD

0

50

100

150

200

120573-Actin

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(d)











ROD

0

50

100

150

200NF120581B

lowast

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(a)

ROD

0

50

100

150

200

TNF120572

lowast

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(b)

ROD

0

50

100

150

200

250

lowast

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

TGF120573

(c)

ROD

0

50

100

150

200

120573-Actin

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(d)







ROD

0

50

100

150

200

250HIF1

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

lowast

lowast

(a)RO

D

0

50

100

150

200

250EPO

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

lowast lowast

(b)

ROD

0

50

100

150

200GLUT1

lowastlowast

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(c)

ROD

0

50

100

150

200VEGF

lowast

lowast

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(d)

ROD

0

50

100

150

200

250120573-Actin

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(e)







2O2(ROIs)




Inflammation

Nrf2

HO1 MT SOD GPx GR

GLUT1 VEGF EPO




Antioxidant genes

Antioxidant enzymes

HIF1


NF120581B

TNF120572 TGF120573

)Hypoxia +











Acknowledgments


References

















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


0

50

100

150

200

250

300

Relat

ive fl

uore

scen

ce u

nits

Con

trol

lowast

lowast

lowast

lowast

lowast

lowastlowast

+5120583

gm

L

+10

120583g

mL

+25

120583g

mL

+50120583

gm

L

+100120583

gm

L

+250120583

gm

L

01

O

2


(a)

0

50

100

150

200

250

Relat

ive fl

uore

scen

ce u

nits

Con

trol


lowast

lowast

+5120583

gm

L

lowast

+10

120583g

mL

lowast

lowast

+25

120583g

mL

+50120583

gm

L

+100120583

gm

L

+250120583

gm

L

05

O

2

(b)

0

50

100

150

200

250

Relat

ive fl

uore

scen

ce u

nits

Con

trol


lowast

lowast

lowast

lowast

+5120583

gm

L

+10

120583g

mL

+25

120583g

mL

+50120583

gm

L

+100120583

gm

L

+250120583

gm

L

1

O2

(c)

0

50

100

150

200

250

Relat

ive fl

uore

scen

ce u

nits

Con

trol


lowastlowast

lowast

+5120583

gm

L

+10

120583g

mL

+25

120583g

mL

+50120583

gm

L

+100120583

gm

L

+250120583

gm

L

5

O2

(d)














(nm

olm

g pr

otei

n)

00

05

10

15

20

25

Con

trol

Lipid per oxidation

CP 2

50 120583

gm

L al

one

lowast

05

O2

(hyp

oxia

)

05

O2

+ CP

250

120583g

mL

(a)

(nm

olm

g pr

otei

n)

0

10

20

30

40

50

60

Con

trol

Protein oxidation

CP 2

50 120583

gm

L al

one

lowast

05

O2

(hyp

oxia

)

05

O2

+ CP

250

120583g

mL

(b)









4 Discussion






ROD

0

50

100

150

200

Nrf2

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

lowastlowast

lowast

(a)

ROD

0

50

100

150

200

250

HO1

lowastlowast

lowast

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(b)

ROD

0

50

100

150

200

250

MT

lowast

lowast

lowast

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(c)

ROD

0

50

100

150

200

120573-Actin

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(d)











ROD

0

50

100

150

200NF120581B

lowast

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(a)

ROD

0

50

100

150

200

TNF120572

lowast

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(b)

ROD

0

50

100

150

200

250

lowast

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

TGF120573

(c)

ROD

0

50

100

150

200

120573-Actin

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(d)







ROD

0

50

100

150

200

250HIF1

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

lowast

lowast

(a)RO

D

0

50

100

150

200

250EPO

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

lowast lowast

(b)

ROD

0

50

100

150

200GLUT1

lowastlowast

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(c)

ROD

0

50

100

150

200VEGF

lowast

lowast

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(d)

ROD

0

50

100

150

200

250120573-Actin

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(e)







2O2(ROIs)




Inflammation

Nrf2

HO1 MT SOD GPx GR

GLUT1 VEGF EPO




Antioxidant genes

Antioxidant enzymes

HIF1


NF120581B

TNF120572 TGF120573

)Hypoxia +











Acknowledgments


References

















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


(nm

olm

g pr

otei

n)

00

05

10

15

20

25

Con

trol

Lipid per oxidation

CP 2

50 120583

gm

L al

one

lowast

05

O2

(hyp

oxia

)

05

O2

+ CP

250

120583g

mL

(a)

(nm

olm

g pr

otei

n)

0

10

20

30

40

50

60

Con

trol

Protein oxidation

CP 2

50 120583

gm

L al

one

lowast

05

O2

(hyp

oxia

)

05

O2

+ CP

250

120583g

mL

(b)









4 Discussion






ROD

0

50

100

150

200

Nrf2

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

lowastlowast

lowast

(a)

ROD

0

50

100

150

200

250

HO1

lowastlowast

lowast

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(b)

ROD

0

50

100

150

200

250

MT

lowast

lowast

lowast

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(c)

ROD

0

50

100

150

200

120573-Actin

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(d)











ROD

0

50

100

150

200NF120581B

lowast

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(a)

ROD

0

50

100

150

200

TNF120572

lowast

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(b)

ROD

0

50

100

150

200

250

lowast

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

TGF120573

(c)

ROD

0

50

100

150

200

120573-Actin

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(d)







ROD

0

50

100

150

200

250HIF1

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

lowast

lowast

(a)RO

D

0

50

100

150

200

250EPO

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

lowast lowast

(b)

ROD

0

50

100

150

200GLUT1

lowastlowast

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(c)

ROD

0

50

100

150

200VEGF

lowast

lowast

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(d)

ROD

0

50

100

150

200

250120573-Actin

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(e)







2O2(ROIs)




Inflammation

Nrf2

HO1 MT SOD GPx GR

GLUT1 VEGF EPO




Antioxidant genes

Antioxidant enzymes

HIF1


NF120581B

TNF120572 TGF120573

)Hypoxia +











Acknowledgments


References

















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


ROD

0

50

100

150

200

Nrf2

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

lowastlowast

lowast

(a)

ROD

0

50

100

150

200

250

HO1

lowastlowast

lowast

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(b)

ROD

0

50

100

150

200

250

MT

lowast

lowast

lowast

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(c)

ROD

0

50

100

150

200

120573-Actin

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(d)











ROD

0

50

100

150

200NF120581B

lowast

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(a)

ROD

0

50

100

150

200

TNF120572

lowast

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(b)

ROD

0

50

100

150

200

250

lowast

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

TGF120573

(c)

ROD

0

50

100

150

200

120573-Actin

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(d)







ROD

0

50

100

150

200

250HIF1

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

lowast

lowast

(a)RO

D

0

50

100

150

200

250EPO

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

lowast lowast

(b)

ROD

0

50

100

150

200GLUT1

lowastlowast

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(c)

ROD

0

50

100

150

200VEGF

lowast

lowast

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(d)

ROD

0

50

100

150

200

250120573-Actin

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(e)







2O2(ROIs)




Inflammation

Nrf2

HO1 MT SOD GPx GR

GLUT1 VEGF EPO




Antioxidant genes

Antioxidant enzymes

HIF1


NF120581B

TNF120572 TGF120573

)Hypoxia +











Acknowledgments


References

















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


ROD

0

50

100

150

200NF120581B

lowast

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(a)

ROD

0

50

100

150

200

TNF120572

lowast

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(b)

ROD

0

50

100

150

200

250

lowast

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

TGF120573

(c)

ROD

0

50

100

150

200

120573-Actin

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(d)







ROD

0

50

100

150

200

250HIF1

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

lowast

lowast

(a)RO

D

0

50

100

150

200

250EPO

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

lowast lowast

(b)

ROD

0

50

100

150

200GLUT1

lowastlowast

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(c)

ROD

0

50

100

150

200VEGF

lowast

lowast

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(d)

ROD

0

50

100

150

200

250120573-Actin

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(e)







2O2(ROIs)




Inflammation

Nrf2

HO1 MT SOD GPx GR

GLUT1 VEGF EPO




Antioxidant genes

Antioxidant enzymes

HIF1


NF120581B

TNF120572 TGF120573

)Hypoxia +











Acknowledgments


References

















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


ROD

0

50

100

150

200

250HIF1

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

lowast

lowast

(a)RO

D

0

50

100

150

200

250EPO

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

lowast lowast

(b)

ROD

0

50

100

150

200GLUT1

lowastlowast

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(c)

ROD

0

50

100

150

200VEGF

lowast

lowast

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(d)

ROD

0

50

100

150

200

250120573-Actin

Con

trol

05

O2

Con

trol +

CS

Hyp

oxia+

CS

(e)







2O2(ROIs)




Inflammation

Nrf2

HO1 MT SOD GPx GR

GLUT1 VEGF EPO




Antioxidant genes

Antioxidant enzymes

HIF1


NF120581B

TNF120572 TGF120573

)Hypoxia +











Acknowledgments


References

















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of



Inflammation

Nrf2

HO1 MT SOD GPx GR

GLUT1 VEGF EPO




Antioxidant genes

Antioxidant enzymes

HIF1


NF120581B

TNF120572 TGF120573

)Hypoxia +











Acknowledgments


References

















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of






Acknowledgments


References

















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of






















Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of





Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

Documents

Research Article Cordyceps sinensis Increases Hypoxia …downloads.hindawi.com/journals/bmri/2013/569206.pdf · 2019-07-31 · Cordyceps sinensis claiming that it gives them energy