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下调GSK3β通过抑制ITPR1-GRP75-VDAC1复合体功能减轻衰老肾小管上皮细胞缺氧/复氧损伤

倪海强, 顾世琦, 彭宣, 等. 下调GSK3β通过抑制ITPR1-GRP75-VDAC1复合体功能减轻衰老肾小管上皮细胞缺氧/复氧损伤[J]. 器官移植. doi: 10.3969/j.issn.1674-7445.2024018
引用本文: 倪海强, 顾世琦, 彭宣, 等. 下调GSK3β通过抑制ITPR1-GRP75-VDAC1复合体功能减轻衰老肾小管上皮细胞缺氧/复氧损伤[J]. 器官移植. doi: 10.3969/j.issn.1674-7445.2024018
Ni Haiqiang, Gu Shiqi, Peng Xuan, et al. Down-regulating GSK3β alleviates hypoxia/reoxygenation-induced injury of senescent renal tubular epithelial cells by inhibiting the function of ITPR1-GRP75-VDAC1 complex[J]. ORGAN TRANSPLANTATION. doi: 10.3969/j.issn.1674-7445.2024018
Citation: Ni Haiqiang, Gu Shiqi, Peng Xuan, et al. Down-regulating GSK3β alleviates hypoxia/reoxygenation-induced injury of senescent renal tubular epithelial cells by inhibiting the function of ITPR1-GRP75-VDAC1 complex[J]. ORGAN TRANSPLANTATION. doi: 10.3969/j.issn.1674-7445.2024018

下调GSK3β通过抑制ITPR1-GRP75-VDAC1复合体功能减轻衰老肾小管上皮细胞缺氧/复氧损伤

doi: 10.3969/j.issn.1674-7445.2024018
基金项目: 国家自然科学基金(82170772、82370759);湖北陈孝平科技发展基金会青年科学专项基金(CXPJJH122001-2210)
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    作者简介:
    通讯作者:

    宫念樵(ORCID 0000-0001-7634-1440),博士,主任医师,研究方向为器官移植、移植免疫、干细胞治疗和器官老化,Email:nqgong@tjh.tjmu.edu.cn

  • 中图分类号: R617,R692

Down-regulating GSK3β alleviates hypoxia/reoxygenation-induced injury of senescent renal tubular epithelial cells by inhibiting the function of ITPR1-GRP75-VDAC1 complex

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  • 摘要:   目的   探讨糖原合成酶激酶3β(GSK3β)对衰老小鼠原代肾小管上皮细胞(RTEC)缺氧/复氧(H/R)损伤的影响及其调控机制。  方法  将RTEC分成为Young组即正常生长的年轻RTEC、Old组即使用Etoposide诱导的衰老RTEC、Old+Ad-shNC+H/R组即使用Etoposide诱导衰老再转染腺病毒阴性对照(Ad-shNC)后进行H/R处理,Old+Ad-shGSK3β+H/R组即使用Etoposide诱导衰老后再转染靶向沉默GSK3β的短发夹RNA腺病毒(Ad-shGSK3β)后进行H/R处理。采用流式细胞术检测各组细胞凋亡水平和线粒体活性氧水平,采用免疫荧光染色法检测各组钙离子水平,采用蛋白质印迹法检测各组GSK3β、线粒体相关的内质网膜结构(MAM)相关蛋白肌醇1,4,5-三磷酸受体1(ITPR1)、电压依赖性阴离子通道1(VDAC1)、葡萄糖调节蛋白75(GRP75)表达及磷酸化水平,采用免疫共沉淀分析GSK3β与MAM相关蛋白的相互作用。  结果  与Young组比较,Old组细胞凋亡水平、线粒体活性氧水平及线粒体钙离子水平均较高;与Old组相比,Old+Ad-shNC+H/R组细胞凋亡水平、线粒体活性氧水平及线粒体钙离子水平均较高;与Old+Ad-shNC+H/R组比较,Old+Ad-shGSK3β+H/R组细胞凋亡水平、线粒体活性氧水平及线粒体钙离子水平均较低,差异均有统计学意义(均为P<0.05)。与Young组比较,Old组ITPR1、GRP75和GSK3β总蛋白表达增多,ITPR1和GRP75磷酸化水平升高,而VDAC1总蛋白和磷酸化蛋白水平均下降;与Old组比较,Old+Ad-shNC+H/R组GSK3β蛋白表达不变,ITPR1和GRP75总蛋白和磷酸化水平升高,VDAC1总蛋白表达不变,磷酸化水平增高;与Old+Ad-shNC+H/R组比较,Old+Ad-shGSK3β+H/R组GSK3β蛋白表达减少,ITPR1、GRP75和VDAC1总蛋白表达不变,磷酸化水平均下降。免疫共沉淀结果显示,GSK3β能够与ITPR1、GRP75和VDAC1蛋白发生相互作用。  结论  GSK3β在衰老RTEC中表达升高,抑制GSK3β表达能够降低ITPR1-GRP75-VDAC1复合体磷酸化水平,限制线粒体钙离子超负荷,保护线粒体功能,减少再灌注时细胞损伤。

     

  • 图  1  小鼠RTEC形态学特征(×100)

    Figure  1.  Morphological characteristics of RTEC of mice

    图  2  衰老细胞鉴定

    注:A图为SA-β-gal染色(×100);B图为蛋白质印迹法。

    Figure  2.  Identification of senescent cell

    图  3  各组细胞凋亡水平

    注:A图为流式细胞术检测各组细胞凋亡水平;B图为各组细胞凋亡率分析。与Young组比较,aP<0.05;与Old组比较,bP<0.05;与Old+Ad-shNC+H/R组比较,cP<0.05。

    Figure  3.  Apoptosis levels in each group

    图  4  各组线粒体活性氧水平

    注:A图为流式细胞术分析线粒体活性氧水平;B图为各组线粒体活性氧水平分析。与Young组比较,aP<0.05,与Old组比较,bP<0.05,与Old+Ad-shNC+H/R组比较,cP<0.05。

    Figure  4.  Mitochondrial reactive oxygen species levels in each group

    图  5  各组线粒体钙离子水平(免疫荧光,×600)

    Figure  5.  Mitochondrial calcium levels in each group

    图  6  各组GSK3β和MAM相关蛋白表达水平

    Figure  6.  The expression levels of GSK3β and MAM-associated proteins in each group

    图  7  GSK3β与MAM相关蛋白免疫共沉淀

    Figure  7.  Co-immunoprecipitation of GSK3β and MAM-associated proteins

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  • 收稿日期:  2023-12-28
  • 网络出版日期:  2024-03-25

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