Zhilong Huoxue Tongyu capsule protects against atherosclerosis by suppressing EndMT via modulating Hippo/YAP signaling pathway

Zhilong Huoxue Tongyu Capsule (ZL capsule) has been demonstrated to be an effective and widely-used traditional Chinese medicine (TCM) formula for the treatment of various diseases, especially for atherosclerosis (AS) related cardiovascular and cerebrovascular diseases. Reversal of endothelial-mesenchymal transition (EndMT) plays a crucial role in the cure of AS. But the curative impact of ZL capsule on EndMT remains obscure during the development of AS. The purpose of this study is to explore the effect of ZL capsule on AS and to study the regulation mechanism on EndMT in AS by ZL capsule in vivo and in vitro. An in vivo model of AS was induced in ApoE−/− mice by administrating them with an 8-week period of high-fat diet (HFD). After oral gavage of different doses of ZL capsule and Atorvastatin calcium tablets (ATO) for 4 weeks, the lipid levels, plaque area, lipid deposition, and EndMT were evaluated using standard assays. In order to simulate EndMT in vitro, human umbilical vein endothelial cells (HUVECs) were subjected to oxidized low-density lipoprotein (ox-LDL). Western blotting (WB) and immunofluorescence techniques were used to evaluate the intervention effect of ZL capsule on EndMT and Hippo/YAP pathways. ZL capsule demonstrated therapeutic effects on dyslipidemia and EndMT among atherosclerotic mice. To be specific, ZL capusle diminished the total cholesterol (TC), total triglyceride (TG) and low-density lipoprotein (LDL-C) levels, whereas increased that of high-density lipoproteins (HDL-C). Meanwhile, ZL capusle upregulated the expression of endothelial markers (CD31 and VE-cadherin) and reduced that of mesenchymal markers (ɑ-SMA and FSP1), indicating that ZL capusle could inhibit EndMT during the development of AS. Furthermore, molecular docking results indicated that active ingredients including formononetin, calycosin, astragaloside III, astragaloside A in ZL capsule have strong affinity with YAP proteins, and ZL capsule can significantly repress the initiation of Hippo/YAP pathway during AS. In conclusion, ZL capsule effectively attenuated AS progression by exerting inhibitory effects on EndMT through modulation of the Hippo/YAP signaling pathway.