Study On The Activity And Mechanism Of NCC Against Coxsackie Virus B3

Background and objective: Viral myocarditis(VMC)is one kind of myocarditis diseases which are induced by virus infection and the incidence of VMC is very high.At present,children and adolescents always fall ill.There are many kinds of virus which could induced viral myocarditis,including coxsackie virus,ECHO virus,intestinal virus,polio virus,asenovirus,etc.Among them,the enterovirus in the Coxsackie virus(Coxsackievirus B3,CVB3)is more common.CVB3 virus is an unencapsulated single-stranded RNA virus,often through the digestive tract or respiratory tract into the human body and make it infected.It could enter the body through the digestive tract and respiratory,then it begins to reproduce,finally damage the myocardial cell.As a result,VMC happens.VMC has a cetain clinical symptoms,mainly as inflammatory cell infiltration and necrosis of myocardial cells.Some paitents,in early stage of VMC,had no obvious symptoms,so that we did not aware of the disease.Unfortunately,with the continuous development of the disease,the patients would have a serious complications,such as angoma pectros,arrhythmia,cardiac,insufficiency,atrioventricular bolck or shock,seveal maybe even sudden death.VMC now is a serious threat to human health,but the pathogenesis of it is not clear,is a disease which severely threat to human health.However,the molecular biological mechanism of VMC is unclear,and there are no effective treatments for it at present.Therefore,the important task for us is to study the pathogenesis and to find some new drugs to treat VMC.NCC is a novel nucleoside analogue that has been shown to have excellent anti-CVB3 activity in vitro cell models in previous studies.In this study,we successfully constructed the in vitro model of viral myocarditis and explore the effects of drugs on CVB3 adsorption,the effect of drugs on CVB3 biosynthesis andthe direct effect of drugs on CVB3 by three different methods.The effect of NCC on the apoptosis of host cells infected with CVB3 was further investigated by detecting the effect of NCC on the stereotactic potential of CVB3 infection.The mechanism of NCC on CVB3 was further studied by RT-PCR and Western-Blot.Method: 1.We use three diffent ways:The first one add the drug solution before virus;The second add the virus before drug solution;The last one the direct effect of drugs on virus.The cells in the normal control group were in good condition,90% of the cells in the virus control group were diseased and some cells in the NCC group appeared.The effect of NCC on CVB3 was detected by MTT in the virus control group after complete lesion.2.We selected CVB3 virus to infect Hela cells which were in good logarithmic growth status.The changes of mitochondrial membrane potential were measured by flow cytometry.The effect of NCC on the expression of TLR3,TLR7 and MDA-5 was detected by reverse transcription-polymerase chain reaction(RT-PCR).The changes of MAPK protein levels were detected by Western blotting.Results: 1.Successful bulid the cell model of viral myocarditis.TCID50 =10-6.3 2.MTT results: The TC50 of NCC is 1.27 m M.Calculate the IC50 value of NCC to CVB3.The first method: first add NCC culture cells,discard the supernatant after 2h,plus CVB3 cultured,then discard the supernatant after 2h,plus culture medium to continue culture.The IC50 value of the first method is 20.04 m M,TI is 0.06;The second method : first plus CVB3 discard the supernatant after 2h,plus NCC liquid continuous culture.The IC50 value of the second method is 25.27μM,TI is 50.26.The third method : first mixed NCC with CVB3 of equal volume,discard the supernatant after 2h,then add the culture medium to continue to culture.The IC50 value of the third method is 223.68μM,TI is 5.68.3.The results of mitochondrial membrane potential: Compared with the virus controlgroup,after the effect of NCC,Mitochondrial membrane potential were significantly increased.4.RT-PCR test results show that compared with the virus control group,the m RNA expression of TLR3,TLR7 and MDA-5 in tissue treated with NCC high and low concentration was significantly inhibited as compared with the control group(P<0.05).Conclusion: 1.NCC has a good activity of resistanting CVB3 in cell model and protectmyocardial cell.2.The effect of NCC on CVB3 may be that NCC affects the biosynthesis of CVB3 and the drug has a direct effect on CVB3.3.The main mechanism of NCC anti-CVB3 may be related to the inhibitory effect of CVB3 infection on the apoptosis of Hela cells.The results showed that the expression of TLR3,TLR7 and MDA-5 could be inhibited at the gene level.And increased p44 / 42 MAPK(Erk1 / 2)expression levels at the protein level.