沉默小窝蛋白1对棕榈酸作用下的胰岛β细胞存活的影响

Effects of caveolin-1 deficiency on palmitic acid-induced apoptosis of islet-βcells

目的探讨小窝蛋白1(Cav1)对棕榈酸作用下的胰岛β细胞存活的影响。方法通过慢病毒载体构建Cav1沉默的小鼠胰岛素瘤NIT-1细胞株和小鼠原代胰岛(Cav1-shRNA组),以空载体病毒组作为对照组(Ctrl-shRNA),并设空白对照组,各组均分别加入脱脂牛血清白蛋白和棕榈酸(0.5 mmol/L)处理24 h及48 h,通过四甲基偶氮唑蓝比色法及Hoechst33342/碘化丙啶(PI)双染色法检测细胞活性及细胞凋亡,实时荧光定量PCR、Western blot检测凋亡相关的mRNA及蛋白表达水平。结果采用t检验和单因素方差分析法进行统计学分析。结果与对照组比较,棕榈酸处理组的细胞存活率显著下降(0.89±0.10比0.24±0.04,t=13.49,P<0.01),P18、P19的mRNA和蛋白表达均上调(1.00±0.09比1.30±0.04、1.00±0.04比1.37±0.13,t=5.28、4.71,均P<0.05;0.87±0.04比1.48±0.05、1.02±0.06比1.41±0.07,t=16.50、7.33,均P<0.01);含半胱氨酸的天冬氨酸蛋白水解酶(Caspase)-6、Caspase-9和Caspase-12的mRNA表达水平明显上调(1.00±0.04比1.57±0.08、1.01±0.15比1.57±0.20、1.02±0.19比1.57±0.09,t=11.04、3.88、4.53,均P<0.05),蛋白表达也明显上调(0.86±0.10比1.28±0.11、0.69±0.01比0.86±0.06、0.70±0.02比1.18±0.01,t=4.89、4.84、37.18,均P<0.01)。而Cav1-shRNA+棕榈酸组细胞存活率较Ctrl-shRNA+棕榈酸组显著上升(0.53±0.10比0.26±0.08,t=4.71,P<0.01),P19的mRNA和蛋白表达均下调(1.53±0.18比0.66±0.04、1.48±0.05比0.70±0.02,t=8.17、25.09,均P<0.01);Caspase-6、Caspase-7、Caspase-9和Caspase-12的mRNA表达水平下调(1.82±0.11比1.03±0.07、1.43±0.24比0.42±0.15、1.41±0.22比0.51±0.18、1.45±0.18比0.42±0.13,t=5.48~10.49,均P<0.01),蛋白表达也下调(0.99±0.14比0.63±0.11、0.90±0.14比0.62±0.04、0.90±0.02比0.60±0.04、1.30±0.01比0.65±0.04,t=3.50~27.31,均P<0.01)。结论Cav1沉默可以通过影响胰岛β细胞Caspase家族,促进其增殖、减少棕榈酸作用下的β细胞凋亡,保护脂毒性下胰岛β细胞活性。

Objective To investigate the role of caveolin-1(Cav1)in palmitic acid-induced survival of pancreatic isletβ-cell.Methods Cav1-deficient NIT-1 cells and mouse primary islets were constructed through lentiviral vector transfection.Control-shRNA was transfected as a control.After fatty-acid-free bovine serum albumin(BSA)and palmitic acid(0.5 mmol/L)incubation for 24 and 48 hours,cell viability and apoptosis werecalculated by methylcyclopentadienyl manganese tricarbonylassay and Hoechst33342/propidium iodide(PI)double staining.Real time PCR and Western blot were used to detect the expression of apoptosis-associated mRNA and protein.The t test and one-way ANOVA were used for statistical analysis.Results Compared with the control group,the cell survival rate of the palmitic acid treatment group was significantly decreased(0.89±0.10 vs 0.24±0.04,t=13.49,P<0.01),and mRNA and protein expressions of P18 and P19 were up-regulated(1.00±0.09 vs 1.30±0.04,1.00±0.04 vs 1.37±0.13,t=5.28,4.71,both P<0.05;0.87±0.04,1.48±0.05,1.02±0.06 vs 1.41±0.07,t=16.50,7.33,both P<0.01);mRNA expression levels of cysteine-containing aspartic proteolytic enzymes(Caspase-6,Caspase-9,and Caspase-12)were significantly up-regulated(1.00±0.04 vs 1.57±0.08,1.01±0.15 vs 1.57±0.20,1.02±0.19 vs 1.57±0.09,t=11.04,3.88,4.53,all P<0.05),and protein expression of them were also significantly increased(0.86±0.10 vs 1.28±0.11,0.69±0.01 vs 0.86±0.06,0.70±0.02 vs 1.18±0.01,t=4.89,4.84,37.18,all P<0.01).The cell survival rate of Cav1-shRNA+palmitic acid group was significantly higher than that of Ctrl-shRNA+palmitic acid group(0.53±0.10 vs 0.26±0.08,t=4.71,P<0.01).The mRNA and protein expression of P19 were all down-regulated(1.53±0.18 vs 0.66±0.04,1.48±0.05 vs 0.70±0.02,t=8.17,25.09,both P<0.01);mRNA expression levels of Caspase-6,Caspase-7,Caspase-9 and Caspase-12 were down-regulated(1.82±0.11 vs 1.03±0.07,1.43±0.24 vs 0.42±0.15,1.41±0.22 vs 0.51±0.18,1.45±0.18 vs 0.42±0.13,t=5.48-10.49,all P<0.01),and protein expression of them were also down-regulated(0.99±0.14 vs 0.63±0.11,0.90±0.14 vs 0.62±0.04,0.90±0.02 vs 0.60±0.04,1.30±0.01 vs 0.65±0.04,t=3.50-27.31,all P<0.01).Conclusion Cav1 deficiency inhibites palmitic acid-induced pancreaticβ-cell apoptosis via regulating Caspase family,and ultimately protectsβcell viability.