过表达ABAD对Aβ_(1-42)刺激的293T细胞线粒体功能和自噬水平的影响

Effects of overexpression of ABAD on mitochondrial function and autophagy of 293T cells stimulated by Aβ_(1-42)

目的探讨β淀粉样蛋白1-42(Aβ_(1-42))刺激下,在293T细胞中过表达淀粉样蛋白结合醇脱氢酶(ABAD)对线粒体功能及自噬相关标记蛋白水平的影响。方法将正常培养的293T细胞随机分为:(1)Control组、过表达ABAD组、Control给药组(Aβ_(1-42)5μmol/L、10μmol/L、20μmol/L)、过表达ABAD给药组(Aβ_(1-42)5μmol/L、10μmol/L、20μmol/L);(2)Control组、sh-ABAD组;(3)Control+DMSO组、Control+CCCP组、过表达ABAD+DMSO组、过表达ABAD+CCCP组(WT/Aβ10μmol/L)。三磷酸腺苷(ATP)试剂盒测定Control组、过表达ABAD组、Control给药组、过表达ABAD给药组细胞的ATP含量;活性氧(ROS)试剂盒测定活性氧生成量。Westernblot法检测Control+DMSO组、Control+CCCP组、过表达ABAD+DMSO组、过表达ABAD+CCCP组线粒体自噬相关蛋白表达水平。通过标准曲线换算ATP细胞含量和活性氧生成量,采用ImageJ对Westernblot条带进行量化。结果Control组与过表达ABAD组ATP含量和活性氧生成量变化不明显,差异无统计学意义(P>0.05);与Control给药组相比,在Aβ_(1-42)(10μmol/L)刺激后,过表达ABAD给药组的ATP含量减少,差异具有统计学意义(P<0.05),但两组的活性氧生成量变化不明显,差异无统计学意义(P>0.05);与Control+CCCP组相比,过表达ABAD+CCCP组在Aβ_(1-42)10μmol/L诱导后线粒体自噬相关蛋白P62降低、LC3B升高,差异均有统计学意义(P<0.05)。结论过表达ABAD不影响293T细胞的线粒体功能,但在特定浓度Aβ_(1-42)刺激下可影响线粒体ATP产生及促进线粒体自噬发生。

Objective To investigate the effect of overexpression of ABAD on mitochondrial function and the level of mitophagy related protein in 293T cells stimulated by Aβ_(1-42).Methods 293T cells were randomly divided into:(1)control group,overexpression of ABAD group,control group(Aβ_(1-42)5μmol/L,10μmol/L,20μmol/L),overexpression of ABAD group(Aβ_(1-42)5μmol/L,10μmol/L,20μmol/L);(2)control group,sh-ABAD group;(3)control+DMSO group,control+CCCP group,overexpression of ABAD+DMSO group,overexpression of ABAD+CCCP group(WT/Aβ10μmol/L).Adenosine triphosphate(ATP)content of control group,overexpression of ABAD group,control group(Aβ_(1-42)5μmol/L,10μmol/L,20μmol/L)and overexpression of ABAD group(Aβ_(1-42)5μmol/L,10μmol/L,20μmol/L)was measured by ATP kit,and the production of reactive oxygen species(ROS)was measured by ROS kit.Western blot was used to detect the expression of mitophagy related proteins in control+DMSO group,control+CCCP group,overexpression of ABAD+DMSO group and overexpression of ABAD+CCCP group.The content of ATP and reactive Oxygen Species(Ros)were calculated by standard curve,and the Western blot was quantified by Image J.Results Compared with the control group,ATP content and reactive oxygen species production in the control group and overexpression ABAD group were not significantly changed(P>0.05);Compared with the control group,the ATP content of 293T cells overexpression ABAD was decreased after Aβ_(1-42)(10μmol/L)stimulation,and the difference was statistically significant(P<0.05),but there was no significant difference in the production of reactive oxygen species between the two groups(P>0.05);Compared with the control+CCCP group,the mitophagy related protein p62 decreased and LC3 increased in the overexpression ABAD+CCCP group after Aβ_(1-42)10μmol/L induction,and the difference was statistically significant(P<0.05).Conclusion Overexpression of ABAD does not affect the mitochondrial function of 293T cells,but can affect the production of mitochondrial ATP and promote the development of mitophagy under the stimulation of Aβ_(1-42).