Mechanism of Mitochondrial Apoptosis Involved in Human Microvascular Endothelial Cell Injury Caused by EV71 Infection
Enterovirus 71(EV71)is a neurotropic enterovirus that is primarily transmitted by the fecal-oral route.However,the mechanism that EV71 infection leads to apoptosis in microvascular endothelial cells has not been fully elucidated.In this study,we infected human microvascular endothelial cells(HMEC-1)in vitro using a clinically isolated EV71 strain to investigate the mechanism of mitochondrial damage.The results of MTT method revealed that the cell viability of HMEC-1 infected with EV71 decreased to 47％after 24 h(P＜0.01);transmission electron microscopy revealed a large number of vacuoles in the cytoplasm of EV71-infected HMEC-1,and mitochondria was observed to be swollen,denatured,and markedly vacuolated with broken mitochondrial cristae,and scattered or free viral particles were seen in the cytoplasm;JC-1 staining showed a significant decrease in mitochondrial membrane potential after 32 h of EV71 infection with HMEC-1;the fluorescent probe DCFH-DA detected a significant increase in mitochondrial reactive oxygen species(ROS)levels in infected cells;the levels of EV71-VP1,Cleaved-caspase 3,Cleaved-caspase 8,Cleaved-caspase 9,and Bax proteins were progressively increased with increasing duration,and the Cyt C protein in the cells was shifted from mitochondria to cytoplasm.Additionally,real-time fluorescence quantitative PCR detected a gradual increase in the mRNA expression levels of TNF-α,IL-6,and IL-1β in HMEC-1 with increasing time of infection.In contrast,the expression of pro-apoptotic proteins and TNF-α,IL-6,and IL-1β mRNA in endothelial cells could be significantly reduced by the intervention of N-acetyl-L-cysteine(P＜0.01).Therefore,in the present study,we found that EV71 infection induced HMEC-1 apoptosis through the mitochondrial damage,which is mediated by ROS.
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