Study On The Mechanism Of Influenza Virus Infection And The Screening Of Therapeutic Drugs

Objective: To study the pathogenesis of influenza virus infection by analyzing the sequence of mouse peripheral blood transcripts and provide the direction for the next step in drug screening;to screen and evaluate the in vivo activity of the candidate drugs and combination,and to obtain the potential for the treatment of influenza virus infection drug.Methods: 1.Pathogenicity of influenza virus infection: First,the establishment of influenza virus infection in mice model,the peripheral blood of mice was taken on the 1st,3rd and 5th day after infection,and the total RNA was extracted to construct c DNA library.The data of mouse peripheral blood transfection group were obtained by high-throughput sequencing,and the differentially expressed genes were obtained by bioinformatics method.Expression of gene functions and pathways were analyzed.2.Drug screening: Mice infected with influenza A virus as a drug screening model,The in vivo activity of 17 anti-inflammatory,anti-oxidative drugs and 21 drug combinations were screened by the clinical symptoms,weight changes,mean survival time and survival rate of mice.The overall protective effect of the tested drugs on infected mice was evaluated effect.3.Study on Anti-influenza Virus Activity of Chalcone Compounds L₂H₁₇: L₂H₁₇ set low,medium and high dose of three doses before the beginning of 24 hours of administration of oral administration,once a day,continuous administration of 6 days,observed to 14 days,the average weight of mice in each group changes,mortality,mean survival time,death protection rate and life extension rate,to observe the protective effect of L₂H₁₇ on influenza virus infected mice.According to the above method,mice lungs were taken on the 3rd and 5th day after modeling,calculate the lung index and lung index inhibition rate.Take part of the lung tissue soaked in 4% formaldehyde,for pathological tissue testing;another part of the lung tissue soaked in RNAstore,real-time quantitative PCR?q PCR?was used to detect the expression of viral M gene in lung tissue,and to explore the effect of L₂H₁₇ on lung injury induced by influenza virus infection and the decrease of lung virus titer.At the same time,mice blood were taken on the 3rd and 5th day after modeling,the levels of IL-6 and TNF-? in serum were measured by double antibody sandwich ELISA,and the effect of L₂H₁₇ on immune cytokines was investigated.Results: 1.Pathogenicity of influenza virus infection: Analysis of differential expression genes in peripheral blood transcriptions showed that: Compared with the normal group,110 genes were up-regulated at the same time on the 1st,3rd and 5th day after influenza virus infection,95 genes were down simultaneously,the difference was more than 2 times.The results of the classic pathway analysis showed that: IL-8 Signaling?Th1 Pathway?IL-6 Signaling?NF-k B Signaling?PI3K/AKT Signaling?Production of Nitric Oxide and Reactive?NRF2-mediated Oxidative Stress Response?Antioxidant Actin of Vitamin C etc.and immune,inflammation,oxidation and other related pathways in the virus infection were significantly affected.Disease and functional analysis showed that: The function of differentially expressed genes in virus-infected mice is more rich in hematopoietic system development,inflammatory response and immune-related functions,and these functions are in the active state,and the activation state gradually increases with the prolongation of time.2.Drug screening: After the mice infected with influenza virus,the mortality of the mice in the model group was 90% 100%,the mean survival time was 6 days;the survival rate of the chalcone compound L₂H₁₇ was increased to 22.2%,the mean survival time was prolonged to 8.13 days;the survival rate of the compound H33L42H3 was increased to 20%,the mean survival time was prolonged to 7.82 days,and the body weight began to rise on the 9th to 10 th day,and the overall protective effect on the influenza virus infected mice was further studied as the target compound.3.Study on Anti-influenza Virus Activity of Chalcone Compounds L₂H₁₇: The mice in the model group showed body tremors,shortness of breath,curled up and so on about the third day after virus infection.The weight of the mice in the model group dropped sharply,and the death rate was 100%.The average survival time was 8.7 days,compared with the normal control group,there was significant difference?P <0.01?.The mice in the administration group showed signs of shortness of breath,chills and colds,weight loss and so on,and died on the 6th day,on the 11 th days or so mental condition began to improve,basically no longer die,weight increased slowly.L2 middle dose group survival rate increased to 16.7%,the average survival time extended to 9.5 days,the survival rate of the high dose group was increased to 50% and the average survival time was prolonged to 10.8 days,which was statistically significant?P <0.01?compared with the model group.Lung index test results showed that: On the third day after infection,the lung index of the model group was 0.83,which was significantly higher than that of the normal group?0.6??P <0.01?,the lung index of middle dose group was 0.73,which was significantly lower than that of model group?P <0.05?,and the inhibition rate of lung index was 43.48%.On the fifth day after infection,the lung index of the model group increased to 0.90,which was significantly higher than that in the normal group?P <0.01?,and the lung index of the high dose group was reduced to 0.75?P <0.05?,and the inhibition rate of lung index was 48.39%.Pathological test showed that the lung tissue of the normal group was normal,the diffuse inflammatory cell infiltration of the lung tissue in the model group was significant,the inflammation of the bronchi and perivascular area was significant,the inflammation range was more than 30%,the middle and high dose group Inflammatory cell infiltration is limited to?fine?bronchial and small blood vessels around the inflammatory lesions relative to the limitations,compared with the model group was significantly improved.The results of real-time fluorescence quantitative PCR showed that the expression of M gene was not detected in the lung tissue of the normal group.The expression of M gene in the model group reached 4.41 and 10.91 at the 3rd and 5th day after infection,?P <0.01?.The relative expression of M gene was decreased to 5.76 on the fifth day after infection in the high dose group?P <0.01?,compared with the model group?P <0.01?.The results of cytokine IL-6 showed that: The levels of IL-6 in the serum of the model group were 245.34 and 262.24 at the 3rd and 5th day after model establishment,which was significantly higher than that of the normal group?167.56,79.81??P <0.01?.The levels of IL-6 in the L2 dose group decreased to 189.97 and 151.73 on the 3rd and 5th day after modeling,and decreased to 134.84 on the fifth day of the high dose group,the difference was significant?P <0.01?.The results of cytokine TNF-? showed that: The levels of TNF-? in the serum of the model group increased to 363.32 and 379.88 at the 3rd and 5th day after model establishment,which was significantly higher than that in the normal group?210.72,204.71??P <0.01?.The dosage of each dose group of L2 was lower than that of model group,but there was no significant difference?P> 0.05?.Conclusion: 1.Influenza virus infection in the body,causing the body and immune,inflammation,oxidation and other related functional gene expression levels change,suggesting that from the anti-inflammatory,anti-oxidation,improve the immune system to start screening for the treatment of influenza virus infection drugs.2.Screening of 38 drugs and combinations showed that chalcone compounds L₂H₁₇ and curcuminoid compounds H33L42H3 had a good overall protective effect on influenza virus-infected mice.3.L₂H₁₇ has a certain degree of death protection against influenza A virus infection in mice in a dose-dependent manner;can reduce lung injury caused by viral infection,reduce lung virus titer;at the same time it can reduce the infected mice serum inflammatory cytokines IL-6,TNF-? expression,it has a certain inhibitory effect on inflammation caused by influenza virus infection.