载脂蛋白A-I模拟肽D-4F对巨噬细胞源性泡沫细胞清道夫受体A1的抑制作用

Inhibitory effect of apolipoprotein A-I mimetic peptide D-4F on scavenger receptor A1 in macrophage-derived foam cells

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 inhibitory effect of apolipoprotein A-I mimetic peptide D-4F on the scavenger receptor A1（ SR-A1） in macrophage-derived foam cells induced by oxidized low-density lipoprotein（ ox-LDL）.METHODS： RAW264. 7 cells were pretreated with different concentrations（ 12. 5,25 and 50 mg /L） of D-4F or 50 mg /L inactive control peptide scrambled D-4F（ sD-4F） for 1 h or endoplasmic reticulum stress（ ERS） inhibitor 4-phenylbutyric acid（ 5 mmol /L） for 30 min,followed by the treatment with 100 mg /L ox-LDL for 12 h. In addition,the cells were pretreated with 50 mg /L D-4F or sD-4F for 1 h,and then stimulated with 2 mg /L tunicamycin（ TM; an ERS inducer）,for 4h. The viability of the cells was measured by MTT assay,and the content of intracellular total cholesterol（ TC） was measured by a tissue /cell TC assay. The protein and mRNA levels of SR-A1 and glucose-regulated protein 78（ GRP78） were analyzed by Western blotting and quantitative real-time PCR,respectively. The fluorescence intensity of DiI-ox-LDL in the cells was detected by a multifunctional microplate reader. RESULTS： D-4F significantly reduced ox-LDL-induced macrophage injury and intracellular cholesterol accumulation,and attenuated the ox-LDL-induced expression of SRA1 and GRP78 in a dose-dependent manner. Additionally,D-4F significantly inhibited the TM-induced protein expression of SR-A1 and GRP78,and attenuated the uptake of ox-LDL by macrophages. CONCLUSION： D-4F reduces ox-LDL-induced macrophage cholesterol accumulation and injury by inhibiting SR-A1 expression. The mechanism may be related to the inhibition of ERS signaling pathway mediated by GRP78.