TFPIct32抑制血管平滑肌细胞增殖迁移及其抗动脉粥样硬化作用

TFPIct32 inhibits proliferation and migration of vascular smooth muscle cells and exerts anti-atherosclerotic effects

摘要:

目的]探究组织因子途径抑制物羧基末端(TFPIct)一段由32个氨基酸序列构成的小肽段TFPIct32对ApoE－/－小鼠动脉粥样硬化(As)病变进展的影响及初步机制。 [方法]采用高脂饲料喂养ApoE－/－小鼠10周,构建As动物模型。将建模成功的小鼠随机分为4组,每组10只(n＝10),分别为:模型组、生理盐水组(模型基础上尾静脉注射生理盐水)、TFPI组(阴性对照肽,每周2.5 nmol/g TFPI尾静脉注射)、TFPIct32组(每周2.5 nmol/g TFPIct32尾静脉注射),各组均持续给药4周。给药结束后,检测各组小鼠尾部出血时间；采集血清样本,检测血清总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白胆固醇(LDLC)及高密度脂蛋白胆固醇(HDLC)水平。分离小鼠主动脉并进行油红O染色,对主动脉窦组织切片行HE、Masson和油红O染色。采用血小板源生长因子BB(PDGF-BB)处理血管平滑肌细胞(VSMC)构建细胞增殖迁移模型,分为对照组、PDGF-BB组、PDGF-BB+TFPI组、PDGF-BB+TFPIct32组以及PDGF-BB+蛋白激酶B(Akt)激动剂SC79组、PDGF-BB+TFPIct32+SC79组。运用CCK-8法检测细胞增殖能力,划痕实验及Transwell法检测细胞迁移能力,Western blot检测增殖细胞核抗原(PCNA)、磷脂酰肌醇3激酶(PI3K)、磷酸化Akt(p-Akt)及总Akt(t-Akt)的蛋白表达水平。 [结果]动物实验结果显示,与生理盐水组和TFPI组相比,TFPIct32组ApoE－/－小鼠血清LDLC水平降低(P＜0.05),同时主动脉壁及主动脉窦斑块面积减小(均P＜0.01)。TFPIct32对PDGF-BB诱导的VSMC增殖与迁移具有显著抑制作用(均P＜0.05),Akt特异性激动剂SC79可部分恢复TFPIct32抑制的VSMC增殖与迁移能力(P＜0.05),提示PI3K/Akt通路在TFPIct32抑制的VSMC增殖与迁移及促进As病变进展中发挥重要作用。 [结论]TFPIct32可显著减缓高脂饮食喂养的ApoE－/－小鼠As病变进展,其机制可能与抑制VSMC增殖和迁移有关。

Abstract:

Aim To investigate the effects of TFPIct32, a 32-amino acid peptide segment of tissue factor pathway inhibitor C-terminal (TFPIct), on the progression of atherosclerosis (As) lesions in ApoE－/－ mice and its preliminary mechanisms. Methods ApoE－/－ mice were fed with a high-fat diet for 10 weeks to establish an As animal model.Mice successfully modeled were randomly divided into four groups of 10 (n＝10) each:model group, saline group (tail vein injection of saline on top of the model), TFPI group (negative control peptide group, 2.5 nmol/g TFPI per week, tail vein injection), TFPIct32 group (2.5 nmol/g TFPIct32 per week, tail vein injection). All groups received continuous treatment for 4 weeks. After treatment completion, tail bleed time was measured in mice from each group; Serum samples were collected to measure serum levels of total cholesterol (TC), triglyceride (TG), low density lipoprotein cholesterol (LDLC), and high density lipoprotein cholesterol (HDLC). Mouse aortas were isolated and stained with oil red O. Sections of aortic sinus tissue were stained with hematoxylin and eosin (HE), Masson's trichrome, and oil red O. A cell proliferation and migration model was established using platelet-derived growth factor-BB (PDGF-BB) treatment on vascular smooth muscle cells (VSMC), divided into control group, PDGF-BB group, PDGF-BB+TFPI group, PDGF-BB+TFPIct32 group, PDGF-BB+protein kinase B (Akt) agonist SC79 group and PDGF-BB+TFPIct32+SC79 group. Cell proliferation was assessed using the CCK-8 assay, while cell migration was evaluated via the scratch assay and Transwell method. Protein expression levels of proliferating cell nuclear antigen (PCNA), phosphoinositide 3-kinase (PI3K), phosphorylated Akt (p-AKT), and total Akt (t-Akt) were detected by Western blot analysis. Results Animal experimental results showed that, compared with the saline group and the TFPI group, the TFPIct32 group significantly reduced serum LDLC levels in ApoE－/－ mice (P＜0.05), while simultaneously decreased plaque area in the aortic wall and aortic sinus (both P＜0.01). TFPIct32 significantly inhibited PDGF-BB-induced proliferation and migration of VSMC (both P＜0.05), while the Akt-specific agonist SC79 partially restored the TFPIct32-inhibited VSMC proliferation and migration (P＜0.05). These findings suggested that the PI3K/Akt pathway played a crucial role in the regulation of TFPIct32-mediated inhibition of VSMC proliferation, migration, and subsequent atherosclerosis progression. Conclusion TFPIct32 significantly slows the progression of atherosclerotic lesions in ApoE－/－ mice fed a high-fat diet, a mechanism likely involving the inhibition of VSMC proliferation and migration.