| Summary: | 碩士 === 國立陽明大學 === 生理學研究所 === 101 === Atherosclerosis is a chronic inflammatory disease accompanied with foamy macrophages formation. Tumor necrosis factor-α (TNF-α), a proinflammatory cytokine produced primarily by monocytes and macrophages, has been shown to induce foam cell formation which is an early step of atherosclerosis. Studies also demonstrated that TNF-α causes increased inducible nitric oxide synthase (iNOS) expression and subsequent NO production in macrophages. However, the regulatory mechanism of TNF-α on foam cell formation is still unclear. The aim of this study was to investigate the roles of iNOS and NO in the regulation of TNF-α on foam cell formation in RAW 264.7 macrophages. Effects of TNF-α on the expression of scavenger receptor SRA, SR-B1, CD36 and ATP-binding cassette transporter ABCA1 and ABCG1 were detected by western blot. Oxidized LDL uptake by macrophages, and cholesterol efflux from macrophages were also evaluated. The involvement of iNOS and NO in the regulatory effect of TNF-α on foam cell formation was clarified by using specific iNOS inhibitor (PBITU) and iNOS shRNA. Our results showed that treatment with TNF-α significantly increased ox-LDL uptake and decreased cholesterol efflux, leading to increase foam cell formation. In addition, TNF-α decreased protein expressions of ABCA1, ABCG1 and SR-B1, but increased SRA expression, which leads to cholesterol accumulation in macrophages. On the other hand, pretreatment with iNOS inhibitor abolished the effect of TNF-α on foam
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cell formation and ABCG1 protein suppression. Moreover, Treatment with TNF-α increased iNOS expression and NO production, and sodium nitroprusside (SNP), a NO donor, showed similar effects to TNF-α on the expressions of SRA, SR-B1, ABCA1 and ABCG1 in RAW 264.7 cells. Transfection of iNOS shRNA in RAW264.7 macrophages was performed to further confirm the role of iNOS in TNF-α-induced foam cell formation. Suppression of TNF-α-induced iNOS expression via shiNOS significantly reduced TNF-α-induced foam cell formation. In conclusion, our data demonstrate that TNF-α treatment induced downregulation of ABCG1 through activation of iNOS/NO signaling, which consequently caused decreased cholesterol efflux, and finally increased foam cell formation in macrophage.
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