【摘要】 【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0²0μmol/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。更多还原

【Abstract】 Objective To investigate the effects of Dihuang Yinzi（DY） on the receptor for advanced glycation end-products（RAGE）/reactive oxygen species（ROS）/apoptosis pathway in SH-SY5 Y cells induced by amyloidbeta1-42(Aβ_1-42)oligomer. Methods Firstly,we adopted methyl thiazolyl tetrazolium（MTT）method to detect the cell vitality in fetal bovine serum（FBS） group,blank serum group,and low-,middle-and high-dose DYcontaining serum groups,so as to confirm the optimal concentration and treatment time of DY-containing serum.Secondly,we applied MTT method to detect cell vitality and applied Annexin V/propidium iodide（PI） staining method to observe the apoptosis of SH-SY5 Y cells treated with 0²0 μmol/L Aβ_1-42 for 24 and 48 h,so as toconfirm the optimal concentration and treatment time of Aβ_1-42 for establishing Alzheimer’s disease（AD） model in vitro. Thirdly,MTT method was used for the detection of cell vitality,and Annexin V/PI staining method was used for detection of the apoptosis of SH-SY5 Y cells in blank serum group, model group, western medicine control group and low-,middle-and high-dose DY-containing serum groups,and Dihydroethidium（DHE） method was used for the assay of ROS contents,so as to observe the effect of DY on the recovery of injured SH-SY5 Y cells induced by Aβ_1-42. Finally,we applied Western blot method to detect the expression level of RAGE in SHSY5 Y cells of blank group,model group and DY-containing serum group;after Aβ_1-42-induced SH-SY5 Y cells were transfected with RAGE gene, we adopted DHE staining method and Annexin V/PI staining method to detect ROS content and cell apoptotic rate in all of the above groups,so as to observe the effect of DY on SHSY5 Y cell apoptosis and RAGE expression. Results The cell vitalities were increased in low-and middle-dose DY-containing serum groups at 24 h（P < 0.05 or P < 0.01 compared with that in the blank serum group）. The conditions for the establishment of AD model in vitro were as follows:the optimal concentration of Aβ_1-42 was 5μmol/L, and the treatment time was 24 h. The cell vitalities were significantly enhanced, the cell apoptotic rate and ROS content were significantly lowered in Aβ_1-42-induced SH-SY5 Y cells of the medication groups（P <0.05 or P < 0.01 compared with those in the model group）, and the cell vitality was the highest and the cell apoptotic rate was the lowest in the middle-dose DY-containing serum group. The RAGE expression level was decreased in Aβ_1-42-induced SH-SY5 Y cells of the middle-dose DY-containing serum group（P < 0.05 compared with that in the model group）. ROS content and cell apoptotic rate were decreased in Aβ_1-42-induced SH-SY5 Y cells transfected with RAGE gene in the middle-dose DY-containing serum group（P < 0.01）. Conclusion DY may play an anti-oxidative role through inhibiting the production of ROS and cell apoptosis, thus to suppress RAGE protein and to achieve the preventive and therapeutic effect for AD.更多还原