Shan Xiao-Meng, Chen Chun-Wei, Zou Da-Wei, Gao Yan-Bin, Ba Yin-Ying, He Jia-Xin, Zhu Zhi-Yao, Liang Jia-Jun
School of Traditional Chinese Medicine, Capital Medical University, #10, Youanmenwai, Xitoutiao, Fengtai District, Beijing, 100069, People's Republic of China.
Beijing Key Lab of TCM Collateral Disease Theory Research, #10, Youanmenwai, Xitoutiao, Fengtai District, Beijing, 100069, People's Republic of China.
Chin Med. 2024 Oct 29;19(1):151. doi: 10.1186/s13020-024-01007-8.
Tangshenning (TSN) is a safe and effective formula to treat diabetic nephropathy (DN), and clinical studies have demonstrated that its therapeutic effects are related to oxidative stress improvements in patients. Herein, this study aims to explore the potential mechanism of how TSN alleviates diabetic renal tubular injury.
The ultrahigh pressure liquid chromatography-quadrupole-time of flight mass spectrometry (UPLC-QTOF/MS) was used to identify the chemical composition and serum components of TSN. KK-Ay mice served to investigate the protective effects and regulatory mechanisms of TSN on tubular damage in DN. Furthermore, inhibitors and inducers of ferroptosis were employed in high glucose-cultured tubular epithelial cells (TECs) to verify the potential mechanisms of TSN. The expressions of proteins related to renal tubular injury, ferroptosis and solute carrier family 7, member 11 (SLC7A11)/glutathione (GSH)/glutathione peroxidase 4 (GPX4) axis were analyzed by western blot and immunofluorescence. Mitochondrial ultrastructure was observed in kidney tissues and TECs by a transmission electron microscope. Pathological changes in the renal tissues were observed by HE, PAS, and Prussian blue staining. Ferroptosis-related reactive oxygen species (ROS), malondialdehyde (MDA), ferrous ion, the intake of cystine, GSH, and oxidized glutathione (GSSG) were evaluated and contrasted in vivo or in vitro.
51 compounds of TSN powder and 11 components in TSN-containing serum were identified by UPLC-QTOF/MS method. Administration of TSN ameliorated the elevated levels of proteinuria, serum creatinine, blood urea nitrogen, abnormal expression of renal tubular injury markers, and pathological damage to the renal tubules in DN mice model. Intriguingly, a strong inhibition of ferroptosis after TSN treatment occurred in both DN mice model and high glucose-cultured TECs. Notably, induction of ferroptosis by erastin attenuated the protective effect of TSN in high glucose-cultured TECs, while the ferroptosis inhibition by ferrostatin-1 treatment protected renal tubular, which was similar to TSN, suggesting the contribution of TSN-mediated by the inhibition of ferroptosis in DN progression. Mechanistically, TSN upregulated the SLC7A11/GSH/GPX4 axis to inhibit ferroptosis.
TSN may delay the DN progression and attenuate the renal tubular injury by inhibiting the ferroptosis regulated by the SLC7A11/GSH/GPX4 axis.
糖肾宁（TSN）是一种治疗糖尿病肾病（DN）安全有效的方剂，临床研究表明其治疗效果与改善患者氧化应激有关。本研究旨在探讨TSN减轻糖尿病肾小管损伤的潜在机制。
采用超高压液相色谱-四极杆-飞行时间质谱（UPLC-QTOF/MS）鉴定TSN的化学成分和血清成分。采用KK-Ay小鼠研究TSN对DN肾小管损伤的保护作用及调节机制。此外，在高糖培养的肾小管上皮细胞（TECs）中使用铁死亡抑制剂和诱导剂，以验证TSN的潜在机制。通过蛋白质免疫印迹法和免疫荧光法分析与肾小管损伤、铁死亡及溶质载体家族7成员11（SLC7A11）/谷胱甘肽（GSH）/谷胱甘肽过氧化物酶4（GPX4）轴相关的蛋白表达。通过透射电子显微镜观察肾脏组织和TECs中的线粒体超微结构。通过苏木精-伊红（HE）、过碘酸雪夫（PAS）和普鲁士蓝染色观察肾组织的病理变化。在体内或体外评估并对比与铁死亡相关的活性氧（ROS）、丙二醛（MDA）、亚铁离子、胱氨酸摄取、GSH和氧化型谷胱甘肽（GSSG）。
采用UPLC-QTOF/MS法鉴定出TSN粉剂中的51种化合物和含TSN血清中的11种成分。给予TSN可改善DN小鼠模型中蛋白尿、血清肌酐、血尿素氮水平的升高，肾小管损伤标志物的异常表达以及肾小管的病理损伤。有趣的是，在DN小鼠模型和高糖培养的TECs中，TSN治疗后均强烈抑制铁死亡。值得注意的是，erastin诱导铁死亡减弱了TSN在高糖培养的TECs中的保护作用，而铁死亡抑制剂ferrostatin-1处理对肾小管的保护作用与TSN相似，表明TSN通过抑制铁死亡在DN进展中发挥作用。机制上，TSN上调SLC7A11/GSH/GPX4轴以抑制铁死亡。
TSN可能通过抑制SLC7A11/GSH/GPX4轴调节的铁死亡来延缓DN进展并减轻肾小管损伤。
