基于NF-κB与NRF2/ARE通路间交互作用探讨络风内动理论指导下的络风宁0号球囊涂层复合物抗PCI术后再狭窄的分子机制研究

How to further reduce the drug balloon / stent restenosis rate is a difficult research in home and abroad, we prepared the Luo Feng Ning-0 (LFN-0) balloon coating compound with the best ratio of Paclitaxel and Hirudin for the research and development of drug balloon. LFN-0 balloon coating compound comes from Taxus Chinensis and leech respectively, which has the function of detoxicating, expelling wind-evil and removing extravasated blood according to the theory of “Stirring of endogenous collateral wind” on atherosclerosis. Our team has successfully developed LFN-0 coated balloon and verified its effectiveness in restenosis in miniature pig experiments. This effect may be related to its regulation of inflammatory and oxidative stress pathways, but the specific mechanism is unknown. Based on the results of pre experiment, we put forward the hypothesis that LFN-0 is able to inhibit the proliferation and migration of smooth muscle cells (SMC) by regulating the interaction between NF-κB and NRF2/ARE pathway. In this study, wild type and NF- κB / NRF2 mutant rats were used to prepare PCI restenosis animal models after balloon injury of left common carotid artery. After LFN-0 intervention, the effect targets of the interaction between NF-κB and NRF2/ARE pathway were screened systematically. Then constructing HCASMC proliferation model by LPS, combined with specific gene knock down or over expression of key proteins in the pathway, exploring the relationship between the interaction of NF-κB and NRF2/ARE pathway and the correlation of SMC proliferation and migration. Then combined with ChIP and Co-IP methods, revealing the effective basis and regulation mechanism of LFN-0 to prevent and control restenosis after PCI from the molecular level.

如何进一步降低介入术后再狭窄率是国内外研究的难点，我们依据络风内动理论，选择祛风解毒逐瘀的红豆杉和水蛭，用其单体紫杉醇和重组水蛭素制备络风宁0号球囊涂层复合物（LFN-0）用于抗再狭窄药物球囊研发。本团队既往研制出LFN-0涂层球囊并在小型猪实验中证实其抗再狭窄的有效性，此效应与调控炎症和氧化应激有关，但具体机制未明。我们基于预实验结果，提出假说：LFN-0抗再狭窄作用是通过调控NF-κB与NRF2/ARE通路间交互作用，抑制SMC增殖迁移实现。本研究选用野生型和NF-κB、NRF2突变型大鼠，采用左颈总动脉球囊拉伤法制备再狭窄动物模型，加入LFN-0干预后对其调控两条通路间交互作用靶点进行筛选。继而用脂多糖构建HCASMC增殖模型，联合特定基因敲减或过表达探讨两条通路间交互作用与SMC增殖迁移的相关性，结合ChIP、Co-IP手段从分子水平揭示LFN-0防治PCI术后再狭窄的微观机理。

当下心脏支架术后管腔再狭窄是介入领域面临的巨大挑战，即便是最新一代的药物支架依旧难以完全避免支架内再狭窄形成风险。针对介入术后再狭窄难题，本项目依据络风内动理论，选择祛风解毒逐瘀的红豆杉和水蛭，用其单体紫杉醇和重组水蛭素制备络风宁0号球囊涂层复合物（LFN-0）用于抗再狭窄药物球囊研发。我们基于预实验结果，提出假说：LFN-0抗再狭窄作用是通过调控NF-κB与NRF2/ARE通路间交互作用，抑制人冠状动脉平滑肌细胞（HCASMC）增殖迁移而实现。为验证该科学假说，本研究选用野生型和NF-κB、NRF2突变型大鼠，采用左颈总动脉拉伤法制备颈动脉再狭窄动物模型，进而对病变血管施加LFN-0灌注干预，探索其抗再狭窄治疗效果并初步筛选出LFN-0调控NF-κB与NRF2/ARE双通路交互作用的可能效应靶点。在此基础上，用脂多糖构建HCASMC炎症和氧化应激双通路活化增殖迁移体外模型，结合初筛靶标的敲减或过表达，验证LFN-0调控NF-κB与NRF2/ARE通路抗HCASMC增殖迁移的精确靶点，从分子水平深度揭示LFN-0防治PCI术后再狭窄的微观机理。本项目结合动物和细胞实验结果发现，LFN-0球囊涂层复合物对左颈总动脉拉伤模型大鼠的血管内膜增生过程具有显著抑制作用，病理切片和分子生物学数据显示LFN-0在有效阻断管腔再狭窄的同时还能拮抗血栓形成，这种治疗效应与其调控NF-κB、NRF2及对应配体有关。在细胞验证实验中，1μmol/L的紫杉醇配比0.2mg/mL比伐卢定（即重组水蛭素）对造模活化后的HCASMC增殖状态具有明显抑制作用，可将活化后分泌型的HCASMC转换为收缩型，且不同程度地减弱了HCASMC的迁移和侵袭能力。此外，LFN-0可通过激活IκBα表达、阻碍NF-κB活化入核，截断炎症过程，同时抑制Keap-1表达、促进NRF2核转位、激活下游NQO-1和HO-1基因和蛋白表达，大幅缓减胞内氧化应激状态。由此可见，LFN-0对NF-κB与NRF2/ARE双通路活化的再狭窄过程和HCASMC胞内炎症和氧化应激状态均具有显著抑制作用，此抑制作用通过双向调控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-0涂层球囊抗PCI术后再狭窄的分子机理提供了科学依据。