Abstract：
BACKGROUND: Atherosclerosis (AS) is commonly regarded as a key driver accounted for the leading causes of morbidity and mortality among cardiovascular and cerebrovascular diseases. A growing body of evidence indicates that autophagy in macrophages involved in AS might be a potential therapeutic target. C1q/TNF-related protein 9 (CTRP9) has been proven to delay the progression of cardiovascular diseases. However, the relations between CTRP9 and Sirt1, as well as their effects on macrophages autophagy have not been fully explored.
METHODS: Macrophages were differentiated from mononuclear cells collected from peripheral blood samples of healthy donors. The in vitro AS models were constructed by ox-LDL treatment. Cell viability was determined by CCK-8 assay. Immunofluorescence assay of LC3 was implemented for evaluating autophagy activity. Oil Red O staining was performed for lipid accumulation detection. ELISA, cholesterol concentration assay and cholesterol efflux analysis were conducted using commercial kits. Cycloheximide assay was implemented for revealing protein stability. RT-qPCR was used for mRNA expression detection, and western blotting was performed for protein level monitoring.
RESULTS: CTRP9 attenuated impaired cell viability, autophagy inhibition and increased lipid accumulation induced by ox-LDL. Moreover, CTRP9 maintained Sirt1 protein level through enhancing its stability through de-ubiquitination, which was mediated by upregulated USP22 level. CRTP9 exerted its protective role in promoting autophagy and reducing lipid accumulation through the USP22/Sirt1 axis.
CONCLUSIONS: Collectively, CTRP9 alleviates lipid accumulation and facilitated the macrophages autophagy by upregulating USP22 level and maintaining Sirt1 protein expression, thereby exerting a protective role in AS progression in vitro.
METHODS: Macrophages were differentiated from mononuclear cells collected from peripheral blood samples of healthy donors. The in vitro AS models were constructed by ox-LDL treatment. Cell viability was determined by CCK-8 assay. Immunofluorescence assay of LC3 was implemented for evaluating autophagy activity. Oil Red O staining was performed for lipid accumulation detection. ELISA, cholesterol concentration assay and cholesterol efflux analysis were conducted using commercial kits. Cycloheximide assay was implemented for revealing protein stability. RT-qPCR was used for mRNA expression detection, and western blotting was performed for protein level monitoring.
RESULTS: CTRP9 attenuated impaired cell viability, autophagy inhibition and increased lipid accumulation induced by ox-LDL. Moreover, CTRP9 maintained Sirt1 protein level through enhancing its stability through de-ubiquitination, which was mediated by upregulated USP22 level. CRTP9 exerted its protective role in promoting autophagy and reducing lipid accumulation through the USP22/Sirt1 axis.
CONCLUSIONS: Collectively, CTRP9 alleviates lipid accumulation and facilitated the macrophages autophagy by upregulating USP22 level and maintaining Sirt1 protein expression, thereby exerting a protective role in AS progression in vitro.
摘要：
背景：动脉粥样硬化（AS）通常被认为是导致心脑血管疾病发病和死亡的主要原因。越来越多的证据表明，参与AS的巨噬细胞自噬可能是一个潜在的治疗靶点。C1q/TNF相关蛋白9(CTRP9)已被证明可延缓心血管疾病的进展。然而,CTRP9与Sirt1的关系及其对巨噬细胞自噬的影响尚未得到充分探讨。
方法：从健康供体的外周血样本中收集的单核细胞分化出巨噬细胞。通过ox-LDL处理构建体外AS模型。通过CCK-8测定确定细胞活力。实施LC3的免疫荧光测定以评估自噬活性。进行油红O染色用于脂质积累检测。ELISA,使用市售试剂盒进行胆固醇浓度测定和胆固醇流出分析.实施环己酰亚胺测定以揭示蛋白质稳定性。RT-qPCR用于mRNA表达检测,蛋白质印迹法用于蛋白质水平监测.
结果：CTRP9减弱了受损的细胞活力，ox-LDL诱导的自噬抑制和脂质积累增加。此外，CTRP9通过去泛素化增强其稳定性来维持Sirt1蛋白水平，这是由上调的USP22水平介导的。CRTP9通过USP22/Sirt1轴在促进自噬和减少脂质积累方面发挥了保护作用。
结论：总的来说，CTRP9通过上调USP22水平和维持Sirt1蛋白表达，减轻脂质积累并促进巨噬细胞自噬，从而在体外AS进展中发挥保护作用。
方法：从健康供体的外周血样本中收集的单核细胞分化出巨噬细胞。通过ox-LDL处理构建体外AS模型。通过CCK-8测定确定细胞活力。实施LC3的免疫荧光测定以评估自噬活性。进行油红O染色用于脂质积累检测。ELISA,使用市售试剂盒进行胆固醇浓度测定和胆固醇流出分析.实施环己酰亚胺测定以揭示蛋白质稳定性。RT-qPCR用于mRNA表达检测,蛋白质印迹法用于蛋白质水平监测.
结果：CTRP9减弱了受损的细胞活力，ox-LDL诱导的自噬抑制和脂质积累增加。此外，CTRP9通过去泛素化增强其稳定性来维持Sirt1蛋白水平，这是由上调的USP22水平介导的。CRTP9通过USP22/Sirt1轴在促进自噬和减少脂质积累方面发挥了保护作用。
结论：总的来说，CTRP9通过上调USP22水平和维持Sirt1蛋白表达，减轻脂质积累并促进巨噬细胞自噬，从而在体外AS进展中发挥保护作用。
