小檗碱改善高糖高脂诱导的胰岛NIT-1细胞凋亡的分子机制

目的观察小檗碱对高脂高糖诱导后NIT-1胰岛β细胞凋亡的改善情况,探讨小檗碱抗凋亡的分子机制。方法采用四甲基偶氮唑蓝(MTT)比色法,在培养基中加入高糖高脂及小檗碱共同培养24h后,比较不同浓度的小檗碱对体外培养及高糖高脂诱导的NIT-1胰岛β细胞活力的影响;AnnexinV/PI双标记法流式细胞术检测细胞凋亡情况;RT-PCR法检测凋亡相关基因bcl-2、bax和caspase-3mRNA表达水平;免疫荧光法检测caspase-3表达。结果与正常对照组相比,模型组NIT-1细胞凋亡率明显升高(P<0.01),bcl-2mRNA表达明显降低(P<0.01),而bax和caspase-3mRNA表达明显增高(P<0.01),且胞质caspase-3表达增加;与模型组相比,小檗碱组NIT-1细胞凋亡率明显降低(P<0.01),bcl-2mRNA表达增高(P<0.01),而bax和caspase-3mRNA表达降低(P<0.01),小檗碱组胞质caspase-3表达亦降低。结论小檗碱抑制高糖高脂诱导NIT-1细胞的凋亡可能与其激活bcl-2和抑制bax和caspase-3的表达有关。

Molecular mechanism of berberine''''s inhibitory effects on the apoptosis of NIT-1 cells induced by high glucose and saturated fatty acids

Aim To investigate whether berberine could inhibite the apoptosis of pancreatic islet β cells strains NIT-1 cells induced by high glucose and saturated fatty acids,and to explore its molecular mechanism.Methods Tetrazolium(MTT) assay was applied to detect the influence of various concentrations ofberberine and high concentration of glucose and free fatty acids on the viability of NIT-1 cells after incubation 24 hrs.Using Annexin V/PI double labeling method,the apoptosis of cultured cells was detected with the technique of flow cytometry.The cellular mRNA expression of apoptosis related genes such as bcl-2,bax and caspase-3 was determined by RT-PCR,further caspase-3 protein expression was determined by immunofluorescence assay.Results Compared with normal group,the apoptotic rate of NIT-1 cells in model group was significantly elevated(P<0.01),bcl-2 mRNA expression significantly decreased(P<0.01),while bax and caspase-3 mRNA expression obviously increased(P<0.01),and cytoplasmic caspase-3 also increased.In comparison with model group,the apoptotic rate of NIT-1 cells in berberine group was significantly declined(P<0.01),bcl-2 mRNA expression significantly increased(P<0.01),while the mRNA expression of bax and caspase-3 evidently reduced(P<0.01),and cytoplasmic caspase-3 also decreased.Conclusion Berberine could inhibit NIT-1 cell apoptosis induced by high glucose and saturated fatty acids,which might be associated with its activation of bcl-2 and inhibiton of bax and caspase-3 expression.