Study On The Anti-influenza Effect Of Chlorogenic Acid Of Yinqiao Powder And Its Related Signaling Pathway Mechanism

ObjectiveTo study the anti-influenza effect of chlorogenic acid and its related cellular signaling mechanism in yinqiao powder.MethodsTo study on the anti-influenza effect of yinqiao powder.1.In vivo anti-influenza effect of the active components of yinqiao powder preparation.A drug for the screening of mouse influenza virus pneumonia was initially screened.Female Balb/c mice were subjected to interval grouping,and all mice were weighed,numbered according to the arrangement,and then randomly grouped.A blank control group,a virus model group,a ribavirin group(100 mg/kg),chlorogenic acid,perillaldehyde,luteolin,luteolin,diosgenin,diosgenin,valerin,vitexin Each group had 10 doses of 100 mg/kg and 0.5%Tween-80.Each group was slightly anesthetized with appropriate amount of ether,and nasal infection with influenza virus FM1 strain 5LD50 was given,4-6 drops per mouse,about 0.05ml.Start dosing 2 hours after the attack.A total of 6 days were given,once a day,by oral gavage at a dose of 0.1ml/10g.The virus model group was given an equal volume of 0.9%sodium chloride injection as the control.Through the method of measuring the anti-influenza virus drug efficacy in mice,the incidence of death in infected mice was taken as the efficacy index,and the effects of the above-mentioned Chinese herbal medicines on the mortality and body weight of mice infected with influenza A virus were observed.Chinese herbal medicines have a good anti-influenza virus effect.2.Anti-influenza mechanism of chlorogenic acid was studied based on antibody chip of cell signaling pathway.On the basis of the above experiments,the effective Chinese herbal active ingredient chlorogenic acid was selected,and the signal pathway target of antiviral effect and its action was further studied by Full MoonJ s signaling pathway phosphorylation antibody chip and chip kit.Follow the standard operating procedures of Full Moon.Sample processing:A.Sample protein extraction B.Sample protein labeling;C.chip detection operations.3.Study on the anti-influenza effect of chlorogenic acid and its mechanism based on cell signal transduction mechanism.Effects on 120 female Balb/c mice infected with influenza virus in vivo all mice were weighed,numbered and randomly assigned using interval grouping method.The normal control group,virus model group,ribavirin group(100mg/kg),chlorogenic acid high dose(200mg/kg),medium dose(100mg/kg)and low dose(50mg/kg)groups were set up.Each group was infected with 5 LD50 nasal drops(i.n.,Intranasal)of influenza virus FM1 strain under mild ether anesthesia,5 drops per mouse,about 0.05 ml.Start dosing 2 hours after attack.The drug was given once a day for 5 days by oral administration at a dose of 0.1ml/10g,and 0.9%sodium chloride injection of the same volume was given to the normal control group and the viral model control group as the control group.Between groups in virus infection after 5 days from mice in each group randomly selected 10 measuring only lung tissue observation lung lesions in mice lung index,observation,and separately antiviral effect(lung weight,lung disease,lung tissue inflammation factors were examined by ELISA method TNF-a,IL-1β,IL-4,IFN-γ,Western blotting of proteins technology to detect IKBα,NF-KB P105 influenza virus infected host cell signaling pathways protein levels)and antiviral mechanism research.The remaining 10 animals continued to observe the death situation within 14 days after infection,and the mortality rate,mortality protection rate,average survival days and life extension rate were calculated.Results1.The anti-influenza virus effect of the active components of yinqiao powder preparation:the study showed that the survival rate of the chlorogenic acid group was higher than that of other groups,and the average body weight decreased slowly on the fifth and sixth day compared with other drug groups,and the difference was statistically significant compared with the virus group(P<0.05).Lung index of chlorogenic acid group was lower than that of other groups,and the difference was statistically significant compared with ribavirin group(P<0.05).2.The results of CSP100 antibody chip:the larger the average signal value of repeat points on the chip,the more reliable the result.Fold change>1.2 has reference significance.The results showed that the corresponding Fold changes of IKBα,NF-κB P105,Src(ab-529)and PDK1(ab-241)were 5.19,5.10,1.49 and 1.42,respectively,greater than 1.2.Fold changes with a large value of both IKB-alpha and NF-κB p105 were selected for verification.3.Based on the anti-influenza effect of chlorogenic acid in the cell signaling pathway and its molecular mechanism,the results showed that compared with the virus group,the lung index of the high-dose and low-dose groups of chlorogenic acid decreased,and the difference was statistically significant(P<0.05).Each dose group of chlorogenic acid could reduce the mortality of mice infected with influenza virus and prolong the average survival time of mice infected with influenza virus,and the difference was statistically significant compared with the virus group(P<0.05),indicating that chlorogenic acid had a protective effect on the death of mice infected with influenza virus.Histological examination of lung disease showed that each dose group of chlorogenic acid improved the pulmonary inflammation degree of mice infected with influenza virus compared with the virus group.The levels of IFN-γ and IL-4 in the high-dose chlorogenic acid group were higher than those in the viral group(P<0.05).The levels of IL-1β and TNF-α in the high-dose group were significantly decreased in the chlorogenic acid group,and the difference was statistically significant(P<0.05)compared with the viral group,indicating that chlorogenic acid could inhibit the viral pneumonia of influenza virus mice.Finally,Western blot was used to detect the protein indicators related to the cell pathway.Compared with the viral group,the expression level of IKB a and NF-κB P105 green protein in the three dose groups of chlorogenic acid was significantly lower than that in the viral group.Compared with the viral group,the difference was statistically significant(P<0.05).ConclusionChlorogenic acid,the main component of yinqiao powder,has a certain therapeutic effect on mice infected with influenza virus.Chlorogenic acid can reduce the lung index of mice infected with influenza virus,improve the rate of life extension,significantly improve the pathology of pneumonia,and inhibit the level of inflammatory cytokines in lung tissue.Chlorogenic acid can inhibit the protein expression levels of IKBα and NF-κ B P105 in lung tissue,and play an anti-influenza role.This study provides a theoretical basis for the development of safe and effective anti-influenza drugs with chlorogenic acid.