METHODS: Human umbilical vein endothelial cells (HUVECs) were treated with oxidized-low density lipoprotein (ox-LDL) to induce cell damage. ApoE-/- mice were fed a Western diet to establish an AS mouse model. Haematoxylin and eosin (H&E) staining and Oil Red O staining evaluated the pathological changes and in lipid deposition in aortic tissues. CCK-8 and flow cytometry detected cell proliferation and apoptosis. Immunohistochemistry, Enzyme linked immunosorbent assay, qRT-PCR and western blotting assessed the levels of mRNA and proteins.
RESULTS: Ox-LDL treatment elevated SPAG5 expression and the expression of autophagy-related proteins, LC3-I, LC3-II, Beclin-1, and p62, in HUVECs. GFP-LC3 dots were increased in ox-LDL-treated HUVECs and LPS-treated HUVECs. SPAG5 knockdown reversed both ox-LDL and LPS treatment-mediated inhibition of cell proliferation and promotion of apoptosis in HUVECs. SPAG5 silencing further elevated autophagy and repressed the expression of PI3K, p-Akt/Akt, and p-mTOR/mTOR in ox-LDL-treated HUVECs. 3-MA (autophagy inhibitor) treatment reversed SPAG5 silencing-mediated increase of cell proliferation and decrease of apoptosis in ox-LDL-treated HUVECs. In vivo, SPAG5 knockdown reduced atherosclerotic plaques in AS mice through activating autophagy and inhibiting PI3K/Akt/mTOR signaling pathway.
CONCLUSIONS: This work demonstrated that SPAG5 knockdown alleviated AS development through activating autophagy. Thus, SPAG5 may be a potential target for AS therapy.
方法:用氧化-低密度脂蛋白(ox-LDL)处理人脐静脉内皮细胞(HUVECs)以诱导细胞损伤。ApoE-/-小鼠饲喂Western饮食以建立AS小鼠模型。苏木精和伊红(H&E)染色和油红O染色评价了主动脉组织中的病理变化和脂质沉积。CCK-8和流式细胞术检测细胞增殖和凋亡。免疫组织化学,酶联免疫吸附测定,qRT-PCR和蛋白质印迹评估mRNA和蛋白质的水平。
结果:Ox-LDL处理可升高SPAG5的表达和自噬相关蛋白的表达,LC3-I,LC3-II,Beclin-1和p62,在HUVEC中。GFP-LC3点在ox-LDL处理的HUVEC和LPS处理的HUVEC中增加。SPAG5敲低可逆转ox-LDL和LPS处理介导的HUVECs细胞增殖抑制和凋亡促进。SPAG5沉默进一步升高自噬并抑制PI3K的表达,p-Akt/Akt,和p-mTOR/mTOR在ox-LDL处理的HUVECs中。3-MA(自噬抑制剂)治疗逆转了SPAG5沉默介导的ox-LDL治疗的HUVEC细胞增殖增加和凋亡减少。在体内,SPAG5敲低通过激活自噬和抑制PI3K/Akt/mTOR信号通路减少AS小鼠动脉粥样硬化斑块。
结论:这项工作证明SPAG5敲低通过激活自噬减轻AS的发展。因此,SPAG5可能是AS治疗的潜在靶点。