Salusin-α通过调控PPARγ抑制THP-1源性泡沫细胞形成

Salusin-α Inhibits THP-1 Derived Foam Cell Formation via PPARγ Pathway

目的 探究Salusin-α是否通过调控过氧化物酶体增殖物激活受体γ(peroxisome pro-liferator-activated receptor gamma,PPARγ)下调酰基辅酶A:胆固醇酰基转移酶1(acyl-coA:cholesterol acyltransferase 1,ACAT1)抑制THP-1源性泡沫细胞形成。方法 THP-1细胞以160 nmol/L佛波醇肉豆蔻酸乙酸酯(Phorbol 12-myristate 13-acetate,PMA)孵育48 h诱导分化为巨噬细胞,再用50 mg/L氧化型低密度脂蛋白(oxidized low density lipoprotein,Ox-LDL)孵育48 h诱导分化为泡沫细胞,Ox-LDL孵育的同时加入不同浓度(0.2、0.4、0.6、0.8、1.0 nmol/L)Salusin-α处理。采用酶偶联比色法检测细胞游离胆固醇(free cholesterol,FC)和总胆固醇(total cholesterol,TC)含量,两者之差即为胆固醇酯(cholesterol ester,CE),比较各组CE/TC的比值;油红O染色法观察胞质脂滴的变化;RT-PCR和Western blot分别检测各组ACAT1、PPARγ mRNA和蛋白相对表达量。用特异性PPARγ抑制剂T0070907进一步验证Salusin-α抑制THP-1源性泡沫细胞形成的分子机制。结果胆固醇检测及油红O染色提示泡沫细胞模型构建成功。随着Salusin-α浓度的增加,CE/TC先降低后稍增加,0.6 nmol/L时最低,差异有统计学意义。Salusin-α可明显减少胞质内脂滴的形成,抑制泡沫细胞形成;同时浓度依赖性降低ACAT1 mRNA和蛋白相对表达量,增加PPARγ mRNA和蛋白相对表达量,差异有统计学意义。T0070907可降低PPARγ mRNA和蛋白相对表达量,增加ACAT1 mRNA和蛋白相对表达量,部分阻断Salusin-α抑制泡沫细胞形成的作用。结论 Salusin-α通过激活PPARγ通路下调ACAT1抑制THP-1源性泡沫细胞形成,发挥抗动脉粥样硬化作用。

Objective To investigate whether or not Salusin-α inhibits THP-1 derived foam cell formation by downregulating acyl-coA:cholesterol acyltransferase 1(ACAT-1)expression via peroxisome pro-liferator-activated receptor gamma(PPARγ)pathway. Methods THP-1 cells were cultured in the presence of 160 nmol/L phorbol 12-myristate 13-acetate(PMA)for 48 h to induce differentiation of monocytes to macrophages,then the cells were incubated with 50 mg/L oxidized low density lipoprotein(Ox-LDL)for 48 h to differentiate into foam cell.When incubated with Ox-LDL,Salusin-α was added at different concentrations(0.2,0.4,0.6,0.8,1.0 nmol/L).The free cholesterol(FC)and total cholesterol(TC)were measured by enzyme linked photometry.The difference between TC and FC is cholesterol ester(CE).The ratio of CE to TC(CE/TC)in each group was compared.The changes of cytoplasmic lipid droplets were observed by oil red O staining.RT-PCR and Western blotting were applied to detect mRNA and protein relative expression levels of ACAT1 and PPARγ in each group.The specific PPARγ inhibitor T0070907 was used to further verify the molecular mechanism by which Salusin-α inhibited the formation of THP-1 derived foam cells. Results Cholesterol test and oil red O staining indicated foam cell model of THP-1 line was established successfully.With the increase of Salusin-α concentration,the CE/TC first decreased and then slightly increased,the lowest was at 0.6 nmol/L,and the difference was statistically significant.Salusin-α significantly reduced intracellular lipid droplets,inhibited the formation of THP-1-derived foam cells.With the increase of Salusin-α concentration,the relative expression level of ACAT1 mRNA and protein decreased,while the relative expression level of PPARγ mRNA and protein increased,the difference was statistically significant.T0070907 reduced the relative expression of PPARγ mRNA and protein,increased the relative expression of ACAT1 mRNA and protein,and partly blocked the effect of Salusin-α on foam cell formation. Conclusion Salusin-α can inhibit macrophage-derived foam cell formation by downregulating ACAT1 expression via PPARγ-dependent pathway,which may contribute to its antiatherogenic properties.