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。 Abstract:Objective To evaluate the effect of glycogen synthase kinase 3β (GSK3β) on hypoxia/reoxygenation (H/R)-induced injury of senescent renal tubular epithelial cell (RTEC) in aged mice and its regulatory mechanism. Methods RTEC were divided into the Young group (young RTEC with normal growth), Old group (aged RTEC induced by Etoposide), Old+Ad-shNC+H/R group [aged RTEC induced by Etoposide and then transfected with adenovirus negative control (Ad-shNC) for H/R treatment], and Old+Ad-shGSK3β+H/R group (aged RTEC induced by Etoposide and then transfected with short-hairpin RNA-expressing adenovirus with targeted silencing GSK3β for H/R treatment), respectively. Apoptosis level and mitochondrial reactive oxygen species level were detected by flow cytometry. Calcium ion level was determined by immunofluorescence staining. The expression and phosphorylation levels of GSK3β, mitochondria-associated endoplasmic reticulum membrane (MAM)-related proteins of inositol 1,4,5-trisphosphate receptor1 (ITPR1), voltage dependent anion-selective channel (VDAC1) and glucose-regulated protein 75 (GRP75) were detected by Western blot. The interaction between GSK3β and MAM-related proteins was analyzed by immunoprecipitation. Results Compared with the Young group, the apoptosis, mitochondrial reactive oxygen species and mitochondrial calcium ion levels were higher in the Old group. Compared with the Old group, the apoptosis, mitochondrial reactive oxygen species and mitochondrial calcium ion levels were higher in the Old+Ad-shNC+H/R group. Compared with the Old+Ad-shNC+H/R group, the apoptosis, mitochondrial reactive oxygen species and mitochondrial calcium ion levels were lower in the Old+Ad-shGSK3β+H/R group, and the differences were statistically significant (all P<0.05). Compared with the Young group, the expression levels of ITPR1, GRP75 and GSK3β proteins were up-regulated, the phosphorylation levels of ITPR1 and GRP75 were increased, whereas the levels of total VDAC1 protein and phosphorylated protein were decreased in the Old group. Compared with the Old group, the expression level of GSK3β protein was unchanged, the total protein and phosphorylation levels of ITPR1 and GRP75 were increased, the expression level of total VDAC1 protein remained unchanged and the phosphorylation level was increased in the Old+Ad-shNC+H/R group. Compared with the Old+Ad-shNC+H/R group, the expression level of GSK3β protein was decreased, the expression levels of total ITPR1, GRP75 and VDAC1 proteins were unchanged, whereas the phosphorylation levels were decreased in the Old+Ad-shGSK3β+H/R group. Immunoprecipitation showed that GSK3β could interact with ITPR1, GRP75 and VDAC1 proteins. Conclusions The expression level of GSK3β is up-regulated in senescent RTEC. Down-regulating GSK3β expression may reduce the phosphorylation level of ITPR1-GRP75-VDAC1 complex, constrain the overload of mitochondrial calcium ion, protect mitochondrial function and mitigate cell damage during reperfusion. -
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 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
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