Objective To investigate the effect of miRNA210 on primary myocardial cells in lipopolysaccharide(LPS)-induced myocarditis.Methods CCK8 method was used to detect the effect of miRNA210 on the viability of primary myocardial cells in normal or LPS-induced myocarditis rats.ELISA was performed to detect the secretion of tumor necrosis factor(TNF)-α and interleukin(IL)-1β after miRNA210 treatment.Flow cytometry was used to detect the apoptosis of primary myocardial cells before and after the intervention.Western blotting was used to detect the expression of TNF-α and IL-1β.The expression of apoptosis-related proteins bcl-2,bax,caspase-3,and hypoxia inducible factor 1 (HIF1)-vascular endothelial growth factor(VEGF)were detected by Western blotting.Results CCK8 detection results showed that,compared with the control group,the effect of miRNA210 mimic(t=0.000,P=1.000)and siRNA(t=0.686,P=0.500)interference had no significant difference on primary rat cardiomyocytes.The viability of rat primary cardiomyocytes significantly decreased after LPS treatment(t=8.764,P<0.001);compared with LPS group,the viability of rat primary cardiomyocytes significantly increased after inhibition of miRNA210(t=3.576, P=0.012).ELISA showed that,compared with the control group,the expressions of IL-1 β(t=4.166,P=0.014)and TNF-α(t=6.309,P=0.003)were significantly up-regulated after LPS induction;compared with the LPS group,the expressions of IL-1 β(t=4.096,P=0.015)and TNF-α(t=4.424,P=0.011)were significantly increased after application of miRNA210 mimic.After silencing miRNA210,IL-1 β(t=4.287,P=0.012)and TNF-α(t=3.577,P=0.023)were significantly down-regulated.Flow cytometry showed that,compared with the control group,the apoptosis of primary cardiomyocytes induced by LPS was significantly increased(t=32.780,P<0.001);compared with LPS group,the apoptosis induced by LPS was significantly aggravated by miRNA210 mimic(t=7.412,P= 0.002),and the apoptosis rate of primary cardiomyocytes was significantly decreased after miRNA210 was silenced(t=11.720,P<0.001).Western blot analysis showed that,compared with the control group,LPS significantly down-regulated the expression of bcl-2(t=8.346,P=0.001)and increased the expressions of bax(t=12.890,P<0.001)and caspase-3(t=4.331,P=0.012).Compared with LPS group,the expression of bcl-2(t=6.074,P<0.001)was significantly decreased,and the expressions of bax(t=5.376,P=0.022)and caspase-3(t=5.859,P=0.004)were increased after miRNA210 mimic.After silencing miRNA210,the expression of bcl-2 significantly increased(t=3.873,P=0.017),the expressions of bax(t=5.205,P=0.006)and caspase-3(t=2.800,P=0.040)significantly decreased.Compared with the control group,the expressions of HIF1(t =10.380,P=0.001)and VEGF(t=4.973,P=0.008)were significantly up-regulated in LPS group.Compared with LPS group,the expressions of HIF1(t=8.952,P<0.001)and VEGF(t=11.203,P=0.001)were significantly up-regulated after miRNA210 mimic was applied,while HIF1(t=3.893,P=0.017)and VEGF expression(t=3.181,P=0.033)were decreased after miRNA210 was silenced.Compared with LPS group,the expressions of bax(t= 4.899,P=0.008),HIF1(t=2.833,P=0.047),caspase-3(t=2.877,P=0.045),and VEGF(t= 2.994, P=0.040)were significantly decreased,and the expression of bcl-2 was increased(t=3.392,P=0.017).Conclusion Silencing miRNA210 can attenuate LPS-induced cardiomyocyte injury through HIF1-VEGF-mediated apoptotic pathway.
Keywords: apoptotic pathway; hypoxia inducible factor 1-vascular endothelial growth factor pathway; miRNA210; myocarditis.