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目的探讨ATP敏感的钾离子(KATP)通道开放药物二氮嗪防治A1-42细胞毒性作用的分子机制。方法采用细胞原代培养的方法,培养大鼠皮层神经元并进行鉴定。将原代培养的细胞随机分为对照组、单纯Aβ1-42干预组、二氮嗪预处理1 h后Aβ1-42干预组、单纯二氮嗪预处理组和单纯Aβ42-1干预组(Aβ1-42反序列对照),各组又分为24、72 h两个亚组。采用免疫荧光双染及免疫印迹法,观察干预后不同培养时间(24、72 h)细胞KATP通道各亚基Kir6.1、Kir6.2、SUR1、SUR2蛋白表达水平的变化。结果与对照组比较,单纯A1-42处理24 h组Kir6.1、SUR2显著升高(P<0.05),二氮嗪预处理后Aβ1-42作用细胞24 h组各亚基表达均无明显变化;二氮嗪预处理后Aβ1-42作用细胞72 h组与单纯A1-42处理72 h组KATP通道各亚基表达均明显升高(P<0.05),而二氮嗪预处理后Aβ1-42作用细胞72 h组与单纯A1-42处理72 h组相比Kir6.1、Kir6.2、SUR2表达显著下调(P<0.05)。结论二氮嗪预处理可完全逆转A1-42作用神经元24 h所引起的Kir6.2及SUR1的表达上调,只能部分逆转A1-42作用神经元72 h所引起的Kir6.1、Kir6.2、SUR2的表达增加,可能会维持神经细胞正常生理功能,起到防治A1-42细胞毒性作用。
Objective To investigate the molecular mechanism of ATP-sensitive potassium channel (KATP) channel-opening drug diazoxide in preventing and treating A1-42 cytotoxicity. Methods Primary cultured rat cortical neurons were cultured and identified. The primary cultured cells were randomly divided into control group, Aβ1-42 intervention group, diazoxide pretreatment 1 h after Aβ1-42 intervention group, diazoxide alone pretreatment group and Aβ42-1 intervention group (Aβ1- 42 reverse sequence control), each group is divided into 24,72 h two subgroups. The expression of Kir6.1, Kir6.2, SUR1 and SUR2 protein in KATP channel of different culture time (24 and 72 h) were observed by immunofluorescence double staining and Western blot. Results Compared with the control group, Kir6.1 and SUR2 in 24 h group treated with A1-42 alone were significantly increased (P <0.05), and no significant changes were found in 24 h subunit expression of Aβ1-42-treated cells after diazoxide preconditioning ; After diazoxide preconditioning, the expression of KATP channel subunit in 72 h group treated with Aβ1-42 for 72 h and Aβ1-42 treated with diazoxide for 72 h were significantly increased (P <0.05) The expression of Kir6.1, Kir6.2 and SUR2 was significantly down-regulated in 72 h treated group compared with 72 h treated with A1 -42 alone (P <0.05). Conclusion Diazoxide preconditioning can completely reverse the expression of Kir6.2 and SUR1 induced by A1-42-induced neurons for 24 h, and only partially reverse the Kir6.1 and Kir6 induced by A1-42-induced neurons for 72 h. 2, SUR2 expression increased, may maintain the normal physiological function of nerve cells, play a role in the prevention and treatment of A1-42 cytotoxicity.