The molecular mechanisms of berberine-mediated inhibitory effect on platelet-derived growth factor-induced proliferation and migration in rat aortic smooth muscle cells

• Main Authors: Sui-Chu Yin, 尹修竹
• Other Authors: Shih-Lan Hsu
• Format: Others
• Language: zh-TW
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/81334110337557420924

碩士 === 國立中興大學 === 生命科學院碩士在職專班 === 95 === Abstract Deregulated proliferation and migration of vascular smooth muscle cells （VSMCs）induced neointima formation plays an important role in many coronary diseases including the pathogenesis of atherosclerosis, vein graft occlusion and post-angioplasty restenosis. Clinically up to date, there were still no effective therapy to cure such vascular diseases. Therefore, it is very imperative to find new drugs and strategies to treat such diseases. Platelet-derived growth factor （PDGF） is released from VSMCs, vascular endothelial cells （VECs）, platelets or macrophages after percutaneous coronary intervention and is strongly associated with neointima formation and restenosis. Berberine, an isoquinolinal plant alkaloid isolated from a well-known Chinese medicinal herb Huanglian (Coptis chinensis), has cholesterol-lowering, hypotensive, cell-growth inhibition, anti-cancer, anti-inflammatory and anti-microbial effects. Berberine could inhibit VSMCs proliferation and migration, yet the exact mechanisms are still unknown. Our previous results showed that berberine is capable of inhibiting cell growth and endogenous PDGF synthesis in rat aortic vascular smooth muscle cells（RASMCs）after in vitro mechanical injury. In the preceeding study, we explore the effects of berberine on RASMCs growth, migration, and downstream signaling events after exogenous PDGF-BB stimulation in vitro in order to mimic a post-angioplasty PDGF shedding condition. By flow cytometry analysis, our results showed that berberine could dose-dependently inhibit PDGF-BB-induced proliferation and cause cell cycle arrest at G0-G1 phase. Western blot analysis showed that PDGF-BB stimulated the activation of MEK1/2 （mitogen-activated protein kinase 1/2）, ERK1/2 （extracellular signal-regulated kinase 1/2）, Akt, and increased the expression of Cyclin-D1, -D3 and Cdk （cyclin dependent kinase）2, 4, and down-regulated Cdk inhibitor p21CIP1/WAF1expression. Semi-quantitative reverse-transcription PCR （RT-PCR） assay further confirmed the increase in cyclin-D1, -D3 and CDK2, 4 expression at transcriptional level. Berberine increased the activity of AMPK （adenosine monophosphate-activated protein kinase, AMP-activated protein kinase）, which led to phosphorylation activation of p53 and up-regulated protein level of Cdk inhibitor p21CIP1/WAF1. Berberine significantly suppressed MEK 1/2, ERK1/2, Akt activation and cyclin-D1, -D3 and Cdk2, 4 expression after PDGF-BB stimulation. Moreover, wound healing assay and Boyden chamber assay showed that berberine prevented PDGF-BB-induced migration in RASMCs. Furthermore, from pull-down assay demonstrated stimulation of RASMCs with PDGF-BB led to a transient increase in Ras, Rac1, Cdc42 activities; however, pretreatment with berberine for 24 h significantly inhibited PDGF-BB-induced Ras, Rac1, Cdc42 activation. These data suggest that berberine inhibited PDGF-BB-induced RASMCs growth via activating the AMPK/p53/p21CIP1/WAF1 signaling, inactivating the Ras/MEK/ ERK- Akt pathway, and suppress PDGF-BB-stimulated migration via inhibition of Rac/Cdc42. These observations offer a molecular explanation for the anti-proliferative and anti-migratory properties of berberine, and suggest that this drug may potentially be used in treating disorders due to unproperly SMCs proliferation.