畅脉乐通过调控PPAR信号通路对动脉粥样硬化大鼠巨噬细胞自噬、血管内皮功能及动脉斑块稳定性相关蛋白影响

Changmaile Affects Macrophage Autophagy, Vascular Endothelial Function and Arterial Plaque Stability-Related Proteins in Atherosclerotic Rats by Regulating the PPAR Signaling Pathway

摘要: 目的：通过观察畅脉乐对过氧化物酶体增殖物激活受体(PPAR)信号通路的调控，研究畅脉乐对动脉粥样硬化大鼠巨噬细胞自噬、血管内皮功能及动脉斑块稳定性相关蛋白的影响。方法：将30只大鼠随机分为对照组(n = 10)、模型组(n = 10)及畅脉乐组(n = 10)；除对照组外，模型组及畅脉乐组大鼠均以高脂饮食为诱变剂建立动脉粥样硬化模型。畅脉乐组按5 mg/(kg•d)剂量服用畅脉乐胶囊，对照组和模型组予以相应饲料喂养，干预周期均为8周。8周后采血和采集血管组织，采用透射电镜观察巨噬细胞中自噬小体数量，蛋白免疫印迹法检测巨噬细胞中酶联相关轻链蛋白3 (LC3)、选择性自噬接头蛋白p62 (p62)的表达水平，免疫荧光吸附法检测大鼠血清血管性血友病因子(vWF)、一氧化氮(NO)、内皮素-1 (ET-1)、一氧化氮合酶(eNOS)含量，Western blot法检测动脉斑块稳定性相关蛋白[基质金属蛋白酶(MMP)-2、MMP-9、MMP抑制剂(TIMP)-1]及PPAR信号通路[PPARγ/肝脏X受体α/ATP结合盒转运体G1 (PPARγ/LXRα/ABCG1)]相关蛋白表达水平，RT-PCR测定细胞内PPARγ、LXRα、ABCG1的mRNA水平。结果：与对照组比较，模型组大鼠自噬小体数量有所减少，LC3II/I表达水平明显下降，p62表达水平明显上升(P < 0.05)；MMP-2、MMP-9表达水平明显上升，TIMP-1表达水平明显下降(P < 0.05)；PPARγ、LXRα、ABCG1蛋白及mRNA表达水平明显下降(P < 0.05)。与模型组比较，畅脉乐组大鼠自噬小体数量增加，LC3II/I表达水平明显上升，p62表达水平明显下降(P < 0.05)；MMP-2、MMP-9表达水平明显下降，TIMP-1表达水平明显上升(P < 0.05)；PPARγ、LXRα、ABCG1蛋白及mRNA表达水平明显上升(P < 0.05)。结论：畅脉乐能够通过调控PPAR信号通路，上调动脉粥样硬化大鼠巨噬细胞自噬水平，改善血管内皮功能，提高动脉斑块的稳定性，进而起到抗脉粥样硬化的作用。

Abstract: Purpose: By observing the regulation of Changmaile on the peroxisome proliferator-activated re-ceptor (PPAR) signaling pathway, study the effect of Changmaile on macrophage autophagy, vascu-lar endothelial function and arterial plaque stability-related proteins in atherosclerotic rats. Meth-ods: 30 rats were randomly divided into control group (n = 10), model group (n = 10) and Changmaile group (n = 10); except for the control group, rats in the model group and Changmaile group Atherosclerosis models were established using high-fat diet as mutagen. The Changmaile group took Changmaile capsules at a dose of 5 mg/(kg•d), and the control group and model group were fed corresponding feeds. The intervention period was 8 weeks. After 8 weeks, blood and vas-cular tissues were collected. Transmission electron microscopy was used to observe the number of autophagosomes in macrophages. Western blotting was used to detect enzyme-linked light chain protein 3 (LC3) and selective autophagy adapter protein p62 expression level in macrophages. Im-munofluorescence adsorption method was used to detect rat serum von Willebrand factor (vWF), nitric oxide (NO), endothelin-1 (ET-1), nitric oxide synthase (eNOS) content, Western blot method was used to detect arterial plaque stability-related proteins [matrix metalloproteinase (MMP)-2, MMP-9, MMP inhibitor (TIMP)-1] and PPAR signaling pathway [PPARγ/liver X receptor α/ATP Binding cassette transporter G1 (PPARγ/LXRα/ABCG1)] related protein expression levels, and RT-PCR measured the intracellular mRNA levels of PPARγ, LXRα, and ABCG1. Results: Compared with the control group, the number of autophagosomes in the model group decreased, the expres-sion level of LC3II/I decreased significantly, and the expression level of p62 increased significantly (P < 0.05); the expression levels of MMP-2 and MMP-9 increased significantly. The expression level of TIMP-1 decreased significantly (P < 0.05); the expression levels of PPARγ, LXRα, ABCG1 protein and mRNA decreased significantly (P < 0.05). Compared with the model group, the number of au-tophagosomes in the Changmaile group increased, the expression level of LC3II/I increased signifi-cantly, and the expression level of p62 decreased significantly (P < 0.05); the expression levels of MMP-2 and MMP-9 decreased significantly, and the expression level of TIMP-1 expression level in-creased significantly (P < 0.05); PPARγ, LXRα, ABCG1 protein and mRNA expression levels in-creased significantly (P < 0.05). Conclusion: Changmaile can upregulate the autophagy level of mac-rophages in atherosclerotic rats, improve vascular endothelial function, increase the stability of ar-terial plaques, and play an anti-atherosclerotic role by regulating the PPAR signaling pathway.