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陈海燕, 潘薇, 刘仲豪, 周淑菁, 阳波, 李丹, 付成效.雷公藤甲素抑制KLF4/SLC7A11/GPX4通路促进铁死亡诱导肾毒性的机制研究[J].湖南中医药大学学报,2026,46(2):248-258[点击复制] |
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| 雷公藤甲素抑制KLF4/SLC7A11/GPX4通路促进铁死亡诱导肾毒性的机制研究 |
| 陈海燕,潘薇,刘仲豪,周淑菁,阳波,李丹,付成效 |
| (南华大学附属第二医院, 湖南 衡阳 421001;南华大学附属第一医院, 湖南 衡阳 421001;南华大学衡阳医学院, 湖南 衡阳 421001) |
| 摘要: |
| 目的 基于体内和体外实验，探讨雷公藤甲素通过诱导铁死亡介导肾毒性的作用，并阐明其分子机制。方法 随机将18只小鼠分为对照组（CON组）、雷公藤甲素低剂量组（LTP组）和雷公藤甲素高剂量组（HTP组），每组6只。CON组、LTP组和HTP组分别腹腔注射含1‰ DMSO的生理盐水、0.2 mg·kg-1雷公藤甲素和0.4 mg·kg-1雷公藤甲素，持续14 d。体外培养人肾小管上皮细胞（HK-2），予以雷公藤甲素、Fer-1和APTO-253干预，将其分为雷公藤甲素0 nmol/L组（TP 0 nmol/L组，无药物处理）、雷公藤甲素40 nmol/L组（TP 40 nmol/L组，40 nmol/L雷公藤甲素处理24 h）、雷公藤甲素80 nmol/L组（TP 80 nmol/L组，80 nmol/L雷公藤甲素处理24 h）和雷公藤甲素160 nmol/L组（TP 160 nmol/L组，160 nmol/L雷公藤甲素处理24 h）、对照组（CON组，无药物处理）、雷公藤甲素组（TP组，80 nmol/L雷公藤甲素处理24 h）、Fer-1组（2 μmol/L Fer-1处理24 h）、Fer-1+雷公藤甲素组（Fer-1+TP组，2 μmol/L Fer-1处理2 h后再加入80 nmol/L雷公藤甲素处理24 h）、APTO-253组（1 μmol/L APTO-253处理24 h）、APTO-253+雷公藤甲素组（APTO-253+TP组，1 μmol/L APTO-253处理24 h后再加入80 nmol/L雷公藤甲素处理24 h）。第14天雷公藤甲素处理后，所有动物称量体质量；采用MTT法检测HK-2细胞活力；生化试剂盒检测小鼠血清中尿素氮（BUN）、肌酐（Cr）含量及肾组织中谷胱甘肽（GSH）、丙二醛（MDA）、Fe2+水平；HE染色观察小鼠肾组织病理变化；透射电镜观察小鼠肾组织线粒体变化；DCFH-DA探针检测HK-2细胞ROS水平；Western blot检测小鼠肾组织中中性粒细胞明胶酶相关脂质运载蛋白（NGAL）、Krüppel样因子4（KLF4）、溶质载体家族7成员11（SLC7A11）、谷胱甘肽过氧化物酶4（GPX4）蛋白的表达水平。结果 与CON组相比，LTP组和HTP组小鼠体质量、肾组织GSH水平、肾组织KLF4、SLC7A11和GPX4蛋白表达水平均降低（P<0.05，P<0.01），血清BUN和Cr含量、肾组织NGAL蛋白表达水平、肾组织ROS、MDA和Fe2+水平均升高（P<0.05，P<0.01）。与LTP组相比，HTP组小鼠体质量、肾组织KLF4、SLC7A11和GPX4蛋白表达水平均降低（P<0.05），血清Cr含量、肾组织NGAL蛋白表达水平、肾组织ROS、MDA和Fe2+水平均升高（P<0.05，P<0.01）。与TP 0 nmol/L组相比，TP 40、80、160 nmol/L组ROS荧光强度增强，GSH水平降低（P<0.05，P<0.01），MDA水平升高（P<0.05，P<0.01）；TP 80、160 nmol/L组Fe2+水平升高（P<0.05，P<0.01），KLF4、SLC7A11、GPX4蛋白表达水平均降低（P<0.05，P<0.01）；TP 40 nmol/L组KLF4蛋白表达水平降低（P<0.05）。与TP 40 nmol/L组相比，TP 80、160 nmol/L组ROS荧光强度增强，GSH水平降低（P<0.05），Fe2+水平升高（P<0.05，P<0.01）；TP 160 nmol/L组MDA水平增加（P<0.01），KLF4、SLC7A11、GPX4蛋白表达水平均降低（P<0.05）；TP 80 nmol/L组、SLC7A11GPX4蛋白表达水平均降低（P<0.05）。与TP 80 nmol/L组相比，TP 160 nmol/L组ROS荧光强度增强，GSH水平降低（P<0.05），MDA水平和Fe2+水平均升高（P<0.05），KLF4、SLC7A11、GPX4蛋白表达水平均降低（P<0.05）。与CON组相比，TP组细胞活力、GSH水平、SLC7A11和GPX4蛋白表达水平均降低（P<0.01），ROS荧光强度增强，MDA和Fe2+水平均升高（P<0.01）；与TP组相比，Fer-1+TP组和APTO-253+TP组细胞活力、GSH水平、SLC7A11和GPX4蛋白表达水平均升高（P<0.05），ROS荧光强度减弱，MDA水平降低（P<0.05）；Fer-1+TP组Fe2+水平降低（P<0.05）。结论 雷公藤甲素通过下调KLF4抑制下游SLC7A11与GPX4的表达，进而诱导肾小管上皮细胞发生铁死亡，这是其引发肾毒性的关键分子机制。 |
| 关键词: 雷公藤甲素 肾毒性 铁死亡 Krüppel样因子4 SLC7A11/GPX4信号通路 |
| DOI：10.3969/j.issn.1674-070X.2026.02.005 |
| 投稿时间：2025-10-31 |
| 基金项目:湖南省自然科学基金项目(2025JJ80131);湖南省卫生健康委员会课题项目(W20243168)。 |
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| Mechanism of triptolide in inhibiting KLF4/SLC7A11/GPX4 pathway to promote ferroptosis-induced nephrotoxicity |
| CHEN Haiyan, PAN Wei, LIU Zhonghao, ZHOU Shujing, YANG Bo, LI Dan, FU Chengxiao |
| (The Second Affiliated Hospital of University of South China, Hengyang, Hunan 421001, China;The First Affiliated Hospital of University of South China, Hengyang, Hunan 421001, China;Hengyang Medical School, University of South China, Hengyang, Hunan 421001, China) |
| Abstract: |
| Objective To investigate the role of triptolide-induced nephrotoxicity mediated through ferroptosis and to elucidate its molecular mechanisms, based on both in vivo and in vitro experiments. Methods Eighteen mice were randomly divided into control group (CON group), low-dose triptolide group (LTP group), and high-dose triptolide group (HTP group), with six mice in each group. The CON, LTP, and HTP groups were intraperitoneally injected with saline containing 1‰ DMSO, 0.2 mg·kg-1 triptolide, and 0.4 mg·kg-1 triptolide, respectively, for 14 consecutive days. Human renal tubular epithelial cells (HK-2) were cultured in vitro and treated with triptolide, Fer-1, and APTO-253. They were divided into 0 nmoI/L triptolide group (TP 0 nmo1/L group, without drug treatment), 40 nmol/L triptolide group (TP 40 nmol/L group, treated with 40 nmol/L concentration of triptolide for 24 h), 80 nmol/L triptolide group (TP 80 nmol/L group, treated with 80 nmol/L concentration of triptolide for 24 h), 160 nmol/L triptolide group (TP 160 nmol/L group, treated with 160 nmol/L concentration of triptolide for 24 h), control group (CON group, without drug treatment)、triptolide group (TP group, treated with 80 nmol/L concentration of triptolide for 24 h)、Fer-1 group (treated with 2 μmol/L concentration of Fer-1 for 24 h), Fer-1+triptolide group (Fer-1+TP group, treated with 2 μmol/L concentration of Fer-1 for 2 h and followed by 80 nmol/L triptolide for 24 h), APTO-253 group (treated with 1 μmol/L concentration of APTO-253 for 24 h), and APTO-253+triptolide group (APTO-253+TP group, treated with 1 μmol/L concentration of APTO-253 for 24 h and followed by 80 nmol/L triptolide for 24 h). On the 14th day after triptolide treatment, the body mass of all animals was measured. HK-2 Cell viability was assessed using the MTT assay. Levels of blood urea nitrogen (BUN) and creatinine (Cr) in mouse serum, as well as levels of glutathione (GSH), malondialdehyde (MDA), and Fe2+ in renal tissue, were measured using biochemical assay kits. Renal histopathological changes in mice were observed by HE staining. Mitochondrial alterations in mouse renal tissue were examined by transmission electron microscopy. Intracellular reactive oxygen species (ROS) levels in HK-2 cells were determined using the DCFH-DA probe. The protein expression levels of neutrophil gelatinase-associated lipocalin (NGAL), Krüppel-like factor 4 (KLF4), solute carrier family 7 member 11 (SLC7A11), and glutathione peroxidase 4 (GPX4) in mouse renal tissue were determined by Western blot. Results Compared with the CON group, the body mass, renal GSH levels, and the protein expression levels of KLF4, SLC7A11, and GPX4 in renal tissues of mice decreased in both the LTP and HTP groups (P<0.05, P<0.01). Serum BUN and Cr levels, renal NGAL protein expression, and renal levels of ROS, MDA, and Fe2+ were increased (P<0.05, P<0.01). Compared with the LTP group, the HTP group showed reduced body mass and renal protein expression levels of KLF4, SLC7A11, and GPX4 (P<0.05), while serum Cr levels, renal NGAL protein expression, and renal levels of ROS, MDA, and Fe2+ were elevated (P<0.05, P<0.01). Compared with the TP 0 nmol/L group, the TP 40, 80, and 160 nmol/L groups exhibited enhanced ROS fluorescence intensity, decreased GSH levels (P<0.05, P<0.01), and increased MDA levels (P<0.05, P<0.01). The TP 80 and 160 nmol/L groups also showed increased Fe2+ levels (P<0.05, P<0.01) and decreased protein expression levels of KLF4, SLC7A11, and GPX4 (P<0.05, P<0.01). The TP 40 nmol/L group showed decreased KLF4 protein expression (P<0.05). Compared with the TP 40 nmol/L group, the TP 80 and 160 nmol/L groups demonstrated enhanced ROS fluorescence intensity, decreased GSH levels (P<0.05), and increased Fe2+ levels (P<0.05, P<0.01). The TP 160 nmol/L group also showed increased MDA levels (P<0.01) and decreased protein expression levels of KLF4, SLC7A11, and GPX4 (P<0.05), while the TP 80 nmol/L group showed decreased protein expression levels of SLC7A11 and GPX4 (P<0.05). Compared with the TP 80 nmol/L group, the TP 160 nmol/L group showed enhanced ROS fluorescence intensity, decreased GSH levels (P<0.05), increased MDA and Fe2+ levels (P<0.05), and decreased protein expression levels of KLF4, SLC7A11, and GPX4 (P<0.05). Compared with the CON group, the TP group showed reduced cell viability, GSH levels, as well as SLC7A11 and GPX4 protein expression levels (P<0.01), along with enhanced ROS fluorescence intensity and increased MDA and Fe2+ levels (P<0.01). Compared with the TP group, both the Fer-1+TP and APTO-253+TP groups exhibited increased cell viability, GSH levels, SLC7A11 and GPX4 protein expression levels (P<0.05), as well as reduced ROS fluorescence intensity and decreased MDA levels (P<0.05). The Fer-1+TP group also showed reduced Fe2+ levels (P<0.05). Conclusion Triptolide induces ferroptosis in renal tubular epithelial cells by downregulating KLF4, which subsequently suppresses the expression of its downstream targets SLC7A11 and GPX4. This mechanism represents a key molecular pathway underlying triptolide-induced nephrotoxicity. |
| Key words: triptolide nephrotoxicity ferroptosis Krüppel-like factor 4 SLC7A11/GPX4 signaling pathway |
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