Molecular Mechanism of Berberine on the Regulation of Vascular Smooth Muscle Cell Growth and Migration

• Main Authors: Kae-Woei Liang, 梁凱偉
• Other Authors: Shih-Lan Hsu
• Format: Others
• Language: en_US
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/57298591368728411498

博士 === 國立陽明大學 === 臨床醫學研究所 === 96 === Vascular smooth muscle cell (VSMC) proliferation and migration play an important role in the pathogenesis of atherosclerosis and post-angioplasty restenosis. Platelet-derived growth factor (PDGF) is released from VSMCs, endothelial cells, or macrophages after percutaneous coronary intervention and is associated with neointimal proliferation and restenosis. Berberine is a well-known component of the Chinese herb medicine Huanglian (Coptis chinensis), and is capable of inhibiting VSMC contraction and proliferation, yet the exact mechanism of action of berberine is unknown. In the first part of the study, we first investigated the effect of berberine on VSMC growth after mechanical injury in vitro. DNA synthesis and cell proliferation assays were performed and showed that berberine inhibits serum-stimulated rat aortic VSMC growth in a concentration-dependent manner. Mechanical injury with a sterile pipette tip stimulated the regrowth of VSMCs. Treatment with berberine prevented the regrowth and migration of VSMCs into the denuded trauma zone. Western blot analysis after mechanic injury in vitro showed the following sequential events: 1) the activation of MEK1/2 (mitogen-activated protein kinase kinase 1/2), 2) the activation of extracellular signal-regulated kinase (ERK), and 3) the up-regulation of early growth response gene (Egr-1), c-Fos and Cyclin D1. Semi-quantitative reverse-transcription polymerase chain reaction (RT-PCR) further confirmed the increase in egr-1, c-fos, PDGF, and cyclin D1 expression at the transcriptional level. However, berberine significantly attenuated MEK/ERK activation and downstream target (Egr-1, c-Fos, and then Cyclin D1, and PDGF-A) expression after mechanical injury in vitro. This study showed that berberine blocked injury-induced VSMC regrowth by inactivating the ERK/Egr-1 signaling pathway, thereby preventing early signaling induced by injury in vitro. In the 2nd part of the study, we analyzed the effects of berberine on VSMC growth, migration, and signaling events after exogenous PDGF stimulation in vitro in order to mimic PDGF shedding post-angioplasty. Pretreatment of VSMCs with berberine inhibited PDGF-induced proliferation causing G1 arrest. Berberine significantly suppressed PDGF-stimulated cyclin, D1/D3, and cyclin-dependent kinases (Cdk)s gene expression. Moreover, berberine increased the activity of AMP-activated protein kinase (AMPK), which led to the phosphorylation and activation of p53 and the increase in expression (protein level) of the Cdk inhibitor p21Cip1. Treatment with compound C, an AMPK inhibitor, partly but significantly attenuated berberine-mediated growth inhibition. In contrast, imidazole-4-carboxamide 1-ribofuranoside (AICAR), an AMPK activator, potentiated berberine-mediated anti-proliferative effects in VSMCs. In addition, stimulation of VSMCs with PDGF transiently increased the GTP-bound, active forms of Ras, Cdc42, and Rac1, as well as enhanced VSMC migration. However, pretreatment with berberine significantly inhibited PDGF-induced Ras, Cdc42, and Rac1 activation and cell migration. Co-treatment with farnesyl pyrophosphate and geranylgeranyl pyrophosphate drastically reversed berberine-mediated anti-proliferative and migratory effects in VSMCs. Based on these findings, we concluded that berberine inhibited PDGF-induced VSMC growth via activation of AMPK/p53/p21Cip1 signaling, while inactivating Ras/Rac1/Cyclin D/Cdks and suppressing PDGF-stimulated migration via inhibition of Cdc42 and Rac1. These observations offer a molecular explanation for the anti-proliferative and anti-migratory properties of berberine.