紫杉醇-比伐卢定球囊涂层复合物调控NF-κB与Nrf2/ARE通路抗HCASMC增殖迁移的机制研究

Mechanism of antagonism of paclitaxel-bivalirudin balloon coating complex on proliferation and migration of human coronary artery smooth muscle cells through regulating pathways of nuclear factor-κB and nuclear factor E2 related factor 2/antioxidant respo

目的 基于人冠状动脉平滑肌细胞(HCASMC)核因子κB(NF-κB)与重组合成蛋白2/抗氧化反应元件(Nrf2/ARE)双通路活化模型探索紫杉醇-比伐卢定球囊涂层复合物(LFN)抑制HCASMC增殖迁移抗介入术后再狭窄的微观机理。方法 选用4~6代的HCASMC,利用脂多糖(LPS)作为诱导剂,最大化激活NF-κB与Nrf2/ARE双通路,构建LPS诱导HCASMC炎性和氧化应激双通路活化细胞模型。在此基础上,将体外培养的HCASMC分为空白对照组、LPS造模组及LPS造模+LFN复合物干预组,用CCK-8法检测LFN对HCASMC增殖率的影响,同时用免疫荧光法检测增殖细胞核抗原(PCNA)表达量确证细胞增殖状态。进而用Transwell法检测LFN对HCASMC迁移、侵袭能力的影响,并用免疫荧光单染法检测α平滑肌肌动蛋白(α-SMA)、骨桥蛋白(OPN)、NF-κB p65和Nrf2的表达变化,探索LFN对HCASMC表型转化的影响,并探究其对HCASMC中活化的NF-κB与Nrf2/ARE通路关键蛋白的调控作用,继而结合Q-PCR和Western blotting明确LFN干预后双通路关键位点基因和蛋白表达的变化,深入挖掘LFN调控炎性和氧化应激活化的HCASMC增殖迁移的潜在机制。结果 1μmol/L的紫杉醇配比0.2 mg/ml比伐卢定对造模活化后的HCASMC增殖状态具有明显抑制作用,减弱了HCASMC迁移和侵袭能力,并将活化后分泌型的HCASMC转换为收缩型。此外,与LPS造模组相比,LFN通过激活人核因子κB抑制蛋白α(IκBα)表达、阻碍NF-κB活化入核,截断了炎症过程,同时抑制胞质接头蛋白1(Keap-1)表达、促进Nrf2核转位、激活下游醌氧化还原酶1(NQO-1)和人血红素加氧酶1(HO-1)基因和蛋白表达(P<0.05),大幅减缓了胞内氧化应激状态。结论 LFN对NF-κB与Nrf2/ARE双通路活化的HCASMC胞内炎症和氧化应激状态具有显著抑制作用,此抑制作用通过调控NF-κB与Nrf2/ARE通路中核转录因子及其配体表达、影响通路下游效应分子活化而实现,为LFN涂层球囊抗经皮冠状动脉介入治疗(PCI)术后再狭窄的分子机理提供了细胞学依据。

Objective To investigate the microscopic mechanism of paclitaxel-bivalirudin balloon coating complex(LFN) in inhibiting proliferation and migration of human coronary artery smooth muscle cells(HCASMC)for preventing restenosis after PCI based on dual pathway activation model of nuclear factor-κB(NF-κB) and nuclear factor E2 related factor 2(Nrf2)/antioxidant response element(ARE) of HCASMC.Methods HCASMC of 4-6 generations was selected to activate dual pathways of NF-κB and Nrf2/ARE by taken lipopolysaccharide(LPS)as an inducer for establishing a dual pathway activated cell model of HCASMC inflammation and oxidative stress.HCASMC,cultured in vitro,were divided into blank control group,LPS group,LPS+LFN group.The influence of LFN on proliferation rate of HCASMC was detected by using CCK-8 method,and expression of proliferating cell nuclear antigen(PCNA) was detected by using immunofluorescence for verifying HCASMC proliferation status.The influence of LFN on migration and invasion ability of HCASMC was detected by using Transwell method,and expression changes of α-smooth muscle actin(α-SMA),osteopontin(OPN),NF-κB p65 and Nrf2.The influence of LFN on phenotype transformation of HCASMC was discussed,and regulation effect of LFN on key proteins in activated NF-κB and Nrf2/ARE pathways in HCASMC was studied.The changes of key site genes and protein expressions after LFN intervention were identified in dual pathways by using fluorescence quantitative polymerase chain reaction(Q-PCR) and Western blotting assay for exploring further the potential mechanism of LFN regulating HCASMC proliferation and migration activated by inflammatory and oxidative stress.Results Paclitaxel(1 μmol/L) combined with bivalirudin(0.2 mg/ml) inhibited significantly HCASMC proliferation after model activation,weakened HCASMC migration and invasion,and reversed activated HCASMC from secreting type to the contractile type.Furthermore,compared with LPS group,LFN inhibited expression of IκBα,blocked activation of NF-κB into the nucleus,At the same time,LFN inhibited expression of Keap-1,improved Nrf2 nuclear translocation,activated gene and protein expressions of downstream NQO-1 and HO-1(P<0.05),and significantly relief intracellular oxidative stress state.Conclusion LFN can significantly inhibit inflammatory and oxidative stress states of HCASMC activated by dual pathways of NF-κB and Nrf2/ARE.This inhibitory effect is achieved through regulating expressions of nuclear transcription factors and their ligands in NF-κB and Nrf2/ARE pathways,as well as influencing activation of downstream effector molecules in the pathways.It provides a cytological basis for molecular mechanism of LFN coated balloon against restenosis after PCI.