L-carnosine 对 NIT-1胰岛细胞的影响及机制

摘要

目的:研究肌肽(L-carnosine)对高糖培养的NIT-1细胞胰岛素分泌、增殖和凋亡的影响及机制.方法:(1)正常和高糖培养细胞72 h,放射免疫法测定葡萄糖刺激的胰岛素分泌,然后分别换不同浓度葡萄糖和L-carnosine培养,测定胰岛素分泌;(2)细胞分为C组(11.1 mmol/L glucose)、H组 (33.3 mmol/L glucose)、H+A组(33.3 mmol/L glucose +1 mmol/L L-carnosine)和H+B(33.3 mmol/L glucose+ 20 mmol/L L-carnosine)组培养72 h,BrdU检测细胞增殖,流式细胞术检测凋亡,RT-PCR测定bcl-2和caspase-3 mRNA的表达,荧光法检测caspase-3活性.结果:(1)高糖组细胞胰岛素分泌减少; 20 mmol/L L-carnosine显著增加正常和高糖组胰岛素分泌(P<0.01),且呈正相关(P<0.01);(2)高糖组细胞增殖和凋亡均增加(均P<0.01),但总体数目减少;1 mmol/L L-carnosine使增殖增加,凋亡减少(P<0.01);(3) 高糖组caspase-3 mRNA表达明显增加,bcl-2 mRNA明显减少(P<0.05);1 mmol/L L-carnosine使前者明显减少(P<0.05),后者明显增加(P<0.01);不同浓度L-carnosine均明显降低caspase-3活性.结论:高浓度L-carnosine可单独刺激细胞分泌胰岛素,低浓度的L-carnosine可增加NIT-1细胞增殖并减少高浓度葡萄糖所导致的凋亡.Caspase-3和Bcl-2可能参与了L-carnosine保护NIT-1细胞的过程.%AIM: To investigate the protective effect of L — carnosine on insulin secretion, proliferation and ap-optosis of (3 — cells impaired by high glucose. METHODS: NIT — 1 cells were pre — treated with glucose at concentrations of 11. 1 mmol/L ( control level ) and 33.3 mmol/L ( high level ) for 72 h, and then the cells were stimulated with various concentrations of glucose (0,5 and 25 mmol/L ) and/or L — carnosine (0,1 and 20 mmol/L ). The level of insulin in the medium was measured by radioimmunoassay. To detect the effect of L — carnosine on proliferation and apoptosis, NIT — 1 cells were divided into 4 groups according to different culture conditions for 72 h: group C ( with 11.1 mmol/L glucose ), group H ( with 33. 3 mmol/L glucose ), group H + A ( with 33. 3 mmol/L glucose + 1 mmol/L L - carnosine ) and group H + B ( with 33. 3 mmol/L glucose + 20 mmol/L L - carnosine ). Proliferous or apoptotic cells were identified by BrdU labeling and flow cytometry ( labeling with annexin V - FITC/PI ), respectively. Total RNA was extracted and the mRNA expression of caspase - 3 and bcl - 2 was measured by RT - PCR. The caspase - 3 activity was also checked by fluorometric assay kit. RESULTS: The insulin in high - level glucose group was lower than that in control - level glucose group. L -carnosine at concentration of 20 mmol/L notably increased the insulin secretion of the cells pre - treated with glucose at control level or high level. The proliferation and apoptosis were both increased in group H compared with group C, but the total cell counts declined because the apoptotic rate was higher than the proliferation rate. L - carnosine at concentration of 1 mmol/L significantly increased the proliferation rate and decreased the apoptotic rate. The mRNA level of caspase — 3 was decreased and the mRNA level of bcl - 2 was increased after the cells were treated with L - carnosine at concentration of 1 mmol/L. L — carnosine at concentrations of both 1 mmol/L and 20 mmol/L significantly decreased the caspase — 3 activity. CONCLUSION: L — carnosine at high level directly stimulates insulin secretion in NIT — 1 cells, and L — carnosine at normal level promotes the cell proliferation and inhibits apoptosis induced by high concentration of glucose. Caspsase — 3 and Bcl - 2 may be partly involved in this process.