载脂蛋白嵌合模拟肽对巨噬细胞胆固醇流出的影响

目的观察载脂蛋白嵌合模拟肽Ac-hE-18A-NH2对RAW264.7巨噬细胞胆固醇流出的影响,并探讨其机制。方法 RAW264.7巨噬细胞种植于24孔板,用0.5μCi/孔3H-胆固醇和含50mg·mL-1氧化低密度脂蛋白共同孵育24h之后,给予不同浓度的Ac-hE-18A-NH2(0～100mg·mL-1)干预24h,收集细胞用液体闪烁计数法检测胆固醇流出。采用ELISA测定细胞内cAMP含量,采用实时荧光定量PCR及Westernblot检测ABCA1、LXRα和PPARγ的mRNA及蛋白表达。结果 Ac-hE-18A-NH2以浓度依赖方式介导胆固醇流出;50mg·mL-1Ac-hE-18A-NH2干预不同时间,其介导的胆固醇流出率分别为(10.86±1.46)%(6h),(13.43±1.55)%(12h),(20.58±1.34)%(18h),和(26.93±4.37)%(24h)。同时,Ac-hE-18A-NH2还以浓度依赖方式增加细胞内cAMP水平,上调ABCA1、LXRα和PPARγmR-NA和蛋白表达。加用cAMP刺激剂8-Br-cAMP,Ac-hE-18A-NH2介导的胆固醇流出率由26.93±4.37增加至35.81±2.73,ABCA1mRNA表达增加了66.67%。而加用PPARγ特异性抑制剂预处理细胞后,PPARγ的表达几乎完全抑制,ABCA1和LXRα的表达也受到一定程度抑制,Ac-hE-18A-NH2介导的胆固醇流出率明显减少。结论模拟肽Ac-hE-18A-NH2可以明显促进巨噬细胞胆固醇流出,其机制可能与cAMP-ABCA1和PPARγ-LXRα-ABCA1两种途径有关。

Effect of Ac-hE-18A-NH2,a novel dual-domain apolipoprotein mimetic peptide,on cholesterol efflux in RAW264.7 macrophages

Aim To explore the effect of a novel dual-domain apolipoprotein mimetic peptide,Ac-hE-18A-NH2,on cholesterol efflux in RAW264.7 macrophages and the possible mechanism.Methods RAW264.7 macrophages were incubated in the medium containing various concentrations of the peptide,Ac-hE-18A-NH2(1,10,50 and 100 mg·L-1)for 24 hours.The intracellular cAMP level was determined by ELISA.ABCA1,LXRα and PPARγ expression in macrophages was quantitated by real time RT-PCR and Western blot analysis.In some experiments,the cells were pretreated with 8-Br-cAMP(a cAMP stimulator)or GW9662(a specific PPARγ inhibitor)for 12 hours.Results The peptide Ac-hE-18A-NH2 significantly increased the cholesterol efflux in a concentration-and time-dependent manner.Cholesterol efflux from macrophages at different time points was(10.86±1.46)%(6 h),(13.43±1.55)%(12 h),(20.58±1.34)%(18 h),and(26.93±4.37)%(24 h)respectively.Concomitantly,Ac-hE-18A-NH2 increased intracellular cAMP level,ABCA1 mRNA and protein expression in a dose-dependent manner,consistent with the changes of cholesterol efflux from macrophages.8-Br-cAMP was a potent activator of cholesterol efflux and ABCA1 expression during the process of cholesterol efflux media-ted by Ac-hE-18A-NH2.Moreover,Ac-hE-18A-NH2 upregulated the expression of LXRα and PPARγ.Addition of GW9662 markedly inhibited the increase of ABCA1 gene expression and Ac-hE-18A-NH2-mediated cholesterol efflux.Conclusions Ac-hE-18A-NH2 affects cholesterol efflux,cAMP level and ABCA1 expression of macrophages.The pathway of cAMP-ABCA1 and LXRα-PPARγ-ABCA1 may be involved in this process.