Liuwei Dihuang soft capsules inhibits the phenotypic conversion of VSMC to prevent the menopausal atherosclerosis by up-regulating the expression of myocardin

Ethnopharmacological relevance: Liuwei Dihuang (LWDH) is a classic prescription that has been used as a traditional medicinal formula for more than 1000 years in China. In clinical, LWDF is used for treating functional decline associated with senile disease and menopausal syndrome. Studies have demonstrated that LWDH could significantly improve estrogen level and ER expression, and suspend the process of atherosclerosis. However, the under mechanism of how LWDH suppressing VSMCs phenotypic conversion and proliferation through ER is still unknown. Aim of the study: This study was to reveal the under mechanism of how LWDH inhibits the phenotypic conversion of VSMCs. Materials and methods: 24 ApoE- / - mice were divided into 4 groups: sham group, model group, E-2 group, and LWDH group, and 6 C57BN/L6 mice were used as control group. The primary VSMCs were divided into control group, model group, E-2 group, LWDH group, LWDH + MPP group, and LWDH + PHTPP group with or without control siRNA, ER alpha siRNA, ER beta siRNA, and myocardin siRNA. Oil red staining was used to evaluate the lipid deposition in the cardiac aorta. Serum chemistry analysis to test serum TG, TC, LDL, and HDL. Immunofluorescence staining was used to test alpha-SMA, osteopontin and F-actin. Immunohistochemical staining was performed to check out the myocardin in the cardiac aorta. The mRNA levels of alpha-SMA, osteopontin, ER alpha, ER beta, SRC3 and myocardin were detected by Real Time-PCR, and the protein expression levels of them were detected by Western blotting. Co-immunoprecipitation was proceed to test the interaction between ER alpha and SRC3 and SRC3 and myocardin. Flow cytometry was used to check out the cell cycle. Wound healing assay and Transwell were managed to evaluate the migration capacity of VSMCs. Results: In vivo administration of LWDH suppressed AS symptoms, decreases phenotypic marker of vascular endothelial cell, and increases phenotypic marker of VSMC in ovariectomized ApoE - / - female mice. Moreover, LWDH significantly increased the mRNA and protein expression levels of ER alpha, ER beta, SRC3 and myocardin in the cardiac aorta of ovariectomized ApoE - / - female mice. In vitro, LWDH altered cell cycle and reduced the elevated cyclinD protein expression migration capacity and in the model VSMCs. In addition, LWDH inhibited phenotypic conversion and promoted the expression of ER, SRC3, and myocardin of the primary VSMC phenotypic conversion model. Inhibition of ER alpha almost completely eliminated the impacts of LWDH on alpha-SMA and osteopontin. Furthermore, LWDH promoted the interaction between ER alpha and SRC3 and up-regulated the coactivation of SRC3 and myocardin. Conclusions: LWDH could inhibit the phenotypic conversion of VSMCs in vitro and in vivo by increasing the activity of myocardin through up-regulating the expression of ER alpha and promoting the interaction between ER alpha and SRC3. Our research reveals the under mechanism of how LWDH inhibits the phenotypic conversion of VSMCs.