Liuwei Dihuang prevents postmenopausal atherosclerosis and endothelial cell apoptosis via inhibiting DNMT1-medicated ERα methylation

Ethnopharmacological relevance: The classical and traditional Chinese medicine prescription, Liuwei Dihuang (LWDH), has been commonly used to treat the menopausal syndrome. It has been reported that LWDH could improve estrogen receptor alpha (ER alpha) expression to prevent atherosclerosis (AS), while the mechanism of LWDH on regulating ER alpha expression was still unknown. Aim of the study: To reveal the mechanism of LWDH on regulating the ER alpha expression. Materials and methods: The protective effect of LWDH on Hcy-induced apoptosis of human umbilical vein endothelial cells (HUVECs) was examined. The expression of ER alpha and DNA methyltransferases 1 (DNMT1) were detected by Western blot and real-time polymerase chain reaction (RT-PCR). The methylation rate of the ER alpha gene was assayed by the bisulfite sequencing PCR (BSP). High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS) was applied to determine the level of S-Adenosyl methionine (SAM) and S-Adenosyl homocysteine (SAH). In vivo, the ApoE(-/-) mice were ovariectomized to establish postmenopausal atherosclerosis (AS) model. Results: In vitro study showed that LWDH protects HUVECs from Hcy-induced apoptosis. Treatment with LWDH significantly increased the ER alpha expression and reduced the methylation rate of the ER alpha gene by inhibiting the DNMT1 expression. The level of main methyl donor SAM and the ration of SAM/SAH were reduced by LWDH. In vivo, LWDH prevented the formation of plaque and reduced the concentration of Hcy. In addition, LWDH up regulated the ER alpha expression, as well as inhibiting the expression of DNMT1 in atherosclerotic mice. Conclusions: LWDH exerted protective effects on postmenopausal AS mice, and HUVECs treated with Hcy. LWDH increased of ER alpha expression via inhibiting DNMT1-dependent ER alpha methylation.