Mechanism Of LY294002and Rapamycin On The Apoptosis Of CVB3-infected HeLa Cells

[Backgroud] viral myocarditis (VMC) is one of the most common acquired heart diseases in children. In the acute phase, VMC can cause d-eath because of cardiogenic shock, acute heart failure or serious arrhyth-mias. While in persistent phase, VMC often leads to left ventricular dys-function or dilated cardiomyopathy. Viral myocarditis is a kind of cardiac inflammatory lesions that due to the viral infection and/or immune respo-nse to virus. A variety of viral infections can lead to VMC. Coxsackievi-rus B3(CVB3) is one of the most common pathogens for viral myocardit-is. CVB3is a virus that belongs to a family of nonenveloped, linear, pos-itive-sense ssRNA viruses, Picornaviridae and the genus Enterovirus. CVB3has high affinity to cardiomyocytes. The mechanisms involved in CVB3-induced VMC include that the virus directly damage to the cardio-myocyte, cell-mediated immune response, the involvement of inflamma-tory cytokines, virus inducing cardiomyocyte apoptosis, production of anti-cardiac autoantibodies, as well as myocardial collagen remodeling, etc. Among which CVB3-induced apoptosis plays a crucial role in the pathogenesis of VMC. Therefore, investigate the mechanism of apoptosis also pave a way for the prevention and treatment of VMC. As there are many signaling pathways involved in regulation of apoptosis, which one do分é'Ÿate CVB3-induced apoptosis of VMC, and how they works is largely unknown. Some studies have found that phospholipid3-kinase protein kinase B-mammalian target of rapamycin (PI3K/Akt/mTOR) signal pathway plays an important role in CVB3-induced VMC. What’s more, PI3K/Akt/mTOR signaling pathway is one of the most importantpathways in the control of apoptosis. In our previous work it was found that PI3K/Akt/mTOR signaling pathways involved in the apoptosis cardiomyocytes, but the precise mechanisms underlying these processes remain uncertain.[Objective] In the present study, we further explored the mechani- sms that underlying CVB3induced apoptosis throgh PI3K/Akt/mTOR si-gnaling pathway in a HeLa cell model, by intervening the different stage of the PI3K/Akt/mTOR signaling system. We designed these experiments to provide more detailed experimental evidence for the mechanisms of CVB3-induced VMC, especially the mechanisms that CVB3infection inducing to apoptosis.[Methods] We employed HeLa cells to be the carrier of CVB3virus replication, then the concentration of CVB3virus was measured. Mean-while, we set up the PI3K inhibitor LY294002and the mTOR inhibitor Rapamycin in the CVB3-infected HeLa cells culture system, to evaluate the cell signaling of apoptosis which caused by CVB3-infected via cell morphology, immunocytochemical stain, flow cytometry, realtime PCR and western blot.[Results](1) The effect of CVB3infection on apoptosis of HeLa cells. CVB3was amplified in HeLa cells and the virus was titrated. We used100TCID50(TCID50=10-5, deter分é'Ÿed by Reed-Muench method) of CVB3to infect HeLa cells. Cytopathic effect (CPE) was observed at6h after CVB3infection. More significant pathological changes were check-ed at48h, and about40%of total cells were changed. While the control group cells did not change significantly at each time point. Using Annex-inV-FITC/PI apoptosis detection kit combined with flow cytometry to detect apoptosis of both CVB3infection group and control group at diff-erent time points, we found a significant higher rate of apoptosis in the CVB3infection group since12h post infection (pi). And while the incu-bation time prolonged, the apoptosis rate of CVB3infection group increa-sed gradually, it reached the peak at48h pi. Then they subsequently stabi-lized. What’s more, Akt and mTOR protein expressions were decreased in HeLa cells after CVB3infection.(2) The effect of LY294002on CVB3infected HeLa cells and the change of downstream signal molecules expression and phosphorylation levels. PI3K expression in HeLa cells was significantly inhibited by10μM and above of LY294002. When LY294002(10μM) was used to treat CVB3infected HeLa cells, HeLa cells showed more obvious morphological changes than untreated CVB3infected HeLa cells, and the apoptosis rate increased significantly. The mRNA expression of Akt, mTOR and the protein levels of Akt, p-Akt and mTOR, p-mTOR were gradually decreased in HeLa cells after CVB3infected. When CVB3infected HeLa cells were treated by LY294002, the expression levels of the above molecules become downregulated more apparent.(3) The effect of Rapamycin on CVB3infected HeLa cells and the changes of the expression and phosphorylation levels of downstream signal molecules. mTOR expression in HeLa cells was significantly inhibited by10nM and above of Rapamycin. When Rapamycin (10nM) was used to treat CVB3infected HeLa cells, HeLa cells showed more obvious morphological changes than untreated CVB3infected HeLa cells, and the apoptosis rate increased significantly, too. The mRNA expression of4EBP1and P70S6K and the protein levels of4EBP1, p-4EBP1, P70S6K and p-P70S6K were gradually decreased in HeLa cells after CVB3infected. Moreover, the expression levels of the above molecules become downregulated more apparent when intervention by Rapamycin.[Conclusions](1) CVB3infection induced apoptosis in HeLa cells with a dose-and time-dependent manner.(2) CVB3infection could induce apoptosis in HeLa cells through inhibiting PI3K/Akt/mTOR signaling pathway.(3) PI3K inhibitor LY294002blocked the PI3K/Akt/mTOR signal thereby promoting apoptosis in CVB3-infected HeLa cells.(4) Rapamycin promoted apoptosis in CVB3-infected HeLa cells by blocking mTOR/4EBP1and mTOR/P70S6K pathway.(5) Inhibition PI3K or mTOR could block the PI3K/Akt/mTOR signaling system and promoting apoptosis in CVB3-infected HeLa cells. And this pro-apoptotic effect might work through phosphorylation of the downstream molecules4EBP1and P70S6K of the PI3K/Akt/mTOR pathway.