PDF(Pubmed)
Abstract：
Atherosclerosis is a huge threaten to the human health, C1q/TNF-related protein 9 (CTRP9) has been previously reported possessing vascular protective functions. Our study is aimed to reveal the mechanism of the regulative effects of CTRP9 on the foam cell formation.
Primary human macrophages were isolated from human monocytes donated by healthy volunteers. CCK-8 assay was performed for determining the cell viability. Oil Red O staining was employed for measuring the lipid accumulation. Cholesterol ester and cholesterol concentration were detected by commercial kits for evaluating the intracellular cholesterol. Ubiquitination assay was performed to reveal the ubiquitination level of CD36, cycloheximide assay was applied for determining the half-life of CD36 protein. Quantitative real-time PCR and western blot assays were performed for detecting the mRNA and protein expression. Pre-treatment with CTRP9 in primary human macrophages markedly suppressed the cholesterol accumulation concentration after oxidized low-density lipoprotein treatment. CD36 was significantly increased after oxidized low-density lipoprotein exposure while was reduced by CTRP9 treatment. Up-regulation of CD36 significantly reversed the CTRP9-mediated protective effects in foam cells. The differential expression levels of several deubiquitinating enzymes preliminarily indicated that USP11 was obviously decreased after CTRP9 treatment. USP11 knockdown decreased the CD36 protein expression and pre-treatment with 10 μg/mL MG132 significantly maintained the CD36 level from USP11 knock down. Up-regulation of CD36 reversed the alterations on the cholesterol metabolism caused by CTRP9 or USP11 knockdown.
CTRP9 regulates the USP11/CD36 axis to protect the macrophages form transforming into foam cells by suppressing intracellular lipid and cholesterol accumulation, which is a potential therapeutic agent for atherosclerosis.
Primary human macrophages were isolated from human monocytes donated by healthy volunteers. CCK-8 assay was performed for determining the cell viability. Oil Red O staining was employed for measuring the lipid accumulation. Cholesterol ester and cholesterol concentration were detected by commercial kits for evaluating the intracellular cholesterol. Ubiquitination assay was performed to reveal the ubiquitination level of CD36, cycloheximide assay was applied for determining the half-life of CD36 protein. Quantitative real-time PCR and western blot assays were performed for detecting the mRNA and protein expression. Pre-treatment with CTRP9 in primary human macrophages markedly suppressed the cholesterol accumulation concentration after oxidized low-density lipoprotein treatment. CD36 was significantly increased after oxidized low-density lipoprotein exposure while was reduced by CTRP9 treatment. Up-regulation of CD36 significantly reversed the CTRP9-mediated protective effects in foam cells. The differential expression levels of several deubiquitinating enzymes preliminarily indicated that USP11 was obviously decreased after CTRP9 treatment. USP11 knockdown decreased the CD36 protein expression and pre-treatment with 10 μg/mL MG132 significantly maintained the CD36 level from USP11 knock down. Up-regulation of CD36 reversed the alterations on the cholesterol metabolism caused by CTRP9 or USP11 knockdown.
CTRP9 regulates the USP11/CD36 axis to protect the macrophages form transforming into foam cells by suppressing intracellular lipid and cholesterol accumulation, which is a potential therapeutic agent for atherosclerosis.
摘要：
目的：动脉粥样硬化是对人类健康的巨大威胁，先前已报道C1q/TNF相关蛋白9(CTRP9)具有血管保护功能。我们的研究旨在揭示CTRP9对泡沫细胞形成的调控作用机制。
结果：从健康志愿者捐赠的人单核细胞中分离原代人巨噬细胞。进行CCK-8测定以确定细胞活力。油红O染色用于测量脂质积累。胆固醇酯和胆固醇浓度通过用于评估细胞内胆固醇的商业试剂盒检测。进行泛素化测定以揭示CD36的泛素化水平，环己酰亚胺测定用于确定CD36蛋白的半衰期。进行定量实时PCR和蛋白质印迹测定以检测mRNA和蛋白质表达。在氧化低密度脂蛋白治疗后，在原代人巨噬细胞中使用CTRP9进行预处理可显着抑制胆固醇积累浓度。氧化低密度脂蛋白暴露后CD36显着增加，而CTRP9治疗后CD36降低。CD36的上调显著逆转了CTRP9介导的泡沫细胞保护作用。几种去泛素化酶的差异表达水平初步表明CTRP9处理后USP11明显降低。USP11敲低降低CD36蛋白表达,并且用10μg/mLMG132预处理显著维持来自USP11敲低的CD36水平。CD36的上调逆转了CTRP9或USP11敲低引起的胆固醇代谢改变。
结论：CTRP9调节USP11/CD36轴，通过抑制细胞内脂质和胆固醇的积累，保护巨噬细胞转化为泡沫细胞，是动脉粥样硬化的潜在治疗剂。
结果：从健康志愿者捐赠的人单核细胞中分离原代人巨噬细胞。进行CCK-8测定以确定细胞活力。油红O染色用于测量脂质积累。胆固醇酯和胆固醇浓度通过用于评估细胞内胆固醇的商业试剂盒检测。进行泛素化测定以揭示CD36的泛素化水平，环己酰亚胺测定用于确定CD36蛋白的半衰期。进行定量实时PCR和蛋白质印迹测定以检测mRNA和蛋白质表达。在氧化低密度脂蛋白治疗后，在原代人巨噬细胞中使用CTRP9进行预处理可显着抑制胆固醇积累浓度。氧化低密度脂蛋白暴露后CD36显着增加，而CTRP9治疗后CD36降低。CD36的上调显著逆转了CTRP9介导的泡沫细胞保护作用。几种去泛素化酶的差异表达水平初步表明CTRP9处理后USP11明显降低。USP11敲低降低CD36蛋白表达,并且用10μg/mLMG132预处理显著维持来自USP11敲低的CD36水平。CD36的上调逆转了CTRP9或USP11敲低引起的胆固醇代谢改变。
结论：CTRP9调节USP11/CD36轴，通过抑制细胞内脂质和胆固醇的积累，保护巨噬细胞转化为泡沫细胞，是动脉粥样硬化的潜在治疗剂。
