The Effect Of Shuqing Granule Against Influenza A Virus H1N1 Based On MAPK Signaling Pathway

Influenza is a pandemic disease which is caused by a global outbreak of influenza viruses that kills between 290 and 650,000 people each year.Influenza,caused several serious complications as pneumonia and ischemic heart disease,can significantly increase the hospitalization and mortality rates of the young and the old,resulting in a huge threat to human health.Influenza viruses are divided into Influenza virus A、B、C and D.Influenza A is the most important pathogen causing seasonal influenza transmission in humans.For the purpose of evading the host’s immune protection,the virus surface antigen has undergone regular mutations.Therefore,the genetic and antigen evolutionary characterization of influenza virus A are exceedingly preventive and therapeutic importance.The Chinese traditional medicine Shuqing Granule is collected by Pharmacopoeia,with its main ingredients Isatidis Folium、Mori Follum、licorice and hragmitis Rhizoma.It has the functions of clearing away heat and detoxifying,relieving cough and promoting lung function.Among them,Isatidis Folium、Mori Follum and licorice have been proved to be anti-influenza virus drugs.This article aims to explore the effect of Shuqing Granules on influenza virus infection and its relative molecular mechanisms.Firstly,this study predicted the relative targets and signaling pathways of Chinese traditional medicine Shuqing Granules by network pharmacology.The TCMSP database was used to filter the active compounds of Isatidis Folium、Mori Follum and licorice.The targets of influenza were searched through the TTD website.The disease-target-compounds network map was established by Cytoscape 3.7.0 software,and KEGG signal pathway enrichment analysis was performed via the DAVID database.The corresponding target genes were calculated according to the STRING database.The results showed that there were 549 active components in the Shuqing Granules and 10 target genes related to influenza.The enrichment results showed that the MAPK signaling pathway played a leading role in the treatment of Shuqing Granules,and the target genes include MAPK14、JUN and PI3RI,etc.Secondly,it is verified in cellular level that Shuqing Granules have a certain inhibitory effect on the influenza virus,it is possible to find out the optimal time and mode of action of the granules on influenza virus.Thirdly,the secretion of type Ⅰinterferon、the phosphorylation levels of MAPK proteins and the expression levels of interferon-stimulated gene(ISG)mRNA were determined via ELISA、PCR and Western blot to study the mechanism of drug anti-influenza virus.The results showed that Shuqing Granules(25.78mg/mL)got the TI value of 4.51 in the best therapeutic effect on influenza virus at 48 hours of action.Shuqing Granules can stimulate the virus-infected cells to secrete IFN-β.The MAPK pathway protein assay showed that virus could induce phosphorylation of ERK、p38 and JNK proteins,and the phosphorylation levels of ERK and p38 protein decreased significantly after drug treatment,while the phosphorylation level of JNK protein increased significantly.The virus can down-regulate the expression levels of ISGs PKR、MxA and OAS1 mRNA,while mRNA expression is significantly increased after drug action.It is speculated that Shuqing Granules may regulate the phosphorylation levels of MAPK proteins,induce the secretion of IFN-β and cause the expression of downstream antiviral proteins to inhibit influenza virus to a certain extent.