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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。
Aim To investigate the protective effects of berberine on Aβ25-35-injured neurons and its mechanism. Methods Primary cultured rat cerebral cortical neurons were treated with 40 μg / mL Aβ25-35 for 36 h. Alzheimer’s disease (AD) cell models were replicated. At the same time, berberine was added for intervention. The experiment was divided into control group, model Group, 0.5μmol / L berberine group and 2μmol / L berberine group. The apoptosis of neurons was observed by Hoechst33258 staining. The apoptosis of neurons was detected by Annexin V-FITC / PI double-labeled flow cytometry. The expression of activated Caspase-3 protein was detected by Western blotting and the expression of rapamycin Target (target of Rapamycin, mTOR) related gene expression. Results Compared with the control group, the apoptosis of neuronal cells in AD cell model was significantly increased. Berberine at the concentration of 2μmol / L could significantly reduce the apoptosis of Aβ-injured neurons (P <0.05), and both at the concentration of 2μmol / L and berberine could inhibit the activation of Caspase-3 protein. 0.5, 2μmol / L berberine significantly down-regulated the mRNA expression of mTOR mRNA and S670706K mRNA (P <0.05,0.01), which were abnormally activated in AD model, and significantly upregulated the expression of AD cells The expression of 4E binding protein 1 (4EBP1) mRNA in the eukaryotic cell model (P <0.01). Conclusion Berberine can protect neurons from primary cultured rat cortical neurons damaged by Aβ25-35, and its protective effect may be through the inhibition of mTOR pathway.