Research On Urolithin A Inhibiting Enterovirus 71 Replication And Its Mechanism

Objects:To study the inhibition of enterovirus71（EV71）proliferation by urolithin A（Uro A）and its preliminary mechanism,providing a new candidate drug for EV71infection and a reference to the development of antiviral drugs.Methods:RD cells were used as the infection model in the following experiments.1、The effect of Uro A on EV71 proliferation（1）CCK8（CCK8）was used to detect the toxicity of Uro A to cells and screen the maximum non-toxic concentration.（2）The expression of EV71-VP1 protein under three administration methods of Uro A（prevention action mode,mixture action mode,treatment action mode）was detected through western blot assay（WB）to screen effective methods.（3）Under the effective mode,the effect of Uro A on the proliferation of EV71 was detected:cytopathic effect（CPE）was observed using inverted microscope.The expression of EV71-VP1 protein,virus titer and virus replication level were detected by WB,50%tissue culture infective dose(TCID₅₀)and quantitative real time polymerase chain reaction（q RT-PCR）;At the same time,effect of Uro A on EV71proliferation in SK-N-SH cells was detected by CCK8,WB and TCID_50.2.Evaluation of Uro A anti-EV71 effect（1）Cytotoxicity evaluation:the half cytotoxic concentration(CC₅₀),half inhibitory concentration(IC₅₀),and selectivity index（SI）were calculated through CCK8 and CPE inhibition assay for anti-EV71 to understand the toxicity of Uro A on cells,and compared with broad-spectrum antiviral drug ribavirin.（2）Effectiveness evaluation:cells infected by EV71 were treated with Uro A,ribavirin and drug solvent,which were divided into three groups,marked Uro A group,ribavirin group and control group,respectively.（1）The expression of EV71-VP1 protein under Uro A and ribavirin was detected by WB.（2）Dose dependent experiment:the expression of EV71-VP1 protein at different concentrations of Uro A and ribavirin was detected by WB,respectively.（3）Effects of Uro B and Uro C on EV71 were compared:the non-toxic concentrations of Uro A,Uro B and Uro C were screened by CCK8,and then the expression of EV71-VP1 was determined through WB.3.Exploration on the mechanism of Uro A inhibiting EV71 proliferationNormal cells were treated by Uro A and drug solvent,respectively.Infected cells were treated by Uro A,drug solvent,respectively.Those were divided into four groups,marked mock group,mock administration group,Uro A group and the virus control group,respectively.（1）The effect of Uro A on virus particles:Uro A and virus particles were incubated separately or co incubated for 1h before infection,the expression of EV71-VP1 protein was detected by WB.（2）Effects of autophagy and apoptosis:the expression of autophagy related proteins such as LC3-Ⅰ,LC3-Ⅱand p62 and apoptosis related proteins including caspase-3 and cleaved-caspase3 were detected by WB.Results:1.The effect of Uro A on EV71 proliferation（1）According to CCK8,the concentration of Uro A solution≤50μM had no obvious toxicity to RD and SK-N-SH cells.（2）Three different action modes of Uro A were compared,the expression of EV71-VP1 protein decreased significantly under treatment action mode.It showed that treatment action mode is an effective way.（2）Under the effective mode of Uro A,CPE was significantly reduced in RD cells,and the surviving cells were in good condition.At the same time,the expression of EV71-VP1 protein,virus titer and virus replication level were significantly reduced;expression of EV71-VP1 protein and virus titer were also significantly decreased in SK-N-SH cells.2.Evaluation of Uro A anti-EV71 effect（1）Cytotoxicity evaluation:The CC₅₀ of Uro A and ribavirin were 88.410±18.290μM,133.415±34.385μM respectively;IC₅₀ was 8.922±1.129μM and 6.402±1.231μM respectively;SI is 9.910 and 20.840 respectively.It could be seen that the toxicity range of Uro A was smaller than ribavirin.（2）Effectiveness evaluation:（1）The expression of EV71-VP1 protein in Uro A group was lower than that in ribavirin group,and they were significantly lower than those in control group.There was no significant difference in EV71-VP1 protein expression between Uro A group and ribavirin group in SK-N-SH cells,but they were significantly lower than those in control group.（2）With the increase of dose concentration of Uro A and ribavirin in RD cells,the expression of EV71-VP1 protein decreased in Uro A group and ribavirin group,and the anti-EV71 effect of Uro A was superior to ribavirin.（3）The expression of EV71-VP1protein decreased in various degrees of Uro A,Uro B and Uro C.Among them,Uro A had the best effect on inhibiting the proliferation of EV71 and the least toxicity.3.Exploration on the mechanism of Uro A inhibiting EV71 proliferation（1）Uro A and virus particles were incubated separately or co incubated for 1h before infection.There was no difference in the expression of EV71-VP1 protein detected through WB.（2）Under the treament of Uro A,the expression of LC3-Ⅱincreased and p62 protein decreased.There was no significant difference in the expression of apoptosis related protein caspase-3 protein,and the expression of cleaved-caspase-3 protein was significantly increased.Conclusion:Uro A exerts more potent-antiviral activity against EV71 in vitro,and it has lower cytotoxicity than ribavirin.The anti-EV71 mechanism of Uro A may be related to the induction of autophagy and apoptosis.