TFP5抑制高糖诱发的细胞周期依赖性激酶5过度激活、减少胰岛β细胞凋亡

TFP5 inhibits high glucose-induced hyperactivity of cyclin dependent kinase 5 and protects pancreatic beta cells from apoptosis in vitro

【摘要】目的 探讨TFP5对高糖诱发的细胞周期素依赖性激酶5（Cdk5）活性的抑制作用及其对胰岛β细胞的保护作用。方法 体外培养小鼠胰岛细胞株Min6。将TFP5转染Min6细胞，分别用免疫印迹和免疫荧光观察其表达。实验分3组：低糖（5mmol/L）组，高糖（25mmol/L）+空病毒载体（EV）组和高糖（25mmol/L）+TFP5组，分别用同位素标记法测定3组细胞内Cdk5激酶活性；用酶联免疫吸附法测定3组细胞胰岛素分泌水平；用免疫印迹法测定胰岛β细胞凋亡。结果 （1）表明TFP5来源和它的分子序列；（2）TFP5在Min6细胞内表达良好；（3）在高糖+TFP5组Cdk5的活性明显低于高糖+EV组（P＜0.01），而且在高糖+TFP5组胰岛素分泌明显高于高糖+EV组（P＜0.01）；（4）在高糖组Min6细胞的Bax表达增加，Bcl-2表达减少，Bax/Bcl-2比值升高，细胞凋亡增加（P＜0.01，vs 低糖组）， 而加入TFP5后，Bax表达减少，Bcl-2表达增加，Bax/Bcl-2的比值减低（P＜0.01，vs 高糖组），细胞的凋亡减少。结论 TFP5可抑制高糖诱发的Cdk5激酶的过度活性、减少高糖刺激下胰岛细胞凋亡，从而恢复胰岛素分泌，具有潜在的以Cdk5为靶点治疗2型糖尿病的前景。

基金项目:国家自然科学基金（81060066）; 宁夏回族自治区自然科学基金（NZ10162）; 宁夏回族自治区科技攻关项目（KGX-19-10-16）;宁夏回族自治区科技攻关国际合作项目（2011ZYH169）

【Abstract】Objective To determine the inhibitory effect of TFP5 on the hyperactivity of cyclin dependent kinase 5 (Cdk5) induced by high glucose and its protection for the pancreatic beta cells from apoptosis. Methods After TFP5 was delivered into mouse insulinoma line 6 (Min 6), the expression of TFP5 was detected in the cells by Western blotting and immunofluorescence staining. Then, the cells were treated by low (5mmol/L), high glucose (25mmol/L)+empty vector (EV), and high glucose +TFP5, respectively. Cdk5 activities were measured by immune precipitation and isotope labeling. The level of insulin secretion were detected with ELISA; and the apoptosis of Min 6 cells were analyzed by Western blotting. Results (1) Our results indicated the derivation of TFP5 peptide from p35 and its sequence. (2) TFP5 was well expressed in the Min 6 cells. (3) Cdk5 activity was significantly lower in the high glucose+TFP5 cells than in the high glucose+EV cells (P＜0.01); the insulin secretion level in the high glucose+TFP5 cells was significantly higher than that in the high glucose cells (P＜0.01). (4) In the high glucose cells, the expression of Bax was increased while that of Bcl-2 was decreased, and the ratio of Bax/Bcl-2 was increased (P＜0.01, vs the low glucose cells), indicating the increase of cell apoptosis. However, the expression of the 2 proteins was opposite, and the ratio was decreased in the high glucose+TFP5 cells, showing lesser apoptotic cells (P＜0.01, vs the high glucose cells). Conclusion TFP5 inhibits the hyperactivity of Cdk5 induced by high glucose, protects pancreatic cells from apoptosis, and recovers the insulin secretion. Therefore, it may have a potential therapeutic agent for type 2 diabetes targeting on Cdk5.