Zhu Xuexue, Meng Xinyu, Du Xinyao, Zhao Chenyang, Ma Xinyu, Wen Yuanyuan, Zhang Shijie, Hou Bao, Cai Weiwei, Du Bin, Han Zhijun, Xu Fei, Qiu Liying, Sun Haijian
Department of Basic Medicine, Wuxi School of Medicine, Jiangnan University, Wuxi 214122, China.
Department of Clinical Research Center, Jiangnan University Medical Center, Wuxi 214001, China.
Acta Biochim Biophys Sin (Shanghai). 2024 Aug 27;56(12):1860-1874. doi: 10.3724/abbs.2024141.
Diabetic nephropathy (DN) is recognized as one of the primary causes of chronic kidney disease and end-stage renal disease. Vaccarin (VAC) confers favorable effects on cardiovascular and metabolic diseases, including type 2 diabetes mellitus (T2DM). Nonetheless, the potential role and mechanism of VAC in the etiology of DN have yet to be completely elucidated. In this study, a classical mouse model of T2DM is experimentally induced via a high-fat diet (HFD)/streptozocin (STZ) regimen. Renal histological changes are assessed via H&E staining. Masson staining and immunohistochemistry (IHC) are employed to assess renal fibrosis. RT-PCR is utilized to quantify the mRNA levels of renal fibrosis, oxidative stress and inflammation markers. The levels of malondialdehyde (MDA) and reactive oxygen species (ROS), as well as the content of glutathione peroxidase (GSH-Px), are measured. The protein expressions of collagen I, TGF-β1, α-SMA, E-cadherin, Nrf2, catalase, SOD3, SOD2, SOD1, p-ERK, p-EGFR (Y845), p-EGFR (Y1173), p-NFκB P65, t-ERK, t-EGFR and t-NFκB P65 are detected by western blot analysis. Our results reveal that VAC has a beneficial effect on DN mice by improving renal function and mitigating histological damage. This is achieved through its inhibition of renal fibrosis, inflammatory cytokine overproduction, and ROS generation. Moreover, VAC treatment effectively suppresses the process of epithelial-mesenchymal transition (EMT), a crucial characteristic of renal fibrosis, in high glucose (HG)-induced HK-2 cells. Network pharmacology analysis and molecular docking identify epidermal growth factor receptor (EGFR) as a potential target for VAC. Amino acid site mutations reveal that Lys-879, Ile-918, and Ala-920 of EGFR may mediate the direct binding of VAC to EGFR. In support of these findings, VAC reduces the phosphorylation levels of both EGFR and its downstream mediator, extracellular signal-regulated kinase 1/2 (ERK1/2), in diabetic kidneys and HG-treated HK-2 cells. Notably, blocking either EGFR or ERK1/2 yields renal benefits similar to those observed with VAC treatment. Therefore, this study reveals that VAC attenuates renal damage via inactivation of the EGFR/ERK1/2 signaling axis in T2DM patients.
糖尿病肾病（DN）被认为是慢性肾脏病和终末期肾病的主要病因之一。刺囊酸（VAC）对心血管和代谢性疾病具有有益作用，包括2型糖尿病（T2DM）。尽管如此，VAC在DN病因学中的潜在作用和机制尚未完全阐明。在本研究中，通过高脂饮食（HFD）/链脲佐菌素（STZ）方案实验性诱导建立经典的T2DM小鼠模型。通过苏木精-伊红（H&E）染色评估肾脏组织学变化。采用Masson染色和免疫组织化学（IHC）评估肾脏纤维化。利用逆转录-聚合酶链反应（RT-PCR）定量肾脏纤维化、氧化应激和炎症标志物的mRNA水平。测定丙二醛（MDA）和活性氧（ROS）水平以及谷胱甘肽过氧化物酶（GSH-Px）含量。通过蛋白质印迹分析检测I型胶原、转化生长因子-β1（TGF-β1）、α-平滑肌肌动蛋白（α-SMA）、E-钙黏蛋白、核因子E2相关因子2（Nrf2）、过氧化氢酶、超氧化物歧化酶3（SOD3）、超氧化物歧化酶2（SOD2）、超氧化物歧化酶1（SOD1）、磷酸化细胞外信号调节激酶（p-ERK）、磷酸化表皮生长因子受体（p-EGFR，Y845）、磷酸化表皮生长因子受体（p-EGFR，Y1173）、磷酸化核因子κB p65（p-NFκB P65）、总细胞外信号调节激酶（t-ERK）、总表皮生长因子受体（t-EGFR）和总核因子κB p65（t-NFκB P65）的蛋白表达。我们的结果表明，VAC通过改善肾功能和减轻组织学损伤对DN小鼠具有有益作用。这是通过抑制肾脏纤维化、炎症细胞因子过度产生和ROS生成来实现的。此外，VAC处理有效抑制了高糖（HG）诱导的人近端肾小管上皮细胞（HK-2细胞）中上皮-间质转化（EMT）过程，而EMT是肾脏纤维化的一个关键特征。网络药理学分析和分子对接确定表皮生长因子受体（EGFR）是VAC的一个潜在靶点。氨基酸位点突变表明，EGFR的赖氨酸-879（Lys-879）、异亮氨酸-918（Ile-918）和丙氨酸-920（Ala-920）可能介导VAC与EGFR的直接结合。支持这些发现的是，VAC降低了糖尿病肾脏和HG处理的HK-2细胞中EGFR及其下游介质细胞外信号调节激酶1/2（ERK1/2）的磷酸化水平。值得注意的是，阻断EGFR或ERK1/2产生的肾脏益处与VAC处理所观察到的相似。因此，本研究表明，VAC通过使T2DM患者的EGFR/ERK1/2信号轴失活来减轻肾脏损伤。
