The Mechanism Of Anti-influenza Virus Of Coptidis Rhizoma-Magnoliae Officinalis Cortex Based On TLRs Signaling Pathway

Objective To research the effect of Coptidis Rhizoma cold-heat couplet Medicines of Coptidis Rhizoma-Magnoliae Officinalison,Coptidis Rhizoma-Perillae Folium,Coptidis Rhizoma-Zingiberis Rhizoma the resistance toinfluenza virus in vitro.On this basis,to furtherinvestigate the overall efficacy of Coptidis Rhizoma-Magnoliae Officinalison against influenza and the mechanism of regulating the TLRs signaling pathway,aiming at providing scientific basis for the clinical application of Coptidis Rhizoma cold-heat couplet Medicines.Methods 1.In order to investigate the toxicity and the effect of Coptidis Rhizoma couplet Medicinesof Coptidis Rhizoma-Magnoliae Officinalison,Coptidis Rhizoma-Perillae Folium,Coptidis Rhizoma-Zingiberis Rhizoma and its single medicines against influenza virus and screen better couplet medicines,the MTT assay was used to detecte its effects of activity of MDCK cells infected withinfluenza virus strain A/PR/8/34.2.The pneumonic model of mice was established by infection with the influenza virus strain A/PR/8/34.The mice were treated with Coptidis Rhizoma-Magnoliae Officinalis Cortex at high,medium and low doses,respectively,and set up Oseltamivir group,normal control group and virus model group.The mice infected with influenza virus in each group were sacrificed on the 5th day.(1)Picking blood from eyeball and separating serum,the levels of cytokines IL-2,IL-6,IFN-γ,TNF-α and the contents of NO,SOD and GSH in the serum of mice infected with influenza virus were detected by ELISA.(2)Lung tissues were taken and weighed,lung index index and lung index inhibition rate were calculated.The pathological changes of lung tissue in each group were recorded and fixed with 10% formalin solution.The pathological changes of lung tissues in each group were observed under light microscope after paraffin embedding and HE staining in routine sections.The spleens and thymus were removed and weighed,spleen index,thymus index and its inhibition rate of each group were calculated.(3)RT-PCR and Western Blot were used to detect the gene and protein expression levels of TLR3,TLR7,My D88,TRAF3 and NF-κBp65 in the lung tissues.Results 1.Coptidis Rhizoma cold-heat couplet Medicines of Coptidis Rhizoma-Magnoliae Officinalison Cortex,Coptidis Rhizoma-Perillae Folium,Coptidis Rhizoma-Zingiberis Rhizoma could significantly increase the activity of MDCK cells infected with H1N1 influenza virus and inhibited the proliferation of H1N1 influenza virus.The maximum non-toxic concentration of MDCK cells was less than Coptidis Rhizoma.2.Coptidis Rhizoma-Magnoliae Officinalis Cortex significantly reduced the lung index,increase the inhibition rate of lung index,and alleviate the pathological changes of pneumonia induced by influenza virus in mice.3.Coptidis Rhizoma-Magnoliae Officinalis Cortex(1)significantly reduced spleen index and thymus index;(2)significantly reduced the contents IL-6,IFN-γ,TNF-α and increased the content of IL-2;(3)reduced the content of NO level,and increased the contents of SOD and GSH in the serum of infected-mice with influenza virus.4.Coptidis Rhizoma-Magnoliae Officinalis Cortex significantly reduced the TLR3,TLR7,My D88,TRAF3 and NF-κBp65 genes and protein expression in the lung tissue of infected-mice with influenza virus.Conlcusion 1.Coptidis Rhizoma cold-heat couplet Medicines had inhibitory activities against influenza virus.Its inhibitory effects on influenza virus are as follows: Coptidis Rhizoma-Magnoliae Officinalison > Coptidis Rhizoma-Perillae Folium > Coptidis Rhizoma-Zingiberis Rhizoma.Coptidis Rhizoma cold-heat couplet Medicines could enhance the ability of its single medicines against H1N1 influenza virus.The cytotoxicity of Coptidis Rhizoma cold-heat couplet Medicines were less than Coptidis Rhizoma to MDCK cells.2.Coptidis Rhizoma-Magnoliae Officinalison Cortex had a significant effect against influenza virus in vivo.3.Coptidis Rhizoma-Magnoliae Officinalis Cortex may play an anti-influenza virus role by regulating the immune function and reducing oxidative stress injury and down-regulating the over-expressions of genes and proteins of TLRs signaling pathway.4.The results can provide a scientific basis for the clinical application and further preparations of Coptidis Rhizoma couplet Medicines,and also provide new research ideas and methods for the mechanism of traditional Chinese medicine against influenza virus.