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目的:研究载脂蛋白A-I模拟肽D-4F对氧化低密度脂蛋白(oxidized low-density lipoprotein,oxLDL)所诱导的巨噬细胞源性泡沫细胞清道夫受体A1(scavenger receptor A1,SR-A1)的抑制作用及其机制。方法:体外培养RAW264.7巨噬细胞,给予不同浓度的D-4F(12.5、25和50 mg/L)、紊乱模拟肽sD-4F(50 mmol/L)处理1h或者5 mmol/L内质网应激(endoplasmic reticulum stress,ERS)抑制剂4-苯丁酸处理30 min后,再加入ox-LDL(100 mg/L)继续培养12 h。另外培养巨噬细胞给予50 mg/L D-4F或sD-4F处理1 h,再加入2 mg/L ERS诱导剂衣霉素(tunicamycin,TM)处理4 h。MTT法检测细胞活力;试剂盒检测细胞内总胆固醇含量;分别采用免疫印迹法和实时荧光定量聚合酶链反应(real-time PCR)技术检测SR-A1和ERS标志分子葡萄糖调节蛋白78(glucose-regulated protein 78,GRP78)蛋白和mRNA表达变化;采用多功能酶标仪检测DiI-ox-LDL摄取情况。结果:D-4F明显减轻ox-LDL所诱导的巨噬细胞损伤和细胞内的胆固醇蓄积。ox-LDL可显著上调SR-A1和GRP78表达,而D-4F对上述变化具有明显抑制作用,且呈浓度依赖性。D-4F显著抑制TM所诱导的SR-A1和GRP78蛋白水平以及巨噬细胞对ox-LDL的摄取。结论:D-4F可通过抑制SR-A1表达减轻ox-LDL所诱导的巨噬细胞内胆固醇蓄积和细胞损伤,其机制可能与抑制GRP78介导的ERS信号途径有关。
AIM: To investigate the effect of apolipoprotein AI mimic peptide D-4F on scavenger receptor A1 (SR-A1) induced by oxidized low-density lipoprotein (oxLDL) in macrophage-derived foam cells ) Inhibition and its mechanism. Methods: RAW264.7 macrophages were cultured in vitro and treated with different concentrations of D-4F (12.5, 25 and 50 mg / L), disrupted peptidomimetic sD-4F (50 mmol / L) for 1 h or 5 mmol / L endoplasmic reticulum After treatment with 4-phenylbutyrate, an inhibitor of endoplasmic reticulum stress (ERS) for 30 min, ox-LDL (100 mg / L) was added to continue the culture for 12 h. In addition, macrophages were treated with 50 mg / L D-4F or sD-4F for 1 h and then treated with 2 mg / L tunicamycin (TM), an ERS inducer, for 4 h. MTT assay was used to detect cell viability. The kit was used to detect the content of total cholesterol in the cells. Western-blotting and real-time PCR were used to detect the expression of SR-A1 and ERS marker glucose- regulated protein 78 and GRP78), and the uptake of DiI-ox-LDL by multifunctional microplate reader. RESULTS: D-4F significantly attenuated ox-LDL-induced macrophage injury and intracellular cholesterol accumulation. Ox-LDL significantly upregulated the expression of SR-A1 and GRP78, while D-4F had a significant inhibitory effect on these changes in a concentration-dependent manner. D-4F significantly inhibited TM-induced SR-A1 and GRP78 protein levels as well as macrophage uptake of ox-LDL. CONCLUSION: D-4F can reduce the cholesterol accumulation and cell injury induced by ox-LDL in macrophages by inhibiting the expression of SR-A1. The mechanism may be related to the inhibition of GRP78-mediated ERS signaling pathway.