目的 考察小檗碱对Aβ25-35损伤的神经元的保护作用,并初步探讨其机制。方法 40µg/mL Aβ25-35作用原代培养大鼠脑皮层神经元36 h,复制阿尔茨海默病(Alzheimer’s disease,AD)细胞模型,同时加入小檗碱进行干预,实验分为对照组、模型组、0.5µmol/L小檗碱组、2µmol/L小檗碱组。通过Hoechst33258染色观察神经元凋亡形态,Annexin V-FITC/PI双标流式细胞术检测早期凋亡情况,Western blotting检测活化的Caspase-3蛋白表达,实时荧光定量RT-PCR检测雷帕霉素靶(target of Rapamycin,mTOR)相关基因表达。结果 与对照组比较,AD细胞模型神经元凋亡明显增加。2µmol/L小檗碱能显著减少Aβ损伤神经元早期凋亡(P<0.05),0.5、2µmol/L小檗碱均能抑制异常活化的Caspase-3蛋白表达。0.5、2µmol/L小檗碱显著下调了AD模型异常活化的mTOR mRNA和下游底物核糖体S6蛋白激酶(S6kinase,S6K)P70S6K mRNA的表达(P<0.05、0.01),同时显著上调了AD细胞模型真核细胞始动因子4E结合蛋白1(4E binding protein,4EBP1)mRNA的表达(P<0.01)。结论 小檗碱对Aβ25-35损伤的原代培养大鼠皮层神经元具有一定神经保护作用,其保护作用可能是通过抑制mTOR途径实现的。

Objective To observe the protective effect of berberin on the cortical neurons of rat injured by Aβ_25-35 and investigate its mechamism. Methods Apzs-ss was used to treat the cortical neurons of primary culture rat, at the final concentration of 40 μg/mL for 36 h, Alzheimer's disease (AD) model was reproduced, and then interferred with berberin together. The experiments were divided into control, model, 0.5 μmol/L berberin, and 2 μmol/L berberin groups. Hoechst33258 stain assay was used to observe the morphological change, Annexin V-FITC/PI double stain flow cytometry assay was used to examine the early neural apoptisis; Western blotting assay was used to observe the activated Caspase-3 protien expression; and RT PCR assay was used to investigate the mRNA expression of target of Rapamycin (mTOR) related genes. Results Neural apoptosis of AD model significantly increased when treated with 40 μg/mL Aβ_25-35 for 36 h compared with the control group (P<0.01). Following 2 μmol/L berberin treatment, the percentage of Aβ_25-35-induced cell apoptosis significantly decreased (P<0.05) compared with the model group. Berberin (0.5 and 2 μmol/mL) inhibited Apzs-3s-induced abnormal activated Caspase-3 expression, down-regulated the expression of abnormal activated mTOR mRNA and S6kinase P70S6K mRNA (P<0.05,0.01), and up-regulated the expression of 4E binding protein (4EBP1) mRNA of AD model (P<0.01). Conclusion Berberin exhibits the protective effects against Aβ_25-35-induced toxicity in cortical neurons of primary cultured rat, which maybe relates with the inhibition of mTOR signal pathway.

教育部科学技术研究重点项目(208009);高等学校博±学科点专项科研基金(20101210110001)