Effects of Gui Zhi Ma Huang Ge Ban Tang on the TLR7

Research ArticleEffects of Gui Zhi Ma Huang Ge Ban Tang on the TLR7 Pathwayin Influenza Virus Infected Mouse Lungs in a Cold Environment

Hong-Qiong Qin 1 Shan-Shan Shi1 Ying-Jie Fu1 Yu-Qi Yan1 ShaWu1 Xiao-Long Tang2

Xiao-Yin Chen 3 Guang-Hui Hou 4 and Zhen-You Jiang 1

1Department of Microbiology and Immunology School of Medicine Jinan University Guangzhou 510632 China2Medical College Anhui University of Science amp Technology Huainan 232001 China3Department of Traditional Chinese Medicine School of Medicine Jinan University Guangzhou 510632 China4Department of Ophthalmic Center Peoplersquos Hospital of Zhuhai City Affiliated Hospital of Zhuhai Medical College Jinan UniversityZhuhai 519000 China

Correspondence should be addressed to Xiao-Yin Chen tchenxiaoyinjnueducn Guang-Hui Hou Houguanghui502qqcomand Zhen-You Jiang tjzhyjnueducn

Received 12 November 2017 Accepted 20 February 2018 Published 23 April 2018

Academic Editor Jose L Rios

Copyright copy 2018 Hong-Qiong Qin et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Objective We wished to investigate the effects of the traditional Chinese medicine Gui Zhi Ma Huang Ge Ban Tang on controllinginfluenza A virus (IAV) infection and improving inflammation in mouse lungsMethod Mice were maintained in normal and coldenvironments and infected with IAV by intranasal application respectively Real-time quantitative polymerase chain reaction wasused to measure mRNA expression of TLR7 myeloid differentiation primary response 88 (MyD88) and nuclear factor-kappa B(NF-120581B)p65 in the TLR7 signaling pathway and virus replication in lungs Western blotting was used to measure expression levelsof TLR7 MyD88 and NF-120581B p65 proteins Flow cytometry was used to detect the proportion of T-helper (Th)1Th2 and Th17T-regulatory (Treg) cells Results Application of Gui Zhi Ma Huang Ge Ban Tang in influenza-infected mice in a cold environmentshowed (i) downregulation of TLR7 MyD88 and NF-120581Bp65 (ii) inhibition of transcriptional activities of promoters coding forTLR7 MyD88 and NF-120581Bp65 (iii) reduction in the proportion of Th1Th2 and Th17Treg cells Conclusions Gui Zhi Ma HuangGe Ban Tang had a good therapeutic effect on mice infected with IAV especially in the cold environment It could reduce lunginflammation in mice significantly and elicit an anti-influenza effect by downregulating expression of the key factors in TLR7signaling pathway

1 Introduction

The influenza A virus (IAV) is an important pathogen inthe respiratory tract that causes seasonal epidemics andpandemics of considerablemorbidity andmortality [1 2] IAVinfection induces the hostrsquos natural immune response [3] andactivation of a Toll-like receptor- (TLR-) mediated antiviralsignaling pathway The TLR family plays an important partin pathogen recognition and activation of innate immunity[4] TLR7 is a member of the TLR family and detects single-stranded RNA (ssRNA) [5]

Currently ribavirin [6] and oseltamivir [7 8] are effectivein suppressing viral replication in IAV infection but theIAV is susceptible to variation The emergence and spread of

adamantane resistance and neuraminidase inhibitors (NAI)resistance would limit therapeutic options Vaccination isknown to be an effective method to prevent influenza How-ever current vaccines are not highly protective if antigenicallydifferent new strains emerge such as the outbreak of thepandemic (H1N1) 2009 virus [9 10]Therefore finding amea-sure that protects against the emergence of an unexpectedinfluenza strain is highly desirable

Many Chinese herbs are used to treat and preventinfluenza [11] Compared withWesternmedicine TraditionalChinese medicines (TCM) have multiple targets againstdifferent types of influenza viruses Yinqiaosan is the rep-resentative solution of xinliangjiebiao a medicine that canrelieve fever and detoxify Yinqiaosan is used for treating

HindawiEvidence-Based Complementary and Alternative MedicineVolume 2018 Article ID 5939720 10 pageshttpsdoiorg10115520185939720

2 Evidence-Based Complementary and Alternative Medicine

viral infectious diseases such as influenza acute tonsillitispharyngitis measles and mumps Xinjiaxiangruyin is arepresentative medicine of qushijiebiao which has the effectof relieving ldquoheatrdquo and ldquodampnessrdquo and is usedmainly to treatsummer colds This recipe has a very good therapeutic effectfor damp-heating flu Gui Zhi Ma Huang Ge Ban Tang is arepresentative medicine of xinwenjiebiao with anti-influenzavirus and anti-inflammatory effects It also has antipyreticanalgesic and other pharmacologic effects and has been usedto treat colds respiratory-tract infections and allergies How-ever studies focusing on the antivirus and anti-inflammatorymechanisms of Gui Zhi Ma Huang Ge Ban Tang are lacking

Wan and colleagues [12] demonstrated that baicalininhibits TLR7MYD88 signaling pathway activation to sup-press lung inflammation in mice infected with influenzaA virus Hence using yinqiaosan and xinjiaxiangruyin ascontrols we investigated the effects of Gui Zhi Ma Huang GeBan Tang on the TLR7 signaling pathway and on the immunebalance of T cells in mouse lungs infected with the IAV in acold environment

2 Materials and Methods

21 Animals and Grouping Experiments were undertakenunder the supervision and assessment of the LaboratoryAnimal Ethics Committee of Jinan University (Jinan Univer-sity Guangzhou China) All experimental procedures werecarried out in accordance with the Guide for the Care andUse of Laboratory Animals (ninth edition National Institutesof Health Bethesda MD USA) and were approved by theAnimal Ethics Committee of Jinan University

Female specific pathogen-free (SPF) C57BL6j mice (20 plusmn1 g) were purchased from the Guangdong Experimental Ani-mal Center (Guangdong China) TLR7 knockout (TLR7minusminus)mice were obtained from Taconic Farms (Germantown NYUSA) TLR7minusminus mice were over 10 generations

Thirty-six female SPF C57BL6j mice (6∘C wild type(WT)) and 36 female SPF TLR7minusminusmice (6∘C TLR7minusminus) weremaintained in a cold room (6 plusmn 1∘C) with humidity of 50and light intensity of 3000 Lx and 0 Lxunder a 12-h lightndashdarkcycle A control group of 36 female SPFC57BL6jmice (20∘CWT) were maintained in a normal environment (20 plusmn 1∘Chumidity of 50 plusmn 5 light intensity of 3000 Lx and 0 Lxunder a 12-h lightndashdark cycle)

Mice were divided randomly into six groups with six ani-mals in each group normal control IAV control (ldquovirus con-trolrdquo) IAV + oseltamivir (ldquooseltamivirrdquo) IAV + yinqiaosan(ldquoyinqiaosanrdquo) IAV + Gui Zhi Ma Huang Ge Ban Tang(ldquoGui Zhi Ma Huang Ge Ban Tangrdquo) IAV + xinjiaxiangruyin(ldquoXinjiaxiangruyinrdquo)

22 IAV and Drugs IAV [FM1147 strain (mouse adapted)]was used for all experiments and grown in the allantoiccavities of 10-11-day-old fertile chicken eggs for 2 days at35∘C Yinqiaosan Gui Zhi Ma Huang Ge Ban Tang andxinjiaxiangruyin were purchased from the First AffiliatedHospital of Jinan University

Yinqiaosan [Detailed Analysis of Epidemic Warm Dis-eases TangWu 1798] contained Forsythia (15 g) honeysuckle

(15 g) Campanulaceae (9 g) mint (9 g) bamboo leaves (6 g)raw licorice (5 g) Nepeta (6 g) dandouchi (6 g) burdock(6 g) and reed rhizome (10 g)

Gui Zhi Ma Huang Ge Ban Tang [Typhoid miscellaneousdiseases theory Zhong-Jing Zhang about 219] containedEphedra Tang [Ephedra (9 g) Guizhi (6 g) almonds (9 g) andZhigancao (6 g)] and Guizhi Tang [Guizhi (9 g) peony (9 g)licorice (6 g) ginger (9 g) and jujube (10 g)]

Xinjiaxiangruyin [Detailed Analysis of Epidemic WarmDiseases Tang Wu 1798] contained Elsholtzia (6 g) honey-suckle (9 g) lentils (9 g)Magnolia (6 g) and Forsythia (6 g)

Oseltamivir phosphate (75mg) was obtained fromYichang Changjiang Pharmaceuticals (Hubei China)

23 Creation of an Influenza Model TLR7minusminus mice and WTC57BL6j mice were infected with the IAV (FM1 strain) onday 11 Mice in the virus control oseltamivir yinqiaosan GuiZhi Ma Huang Ge Ban Tang and xinjiaxiangruyin groupswere anesthetized using chloral hydrate and then infected byintranasal application of 40 LD50 (Half the lethal dose)This procedure leads to infection in the upper and lowerrespiratory tracts Mice in the normal control group wereanesthetized with chloral hydrate and then given an identicalamount of distilled water The physical status and weight ofmice were recorded daily

24 Anti-IAV Experiments The drug dose was calculatedbased on body weight differences between humans andmice The value for yinqiaosan was 112 gmL body weight0940 gmL for Gui Zhi Ma Huang Ge Ban Tang 0465 gmLfor xinjiaxiangruyin and 3mgmL for oseltamivir respec-tively Mice in the normal control group and virus controlgroup were given physiologic (09) saline instead Thecorresponding drug concentration (or 09 saline) wasadministered (02mLday ig) to each mouse 24 h after viruschallenge for 5 days (day 12 to day 16) (Figure 1)

25 Tissue Harvesting Six days after infection (day 17) afterdocumenting the body weight mice were anesthetized andsacrificed The spleen and lung were harvested The lungweight was obtainedThen the lung weightbody weight per-centage was calculated

26 Histopathology Lung tissue was fixed with 4 para-formaldehyde and then trimmed by a scalpel The appropri-ate lung portion was selected and rinsed dehydrated andtreated by a transparent agent xylene immersing After thesections had been dried and dewaxed they were stained withhematoxylin and eosin The tissues were sectioned and theorganization of tissue was observed at times100 magnification

27 Quantitative Real-Time Reverse Transcription-PolymeraseChain Reaction (RT-qPCR) of TLR7 MyD88 and NF-120581Bp65 mRNA and Relative Replication of IAV in Lung TissueTotal RNA was extracted with RNAiso Plus according tomanufacturer (TaKaRa Bio Shiga Japan) instructions cDNAsynthesis and real-time PCRs were carried out using a CFXConnect Real-Time PCR Detection system (Bio-Rad Labo-ratories Hercules CA USA) with PrimeScript RT Reagent

Evidence-Based Complementary and Alternative Medicine 3

0

FM1 treated

G avage 02 mloneday

11 12 17

Harvest

16

20∘C-WT

6∘C-WT

6∘C-TLR-7minusminus

Figure 1 Influenza model in mice

Table 1 Primers used for RT-qPCR analysis

Gene Primer Sequence

GAPDH Forward 51015840-CTGAGCAAGAGAGGCCCTATCC-31015840

Reverse 51015840-CTCCCTAGGCCCCTCCTGTT-31015840

FM1 Forward 51015840-GACCAATCCTGTCACCTCTGAC-31015840

Reverse 51015840-AGGGCATTTGGACAAAGCGTCTA-31015840

TLR-7 Forward 51015840-GGGTCCAAAGCCAATGTG-31015840

Reverse 51015840-TGTTAGATTCTCCTTCGTGATG-31015840

MyD88 Forward 51015840-CGATTATCTACAGAGCAAGGAATG-31015840

Reverse 51015840-ATAGTGATGAACCGCAGGATAC-31015840

NF-120581B p65 Forward 51015840-ATTCTGACCTTGCCTATCTAC-31015840

Reverse 51015840-TCCAGTCTCCGAGTGAAG-31015840

kits and SYBR Premix EX Taq II according to manufacturer(TaKaRa Bio) instructions Primers were synthesized byGenerayBiotech (Shanghai China) All primers for RT-qPCRare presented in Table 1

After RT-qPCR the corresponding relative mRNAexpression was normalized to that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and was calculatedusing the 2minusΔΔCq method Each sample was measured thriceand averaged qPCR results were representative of three inde-pendent experiments Gene expression levels in the viruscontrol oseltamivir yinqiaosan Gui Zhi Ma Huang Ge BanTang and xinjiaxiangruyin groups were expressed relative tothat of the normal control group

28 Western Blotting for TLR7 MyD88 and NF-120581B p65 West-ern BlottingWas Performed According to Standard ProceduresProteins were extracted from lung tissues and quantifiedusing a bicinchoninic acid protein assay kit (MultiSciencesBiotech Hangzhou China) An equal amount of protein(40 120583glane) was fractionated using an electrophoresis system(Bio-Rad Laboratories) on 10 polyacrylamide gels and thentransferred to polyvinylidene fluoride (PVDF) membranes(Millipore Billerica MA USA) PVDF membranes wereblocked with 5 nonfat milk and incubated with TLR7(1 1000 dilution Cell Signaling Technology Danvers MAUSA) MyD88 (1 1000 Cell Signaling Technology) or NF-120581B p65 (1 1000 Cell Signaling Technology) in 5 BSA-TBSTbuffer overnight at 4∘C The loading control was GAPDH(1 1000 Cell Signaling Technology) After incubation withthe secondary antibody (horseradish peroxidase-conjugatedgoat anti-rabbit IgG 1 5000 MultiSciences Biotech) protein

bands were detected using an electrochemiluminescence kitaccording to manufacturer instructions and analyzed usingImageJ software (NIH)

29 Immunofluorescence Labeling and Flow CytometryPeripheral blood mononuclear cells (PBMCs) were isolatedfrom the spleen by lymphocyte separationmedium accordingtomanufacturer (MultiSciences Biotech) instructions Differ-ent subsets of T cells were evaluated by flow cytometry Allanti-mouse-specific antibodies used were obtained fromeBioscience (San Diego CA USA) PBMCs were stimulatedwith phorbol myristate acetate (125 120583gmL MultiSciencesBiotech) and ionomycin (025mgmL MultiSciences Bio-tech) in the presence of an FC receptor blocker (MultiSci-ences Biotech) for 5 h PBMCs were washed and thenfixedpermeabilized in fixationpermeabilization buffers(eBioscience) and stained with antibodies against cluster ofdifferentiation (CD)4-Phycoerythrin (PE) CD25-Allophy-cocyanin (APC) interleukin (IL)-4-APC interferon (IFN)gamma-biotin IL-17A-Alexa Fluor 488 and forkhead box P3(Foxp3)-PE-cyanine55 Appropriate isotype controls wereused Flow cytometry was undertaken on a FACSVerse flowcytometer (Becton Dickinson Biosciences Franklin LakesNJ USA) and analyzed using FlowJo v10 (FlowJo AshlandOR USA)

210 Statistical Analyses Statistical analyses were carried outusing SPSS v22 (IBM Armonk NY USA) Results are themean plusmn SD Data were analyzed using ANOVA or Studentrsquost-test 119901 lt 005 was considered significant and 119901 lt 001 wasconsidered to be statistically significant


00

01

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04

Body

wei

ght l

oss p

erce

nt

ns nsns

nsns

ns

20∘C-WT 6

∘C-WT 6∘C-TLR-7minusminus

Normal controlVirus controlOseltamivir

YinqiaosanGui Zhi Ma Huang Ge Ban TangXinjiaxiangruyin

lowastlowastlowast lowastlowastlowast

lowastlowastlowast lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

Figure 2 Changes in body weight of miceThe body weight of micein virus control oseltamivir yinqiaosan Gui Zhi Ma Huang Ge BanTang and xinjiaxiangruyin groups lowastlowastlowast119901 lt 0001 ns 119901 gt 005

3 Results

31 Physical Status of Mice in Each Group Eighteen hoursafter the induction of infection in addition to the normalcontrol group mice showed varying degrees of influenzasymptoms lying down in a curled up position dyspnea beingapathetic loss of luster of body hair and reduced food intakeDuring the administration period influenza symptoms in theoseltamivir group were mild and mice in the yinqiaosan andGui Zhi Ma Huang Ge Ban Tang groups had good physicalstatus in the normal environment In the cold environmentcompared with the virus control group the physical status ofmice in the Gui ZhiMaHuangGe Ban Tang groupwas better

32 Changes in Body Weight The percentage loss in bodyweight was calculated using the following formula Percent-age loss in body weight = [(bodyweight at day 11 ndash bodyweight at day 17)bodyweight at day 11] times 100

The body weight changes of mice in the experimentalgroups are shown in Figure 2 Body weight of mice inthe normal control group showed no changes After IAVinfection except for the normal control group the physicalstatus of the mice in other groups started to deteriorate andthey began to lose body weight The weight of mice in thevirus control group decreased more quickly There were nosignificant differences (119901 gt 005) in body weight between theoseltamivir yinqiaosan and Gui Zhi Ma Huang Ge Ban Tanggroups in the normal environment In the cold environmentthe oseltamivir group and Gui Zhi Ma Huang Ge Ban Tanggroup had higher body weight There were no significantdifferences (119901 gt 005) between the virus control group andxinjiaxiangruyin yinqiaosan and Gui Zhi MaHuang Ge BanTang groups of TLR7minusminus mice

33 Changes in Lung WeightBody Weight Changes in thelung weightbody weight of mice in the different experi-mental groups are shown in Figure 3 The lung weightbodyweight of mice in the virus control oseltamivir and xinjiax-iangruyin yinqiaosan and Gui Zhi Ma Huang Ge Ban Tanggroups increased compared with that in the normal controlgroup In the normal environment the lung weightbody

0000

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Lung

Bod

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eigh

t (g

g)

ns ns

nsns

nsns

20∘C-WT 6


lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

Figure 3 Changes in lung weightbody weight of mice lowastlowastlowast119901 lt0001 ns 119901 gt 005

weight of mice in the virus control group increased morethan that in the oseltamivir yinqiaosan and Gui Zhi MaHuang Ge Ban Tang groups In the cold environment thelung weightbody weight of mice in the virus control groupincreased more than that in the oseltamivir and Gui ZhiMa Huang Ge Ban Tang groups There were no significantdifferences (119901 gt 005) between the virus control group andthe oseltamivir yinqiaosan and Gui Zhi Ma Huang Ge BanTang groups of TLR7minusminus mice

34 Morphologic Changes in Lung Tissue The integrity ofthe structure of alveoli before IAV infection was shown byexamination of pathologic tissue sections (Figure 4(a)) AfterIAV infection diffuse damage was seen in the alveoli alve-olar sacs alveolar tubes alveolar septa and bronchi Therewas considerable lymphocyte infiltration in the pulmonaryinterstitium (Figure 4(b)) Compared with the virus controlgroup WT mice in yinqiaosan and Gui Zhi Ma Huang GeBan Tang groups in the normal environment and WT micein the Gui Zhi Ma Huang Ge Ban Tang group in the coldenvironment showed a clear alveolar structure with only asmall number of inflammatory cells In the TLR7minusminus miceof xinjiaxiangruyin yinqiaosan and Gui Zhi Ma Huang GeBan Tang groups there was vascular congestion alveolarexpansion and pulmonary interstitial edema and alveolarsepta were filled with inflammatory cells There were noobvious differences between the virus control group andxinjiaxiangruyin yinqiaosan and Gui Zhi MaHuang Ge BanTang groups in TLR7minusminus mice

35 Changes in IAV Replication in the Lungs There was noIAV replication in the normal control group IAV-infectedmice in the virus control group had significantly greaterreplication than that in the normal control group (119901 lt 0001)Oseltamivir reduced virus replication significantly comparedwith the virus control group (119901 lt 0001) In the normalenvironment IAV replication in the yinqiaosan and Gui ZhiMa Huang Ge Ban Tang groups was significantly differentcompared with that in the oseltamivir group (119901 lt 005) butthere were no significant differences between the virus con-trol group and xinjiaxiangruyin group (119901 gt 005) In the cold


(a) (b) (c) (d) (e) (f)

20∘C WT

6∘C WT

6∘C TLR7minusminus

(a) (b) (c) (d) (e) (f)

(a) (b) (c) (d) (e) (f)

Figure 4 Effects of yinqiaosan Gui Zhi Ma Huang Ge Ban Tang and xinjiaxiangruyin on histology in mice (A-F) Representative staining(hematoxylin and eosin) of histologic sections of all groups at day 17 (A) normal control (B) virus control (C) oseltamivir (D) yinqiaosan(E) Gui Zhi Ma Huang Ge Ban Tang and (F) xinjiaxiangruyin All images were obtained at times200 magnification scale bar = 100 120583m

0

500

1000

1500

2000

Relat

ive m

RNA

expr

essio

nof

FM

1

nsns ns

nsnsns



20∘C-WT 6


lowastlowastlowastlowast

lowastlowastlowastlowastlowast

lowastlowastlowast lowastlowastlowastlowastlowastlowastlowastlowastlowast

lowast

Figure 5 Effect of yinqiaosan Gui Zhi Ma Huang Ge Ban Tangand oseltamivir on IAV (FM1) replication lowast119901 lt 005 lowastlowast119901 lt 001lowastlowastlowast119901 lt 0001 ns 119901 gt 005

environment IAV replication in the Gui Zhi Ma Huang GeBan Tang group was significantly higher than that in theoseltamivir group (119901 lt 005) but there were no significantdifferences between the virus control group and yinqiaosanand xinjiaxiangruyin groups (119901 gt 005) There were nosignificant differences (119901 gt 005) between the virus controlgroup and xinjiaxiangruyin yinqiaosan and Gui Zhi MaHuang Ge Ban Tang groups of TLR7minusminus mice (Figure 5)

36 mRNA Expression Levels of TLR7 MyD88 and NF-120581BExpression of the mRNA of TLR7 (A) MyD88 (B) andNF-120581B (C) via the TLR7 signaling pathway in pulmonaryimmunocytes was increased significantly in the virus controlgroup (119901 lt 0001) (Figure 6) In the normal environmenttreatment with oseltamivir yinqiaosan or Gui ZhiMaHuangGe Ban Tang reduced mRNA expression in the TLR7 signal-ing pathway Compared with the virus control group mRNAexpression in the TLR7 signaling pathwaywas downregulatedin the oseltamivir yinqiaosan andGui ZhiMaHuangGe Ban

Tang groups (119901 lt 005) In the cold environment comparedwith the virus control group the relative mRNA expressionof TLR7 MyD88 and NF-120581B p65 in the oseltamivir and GuiZhi Ma Huang Ge Ban Tang groups was downregulated (119901 lt005) There were no significant differences between the viruscontrol and the yinqiaosan and xinjiaxiangruyin groups (119901 gt005) (Figure 6)

37 Relative Protein Expression of TLR7 MyD88 and NF-120581Bp65 Western blotting revealed that expressions of proteinlevels of TLR7 (A) MyD88 (B) and NF-120581B p65 (C) wereenhanced gradually in the virus control group comparedwiththe normal control group Compared with the virus controlgroup the relative protein expression of TLR7 MyD88 andNF-B p65 was downregulated in the oseltamivir yinqiaosanand Gui Zhi Ma Huang Ge Ban Tang groups in the normalenvironment In the cold environment compared with thevirus control group the relative protein expression of TLR7MyD88 and NF-120581B p65 of the oseltamivir and Gui ZhiMa Huang Ge Ban Tang groups was downregulated (119901 lt005) There were no significant differences between the viruscontrol group and yinqiaosan and xinjiaxiangruyin groups(119901 gt 005) Western blotting results were in accordance withRT-qPCR results (Figure 7)

38 Detection ofTh1Th2Th17 and T-Regulatory (Treg) CellsFlow cytometry was used to detect the proportion of theTh1Th2 and Th17Treg cells These proportions increasedafter the induction of infection Th1Th2 cells differentiatedtowards Th1 cells and Th17Treg cells differentiated towardsTh17 cells IAV-infected mice in the oseltamivir as well as thexinjiaxiangruyin yinqiaosan and Gui Zhi MaHuang Ge BanTang groups had lower proportions of these T-cell subsetsthan the IAV-infected mice of the virus control group The Tcells from the xinjiaxiangruyin yinqiaosan and Gui Zhi MaHuang Ge Ban Tang groups differentiated into Th1 or Th17cells and the proinflammatory effects of T cellswere inhibited(Figure 8)


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ns ns nsns

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20∘C-WT 6



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20∘C-WT 6


lowastlowastlowastlowastlowastlowast lowastlowastlowastlowastlowastlowast lowastlowastlowast

lowastlowast


(c)

Figure 6 mRNA expression of TLR7 (a) NF-120581B p65 (b) and MyD88 (c) in the mouse lung tissue lowastlowast119901 lt 001 lowastlowastlowast119901 lt 0001 ns 119901 gt 005

4 Discussion

Humans may be infected by the influenza virus at any timeof the year but infection happens more likely in winter Inaddition most people are indoors during winter and thewindows are often closed resulting in poor air circulationwhich increases the chance of the virus spreading

In the present study compared with yinqiaosan andxinjiaxiangruyin Gui Zhi Ma Huang Ge Ban Tang hada good therapeutic effect on mice infected with the IAV(FM1 strain) especially in the cold environment Gui ZhiMa Huang Ge Ban Tang could reduce lung inflammationin mice significantly and elicit an anti-influenza effect bydownregulating expression of the key factors of the TLR7signaling pathway

TLR7 [12 13] is an important member of the TLR familyTLRs can identify the pathogen-related molecular patternsof various microbes including bacteria viruses protozoaand fungi and have an important role in natural immunityTLRs can induce the expression of IFN-120572 IL-12 and othercytokines by identifying ssRNA IAV infection triggers aseries of immune responses that can limit viral replicationand viral infection

The cells that express TLR7 are mainly immunocytessuch as plasmacytoid dendritic cells and macrophages Theseimmunocytes can recognize viral ssRNA through TLR7 and

elicit an antiviral immune response through theMyD88 path-way which leads to activation of costimulatorymolecules andcytokine production followed by a specific immune responseIf activation of this pathway is dysfunctional it leads toexpression of many proinflammatory mediators resulting ininflammation [12 14 15] TLR7 has been shown to mediatemultiple viral immune responses [16ndash18] including thoseto the human immunodeficiency virus-1 influenza virusvesicular stomatitis virus and hepatitis-C virus

The present study showed that xinjiaxiangruyin yin-qiaosan andGui ZhiMaHuangGe Ban Tang could lower theratio of Th1Th2 cells andTh17Treg cells in normal and coldenvironments as well as in TLR7minusminus mice but Gui Zhi MaHuang Ge Ban Tang is better Th1 Th2 Th17 and Treg cellsare different subpopulations differentiated by CD4+ T cellsand have different roles in the immune response [19ndash22]Th1 cells mainly secrete IL-2 IFN-120574 and other cytokines andare mainly involved in cellular immune responses mediatingcytotoxic delayed-type hypersensitivity and local inflamma-tionThey can also inhibit the formation ofTh2 cellsTh2 cellsmainly secrete IL-4 and IL-6 and are associated with humoralimmunity stimulating the activation and proliferation of Bcells and antibody secretion Th17 cells can secrete IL-17 IL-21 and other cytokines IL-17 is a strong and effective proin-flammatory cytokine [22 23] and can stimulate the secretionof granulocyte-colony stimulating factor and prostaglandin


TLR-7

MyD88

NF-휅B (p65)

GAPDH

A B C D E F A B C D E F A B C D E F

20∘C WT 6

∘C WT 6∘C TLR7minusminus

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TLR7

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tein

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∘C-WT

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NF-휅

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20∘C-WT 6


lowastlowastlowast lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast lowastlowast


(d)

Figure 7 Relative protein expression of TLR7 NF-120581B p65 andMyD88 (a) A normal control B virus control C oseltamivir D yinqiaosanE Gui Zhi Ma Huang Ge Ban Tang and F xinjiaxiangruyin Protein expression of TLR7 (b) MyD88 (c) and NF-120581B p65 (d) lowastlowast119901 lt 001lowastlowastlowast119901 lt 0001 ns 119901 gt 005

(PG)E2 enhance the function of IL-1 and tumor necrosisfactor (TNF) induce the secretion of NF-120581B IL-6 IL-8 andother factors in promoting the inflammatory response andplay an important part in autoimmune injury [24 25] Tregcells secrete TGF-120573 and IL-10 which inhibit the differenti-ation and function of Th17 cells In the infection processespecially in later stages Treg cells can regulate and controlinflammatory reactions to prevent autoimmune injury

In healthy individuals the ratios of Th1Th2 cells andTh17Treg cells [26 27] are in a dynamic balance We foundthat IAV (FM1 strain) infection could activate the immunesystem increase the ratios of Th1Th2 cells and Th17Tregcells and lead to an increased proinflammatory responseTheratios ofTh1Th2 cells andTh17Treg cells in the virus controlgroup were significantly higher than those in the normalcontrol group suggesting that IAV infection could promotethe differentiation of CD4+ T cells toTh1 cells andTh17 cells

TCM has been employed for thousands of years [28]Studies have shown that Gui Zhi Ma Huang Ge Ban Tang[29] has effects on infection of the upper respiratory tractpneumonia fever urticaria allergic purpura and severepsoriasis Guizhi [30] and Ephedra have anti-inflammatoryantiviral antitumor antibacterial and immunoregulatoryeffects Ginger [31] has anti-inflammatory antimicrobial

and antioxidant effects Ginger contains gingerol whichcan inhibit production of nitric oxide IL-6 and TNF inglial cells Peony [32] Guizhi [33] and ginger have anti-inflammatory effects and can reduce expression of PGE2NF-120581B and other mediators of the inflammatory responseThe chemical constituents in Gui-Zhi-Tang [34] were studiedby rapid resolution liquid chromatography quadrupole time-of-flight mass spectrometry combined with rapid resolutionliquid chromatography-diode array detector-ion trap massspectrometry and a total number of 187 compounds weredetected 3-phenyl-propenal is one of the principle com-pounds isolated from Guizhi 3-phenyl-propenal blocked theoverexpression of TLR3 TLR4 and their downstream signal-ing componentsMyD88 andTRAF-6 [35]There are apparentchanges not only in quantity but also in quality betweendecoction and its major constituted herbs Meanwhile anychanges in composition may lead to a change in efficacy [36]

5 Conclusions

We demonstrated that compared with yinqiaosan and xin-jiaxiangruyin Gui Zhi Ma Huang Ge Ban Tang had a goodtherapeutic effect on mice infected with IAV (FM1 strain)especially in the cold environment Gui Zhi Ma Huang Ge


th17 cells(CD4+IL-17+)

treg cells (CD25+FoxP3+)

th1 cells(CD4+IFN-gamma)


cd4+ cells

Lymphocytes gating

105

104

103

0minus103

105

104

103

0

minus103

Com

p-pE

-Cy5

-A

pE-

Cy5-

A

250K

200K

150K

100K

50K

0

25

0K

200

K

150

K

100

K

50

K0

SSC-

A

FSC-A

250K

200K

150K

100K

50K

0

SSC-

A

Comp-PE-A PE-A

Com

p-A

PC-A

A

PC-A

105

104

1030minus10

3

105

104

103

0

minus103

Comp-PE-A PE-A

Com

p-A

PC-A

A

PC-A

105

104

103

0minus103

105

104

103

0

minus103

Comp-PE-A PE-A

Com

p-FI

TC-A

F

ITC-

A

105

104

103

0minus103

Comp-PE-A PE-A

Comp-APC-A APC-A

105

104

103

0minus103

105

104

103

0

minus103

(a)

012345

th1

th2

in sp

leen

tiss

ue

ns



20∘C-WT 6


lowastlowast lowastlowast



lowast lowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

(b)

00

02

04

06

th17

treg

in sp

leen

tiss

ue



20∘C-WT 6




lowastlowastlowastlowastlowastlowastlowastlowastlowast


lowastlowastlowastlowastlowastlowastlowast

(c)

Figure 8 Flow cytometry of CD4+ T-cell subsets (a) Changes in the proportions of Th1Th2 cells (b) and Th17Treg cells (c) lowast119901 lt 005lowastlowast119901 lt 001 lowastlowastlowast119901 lt 0001 ns 119901 gt 005


Ban Tang could reduce lung inflammation in mice signifi-cantly and elicit an anti-influenza effect by downregulatingexpression of the key factors of the TLR7 signaling pathway

Conflicts of Interest

The authors declare that there are no financial and commer-cial conflicts of interest

Authorsrsquo Contributions

Hong-Qiong Qin and Shan-Shan Shi contributed equally tothis work

Acknowledgments

The authors thank Animal Experimental Center (Jinan Uni-versity) for providing experimental placeThis work was sup-ported by the National Natural Science Foundation of China[Grant nos 81473454 81473557] the Natural Science Foun-dation of Guangdong China [Grant no 2015A030313331]Science and Technology Project of Guangdong Province(2013B021800114) and Science and Technology Project ofZhuHai City (2013D0404990017)

References

[1] C Panayiotou J Richter S Bashiardes D Koptides C Try-fonos andC Christodoulou ldquoMolecular epidemiology of influ-enza A virus infection in Cyprus in four consecutive seasons(2009 pandemic-2013)rdquoEpidemiology and Infection vol 142 no5 pp 975ndash983 2014

[2] S S Chaves A Perez M M Farley et al ldquoThe burden ofinfluenza hospitalizations in infants from 2003 to 2012 UnitedStatesrdquoThePediatric Infectious Disease Journal vol 33 no 9 pp912ndash919 2014

[3] L Greiff A Cervin C Ahlstrom-Emanuelsson et al ldquoRepeatedintranasal TLR7 stimulation reduces allergen responsiveness inallergic rhinitisrdquo Respiratory Research vol 13 article no 532012

[4] S Harsini M Beigy M Akhavan-Sabbagh and N RezaeildquoToll-like receptors in lymphoid malignancies Double-edgedswordrdquo Critical Review in OncologyHematology vol 89 no 2pp 262ndash283 2014

[5] T Kawai and S Akira ldquoAntiviral signaling through patternrecognition receptorsrdquoThe Journal of Biochemistry vol 141 no2 pp 137ndash145 2007

[6] S-H Liao Y Li Y-N Lai N Liu F-X Zhang and P-PXu ldquoRibavirin attenuates the respiratory immune responses toinfluenza viral infection in micerdquo Archives of Virology vol 162no 6 pp 1661ndash1669 2017

[7] T Jefferson M Jones P Doshi E A Spencer I Onakpoyaand C J Heneghan ldquoOseltamivir for influenza in adults andchildren Systematic review of clinical study reports and sum-mary of regulatory commentsrdquo BMJ vol 348 Article ID g25452014

[8] C J Heneghan I OnakpoyaM A Jones et al ldquoNeuraminidaseinhibitors for influenza A systematic review and meta-analysisof regulatory andmortality datardquoHealth TechnologyAssessmentvol 20 no 42 pp 1ndash242 2016

[9] Y Itoh K Shinya M Kiso et al ldquoIn vitro and in vivo charac-terization of new swine-origin H1N1 influenza virusesrdquo Naturevol 460 no 7258 pp 1021ndash1025 2009

[10] Writing Committee of the WHO Consultation on ClinicalAspects of Pandemic (H1N1) 2009 Influenza ldquoClinical aspectsof pandemic 2009 influenza A (H1N1) virus infectionrdquoTheNewEngland Journal ofMedicine vol 362 no 18 pp 1708ndash1719 2010

[11] W He H Han W Wang and B Gao ldquoAnti-influenza viruseffect of aqueous extracts fromdandelionrdquoVirology Journal vol8 article no 538 2011

[12] Q Wan H Wang X Han et al ldquoBaicalin inhibits TLR7MYD88 signaling pathway activation to suppress lung inflam-mation in mice infected with influenza A virusrdquo BiomedicalReports vol 2 no 3 pp 437ndash441 2014

[13] A Boneparth W Huang R Bethunaickan et al ldquoTLR7 influ-ences germinal center selection in murine SLErdquo PLoS ONE vol10 no 3 Article ID e0119925 2015

[14] H Zhu X Shi D Ju H Huang W Wei and X Dong ldquoAnti-Inflammatory Effect of Thalidomide on H1N1 Influenza Virus-Induced Pulmonary Injury in Micerdquo Inflammation vol 37 no6 pp 2091ndash2098 2014

[15] Z Yunshi Y Jing Q Xian L Xing L Xieqin and F GanzhuldquoGeniposide demonstrates anti-inflammatory and antiviralactivity against pandemic AJiangsu12009 (H1N1) influenzavirus infection in vitro and in vivordquo Antiviral Therapy vol 22no 7 pp 599ndash611 2017

[16] C-W Yang and S-M Chen ldquoA comparative study of humanTLR 78 stimulatory trimer compositions in influenza a viralgenomesrdquo PLoS ONE vol 7 no 2 Article ID e30751 2012

[17] S Wu Z-Y Jiang Y-F Sun et al ldquoMicrobiota regulates theTLR7 signaling pathway against respiratory tract influenza avirus infectionrdquo Current Microbiology vol 67 no 4 pp 414ndash422 2013

[18] R A Benson J C Lawton and M K L MacLeod ldquoT cellresponse in the lung following influenza virus infectionrdquoMeth-ods in Molecular Biology vol 1591 pp 235ndash248 2017

[19] M Galgani V De Rosa and G Matarese ldquoT cell metabolismand susceptibility to autoimmune diseasesrdquo Molecular Immu-nology vol 68 no 2 pp 558ndash563 2015

[20] G H Zhao Y Q Fang U Ryan et al ldquoDynamics of Th17associating cytokines in Cryptosporidium parvum-infectedmicerdquo Parasitology Research vol 115 no 2 pp 879ndash887 2016

[21] M Huckans B E Fuller A L N Chalker M Adams andJ M Loftis ldquoPlasma inflammatory factors are associated withanxiety depression and cognitive problems in adults withand without methamphetamine dependence An exploratoryprotein array studyrdquo Frontiers in Psychiatry vol 6 article no178 2015

[22] T Nakahara M Kido-Nakahara F Ohno et al ldquoThe pru-ritogenic mediator endothelin-1 shifts the dendritic cell-T-cell response toward Th17Th1 polarizationrdquo Allergy EuropeanJournal of Allergy and Clinical Immunology 2017

[23] B Nedoszytko M Lange M Sokolowska-Wojdylo et al ldquoTherole of regulatory T cells and genes involved in their differenti-ation in pathogenesis of selected inflammatory and neoplasticskin diseases Part I Treg properties and functionsrdquo PostepyDermatologii i Alergologii vol 34 no 4 pp 285ndash294 2017

[24] Y Wang G Xu J Wang et al ldquoRelationship of Th17tregcells and radiation pneumonia in locally advanced esophagealcarcinomardquo Anticancer Reseach vol 37 no 8 pp 4643ndash46472017


[25] C Li A Sheng X Jia et al ldquoTh17Treg dysregulation in allergicasthmatic children is associated with elevated notch expres-sionrdquo Journal of Asthma amp Allergy Educators pp 1ndash7 2017

[26] LQKe FMWang andYC Luo ldquoEffects of vasoactive intesti-nal peptide on airway inflammation and Th17Treg balance inasthmatic micerdquo Zhongguo Dang Dai Er Ke Za Zhi vol 19 pp699ndash704 2017

[27] Q Liu J Wang Y Ma and L Gu ldquoA research on the influenceof two herbal concoctions on Toll-like receptor signal pathwaysof influenza virus induced pneumonia in micerdquo Zhonghua WeiZhong Bing Ji Jiu Yi Xue vol 26 no 5 pp 321ndash324 2014

[28] N-D Zhang T Han and B-K Huang ldquoTraditional Chi-nese Medicine formulas for the treatment of osteoporosisimplication for antiosteoporotic drug discoveryrdquo Journal ofEthnopharmacology vol 189 pp 61ndash80 2016

[29] Z-Z Zhang X-Z Zhang N Li et al ldquoStudy on anti-inflam-mation effect and involvedmechanism of Guizhi Fuling capsuleand its active complexrdquo Zhongguo Zhong yao za zhi = Zhongguozhongyao zazhi = China journal of Chinese materia medica vol40 no 6 pp 993ndash998 2015

[30] R B Semwal D K Semwal S Combrinck and A M ViljoenldquoGingerols and shogaols Important nutraceutical principlesfrom gingerrdquo Phytochemistry vol 117 Article ID 11221 pp 554ndash568 2015

[31] D Wang W Wang Y Zhou et al ldquoStudies on the regulatoryeffect of peony-glycyrrhiza decoction onprolactin hyperactivityand underlying mechanism in hyperprolactinemia rat modelrdquoNeuroscience Letters vol 606 pp 60ndash65 2015

[32] L Kishore N Kaur and R Singh ldquoNephroprotective effect ofPaeonia emodi via inhibition of advanced glycation end pro-ducts and oxidative stress in streptozotocinndashnicotinamideinduced diabetic nephropathyrdquo Journal of Food andDrugAnaly-sis vol 25 no 3 pp 576ndash588 2017

[33] H-H Chen P Zhao J Tian et al ldquoTheEffects ofGuizhiGancaoDecoction on Pressure Overload-Induced Heart Failure andPosttranslational Modifications of Tubulin in Micerdquo Evidence-Based Complementary and Alternative Medicine vol 2017Article ID 2915247 2017

[34] S Wang L Chen J Leng P Chen X Fan and Y ChengldquoFragment ion diagnostic strategies for the comprehensiveidentification of chemical profile ofGui-Zhi-Tang by integratinghigh-resolution MS multiple-stage MS and UV informationrdquoJournal of Pharmaceutical and Biomedical Analysis vol 98 pp22ndash35 2014

[35] B-S Zhao H-R Huo Y-Y Ma et al ldquoEffects of 3-phenyl-propenal on the expression of toll-like receptors and down-stream signaling components on raw2647 murine macro-phagesrdquo American Journal of Chinese Medicine vol 36 no 1pp 159ndash169 2008

[36] S-Y Liang X-M Tan F-L Chen and J-B Luo ldquoComparativeanalysis of volatile oils of Mahuang Fuzi Xixin decoctionand its major constituted herbs by GC-MSrdquo Zhong yao cai =Zhongyaocai = Journal of Chinese medicinal materials vol 34no 6 pp 896ndash901 2011

Stem Cells International

Hindawiwwwhindawicom Volume 2018


MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine


Behavioural Neurology

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom


viral infectious diseases such as influenza acute tonsillitispharyngitis measles and mumps Xinjiaxiangruyin is arepresentative medicine of qushijiebiao which has the effectof relieving ldquoheatrdquo and ldquodampnessrdquo and is usedmainly to treatsummer colds This recipe has a very good therapeutic effectfor damp-heating flu Gui Zhi Ma Huang Ge Ban Tang is arepresentative medicine of xinwenjiebiao with anti-influenzavirus and anti-inflammatory effects It also has antipyreticanalgesic and other pharmacologic effects and has been usedto treat colds respiratory-tract infections and allergies How-ever studies focusing on the antivirus and anti-inflammatorymechanisms of Gui Zhi Ma Huang Ge Ban Tang are lacking

Wan and colleagues [12] demonstrated that baicalininhibits TLR7MYD88 signaling pathway activation to sup-press lung inflammation in mice infected with influenzaA virus Hence using yinqiaosan and xinjiaxiangruyin ascontrols we investigated the effects of Gui Zhi Ma Huang GeBan Tang on the TLR7 signaling pathway and on the immunebalance of T cells in mouse lungs infected with the IAV in acold environment

2 Materials and Methods

21 Animals and Grouping Experiments were undertakenunder the supervision and assessment of the LaboratoryAnimal Ethics Committee of Jinan University (Jinan Univer-sity Guangzhou China) All experimental procedures werecarried out in accordance with the Guide for the Care andUse of Laboratory Animals (ninth edition National Institutesof Health Bethesda MD USA) and were approved by theAnimal Ethics Committee of Jinan University

Female specific pathogen-free (SPF) C57BL6j mice (20 plusmn1 g) were purchased from the Guangdong Experimental Ani-mal Center (Guangdong China) TLR7 knockout (TLR7minusminus)mice were obtained from Taconic Farms (Germantown NYUSA) TLR7minusminus mice were over 10 generations

Thirty-six female SPF C57BL6j mice (6∘C wild type(WT)) and 36 female SPF TLR7minusminusmice (6∘C TLR7minusminus) weremaintained in a cold room (6 plusmn 1∘C) with humidity of 50and light intensity of 3000 Lx and 0 Lxunder a 12-h lightndashdarkcycle A control group of 36 female SPFC57BL6jmice (20∘CWT) were maintained in a normal environment (20 plusmn 1∘Chumidity of 50 plusmn 5 light intensity of 3000 Lx and 0 Lxunder a 12-h lightndashdark cycle)

Mice were divided randomly into six groups with six ani-mals in each group normal control IAV control (ldquovirus con-trolrdquo) IAV + oseltamivir (ldquooseltamivirrdquo) IAV + yinqiaosan(ldquoyinqiaosanrdquo) IAV + Gui Zhi Ma Huang Ge Ban Tang(ldquoGui Zhi Ma Huang Ge Ban Tangrdquo) IAV + xinjiaxiangruyin(ldquoXinjiaxiangruyinrdquo)

22 IAV and Drugs IAV [FM1147 strain (mouse adapted)]was used for all experiments and grown in the allantoiccavities of 10-11-day-old fertile chicken eggs for 2 days at35∘C Yinqiaosan Gui Zhi Ma Huang Ge Ban Tang andxinjiaxiangruyin were purchased from the First AffiliatedHospital of Jinan University

Yinqiaosan [Detailed Analysis of Epidemic Warm Dis-eases TangWu 1798] contained Forsythia (15 g) honeysuckle

(15 g) Campanulaceae (9 g) mint (9 g) bamboo leaves (6 g)raw licorice (5 g) Nepeta (6 g) dandouchi (6 g) burdock(6 g) and reed rhizome (10 g)

Gui Zhi Ma Huang Ge Ban Tang [Typhoid miscellaneousdiseases theory Zhong-Jing Zhang about 219] containedEphedra Tang [Ephedra (9 g) Guizhi (6 g) almonds (9 g) andZhigancao (6 g)] and Guizhi Tang [Guizhi (9 g) peony (9 g)licorice (6 g) ginger (9 g) and jujube (10 g)]

Xinjiaxiangruyin [Detailed Analysis of Epidemic WarmDiseases Tang Wu 1798] contained Elsholtzia (6 g) honey-suckle (9 g) lentils (9 g)Magnolia (6 g) and Forsythia (6 g)

Oseltamivir phosphate (75mg) was obtained fromYichang Changjiang Pharmaceuticals (Hubei China)

23 Creation of an Influenza Model TLR7minusminus mice and WTC57BL6j mice were infected with the IAV (FM1 strain) onday 11 Mice in the virus control oseltamivir yinqiaosan GuiZhi Ma Huang Ge Ban Tang and xinjiaxiangruyin groupswere anesthetized using chloral hydrate and then infected byintranasal application of 40 LD50 (Half the lethal dose)This procedure leads to infection in the upper and lowerrespiratory tracts Mice in the normal control group wereanesthetized with chloral hydrate and then given an identicalamount of distilled water The physical status and weight ofmice were recorded daily

24 Anti-IAV Experiments The drug dose was calculatedbased on body weight differences between humans andmice The value for yinqiaosan was 112 gmL body weight0940 gmL for Gui Zhi Ma Huang Ge Ban Tang 0465 gmLfor xinjiaxiangruyin and 3mgmL for oseltamivir respec-tively Mice in the normal control group and virus controlgroup were given physiologic (09) saline instead Thecorresponding drug concentration (or 09 saline) wasadministered (02mLday ig) to each mouse 24 h after viruschallenge for 5 days (day 12 to day 16) (Figure 1)

25 Tissue Harvesting Six days after infection (day 17) afterdocumenting the body weight mice were anesthetized andsacrificed The spleen and lung were harvested The lungweight was obtainedThen the lung weightbody weight per-centage was calculated

26 Histopathology Lung tissue was fixed with 4 para-formaldehyde and then trimmed by a scalpel The appropri-ate lung portion was selected and rinsed dehydrated andtreated by a transparent agent xylene immersing After thesections had been dried and dewaxed they were stained withhematoxylin and eosin The tissues were sectioned and theorganization of tissue was observed at times100 magnification

27 Quantitative Real-Time Reverse Transcription-PolymeraseChain Reaction (RT-qPCR) of TLR7 MyD88 and NF-120581Bp65 mRNA and Relative Replication of IAV in Lung TissueTotal RNA was extracted with RNAiso Plus according tomanufacturer (TaKaRa Bio Shiga Japan) instructions cDNAsynthesis and real-time PCRs were carried out using a CFXConnect Real-Time PCR Detection system (Bio-Rad Labo-ratories Hercules CA USA) with PrimeScript RT Reagent


0

FM1 treated

G avage 02 mloneday

11 12 17

Harvest

16

20∘C-WT

6∘C-WT






















00

01

02

03

04

Body

wei

ght l

oss p

erce

nt

ns nsns

nsns

ns

20∘C-WT 6







3 Results





0000

0005

0010

0015

0020

0025



Lung

Bod

y w

eigh

t (g

g)

ns ns

nsns

nsns

20∘C-WT 6



lowastlowastlowast

lowastlowastlowast







(a) (b) (c) (d) (e) (f)

20∘C WT

6∘C WT


(a) (b) (c) (d) (e) (f)

(a) (b) (c) (d) (e) (f)


0

500

1000

1500

2000

Relat

ive m

RNA

expr

essio

nof

FM

1

nsns ns

nsnsns



20∘C-WT 6





lowast








0

2

4

6

8

Relat

ive m

RNA

expr

essio

nof

TLR

7ns ns

ns



20∘C-WT 6

∘C-WT

lowastlowastlowast

lowastlowastlowast


lowastlowast

lowastlowast

(a)

0

1

2

3

Relat

ive m

RNA

expr

essio

nof

MyD

88

ns ns nsns

nsns



20∘C-WT 6



lowastlowastlowast


(b)

00

05

10

15

20

Relat

ive m

RNA

expr

essio

nof

NF-휅

Bp65

ns ns

ns

nsns

ns



20∘C-WT 6



lowastlowast


(c)


4 Discussion








TLR-7

MyD88

NF-휅B (p65)

GAPDH


20∘C WT 6


(a)

00

05

10

15

20

TLR7

Pro

tein

GA

PDH

Rat

io

ns ns

ns



20∘C-WT 6

∘C-WT

lowastlowastlowast




(b)

00

05

10

15

20

MyD

88 P

rote

in

GA

PDH

Rat

io

ns ns

ns

nsns

ns



20∘C-WT 6




(c)

00

05

10

15

NF-휅

B p6

5 Pr

otei

nG

APD

H R

atio

ns ns

nsns

nsns



20∘C-WT 6





(d)






5 Conclusions







cd4+ cells

Lymphocytes gating

105

104

103

0minus103

105

104

103

0

minus103

Com

p-pE

-Cy5

-A

pE-

Cy5-

A

250K

200K

150K

100K

50K

0

25

0K

200

K

150

K

100

K

50

K0

SSC-

A

FSC-A

250K

200K

150K

100K

50K

0

SSC-

A

Comp-PE-A PE-A

Com

p-A

PC-A

A

PC-A

105

104

1030minus10

3

105

104

103

0

minus103

Comp-PE-A PE-A

Com

p-A

PC-A

A

PC-A

105

104

103

0minus103

105

104

103

0

minus103

Comp-PE-A PE-A

Com

p-FI

TC-A

F

ITC-

A

105

104

103

0minus103

Comp-PE-A PE-A

Comp-APC-A APC-A

105

104

103

0minus103

105

104

103

0

minus103

(a)

012345

th1

th2

in sp

leen

tiss

ue

ns



20∘C-WT 6





lowast lowast


(b)

00

02

04

06

th17

treg

in sp

leen

tiss

ue



20∘C-WT 6







(c)








Acknowledgments


References










































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















0

FM1 treated

G avage 02 mloneday

11 12 17

Harvest

16

20∘C-WT

6∘C-WT






















00

01

02

03

04

Body

wei

ght l

oss p

erce

nt

ns nsns

nsns

ns

20∘C-WT 6







3 Results





0000

0005

0010

0015

0020

0025



Lung

Bod

y w

eigh

t (g

g)

ns ns

nsns

nsns

20∘C-WT 6



lowastlowastlowast

lowastlowastlowast







(a) (b) (c) (d) (e) (f)

20∘C WT

6∘C WT


(a) (b) (c) (d) (e) (f)

(a) (b) (c) (d) (e) (f)


0

500

1000

1500

2000

Relat

ive m

RNA

expr

essio

nof

FM

1

nsns ns

nsnsns



20∘C-WT 6





lowast








0

2

4

6

8

Relat

ive m

RNA

expr

essio

nof

TLR

7ns ns

ns



20∘C-WT 6

∘C-WT

lowastlowastlowast

lowastlowastlowast


lowastlowast

lowastlowast

(a)

0

1

2

3

Relat

ive m

RNA

expr

essio

nof

MyD

88

ns ns nsns

nsns



20∘C-WT 6



lowastlowastlowast


(b)

00

05

10

15

20

Relat

ive m

RNA

expr

essio

nof

NF-휅

Bp65

ns ns

ns

nsns

ns



20∘C-WT 6



lowastlowast


(c)


4 Discussion








TLR-7

MyD88

NF-휅B (p65)

GAPDH


20∘C WT 6


(a)

00

05

10

15

20

TLR7

Pro

tein

GA

PDH

Rat

io

ns ns

ns



20∘C-WT 6

∘C-WT

lowastlowastlowast




(b)

00

05

10

15

20

MyD

88 P

rote

in

GA

PDH

Rat

io

ns ns

ns

nsns

ns



20∘C-WT 6




(c)

00

05

10

15

NF-휅

B p6

5 Pr

otei

nG

APD

H R

atio

ns ns

nsns

nsns



20∘C-WT 6





(d)






5 Conclusions







cd4+ cells

Lymphocytes gating

105

104

103

0minus103

105

104

103

0

minus103

Com

p-pE

-Cy5

-A

pE-

Cy5-

A

250K

200K

150K

100K

50K

0

25

0K

200

K

150

K

100

K

50

K0

SSC-

A

FSC-A

250K

200K

150K

100K

50K

0

SSC-

A

Comp-PE-A PE-A

Com

p-A

PC-A

A

PC-A

105

104

1030minus10

3

105

104

103

0

minus103

Comp-PE-A PE-A

Com

p-A

PC-A

A

PC-A

105

104

103

0minus103

105

104

103

0

minus103

Comp-PE-A PE-A

Com

p-FI

TC-A

F

ITC-

A

105

104

103

0minus103

Comp-PE-A PE-A

Comp-APC-A APC-A

105

104

103

0minus103

105

104

103

0

minus103

(a)

012345

th1

th2

in sp

leen

tiss

ue

ns



20∘C-WT 6





lowast lowast


(b)

00

02

04

06

th17

treg

in sp

leen

tiss

ue



20∘C-WT 6







(c)








Acknowledgments


References










































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















00

01

02

03

04

Body

wei

ght l

oss p

erce

nt

ns nsns

nsns

ns

20∘C-WT 6







3 Results





0000

0005

0010

0015

0020

0025



Lung

Bod

y w

eigh

t (g

g)

ns ns

nsns

nsns

20∘C-WT 6



lowastlowastlowast

lowastlowastlowast







(a) (b) (c) (d) (e) (f)

20∘C WT

6∘C WT


(a) (b) (c) (d) (e) (f)

(a) (b) (c) (d) (e) (f)


0

500

1000

1500

2000

Relat

ive m

RNA

expr

essio

nof

FM

1

nsns ns

nsnsns



20∘C-WT 6





lowast








0

2

4

6

8

Relat

ive m

RNA

expr

essio

nof

TLR

7ns ns

ns



20∘C-WT 6

∘C-WT

lowastlowastlowast

lowastlowastlowast


lowastlowast

lowastlowast

(a)

0

1

2

3

Relat

ive m

RNA

expr

essio

nof

MyD

88

ns ns nsns

nsns



20∘C-WT 6



lowastlowastlowast


(b)

00

05

10

15

20

Relat

ive m

RNA

expr

essio

nof

NF-휅

Bp65

ns ns

ns

nsns

ns



20∘C-WT 6



lowastlowast


(c)


4 Discussion








TLR-7

MyD88

NF-휅B (p65)

GAPDH


20∘C WT 6


(a)

00

05

10

15

20

TLR7

Pro

tein

GA

PDH

Rat

io

ns ns

ns



20∘C-WT 6

∘C-WT

lowastlowastlowast




(b)

00

05

10

15

20

MyD

88 P

rote

in

GA

PDH

Rat

io

ns ns

ns

nsns

ns



20∘C-WT 6




(c)

00

05

10

15

NF-휅

B p6

5 Pr

otei

nG

APD

H R

atio

ns ns

nsns

nsns



20∘C-WT 6





(d)






5 Conclusions







cd4+ cells

Lymphocytes gating

105

104

103

0minus103

105

104

103

0

minus103

Com

p-pE

-Cy5

-A

pE-

Cy5-

A

250K

200K

150K

100K

50K

0

25

0K

200

K

150

K

100

K

50

K0

SSC-

A

FSC-A

250K

200K

150K

100K

50K

0

SSC-

A

Comp-PE-A PE-A

Com

p-A

PC-A

A

PC-A

105

104

1030minus10

3

105

104

103

0

minus103

Comp-PE-A PE-A

Com

p-A

PC-A

A

PC-A

105

104

103

0minus103

105

104

103

0

minus103

Comp-PE-A PE-A

Com

p-FI

TC-A

F

ITC-

A

105

104

103

0minus103

Comp-PE-A PE-A

Comp-APC-A APC-A

105

104

103

0minus103

105

104

103

0

minus103

(a)

012345

th1

th2

in sp

leen

tiss

ue

ns



20∘C-WT 6





lowast lowast


(b)

00

02

04

06

th17

treg

in sp

leen

tiss

ue



20∘C-WT 6







(c)








Acknowledgments


References










































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















(a) (b) (c) (d) (e) (f)

20∘C WT

6∘C WT


(a) (b) (c) (d) (e) (f)

(a) (b) (c) (d) (e) (f)


0

500

1000

1500

2000

Relat

ive m

RNA

expr

essio

nof

FM

1

nsns ns

nsnsns



20∘C-WT 6





lowast








0

2

4

6

8

Relat

ive m

RNA

expr

essio

nof

TLR

7ns ns

ns



20∘C-WT 6

∘C-WT

lowastlowastlowast

lowastlowastlowast


lowastlowast

lowastlowast

(a)

0

1

2

3

Relat

ive m

RNA

expr

essio

nof

MyD

88

ns ns nsns

nsns



20∘C-WT 6



lowastlowastlowast


(b)

00

05

10

15

20

Relat

ive m

RNA

expr

essio

nof

NF-휅

Bp65

ns ns

ns

nsns

ns



20∘C-WT 6



lowastlowast


(c)


4 Discussion








TLR-7

MyD88

NF-휅B (p65)

GAPDH


20∘C WT 6


(a)

00

05

10

15

20

TLR7

Pro

tein

GA

PDH

Rat

io

ns ns

ns



20∘C-WT 6

∘C-WT

lowastlowastlowast




(b)

00

05

10

15

20

MyD

88 P

rote

in

GA

PDH

Rat

io

ns ns

ns

nsns

ns



20∘C-WT 6




(c)

00

05

10

15

NF-휅

B p6

5 Pr

otei

nG

APD

H R

atio

ns ns

nsns

nsns



20∘C-WT 6





(d)






5 Conclusions







cd4+ cells

Lymphocytes gating

105

104

103

0minus103

105

104

103

0

minus103

Com

p-pE

-Cy5

-A

pE-

Cy5-

A

250K

200K

150K

100K

50K

0

25

0K

200

K

150

K

100

K

50

K0

SSC-

A

FSC-A

250K

200K

150K

100K

50K

0

SSC-

A

Comp-PE-A PE-A

Com

p-A

PC-A

A

PC-A

105

104

1030minus10

3

105

104

103

0

minus103

Comp-PE-A PE-A

Com

p-A

PC-A

A

PC-A

105

104

103

0minus103

105

104

103

0

minus103

Comp-PE-A PE-A

Com

p-FI

TC-A

F

ITC-

A

105

104

103

0minus103

Comp-PE-A PE-A

Comp-APC-A APC-A

105

104

103

0minus103

105

104

103

0

minus103

(a)

012345

th1

th2

in sp

leen

tiss

ue

ns



20∘C-WT 6





lowast lowast


(b)

00

02

04

06

th17

treg

in sp

leen

tiss

ue



20∘C-WT 6







(c)








Acknowledgments


References










































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















0

2

4

6

8

Relat

ive m

RNA

expr

essio

nof

TLR

7ns ns

ns



20∘C-WT 6

∘C-WT

lowastlowastlowast

lowastlowastlowast


lowastlowast

lowastlowast

(a)

0

1

2

3

Relat

ive m

RNA

expr

essio

nof

MyD

88

ns ns nsns

nsns



20∘C-WT 6



lowastlowastlowast


(b)

00

05

10

15

20

Relat

ive m

RNA

expr

essio

nof

NF-휅

Bp65

ns ns

ns

nsns

ns



20∘C-WT 6



lowastlowast


(c)


4 Discussion








TLR-7

MyD88

NF-휅B (p65)

GAPDH


20∘C WT 6


(a)

00

05

10

15

20

TLR7

Pro

tein

GA

PDH

Rat

io

ns ns

ns



20∘C-WT 6

∘C-WT

lowastlowastlowast




(b)

00

05

10

15

20

MyD

88 P

rote

in

GA

PDH

Rat

io

ns ns

ns

nsns

ns



20∘C-WT 6




(c)

00

05

10

15

NF-휅

B p6

5 Pr

otei

nG

APD

H R

atio

ns ns

nsns

nsns



20∘C-WT 6





(d)






5 Conclusions







cd4+ cells

Lymphocytes gating

105

104

103

0minus103

105

104

103

0

minus103

Com

p-pE

-Cy5

-A

pE-

Cy5-

A

250K

200K

150K

100K

50K

0

25

0K

200

K

150

K

100

K

50

K0

SSC-

A

FSC-A

250K

200K

150K

100K

50K

0

SSC-

A

Comp-PE-A PE-A

Com

p-A

PC-A

A

PC-A

105

104

1030minus10

3

105

104

103

0

minus103

Comp-PE-A PE-A

Com

p-A

PC-A

A

PC-A

105

104

103

0minus103

105

104

103

0

minus103

Comp-PE-A PE-A

Com

p-FI

TC-A

F

ITC-

A

105

104

103

0minus103

Comp-PE-A PE-A

Comp-APC-A APC-A

105

104

103

0minus103

105

104

103

0

minus103

(a)

012345

th1

th2

in sp

leen

tiss

ue

ns



20∘C-WT 6





lowast lowast


(b)

00

02

04

06

th17

treg

in sp

leen

tiss

ue



20∘C-WT 6







(c)








Acknowledgments


References










































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















TLR-7

MyD88

NF-휅B (p65)

GAPDH


20∘C WT 6


(a)

00

05

10

15

20

TLR7

Pro

tein

GA

PDH

Rat

io

ns ns

ns



20∘C-WT 6

∘C-WT

lowastlowastlowast




(b)

00

05

10

15

20

MyD

88 P

rote

in

GA

PDH

Rat

io

ns ns

ns

nsns

ns



20∘C-WT 6




(c)

00

05

10

15

NF-휅

B p6

5 Pr

otei

nG

APD

H R

atio

ns ns

nsns

nsns



20∘C-WT 6





(d)






5 Conclusions







cd4+ cells

Lymphocytes gating

105

104

103

0minus103

105

104

103

0

minus103

Com

p-pE

-Cy5

-A

pE-

Cy5-

A

250K

200K

150K

100K

50K

0

25

0K

200

K

150

K

100

K

50

K0

SSC-

A

FSC-A

250K

200K

150K

100K

50K

0

SSC-

A

Comp-PE-A PE-A

Com

p-A

PC-A

A

PC-A

105

104

1030minus10

3

105

104

103

0

minus103

Comp-PE-A PE-A

Com

p-A

PC-A

A

PC-A

105

104

103

0minus103

105

104

103

0

minus103

Comp-PE-A PE-A

Com

p-FI

TC-A

F

ITC-

A

105

104

103

0minus103

Comp-PE-A PE-A

Comp-APC-A APC-A

105

104

103

0minus103

105

104

103

0

minus103

(a)

012345

th1

th2

in sp

leen

tiss

ue

ns



20∘C-WT 6





lowast lowast


(b)

00

02

04

06

th17

treg

in sp

leen

tiss

ue



20∘C-WT 6







(c)








Acknowledgments


References










































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of
























cd4+ cells

Lymphocytes gating

105

104

103

0minus103

105

104

103

0

minus103

Com

p-pE

-Cy5

-A

pE-

Cy5-

A

250K

200K

150K

100K

50K

0

25

0K

200

K

150

K

100

K

50

K0

SSC-

A

FSC-A

250K

200K

150K

100K

50K

0

SSC-

A

Comp-PE-A PE-A

Com

p-A

PC-A

A

PC-A

105

104

1030minus10

3

105

104

103

0

minus103

Comp-PE-A PE-A

Com

p-A

PC-A

A

PC-A

105

104

103

0minus103

105

104

103

0

minus103

Comp-PE-A PE-A

Com

p-FI

TC-A

F

ITC-

A

105

104

103

0minus103

Comp-PE-A PE-A

Comp-APC-A APC-A

105

104

103

0minus103

105

104

103

0

minus103

(a)

012345

th1

th2

in sp

leen

tiss

ue

ns



20∘C-WT 6





lowast lowast


(b)

00

02

04

06

th17

treg

in sp

leen

tiss

ue



20∘C-WT 6







(c)








Acknowledgments


References










































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of

























Acknowledgments


References










































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of























of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



















Documents

Effects of Gui Zhi Ma Huang Ge Ban Tang on the TLR7