| Purpose:Through the application of new Fuzheng Chuyi Granules of to intervention therapy for Coxsackie virus B3?CVB3?animal model of infection,observe and evaluate the effects of the granules on general situation,mortality,viscera virus drops and tissue pathology change,IFN-gamma and IL-4,serum IL-1 beta,IL-6 and TNF-alpha protein expression,apoptosis factors Caspase-9 and the myocardial tissue of phosphorylated p-AKT and NF–kB of mice infected with CVB3.In addition,explore the effect and possible mechanism of action of the new Fuzheng Chuyi Granules against Coxsackie virus B3.Method:It consists of two parts:in vitro and in vivo.1.Study the effect of the new Fuzheng Chuyi Granules on Coxsackie virus B3 in Vitro.After cultured cardiomyocytes,CVB3 will be cultured and prepared,and the titer of CVB3virus will be determined.TCID50 will be calculated by Reed-Muench method.The antiviral adsorption of the Granules,the protective effect of XFZG on the exposed cardiomyocytes and the inhibition of CVB3 proliferation will be observed in the culture plate.2.In vivo experiment2.1 Establishment of Coxsackie virus B3?CVB3?Animal Model:144 BALB/c mice will be randomly divided into 6 groups with 24 mice in each group.According to the method of surface area conversion,group builds as follows:virus model group:normal saline intragastric perfusion.0.4ml/dose,1 time a day for 7 da ys.The low dose group is given intragastric administration with the dosage of 4.5g/?kg·d?of the new Fuzheng Chuyi Granules for 7 days,once a day.Medium dose of the Granules:intragastric administration with the dosage of 9g/?kg·d?of the Granules,once a day for 7 days,With high dose of the Granules:intragastric administration with the dosage of 18 g/?kg·d?,once a day for 7 days.Ribavirin control group:ribavirin solution 0.025g/?kg·d?is administered once daily for 7 days.Each mouse is given intraperitoneal injection of 50ul CVB3 solution(10×TCID50)on the third day.One hour after the model is made,each group continues to be given drugs.if they survived more than 72hours,and body weight significantly reduced,it would confirm the successful establishment of the virus infection model.2.2 Study the effect of the new Fuzheng Chuyi Granules on Coxsackie virus B3 infection and its mechanism.From the day of infection?that is,the 3rd day of administration?,10 mice in each group will be randomly selected and observed for 7 days to evaluate the general state and death protective effect of the new Fuzheng Chuyi Granules on CVB3 infected mice,and Viral titer and pathological sections of heart,lung and other organs of the remaining mice will be observed by HE staining.The effects of the new Fuzheng Chuyi Granules on the expression of IFN-?and IL-4 in mice exposed to CVB3 are observed by flow cytometry.The expression of IL-1?,IL-6 and TNF-?in serum is determined by ABC-ELISA method,and the effect of the new Fuzheng Chuyi Granules on apoptosis factor Caspase-9 is observed by flow cytometry.Western blot method is used to observe the effect of the Granules on the contents of phosphorylated p-AKT and NF-kB in myocardial tissue.Result:1.The new Fuzheng Chuyi Granules shows a good effect of the anti-CVB3 infection in vitro.New granules have very good pr otective effect on the infected myocardial cells,especially the 0.8mg/ml granules.The granules can significantly prevent the adsorption of CVB3,which is comparable to ribavirin.In addition,it can suppress CVB3 proliferation.The effect and drug concentrations are positively correlated.2.The results of the study on t he effect of the new Fuzheng Chuyi Granules on Coxsackie virus B3 in vivo:the cytotoxicity of the Granules is low,and the symptoms and signs of hypoxia,ischemia and necrosis of the exposed mice can be effectively alleviated,thus reducing the mortality of mice exposed to the virus.Virus titer:7 days after infection,virus titers were detected in different groups of heart,lung,and kidney tissues.It was found that the middle dose group was lower than the virus model group?P<0.05?,the high dose group was significantly lower than the virus model group and the low dose group?P<0.01?.Pathological Changes of Heart and Lung Tissue:as for Myocardial pathological changes,the infiltration of inflammatory cells in the middle and high dose experimental groups was obviously reduced,and the protective effect of high dose group on myocardium was more obvious,similar to that of ribavirin drug control group;as to the pathological changes of lung tissue,the inflammatory lesions in the middle and high dose test groups were obviously alleviated,and the results of the high dose granules group were better than those of the ribavirin positive drug control group.3.Results of the discussions about the mechanism of anti-CVB3 infection of the new Fuzheng Chuyi Granules:the relative immunological indexes were detected.The values of IFN-?and IL-4 in the middle,high dose group and ribavirin group were significantly increased.There was significant difference in comparison to the virus model group?P<0.01or P<0.05?,but there was no significant difference between the low dose group and the virus model group?P>0.05?.The expression of IL-1?,IL-6 and TNF-?in serum was detected,and the expression of IL-1?,IL-6 and TNF-?in the middle,high dose group and ribavirin group was significantly decreased.Compared with the virus model group,there was significant difference?P<0.01 or P<0.05?.There was no significant difference between low dose group and virus model group?P>0.05?.The expression of apoptosis factor Caspase-9protein was detected.The expression of apoptosis factor Caspase-9 protein in middle,high dose group and ribavirin group was significantly higher than that in virus model group?P<0.01?.There was significant difference between high dose group and virus group?P<0.001?,There was significant difference between high dose group and ribavirin group?P<0.01?.The expression of p-AKT and NF-?B protein in cardiomyocytes was detected and compared with that of virus group,the protein expression of normal saline group was the highest,followed by the low dose group and the middle dose group,while the high dose group of the Granules had the lowest expression,which was similar to the result of ribavirin group.Conclusion:1.The new Fuzheng Chuyi Granules showed good effect in the experiment of resisting CVB3 infection in vitro.The observation of the infected cardiomyocytes showed that the Granules could obviously reduce the degree of cytopathic changes of CVB3 infected cells and inhibit the proliferation of CVB3 virus.It had a good pr otective effect on t he exposed cardiomyocytes,and the effect was positively correlated with the concentration of the drug,and the cytotoxicity of the Granules was very low.2.The effect of the new Fuzheng Chuyi Granules on anti-infection of CVB3 mice was obvious.The Granules could effectively alleviate the symptoms and signs of hypoxia,ischemia and necrosis in the tissues of the exposed mice,and could reduce the mortality of the infected mice.It can inhibit the proliferation of virus in heart,lung and kidney and can also effectively inhibit the excessive inflammation of heart and lung tissues in mice,and has a certain protective effect on the organs of infected mice.3.The new Fuzheng Chuyi Granules can improve the cellular immune function of mice exposed to CVB3,mediate the cellular immune response and improve the ability of virus clearance in cells.The mechanism may be related to the increased expression of IFN-?and IL-4.4.The new Fuzheng Chuyi Granules could reduce the expression of TNF-?,IL-6 and IL-1?in the serum of mice exposed to Coxsackie virus B3.These cytokines could be expressed in the form of infiltrating cells in cardiomyocytes at the early stage of virus infection,which can aggravate the inflammatory manifestations of cardiomyocytes.5.The new Fuzheng Chuyi Granules could increase the expression of cardiomyocyte apoptosis factor?Caspase-9?in mice exposed to Coxsackie virus B3.Caspase-9 was the promoter of apoptosis,but it could not directly promote apoptosis.Hence,it needs to firstly activate the core apoptotic protease Caspase-3,and then induces apoptosis by t he cascade reaction of apoptosis induced by C aspase-3.The mechanism of the Granules promoting apoptosis may be to promote the apoptosis of exposed cells by increasing the expression of Caspase-9,thus inhibiting the replication and proliferation of the virus in the body.6.The new Fuzheng Chuyi Granules can reduce the expression of p-AKT and NF-kB in myocardium.PI3K-AKT-mTOR is an important immune signal pathway in mammalian cells.The pathway is composed of upstream signal transduction factor PI3K,downstream signal factor Akt and substrate factor mTOR.Akt plays an important role in the process of viral replication.During the process of CVB3 infection,the phosphorylated Akt has a definite anti-apoptosis effect.Activated Akt can induce the downstream transcription factor NF-?B to be transferred into the nucleus,which can cause NF-?B to inhibit the apoptosis of cells,promote cell proliferation and survival.The mechanism of the Granules against CVB3infection may be related to the inhibition of PI3K-AKT-mTOR in the cell pathway by the action. |
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