南方医科大学学报

南方医科大学学报 ›› 2023, Vol. 43 ›› Issue (12): 2078-2085.doi: 10.12122/j.issn.1673-4254.2023.12.12

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清肾颗粒含药血清通过miR-23b-5p靶向Nrf2通路减轻NRK-52E细胞转分化

刘 敏，金 华，呼 琴，陈 诺，张叶青，王亿平   

  1. 安徽中医药大学第一临床医学院，安徽 合肥 230000；安徽中医药大学第一附属医院肾病科，安徽 合肥 230000；合肥综合性国家科学中心大健康研究院，新安医学与中医药现代化研究所，安徽 合肥 230000
  • 出版日期:2023-12-20 发布日期:2023-12-29

Qingshen Granules-medicated serum reduces transdifferentiation of NRK-52E cells by miR-23b-5p-mediated activation of the Nrf2 pathway

LIU Min, JIN Hua, HU Qin, CHEN Nuo, ZHANG Yeqing, WANG Yiping   

  1. The First Clinical Medical College of Anhui University of Traditional Chinese Medicine, Hefei 230000, China; Department of Nephrology, First Affiliated Hospital of Anhui University of Traditional Chinese Medicine, Hefei 230000, China; Center for Xin'an Medicine and Modernization of Traditional Chinese Medicine of IHM, Anhui University of Traditional Chinese Medicine, Hefei 230000, China
  • Online:2023-12-20 Published:2023-12-29

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摘要： 目的 探讨转化生长因子β1（TGF-β1）诱导NRK-52E（大鼠肾小管上皮细胞）细胞转分化模型中miR-23b-5p是否能对Nrf2通路靶向调节，并阐明清肾颗粒含药血清减轻NRK-52E细胞转分化的机制。方法 构建TGF-β1诱导的NRK-52E细胞转分化模型，分别使用miR-23b-5p mimic、inhibitor以及阴性对照（NC）siRNA转染细胞；采用清肾颗粒含药血清进行干预，分为正常组、TGF-β1组、清肾颗粒组、miR-23b-mimic-NC组、miR-23b-mimic组、miR-23b-mimic+清肾颗粒组。采用CCK8法测定NRK-52E细胞活性；双荧光素酶报告基因实验检测miR-23b-5p与Nrf2间的靶向关系；Western blot法检测细胞中Nrf2、Keap1、α-SMA蛋白表达；RT-qPCR法检测细胞中miR-23b、Keap1、Nrf2、α-SMA mRNA表达；流式细胞术检测ROS。结果 根据CCK8结果筛选得出清肾颗粒含药血清的最佳浓度和时间分别为20%和24 h，TGF-β1刺激的最佳浓度和时间分别为10 ng/mL和24 h。转染miR-23b-5p mimic 和 inhibitor 发现，当 miR-23b-5p 过表达后，Nrf2 mRNA 表达量也增加；而 miR-23b-5p 表达沉默后，Nrf2mRNA表达量也减少。双荧光素酶报告显示，Rno-miR-23b-5p能显著下调Rno-Nrf2-wt荧光素酶活性（P<0.05），但未能下调突变Rno-Nrf2-mu荧光素酶活性（P>0.05）。清肾颗粒含药血清的干预实验发现，与正常组比较，TGF-β1组的miR-23b-5p、Nrf2表达降低，Keap1、α-SMA和ROS表达升高（P<0.05）。与TGF-β1组比较，清肾颗粒组的miR-23b-5p、Nrf2表达升高，Keap1、α-SMA和ROS表达降低（P<0.05）；miR-23b-mimic组miR-23b-5p、Nrf2表达升高，Keap1、α-SMA和ROS降低（P<0.05）。与miR-23b-mimic-NC组比较，miR-23b-mimic组miR-23b-5p、Nrf2表达升高，Keap1、α-SMA和ROS降低（P<0.05）。与miR-23b-mimic组比较，miR-23b-mimic+清肾颗粒组的miR-23b-5p、Nrf2表达升高，Keap1、α-SMA和ROS降低（P<0.05）。结论 清肾颗粒含药血清能通过miR-23b-5p靶向Nrf2通路机制减轻NRK-52E细胞转分化。

关键词: miR-23b-5p；Nrf2信号通路；NRK-52E细胞；肾小管EMT；清肾颗粒

Abstract: Objective TTo investigate the targeted regulation of the Nrf2 pathway by miR-23b-5p in transdifferentiation of rat renal tubular epithelial NRK-52E cells induced by transforming growth factor β1 (TGF-β1) and the effect of Qingshen Granules-medicated serum for alleviating transdifferentiation of NRK-52E cells. Methods NRK-52E cells with TGF-β1-induced transdifferentiation were transfected with miR-23b-5p mimic, miR- 23b-5p inhibitor or the negative control (NC) siRNA and then treated with of Qingshen Granules-medicated serum. CCK8 assay was used to detectthe changes in viability of NRK-52E cells. The targeting relationship between miR-23b-5p and Nrf2 was verified using a dual luciferase reporter gene assay. The expressions of Nrf2, Keap1 and α- SMA mRNAs and proteins in the treated cells were detected with RT- qPCR and Western blotting, and ROS production in the cells was detected with flow cytometry. Results Transfection of NRK-52E cells with miR-23b-5p mimic significantly increased the expression of Nrf2 mRNA, while inhibition of miR-23b-5p obviously lowered Nrf2 mRNA in the cells. Rno-miR-23b-5p significantly down-regulated the luciferase activity of Rno-Nrf2-wt but not that of Rno-Nrf2-mu (P<0.05). Treatment with TGF-β1 significantly decreased the expressions of miR-23b-5p and Nrf2 and increased the expressions of Keap1, α-SMA and ROS in NRK-52E cells (P<0.05), and these changes were obviously ameliorated by treatment with 20% Qingshen Granules-medicated serum for 24 h. Transfection of the cells with miR-23b-mimic significantly decreased the expressions of Keap1, α-SMA and ROS (P<0.05), which were further decreased by treatment with the medicated serum (P<0.05). Conclusion Qingshen Granules-medicated serum reduces transdifferentiation of NRK-52E cells via miR-23b-5p-mediated activation of the Nrf2 pathway.

Key words: miR-23b-5p; Nrf2 signaling pathway; NRK-52E cells; epithelial-mesenchymal transition of renal tubular; Qingshen Granules