Study On The Antiviral Effects And Preliminary Mechanism Of Phyllaemblicin B Against Coxsackievirus B3

Background and ObjectiveViral myocarditis is one of the most common infective one in cardiovascular diseases. Acute viral myocarditis is a principal cause of heart failure in young adults and often progresses to chronic myocarditis,dilated cardiomyopathy and congestive heart failure requiring heart transplantation.Coxsackievirus B3 infection is believed to be a principle etiologic agent in human viral myocarditis.But until now the mechanisms how CVB3 causes myocarditis are not well characterized and there are no virus-specific preventive or therapeutic procedures available to protect humans against CVB3-induced heart diseases.So it is very important to focus on studying its treatment and medicine development.It is a new way to find a medicine to cure VMC in Chinese herbal medicine. Phyllaemblicin B and its analogues are the main ellagitannin compound isolated from the roots of Phyllanthus em blica L.(Euphorbiaceae).As its antiinflammatory and antipyretic effects,it has been widely used in many local traditional medicinal systems.However,little information is available in the literature about the anti-CVB3 activities of Phyllaemblicin B and the underlying mechanisms.The objective of this study is through simulating VMC model infected by CVB3 in vitro and vivo to observe the protect effects of Phyllaemblicin B on myocardiocytes and to approach the action mechanism on VMC infected by CVB3.The therapeutic effects of Phyllaemblicin B have be investigated through apoptosis and apoptosis pathway in order to provide an experimental base to the safe,reasonable and target application in clinic.Methods1.In vitro experiment(1) ObservationConstruct VMC model in vitro by infecting Hela cells.They were also divided into three kinds of style which are simultaneous medicine and virus,medicine before and after virus in vitro.Cell survival rate was measured by MTT assay,and observe the protection from cytopathic effects(CPE). Construct VMC model in vitro by infecting myocardiocytes of newborn SD rat.Cell survival rate was measured by CPE assay,the level of aspartate transferase(AST) and lactate dehydrogenase(LDH) were detcted.(2) Investigation on mechanismFurther,detecting apoptosis rate by Annexin V/PI through FACS,measuring protein expression by proteomics,detecting expression of caspase-3 and bcl-2 by real-time PCR and Western-blot,to search into the anti-apoptosis mechanism of Phyllaemblicin B on Hela cells infected by CVB3.2.In vivo experiment(1) ObservationSimilarly,VMC Balb/c also were divided into different groups to detect survival status,the level of LDH and Creatine kinase(CK) in serum,TCID₅₀,histopathology changes.(2) Investigation on mechanismWhile in vivo,apoptosis of myocardiocytes was detected with TUNEL kit.Expression of caspase-3 and bcl-2 were detected by immunohistochemical method to search into the anti-apoptosis mechanism of Phyllaemblicin B on VMC Balb/c.Results1.In vitro experiment(1) Results of observationPhyllaemblicin B and its analogues inhibited CVB3-mediated cytopathic effects on Hela cells effectively with IC₅₀ value of 50.30μg/ml,195.62μg/ml,67.65μg/ml and 50.23μg/ml,TI value of 9.21,8.96,6.20 and 6.46.By three kinds of style the replication of virus was reduced by Phyllaemblicin B and its analogues.Phyllaemblicin B inhibited cytopathic effects on myocardiocytes of newborn SD rat induced by CVB3.It can be observed that Phyllaemblicin B could reduce CPE and AST,LDH of myocardiocytes significantly.(2) Investigation on mechanismApoptosis of CVB3-infected Hela cells can be inhibited by Phyllaemblicin B significantly. Proteins related with apoptosis were increased,while proteins related CVB3 were decreased. Phyllaemblicin B can suppress the expression of caspase-3,promote the expression of bcl-2. 2.In vivo experiment(1) Results of observationIn the groups of Phyllaemblicin B treatment,survival status can be significantly improved, the level of LDH and CK in serum,TCID₅₀ and histopathology changes in mice myocardium were reduced.(2) Investigation on mechanismA large number of apoptotic cells can be observed in the inffected groups.Phyllaemblicin B can significantly inhibit the apoptosis of myocardiocytes of VMC animals.The level of caspase-3 protein expression can be decreased,while bcl-2 protein expression can be increased in Phyllaemblicin B treated group.Conclusion1.It was proved that Phyllaemblicin B and its analogues could inhibit cytopathic effect resulted from CVB3.2.The study confirms that Phyllaemblicin B could inhibit cytopathic effect resulted from CVB3 in vitro and in vivo,and greatly protect myocardiocytes infected by CVB3.3.Phyllaemblicin B can inhibit apoptosis resulted from CVB3 in vitro and in vivo. Phyllaemblicin B can restrain obviously the protein expression of caspase-3 and promote the expression of bcl-2.To our knowledge,this is the first report of such effects of Phyllaemblicin B and conveys new insights in our efforts to characterize a novel therapeutic target for treatment of enteroviral myocarditis.