NADPH氧化酶活性不影响主动脉平滑肌细胞负荷胆固醇

NADPH oxidase activity does not affect cellular cholesterol loading in vascular smooth muscle cells

NADPH氧化酶产生的活性氧促进血管平滑肌细胞的增殖和迁移,与动脉粥样硬化的发生密切相关。为了观察NADPH氧化酶的亚基p47phox对血管平滑肌细胞胆固醇代谢的影响,把p47phox基因敲除小鼠的主动脉血管平滑肌细胞与10mg/L水溶性胆固醇共孵育72h,然后用0.3mg/L凝血酶处理10min,采用免疫组织化学和油红O染色、实时定量逆转录PCR、免疫蛋白印迹、细胞内胆固醇测定等方法,观察细胞内胆固醇的改变,与平滑肌细胞、巨噬细胞、炎症反应细胞内胆固醇代谢相关蛋白的表达。结果显示,与未孵育的对照组相比,水溶性胆固醇孵育过的主动脉血管平滑肌细胞内胆固醇明显增加,差别有显著性意义;细胞内中性脂滴明显增加;α-肌动蛋白的表达下降,半乳糖凝集素3表达升高,单核细胞趋化蛋白1及血管细胞黏附分子1的表达不变;ATP结合盒转运体A1、酰基辅酶A:胆固醇酰基转移酶1及脂肪分化相关蛋白的表达增加。但是,与野生型血管平滑肌细胞相比,敲除p47phox基因并不能使所测定的指标发生变化。结果提示,负荷胆固醇后,p47phox依赖的NADPH氧化酶并不能改变血管平滑肌细胞向泡沫细胞的转变。单纯敲除p47phox基因不能改变细胞内胆固醇代谢的状态。

Reactive oxygen species generated by NADPH oxidase enhance aortic vascular smooth muscle cell proliferation and migration which play an important role in the pathophysiology of atherosclerosis. We investigated the role of NADPH oxidase in the cellular cholesterol metabolism in vascular smooth muscle cells using p47phox-deficient cells. Wild-type and p47phox knockout vascular smooth muscle cells were loaded with cholesterol for 72 h by using 10 mg/L cholesterol：methyl-β-cyclodextrin complexes and then incubated with or without 0.3 mg/L thrombin for 10 min. Foam cell formation was determined by accumulation of intracellular cholesterol, oil Red O-stained lipid droplets. After cholesterol loading, cellular lipid droplets raised sharply, cellular cholesterol increased from （31.4±2.0） to （61.0±2.1） mg/g protein （P〈0.05） in wild-type cells, and from （29.8±2.5） to （51.3±3.1） mg/g protein （P〈 0.05） in p47phox deficient cells, but the difference between the two cell types was not significant. Immunostaining showed decreased levels of smooth muscle α-actin and increased levels of macrophage marker Mac-2 in both wild-type and p47phox deficient vascular smooth muscle cells. One of the macrophage-related inflammation genes, monocyte chemoattractant protein-1 （MCP-1） expression didnot change in both two cell types detected by immunostaining. Although additional incubating with thrombin, another macrophagerelated inflammation gene, vascular cell adhesion molecule-1 （VCAM-1） expression was similar in all groups analyzed by real-time RT- PCR. However, the expression of ATP-binding cassette transporter A1 （ABCA1）, acyl-coenzyme A：cholesterol acyltransferase 1 （ACAT1）, the key proteins in cellular cholesterol metabolism, were similarly increased （P〈0.05） in both two cell types as determined by quantitative real-time RT-PCR and Western blot, and it was not related to the state of oxidative stress. Interestingly, the expression of adipophilin, the lipid droplet related protein, had the similar results with ABCA1 and ACAT1, but, in wild-type cells, its expression also increased merely incubating with thrombin as determined by quantitative real-time RT-PCR. Together, these results suggest that p47phox-dependent NADPH oxidase is not involved in transdifferentitation of vascular smooth muscle cells into macrophage-like state after cholesterol loading. Deleting p47phox gene does not affect the cellular cholesterol metabolism in vascular smooth muscle cells.