High Expression Of Sptlc2 Gene Promote The Apoptosis And Autophagy Of HSC-T6 Cells Induced By Oleic Acid

Objective: In this study, we proposed that the effects of the key enzyme Sptlc2 on the HSC-T6 cells apoptosis and autophagy from the Sptlc2 gene, which regulated the de novo synthesis pathway of ceramide, confirmed the new target for intervention of the apoptosis and autophagy in HSC-T6 cells.Methods:1.Cultured HSC-T6 cell line of SD rat in vitro,and were divided into four groups:HSC-T6 cells group(normal control group),induced by oleic acid in HSC-T6 cells group(oleic acid group), induced by oleic acid in pc DNA3.1(+)- NC group(empty vector group), induced by oleic acid in pc DNA3.1(+)-Sptlc2 group(Sptlc2 transfection group).In addition to the normal control group,HSC-T6 cells were co-cultured with 100umol/l oleic acid in the other groups.2.Used q PCR technology to detect the expression of Sptlc2 m RNA in HSC-T6 cells after transfection for 48 hours.3.Used Flow cytometry(FCM) to detect the apoptosis of HSC-T6 cells.4.Used MDC staining under fluorescence microscope to observe the number of autophagy of HSC-T6 cells. 5.Used Western blot to detect the expression of apoptosis related protein Bax, Bcl-2, autophagy marker protein LC3 B, autophagy related protein Atg5, Beclin-1.Results:1.The expression of Sptlc2 m RNA of oleic acid group compared with normal control group, Sptlc2 m RNA expression was significantly increased(P<0.01). Sptlc2 transfection group compared with oleic acid group and empty vector group,Sptlc2 m RNA expression was significantly increased(P<0.01).2.Oleic acid group compared with normal control group,the total apoptosis rate was significantly increased(P<0.01).Sptlc2 transfection group compared with oleic acid group and empty vector group,the total apoptosis rate was significantly increased(P<0.01).3.Under fluorescence microscope, oleic acid group compared with normal control group,the number of autophagy increased significantly.Sptlc2 transfection group compared with oleic acid group and empty vector group,autophagy number also increased significantly.4.Oleic acid group compared with normal control group,apoptosis related protein Bax expression was significantly increased,apoptosis related protein Bcl-2 expression was significantly decreased, Bax / Bcl-2 ratio was significantly larger(P<0.01); Sptlc2 transfection group compared with oleic acid group and empty vector group, apoptosis related protein Bax expression was significantly increased,apoptosis related protein Bcl-2 expression was significantly decreased, Bax / Bcl-2 ratio was significantly larger(P<0.01);5.Oleic acid group compared with normal control group,autophagy marker protein LC3B-II expression was significantly increased, autophagy marker protein LC3B-I expression decreased, LC3B-II/LC3B-I ratio was significantly larger(P<0.01); autophagy related protein Beclin-1 expression was significantly increased(P<0.01); autophagy related protein Atg5 expression was increased(P<0.05). Sptlc2 transfection group compared with oleic acid group and empty vector group,autophagy marker protein LC3B-II expression was significantly increased, autophagy marker protein LC3B-I expression decreased, LC3B-II/LC3B-I ratio was significantly larger(P<0.01); autophagy related protein Beclin-1 expression was significantly increased(P<0.01); autophagy related protein Atg5 expression was increased(P<0.05).Conclusions: 1.Oleic acid can induce the apoptosis and autophagy of HSC-T6 cells. 2. Up regulation of Sptlc2 which was the key enzymes of the de novo synthesis pathway of ceramide can promote the apoptosis and autophagy of HSC-T6 cells induced by oleic acid.