Effects Of CBS Gene Interference And OGD/R On Autophagy And Apoptosis And Related Signal Pathway In SH-SY5Y Cells

Objective: In order to investigate the changes of autophagy and apoptosis and related signal pathways in SH-SY5 Y cells after OGD/R and CBS gene interference.Methods: The siRNA pathway was used to interference the cystathionine β-synthase(CBS)gene and the Oxygen-glucose deprivation/reperfusion(OGD/R)model was constructed.According to the different time of oxygen glucose deprivation/reperfusion,the cells were divided into nine groups to construct OGD/R models,respectively,the normal control group,oxygen glucose deprivation4h/reperfusion 12 h group(4h/12 h group),oxygen glucose deprivation 4h/reperfusion24 h group(4h/24 h group),oxygen glucose deprivation 8h/reperfusion 12 h group(8h/12 h group),oxygen glucose deprivation 8h/reperfusion 24 h group(8h/24 h group),oxygen glucose deprivation 12h/reperfusion 12 h group(12h/12 h group),oxygen glucose deprivation 12h/reperfusion 24 h group(12h/24 h group),oxygen glucose deprivation 24h/reperfusion 12 h group(24h/12 h group),oxygen glucose deprivation24h/reperfusion 24 h group(24h/24 h group).The autophagosomes were observed by transmission electron microscopy(TEM);The siRNA targeting the CBS gene was transferred into SH-SY5 Y cells by liposome transfection,at the same time,OGD/R model was constructed,oxygen glucose deprivation 4 h/reperfusion 24 h.The cells were divided into three groups,only interference group,only OGD/R group and interference + OGD/R group.In only interference group,negative control was treated as control group;in only OGD/R group,normal cells was treated as control group;in interference + OGD/R group,negative control + OGD/R was treated as control group.To observe the toxic effects of transfection reagents on normal cells,the normal control group was seted as a control specially.Each of the above groups has three wells and three independent experiments;The interference efficiency of CBS gene was identificated by Western blotting,and the expression levels of protein LC3Ⅱ/LC3Ⅰ,Beclin-1,Akt,p-Akt,mTOR and p-mTOR in each group were detected;The expressions of protein NF-κBp65 and COX-2 were detected by Western blotting after OGD/R;The apoptosis levels of cells were measured by flow cytometry.Results: 1.Results of autophagosomes by transmission electron microscopy:Autophagosomes were Significantly observed in all groups in addition to the normal control group and oxygen glucose deprivation 4h/reperfusion 12 h group(4h/12 h group).2.The interference efficiency of CBS gen: Compared with the negative control group,the expression of CBS protein was significantly decreased in the interference group(P<0.05)and there was no significant difference between the negative control group and the normal control group(P>0.05).3.Result of Western Blotting: 3.1 The expression of LC3Ⅱ/LC3Ⅰ,Beclin-1,p-Akt/Akt andp-mTOR/mTOR proteins in the interference group were not significantly different from that in the negative control group(P>0.05).3.2 In OGD/R group,LC3Ⅱ/LC3Ⅰwas significantly higher than the normal control group(P<0.05).There was no significant difference in the expression of Beclin-1 protein between two groups(P>0.05).p-Akt/Akt and p-mTOR/mTOR of OGD/R group were significantly decreased than that in normal group(P<0.05).The expression of NF-κBp65 and COX-2 protein of OGD/R group was significantly increased than that of normal control group(P<0.05).3.3 The expression of LC3Ⅱ/LC3Ⅰ,Beclin-1,p-Akt / Akt and p-mTOR/mTOR proteins in the interference +OGD/R group were not significantly different from that in the negative control +OGD/R group(P> 0.05).4.Results of Apoptosis: 4.1 There was no significant difference in the apoptotic rate between the CBS group and the negative control group(P>0.05).4.2 Compared with the normal control group,OGD / R group cell apoptosis rate was significantly higher than the normal control group(P<0.05).4.3 The apoptotic rate between CBS interference + OGD / R group and negative control + OGD / R group was not significantly different(P>0.05).Conclusions: OGD / R can significantly induce autophagy and apoptosis in SH-SY5 Y cells and may be associated with Akt / mTOR and NF-κBp65 / COX-2 signaling pathways;CBS gene interference had no significant effect on cell autophagy and apoptosis.