六味地黄丸对去卵巢ApoE^(-/-)AS小鼠动脉粥样硬化的作用及机制研究（英文）

Liuweidihuang Formula(LWDHF) Inhibits Vascular Smooth Muscle Cell Proliferation and Migration via Estrogen Receptorβ-mediated pHSP27 Dephosphorylation and Attenuatesatherosclerosis in Ovariectomized ApoE^(-/-) Mice

目的探讨六味地黄丸(LWDHF)对更年期动脉粥样硬化的影响,阐明雌激素受体β和热休克蛋白27磷酸化在其中的作用。方法 HE染色观察ApoE-/-小鼠动脉损伤情况,免疫组织化学法检测血管内HSP27磷酸化水平。MTT法和流式细胞术分别检测VSMC增殖和细胞周期。通过伤口愈合实验和F-actin染色观察VSMC迁移。Western blot检测HSP27、pHSP27、cyclin D1、蛋白磷酸酶2和ERβ的表达。分别采用ERβsiRNA和ERαshRNA转染研究它们在LWDHF效应中的作用。结果 LWDHF改善了去卵巢ApoE-/-AS小鼠的动脉损伤,抑制了HSP27的磷酸化,并增加了PP2A和ERβ的表达。在体外实验中,1×10-7 mol/L浓度的血管紧张素Ⅱ明显诱导VSMC增殖和迁移,增加cyclin D1的表达和S期细胞数,但12μg/mL浓度的LWDHF可以明显抑制上述作用。进一步机制研究表明LWDHF通过ERβ上调PP2A的表达,抑制HSP27磷酸化,而抑制VSMC增殖和迁移。结论 LWDHF可以减轻更年期动脉粥样硬化,且通过调节ERβ抑制HSP27磷酸化,抑制VSMCs的增殖迁移。这些发现为LWDHF作为动脉粥样硬化的潜在治疗提供了新的理论基础。

OBJECTIVE To study the effects of Liuweidihuang Formula （LWDHF） on the ovariectomized ApoE^-/- AS mice and the proliferation and migration of vascular smooth muscle cell （VSMCs） in vitro , and to elucidate the roles of estrogen receptor β （ERβ） and heat shock protein 27 （HSP27） phosphorylation in the context. METHODS The artery injury of ApoE^-/- mice was observed by HE staining and the phosphorylation of HSP27 in vessels was detected by immunohistochemistry. VSMC proliferation and cell cycle were examined by MTT assay and flow cytometry, respectively. VSMC migration was observed by wound-healing assay and F actin staining. The levels of HSP27, pHSP27, cyclin D1, protein phosphatase 2 （PP2A） and ERβ were determined by Western blot analyses. Transfection with ERβ siRNA and ERα shRNA was carried out to investigate their roles in LWDHF effects, respectively. RESULTS LWDHF improved the arterial lesion, inhibited HSP27 phosphorylation, and increased the expression of PP2A and ERβ in ovariectomized ApoE^-/- AS mice. In in vitro experiments, angiotensin II （Ang Ⅱ） at 1×10^-7 mol/L obviously induced VSMC proliferation and migration, increased the expression of cyclin D1 and cell number in the S phase, but these effects were significantly inhibited by LWDHF （12 μg/mL）. Further molecular examinations showed that LWDHF up regulated PP2A expression through ERβ to inhibit HSP27 phosphorylation, contributing to inhibition of VSMC proliferation and migration. CONCLUSION LWDHF reduced menopause AS in vivo and inhibited proliferation and migration of VSMCs by regulating ERβ inhibition of HSP27 phosphorylation in vitro . These discoveries provided novel molecular insights into LWDHF as potential therapy for AS.