Li Ying-Ying, Tian Zeng-Hui, Pan Guang-Hui, Zhao Ping, Pan De-Jun, Zhang Jun-Qing, Ye Li-Ying, Zhang Fa-Rong, Xu Xiang-Dong
College of First Clinical Medical, Shandong University of Traditional Chinese Medicine, Jinan, China.
Department of Nephrology, Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Jinan, China.
Front Pharmacol. 2022 Sep 9;13:977284. doi: 10.3389/fphar.2022.977284. eCollection 2022.
Renal fibrosis is a common pathway for the progression of various chronic kidney diseases (CKD), and the formation and deterioration will eventually lead to end-stage renal failure, which brings a heavy medical burden to the world. HeidihuangWan (HDHW) is a herbal formulation with stable and reliable clinical efficacy in the treatment of renal fibrosis. However, the mechanism of HDHW in treating renal fibrosis is not clear. In this study, we aimed to investigate the mechanism of HDHW to improve renal fibrosis. Wistar rats were randomly divided into the normal control group, 5/6 Nephrectomy group, astragaloside IV (AS-IV) group, HDHW group, and HDHW + IGF-1R inhibitor (JB1) group. Except for the normal control group, the rat renal fibrosis model was established by 5/6 nephrectomy and intervened with drugs for 8 weeks. Blood samples were collected to evaluate renal function. Hematoxylin-Eosin (HE), Periodic Acid-Schiff (PAS), Modified Masson's Trichrome (Masson) staining were used to evaluate the pathological renal injury, and immunohistochemistry and Western blotting were used to detect the protein expression of renal tissue. The results showed that HDHW was effective in improving renal function and reducing renal pathological damage. HDHW down-regulated the levels of fibrosis marker proteins, including α-smooth muscle actin (α-SMA), vimentin, and transforming growth factors-β(TGF-β), which in turn reduced renal fibrosis. Further studies showed that HDHW down-regulated the expression of autophagy-related proteins Beclin1 and LC3II, indicating that HDHW inhibited autophagy. In addition, we examined the activity of the class I phosphatidylinositol-3 kinase (PI3K)/serine-threonine kinase (Akt)/mTOR pathway, an important signaling pathway regulating autophagy, and the level of insulin-like growth factor 1 (IGF-1), an upstream activator of PI3K/Akt/mTOR. HDHW upregulated the expression of IGF-1 and activated the PI3K/Akt/mTOR pathway, which may be a vital pathway for its inhibition of autophagy. Application of insulin-like growth factor 1 receptor (IGF-1R) inhibitor further confirmed that the regulation of autophagy and renal fibrosis by HDHW was associated with IGF-1-mediated activation of the PI3K/Akt/mTOR pathway. In conclusion, our study showed that HDHW inhibited autophagy by upregulating IGF-1 expression, promoting the binding of IGF-1 to IGF-1R, and activating the PI3K/Akt/mTOR signaling pathway, thereby reducing renal fibrosis and protecting renal function. This study provides support for the application and further study of HDHW.
肾纤维化是各种慢性肾脏病（CKD）进展的共同途径，其形成和恶化最终将导致终末期肾衰竭，给全球带来沉重的医疗负担。黑地黄丸（HDHW）是一种在治疗肾纤维化方面临床疗效稳定可靠的中药制剂。然而，HDHW治疗肾纤维化的机制尚不清楚。在本研究中，我们旨在探讨HDHW改善肾纤维化的机制。将Wistar大鼠随机分为正常对照组、5/6肾切除组、黄芪甲苷（AS-IV）组、HDHW组和HDHW +胰岛素样生长因子1受体（IGF-1R）抑制剂（JB1）组。除正常对照组外，其余大鼠通过5/6肾切除建立肾纤维化模型，并用药干预8周。采集血样评估肾功能。采用苏木精-伊红（HE）染色、过碘酸-希夫（PAS）染色、改良Masson三色染色（Masson）评估肾脏病理损伤，采用免疫组化和蛋白质印迹法检测肾组织蛋白表达。结果显示，HDHW能有效改善肾功能，减轻肾脏病理损伤。HDHW下调了包括α-平滑肌肌动蛋白（α-SMA）、波形蛋白和转化生长因子-β（TGF-β）在内的纤维化标志物蛋白水平，从而减轻了肾纤维化。进一步研究表明，HDHW下调了自噬相关蛋白Beclin1和LC3II的表达，表明HDHW抑制了自噬。此外，我们检测了调节自噬的重要信号通路I类磷脂酰肌醇-3激酶（PI3K）/丝氨酸-苏氨酸激酶（Akt）/哺乳动物雷帕霉素靶蛋白（mTOR）通路的活性，以及PI3K/Akt/mTOR的上游激活剂胰岛素样生长因子1（IGF-1）的水平。HDHW上调了IGF-1的表达并激活了PI3K/Akt/mTOR通路，这可能是其抑制自噬的关键通路。应用胰岛素样生长因子1受体（IGF-1R）抑制剂进一步证实，HDHW对自噬和肾纤维化的调节与IGF-1介导的PI3K/Akt/mTOR通路激活有关。总之，我们的研究表明，HDHW通过上调IGF-1表达、促进IGF-1与IGF-1R结合以及激活PI3K/Akt/mTOR信号通路抑制自噬，从而减轻肾纤维化并保护肾功能。本研究为HDHW的应用和进一步研究提供了依据。
