Cdk5活性抑制肽CIP对糖尿病治疗作用机制的相关研究

The study of effects and mechanism of the Cdk5 inhibitory peptide in type 2 diabetes

目的探讨周期性依赖性蛋白激酶5(Cdk5)的活性抑制肽(CIP)在糖尿病中的治疗作用及其机制。方法体外培养Min6小鼠胰岛细胞(以下简称胰岛细胞),应用基因克隆CIP质粒,转入腺病毒,转染细胞。根据是否导入CIP基因分为空载体组(EV组)和CIP转染组。应用不同浓度的葡萄糖(5 mmoL/l和25 mmol/L)刺激胰岛细胞,分为EV+5 mM组、EV+25 mM组、CIP+5 mM组、CIP+25 mM组。用免疫印迹和免疫荧光检测基因的表达和细胞凋亡情况,同位素标记测定酶的活性,酶联免疫吸附试验(ELISA)测定胰岛素的分泌水平。体内试验,正常小鼠、糖尿病小鼠组、CIP+糖尿病小鼠组,喂养14周时腹腔内注射EV和CIP病毒,检测各组小鼠体重、血糖、糖化血红蛋白指标及各种分子病理实验。结果在同样浓度的高糖刺激Min6细胞下,Cdk5的活性在CIP组较EV组明显受到抑制(P<0.01);高糖(HG)+EV组较低糖(LG)+EV组细胞凋亡相关蛋白表达增加(P<0.01),而转染了CIP基因细胞的凋亡相关蛋白较HG+EV细胞明显下降(P<0.01);胰岛素基因的表达和分泌在EV组高糖刺激6 h的细胞较2 h的细胞明显降低,而转染CIP的细胞较EV细胞胰岛素基因的表达和分泌均明显升高(P<0.01);体内试验结果显示,体重、血糖和糖化血红蛋白指标,在db/db组与db/db+CIP组差异无统计学意义(P>0.05);细胞凋亡蛋白在db/db+CIP组较db/db组明显降低;胰岛素基因的表达db/db+CIP组较db/db组明显增高。结论 CIP可以降低Min6细胞由于高糖导致的Cdk5的过度活性,抑制胰岛细胞的凋亡,恢复胰岛素的分泌,这可能是CIP在糖尿病中起到保护性作用的机制之一。

Objective To explore the treatment effect and possible mechanism of Cdk5 inhibitory peptide （CIP） in type 2 dia- betes （T2DM）. Methods In vitro ： mouse insulinoma line 6 （MIN6） cells was cultured in vitro. Cloning CIP plasmid and transferred it into the adenovirus virus, and then infected it into cells. Experiment groups： according whether infection of empty vector （EV） or CIP, and the concentration of glucose treatment, the cells was divided into three groups： 5mM （LG） ＋ EV group; 25mM （HG） ＋ EV group; 5mM ＋ CIP group; 25mM ＋ CIP group for Min6 cells. The gene expression and apoptosis were detected by using immunoblot- ting and immunofluorescence. Cdk5 activity was measured by using isotope - labeled method, and level of insulin secretion was meas- ured by ELISA. In vivo： there were three groups of mice： control group, db ＋ EV group and db＋ CIP group. The mice were injected with EV and CIP - virus by Intraperitoneal injection at age 14 week and sacrificed for the experiments. Results Cdk5 activity in the HG ＋ CIP cells was significantly inhibited than in the cells with HG ＋ EV （P 〈 0.01 ）. The cell apoptosis related protein expression was sig- nificantly increased in the HG ＋ EV cells, but it was remarkably decreased in the HG ＋ CIP cells （ comparing to HG ＋ EV cells, P 〈0.01 ）. The insulin gene expression and secretion were inhibited in the HG ＋ EV cells at the 6hrs HG treatment, however, they were increased in the cells with HG ＋ CIP group （P 〈0.01 ）. In vivo, the changes of mice body weight, blood glucose and glycosylated he- moglobin were no significantly different between db/db mice and db/db ＋ CIP mouse, but the reduction tendency was showed （ P value were 0. 073, 0.062, 0.060, respectively）. The cell apoptosis protein expression in the pancreatic islet was decreased in db/db ＋ CIP mice, and insulin gent expression was increased in db/db ＋ CIP mice. Conclusion CIP can inhibit the abnormal Cdk5 activity and in- hibit cell apoptosis, meanwhile, recover the expression and secretion of insulin, which is induced by high concentrations of glucose. Therefore, CIP may develop a Cdk5 inhibition strategy in the treatment of T2DM.