Study On The Activity And Mechanism Of Qingwen Jiere Mixture Against Influenza A Virus

Influenza is an acute respiratory infectious disease caused by influenza virus infection.The World Health Organization estimates that nearly 10%of the global population is infected with this virus each year,resulting in 200,000 to 500,000 deaths.Among the four influenza viruses that have been discovered,influenza A virus（IAV）is one of the most important pathogens clinically.Since the 20th century,there have been several IAV pandemics,such as the"Spanish flu"in 1918 and the"swine flu"in2009,which posed a great threat to public health.Studies have shown that IAV infection can activate signaling pathways such as TLRs/NF-κB,induce large expressions of inflammatory factors and even lead to cytokine storms,which may be one of the pathogenic mechanisms of severe pneumonia caused by IAV infection.Antiviral drugs are effective means to combat influenza virus infection.M2 ion channel inhibitors represented by amantadane,NA inhibitors（NAIs）represented by oseltamivir and RNA polymerase inhibitors represented by baloxavir marboxil are small molecule targeted drugs widely used in influenza treatment.They can improve symptoms and outcomes in patients infected with influenza virus such as H1N1,H5N1 and H7N9.However,a growing number of studies have shown that multiple circulating strains are resistant to these drugs,and a large number of patients still suffer from severe sequelae and even death.Traditional Chinese medicine（TCM）has been used to treat influenza for thousands of years in China,such as Yinqiao Powder and Mayxingshigan Decoction.They have many ingredients,numerous targets and are not easy to develop drug resistance.However,the mechanism of action of most antiviral Chinese medicines remains unclear.Qingwenjiere Mixture（QJM）is a TCM mixture prepared by Wen Weibo,president of Yunnan Hospital of Traditional Chinese Medicine,based on the"plague prevention theory".It has good clinical therapeutic effect on patients with COVID-19 and influenza.Prvious studies found that QJM has broad-spectrum antiviral activity and anti-inflammatory activity against SARS-Co V-2and other three human coronaviruses.However,the therapeutic mechanism of QJM against IAV remains unclear.The purpose of this study was to clarify the activity and mechanism of QJM against IAV in vivo and in vitro,which would provide theoretical reference for the clinical application of QJM in the treatment of influenza.Firstly,the anti-IAV effect of QJM was studied in vitro.The canine renal cell line（MDCK）models infected with influenza A viruses A/PR/8/34（H1N1）（PR8）and A/duck/Hong Kong/Y280/97（H9N2）（Y280）were established.The virus infection caused the cells to become round and wrinkled,and the virus replication level increased with time.Lianhua Qingwen（LH）extract and Oseltamivir Phosphate（OSE）were used as positive control drugs.Half cytotoxic concentration(CC₅₀),half inhibitory concentration(IC₅₀)and drug selection index（SI）of QJM,LH and OSE in MDCK cells were detected.The CC₅₀values of QJM on MDCK were 6.33±0.25mg/m L,the IC₅₀values of QJM on PR8 and Y280 were 0.33±0.009 mg/m L and0.65±0.007 mg/m L,respectively,and the SI values were 19±1.26 and 9.6±0.51,respectively.These results indicated that QJM had low toxicity and high efficiency to IAV.QJM inhibited the proliferation of IAV by plaque test and hemagglutination inhibition test.In order to clarify the stage of action of QJM,2 mg/m L of this drug was administered before infection and at 0,2,4,6 and 8 h after infection.Virus titer detection showed that QJM mainly acted on the early stage of virus replication（0-4 h）.At the early stage of IAV replication,nucleoprotein（NP）packages are required for positive RNA to synthesize new v RNAs,so the expression and subcellular localization of NP is analyzed at 6 hpi and 8 hpi.In virus-infected cells,NP is highly expressed in the nucleus and cytoplasm.After the treatment of QJM,the expression of NP is decreased and its protein is restricted in the nucleus,suggesting that QJM can inhibit the nuclear output of NP protein.In addition,real-time quantitative fluorescent PCR（q PCR）showed that viral infection up-regulated the m RNA expression levels of inflammatory factors such as TNF-α,IL-6,IL-1α,IFN-γ,TRAIL and RANTES,while QJM treatment significantly inhibited the expression of these inflammatory factors（P<0.05）.These results suggest that QJM has anti-i AV and anti-inflammatory activity at the cellular level.Secondly,the efficacy of QJM against IAV in vivo was evaluated using an influenza mouse model.The LD₅₀of PR8 strain was 103PFU/m L in BALB/c mice infected by nasal drops.All the mice died at 8 dpi after infection with 10LD₅₀,and severe pulmonary lesions were observed.The virus titer in lung tissues was about105TCID₅₀/0.1 m L.Through the death protection experiment,QJM high-dose group（17 g/kg/d）was found to significantly improve the survival rate of infected mice（71.25%,P<0.01）.Although QJM medium-dose（8.5 g/kg/d）and low-dose（4.25g/kg/d）treatment could not significantly improve the survival rate of mice,the survival time of mice was extended.The virus titer and lung index of lung tissue were detected at 6 dpi,and QJM high-dose group（17 g/kg/d）significantly inhibited virus replication level（P<0.01）and reduced lung index（P<0.05）.Further,QJM reduced the secretion of inflammatory factors including TNF-α,IL-6,TRAIL and IP-10,reduced the number of inflammatory cells and neutrophilic granulocyte infiltration in lung tissue,and improved the pathological features of pulmonary hemorrhage,emphysema and diffuse lung injury.These results proved that QJM has anti-H1N1virus and anti-inflammatory activity at the animal level.Finally,IAV infected A549 cell model was used to explore the anti-influenza mechanism of QJM.Since the activation of NF-κB signaling pathway mediated by pattern recognition receptors（PRRs）plays an essential role in expression of cytokines and chemokines,the m RNA expression levels TLR3,TLR7,RIG-1,LGP2 and NLRP3 were detected by q PCR.It was found that QJM inhibited IAV’s up-regulation of these PRRs m RNA in a dose-dependent manner,and the inhibitory effect of QJM on TLR7 was more obvious（P<0.001）.Western blotting assay showed that QJM inhibited the phosphorylation of NF-κB p65,suggesting that this drug may play an anti-IAV role by inhibiting the activation of NF-κB signaling pathway mediated by TLR7.The TLR7 inhibitor Hydroxychloroquine and the TRAF6,Phospho-IKK,Phospho-IκB,NF-κB dimer p65/p50inhibitors including 6877002,TPCA-1,BAY11-7082 and JSH-23 were used to explore the effect of QJM on the excessive production of inflammatory factors caused by IAV infection.It was found that TLR7/NF-κB signaling pathway significantly decreased the viral replication level and the expression of inflammatory factors,with TLR7 and Phospho-IKK inhibitors having the most significant effects（P<0.001;P<0.05）.To further verify the effect of QJM on the NF-κB pathway,NF-κB ws stimulated with Phorbol 12-myristate13-acetate（PMA）treatment which is an agonist of this transcription factor.It showed that QJM could significantly inhibit the m RNA expression levels of IL-6,TRAIL and TNF-αinduced by PMA treatment.These results suggest that QJM exerts anti-i AV and anti-inflammatory effects by inhibiting the activation of TLR7/NF-κB signaling pathway.In conclusion,this study demonstrated the anti-IAV and anti-inflammatory activity of QJM at both cellular and animal levels,and it may inhibit the occurrence of viral pneumonia by inhibiting the activation of TLR7/NF-κB signaling pathway.This study provides clues for the in-depth exploration of the anti-influenza mechanism of QJM,and provides theoretical reference for the clinical treatment of influenza with QJM and the development of Chinese herbal medicine resources in Yunnan.