research article the modified jiuwei qianghuo decoction...

Research ArticleThe Modified JiuWei QiangHuo Decoction Alleviated SevereLung Injury Induced by H1N1 Influenza Virus by Regulating theNF-120581B Pathway in Mice

Lijuan Chen1 Xin Yan2 Qianlin Yan1 Jiajun Fan3 Hai Huang3 Xunlong Shi3 Lei Han3

Tianxiong Han1 and Haiyan Zhu3

1 Department of Chinese Medicine Shanghai Tenth Peoplersquos Hospital Tongji University School of Medicine Shanghai 200092 China2 Institute of Chinese Medicine Tongji University School of Medicine Shanghai 200092 China3Department of Biosynthesis School of Pharmacy Fudan University 826 Zhangheng Road Shanghai 201203 China

Correspondence should be addressed to Haiyan Zhu hyzhumailshcncaccn

Received 4 May 2014 Revised 22 September 2014 Accepted 23 September 2014

Academic Editor Yi-tao Wang

Copyright copy 2015 Lijuan Chen et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

A new approach to treat infections of highly pathogenic influenza virus is to inhibit excessive innate immune response JiuWeiQiangHuo decoction has been used for centuries for the treatment of pulmonary disorders in China In this study we evaluated theanti-inflammatory activities of the modified JiuWei QiangHuo (MJWQH) decoction in the treatment of influenza A (H1N1) virus-induced severe pneumonia in mice The results showed that MJWQH significantly increased the survival rate of H1N1-infectedmice and suppressed the production of TNF-120572 IL-1 IL-6 MCP-1 RANTES and IFN-120572 on day 4 after infection Moreover oraladministration ofMJWQH efficiently inhibited virus replication and alleviated the severity of lung injuriesThe results also showedthat MJWQHmay have potential therapeutic effect on severe lung injury induced by H1N1 virus by regulating the NF-120581B pathwayOur study suggested that MJWQHmight be an alternative therapy for the treatment of viral pneumonia

1 Introduction

Influenza A (H1N1) virus can cause severe respiratory dis-eases and lead to pandemic outbreaks in humans Somesevere patients may develop acute lung injury and even acuterespiratory distress syndrome which are the most commoncauses of death in these H1N1-infected patients [1] Howeverinfluenza continues to evolve and antiviral drugs are usuallynot given in the early stage of virus infection thus renderingthe antiviral therapy ineffective It is suggested that modula-tion of the host immune response has the potential advantageof exerting less-selective pressure on viral populations [2]Many traditional Chinese medicines (TCM) and herbs havebeen shown to have immunomodulatory anti-inflammatoryantiviral and antioxidant activities [3ndash5]There are a numberof clinical trials evaluating the potential benefit of TCM inthe treatment of H1N1-induced pneumonia [6] It seems tobe a promising approach to extract effective ingredients from

TCM for the treatment of influenza virus-induced severepneumonia

The recruitment of innate immune cells into the lungand excessive proinflammatory cytokines and chemokinesare hallmarks of influenza virus infection [7] It has beenshown that early recruitment of inflammatory leukocytesto the lung followed by excessive early cytokine responsesis the key contributor to the morbidity and mortality ofH1N1 virus infections [8 9] Toll-like receptors (TLRs) arethe main intracellularextracellular immune cell receptorsthat recognize pathogen-associated molecular patterns ofthe microbes and foreign particles including viruses toinduce several immune cell functions ranging frommigrationand phagocytosis to inflammatory cytokine expression [10ndash13] TLR7 is located in the membranes of the endosomalcompartment and can recognize viral single-stranded RNA(ssRNA) [14] TLR7 expression is upregulated in pulmonaryepithelial cells and macrophages after virus infection Upon

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2015 Article ID 790739 12 pageshttpdxdoiorg1011552015790739

2 Evidence-Based Complementary and Alternative Medicine

Table 1 Composition of MJWQH

Chinese name Latin name Amount (g) Place of originQiang Huo Rhizoma et Radix Notopterygii 5 Inner Mongolia ChinaCang Zhu Rhizoma Atractylodis 10 Sichuan ChinaPu Gong Yin Herba Taraxaci 20 Jiangsu ChinaHuang Qin Radix Scutellariae 10 Inner Mongolia ChinaHuang Qi Radix Astragali 20 Gansu ChinaFang Feng Radix Saposhnikoviae 5 Hebei ChinaYu Xing Cao Herba Houttuyniae 10 Gansu ChinaTian Hua Fen Radix Trichosanthis 15 Henan China

stimulation cells recognize viral ssRNA via TLR7 and acti-vate downstream signaling molecules through a MyD88dependent pathway causing subsequent cytokine storm andtriggering inflammatory lung injury [15 16]

JiuWei QiangHuo (JWQH) decoction was first intro-duced in themonographCishinanzhiwritten by an influentialTCM physician Zhang Yuansu of Jin Dynasty (1151ndash1234)It has been used for over 900 years for the treatment ofpulmonary disorders such as common cold bronchial infec-tions and pneumoniaThis decoction has recently beenmod-ified by Dr Yan Dexin a Grandmaster of Chinese Medicinegranted by the Ministry of Health of China which consistsof eight herbs including Rhizoma et Radix NotopterygiiHerba Taraxaci Radix Scutellariae Radix Astragali RhizomaAtractylodis Radix Saposhnikoviae Herba Houttuyniae andRadix Trichosanthis The modified JWQH (MJWQH) hasbeen shown to be effective in patients with severe pneumoniainduced by influenza virus It may reverse H1N1-inducedpneumonia in mice by regulating the metabolism of arachi-donic acid fatty acid and amino acid and is thus involvedin anti-inflammatory activity and cell protection [17] Basedon previous research findings and the pathogenesis of H1N1-induced pneumonia we hypothesize that MJWQH has animmunoregulatory effect in the early stages of the disease byinhibiting excessive immune response thus inhibiting virusreplication lung inflammation and severe lung injury

2 Materials and Methods

21 Preparation of MJWQH MJWQH was prepared fromeight dried raw materials (Table 1) purchased from ShanghaiShuguang Hospital (Shanghai China) and authenticated bya physician in Fudan University Voucher specimens weredeposited in the Herbarium Center of the Department ofPharmacognosy School of Pharmacy Fudan University Allraw materials were extracted by boiling in distilled water(about 6-fold the weight of the mixture) at 100∘C for 20minand then filtered The filtrates from two decoctions werecombined and the decoction was dried in vacuo at 70∘Cand ground into powder for use The yield of the extractionwas 24 The extracts were stored in an airtight container atminus80∘C until further analysis

22 Chromatographic Analysis of MJWQH MJWQH wasdissolved in pyrogen-free isotonic saline and filtered through

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Figure 1 HPLC chromatogram of MJWQH (280 nm) Calycosin-7-O-120573-D-glucopyranoside (1) and baicalin (2) were separated fromMJWQH at a retention time of 316 and 434min respectively

a 02mm filter (Microgen Laguna Hills CA USA) beforehigh performance liquid chromatography (HPLC) analysis(Figure 1) HPLC analysis was performed on an Agilentseries 1100 HPLC system (Agilent Waldbronn Germany)equipped with quaternary pump diode-array detectorautosampler and column compartment Calycosin-7-O-120573-D-glucopyranoside and baicalin purchased from the NationalInstitute for the Control of Pharmaceutical and BiologicalProducts (Beijing China) were used as the external standardsin HPLC Chromatographic separation was performed on aZorbax XDB-C18 column (5 120583m 120593 46 times 250mm AgilentTechnologies USA) The mobile phase consisted of acetoni-trile (A) and water containing 02 acetic acid (B) and thefollowing gradient programwas used 5A in the first 10minthen a linear gradient to 36A over 60min and then a lineargradient to 85 A over 30min The mobile phase flow ratewas 08mLmin the detector was monitored at 280 nm thespectral data for all peaks were accumulated in the range of190ndash400 nm and the column temperature was set at 25∘C

23 Mice and Virus Male BALBc mice 5 to 6 weeks oldwere purchased from Shanghai SLAC Laboratory Animal CoLtd (Certificate number 2007000548167 SCXK (Hu) 2012-0002 Shanghai China) and bred and maintained in a closedbreeding facility at the Animal Center of Fudan UniversityAll animal experiments were performed in accordance withthe guidelines of the Ethics Committee for Animal Use ofFudan University The mouse adapted influenza AFM147

Evidence-Based Complementary and Alternative Medicine 3

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Day 0 infect mice inwith 10 LD50

Day 14end assay

Monitor clinical signs weight loss and survival

ig administration with CMCMJWQH or Rib (02mL per mouse)

AFM147

Figure 2 Experimental protocol All BALBc mice except the NCones were challenged intranasally with influenza AFM147 (H1N1)virus (10 LD

50

permouse) and then treated with 05CMC solution(model control MC) MJWQH or Rib one hour later for 2 4 or 7days respectively

(H1N1) virus was provided by the Institute of MedicinalBiotechnology Chinese Academy of Medical Sciences (Bei-jing China) and stored at minus80∘C until use Mice wereinfected intranasally (in) with 10 LD

50doses of H1N1 virus

in 1640 culture medium

24 Experimental Design The BALBc mice were randomlydivided into four groups normal control (NC)model control(MC) MJWQH and ribavirin (Rib) The ribavirin was sup-plied by Star Lake Bioscience Co Inc (Guangdong China)Figure 2 illustrates our initial protocol On day 0 BALBcmice (except for the NC) were anesthetized with isofluraneand infected in with 10 LD

50doses of influenza AFM147

H1N1 virus in a volume of 30 120583L One hour later theMJWQHand Rib mice were intragastrically (ig) administered withMJWQH (18 gkgd) or ribavirin (100mgkgd dissolved in05 carboxyl methyl cellulose (CMC) solution) The NCand MC mice were treated with the same volume of 05CMC solution All mice were monitored daily for clinicalsymptoms body weight and survival for 14 consecutive days[2] Mice showing more than 25 of body weight loss wereconsidered to have reached the experimental endpoint andwere then humanely euthanized by CO

2asphyxiation

25 Pathological Examination and ImmunohistochemistryLungs were removed on day 4 after infection weighed andthen inflated with 10 phosphate-buffered formalin to theirnormal volumeThe lung index (ratio of lung weight to bodyweight) was calculated as a parameter of lung edema Theleft lobes were paraffin embedded and cut into 5 120583m thicksections and one section from each tissue sample was stainedusing a standard haematoxylin-and-eosin (HE) procedureLung sections were examined to determine the extent ofpneumonia in a blinded fashion as previously described [18]0 = no pneumonia 1 = mild interstitial pneumonia (lt25 ofthe lung) 2 = moderate interstitial pneumonia (25ndash50 ofthe lung) and 3 = severe interstitial pneumonia (gt50 of thelung) The scores of the individual samples were summed upto yield a composite score

The tissue sections were immunostained using thestreptavidin-biotin-horseradish peroxidase methodThe sec-tions were deparaffinized and rehydrated through a graded

series of alcohols and then microwaved in EDTA buffer (pH80) at 97∘C for 12min to unmask antigen epitopes Thesections were treated with 3 hydrogen peroxide methanolsolution for 10min to block endogenous peroxidase washedin PBS and incubated with rabbit anti-NF-120581B p65 antibody(Assay Biotech USA) at 1 200 dilution They were washedagain in PBS and incubated with avidin-biotin complex-horseradish peroxidase for 1 h at room temperature The sec-tions were incubatedwith chromogen-fast diaminobenzidine(DAB) for 1ndash5min after which they were counterstained inhaematoxylin and mounted on aqueous mounting medium

26 Hemagglutination (HA) Test Lung tissues pooled fromall mice in each groupwere homogenized in 1mL of sterilizedPBS The homogenates were centrifuged at 10000timesg for10min and the resulting supernatant was analyzed by HAassay Serial 2-fold dilutions of each viral preparation weremade in PBS (pH 715) in 96-well V-bottomed microplatesand 25 120583L of 05 suspension of chicken red blood cells(RBCs) was added to each well The contents in plates weremixed and incubated at room temperature (22ndash25∘C) untilcomplete agglutination of erythrocytes The HA titer wasthe reciprocal of the dilution of virus in the last well withcomplete hemagglutination

27 ELISA Lungs were homogenized in PBS and the super-natants were collectedThe inflammatory cytokines (IL-1 IL-6 TNF-120572 and IFN-120572) and chemokines (MCP-1 RANTESMIP-1 and IP-10) in the supernatants of each group weretested by ELISA kits (Abcam England) supplied by JinmaLaboratory Equipment Co Ltd (Lot number 201303 Shang-hai China) which was performed according to the manu-facturerrsquos instructions ELISAs were read on a 96-well platereader and concentrations were determined using Revelationsoftware (BioTek Epoch USA)

28 Western Blotting Lung protein extracts were preparedusing the Nuclear Extract Kit (BestBio China) followingthe manufacturerrsquos instruction Equal amounts of proteinwere separated by SDS-PAGE and subsequently blotted onpolyvinylidene fluoride membranes (220mA 65min) Blotswere blocked in TBS solution containing 01 Tween 20and 5 nonfat dry milk overnight at 4∘C The follow-ing antibodies and dilutions were used TLR7 (Catalognumber 2633 Cell Signaling Technology 1 1000) MyD88(D80F5) (Catalog number 4283 Cell Signaling Technology1 1000) TRAF6 (Catalog number 04-451 Millipore USA1 1000) I120581B-120572 (c-21) (sc-371 Santa Cruz Biotechnology1 200) p-IKK120572120573 (Ser 176) (sc-21661 Santa Cruz Biotech-nology 1 200) NF-120581B p65 (sc-109 Santa Cruz Biotech-nology 1 200) p-NF-120581B p65 (Ser 276) (sc-101749 SantaCruz Biotechnology 1 200) and GAPDH (14C10) (Catalognumber 2118 Cell Signaling Technology 1 1000) The mem-branes were blotted with appropriate secondary antibodies(Immunology Consultants Laboratory Inc USA 1 5000)and the blotted proteins were visualized by enhanced chemi-luminescence using a commercially available kit (MilliporeUSA)


Table 2 RT-PCR primer sequence

Gene SequenceInfluenza Avirus M

Forward 51015840-GACCGATCCTGTCACCTCTGAC-31015840

Reverse 51015840-AGGGCATTCTGGACAAAGCGTCTA-31015840

GAPDHForward 51015840-ACCACCATGGAGAAGGCTGG-31015840

Reverse 51015840-CTCAGTGTAGCCCAGGATGC-31015840

TLR7Forward 51015840-GGTGGCAAAATTGGAAGATCC-31015840

Reverse 51015840-AGCTGTATGCTCTGGGAAAGGTT-31015840

MyD88Forward 51015840-CCAGAGTGGAAAGCAGTGTC-31015840

Reverse 51015840-GTCCTTCTTCATCGCCTTGT-31015840

TRIFForward 51015840-CCACGTCCTACACGGAAGAT-31015840

Reverse 51015840-AACAGCATCTGCAGCTACCA-31015840

NF-120581B P65Forward 51015840-ATGTGCATCGGCAAGTGG-31015840

Reverse 51015840-CAGAAGTTGAGTTTCGGGTAG-31015840

29 Real-Time Reverse Transcription-PCR Lungs were ho-mogenized with a homogenizer to prepare the suspensionwhich was serially diluted in Trizol reagent (1mL of Trizol for100mg tissue) Total RNA was prepared using the RNA fastisolation kit (Shanghai Generay Biotech Co Ltd ShanghaiChina) For first-strand cDNA synthesis 1 120583g of total RNAwas primed with random primers by reverse transcriptase(Promega USA) and then 120 volume of cDNA was ampli-fied on a StepOne Plus instrument using SYBR Green Real-Time PCR Master Mix Reagent (Toyobo Japan) The PCRwas performed at 95∘C for 10min followed by 35 cycles of95∘C for 10 s 58∘C for 15 s and 72∘C for 30 s Amplificationwas followed by melting curve analysis The primers weredesignedwith BeaconDesigner 7 software (Table 2)The levelof gene transcription was determined by comparing with theNC group

210 Statistical Analyses All statistical analyses were per-formed using GraphPad Prism 50 software (GraphPad Soft-ware Inc CA USA) Survival curves were estimated by theKaplan-Meier method and their homogeneity was estimatedby the log-rank test Multiple group comparisons wereperformed using one-way analysis of variance (ANOVA)followed byDunnettrsquos test to determine significant differencesfrom the control A 119875 value less than 005 was consideredsignificant for all tests

3 Results

31 MJWQH Increased the Survival Rate of the H1N1-InfectedMice Mice receiving 05 CMC developed the most severesymptoms that is lethargy ruffled fur hunched posture

piloerection rapid shallow breathing and audible rattlingwhereas some MJWQH and Rib treated mice presented suchsymptoms over the 14 days of treatment The CMC-treatedmice showed a progressive weight loss from day 3 afterinfection onward (Figure 3(a)) while the MJWQH and Ribtreated mice showed a comparable weight loss from day 3through day 6 after infection but followed by a steady weightgain The survival curves further confirmed the efficacy ofMJWQH against the lethal influenza infection (Figure 3(b))It showed that all MC mice died by day 10 after infectiontheMJWQH treatment significantly increased the survival ofH1N1-infected mice (42 119875 lt 001 versus the MC group)and the Rib treatment further increased the survival rate upto 83 (119875 lt 001 versus the MC group) The survival curveswere ended on day 14 after infection as no further mortalityoccurred after this time point

32 MJWQH Alleviated the Severity of Lung Injuries Lungswere removed on day 4 after infection for gross observationand histopathological examination Edema consolidationand profuse hemorrhage were observed in the MC micewhereas significantly less lung damage was observed in theRib orMJWQH treatedmice (Figure 4(a)) Histopathologicalanalysis also revealed a significant reduction in the thicknessand congestion of alveolar walls intra-alveolar edema andinfiltrated neutrophils in lung tissues of mice treated withRib and MJWQH (Figure 4(b)) In agreement with thesehistopathological findings the Rib andMJWQH treatedmicehad significantly lower pathological scores (Figure 4(c)) andlung index (Figure 4(d)) than the MC group on day 4after infection In summary MJWQH treated mice had areduced lung injury after a lethal inoculumof pandemicH1N1influenza virus

33 MJWQH Inhibited Influenza Virus Replication In VivoThe antiviral effect of MJWQH was determined by HAtiters and the relative quantitation (RQ) of influenza virusreplication in the lungs By day 4 after infection the viral titerwas increased in all infected mice as compared to the NCgroup (Figure 5(a)) However MJWQH or Rib significantlyinhibited virus replication as evidenced by a significantreduction in HA titer (119875 lt 001 versus the MC groupFigure 5(a)) and the RQ of influenza A virus in the lungs(119875 lt 005 versus the MC group Figure 5(b)) indicating thatboth of them were effective in decreasing the viral load inmice infected by H1N1

34 MJWQH Suppressed the Secretion of CytokineChemokineInduced by H1N1 Influenza Virus Early dysregulated innateimmune responses in the lung are associated with morbidityandmortality during infectionwith highly pathogenic strainsof influenza virus [8 19] Robust innate proinflammatorycytokine expression is believed to cause direct tissue insultand to recruit potentially tissue destructive inflammatorycellsThe results showed significant reductions in TNF-120572 IL-1 IL-6 MCP-1 RANTES and IFN-120572 in the MJWQH treatedmice on day 4 after infection However in comparison to theRib treatment MJWQH failed to suppress MIP-1 and IP-10


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Figure 3 MJWQH treatment protected mice from lethal influenza challenge BALBc mice (119899 = 12micegroup) were treated as described inFigure 2 for 7 days and monitored daily for signs and symptoms body weight and survival for 14 consecutive days (a) Body weight (meansplusmn SEM) (b) survival (number of survivorstotal number of mice) lowastlowast119875 lt 001 versus the MC group

on day 4 after infection (Figures 6(a)ndash6(c)) All these resultsindicated that MJWQH suppressed cytokine production insevere pneumonia

35 MJWQH Downregulated TLR7-MyD88-Dependent NF-120581B Signaling Pathway NF-120581B is an important transcriptionfactor for the induction of various inflammation-associatedgenes including cytokines and chemokines To better under-stand the therapeutic mechanism of MJWQH in miceinfected with H1N1 virus we measured the expressions ofTLR7 MyD88 TRAF6 p65 p-p65 I120581B-120572 and p-IKK120572120573in lung tissues on days 2 and 4 after infection by usingWestern blot analysis Figure 7 showed that influenza H1N1infection significantly increased the protein expressions ofTLR7 MyD88 and TRAF6 on days 2 and 4 after infectionwhich could be downregulated by the Rib or MJWQHtreatment We then examined whether MJWQH regulatedthe nuclear translocation of NF-120581B as well as the phos-phorylation and proteolytic degradation of I120581B-120572 No effectwas observed on the NF-120581B signaling pathway on day 2after infection However NF-120581B subunit p65 and p-p65proteins in nucleus were significantly increased in virus-infected mice on 4 day after infection but this increase couldbe suppressed by the MJWQH or Rib treatment MJWQHalso significantly inhibited the proteolytic degradation ofI120581B-120572 in mice IKK120572120573 was expected to make an essentialcontribution to I120581B phosphorylation so we examined the p-IKK120572120573 level The results showed that MJWQH significantlyinhibited p-IKK120572120573 These results together suggested thatMJWQH inhibited H1N1 infection by inhibiting NF-120581Bsignaling pathway on day 4 after infection

To further demonstrate that MJWQH inhibited the NF-120581B signaling pathway in the RNA level we quantified themRNA levels of TLR7 MyD88 and p65 in lung tissues on

day 4 after infection Figure 8 showed that all of them weresignificantly reduced in the Rib and MJWQH treated mice

Finally we examined NF-120581B signaling in lungs of H1N1-infected mice using immunohistochemistry with antibodiesagainst p65 on day 4 after infection Figure 9 showed that p65could be hardly detected in the NC mice but it was abundantin the lungs of MC mice However there was a significantlower expression of p65 in the lungs of the Rib and MJWQHtreated mice

All these results clearly demonstrated that MJWQHblocked influenza virus infection by inhibiting the activationof TLR7MyD88TRAF6IKK120572120573NF-120581B signaling pathway

4 Discussion

MJWQH is composed of eight herbs each having specificantiviral anti-inflammatory or antioxidant activities RadixScutellariae has inhibitory effects on the influenza virus hep-atitis B virus and human immunodeficiency virus [20ndash22]Radix Notopterygii has antioxidant and anti-inflammatoryactivities [23] Herba Taraxaci has antiviral antiseptic andanti-inflammatory activities [24] Radix Astragali has antiox-idant immunopotentiating and antistress activities [25 26]Radix Saposhnikoviaehas immunoregulatory and antioxidantactivities [27] Rhizoma Atractylodis has potent antiviral andantioxidant activities [28] Herba Houttuyniae has inhibitoryeffects on acute inflammation [29 30] and Radix Trichosan-this has antiviral activities and inhibits the replication ofhuman immunodeficiency virus type 1 (HIV-1) [31 32]Thesebiological activities would be helpful for severe pneumoniainduced by influenza virus However the mechanism of theircombined actions has not yet been verified

AFM147 is a mouse-adapted influenza virus whichexhibits high virulence and can induce strong immuneresponses and inflammation in the murine lung [33] The


NC MC MJWQH Rib

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Figure 4 MJWQH alleviated the severity of H1N1-induced lung injuries BALBc mice (119899 = 6 micegroup) were treated as described inFigure 2 for 4 days (a) Macroscopic appearance of lungs (b) pathological changes of lung tissues (HE times200) (c) pathological scores and (d)lung index Data were presented as mean plusmn SD lowastlowast119875 lt 001 versus the MC group

effect of MJWQH on the H1N1-induced severe pneumoniawas evaluated in a murine acute lung injury model in thisstudyThe results showed that MJWQH significantly relievedthe signs and symptoms reduced body weight loss andimproved the survival rate of H1N1-infected mice It alsosignificantly inhibited virus replication as evidenced by a

significant reduction in HA titer and the relative quantitationof influenza A virus in the lungs Altogether these resultssuggested that MJWQH had potent antiviral activity againstH1N1 influenza A virus infection in mice Our previousstudies showed that there was no reduction in the virus titerin the A549 cells infected by influenza A virus at 24 hours


NC MC MJWQH Rib

lowastlowast lowast

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Figure 5 MJWQH inhibited virus replication BALBc mice (119899 = 6 micegroup) were treated as described in Figure 2 for 4 days (a) HAtiters of lung homogenates (b) relative quantitation of influenza A virus Data were presented as mean plusmn SD lowast119875 lt 005 lowastlowast119875 lt 001 versusthe MC group

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MJWQHRib

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Figure 6 MJWQH suppressed innate immune responses Proinflammatory cytokine and chemokine levels were analyzed in lunghomogenates of mice on day 4 after infection by ELISA Data were presented as mean plusmn SD lowast119875 lt 005 lowastlowast119875 lt 001 versus the MC group


TLR7

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minus + + +

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GAPDH

MJWQH Ribminus + + +H1N1

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P-P65

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p-IKK120572120573

I120581B-120572

P65

P-P65

GAPDH

p-IKK120572120573

I120581B-120572

Figure 7 MJWQH inhibited the NF-120581B signaling pathway BALBc mice (119899 = 6 micegroup) were treated as described in Figure 2 andkilled on day 2 or 4 after infection Cytosolic fractions and nuclear extracts were prepared from lung homogenates NF-120581B p65 I120581B-120572 TLR7MyD88 and TRAF6 levels were detected by specific antibodies in cytosolic fractions usingWestern blot analysis Phosphorylation of IKK120572120573and p65 was determined in the nuclear extracts GAPDH protein was used as a loading control

after infection (data not shown here) However in this studywe found that the MJWQH treatment significantly reducedlung HA titers Therefore we speculated that the antiviralactivity of MJWQHwas accomplished by inhibiting the earlyrecruitment of inflammatory leukocytes to the lungs andsuppressing excessive innate inflammatory responses

Highly pathogenic flu can trigger excessive immuneresponse or cytokine storm which in turn leads to immunedamage to the lung In the case of infection inflamma-tion begins when the cells of the innate immune systemrecognize a pathogen-associated molecular pattern thencertain host cells begin to secrete chemokines Chemokinesare small proteins (lt10 kDa) that can activate and mediatethe migration of leukocytes to the site of infection orinflammation [34] Cytokines can be secreted by a varietyof cells including phagocytic cells such as macrophages andneutrophils despite the fact that the endothelial cells areresponsible for over half of all produced various cytokinesand chemokines during inflammatory processes [35] The

early induction of cytokines and chemokines is associatedwith symptom formation in humans [36ndash38] IFN-120572 activatesinflammatory cells and stimulates expression of multiplecytokines and chemokines [39ndash41] TNF-120572 IL-1 and IL-6 have multifunctional activities and are associated withmorbidity during influenza virus infection Chemokines suchasMCP-1MIP-1 RANTES and IP-10 induce the recruitmentof innate immune cells into the lung which can release morecytokines exacerbating cytokine storm and further damagethe lung [42ndash44] Therefore balancing the inflammatorynetwork may represent an effective treatment for influenzavirus infection We demonstrated in this study that theproduction of proinflammatory cytokines and chemokinesfrom lung tissues following influenza infection was regulatedby MJWQH Thus the efficacy of MJWQH is likely due toits ability to inhibit excessive innate immune response andimmune-mediated pulmonary tissue injury

TLRs are key receptors in innate immune recognition andplay an important role in the initiation of acquired immune


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15

Figure 8 MJWQH inhibited the NF-120581B signaling pathway BALBc mice (119899 = 6micegroup) were treated as described in Figure 2 and killedon day 4 after infection Lungs were processed for total RNA and subjected to real-time PCR for detection of TLR7 MyD88 and NF-120581B p65gene expressions using GAPDH as a housekeeping gene Data were presented as mean plusmn SD lowast119875 lt 005 lowastlowast119875 lt 001 versus the MC group

response [45] TLR7 has been demonstrated to be an intracel-lular receptor that recognizes ssRNA after the ribonucleopro-tein complex has been degraded inside acidified endosomes[46 47] and triggers cytokine secretion through the adapterMyD88 which recruits signaling mediators that activate NF-120581B [48] NF-120581B could regulate the gene transcription ofgrowth factors transcription factors cytokines chemokinesand interferons [49] Recognition and rapid clearance ofpathogens by the innate immune system provide the firstline of defense against infection Overexpression of cytokinesand chemokines induced by influenza virus infection isdependent on NF-120581B signaling pathway It has been welldocumented that inhibition of NF-120581B or proteasome leadsto I120581B accumulation and inhibits influenza virus replicationin vitro and in vivo [50ndash52] The signaling cascade of allactivated TLRs starts with the activation of MyD88 whichactivates TRAF6 and eventually leads to the activation ofNF-120581B [53] MyD88 is a crucial adaptor protein downstreamof TLRs whereas TRAF6 is a signal transducer in the NF-120581B pathway both of which are essential for the productionof inflammatory cytokines [54ndash56] IKK stimulates NF-120581Bactivity by causing phosphorylation and degradation of I120581Bproteins the cytoplasmic inhibitors of NF-120581B [57] ActivatedIKK promotes the dissociation of the cytosolic inactiveNF-120581B-I120581B complexes via the serine phosphorylation and

degradation of I120581B leading to NF-120581B translocation to thenucleus and transcriptional upregulation of inflammatorygenes [58] The inhibitory effect of MJWQH on the NF-120581Bsignaling pathway could be associated with the inhibitionof IKK activation We found that I120581B and p65 levels werenot affected on day 2 after infection but p65 and p-p65expressions were significantly increased on day 4 after infec-tionThe real time-PCR and immunohistochemistry analysisrevealed similar results Therefore it can be concluded thatMJWQH modulates the immune response by inhibitingthe TLR7-MyD88-dependent NF-120581B signaling pathway Theantiviral effects of MJWQH might relate to the inhibitionof TLR7MyD88TRAF6IKK120572120573 NF-120581B signaling pathwayHowever as MJWQH contains numerous compounds withvarious pharmacological activities it is not clear whichcompounds are responsible for the immune regulation Thisunderscores the need to further determine whetherMJWQHdirectly blocks the combination of virus and TLR7 and whatthe active components are and how they work

Taken together the present study showed that earlyMJWQH treatment contributed to recovery from severepneumonia due to its anti-inflammatory activity through reg-ulating TLR7MyD88NF-120581B signaling pathway The resultspresented in this study may provide compelling evidence forthe therapeutic potential ofMJWQHas an alternative therapy


NC MC

MJWQH Rib

Figure 9 MJWQH inhibited the NF-120581B expression in lung tissue (times200) BALBc mice (119899 = 6 micegroup) were treated as described inFigure 2 and killed on day 4 after infection Lung sections were stained with rabbit anti-p65 (brown) and counterstained with hematoxylin

to the western medicine in the treatment of influenza virusinfections

Abbreviations

BALF Bronchoalveolar lavage fluidIFN InterferonIL InterleukinTNF Tumor necrosis factorMCP-1 Macrophage chemotactic protein-1RANTES Regulated upon activation normal T cell

expressed and presumably secretedMIP-1 Macrophage inflammatory protein-1IP-10 IFN-120574-inducible protein-10NF-120581B Nuclear factor

kappa-light-chain-enhancer of activated Bcells

TLR Toll-like receptorMyD88 Myeloid differentiation factor 88TRAF6 Tumor necrosis factor receptor-associated

factor 6I120581B Inhibitor of NF-120581BIKK I120581B kinase

Conflict of Interests

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

Acknowledgments

This work was supported by the National Natural ScienceFoundation of China (Grant no 81173173)The authors wouldlike to thank all the members of their department for theexperimental support

References

[1] J K Louie M Acosta K Winter et al ldquoFactors associatedwith death or hospitalization due to pandemic 2009 influenzaA(H1N1) infection in Californiardquo The Journal of the AmericanMedical Association vol 302 no 17 pp 1896ndash1902 2009

[2] K A Shirey W Lai A J Scott et al ldquoThe TLR4 antagonistEritoran protects mice from lethal influenza infectionrdquo Naturevol 497 no 7450 pp 498ndash502 2013

[3] D Wang Y Hu J Sun X Kong B Zhang and J LiuldquoComparative study on adjuvanticity of compound Chineseherbal medicinal ingredientsrdquoVaccine vol 23 no 28 pp 3704ndash3708 2005

[4] C-C Yeh C-C Lin S-D Wang et al ldquoProtective andimmunomodulatory effect of Gingyo-san in a murine model ofacute lung inflammationrdquo Journal of Ethnopharmacology vol111 no 2 pp 418ndash426 2007

[5] C-HHsu K-C Hwang C-L Chao et al ldquoAn evaluation of theadditive effect of natural herbal medicine on SARS or SARS-like infectious diseases in 2003 a randomized double-blindand controlled pilot studyrdquo Evidence-Based Complementary andAlternative Medicine vol 5 no 3 pp 355ndash362 2008

[6] W Chen C E D Lim H-J Kang and J Liu ldquoChineseherbal medicines for the treatment of type A H1N1 influenza a


systematic review of randomized controlled trialsrdquo PLoS ONEvol 6 no 12 Article ID e28093 2011

[7] N L la Gruta K Kedzierska J Stambas and P C DohertyldquoAquestion of self-preservation immunopathology in influenzavirus infectionrdquo Immunology and Cell Biology vol 85 no 2 pp85ndash92 2007

[8] D Kobasa S M Jones K Shinya et al ldquoAberrant innateimmune response in lethal infection of macaques with the 1918influenza virusrdquo Nature vol 445 no 7125 pp 319ndash323 2007

[9] D M Tscherne and A Garcıa-Sastre ldquoVirulence determinantsof pandemic influenza virusesrdquo The Journal of Clinical Investi-gation vol 121 no 1 pp 6ndash13 2011

[10] T-N Nguyen-Pham M-S Lim T A T Nguyen et al ldquoTypeI and II interferons enhance dendritic cell maturation andmigration capacity by regulating CD38 and CD74 that havesynergistic effects with TLR agonistsrdquo Cellular amp MolecularImmunology vol 8 no 4 pp 341ndash347 2011

[11] Y S Bae J H Lee S H Choi et al ldquoMacrophages generatereactive oxygen species in response to minimally oxidized low-density lipoprotein toll-like receptor 4- and spleen tyrosinekinase-dependent activation of NADPH oxidase 2rdquo CirculationResearch vol 104 no 2 pp 210ndash218 2009

[12] T Hirayama Y Tamaki Y Takakubo et al ldquoToll-like receptorsand their adaptors are regulated in macrophages after phagocy-tosis of lipopolysaccharide-coated titaniumparticlesrdquo Journal ofOrthopaedic Research vol 29 no 7 pp 984ndash992 2011

[13] A A I Fooladi S FMousavi S Seghatoleslami S Yazdani andM R Nourani ldquoToll-like receptors role in inflammation andcommensal bacteriardquo InflammationampAllergy-Drug Targets vol10 no 3 pp 198ndash207 2011

[14] K Takeda and SAkira ldquoToll-like receptors in innate immunityrdquoInternational Immunology vol 17 no 1 pp 1ndash14 2005

[15] S Akira and K Takeda ldquoToll-like receptor signallingrdquo NatureReviews Immunology vol 4 no 7 pp 499ndash511 2004

[16] W Barchet A Krug M Cella et al ldquoDendritic cells respondto influenza virus through TLR7- and PKR-independent path-waysrdquo European Journal of Immunology vol 35 no 1 pp 236ndash242 2005

[17] L Chen J Fan Y Li et al ldquoModified Jiu Wei Qiang Huodecoction improves dysfunctional metabolomics in influenzaA pneumonia-infected micerdquo Biomedical Chromatography vol28 no 4 pp 468ndash474 2014

[18] M-H Ji G-M Li M Jia et al ldquoValproic acid attenuateslipopolysaccharide-induced acute lung injury in micerdquo Inflam-mation vol 36 no 6 pp 1453ndash1459 2013

[19] C Cilloniz K Shinya X Peng et al ldquoLethal influenza virusinfection in macaques is associated with early dysregulationof inflammatory related genesrdquo PLoS Pathogens vol 5 no 102009

[20] Y P Tseng Y C Wu Y L Leu S F Yeh and C K ChouldquoScutellariae radix suppresses hepatitis B virus production inhuman hepatoma cellsrdquo Frontiers in Bioscience vol 2 no 4 pp1538ndash1547 2010

[21] J Dou L Chen G Xu et al ldquoEffects of baicalein on Sendaivirus in vivo are linked to serum baicalin and its inhibition ofhemagglutinin-neuraminidaserdquo Archives of Virology vol 156no 5 pp 793ndash801 2011

[22] Q Zhang B Yang N Wang L Duan S He and J SunldquoEffect of total flavonoids of Scutellaria baicalensis Georgi onexpression of influenza A virus nucleoprotein in HeLa cellsrdquoNan Fang Yi Ke Da Xue Xue Bao vol 32 no 7 pp 966ndash9692012

[23] X-W Yang Z-M Gu B-X Wang M Hattori and T NambaldquoComparison of anti-lipid peroxidative effects of the under-ground parts ofNotopterygium incisum andN forbesii inmicerdquoPlanta Medica vol 57 no 5 pp 399ndash402 1991

[24] M Zheng ldquoExperimental study of 472 herbs with antiviralaction against the herpes simplex virusrdquo Chinese Journal ofModern Developments in Traditional Medicine vol 10 no 1 pp39ndash41 1990

[25] G Ye Y-H Tang G-X Xia Z-L Sun Z-X Li and C-G Huang ldquoCharacterization of anti-Coxsackie virus B3 con-stituents of Radix Astragali by high-performance liquid chro-matography coupled with electrospray ionization tandem massspectrometryrdquo Biomedical Chromatography vol 24 no 11 pp1147ndash1151 2010

[26] S Wang J Li H Huang et al ldquoAnti-hepatitis B virus activitiesof astragaloside IV isolated fromRadixAstragalirdquoBiological andPharmaceutical Bulletin vol 32 no 1 pp 132ndash135 2009

[27] Z Q Zhang Y J Tian and J Zhang ldquoStudies on the antiox-idative activity of polysaccharides from radix SaposhnikoviaerdquoZhong Yao Cai vol 31 no 2 pp 268ndash272 2008

[28] N Inagaki Y Komatsu H Sasaki et al ldquoAcidic polysaccharidesfrom rhizomes of Atractylodes lancea as protective principle inCandida-infected micerdquo Planta Medica vol 67 no 5 pp 428ndash431 2001

[29] K Taguchi Y Hagiwara K Kajiyama and Y Suzuki ldquoPharma-cological studies of Houttuyniae Herba the anti-inflammatoryeffect of quercitrinrdquoYakugakuZasshi vol 113 no 4 pp 327ndash3331993

[30] S Li R Wang Y Zhang et al ldquoSymptom combinationsassociated with outcome and therapeutic effects in a cohort ofcases with SARSrdquoAmerican Journal of ChineseMedicine vol 34no 6 pp 937ndash947 2006

[31] J-H Wang H-L Nie S-C Tam H Huang and Y-T ZhengldquoAnti-HIV-1 property of trichosanthin correlates with its ribo-some inactivating activityrdquo FEBS Letters vol 531 no 2 pp 295ndash298 2002

[32] J-H Wang H-L Nie H Huang S-C Tam and Y-TZheng ldquoIndependency of anti-HIV-1 activity from ribosome-inactivating activity of trichosanthinrdquo Biochemical and Biophys-ical Research Communications vol 302 no 1 pp 89ndash94 2003

[33] L Xu L Bao F Li et al ldquoAdaption of seasonal H1N1 influenzavirus in micerdquo PLoS ONE vol 6 no 12 Article ID e28901 2011

[34] C L Speyer and P A Ward ldquoRole of endothelial chemokinesand their receptors during inflammationrdquo Journal of Investiga-tive Surgery vol 24 no 1 pp 18ndash27 2011

[35] R V Drsquoelia K Harrison P C Oyston R A Lukaszewski andG C Clark ldquoTargeting the ldquocytokine stormrdquo for therapeuticbenefitrdquo Clinical and Vaccine Immunology vol 20 no 3 pp319ndash327 2013

[36] S Matsukura F Kokubu H Kubo et al ldquoExpression ofRANTES by normal airway epithelial cells after influenza virusA infectionrdquoAmerican Journal of Respiratory Cell andMolecularBiology vol 18 no 2 pp 255ndash264 1998

[37] F G Hayden R S Fritz M C Lobo W G Alvord W Stroberand S E Straus ldquoLocal and systemic cytokine responses duringexperimental human influenza A virus infection Relation tosymptom formation and host defenserdquo The Journal of ClinicalInvestigation vol 101 no 3 pp 643ndash649 1998

[38] L Kaiser R S Fritz S E Straus L Gubareva and F G HaydenldquoSymptom pathogenesis during acute influenza interleukin-6and other cytokine responsesrdquo Journal of Medical Virology vol64 no 3 pp 262ndash268 2001


[39] J Siren T Sareneva J Pirhonen et al ldquoCytokine and contact-dependent activation of natural killer cells by influenza Aor Sendai virus-infected macrophagesrdquo Journal of GeneralVirology vol 85 no 8 pp 2357ndash2364 2004

[40] C L Galligan T T Murooka R Rahbar E Baig B Majchrzak-Kita and E N Fish ldquoInterferons and viruses signaling forsupremacyrdquo Immunologic Research vol 35 no 1-2 pp 27ndash402006

[41] A Billiau H Heremans K Vermiere and P Matthys ldquoImm-unomodulatory properties of interferon-120574 An updaterdquo Annalsof the New York Academy of Sciences vol 856 pp 22ndash32 1998

[42] J R Teijaro K B Walsh S Cahalan et al ldquoEndothelial cells arecentral orchestrators of cytokine amplification during influenzavirus infectionrdquo Cell vol 146 no 6 pp 980ndash991 2011

[43] H Sprenger R G Meyer A Kaufmann D Bussfeld ERischkowsky and D Gemsa ldquoSelective induction of monocyteand not neutrophil-attracting chemokines after influenza Avirus infectionrdquo Journal of Experimental Medicine vol 184 no3 pp 1191ndash1196 1996

[44] VAArankalle K S Lole R PArya et al ldquoRole of host immuneresponse and viral load in the differential outcome of pandemicH1N1 (2009) influenza virus infection in indian patientsrdquo PLoSONE vol 5 no 10 Article ID e13099 2010

[45] H Hemmi T Kaisho O Takeuchi et al ldquoSmall-antiviral com-pounds activate immune cells via the TLR7 MyD88-dependentsignaling pathwayrdquo Nature Immunology vol 3 no 2 pp 196ndash200 2002

[46] J M Lund L Alexopoulou A Sato et al ldquoRecognition ofsingle-stranded RNA viruses by Toll-like receptor 7rdquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 101 no 15 pp 5598ndash5603 2004

[47] S S Diebold T Kaisho H Hemmi S Akira and C R eSousa ldquoInnate antiviral responses by means of TLR7-mediatedrecognition of single-stranded RNArdquo Science vol 303 no 5663pp 1529ndash1531 2004

[48] K Takeda and S Akira ldquoTLR signaling pathwaysrdquo Seminars inImmunology vol 16 no 1 pp 3ndash9 2004

[49] H L Pahl ldquoActivators and target genes of RelNF-120581B transcrip-tion factorsrdquo Oncogene vol 18 no 49 pp 6853ndash6866 1999

[50] S E Dudek C Luig E-K Pauli U Schubert and S LudwigldquoThe clinically approved proteasome inhibitor PS-341 efficientlyblocks influenza A virus and vesicular stomatitis virus propaga-tion by establishing an antiviral staterdquo Journal of Virology vol84 no 18 pp 9439ndash9451 2010

[51] E Haasbach E-K Pauli R Spranger et al ldquoAntiviral activityof the proteasome inhibitor VL-01 against influenza A virusesrdquoAntiviral Research vol 91 no 3 pp 304ndash313 2011

[52] I Mazur W J Wurzer C Ehrhardt et al ldquoAcetylsalicylicacid (ASA) blocks influenza virus propagation via its NF-120581B-inhibiting activityrdquo Cellular Microbiology vol 9 no 7 pp 1683ndash1694 2007

[53] C E van der Sandt J H C M Kreijtz and G F RimmelzwaanldquoEvasion of influenza A viruses from innate and adaptiveimmune responsesrdquo Viruses vol 4 no 9 pp 1438ndash1476 2012

[54] Z Cao J Xiong M Takeuchi T Kurama and D V GoeddelldquoTRAF6 is a signal transducer for interleukin-1rdquo Nature vol383 no 6599 pp 443ndash446 1996

[55] E Kopp and R Medzhitov ldquoRecognition of microbial infectionby Toll-like receptorsrdquo Current Opinion in Immunology vol 15no 4 pp 396ndash401 2003

[56] S Akira and S Sato ldquoToll-like receptors and their signalingmechanismsrdquo Scandinavian Journal of Infectious Diseases vol35 no 9 pp 555ndash562 2003

[57] E Zandi D M Rothwarf M Delhase M Hayakawa and MKarin ldquoThe I120581B kinase complex (IKK) contains two kinasesubunits IKK120572 and IKK120573 necessary for I120581b phosphorylationand NF-120581B activationrdquo Cell vol 91 no 2 pp 243ndash252 1997

[58] S S Makarov ldquoNF-ΚB in rheumatoid arthritis a pivotalregulator of inflammation hyperplasia and tissue destructionrdquoArthritis Research vol 3 no 4 pp 200ndash206 2001

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


Table 1 Composition of MJWQH

Chinese name Latin name Amount (g) Place of originQiang Huo Rhizoma et Radix Notopterygii 5 Inner Mongolia ChinaCang Zhu Rhizoma Atractylodis 10 Sichuan ChinaPu Gong Yin Herba Taraxaci 20 Jiangsu ChinaHuang Qin Radix Scutellariae 10 Inner Mongolia ChinaHuang Qi Radix Astragali 20 Gansu ChinaFang Feng Radix Saposhnikoviae 5 Hebei ChinaYu Xing Cao Herba Houttuyniae 10 Gansu ChinaTian Hua Fen Radix Trichosanthis 15 Henan China

stimulation cells recognize viral ssRNA via TLR7 and acti-vate downstream signaling molecules through a MyD88dependent pathway causing subsequent cytokine storm andtriggering inflammatory lung injury [15 16]

JiuWei QiangHuo (JWQH) decoction was first intro-duced in themonographCishinanzhiwritten by an influentialTCM physician Zhang Yuansu of Jin Dynasty (1151ndash1234)It has been used for over 900 years for the treatment ofpulmonary disorders such as common cold bronchial infec-tions and pneumoniaThis decoction has recently beenmod-ified by Dr Yan Dexin a Grandmaster of Chinese Medicinegranted by the Ministry of Health of China which consistsof eight herbs including Rhizoma et Radix NotopterygiiHerba Taraxaci Radix Scutellariae Radix Astragali RhizomaAtractylodis Radix Saposhnikoviae Herba Houttuyniae andRadix Trichosanthis The modified JWQH (MJWQH) hasbeen shown to be effective in patients with severe pneumoniainduced by influenza virus It may reverse H1N1-inducedpneumonia in mice by regulating the metabolism of arachi-donic acid fatty acid and amino acid and is thus involvedin anti-inflammatory activity and cell protection [17] Basedon previous research findings and the pathogenesis of H1N1-induced pneumonia we hypothesize that MJWQH has animmunoregulatory effect in the early stages of the disease byinhibiting excessive immune response thus inhibiting virusreplication lung inflammation and severe lung injury

2 Materials and Methods

21 Preparation of MJWQH MJWQH was prepared fromeight dried raw materials (Table 1) purchased from ShanghaiShuguang Hospital (Shanghai China) and authenticated bya physician in Fudan University Voucher specimens weredeposited in the Herbarium Center of the Department ofPharmacognosy School of Pharmacy Fudan University Allraw materials were extracted by boiling in distilled water(about 6-fold the weight of the mixture) at 100∘C for 20minand then filtered The filtrates from two decoctions werecombined and the decoction was dried in vacuo at 70∘Cand ground into powder for use The yield of the extractionwas 24 The extracts were stored in an airtight container atminus80∘C until further analysis

22 Chromatographic Analysis of MJWQH MJWQH wasdissolved in pyrogen-free isotonic saline and filtered through

0

25

50

75

100

125

150

(mAU

)0 10 20 30 40 50 60 70 80

(min)

(1)

tR = 434min(2)

tR = 316min

PDA 280nm 4nm

Figure 1 HPLC chromatogram of MJWQH (280 nm) Calycosin-7-O-120573-D-glucopyranoside (1) and baicalin (2) were separated fromMJWQH at a retention time of 316 and 434min respectively

a 02mm filter (Microgen Laguna Hills CA USA) beforehigh performance liquid chromatography (HPLC) analysis(Figure 1) HPLC analysis was performed on an Agilentseries 1100 HPLC system (Agilent Waldbronn Germany)equipped with quaternary pump diode-array detectorautosampler and column compartment Calycosin-7-O-120573-D-glucopyranoside and baicalin purchased from the NationalInstitute for the Control of Pharmaceutical and BiologicalProducts (Beijing China) were used as the external standardsin HPLC Chromatographic separation was performed on aZorbax XDB-C18 column (5 120583m 120593 46 times 250mm AgilentTechnologies USA) The mobile phase consisted of acetoni-trile (A) and water containing 02 acetic acid (B) and thefollowing gradient programwas used 5A in the first 10minthen a linear gradient to 36A over 60min and then a lineargradient to 85 A over 30min The mobile phase flow ratewas 08mLmin the detector was monitored at 280 nm thespectral data for all peaks were accumulated in the range of190ndash400 nm and the column temperature was set at 25∘C

23 Mice and Virus Male BALBc mice 5 to 6 weeks oldwere purchased from Shanghai SLAC Laboratory Animal CoLtd (Certificate number 2007000548167 SCXK (Hu) 2012-0002 Shanghai China) and bred and maintained in a closedbreeding facility at the Animal Center of Fudan UniversityAll animal experiments were performed in accordance withthe guidelines of the Ethics Committee for Animal Use ofFudan University The mouse adapted influenza AFM147


0 1 2 3 4 5 6 7 8 9 10 11 12 13 14


Day 14end assay



AFM147


50








2asphyxiation
























3 Results







65

75

85

95

105

115

125

135

145Bo

dy w

eigh

t ()

0 1 2 3 4 5 6 7 8 9 10 11 12 13


NCMC

MJWQHRib

(a)

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14


0

20

40

60

80

100

Surv

ival

rate

()

mice 100)



mice)

NCMC

MJWQHRib

(1212

(1012

(512

(012

(b)








4 Discussion




NC MC MJWQH Rib

(a)

NC MC

MJWQH Rib

(b)

NC MC MJWQH Rib0

1

2

3

4

5

Path

olog

ical

scor

e

lowastlowast

lowastlowast

lowastlowast

(c)

NC MC MJWQH Rib


lowastlowast

00

05

10

15

20

Lung

inde

x (

)

(d)





NC MC MJWQH Rib

lowastlowast lowast

lowast

0

2

4

6

HA

tite

r (lo

g 2)

(a)

NC MC MJWQH Rib


lowast

0

200

400

600

800

3000

4000

5000

6000

Influ

enza

viru

s A R

Q

(b)


0

10

20

3050

100

150

200

250

IL-6 IFN-120572

NCMC

MJWQHRib



Lung

hom

ogen

ate o

f mic

e (pg

mL)

(a)

Lung

hom

ogen

ate o

f mic

e (pg

mL)




0

10

20

30

40

150

300

450

600

MIP-1 RANTES MCP-1

NCMC

MJWQHRib

(b)

0

20

40

60

100

150

200

250

TNF-120572 IP-10 IL-1

lowastlowast


lowast

lowast lowastlowast

lowast

NCMC

MJWQHRib

Lung

hom

ogen

ate o

f mic

e (ng

mL)

(c)



TLR7

MyD88

TRAF6

GAPDH

MJWQH Rib

minus + + +

MJWQH Rib

minus + + +

MJWQH Rib

minus + + +

H1N1

Day 2

Day 2

TLR7

MyD88

TRAF6

GAPDH


H1N1H1N1

Day 4

Day 4

P65

P-P65

GAPDH

p-IKK120572120573

I120581B-120572

P65

P-P65

GAPDH

p-IKK120572120573

I120581B-120572







0

1

2

3

4

5

RQ (r

elat

ive q

uant

ifica

tion)

RQ (r

elat

ive q

uant

ifica

tion)

0

1

2

3

4

5RQ

(rel

ativ

e qua

ntifi

catio

n)



lowastlowast

lowast

lowast

lowast

lowastlowast


lowastlowast

TLR7 MyD88

P65

00

05

10

15






NC MC

MJWQH Rib



Abbreviations








Acknowledgments


References



































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0 1 2 3 4 5 6 7 8 9 10 11 12 13 14


Day 14end assay



AFM147


50








2asphyxiation
























3 Results







65

75

85

95

105

115

125

135

145Bo

dy w

eigh

t ()

0 1 2 3 4 5 6 7 8 9 10 11 12 13


NCMC

MJWQHRib

(a)

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14


0

20

40

60

80

100

Surv

ival

rate

()

mice 100)



mice)

NCMC

MJWQHRib

(1212

(1012

(512

(012

(b)








4 Discussion




NC MC MJWQH Rib

(a)

NC MC

MJWQH Rib

(b)

NC MC MJWQH Rib0

1

2

3

4

5

Path

olog

ical

scor

e

lowastlowast

lowastlowast

lowastlowast

(c)

NC MC MJWQH Rib


lowastlowast

00

05

10

15

20

Lung

inde

x (

)

(d)





NC MC MJWQH Rib

lowastlowast lowast

lowast

0

2

4

6

HA

tite

r (lo

g 2)

(a)

NC MC MJWQH Rib


lowast

0

200

400

600

800

3000

4000

5000

6000

Influ

enza

viru

s A R

Q

(b)


0

10

20

3050

100

150

200

250

IL-6 IFN-120572

NCMC

MJWQHRib



Lung

hom

ogen

ate o

f mic

e (pg

mL)

(a)

Lung

hom

ogen

ate o

f mic

e (pg

mL)




0

10

20

30

40

150

300

450

600

MIP-1 RANTES MCP-1

NCMC

MJWQHRib

(b)

0

20

40

60

100

150

200

250

TNF-120572 IP-10 IL-1

lowastlowast


lowast

lowast lowastlowast

lowast

NCMC

MJWQHRib

Lung

hom

ogen

ate o

f mic

e (ng

mL)

(c)



TLR7

MyD88

TRAF6

GAPDH

MJWQH Rib

minus + + +

MJWQH Rib

minus + + +

MJWQH Rib

minus + + +

H1N1

Day 2

Day 2

TLR7

MyD88

TRAF6

GAPDH


H1N1H1N1

Day 4

Day 4

P65

P-P65

GAPDH

p-IKK120572120573

I120581B-120572

P65

P-P65

GAPDH

p-IKK120572120573

I120581B-120572







0

1

2

3

4

5

RQ (r

elat

ive q

uant

ifica

tion)

RQ (r

elat

ive q

uant

ifica

tion)

0

1

2

3

4

5RQ

(rel

ativ

e qua

ntifi

catio

n)



lowastlowast

lowast

lowast

lowast

lowastlowast


lowastlowast

TLR7 MyD88

P65

00

05

10

15






NC MC

MJWQH Rib



Abbreviations








Acknowledgments


References



































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

































3 Results







65

75

85

95

105

115

125

135

145Bo

dy w

eigh

t ()

0 1 2 3 4 5 6 7 8 9 10 11 12 13


NCMC

MJWQHRib

(a)

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14


0

20

40

60

80

100

Surv

ival

rate

()

mice 100)



mice)

NCMC

MJWQHRib

(1212

(1012

(512

(012

(b)








4 Discussion




NC MC MJWQH Rib

(a)

NC MC

MJWQH Rib

(b)

NC MC MJWQH Rib0

1

2

3

4

5

Path

olog

ical

scor

e

lowastlowast

lowastlowast

lowastlowast

(c)

NC MC MJWQH Rib


lowastlowast

00

05

10

15

20

Lung

inde

x (

)

(d)





NC MC MJWQH Rib

lowastlowast lowast

lowast

0

2

4

6

HA

tite

r (lo

g 2)

(a)

NC MC MJWQH Rib


lowast

0

200

400

600

800

3000

4000

5000

6000

Influ

enza

viru

s A R

Q

(b)


0

10

20

3050

100

150

200

250

IL-6 IFN-120572

NCMC

MJWQHRib



Lung

hom

ogen

ate o

f mic

e (pg

mL)

(a)

Lung

hom

ogen

ate o

f mic

e (pg

mL)




0

10

20

30

40

150

300

450

600

MIP-1 RANTES MCP-1

NCMC

MJWQHRib

(b)

0

20

40

60

100

150

200

250

TNF-120572 IP-10 IL-1

lowastlowast


lowast

lowast lowastlowast

lowast

NCMC

MJWQHRib

Lung

hom

ogen

ate o

f mic

e (ng

mL)

(c)



TLR7

MyD88

TRAF6

GAPDH

MJWQH Rib

minus + + +

MJWQH Rib

minus + + +

MJWQH Rib

minus + + +

H1N1

Day 2

Day 2

TLR7

MyD88

TRAF6

GAPDH


H1N1H1N1

Day 4

Day 4

P65

P-P65

GAPDH

p-IKK120572120573

I120581B-120572

P65

P-P65

GAPDH

p-IKK120572120573

I120581B-120572







0

1

2

3

4

5

RQ (r

elat

ive q

uant

ifica

tion)

RQ (r

elat

ive q

uant

ifica

tion)

0

1

2

3

4

5RQ

(rel

ativ

e qua

ntifi

catio

n)



lowastlowast

lowast

lowast

lowast

lowastlowast


lowastlowast

TLR7 MyD88

P65

00

05

10

15






NC MC

MJWQH Rib



Abbreviations








Acknowledgments


References



































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















65

75

85

95

105

115

125

135

145Bo

dy w

eigh

t ()

0 1 2 3 4 5 6 7 8 9 10 11 12 13


NCMC

MJWQHRib

(a)

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14


0

20

40

60

80

100

Surv

ival

rate

()

mice 100)



mice)

NCMC

MJWQHRib

(1212

(1012

(512

(012

(b)








4 Discussion




NC MC MJWQH Rib

(a)

NC MC

MJWQH Rib

(b)

NC MC MJWQH Rib0

1

2

3

4

5

Path

olog

ical

scor

e

lowastlowast

lowastlowast

lowastlowast

(c)

NC MC MJWQH Rib


lowastlowast

00

05

10

15

20

Lung

inde

x (

)

(d)





NC MC MJWQH Rib

lowastlowast lowast

lowast

0

2

4

6

HA

tite

r (lo

g 2)

(a)

NC MC MJWQH Rib


lowast

0

200

400

600

800

3000

4000

5000

6000

Influ

enza

viru

s A R

Q

(b)


0

10

20

3050

100

150

200

250

IL-6 IFN-120572

NCMC

MJWQHRib



Lung

hom

ogen

ate o

f mic

e (pg

mL)

(a)

Lung

hom

ogen

ate o

f mic

e (pg

mL)




0

10

20

30

40

150

300

450

600

MIP-1 RANTES MCP-1

NCMC

MJWQHRib

(b)

0

20

40

60

100

150

200

250

TNF-120572 IP-10 IL-1

lowastlowast


lowast

lowast lowastlowast

lowast

NCMC

MJWQHRib

Lung

hom

ogen

ate o

f mic

e (ng

mL)

(c)



TLR7

MyD88

TRAF6

GAPDH

MJWQH Rib

minus + + +

MJWQH Rib

minus + + +

MJWQH Rib

minus + + +

H1N1

Day 2

Day 2

TLR7

MyD88

TRAF6

GAPDH


H1N1H1N1

Day 4

Day 4

P65

P-P65

GAPDH

p-IKK120572120573

I120581B-120572

P65

P-P65

GAPDH

p-IKK120572120573

I120581B-120572







0

1

2

3

4

5

RQ (r

elat

ive q

uant

ifica

tion)

RQ (r

elat

ive q

uant

ifica

tion)

0

1

2

3

4

5RQ

(rel

ativ

e qua

ntifi

catio

n)



lowastlowast

lowast

lowast

lowast

lowastlowast


lowastlowast

TLR7 MyD88

P65

00

05

10

15






NC MC

MJWQH Rib



Abbreviations








Acknowledgments


References



































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















NC MC MJWQH Rib

(a)

NC MC

MJWQH Rib

(b)

NC MC MJWQH Rib0

1

2

3

4

5

Path

olog

ical

scor

e

lowastlowast

lowastlowast

lowastlowast

(c)

NC MC MJWQH Rib


lowastlowast

00

05

10

15

20

Lung

inde

x (

)

(d)





NC MC MJWQH Rib

lowastlowast lowast

lowast

0

2

4

6

HA

tite

r (lo

g 2)

(a)

NC MC MJWQH Rib


lowast

0

200

400

600

800

3000

4000

5000

6000

Influ

enza

viru

s A R

Q

(b)


0

10

20

3050

100

150

200

250

IL-6 IFN-120572

NCMC

MJWQHRib



Lung

hom

ogen

ate o

f mic

e (pg

mL)

(a)

Lung

hom

ogen

ate o

f mic

e (pg

mL)




0

10

20

30

40

150

300

450

600

MIP-1 RANTES MCP-1

NCMC

MJWQHRib

(b)

0

20

40

60

100

150

200

250

TNF-120572 IP-10 IL-1

lowastlowast


lowast

lowast lowastlowast

lowast

NCMC

MJWQHRib

Lung

hom

ogen

ate o

f mic

e (ng

mL)

(c)



TLR7

MyD88

TRAF6

GAPDH

MJWQH Rib

minus + + +

MJWQH Rib

minus + + +

MJWQH Rib

minus + + +

H1N1

Day 2

Day 2

TLR7

MyD88

TRAF6

GAPDH


H1N1H1N1

Day 4

Day 4

P65

P-P65

GAPDH

p-IKK120572120573

I120581B-120572

P65

P-P65

GAPDH

p-IKK120572120573

I120581B-120572







0

1

2

3

4

5

RQ (r

elat

ive q

uant

ifica

tion)

RQ (r

elat

ive q

uant

ifica

tion)

0

1

2

3

4

5RQ

(rel

ativ

e qua

ntifi

catio

n)



lowastlowast

lowast

lowast

lowast

lowastlowast


lowastlowast

TLR7 MyD88

P65

00

05

10

15






NC MC

MJWQH Rib



Abbreviations








Acknowledgments


References



































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















NC MC MJWQH Rib

lowastlowast lowast

lowast

0

2

4

6

HA

tite

r (lo

g 2)

(a)

NC MC MJWQH Rib


lowast

0

200

400

600

800

3000

4000

5000

6000

Influ

enza

viru

s A R

Q

(b)


0

10

20

3050

100

150

200

250

IL-6 IFN-120572

NCMC

MJWQHRib



Lung

hom

ogen

ate o

f mic

e (pg

mL)

(a)

Lung

hom

ogen

ate o

f mic

e (pg

mL)




0

10

20

30

40

150

300

450

600

MIP-1 RANTES MCP-1

NCMC

MJWQHRib

(b)

0

20

40

60

100

150

200

250

TNF-120572 IP-10 IL-1

lowastlowast


lowast

lowast lowastlowast

lowast

NCMC

MJWQHRib

Lung

hom

ogen

ate o

f mic

e (ng

mL)

(c)



TLR7

MyD88

TRAF6

GAPDH

MJWQH Rib

minus + + +

MJWQH Rib

minus + + +

MJWQH Rib

minus + + +

H1N1

Day 2

Day 2

TLR7

MyD88

TRAF6

GAPDH


H1N1H1N1

Day 4

Day 4

P65

P-P65

GAPDH

p-IKK120572120573

I120581B-120572

P65

P-P65

GAPDH

p-IKK120572120573

I120581B-120572







0

1

2

3

4

5

RQ (r

elat

ive q

uant

ifica

tion)

RQ (r

elat

ive q

uant

ifica

tion)

0

1

2

3

4

5RQ

(rel

ativ

e qua

ntifi

catio

n)



lowastlowast

lowast

lowast

lowast

lowastlowast


lowastlowast

TLR7 MyD88

P65

00

05

10

15






NC MC

MJWQH Rib



Abbreviations








Acknowledgments


References



































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















TLR7

MyD88

TRAF6

GAPDH

MJWQH Rib

minus + + +

MJWQH Rib

minus + + +

MJWQH Rib

minus + + +

H1N1

Day 2

Day 2

TLR7

MyD88

TRAF6

GAPDH


H1N1H1N1

Day 4

Day 4

P65

P-P65

GAPDH

p-IKK120572120573

I120581B-120572

P65

P-P65

GAPDH

p-IKK120572120573

I120581B-120572







0

1

2

3

4

5

RQ (r

elat

ive q

uant

ifica

tion)

RQ (r

elat

ive q

uant

ifica

tion)

0

1

2

3

4

5RQ

(rel

ativ

e qua

ntifi

catio

n)



lowastlowast

lowast

lowast

lowast

lowastlowast


lowastlowast

TLR7 MyD88

P65

00

05

10

15






NC MC

MJWQH Rib



Abbreviations








Acknowledgments


References



































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

1

2

3

4

5

RQ (r

elat

ive q

uant

ifica

tion)

RQ (r

elat

ive q

uant

ifica

tion)

0

1

2

3

4

5RQ

(rel

ativ

e qua

ntifi

catio

n)



lowastlowast

lowast

lowast

lowast

lowastlowast


lowastlowast

TLR7 MyD88

P65

00

05

10

15






NC MC

MJWQH Rib



Abbreviations








Acknowledgments


References



































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















NC MC

MJWQH Rib



Abbreviations








Acknowledgments


References



































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of












































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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