| Objective:To explore the effect of Maxingshigan decoction on type A influenza virus immune regulation mechanism,By using cell culture,animal experiment and pathology,immunology and molecular biology experimental technique and so on,research the effect on the type A influenza virus mediated Maxingshigan decoction of IL-6 and MCP-1 protein expression level.Methods:1.Using MTT method to detect Maxingshigan decoction serum containing of type A influenza virus infection RAW264.7 cell activity;Using ELISA method to detect the IL-6 and MCP-1 levels in RAW264.7 cell supernatant.2.Establishment of animal models,after 3 days and 7 days irrigation with drug or physiological saline respectively,the mice were killed,and get out the lung tissue,spleen tissue,thymus tissue and colon tissue in mice.Compared with groups of mice body weight,weight loss rate,lung index,lung index inhibition rate,spleen index and thymus index;To observe the pathological changes of lung tissue and colon tissue in mice with HE staining;Using ELISA method to detect the IL-6 and MCP-1 levels in serum;Using Immunohistochemistry to detect the IL-6 and MCP-1 protein expression level in mouse lung tissue and colon tissue;Using Western Blotting Technology to detect the IL-6 and MCP-1 protein expression level in mouse lung tissue.Results:1.Compared with concentration of 10% fetal bovine serum normal cells in the control group,the concentration of 50%,25%,12.5%,6.25% of the blank serum in rats for 24 h and 48 h corresponding cell activity OD values were significantly increased(p< 0.01),suggesting that the blank serum of rats in RAW264.7 cells has no toxic effect,RAW264.7 cells can still continue to grow.Compared with the blank serum group of rats,the concentration of 50%,25%,12.5%,6.25% of the medicatedserum in rats 24 h,48h cell activity OD value of the corresponding difference has no statistical significance(p> 0.05),shows that the drug-containing serum of rats have no toxic effect of RAW264.7 cells.Considering the conventional cell culture of fetal bovine serum concentration of 10%,so this study choose the concentration of 12.5%medicated serum of rats in the experimental follow-up experiments adding sample testing related indicators.2.Compared with normal control group,after the infected with the type A influenza virus,the virus control group 24 h and 48 h of RAW264.7 cells in supernatant of IL-6?MCP-1 levels rise,the significant difference(p < 0.01);Compared with the virus control group,medication for 24 h and 48 h the Maxingshigan decoction group of IL-6?MCP-1 levels are drop,there are significantly different(p<0.05).3.Maxingshigan decoction on type A influenza virus infection in mice,and the impact on mice body weight,lung pathological changes and each organ indexes:(1)The effect of Maxingshigan decoction on type A influenza virus infection in mice body weight: compared with normal control group,after type A influenza virus attack mice,the model group mince body weight are loss,there are significant differences(p < 0.01).Compared with the model group,each treatment group of mince body weight are significantly increased(p< 0.01),after drug treatment for 3days of Maxingshigan decoction group,the body weight decreased inhibition rate was23.39%;after drug treatment for 7 days of Maxingshigan decoction group,the body weight decreased inhibition rate was 26.68%.(2)The effect of Maxingshigan decoction on type A influenza virus infection in mice the lung index and the inhibitory rate of lung index: compared with normal control group,after type A influenza virus attack mice,the lung index of model control group are increased,there are significant differences(p < 0.01).Compared with the model group,each treatment group of mice the lung index are decreased,there are significant differences(p < 0.01),after drug treatment for 3 days of Maxingshigan decoction group,the lung index inhibition rate was 28.67%;after drug treatment for 7 days of Maxingshigan decoction group,the lung index inhibition rate was 29.47%.(3)The effect of Maxingshigan decoction on type A influenza virus infection in mice the spleen index and the thymus index: compared with normal controlgroup,after type A influenza virus attack mice,the spleen index and the thymus index of model control group are decreased,there are significant differences(p < 0.01).Compared with the model group,after drug treatment for 3 days and 7 days of Maxingshigan decoction group of the mice spleen index and the thymus index are increased,there are significant differences(p<0.01).(4)The effect of Maxingshigan decoction on type A influenza virus infection on lung pathological changes in mice: the mice in normal control group have normal alveolar size,alveolar sac,alveolar ducts,alveolar septum,normal bronchioles tissue structures,no inflammatory cell infiltration;the mice in model group have alveolar size disparity,alveolar sac,alveolar ducts,alveolar septum,the normal structure of the bronchioles had been damaged,there are a large number of inflammatory cell infiltration,pulmonary vascular congestion;after drug intervention of Maxingshigan decoction group to reduce the severity of varying degrees.4.The effect of Maxingshigan decoction on type A influenza virus infection on colon pathological changes in mice: normal colon epithelial integrity,gland arrangement rules;interstitial congestion,edema,inflammatory cell infiltration;after type A influenza virus attack mice,colon mucosa edema,or even fall off,gland irregular arrangement,mucous glands damage,mucosa hyperemia,edema,inflammatory cell infiltration.5.The effect of Maxingshigan decoction on type A influenza virus infection on IL-6and MCP-1 levels in serum of mice: compared with normal control group,after type A influenza virus attack mice,the IL-6 levels in the serum of mice in model control group are increased,there are significant differences(p < 0.01);compared with the model group,after drug treatment for 3 days and 7 days of Maxingshigan decoction group the IL-6 levels are decreased,there was significant difference(p<0.01 or p<0.05);after drug treatment for 3 days of Maxingshigan decoction group the IL-6levels are decreased,there are obvious differences(p<0.05).after drug treatment for3 days of Maxingshigan decoction group the IL-6 levels are decreased,there are significant differences(p< 0.01).compared with normal control group,after type A influenza virus attack mice,the MCP-1 levels in the serum of mice in model control group are increased,there are significant differences(p < 0.01);compared with themodel group,after drug treatment for 3 days and 7 days of Maxingshigan decoction group the MCP-1 levels are decreased,there was significant difference(p < 0.05).6.The effect of Maxingshigan decoction on type A influenza virus infection on IL-6and MCP-1 protein expression levels in the lung tissue of mice:(1)The result of immunohistochemistry: compared with normal control group,after type A influenza virus attack mice,the IL-6 protein expression levels in the lung tissue of mice in model control group are increased,there are significant differences(p < 0.01);compared with the model group,after drug treatment for 3 days and 7 days of Maxingshigan decoction group the IL-6 protein expression levels are decreased,there was significant difference(p< 0.05).compared with the normal control group,after type A influenza virus attack mice,the MCP-1 protein expression levels in the lung tissue of mice in model control group are increased,there are significant differences(p < 0.01);compared with the model group,after drug treatment for 3days and 7 days of Maxingshigan decoction group the MCP-1 protein expression levels are decreased,there was significant difference(p<0.01 or p<0.05);after drug treatment for 3 days of Maxingshigan decoction group the MCP-1 protein expression levels are decreased,there are obvious differences(p<0.05).after drug treatment for7 days of Maxingshigan decoction group the MCP-1 protein expression levels are decreased,there are significant differences(p<0.01).(2)The results of Western blotting: compared with the normal control group,after type A influenza virus attack mice,the IL-6 protein expression levels in the lung tissue of mice in model control group are increased,there are significant differences(p<0.01);compared with the model group,after drug treatment for 3 days and 7 days of Maxingshigan decoction group the IL-6 protein expression levels are decreased,there was significant difference(p<0.01 or p<0.05);after drug treatment for 3 days of Maxingshigan decoction group the IL-6 protein expression levels are decreased,there are significant differences(p < 0.01).after drug treatment for 7 days of Maxingshigan decoction group the IL-6 protein expression levels are decreased,there are obvious differences(p < 0.05).compared with the normal control group,after type A influenza virus attack mice,the MCP-1 protein expression levels in the lung tissue of mice in model control group are increased,there are significant differences(p<0.01);compared with the model group,after drug treatment for 3 days and 7 days of Maxingshigan decoction group the MCP-1 protein expression levels are decreased,there are significant differences(p<0.01).7.The effect of Maxingshigan decoction on type A influenza virus infection on IL-6and MCP-1 protein expression levels in the colon tissue of mice: compared with the normal control group,after type A influenza virus attack mice,the IL-6 and MCP-1protein expression levels in the colon tissue of mice in model control group are increased,there are significant differences(p< 0.01 or p< 0.05);compared with the model group,after drug treatment for 3 days and 7 days of Maxingshigan decoction group the IL-6 and MCP-1 protein expression levels in the colon tissues of mice was not statistically significant(p>0.05).8.Type A influenza virus infection in mice lung index and the IL-6 and MCP-1protein expression level in lung tissue correlation analysis: after drug treatment for 3and 7 days,the lung index and the IL-6 protein expression level in lung tissue and the lung index and the MCP-1 protein expression level in lung tissue,there are have correlation.9.The IL-6 and MCP-1 protein expression level in lung tissue and the IL-6 and MCP-1 protein expression level in colon tissue correlation analysis:after drug treatment for 3 days the IL-6 protein expression level in lung tissue and the IL-6protein expression level in colon tissue and the MCP-1 protein expression level in lung tissue and the MCP-1 protein expression level in colon tissue,there are have correlation.Conclusion:1.After type A influenza virus attack RAW264.7 cells,virus can infect and adsorption cells,establish a good model for the study of type A influenza virus;type A influenza virus attack RAW264.7 cells,the IL-6 and MCP-1 levels in supernatant are increased,indicating that after type A influenza virus into the body,could caused the much number of cytokines and chemokines release.after treatment with Maxingshigan decoction,the IL-6 and MCP-1levels are decreased,and indicating that Maxingshigan decoction can inhibit inflammation,regulating immune function.2.After type A influenza virus attack,the mice are weight loss,severe pulmonary congestion,pulmonary index increased,indicating that the model was successful inmice.after treatment with Maxingshigan decoction,the body weight of mice are increased;influenza virus attacks the lung index rise,there is obvious inflammation in lungs of mice,after type A influenza virus attack,the spleen index,thymus index are decreased.indicating that the immune organs of mice suffered injury,and decreased immune function;after treatment with Maxingshigan decoction,the lung index of mice are decreased,indicating that Maxingshigan decoction can relieve inflammatory reaction;after treatment with Maxingshigan decoction,the spleen index and the thymus index are increased,indicating that Maxingshigan decoction has a protective effect on the immune function in mice.3.After type A influenza virus attack,the normal structure of lung in mice has been destroyed,a large number of inflammatory cell infiltration,after drug treatment,the lung inflammatory reaction has alleviated;after type A influenza virus attack,mucosal congestion,edema and even epithelial shedding,after drug treatment,the inflammatory reaction has reduced.4.After type A influenza virus attack,the IL-6 and MCP-1 levels in serum content are increased,indicating that the type A influenza virus into the body,could caused the cytokines and chemokines released,after treatment with Maxingshigan decoction,the IL-6 and MCP-1 levels in serum content are decreased,indicating that Maxingshigan decoction can regulate the immune response.5.After type A influenza virus attack,the IL-6 protein expression level and MCP-1protein expression level in lung tissue in mice are increased,indicating that the type A influenza virus into the body,could caused the cytokines and chemokines increased in the lung tissue.after treatment with Maxingshigan decoction,the IL-6 and MCP-1protein expression level are decreased,indicating that Maxingshigan decoction can regulate cytokines and chemokines protein expression levels in lung tissue,reduced inflammation in the Lung.6.After the invasion of type A influenza virus,the IL-6 protein expression level and MCP-1 protein expression level in colon tissue in mice are increased,indicating that the type A influenza virus into the body,could caused the cytokines and chemokines increased in the colon tissue.after treatment with Maxingshigan decoction and oseltamivir,the IL-6 and MCP-1 protein expression levels in the colon tissues of mice was not statistically significant.7.There was a correlation between lung index and the expression of IL-6 and MCP-1 protein in lung tissue.The correlation between the expression of IL-6 and MCP-1 protein in lung tissue was correlated with the time of administration.8.After type A influenza virus attack,inflammation of the lung is likely to spread,causing the spread of the disease,the material basis of which may be inflammatory cytokines,our team needs further study. |
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