The Molecular Mechanism For Emodin In The Treatment Of Viral Myocarditis In Mice

Objectives:1.To explore the efficacy of emodin in mice with viral myocarditis;2.After treatment with emodin to investigate whether emodin can regulate TLR4 induced viral myocardium and myocardial injury;3.To explore the molecular mechanism of emodin in the regulation of viral infection by using CVB3-induced mouse viral myocarditis model;4.To provide new strategies for the clinical application of VMC and the clinical application value of emodin in future.And provide the theoretical basis and experimental basis for emodin on the treatment of viral myocarditis.Methods:90 male BALB/c mice were randomly divided into 3 groups,A group:control group,B group:model group,C group:emodin group.And 30 mice in every group.Group B and group C were injected intraperitoneally with Coxsackievirus B3(CVB3).The mice were sacrificed in C group by administration of emodin 30mg/kg/d,once/d for 14 days.Other mice in A and B groups were fed food and water in normal.Then investigated all the condition of mice in all.The mortality of mice was recorded during the experiment.On the 7th day,5 mice were killed in every group.And removed the hearts after be killed.Then the virus titer was measured on the 7th day.The hearts would be cut grinding,centrifugated supernatant,diluted into different concentrations.Then the various concentration(10-1-10-6)0.2ml diluent were be extracted,and joined the 96 orifice(hela cells),set the 4 holes.Observed the cells,lesion hole:pathological cells>50%,on the basis of reed-muench determination by lgtcid50.Then,Fluorescence quantitative PCR was used to determine the copy number of CVB3 mRNA.Total RNA was extracted from the virus in myocardial tissue,the determination of the ratio of d260/d280 by ultraviolet spectrophotometry(1.8-2.0),analysize of agarose gel electrophoresis of PCR products.repeated for 3 times.The relative expression were be set.Eight mice were sacrificed at 7 and 14 days.And then removed the heart after anatomy.After fixation washing,dehydration,transparen,embedded in paraffin,paraffin production,mount HE staining and other steps to made the pathological section of mice myocardium.The pathological changes of myocardium were observed by HE staining.And calculated the pathological integral.The method was as follows.Prepared slices every piece pathological section randomly selected five at high magnification.Observation and calculation were proceed In view of the high magnification.The necrotic area in the view of the area,the infiltration of inflammatory cells and the area occupied by the ratio of the all view area were be detected.0:no lesions;1 points:the lesion area is less than 25%;2:25%-50%lesion area;3:51%-75%lesion area;4 points:the lesion size is more than 75%.TLR4 mRNA was detected by RT-PCR.The total RNA was be extracted from myocardial tissue,then the total RNA reversed transcription into cDNA.RT-PCR based on trizol method of operation.Quantitated using ultraviolet spectrophotometer.The PCR products were analyzed by agarose gel electrophoresis,repeated for 3 times.The relative expression were be set.The gray value and the ratio of beta gene-actin gray value were be detected.The expression of inflammatory factor,TLR4-NF-kB,P38MAPK expression,ELISA to detect changes in serum levels of inflammatory factors.The expression of TLR4-NF-kB was detected by Western-blot and the expression of inflammatory cytokines was detected by ELISA.Primary cultured the myocardial cells of neonatal mice in first step.Then the primary cultured myocardial cells of neonatal mice were randomly divided to 4 group.They were A group:control group,group B:model group,C group:emodin group,D group:lps+ emodin group.The C group pretreated with 2h in myocardial cell of emodin 100ug/ml,application of CVB3 myocardial cells infected with 8h.D group of pretreated activation of myocardial cell virus by LPS.Then blocked with emodin.At last,checked the changes of protein levels and determination of TLR4 and NF-kB and the changes of its downstream key factors IkBa and p65.Inflammatory factors were be detected by western-blot method.TLR4 and NF-kB and related molecules such as p-IkBaand p-p65 and the expression of TNF-?,IL-1,IL-6 and MCP-1 were be detected by western-blot method too.Results:1.Compared with group B,the expression of P38MAPK and TLR4 mRNA in group B was significantly higher than that in group B(P<0.05).Compared with group B,the death rate,virus titer,CVB3 mRNA copy number and myocardial pathological score of group C were significantly decreased(P<0.05).The expression of P38MAPK and TLR4 mRNA in myocardium was significantly decreased(P<0.05).2.The serum concentrations of TNF-a,IL-6,IL-1? and MCP-1 in the model group were significantly higher than those in the control group,and there was significant difference between the two groups.After treatment with emodin,their serum The concentration was significantly lower than the model group,and there were statistically significant differences.3.The protein levels of TNF-a,IL-6,IL-1? and MCP-1 in the model group were significantly higher than those in the control group,and there was significant difference between the two groups.After the emodin intervention,their protein The level was significantly lower than the model group,and there were statistically significant differences.4.The expression of TLR4 and NF-kB protein was significantly increased in only infected cells,and the phosphorylation levels of IkBa and p65 were also significantly up-regulated.In the pretreatment group,the levels of these proteins were significantly down-regulated.5.The levels of TNF-a,IL-6,IL-1? and MCP-1 in the LPS + Emodin group were not significantly different from those in the model group,and the levels of TNF-a,IL-6,IL-1? and MCP-1 in the LPS + Emodin group were not significantly different from those in the model group Group comparison,there is a significant increase.Conclusions:1.TLR4—NF-kB and TLR4—P38MAPK signaling pathway maybe had very important function in viral myocarditis.2.Inflammatory reaction was persisted in viral myocarditis.Very much inflammatory factors had important function,such as TNF-a,1L-6,IL-1? and MCP-1.3.The molecular mechanism of emodin in the treatment of viral myocarditis was be found.Emodin was activated by inhibiting the activation of TLR4-NF-kB signaling pathway,thereby reducing the concentration of inflammatory factors and reducing the infiltration of inflammatory cells,reducing myocardial injury.