| Objective: To explore the effect and mechanism of Maxingshigan decoction and oseltamivir combination in vitro and in vivo anti-influenza A virus,and provide experimental support for the clinical application of integrated traditional Chinese and Western medicine in the treatment of influenza.Methods:1.Using serum pharmacological method prepare MaXingShiGan decoction and Oseltamivir combination serum containing.Using tetrazolium blue(MTT) colorimetric assay Ma Xing Shi Gan decoction and Oseltamivir combination serum containing on MDCK cells(MDCK) the maximum non-toxic concentration.Synchronous design of Ma Xing Shi Gan decoction containing serum group, Oseltamivir medicated serum group, blank serum control group.2.Using tetrazolium blue(MTT) colorimetric assay Ma Xing Shi Gan decoction and Oseltamivir combination serum containing in the direct mode of administration inactivated virus, prevent infection mode of administration, the mode of administration in the treatment of infections, influenza A virus on infected MDCK cell activity. At the same time, use MUNANA as substrate, detect the influence of Ma Xing Shi Gan decoction and Oseltamivir combination serum containing and its corresponding drug-containing serum on the influenza virus neuraminidase activity.3.Mice were inoculated with influenza A virus in nasal cavity to establish an animal model of infection. After drug or physiological saline irrigation, detection the effect of Ma Xing Shi Gan Decoction and Oseltamivir combination therapy in a mouse model of influenza A virus.Results: 1.Concentration of 25%~3.125% medicated serum no significant cytotoxicity.Compared with the blank serum group, the concentration of 6.25%~25% Ma Xing Shi Gan decoction and Oseltamivir combination serum containing group(combination drug containing serum group) of MDCK cells activities OD value had no obvious difference.2.The effects of Ma Xing Shi Gan decoction and Oseltamivir combination serum containing anti-influenza A virus in vitro:(1) Inactivated virus directly to medicine, Ma Xing Shi Gan decoction and Oseltamivir combination serum containing in vitro effect of anti influenza A viruses:1) The impact on the influenza viruses infect MDCK cells activity: Compared with normal control group, the virus control group OD value decreased significantly(P < 0.01); Compared with the virus control group, the concentration of 6.25%,12.5%,25% Ma Xing Shi Gan decoction group,Oseltamivir group, Ma Xing Shi Gan decoction and Oseltamivir combination group(combination drug containing serum group) MDCK cell activity increased significantly, the OD value increased(P < 0.01);Compared with Ma Xing Shi Gan decoction group, the OD value of combination drug containing serum group increased significantly(P < 0.05).2) The impact on the influenza virus neuraminidase activity: Fluorescence value compared with the normal control group, the virus control group significantly increased(P < 0.01); Compared with the virus control group, blank serum group and drug group OD value decreased significantly(P < 0.01); Compared with Ma Xing Shi Gan decoction group, combination drug containing serum group corresponding OD value decreased significantly(P < 0.01).(2) To prevent infection to medicine mode, Ma Xing Shi Gan decoction and Oseltamivir combination serum containing against influenza A virus in vitro effect of impact:1) The impact on the influenza viruses infect MDCK cells activity: Compared with normal control group, the virus control group OD value decreased significantly(P < 0.01); Compared with the virus control group, the concentration of 6.25%,12.5%,25% Ma Xing Shi Gan decoction group,Oseltamivir group, combination drug containing serum group MDCK cell activity increased significantly, the OD value increased(P < 0.01);Compared with Ma Xing Shi Gan decoction group, the OD value of combination drug containing serum group increased significantly(P < 0.05).2) The impact on the influenza virus neuraminidase activity:Fluorescence value compared with the normal control group, the virus control group significantly increased(P < 0.01); Compared with the virus control group, blank serum group and drug group OD value decreased significantly(P < 0.01); Compared with Ma Xing Shi Gan decoction group, combination drug containing serum group corresponding OD value decreased significantly(P < 0.01).(3) Treatment dosing mode of infection, Ma Xing Shi Gan decoction and Oseltamivir combination serum containing against influenza A virus in vitro effect of impact:1) The impact on the influenza viruses infect MDCK cells activity: Compared with normal control group, the virus control group OD value decreased significantly(P < 0.01); Compared with the virus control group, the concentration of 6.25%,12.5%,25% Ma Xing Shi Gan decoction group,Oseltamivir group, combination drug containing serum group MDCK cell activity increased significantly, the OD value increased(P < 0.01);Compared with Ma Xing Shi Gan decoction group, the OD value of combination drug containing serum group increased significantly(P < 0.05).2) The impact on the influenza virus neuraminidase activity:Fluorescence value compared with the normal control group, the virus control group significantly increased(P < 0.01); Compared with the virus control group, blank serum group and drug group OD value decreased significantly(P<0.01); Compared with Ma Xing Shi Gan decoction group, combination drug containing serum group corresponding OD value decreased significantly(P<0.01).3.Therapeutic effect of Maxingshigan decoction and Oseltamivir combination in mice infected for influenza A virus:(1)The effect of Ma Xing Shi Gan decoction and Oseltamivir combined weight of influenza virus infected mice:Compared with normal control group,the weight of model control group was significantly decreased(P<0.01).Compared with model control group,the weight of Ma Xing Shi Gan decoction group,Oseltamivir group, Ma Xing Shi Gan decoction and Oseltamivir combination group(combination drug containing serum group) obviously increased(P<0.01),the inhibition rates of weight loss respectively were 27.08%, 29.32%, 29.41%.Compared with Ma Xing Shi Gan decoction group,the weight of combination drug group obviously increased(P<0.01).(2)The effect of Ma Xing Shi Gan decoction and Oseltamivir combined treatment of lung index of mice infected by influenza virus:Compared with normal control group,the lung index of model control group was significantly decreased(P<0.01).Compared with model control group,the lung index ofMa Xing Shi Gan decoction group,Oseltamivir group,combination drug containing serum group significantly reduced(P<0.01),the inhibition rates of lung index respectively were 33.64%, 40.21%, 40.50%.Compared with Ma Xing Shi Gan decoction group,the lung index of combination drug group ignificantly reduced(P<0.01).(3)The effect of Ma Xing Shi Gan decoction and Oseltamivir combined infection of the spleen index and thymus index of mice of influenza virus:Compared with normal control group,the spleen index and thymus index of model control group was significantly reduced(P<0.01).Compared with model control group,the spleen index and thymus index of Ma Xing Shi Gan decoction group,Oseltamivir group,combination drug containing serum group obviously increased(P<0.01).Compared with Oseltamivir group,the spleen index and thymus index of combination drug group increased(P<0.05).(4)The effect of Ma Xing Shi Gan Decoction and Oseltamivir combined infection of the pathological changes of lung tissue of mice to the flu virus:The normal control group mice lungs were pale pink, soft texture, lung surface smooth, no gas, the area of consolidation.The model control group was found in the lung pathological changes of pneumonia in different degree, lung congestion, dark brown appearance.After drug intervention, the degree of lesion in each group were reduced to varying degrees.The degree of improvement in turn is combined drug group, oseltamivir group, Maxingshigan Decoction group.Conclusion:1. MDCK cells can well type A influenza virus infection,and it can be used as an ideal carrier of viral replication;2. Ma Xing Shi Gan decoction and Oseltamivir combination serum containing can intervent type A influenza virus adsorption and proliferation by directly kill the virus, prevent the virus infection, inhibite viral multiplication, and inhibit the neuraminidase activity of influenza virus,and its effect is better than that of Ma Xing Shi Gan decoction group.3. The antiviral action of drug-containing serum is not only related to the active components of drugs directly absorbed into the bloodstream and concentration,but also associated with the level of endogenous active substances such as complement and enzyme in rat's body.So in this experiment,we did not inactivate the drug-containing serum,but using blank serum of rats as a control group to eliminate serum non drug interference.4.Ma Xing Shi Gan decoction and Oseltamivir combination has a protective effect on influenza A virus infection in mice,and can inhibit influenza virus infection caused by weight loss, and decrease the lung index, and reduce the pathological injury of lung tissue,and its effect is better than that of Ma Xing Shi Gan decoction group, ang increased spleen index and thymus index, and its effect is better than that of oseltamivir group. |
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