下调XBP1s通过Sirt3/SOD2/mtROS轴减轻缺氧/复氧诱导的肾小管上皮细胞衰老

Down-regulation of XBP1s alleviates the senescence of renal tubular epithelial cells induced by hypoxia/reoxygenation through Sirt3/SOD2/mtROS signaling pathway

    摘要
  • 摘要:
      目的   探讨剪接型X-盒结合蛋白1（XBP1s）在缺氧/复氧（H/R）诱导的原代肾小管上皮细胞衰老中的作用及机制。
      方法   将原代肾小管上皮细胞分为空白对照组（NC组）、H/R组、空载腺病毒阴性对照组（Ad-shNC组）、靶向沉默XBP1s腺病毒组（Ad-shXBP1s组）、空载腺病毒+H/R处理组（Ad-shNC+H/R组）、靶向沉默XBP1s腺病毒+H/R处理组（Ad-shXBP1s+H/R组）。检测NC组、H/R组、Ad-shNC组、Ad-shXBP1s组XBP1s的表达情况。检测Ad-shNC组、Ad-shNC+H/R组、Ad-shXBP1s+H/R组β-半乳糖苷酶染色情况，细胞衰老标志物p53、p21、γH2AX表达情况，氧化应激相关指标活性氧（ROS）、丙二醛（MDA）和超氧化物歧化酶（SOD）水平。采用染色质免疫共沉淀验证XBP1s转录调控沉默信息调节因子3（Sirt3），检测下调XBP1s后Sirt3及下游SOD2表达，采用流式细胞术检测线粒体活性氧簇（mtROS）。
      结果   与NC组比较，H/R组XBP1s表达增多；与Ad-shNC组比较，Ad-shXBP1s组XBP1s表达减少（均为P<0.001）。与Ad-shNC组比较，Ad-shNC+H/R组β-半乳糖苷酶染色阳性细胞数增加，p53、p21、γH2AX表达增多，ROS、MDA、mtROS水平升高，SOD活性下降，Sirt3表达量降低，Ac-SOD2/SOD2比值升高；与Ad-shNC+H/R组相比，Ad-shXBP1s+H/R组β-半乳糖苷酶染色阳性细胞数减少，p53、p21、γH2AX表达减少，ROS、MDA、mtROS水平下降，SOD活性升高，Sirt3表达量升高，Ac-SOD2/SOD2比值下降（均为P<0.05）。
      结论   下调XBP1s可减轻H/R诱导的原代肾小管上皮细胞衰老，可能是通过Sirt3/SOD2/mtROS信号轴发挥作用。

     

    Abstract:
      Objective  To investigate the role and mechanism of spliced X-box binding protein 1 (XBP1s) in the senescence of primary renal tubular epithelial cells induced by hypoxia/reoxygenation (H/R).
      Methods  Primary renal tubular epithelial cells were divided into the normal control group (NC group), H/R group, empty adenovirus negative control group (Ad-shNC group), targeted silencing XBP1s adenovirus group (Ad-shXBP1s group), empty adenovirus+H/R treatment group (Ad-shNC+H/R group) and targeted silencing XBP1s adenovirus+H/R treatment group (Ad-shXBP1s +H/R group), respectively. The expression levels of XBP1s in the NC, H/R, Ad-shNC and Ad-shXBP1s groups were measured. The number of cells stained with β-galactosidase, the expression levels of cell aging markers including p53, p21 and γH2AX, and the levels of reactive oxygen species (ROS), malondialdehyde (MDA) and superoxide dismutase (SOD) were determined in the Ad-shNC, Ad-shNC+H/R and Ad-shXBP1s+H/R groups. Chromatin immunoprecipitation was employed to verify Sirtuin 3 (Sirt3) of XBP1s transcription regulation, and the expression levels of Sirt3 and downstream SOD2 after down-regulation of XBP1s were detected. Mitochondrial reactive oxygen species (mtROS) were detected by flow cytometry.
      Results  Compared with the NC group, the expression level of XBP1s was up-regulated in the H/R group. Compared with the Ad-shNC group, the expression level of XBP1s was down-regulated in the Ad-shXBP1s group (both P<0.001). Compared with the Ad-shNC group, the number of cells stained with β-galactosidase was increased, the expression levels of p53, p21 and γH2AX were up-regulated, the levels of ROS, MDA and mtROS were increased, the SOD activity was decreased, the expression level of Sirt3 was down-regulated, and the ratio of Ac-SOD2/SOD2 was increased in the Ad-shNC+H/R group. Compared with the Ad-shNC+H/R group, the number of cells stained with β-galactosidase was decreased, the expression levels of p53, p21 and γH2AX were down-regulated, the levels of ROS, MDA and mtROS were decreased, the SOD activity was increased, the expression level of Sirt3 was up-regulated and the ratio of Ac-SOD2/SOD2 was decreased in the Ad-shXBP1s+H/R group (all P<0.05).
      Conclusions  Down-regulation of XBP1s may ameliorate the senescence of primary renal tubular epithelial cells induced by H/R, which probably plays a role through the Sirt3/SOD2/mtROS signaling pathway.

     

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