Vascular injury-induced endothelium-denudation and profound vascular smooth muscle cells (VSMCs) proliferation and dis-regulated apoptosis lead to post-angioplasty restenosis. Coptisine (CTS), an isoquinoline alkaloid, has multiple beneficial effects on the cardiovascular system. Recent studies identified it selectively inhibits VSMCs proliferation. However, its effects on neointimal hyperplasia, re-endothelialization, and the underlying mechanisms are still unclear. Cell viability was assayed by 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and cell counting kit-8 (CCK-8). Cell proliferation and apoptosis were measured by flow cytometry and immunofluorescence of Ki67 and TUNEL. Quantitative phosphoproteomics (QPP) was employed to screen CTS-responsive phosphor-sites in the key regulators of cell proliferation and apoptosis. Neointimal hyperplasia was induced by balloon injury of rat left carotid artery (LCA). Adenoviral gene transfer was conducted in both cultured cells and LCA. Re-endothelialization was evaluated by Evan's blue staining of LCA. 1) CTS had strong anti-proliferative and pro-apoptotic effects in cultured rat VSMCs, with the EC 4∼10-folds lower than that in endothelial cells (ECs). 2) Rats administered with CTS, either locally to LCA's periadventitial space or orally, demonstrated a potently inhibited balloon injury-induced neointimal hyperplasia, but had no delaying effect on re-endothelialization. 3) The QPP results revealed that the phosphorylation levels of Pak1, Pak2, Erk1, Erk2, and Bad were significantly decreased in VSMCs by CTS. 4) Adenoviral expression of phosphomimetic mutants Pak1/Pak2 enhanced Pak1/2 activities, stimulated the downstream pErk1/pErk2/pErk3/pBad, attenuated CTS-mediated inhibition of VSMCs proliferation and promotion of apoptosis , and potentiated neointimal hyperplasia . 5) Adenoviral expression of phosphoresistant mutants Pak1/Pak2 inactivated Pak1/2 and totally simulated the inhibitory effects of CTS on platelet-derived growth factor (PDGF)-stimulated VSMCs proliferation and PDGF-inhibited apoptosis and neointimal hyperplasia . 6) LCA injury significantly enhanced the endogenous phosphorylation levels of all but pBad. CTS markedly attenuated all the enhanced levels. These results indicate that CTS is a promising medicine for prevention of post-angioplasty restenosis without adverse impact on re-endothelialization. CTS-directed suppression of pPak1/pPak2 and the subsequent effects on downstream pErk1/pErk2/pErk3 and pBad underline its mechanisms of inhibition of VSMCs proliferation and stimulation of apoptosis. Therefore, the phosphor-sites of Pak1/Pak2 constitute a potential drug-screening target for fighting neointimal hyperplasia restenosis.

中文翻译：

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黄连碱通过减弱血管平滑肌细胞中的 Pak1/Pak2 信号传导来抑制新生内膜增生，而不延迟再内皮化

血管损伤诱导的内皮剥脱和深层血管平滑肌细胞（VSMC）增殖和失调的细胞凋亡导致血管成形术后再狭窄。黄连碱 (CTS) 是一种异喹啉生物碱，对心血管系统具有多种有益作用。最近的研究发现它选择性地抑制 VSMC 增殖。然而，其对内膜增生、再内皮化的影响及其潜在机制仍不清楚。通过 3-[4,5-二甲基噻唑-2-基]-2,5-二苯基溴化四唑 (MTT) 和细胞计数试剂盒-8 (CCK-8) 测定细胞活力。通过流式细胞术以及Ki67和TUNEL免疫荧光检测细胞增殖和凋亡。采用定量磷酸蛋白质组学 (QPP) 筛选细胞增殖和凋亡关键调节因子中 CTS 响应的磷酸位点。大鼠左颈动脉（LCA）球囊损伤诱导新生内膜增生。腺病毒基因转移在培养细胞和LCA中进行。通过LCA的埃文蓝染色来评估再内皮化。 1）CTS对培养的大鼠VSMC具有较强的抗增殖和促凋亡作用，其EC比内皮细胞（EC）低4~10倍。 2)向大鼠局部给予LCA外膜周围空间或口服CTS，表现出有效抑制球囊损伤诱导的新内膜增生，但对再内皮化没有延迟作用。 3）QPP结果显示，CTS显着降低了VSMC中Pak1、Pak2、Erk1、Erk2和Bad的磷酸化水平。 4) 磷酸模拟突变体Pak1/Pak2的腺病毒表达增强了Pak1/2活性，刺激下游pErk1/pErk2/pErk3/pBad，减弱CTS介导的VSMC增殖抑制和促进细胞凋亡，并增强内膜增生。 5）抗磷酸突变体Pak1/Pak2的腺病毒表达使Pak1/2失活，并完全模拟CTS对血小板源性生长因子（PDGF）刺激的VSMC增殖以及PDGF抑制的细胞凋亡和内膜增生的抑制作用。 6) LCA损伤显着增强了除pBad之外的所有蛋白的内源磷酸化水平。 CTS 显着减弱了所有增强的水平。这些结果表明，CTS 是一种有前景的药物，用于预防血管成形术后再狭窄，且不会对再内皮化产生不利影响。 CTS 定向抑制 pPak1/pPak2 以及随后对下游 pErk1/pErk2/pErk3 和 pBad 的影响，强调了其抑制 VSMC 增殖和刺激细胞凋亡的机制。因此，Pak1/Pak2的磷位点构成了对抗内膜增生再狭窄的潜在药物筛选靶点。