Down-regulation of XBP1s alleviates the senescence of renal tubular epithelial cells induced by hypoxia/reoxygenation through Sirt3/SOD2/mtROS signaling pathway
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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. -
图 1 H/R对肾小管上皮细胞XBP1s表达的影响
注：A、C图为各组XBP1s mRNA相对表达量；B、D图为蛋白质印迹法检测各组XBP1s蛋白表达。与NC组相比，aP<0.001；与Ad-shNC组比较，bP<0.001。
Figure 1. The effect of H/R on XBP1s expression in renal tubular epithelial cells
图 2 下调XBP1s减轻H/R诱导的细胞衰老
注：A图为检测各组β-半乳糖苷酶阳性细胞数（×200）；B图为蛋白质印迹法检测各组p53、p21、γH2AX蛋白表达。
Figure 2. Downregulation of XBP1s mitigates cell senescence induced by H/R
图 3 下调XBP1s减轻H/R诱发的氧化应激
注：A图为用DCFH-DA荧光探针检测各组ROS水平（免疫荧光，×200）；B图为各组ROS荧光强度统计结果；C图为各组MDA含量检测结果；D图为各组SOD活性检测结果。与Ad-shNC组比较，aP<0.01；与Ad-shNC+H/R组比较，bP<0.05。
Figure 3. Downregulation of XBP1s alleviates oxidative stress induced by H/R
图 4 下调XBP1s对Sirt3/SOD2/mtROS信号轴的影响
注：A图为ChIP-seq峰图；B图为ChIP-qPCR结果，与IgG组比较，aP<0.01；C图为各组Sirt3 mRNA相对表达量；D图为蛋白质印迹法检测各组Sirt3、Ac-SOD2、SOD2蛋白表达；E图为各组Ac-SOD2/SOD2蛋白相对表达量；F图为流式细胞术分析mtROS变化；G图为各组mtROS荧光强度分析结果。与Ad-shNC组比较，aP<0.01，与Ad-shNC+H/R组比较，bP<0.01。
Figure 4. The impact of downregulating XBP1s on the Sirt3/SOD2/mtROS signaling axis
表 1 引物序列
Table 1. Primer sequences
名称 正义链（5'-3'） 反义链（5'-3'） XBP1s AAGAACACGCTTGGGAATGG CTGCACCTGCTGCGGAC Sirt3 ATCCCGGACTTCAGATCCCC CAACATGAAAAAGGGCTTGGG β-actin AGGCCAACCGTGAAAGATG TGGCGTGAGGGAGAGCATAG 引物1 TGGTGACTTATCTACGTCTGCT ATCACTTTGTGCCACGGACT 引物2 CATCGTCTGAGCTGTCTGGTA TGTGGGACTGAGGGCTTTTTG 引物3 CAGCGTCAACTCCCACTCT AAGGCTGAAGGCATCCGTTTC 引物4 CCTGACCCTCCGGACTGAT TTGTTCCCAGCTCTCTGCAAT 引物5 ACTAAGCAGTCAGAACCGGC GTTACCTTGACGACCGCAAC 
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