Neuronal apoptosis and expressions of Ire1α and caspase-12 in rats with spinal cord ischemia reperfusion injury / 中华创伤杂志

Objective To investigate the expression changes of Ire1α and caspase-12 in rats with spinal cord ischemia-reperfusion injury.Methods Fifty-five adult SD rats(250-300 g)were randomly divided into control group(re=5)and operation group(n=50).The spinal cord ischemia-reperfusion models were established and the neuronal apoptosis was detected by terminal deoxynucleotidyl transferasemediated dUTP nick end labeling(TUNEL).The expressions of Ire1α and caspase-12 in spinal cord tissue were detected by immunohistochemistry,immunofluorescence and Western blot analysis at 1,4,8,16and 24 hours after ischemia-reperfusion.Results TUNEL staining showed that the number of apoptotic cells was gradually elevated with time.The expressions of Ire 1α and caspase-12 were increased at 1 hour after reperfusion,and peaked at 16 hours,but began to decline at 24 hours after reperfusion.The number of neurons with positive expressions of Irelaand caspase-12 was significantly higher than that of control group(P＜0.05).Conclusion Ire 1α and caspase-12 synergistically participate in the neuronal apoptosis induced by the endoplasmic reticulum stress.

The effect of proteasomal inhibitors on anterior pharynx decfective-1 expression in neuronal cells / 中华神经科杂志

Objectives To investigate whether degradation of anterior pharynx decfective-1(Aph-1) goes through proteasomal pathway or lysosomal pathway.Methods Various methods such as cell culture,Western blotting,pulse-chase metabolic labeling technique,double immunofluoresecnt staining,combined with proteasomal and lysosomal inhibition were used to check Aph-1 expression level in stable Aph-1-transfected or non-transfected neuronal(SH-SY5Y)cell line.Results Using Western blotting,treating the neuronal cells with proteasome specific inhibitors significantly increased the expression of both endogenous and exogenous Aph-1.The effect of the proteasome inhibitors on Aph-1 expression was dose-and time-dependent Lysosomal pathway was not involved in Aph-1 degradation. Pulse-chase metabolic labeling experiment showed that the turnover of newly-synthesized radiolabeled Aph-1 protein was blocked by Lactacystin.Double immunofluorescent staining revealed colocalization of Aph-1 and ubiquitin in the same cells.Conclusion Degradation of Aph-1 protein is mediated by proteasomal pathway in neuronal cells,and is not related to lysosomal pathway.Aph-1 protein is ubiquitinated before degradation.

Effect of proteasomal inhibitors on nicastrin expression in neuronal cells / 中国病理生理杂志

AIM: To explore the possibility that proteasome is involved in nicastrin(NCT) degradation and NCT is ubiquitinated before degradation.METHODS: Following the generation of NCT stable cell lines,the methods of Western blotting,pulse-chase metabolic labeling technique,double immunofluorescent staining,combined with proteasomal inhibition were used to investigate the NCT expression in NCT stable cell line.RESULTS: Treatment of the cells with proteasomal inhibitors significantly increased both endogenous NCT(produced by the cell itself) and exogenous NCT(produced by the gene transfection) in SH-SY5Y cells.The effect of specific proteasomal inhibitor lactacystin on NCT expression was in time-and dose-dependent manners.Pulse-chase metabolic labeling experiment showed that the turnover of newly-synthesized radio-labeled nicastrin protein was blocked by lactacystin.The results of double immunofluorescent staining showed that NCT and ubiquitin were co-located in the cells.CONCLUSION: The proteasome is involved in the degradation of NCT in neuronal cells,and NCT is ubiquitinated before degradation.