PI3K抑制剂对2型糖尿病小鼠脑组织中乙酰胆碱酯酶表达的影响

Effect of PI3K inhibitor on AchE expression in brain tissues of T2DM mice

目的研究磷脂酰肌醇3-激酶(PI3K)抑制剂对2型糖尿病小鼠脑组织中乙酰胆碱酯酶(Ach E)表达的影响,并初步探讨其可能的分子机制。方法将16只自发性2型糖尿病模型动物db/db小鼠随机分为抑制组和模型组各8只,分别腹腔注射溶于5%DMSO中的PI3K抑制剂LY294002 3 mg/kg和相同体积的5%DMSO溶液;将6只同周龄非糖尿病动物db/m小鼠作为正常对照组,腹腔注射相同体积的生理盐水。各组小鼠连续给药7 d,取血和肾脏组织;比色法检测血糖,HPLC法检测血清丙二醛(MDA),酶底物法测定大脑皮质Ach E,Western blotting法检测大脑皮质中哺乳动物雷帕霉素靶蛋白(m TOR)及其下游蛋白70 k D核糖体S6蛋白激酶(p70S6K)磷酸化水平。结果与正常对照组比较,模型组血糖和血清MDA水平均增高(P均<0. 01);与模型组比较,抑制组血清MDA水平降低(P <0. 05),而血糖水平无明显变化。模型组大脑皮质中Ach E表达量为0. 957±0. 089,高于正常对照组的0. 508±0. 222(P <0. 05);模型组大脑皮质中Ach E表达量(0. 506±0. 161)低于模型组(P <0. 05),而与正常对照组差异无统计学意义。与正常对照组比较,模型组大脑皮质中m TOR和p70S6K磷酸化水平均增加(P均<0. 05);与模型组比较,抑制组大脑皮质中m TOR和p70S6K磷酸化水平降低(P <0. 05或<0. 01)。结论 PI3K抑制剂LY294002可降低2型糖尿病小鼠脑组织中Ach E表达水平,该作用可能与其降低氧化应激水平从而抑制mTOR相关通路的活性有关。

Objective To investigate the effects of phosphatidylinositide 3-kinases (PI3K) inhibitor on mammalian target of rapamycin (mTOR) signaling and acetylcholinesterase (AchE) protein expression in the brain tissues of mice with type 2 diabetes mellitus (T2DM) and to explore its possible molecular mechanism. Methods Sixteen db/db mice were randomly divided into two groups:the inhibition group and model group,with 8 rats in each,which were injected intraperitoneally with PI3K inhibitor LY294002 3 mg/kg (dissolved in 5% DMSO) and the same volume of 5 % DMSO solution;meanwhile,the 6 age-matched db/m mice were used as the control group.After treatment for 7 days,blood and brain samples were collected for biochemical analysis.Blood glucose was measured by glucose oxidase method,blood MDA level was measured by HPLC,acetylcholinesterase (AchE) activity was measured by enzyme substrate method,and the protein expression levels of mammalian target of rapamycin (mTOR) and the downstream protein p70 ribosomal S6 protein kinase (p70S6K) were detected by Western blotting. Results Compared with the control group,the blood glucose and MDA levels increased in the model group (both P <0.01).Compared with the model group,the serum MDA level in the inhibition group decreased ( P <0.05),while the blood glucose level did not change significantly.The activity of AchE in the cerebral cortex of the model group was 0.957±0.089,which was higher than that of the normal control group (0.508± 0.222 )( P <0.05).The expression of AchE in the cerebral cortex of the inhibition group (0.506±0.161) was lower than that of the model group ( P <0.05);and there was no significant difference between the inhibition group and the control group.Compared with the control group,the expression of AchE protein,mTOR and phospho-p70S6K protein in the cerebral cortex of the model group increased (all P <0.05).Compared with the model group,AchE protein expression,mTOR and phospho-p70S6K protein decreased in the cerebral cortex of the inhibition group ( P <0.05 or P <0.01). Conclusion The PI3K inhibitor LY294002 could elevate the AchE expression in the brain tissues of db/db mice by decreasing the oxidative stress and thus inhibiting the activity of mTOR signaling pathway.