HGK在血管再狹窄化的治療效果及相關機制

Abstract

血管再狹窄(restenosis)多好發於經皮冠狀動脈介入治療後，儘管有藥物釋放型支架的發現，支架再狹窄的復發率仍居高不下。而血管中層平滑肌細胞不正常的增生及移行是主要的致病機制，如何減緩這樣的病徵為目前主要研究方向。芫花素(Hydroxygenkwanin, HGK)是從瑞香科植物芫花中萃取出來的黃酮類化合物，在過去抗癌藥物研究已被證實具有抗發炎、抑制細胞增生及移行等效果，但其對於心血管疾病的作用尚未明瞭。本篇研究目的是探討HGK在血管再狹窄病症中，是否具有療效。在細胞實驗以大鼠動脈平滑肌細胞和U937單核球細胞為細胞模式，觀察HGK對於平滑肌細胞增生、移行、黏附及發炎的作用；在動物實驗部分，利用wire injury誘發小鼠股動脈內皮細胞受損為動物損傷模式，觀察HGK是否具有抑制血管內層增生的作用。細胞實驗結果顯示，HGK具有抑制經腫瘤壞死因子刺激細胞發炎及因血小板衍生生長因子誘導細胞增生及移行的作用。此外，得知HGK能透過降低AKT磷酸化而抑制平滑肌細胞的細胞週期調控蛋白表現、肌動蛋白(F-actin)經生長因子誘導的重組反應，及參與降低黏附因子產生的生理機制。後以分子嵌合分析(Molecular docking)發現，HGK與AKT的上游蛋白PDK1之間可能有交互作用關係，故推測HGK可與PDK1作用而抑制AKT磷酸位點的活化，進而抑制平滑肌細胞的生理機制。在動物實驗結果方面，HGK能夠有效抑制血管內皮損傷28天後的內層增生及黏附蛋白的表現，且從伊文斯藍染色及組織免疫化學染色結果，證實HGK能促進血管再內皮化。綜合上述的結果，我們推測HGK可作為支架上塗藥的新選擇。

Blood vessel restenosis is commonly defined as the recurrence of the narrowing of arteries after percutaneous coronary intervention. Despite on-going evolution of drug-eluting stent (DES) technology, the occurrence of in-stent restenosis (ISR) remains relatively unchanged. The abnormal proliferation and migration of smooth muscle cells in the vascular medial layer is the main pathogenesis of the disease. How to slow down such symptoms becomes the core research direction at present. Hydroxygenkwanin (HGK), a nature flavonoid extracted from Daphne genkwa, has been proved to exhibit anti-inflammation, anti-cell proliferation, and cell migration inhibition in previous anti-cancer studies; however, its effects on cardiovascular disease (CVD) still remain unclear. Given to this, this study aims at investigating whether HGK has therapeutic effects on ISR. Rat aortic smooth muscle cells (RASMCs) and U937 cells were used to examine the therapeutic effects of HGK on cell proliferation, migration, adhesion, and anti-inflammation. In addition, supported with the vascular endothelial damage mouse model with C57B/6 mice, we investigated the effect of HGK on neointimal hyperplasia. In vitro, we observed that HGK inhibited the cell inflammation stimulated by tumor necrosis factor (TNF-α), and the proliferation and migration induced by platelet-derived growth factor (PDGF). The results also demonstrated that HGK could hinder the expression of cell cycle regulatory proteins, F-actin rearrangement at the leading edge, and participate in the attenuation of adhesion proteins by reducing AKT phosphorylation. Afterwards, molecular docking analysis suggested that HGK might interact with PDK1, a AKT upstream protein; hence, we supposed that HGK reduce the AKT phosphorylation through PDK1 interaction, which in turn inhibited the physiological mechanism of smooth muscle cells. In vivo, the results showed that neointimal hyperplasia and adhesion proteins expressions were effectively inhibited by HGK intraperitoneal injection on 28 days after injury. In addition, IHC analysis showed that HGK facilitated re-endothelialization in denuded-vessel. Based on the above results, HGK can be used as a new strategy for developing DES.