Objective: To evaluate the efficacy of Liuwei Dihuang formula ( LWDHF) on endothelial cells, and to study the mechanism behind the action of modulating expression of estrogen receptors.
Methods: Hydrogen peroxide (H2O2) was applied to induce the apoptosis of human umbilical vein endothelial cells (HUVECs). The concentration of nitric oxide (NO), endothelial nitric oxide synthase (eNOS) and inducible NOS (iNOS) were measured by assay kits. Western blot and real-time polymerase chain reaction (RT-PCR) were used to detect the expression of iNOS, eNOS, b-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), estrogen receptor (ER) α and ERβ. Also, small interfering RNA (siRNA) was involved to confirm whether the protective effects of LWDHF was medicated by ERs. In vivo, the female rats were ovariectomized to establish postmenopausal vascular injury model. Then the model rats were divided into three groups and treated with saline, estradiol and LWDHF respectively. The concentration of NO and NOS in serum were measured by assay kits, and the expression of Bax, Bcl-2, ERα and ERβ were detected by western blot and immunohistochemistry.
Results: In vitro study, LWDHF significantly protected HUVECs from H2O2-induced apoptosis, with the increase of Bcl-2 and the decrease of Bax. The treatment with LWDHF inhibited concentration of NO and iNOS, and upregulated the expression of eNOS, ERα and ERβ. In addition, ERα siRNA could block the protective effects of LWDHF, while ERβ siRNA showed little influence. In vivo, the treatment with LWDHF suppressed the vascular injury and reduced the level of NO and NOS. LWDHF increased the expression of Bcl-2, ERα and ERβ, as well as inhibiting the Bax expression.
Conclusion: LWDHF could improve endothelial function and protect HUVECs from apoptosis via increasing the expression of ERα.
Keywords: Liuwei Dihuang; apoptosis; human umbilical vein endothelial cells; receptors, estrogen.