炎症干扰胆固醇调节元件结合蛋白2致ApoE/SRA/CD36三基因敲除小鼠肝脏脂质的异常积聚

Inflammation enhances the accumulation of lipid in ApoE/SRA/CD36 KO mice liver

目的 研究炎症是否通过干扰核转录因子胆固醇调节元件结合蛋白2（SREBP-2）而致ApoE/SRA/CD36三基因敲除小鼠肝脏胆固醇的异常积聚.方法 将8周龄ApoE/SRA/CD36三基因敲除雄性小鼠随机分为对照组（n=8）和炎症组（n=8）,2组均喂以西方饮食（Western diet）,炎症组小鼠皮下注射10%酪蛋白建立慢性炎症模型,对照组注射相应量磷酸盐缓冲液,14周后处死,测定血清中炎症介质和脂质的水平及肝组织中胆固醇含量,油红O、免疫组织化学染色后观察肝脂质沉积程度以及组织形态变化,实时定量PCR法检测肝脏SREBP裂解激活蛋白（SCAP）、SREBP-2及其下游基因低密度脂蛋白受体（LDLr）mRNA水平.对计量资料采用两样本均数比较的t检验进行统计学分析.结果 炎症状态下,血清中总胆固醇[（7.72±1.70）mmol/L]、低密度脂蛋白胆固醇[（2.94±0.44）mmol/L]、高密度脂蛋白胆固醇[（2.24±0.63）mmol/L]水平均显著降低,与对照组[分别为（13.23±3.61）mmol/L、（9.28±3.66）mmol/L、（4.13±0.42）mmol/L]比较,t值分别为3.383、4.245、5.937,P值均＜0.05;肝组织中胆固醇含量显著增加;油红O染色表明,胆固醇在肝脏中的沉积异常增多（t=2.707,P＜0.05）;实时定量PCR和免疫组织化学检测结果表明胆固醇代谢相关基因SREBP2、LDLr和SCAP的mRNA和蛋白质表达水平显著增加. 结论炎症可以通过干扰SREBP-2导致低密度脂蛋白胆固醇摄取异常,造成肝脏脂质沉积增多和损害.

Objective To investigate if inflammatory stress enhances liver lipid accumulation via SREBPs mediated dysregulation of low density protein receptor （LDLr） expression in apolipoprotein E, scavenger receptors class A and CD36 triple knockout （ApoE/SRA/CD36 KO） mice. Methods 16 Male ApoE/SRA/CD36 KO mice were subcutaneously injected with 0.5 ml 10% casein or PBS. The mice were fed a Western diet （Harlan, TD88137） containing 21% fat and 0.15% of cholesterol for 14 weeks. Animals were sacrificed and blood samples were collected. The serum amyloid A （SAA）, IL-6, total cholesterol （TC）, LDL and high density protein （HDL） were assayed. The lipid accumulation in liver was evaluated by Oil Red O staining. The mRNA and protein expression of SREBP-2, SREBPs cleavage activating protein （SCAP） and LDLr were analyzed by Real-Time Polymerase Chain Reaction （RT-PCR） and immunohistochemistry staining. Results Blood levels of SAA [（26.60 ± 3.24） ng/ml vs （14.35 ± 1.73） ng/ml, P 〈 0.01] and IL-6 [（36.37 ± 2.20） pg/ml vs （18.02 ± 4.87） pg/ml, P 〈 0.01] were higher, while TC [（7.72 ± 1.70） mmol/L vs （13.23 ± 3.61）mmol/L, P 〈 0.01], LDL-cholesterol [（2.94 ± 0.44） mmol/L vs （9.28 ± 3.66） mmol/L, P 〈 0.01] and HDL cholesterol [（2.24 ± 0.63） mmol/L vs （4.13 ± 0.42） mmol/L, P 〈 0.01] were lower in inflamed mice compared to controls. ORO staining showed that lipid accumulation in the liver was more extensive in inflamed group despite lower blood lipid levels. Meanwhile, Real Time PCR data showed inflammation induced the expression of LDLr （4.56 fold）, SCAP （3.14 fold） and SREBP-2 （14.72 fold） in liver. Immunohistochemical staining also indicated increased proteins expression in the liver, which was consistent with mRNA data.Conclusions Inflammation causes lipid accumulation in liver via disrupting SREBP-2 and LDLr expression.