GLP-1Ra减少高糖诱导的β细胞凋亡作用机制探讨

Glucagon-like peptide 1 receptor agonist protects high-glucose induced β cells apoptosis via inhibition of NOX2-dependent ROS production

目的探讨胰高血糖素样肽-1(GLP-1)受体激动剂(GLP-1Ra)减少高糖诱导的β细胞凋亡作用的可能机制。方法正常对照(N,普通饲料喂养)组、2型糖尿病(T2DM)组和GLP-1 Ra组[利拉鲁肽200μg/(kg·d)]大鼠干预12周。比较各组大鼠造模前、造模后给药前(0周)及12周末血糖水平。高压液相色谱分析法测定糖化血红蛋白(Hb A1c);全自动生化分析仪测定天冬氨酸转氨酶(AST)、肌酐(CR)及尿素氮(BUN)等;TUNEL染色观察胰岛细胞凋亡情况;免疫组化法测定cleaved caspase 3;DCFH-DA荧光探针检测胰岛活性氧簇(ROS);免疫组织化学法检测NADPH氧化酶(NOX)催化亚基(NOX2)。结果 12周时,GLP-1Ra组的血糖、Hb A1c、总胆固醇(TC)及低密度脂蛋白胆固醇(LDL-c)水平均低于T2DM组(P<0.05);GLP-1Ra组胰岛细胞凋亡率和cleaved caspase 3水平较T2DM组下降(P<0.05);应用Apocynin抑制前,GLP-1Ra组胰岛ROS水平明显低于T2DM组,并与N组差异无统计学意义(P>0.05),应用Apocynin抑制后,各组间差异均无统计学意义(P>0.05)。GLP-1Ra组胰岛NOX2水平较T2DM组下降(P<0.05)。结论 GLP-1Ra能抑制糖尿病大鼠β细胞的凋亡,抑制NOX2来源的ROS产生可能是重要的潜在机制之一。

Objective To investigate the possible mechanisms of glucagon-like peptide 1 receptor agonists （GLP- 1Ra） protection against hyperglycemic induced beta cell apoptosis through depression of NOX2-dependent ROS production. Methods The rat model of type 2 diabetes （T2DM） was established by injecting small doses of streptozotocin （STZ） fol- lowed by 8-week high fat diet. The experimental animals were divided into three groups： normal control （N） group, diabetes （T2DM） group and GLP-IRa group [treated with liraglutide 200 μg/（kg, d） for 12 weeks]. The blood glucose levels were compared before and after modeling, before treatment and 12-week after treatment with GLP- 1Ra. The level of glycosylated hemoglobin （HbAlc） was detected by high-pressure liquid chromatography. Automatic biochemical analyzer was used to detect levels of aspertate aminotransferase （AST）, creatinine （CR） and urea nitrogen （BUN）. The apoptotic rates of islets were determined by TUNEL method and cleaved caspase 3 was detected by immunohistochemistry. DCFH-DA fluorescent probe was used to detect reactive oxygen species （ROS） levels of islets. Levels of NADPH oxidase （NOX） catalytic subunit （NOX2） in islets were measured by immunohistochemistry. Results At the end of the study, glycemic control （average blood glucose/ week and HbAlc） and lipid situation were improved significantly in the GLP-IRa group than those of N group （P〈 0.05）. TUNEL staining and displayed that β cell apoptotic and cleaved caspase 3 level were significantly decreased in GLP-IRa group compared to those of T2DM group （P 〈 0.05）. ROS levels were significantly decreased in GLP- 1Ra group than those of T2DM group before treatment with Apocynin, but no significant difference between GLP1-Ra group and N group （P 〉 0.05）. After application Apocynin for inhibition, there were no significant differences between three groups （P 〉 0.05）. The level of NOX2 was significantly lower in GLP- 1Ra group compared to that of T2DM group （P 〈 0.05）. Conclusion GLP- IRa can inhibit apoptosis of β cells in diabetes rat, and the depression of NOX2-dependent ROS may be one of the important underlying mechanisms.