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【目的】探讨地黄饮子对Aβ_(1-42)诱导的SH-SY5Y细胞中RAGE/ROS/凋亡通路的影响。【方法】(1)采用四甲基偶氮唑盐(MTT)法检测胎牛血清组、空白组及地黄饮子含药血清低、中、高剂量组的细胞活力,确定地黄饮子含药血清的最佳浓度和作用时间。(2)以0~20μmol/L Aβ_(1-42)寡聚体处理SH-SY5Y细胞24 h和48 h后,采用MTT法检测细胞活力,Annexin V/碘化丙啶(PI)双染法观察细胞凋亡,确定Aβ_(1-42)作用细胞的最佳浓度及时间,以建立阿尔茨海默病(AD)细胞模型。(3)采用MTT法检测空白组、模型组、西药对照组及地黄饮子含药血清低、中、高剂量组细胞活力,采用Annexin V/PI双染法观察各组细胞凋亡,采用二氢乙啶(DHE)染色法检测各组活性氧(ROS)含量,观察地黄饮子对Aβ_(1-42)诱导的SH-SY5Y的细胞损伤的修复作用。(4)采用Western blot法检测空白组、模型组、地黄饮子含药血清中剂量组RAGE蛋白;进一步将Aβ_(1-42)诱导的SH-SY5Y细胞进行RAGE转染后,采用DHE染色法检测各组ROS含量,采用Annexin V/PI双染法检测各组细胞凋亡率,观察地黄饮子对Aβ_(1-42)诱导的SH-SY5Y细胞凋亡及RAGE表达的影响。【结果】作用时间为24 h时,地黄饮子含药血清低、中剂量组细胞活力较空白组均显著增强(P<0.05或P<0.01)。建立AD体外模型的Aβ_(1-42)浓度为5μmol/L,作用时间为24 h。各给药组Aβ_(1-42)诱导细胞活力较模型组显著增强,细胞凋亡率和ROS含量显著下降(P<0.05或P<0.01),而地黄饮子中剂量组细胞活力最强,细胞凋亡率最低。地黄饮子中剂量组Aβ1-42诱导细胞RAGE蛋白表达较模型组显著降低(P<0.05)。地黄饮子中剂量组能降低RAGE转染的Aβ_(1-42)诱导SH-SY5Y细胞的ROS生成和细胞凋亡率(P<0.01)。【结论】地黄饮子可能通过抑制ROS产生和细胞凋亡发挥抗氧化作用,进而抑制RAGE蛋白来防治AD。
【Objective】 To investigate the effect of Dihuang Yinzi on RAGE / ROS / Apoptosis pathway induced by Aβ 1-42 in SH-SY5Y cells. 【Methods】 (1) MTT assay was used to detect cell viability in fetal bovine serum, blank group and Rehmanniae Decoction-containing serum in low, medium and high dose groups, The best serum concentration and duration of action. (2) SH-SY5Y cells were treated with 0 ~ 20μmol / L Aβ 1-42 oligomers for 24 h and 48 h, MTT assay was used to determine the cell viability. Annexin V / PI double staining Apoptosis was observed to determine the optimal concentration of Aβ 1-42-acting cells and time to establish an Alzheimer’s disease (AD) cell model. (3) MTT assay was used to detect cell viability in blank, model group, western medicine control group and Rehmanniae Decoction drug-containing serum in low, medium and high dose groups. Cell apoptosis was observed by Annexin V / PI double staining method. The content of reactive oxygen species (ROS) in each group was detected by DHE staining, and the repair effect of Dihuang Yinzi on cell injury of SH-SY5Y induced by Aβ 1-42 was observed. (4) Western blot was used to detect the RAGE protein in middle dose group, model group and Rehmanniae Decoction containing serum. The SHAGE-SY5Y cells induced by Aβ 1-42 were further transfected with RAGE by DHE staining The ROS levels in each group were detected. The apoptosis rate of each group was detected by Annexin V / PI double staining method. The effect of Dihuang Yinzi on Aβ 1-42-induced apoptosis and RAGE expression in SH-SY5Y cells was observed. 【Result】 When the action time was 24 h, the cell viability of Rehmanniae Decoction containing serum of low and medium dose groups was significantly higher than that of the blank group (P <0.05 or P <0.01). The concentration of Aβ_ (1-42) in AD model was 5μmol / L and the effect time was 24 h. Compared with the model group, the cell viability induced by Aβ 1-42 in each administration group was significantly increased, and the apoptosis rate and ROS content were significantly decreased (P <0.05 or P <0.01) The lowest rate of apoptosis. Compared with model group, the expression of RAGE in Aβ1-42 induced by Rehmanniae Decoction of medium dosage group was significantly lower (P <0.05). Rehmanniae Decoction could reduce the ROS generation and apoptosis rate of SH-SY5Y cells induced by AAGE_ (1-42) induced by RAGE (P <0.01). 【Conclusion】 Rehmanniae Decoction may exert anti-oxidative effects by inhibiting ROS production and apoptosis, thereby inhibiting RAGE protein to prevent and treat AD.