The Study On The Effect Of Yinqiaochaigui Granule On Mouse Pneumonia Caused By FM1Strain Of Type A Influenza Virus

Objective:1. By investigating the anti-virus effect of Yinqiaochaigui Granule on Type A influenza land III virus in vitro,we selecte effectively strains and provid a reference for further experiments in vivo.2. We observe the protective effect on infected mice of Yinqiaochaig ui Granule on FM1strain of Type A influenza virus in vivo.3. With lung wet to dry weight ratio, pathological changes in the lung tissue and lavage fluid protein content as an indicator, we research Yinqiaochaigui Granule protective effect of lung injury on influenza mouse model.4.We explore the anti-inflammatory effects of Yinqiaochaigui Granule by detecting cytokine IL-6and levels in mice lavage fluid.5.We explore antioxidant effect of Yinqiaochaigui Granule by compari son the change of superoxide dismutase (SOD) activity and malondialdehyde (MDA) content of mice infected by FM1strain of Type A influenza virus.6. We explore the immune function by observing the changes of spleen index, thymus index and T cell subsets ratio of mice infected with FM1strain of Type A influenza virus.Methods:1. We determinat of antiviral effect in cell culture method. We use hemagglutination and MTT method to explore the role of anti-influenza virus on Yinqiaochaigui Granule.2. Intranasally infected NIH mice with FM1strain of Type A influenza virus, we observe the incidence and the number of deaths in mice.3. We weighe the lung wet weight and dry weight and calculate lung wet-dry weight ratio, we observe pathological changes in the lung tissue with HE staining (hematoxylineosin staining), and we determinat of the total protein content in bronchoalveolar lavage fluid with Bradford method.4. We determine IL-6and TNF-α content in mouse lavage fluid by enzyme-linked immunosorbent assay (ELISA).5. We use the superoxide dismutase (SOD) test kit and malondialdehyde (MDA) test kit to detect the activity of SOD and the content of MDA.6. We detecte the immunized index that infected by FM1strain of Type A influenza virus and determining T lymphocyte CD4+/CD8+ratio using flow cytometry.Results:1. In vitro experiments, hemagglutination experiment results showed that Yinqiaochaigui Granule had no effect on Type A influenza Ⅲ virus in MDCK cell;At a concentration of2.5mg/ml, it had inhibition rate of100%to PR-8influenza virus;At the maximum non-toxic concentrations0.625mg/ml, there was inhibition rate57.43%to FM1. MTT assay showed that the therapeutic of Yinqiaochaigui Granule to Type A influenza Ⅰ and Ⅲ virus are:TI<0.68, TI=3.59, TI=4.20. The results are consistent with hemagglutin ation.2. No death of mice in normal control group, model group mortality was up to90%. Yinqiaochaigui Granule each dose group could significantly reduce mortality in mice caused by FM1, prolong survival time of mice, and inhibition of mortality with dose increasing.3. Lung disease was extremely obvious of model group mice. Compared with the normal group, the wet to dry weight ratio of lung lavage fluid and total protein content of model group were significant differences(P<0.01). Yinqiaochaigui Granule each dose group can reduce the lung disease, significantly reduce lung wet and dry weight ratio and the content of total protein.4. The content of IL-6and TNF-αin lavage fluid of normal mice had no significant change. Compared with the normal group, the levels of IL-6and TNF-αin lavage fluid of model group was increased, there was a significant difference(F<0.01). Compared with the model group, Yinqiaochai gui Granule each dose group was significantly decreased the levels of IL-6and TNF-ain mice lavage fluid (P<0.01).5. The activity of SOD and the content of MDA in lung tissue homogenates of normal mouse was no significant change. The SOD activity decreased and the MDA content increased in model group, compared with the normal group, there was a significant difference(P<0.01). Yinqiaochaigui Granule in each dose group can significantly reduced the amount of MDA, and increased the activity of SOD.6. The immune organ of model group was atrophy, and the ratio of CD4+/CD8+was decreased, there was significant differences(P<0.01)compared with the normal control group;Compared with the model group, Yinqiaochaigui Granule in each dose group could inhibit atrophy of immune organs, signi ficantly increased CD4+/CD8+ratio(P<0.01).Conclusion:1. Yinqiaochaigui Granule showed significant inhibition to Type A influenza Ⅰ virus, while has no inhibition to Type A influenza Ⅲ virus. Establish a platform for the following vivo experiments that is important to evaluation treatment and mechanism on Type A influenza Ⅰ virus.2. Yinqiaochaigui Granule has a protective effect death to mice infected with FM1virus.3. Yinqiaochaigui Granule can significantly reduce the tissue lesio ns of mice infected with FM1virus, and reduce lung wet and dry weight ratio lavage fluid total protein content. It showed that the drug can signifi cantly inhibited lung injury of mice infected with FM1virus.4. The content of IL-6and TNF-α on test group lavage fluid were significantly reduced, indicating that Yinqiaochaigui Granule can improve lung injury with anti-inflammatory effects.5. After treated, the activity of SOD was significantly increased and the levels of MDA were significantly reduced in mice lung homogenates, indicating that the drug can inhibit lung injury by exerting anti-oxidati ve.6. Yinqiaochaigui Granule also can improve the ratio of T lymphocyte subsets and inhibit the atrophy of immune organs that influenza virus-induced lung injury.