脑肠肽Ghrelin通过生长激素促分泌素受体下调人源单核细胞株THP-1源性泡沫细胞酰基辅酶A：胆固醇酰基转移酶1表达的实验研究

Ghrelin down-regulates ACAT-1 in THP-1 derived foam cells via growth hormone secretagogue receptor-dependent pathway

目的探讨脑肠肽Ghrelin对人源单核细胞株THP-1源性泡沫细胞酰基辅酶A：胆固醇酰基转移酶1（ACAT-1）表达的调控作用及其调控途径。方法体外培养人源单核细胞株THP-1，由佛波酯（PMA）作用使其分化为巨噬细胞，继而在氧化低密度脂蛋白（ox—LDL）存在条件下进一步转变为泡沫细胞，油红O染色法鉴定泡沫细胞形成。运用PT—PCR法和Western blot法分别检测Ghrelin干预后及拮抗生长激素促分泌素受体（GHS—R）后ACAT-1 mRNA水平与ACAT-1蛋白表达，采用酶法，通过荧光分光光度计检测细胞内胆固醇含量并计算胆固醇酯含量。结果Ghrelin可明显减少细胞内胆固醇酯的含量，并能显著降低单核／巨噬细胞泡沫化过程中ACAT-1 mRNA水平和蛋白表达（P均〈0．01）。拮抗GHS—R后，Ghrelin抑制泡沫细胞形成的效应被阻断，且GHS-R特异性拮抗剂浓度越高，该阻断作用越明显。浓度为10^-5、5×10^-5、10^-4mol／L的GHS—R特异性拮抗剂组ACAT-1 mRNA水平与蛋白表达分别为1．14±0．04、1．58±0．03、2．40±0．16和1．25±0．09、1．77±0．11、2．30±0．09，明显高于Ghrelin组0．89±0．05和0．86±0．08（P均〈0．05）。结论Ghrelin可能通过ACAT-1转录翻译水平的下调抑制泡沫细胞的形成，从而延缓动脉粥样硬化的发生，且该效应通过GHS—R途径发挥作用。

Objective To investigate the effects of Ghrelin on the expression of acyl coenzyme A： cholesterol acyhransferases-1 （ ACAT-1 ） in THP-1 derived foam cells. Methods The human monocytic leukemia cell line （ THP-1 ） was chosen in our study. The differentiation of THP-1 cells into macrophages was induced by phorbol 12-myristate 13-acetate. Maernphages were then incubated with oxidized LDL （ox- LDL） to generate foam cells. Ghrelin and [ D-Lys3 ]-GHRP-6, the special antagonist of growth hormone secretagogue receptor （GHS-R） , were treated during foam cells formation. The ACAT-1 protein and mRNA levels were detected by Western blot and RT-PCR. The effect of variance of cholesterol content was measured by zymoehemistry via-fluorospectrophotometer. Results Ghrelin reduced the content of cholesterol ester in foam cells obviously. ACAT-1 protein and mRNA levels were also decreased. The antagonist of GHS-R inhibited the effects of Ghrelin on ACAT-1 expression in dose-dependent manner. The ACAT-1 mRNA levels of the GHS-R specific antagonist groups （ 10^-5, 5 × 10^-5, 10^-4 mol/L） were 1. 14 ± 0.04,1.58 ± 0.03, 2.40 ± 0.16, significantly higher than that of the Ghrelin group （0.89 ± 0.05 ）. And the protein expressions were 1.25 ± 0.09,1.77 ± 0.11,2.30 ± 0.09, also higher than that of the Ghrelin group （0.86 ± 0.08 ）. Conclusions Ghrelin might interfere atherosclerosis by down-regnlating the expression of ACAT-1 via GHS-R pathway.