温肺降浊方对阿尔茨海默病体外模型Aβ25-35诱导PC12细胞凋亡的影响

Effects of Wenfei Jiangzhuo Formula on apoptosis of PC12 cells induced by Aβ25-35 in an in vitro model of Alzheimerdisease

目的:探讨温肺降浊方对阿尔茨海默病(AD)体外模型Aβ25-35诱导PC12细胞凋亡的影响。方法:通过β-神经生长因子(β-NGF)分化PC12细胞,Aβ25-35干预其成为AD细胞模型,CCK-8法确定Aβ25-35干预的最佳浓度和时间点,设立温肺降浊方含药血清低、中、高剂量组进行干预;Transwell检测细胞迁移活性;流式细胞术确定细胞凋亡周期及凋亡比率;qRT-PCR和Western Blot分别检测各组细胞中Bcl-xl、Bad、p53、Caspase-3 mRNA和蛋白的表达情况。结果:β-NGF分化PC12细胞在第6天后成为海马神经元细胞形态结构,制备AD细胞模型以5μmol/L终浓度的Aβ25-35干预48 h最佳。与空白组比较,模型组细胞迁移数量显著减少(P<0.05),细胞凋亡比率显著增加(P<0.05),Bad、Caspase-3 mRNA及蛋白表达显著上升(P<0.05),Bcl-xl、p53 mRNA及蛋白表达显著下降(P<0.05);与模型组比较,温肺降浊方含药血清中、高剂量组细胞迁移数量增加(P<0.05),细胞凋亡比率显著下降(P<0.05),温肺降浊方含药血清高剂量组Bad、Caspase-3 mRNA及蛋白表达显著下降(P<0.05),Bcl-xl、p53 mRNA及蛋白表达显著上升(P<0.05)。结论:温肺降浊方在抑制Aβ25-35诱导PC12细胞损伤中发挥重要作用,其机制可能与上调Bcl-xl和p53表达并抑制细胞凋亡有关,从而发挥神经保护效应。

Objective:To explore the effect of Wenfei Jiangzhuo Formula on the apoptosis of PC12 cells induced by Aβ25-35 in an in vitro model of Alzheimer disease(AD).Methods:PC12 cells were differentiated byβ-nerve growth factor(β-NGF),and Aβ25-3s intervened to become an AD cell model.CCK-8 method was used to determine the optimal concentration and time point of Aβ25-3s intervention,and low,medium and high dose groups of Wenfei Jiangzhuo Formula-containing serums were set up to intervene;Transwell assay was performed to detect the migration activity of the cells:Flow cytometry was performed to determine the apoptotic cycle and apoptotic ratio;the mRNA and protein expression of Bcl-xl,Bad,p53 and Caspase-3 were detected by qRT-PCR and Western Blot in the cells of each group,respectively.Results:β-NGF differentiated PC12 cells became hippocampal neuronal cell morphology and structure after day 6,and the AD cell model was prepared with 5μmol/L final concentration of Aβ25-3s intervention for 48 h optimally.Compared with the blank group,the model group showed a significant decrease in the number of cell migration(P<0.05),an increase in the cell apoptosis rate(P<0.05),an increase in the expression of Bad and Caspase-3 mRNA and protein(P<0.05),and a decrease in the expression of Bcl-xl and p53 mRNA and protein(P<0.05).Compared with the model group,the medium and high doses of Wenfei Jiangzhuo Formula serum-containing showed an increase in the number of cell migration and a decrease in the cell apoptosis rate(P<0.05);the expression of Bad,Caspase-3 mRNA and protein decreased,and the expression of Bcl-xl,p53 mRNA and protein were increased in the high-dose group of Wenfei Jiangzhuo Formula serum-containing(P<0.05).Conclusion:Wenfei Jiangzhuo Formula plays an important role in inhibiting Aβ25-3 induced PC12 cell injury,and its mechanism may be related to the up-regulation of Bcl-xl and p53 expression and the inhibition of apoptosis,thus exerting neuroprotective effects.