酰基辅酶A胆固醇酰基转移酶抑制剂对人系膜细胞细胞内脂质稳态的影响

目的 研究酰基辅酶A胆固醇酰基转移酶抑制剂（ACATI，58-035）对脂质负荷人系膜细胞系（HMCL）细胞内脂质稳态的影响。 方法 油红O染色观察细胞内脂滴变化。偶氮四唑盐（MTT）法了解细胞增殖。高效液相色谱法（HPLC）测定细胞内游离胆固醇(FC)和胆固醇酯（CE）。Western印迹法检测ACATI对HMCL ACAT1和腺苷三磷酸结合盒转运体A1(ABCA1)的蛋白表达。荧光实时定量PCR检测ACATI对HMCL ACAT1、ABCA1和低密度脂蛋白（LDL）受体的影响。瞬时转染检测ACATI对HMCL ACAT1 p1启动子的影响。 结果 100 mg/L LDL明显增加HMCL内脂滴和CE含量。10 mg/L ACATI 58-035没有细胞毒性，且能显著抑制LDL导致的脂滴形成和CE含量增加(相对对照比值，分别是1.91±0.36和1.07±0.30,P < 0.01)。100 mg/L LDL主要在蛋白水平增加ACAT1表达（为对照1.27倍），在10 mg/L ACATI 58-035共同作用时，ACAT1蛋白表达进一步增加（为对照1.77倍）；100 mg/L LDL明显上调HMCL ABCA1 mRNA表达为对照（2.97±0.39）倍，P < 0.01]，明显下调LDL受体mRNA表达为对照（0.08±0.02）倍, P < 0.01]，在10 mg/L ACATI 58-035共同作用时，HMCL ABCA1的蛋白和mRNA表达进一步上调为对照（4.41±1.27）倍，与LDL作用组比较，P < 0.05]，LDL受体mRNA表达进一步下调为对照（0.04±0.005）倍，与LDL作用组比较，P < 0.01]。 结论 脂质负荷HMCL在一定剂量ACATI作用下，细胞内CE含量明显减少，FC并没有明显增加，这与HMCL上调ABCA1蛋白和mRNA表达、下调LDL受体mRNA表达有关。脂质负荷主要引起HMCL ACAT1蛋白表达增加。

Impact of acyl-coenzyme A cholesterol acyltransferase inhibitor on cholesterol homeostasis in human mesangial cells

Objective To study the effects of acyl-coenzyme A cholesterol acyltransferase inhibitor(ACATI, 58-035) on cholesterol homeostasis in lipid-loaded human mesangial cell line(HMCL) . Methods Oil red O was used to examine the lipid droplet. MTT was performed to detect the cell proliferation. High performance liquid chromatography (HPLC) was used to measure intracellular free cholesterol (FC) and cholesterol ester(CE). Western blot was used to demonstrate the protein level. Real-time PCR was performed to detect the mRNA expression. Results In HMCL loaded with 100 mg/L of low density lipoprotein(LDL), 10 mg/L 58-035 significantly inhibitted the formation of lipid droplets and CE mass (ratios over control, 1.91±0.36 and 1.07±0.30 respectively, P<0.01) without toxic effect. It further enhanced the ACAT1 protein expression (ratios over control, 1.27 and 1.77 respectively) without significant influence on mRNA level, since the activity of ACAT1 p1 promoter by transient transfection was not affected by either LDL or ACATI. 100 mg/L LDL down-regulated the mRNA of LDL receptor(LDLR) (ratio over control, 0.08±0.02, P<0.01)and up-regulated that of ATP binding cassette transporter 1(ABCA1) (ratio over control, 2.97±0.39, P<0.01). The mRNA expression of ABCA1 was further up-regulated (ratio over control 4.41±1.27, compared with LDL group P<0.05), and LDLR was further down-regulated(ratio over control 0.04±0.005, compared with LDL group P<0.01) in the presence of 10 mg/L ACATI 58-035. Conclusions The CE mass in lipid-loaded HMCL can be decreased in the presence of ACATI, accompanied by further down-regulation of LDLR mRNA expression and up-regulation of ABCA1 protein and mRNA expression. It is physiological feedback to inhibit free cholesterol accumulation to maintain cholesterol homeostasis.