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目的:研究大鼠更年春含药血清对β淀粉样蛋白25-35(amyloid beta protein 25-35,Aβ_(25-35))导致的大鼠肾嗜铬细胞瘤细胞(pheochromocytoma cell,PC12)损伤的抗凋亡作用。方法:通过对去势雌性大鼠灌服中药更年春制备含药血清,细胞计数试剂盒(cell counting kit-8,CCK-8)检测不同浓度含药血清对PC12细胞存活率的影响;不同浓度Aβ_(25-35)作用于PC12细胞,建立阿尔茨海默病细胞模型,CCK-8法检测含药血清对损伤细胞的保护作用,流式细胞术检测PC12细胞在Aβ_(25-35)和含药血清共同培养下的细胞凋亡率;蛋白印迹法检测各组细胞Bcl-2、Bax和半胱氨酸天冬氨酸特异性蛋白酶3(caspase-3)的蛋白表达情况。结果:与空白血清相比,20%含药血清培养PC12细胞24 h或48 h后可促进其增殖(P<0.05)。Aβ_(25-35)对PC12细胞有细胞毒性,并呈剂量依赖性地降低细胞存活率(P<0.05),导致细胞凋亡。CCK-8法和流式细胞术检测均显示,20%更年春含药血清对Aβ_(25-35)诱导的PC12细胞损伤有保护作用(P<0.05),其作用效果类似于阳性对照药神经生长因子。蛋白印迹法检测显示,与模型组比较,含药血清组Bax和Bcl-2蛋白表达的比值和caspase-3蛋白表达均降低。结论:更年春含药血清可提高Aβ_(25-35)损伤的PC12细胞的存活率,并减少PC12细胞凋亡。其机制可能与调控Bcl-2家族蛋白的表达而实现抗凋亡作用有关。
OBJECTIVE: To investigate the effect of serum of rats containing more year-of-spring on injury of rat renal pheochromocytoma cell (PC12) induced by amyloid beta protein 25-35 (Aβ 25-35) Anti-apoptotic effect. Methods: The ovaries of ovariectomized female rats were treated with Chinese medicine to prepare the drug-containing serum. Cell counting kit-8 (CCK-8) was used to detect the effect of serum containing different concentrations on the survival rate of PC12 cells. Aβ_ (25-35) was induced in PC12 cells to establish a cell model of Alzheimer’s disease. CCK-8 assay was used to detect the protective effects of the drug-containing serum on injured cells. Flow cytometry was used to detect the expression of PC12 in Aβ_ (25-35) and The apoptosis rates of the cells were detected by Western blotting. The protein expressions of Bcl-2, Bax and caspase-3 in each group were detected by Western blotting. Results: Compared with blank serum, PC12 cells cultured in 20% drug serum could promote the proliferation of PC12 cells for 24 h or 48 h (P <0.05). Aβ_ (25-35) is cytotoxic to PC12 cells and decreases cell survival rate (P <0.05) in a dose-dependent manner, leading to apoptosis. CCK-8 assay and flow cytometry showed that 20% Gengnianchun serum had a protective effect on PC12 cells induced by Aβ_ (25-35) (P <0.05), and its effect was similar to that of positive control drug Growth factor. Western blotting showed that compared with the model group, the ratio of Bax and Bcl-2 protein expression and caspase-3 protein expression in the serum-containing group decreased. CONCLUSION: Gengnianchun serum can increase the survival rate of PC12 cells damaged by Aβ_ (25-35) and reduce the apoptosis of PC12 cells. The mechanism may be related to the regulation of Bcl-2 family protein expression and anti-apoptotic effect.