Immune Regulation Of Maxing Shigan Decoction On Mice Infected By A-type Influenza Virus

Objective:By studying Immune regulation of Maxing Shigan Decoction on mice infected by A-type influenza virus with body weight, lung index, the weight of immune organs index, pulmonary pathological changes and IL-2,IL-4, TNF-a secretion levels in serum and location and quantitative protein expression levels in lung tissue,we preliminarily investigate its influence on controling the lung injury and regulating immune function and clarify its mechanism, then It provide a theoretical basis for clinical application.Methods:Use nasal inoculation with A-type influenza virus adapted lungs of mice (A/PR/8/34) to establish A-type influenza virus infected animal models.Experimental set up the normal control group, model control group, ribavirin group, anti-virus particles group and Maxing Shigan Decoction(MXSGD)clinical equivalent dose group, two times clinical equivalent dose group,and give the corresponding drug or saline by gavage for 7 days.Compare:these mice body weight, lung index, spleen index and thymus index in each group, the pathological changes of lung tissue by HE staining under light microscope,the IL-2,IL-4,TNF-αsecretion levels in serum by ELISA, they protein expression in lung tissue by immune histochemistry positioning and semi-quantitative detection and they protein accurate expression in lung tissue by western blotting technique. Result:1.the general conditions of MXSGD on mice infected by A-type influenza virusBy observing the impact of the general conditions(such as weight, activity, appetite, etc.)of mice which are infected by A-type influenza virus, analysis and compare these mice's body weight before and after infection to determine MXSGD on the therapeutic effect of mice by infected with A-type influenza virus.The results showed that these mice perform the symptoms such as gloomy spirit, humped hair, curly, hair dull,activity reduction, weight loss,chills afraid of the cold, shortness of breath, obvious abdominal breathing by 48h post-infection of A-type influenza virus.Model control group is the most significant in weight loss, compared with the normal control group, there is a very significant difference (P<0.01).Mice body weight of in MXSGD clinical equivalent dose group and two times clinical equivalent dose group increased, the weight loss is effectively inhibited, compared with the model control group, there are significant differences (P<0.01).2.the changes of lung index, inhibition rate of lung index and pathological in lung tissue of MXSGD on mice infected by A-type influenza virusA-type influenza virus infection in mice led to the lung index was significantly increased, lung tissue showed a visible pathological damage, disappearance of the normal structure such as alveolar, alveolar sac, alveolar duct, alveolar septa, bronchioles,etc, proliferation of connective tissue, a large number of lymphocyte infiltration, intravascular congestion. The lung index of model control group increased compared with the normal control group, there was a significant difference (P<0.01). MXSGD clinical equivalent dose group and two times clinical equivalent dose group can significantly reduce pathological damage of mice lung tissue by infected with A-type influenza virus,the lung index decreased significantly (P<0.01),the lung tissue pathomorphology showed lung tissue morphology is more complete,even visible a small amount of lymphocytes infiltration, alveolar, alveolar sac, lung saturated tube, alveolar septa intact, cells were red, nuclei blue.3.The changes of thymus index and spleen index of MXSGD on mice infected by A-type influenza virusThymus and spleen are the body's immune organ, their function is closely related to the body's immune response level and immune regulation. The results showed that infected by A-type influenza virus infection, mice's thymus index and spleen index decreased compared with the normal control group, there was a significant difference (P<0.01). MXSGD clinical equivalent dose group and two times clinical equivalent dose group significantly increased the thymus index and spleen index (P<0.01,P<0.05).4.The levels of IL-2,IL-4,TNF-αin serum and lung of mice infected by A-type influenza virusBy studying MXSGD on influenza virus infected mice above the levels of cytokines in serum and lung tissue protein levels in the initial study Ma Xing Shi Gan Tang mechanism of action of anti-influenza virus4.1 ELISA assay results showed:after infected by A-type influenza virus, the serum levels of IL-2 reduced,while the levels of IL-4 and TNF-a increased, compared with the normal control group, significant difference (P<0.01,P<0.05).The serum levels of IL-2 increased in MXSGD clinical equivalent dose group and two times clinical equivalent dose group, while levels of IL-4 and TNF-a reduced significantly different (P<0.01, P<0.05).4.2 Immunohistochemistry location and semi-quantitative results showed: After infected by A-type influenza virus, the lung tissue levels of IL-2 protein expression reduced, IL-4 and TNF-a protein levels increased compared with the normal control group, significant difference (P<0.01). The protein expression of IL-2 increased in MXSGD clinical equivalent dose group and two times clinical equivalent dose group, while levels of IL-4 and TNF-a reduced significantly different (P<0.01,P<0.05).4.3 Western blotting for quantification of results showed that:After infected by A-type influenza virus,the lung tissue levels of IL-2 protein expression reduced, IL-4 and TNF-a protein levels increased compared with the normal control group, significant difference (P<0.01).The protein expression of IL-2 increased in MXSGD clinical equivalent dose group and two times clinical equivalent dose group, while levels of IL-4 and TNF-a reduced significantly different (P<0.01).Conclusion:1.MXSGD on mice infected by A-type influenza virus has a protective effect:MXSGD clinical equivalent dose group and two times clinical equivalent dose group can inhibit weight loss which led by infection with A-type influenza virus, and can significantly improve the flu-like symptoms,suggesting:MXSGD on mice infected by A-type influenza virus has a protective effect.2.MXSGD can inhibit viral replication and reduce pathological damage:MXSGD clinical equivalent dose group and two times clinical equivalent dose group can significantly reduce pathological damage of mice lung tissue by infected with A-type influenza virus, decrease the lung index, make lung tissue morphology more complete.suggesting: MXSGD can inhibit A-type influenza virus replication, obviously reduce lung pathological damage.3.MXSGD has a protective effect of immune organs in mice: MXSGD clinical equivalent dose group and two times clinical equivalent dose group significantly increased thymus index and spleen index, suggesting:MXSGD has a protective effect to mice infected by A-type influenza virus,and it should be the party base that it regulate the immune response.4.MXSGD have a moderating effect the levels of IL-2,IL-4, TNF-αin mice serum:by testing serum cytokines in mice after infected seven days, MXSGD can promote IL-2 secretion, inhibit IL-4 and TNF-αsecretion,suggesting:MXSGD on mice infected by A-type influenza virus has a certain inhibition of inflammation, at the same time play a role in immune regulation and enhancing the cellular immune response levels and anti-virus capabilities.5.MXSGD on mice infected by A-type influenza virus have a regulatory role in expression of cytokines in lung tissue:Analysis of immunohistochemistry and Western blotting to detect protein expression in the lung, suggest that after mice are infected by A-type influenza virus pulmonary immune response to more intense.Although the expression of cytokines in serum was not significantly better than the lung, but the overall trend is basically the same, indicating MXSGD can not only regulate disorders of inflammatory cytokines in serum, but also to in lung, it's protective effect is close to it inhibited circulation of blood and local tissue IL-4, TNF-a and enhance the expression of IL-2.Which can show that MXSGD can adjust the body's immune function, and fight the body's inflammatory injury infected by the virus.In conclusion,MXSGD can protect the thymus and spleen, while mice infected with influenza virus to improve the immune function; By regulating cytokine secretion of inflammatory factors, including ease the lungs of mice infected with influenza virus pathological damage.As the complexity of Chinese herbal compound composition and role of target range, cytokines in the body and promote each other again and check each other, forming a complex network of cytokine regulation. Therefore, Maxing Shigan Decoction prevention and treatment of influenza virus infection in-depth study of the immunological mechanism has yet to be carried out.