Chen Yongyan, Han Xu, Hao Yuxuan, Wang Xu, Wu Fupeng, Tan Sheng, Ye Huijing, Hong Yang, Shen Xiaoyan
Department of Pharmacology, School of Pharmacy, Fudan University, Shanghai, PR China.
Department of Orthopedics, The Fifth People's Hospital of Shanghai, Shanghai Clinical Research Center for Aging and Medicine, Center of Community-Based Health Research, Fudan University, Shanghai, PR China.
Phytomedicine. 2025 Jun 6;145:156915. doi: 10.1016/j.phymed.2025.156915.
Vascular calcification is a major cause of death in chronic kidney disease (CKD), with osteoblastic transdifferentiation of vascular smooth muscle cells (VSMCs) considered to be the crucial pathologic process. However, there remains a significant deficiency in effective prevention and treatment strategies.
In an in vitro calcification screening model, we observed an inhibitory activity of Emodin on osteoblastic transdifferentiation of VSMCs. In the present study, we therefore aimed to evaluate its efficacy in CKD-induced medial vascular calcification in vitro and in vivo, and to further explore the underlying mechanisms.
A7R5 and MOVAS cells were treated with sodium phosphate to induce osteogenic transdifferentiation as in vitro models, while mice were fed with adenine and a high-phosphorus diet, and additionally received an intraperitoneal injection of 10,000 IU Vitamin D3 to induce chronic kidney disease (CKD) as an in vivo model. Vitamin K2 served as a positive control.
Alizarin Red S staining and VON-KOSSA staining were used to evaluate the effects of Emodin on osteogenic transdifferentiation. Western blotting, RT-qPCR and Von-Kossa staining were used to detect the effects of Emodin on aortic calcification in CKD mice in vivo. Chemical biology techniques including ITC, fluorescence titration, dual fluorescein reporter genes, CETSA, and DARTS, were used to detect the binding activity of Emodin to ERα and SYVN1. Immunoprecipitation, immunostaining, etc. were used to explore the mechanisms, and small molecule inhibitors and small RNA interference were used to verify the target of Emodin.
Emodin could effectively inhibit the osteogenic transdifferentiation of A7R5 and MOVAS cells in vitro, and alleviate aortic calcification in CKD mice in vivo. Mechanism study revealed that Emodin could act as a molecular glue that binds directly to ERα and SYVN1 and enhances their interaction, thereby accelerating the ubiquitination degradation process of ERα. The decrease in ERα diminished the inhibition of ERα on the deacetylation of HIF-1α by SIRT6, thereby inhibiting VSMC osteogenic transdifferentiation and relieving the vascular cells calcification.
Our study demonstrates that ERα has a non-genomic effect to inhibit the deacetylation of HIF-1α by SIRT6, which can be abrogated by Emodin through SYVN1-mediated ERα degradation. These results provide evidences for Emodin to serve as a candidate drug for controlling clinical vascular calcification in CKD.
血管钙化是慢性肾脏病（CKD）患者死亡的主要原因，血管平滑肌细胞（VSMCs）向成骨细胞转分化被认为是关键的病理过程。然而，在有效预防和治疗策略方面仍存在重大不足。
在体外钙化筛选模型中，我们观察到大黄素对VSMCs成骨细胞转分化具有抑制活性。因此，在本研究中，我们旨在评估其在体外和体内对CKD诱导的血管中层钙化的疗效，并进一步探索其潜在机制。
使用A7R5和MOVAS细胞，用磷酸钠处理以诱导成骨转分化作为体外模型，而给小鼠喂食腺嘌呤和高磷饮食，并额外腹腔注射10,000 IU维生素D3以诱导慢性肾脏病（CKD）作为体内模型。维生素K2作为阳性对照。
采用茜素红S染色和冯·科萨染色评估大黄素对成骨转分化的影响。使用蛋白质免疫印迹法、逆转录定量聚合酶链反应（RT-qPCR）和冯·科萨染色检测大黄素对体内CKD小鼠主动脉钙化的影响。使用包括等温滴定量热法（ITC）、荧光滴定、双荧光素报告基因、热蛋白质组分析（CETSA）和药物亲和反应靶标稳定性（DARTS）等化学生物学技术检测大黄素与雌激素受体α（ERα）和含SYVN1的E3泛素连接酶（SYVN1）的结合活性。采用免疫沉淀、免疫染色等方法探索其机制，并使用小分子抑制剂和小RNA干扰来验证大黄素的作用靶点。
大黄素可有效抑制体外A7R5和MOVAS细胞的成骨转分化，并减轻体内CKD小鼠的主动脉钙化。机制研究表明大黄素可作为分子胶直接结合ERα和SYVN1并增强它们的相互作用，从而加速ERα的泛素化降解过程。ERα的减少减弱了其对沉默调节蛋白6（SIRT6）介导的低氧诱导因子-1α（HIF-1α）去乙酰化的抑制作用，从而抑制VSMC成骨转分化并减轻血管细胞钙化。
我们的研究表明，ERα具有非基因组效应，可抑制SIRT6介导的HIF-1α去乙酰化，大黄素可通过SYVN1介导的ERα降解消除这种效应。这些结果为大黄素作为控制CKD临床血管钙化的候选药物提供了证据。
