六味地黄丸含药血清对Aβ诱导SH-SY5Y细胞的自噬影响及机制研究

Study on Effect and Mechanism of Liuwei Dihuang Pill on Autophagy Induced by Aβ in SH-SY5Y Cells

目的研究六味地黄丸含药血清对Aβ诱导SH-SY5Y细胞的自噬影响及其机制,探讨阿尔茨海默病的病理机制及补肾填精法的作用机制。方法30只大鼠随机分成对照组和六味地黄丸组两组,分别予以等量的双蒸水和含六味地黄丸粉末水溶液进行灌胃,末次灌胃结束后取血并分离血清备用。将SHSY5Y细胞分为对照组、模型组和药物组,对照组和模型组给予含空白血清的完全培养液,药物组给予含含药血清的完全培养液,模型组和药物组加入Aβ1-42寡聚体,培养48 h。应用CCK-8法对各组进行细胞增殖率分析;免疫荧光法分析各组细胞内Aβ沉积情况;Western Blot法检测各组细胞BACE1、p62、LC3-Ⅰ、LC3-Ⅱ、Beclin1蛋白表达情况。结果CCK-8结果显示,与对照组相比,模型组细胞增殖率显著降低(P<0.01);与模型组相比,药物组细胞增殖率显著增高(P<0.01)。与对照组相比,模型组细胞内Aβ的沉积显著增加(P<0.01);与模型组相比,药物组细胞内的Aβ沉积显著减少(P<0.01)。Western Blot结果显示,与对照组相比,模型组细胞的BACE1、Beclin1的蛋白表达量和LC3-Ⅱ/LC3-Ⅰ的比值显著升高(P<0.01),p62的蛋白表达量显著降低(P<0.01);与模型组相比,药物组细胞的Beclin1的蛋白表达量和LC3-Ⅱ/LC3-Ⅰ的比值显著升高(P<0.01),BACE1和p62的蛋白表达量显著降低(P<0.01)。结论六味地黄丸含药血清可以增加Aβ诱导的SH-SY5Y细胞自噬相关基因的表达,上调自噬水平,减轻细胞损伤,对细胞具有明显的保护作用。

Objective To study the effect of Liuwei Dihuang pill containing serum on autophagy of SH-SY5Y cells induced by Aβ and its mechanism, and to explore the pathological mechanism of Alzheimer’s disease and the action mechanism of tonifying kidney and filling essence method.Methods 30 rats were randomly divided into control group and Liuwei Dihuang Pill group. They were given the same amount of double distilled water and Liuwei Dihuang Pill powder solution by gavage respectively. After the last gavage, the blood was taken and the serum was separated for standby. SH-SY5Y cells were divided into control group, model group and drug group. The control group and model group were given complete culture medium containing blank serum. The drug group was given complete culture medium containing drug serum. The model group and drug group were added Aβ1-42 oligomer and cultured for 48 hours. CCK-8 method was used to analyze the cell proliferation rate;immunofluorescence method was used to analyze the deposition of Aβ;Western blot was used to detect the protein expression of BACE1, p62, LC3-Ⅰ, LC3-Ⅱ and Beclin1.Results CCK-8 results showed that compared with the control group, the cell proliferation rate of the model group was significantly decreased(P<0.01);compared with the model group, the cell proliferation rate of the drug group was significantly increased(P<0.01). Compared with the control group, the deposition of Aβ in the model group increased significantly(P<0.01);compared with the model group, the deposition of Aβ in the drug group decreased significantly(P<0.01). Compared with the control group, the protein expression of BACE1, Beclin1 and the ratio of LC3-Ⅱ/LC3-Ⅰ in the model group were significantly increased(P<0.01), and the protein expression of p62 was significantly decreased(P<0.01);compared with the model group, the protein expression of Beclin1 and the ratio of LC3-Ⅱ/LC3-Ⅰ in the drug group were significantly increased(P<0.01), and the protein expression of BACE1 and p62 were significantly decreased(P<0.01). Conclusion Liuwei Dihuang pill containing serum can increase the expression of autophagy related genes induced by Aβ, up regulate autophagy level, reduce cell damage, and has obvious protective effect on SH-SY5Y cells.