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投稿时间:2022-10-24 网络发布日期:2023-07-19
投稿时间:2022-10-24 网络发布日期:2023-07-19
中文摘要: 目的 探讨分泌型卷曲相关蛋白5(SFRP5)在肾小管上皮细胞上皮—间质转化(EMT)中的作用及机制。
方法 体外以转化生长因子(TGF)-β1刺激人近端肾小管上皮细胞系HK-2细胞,给予SFRP5与细胞共孵育,应用Western Blot法及RT-qPCR检测细胞中的EMT指标[E-钙黏蛋白(E-cadherin)和α平滑肌肌动蛋白(α-SMA)]的mRNA和蛋白表达水平,Western Blot法检测纤维化指标[Fibronectin(Fn)和Collagen I(Col-I)]的蛋白水平;并检测Wnt/β-catenin信号通路中细胞质中磷酸化β-catenin或细胞核中活化β-catenin蛋白水平,TOP/FOP-Flash荧光素酶试验评估β-catenin激活介导的转录及下游靶基因c-Myc 和cyclin D1的mRNA水平。结果 TGF-β1刺激后上皮细胞固有E-cadherin水平下降,而α-SMA、Fn和Col-I表达增多(P<0.05);胞质中磷酸化(p-S37位点)β-catenin减少且核内β-catenin水平增加,TOP/FOP比率上升,靶基因c-Myc 和cyclin D1转录增加(P<0.05)。加入SFRP5共培养细胞后,上述EMT及纤维化指标变化被逆转,异常激活的Wnt/β-catenin通路中各标志物被抑制,再加入SFRP5特异性中和抗体后SFRP5的抑制及保护作用被取消(P<0.05)。结论 本研究首次发现SFRP5可以减轻TGF-β1诱导的肾小管上皮细胞EMT和纤维化,其机制与抑制异常激活的Wnt/β-catenin信号通路相关,这可能成为临床诊治及延缓慢性肾脏病进展的新靶点。
中文关键词: 肾间质纤维化 上皮—间质转化 分泌型卷曲相关蛋白5 Wnt/β-catenin信号通路
Abstract:Objective To explore the role and mechanism of secreted frizzled related protein 5(SFRP5) in epithelial to mesenchymal transition(EMT) of renal tubular epithelial cells.
Methods The humam proximal renal tubular epithelial cell line HK-2 cells were stimulated by transforming growth factor (TGF)-β1 in vitro and incubated with or without SFRP5. The mRNA and protein expression of EMT indexes including E-cadherin and α-SMA were detected by quantitative real-time PCR and Western Blot. The protein levels of fibrosis indexes [Fibronectin(Fn) and Collagen I(Col-I)] were also evaluated. The level of phosphorylated β-catenin in cytoplasm or activated β-catenin in nucleus of Wnt/β-catenin signaling pathway was detected by WB. The TOP/FOP-flash luciferase assay was used to evaluate β-catenin-mediated transcription, in addition, the mRNA levels of downstream target genes c-Myc and cyclin D1 were measured.
Results The original E-cadherin level of renal epithelial cells decreased, while α-SMA, Fn and Col-I increased after TGF-β1 stimulation. The cytoplasmic level of phosphorylation(site p-S37) of β-catenin decreased and nuclear level of β-catenin increased (P<0.05). The TOP/FOP ratio was up-regulated, and the transcription of the target genes c-Myc and cyclin D1 increased (P<0.05). After adding SFRP5 to treat cells, the changes of EMT and fibrosis indexes were reversed, and the above-mentioned markers in abnormally activated Wnt/β-catenin pathway were inhibited, and the inhibiting and protecting effects of SFRP5 were cancelled by SFRP5 specific neutralizing antibody (P<0.05).
Conclusion In this study, it has been found for the first time that SFRP5 can ameliorate the EMT and fibrosis in renal tubular epithelial cells induced by TGF-β1. The mechanism is related to the inhibition of abnormally activated Wnt/β-catenin signaling pathway, which may become a new target for clinical diagnosis and treatment and delaying the progress of chronic kidney disease.
keywords: Renal interstitial fibrosis Epithelial-mesenchymal transition Secreted frizzled related protein 5 Wnt/β-catenin sigalling pathway
文章编号: 中图分类号:R594 文献标志码:B
基金项目:国家自然科学基金青年科学基金资助项目(82000700)
Author Name | Affiliation |
DENG Dai, WU Yiru, BAI Yu, DIAO Zongli, HUANG Hongdong, LIU Wenhu | Department of Nephrology, Beijing Friendship Hospital, Capital Medical University, Beijing 100050, China |
引用文本:
邓岱,吴逸如,白雨,刁宗礼,黄红东,刘文虎.SFRP5抑制Wnt/β-catenin通路减轻肾小管上皮细胞上皮—间质转化[J].中国临床研究,2023,36(7):1022-1026.
邓岱,吴逸如,白雨,刁宗礼,黄红东,刘文虎.SFRP5抑制Wnt/β-catenin通路减轻肾小管上皮细胞上皮—间质转化[J].中国临床研究,2023,36(7):1022-1026.