高磷环境下胆固醇敏感器SCAP功能失调促进巨噬细胞内胆固醇蓄积  

Dysfunction of cholesterol sensor SCAP under high phosphate condition promotes cholesterol accumulation in macrophage

作　　者：刘巧[1,2] 欧阳南 何泉[1] 周超[1] LIU Qiao;OUYANG Nan;HE Quan;ZHOU Chao(Department of Cardiology, the First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China;Chongqing Key Laboratory of Translational Medicine in Major Metabolic Diseases, Chongqing 400016, China;Department of Nephrology, the First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China)

机构地区：[1]重庆医科大学附属第一医院心血管内科,重庆400016 [2]重庆代谢性疾病转化医学重点实验室,重庆400016 [3]重庆医科大学附属第一医院肾脏内科,重庆400016

出　　处：《第二军医大学学报》2017年第12期1514-1520,共7页Academic Journal of Second Military Medical University

基　　金：国家自然科学基金青年科学基金(81500341)~~

摘　　要：目的观察高磷环境对巨噬细胞内胆固醇蓄积的影响及其分子机制。方法将人单核细胞株(THP-1)源性巨噬细胞分为对照组(磷1.0mmol/L)、高磷处理组(磷3.0mmol/L)、磷甲酸钠(PFA)处理组(磷1.0mmol/L加PFA 1.0mmol/L)以及高磷联合PFA处理组(磷3.0mmol/L加PFA 1.0mmol/L),按不同要求分别处理各组细胞。培养24h后,油红O染色观察细胞内中性脂质的分布,酶催化比色法定量测定细胞内的胆固醇含量,qPCR检测细胞内羟甲基戊二酸单酰辅酶A还原酶(HMGCoAR)、低密度脂蛋白受体(LDLR)、固醇调节元件结合蛋白裂解激活蛋白(SCAP)mRNA的表达,蛋白质印迹法测定细胞内SCAP、LDLR、HMGCoAR及核内固醇调节元件结合蛋白2(N-SREBP2)的蛋白水平,激光共聚焦法检测SCAP从内质网向高尔基体的移位情况。结果与对照组相比,高磷处理组巨噬细胞内中性脂质明显聚集,细胞内总胆固醇与胆固醇酯的含量增加(P<0.05),LDLR、HMGCoAR mRNA与蛋白的表达增高(P<0.05,P<0.01),细胞核内N-SREBP2的蛋白表达水平增加(P<0.05),SCAP从内质网向高尔基体的移位增加;PFA处理(高磷联合PFA处理组)可阻断高磷引起的上述作用(P<0.05,P<0.01)。高磷处理组巨噬细胞内SCAP的蛋白水平相比对照组增高(P<0.05),PFA处理能抑制高磷导致的SCAP蛋白水平增高,但SCAP mRNA的表达在各组间差异均无统计学意义(P>0.05)。结论高磷环境下,钠磷转运体介导磷离子进入巨噬细胞内,通过基因转录后机制增加SCAP的蛋白水平,诱导其功能失调并促使其异常转运SREBP2至高尔基体裂解释放NSREBP2,后者转位入核促进HMGCoAR和LDLR的表达,促使细胞内源性胆固醇合成和外源性LDL经LDLR摄入的增加,最终导致细胞内胆固醇异常蓄积。ObjectiveTo explore the effects of high phosphate(Pi)condition on the cholesterol accumulation in macrophage and its underlying mechanisms.MethodsThe human monocytic cell line THP1derived macrophages were divided into control group(concentration of Pi being1.0mmol/L),high Pi exposure group(concentration of Pi being3.0mmol/L),phosphonoformic acid(PFA)treatment group(concentration of Pi and PFA being1.0mmol/L)and high phosphate plus PFA treatment group(concentration of Pi and PFA being3.0mmol/L and1.0mmol/L,respectively).Intracellular neutral lipids were observed by Oil Red O staining.Cholesterol contents were quantified by enzyme catalyzed colorimetry.qPCR was used to dectect the relative mRNA expressions of3hydroxy3methyl glutaryl coenzyme A reductase(HMGCoAR),low density lipoprotein receptor(LDLR)and sterol regulatory element binding protein(SREBP)cleavage activating protein(SCAP),and their proteins and nuclear SREBP2(N SREBP2)protein expressions were assessed by Western blotting.The translocation of SCAP from endoplasmic reticulum(ER)to Golgi body was observed by laser confocal microscope.ResultsCompared with the control group,the macrophages in the high Pi exposure group showed an obvious aggregation of neutral lipids and significantly increased contents intracellular cholesterol ester and total cholesterol(P<0.05).The mRNA and protein expressions of LDLR and HMGCoAR,and protein level of N SREBP2in macrophage nucleus in the high Pi exposure group were increased significantly in comparison to the control group(P<0.05,P<0.01).The SCAP translocated obviously from ER to Golgi body.However,above mentioned changes were significantly suppressed by PFA treatment(high Pi plus PFA treatment group)(P<0.05,P<0.01).Furthermore,the significant increase of protein level of SCAP induced by high Pi treatment(P<0.05)was suppressed by PFA treatment,but there were no differences in SCAP mRNA expressions in each group(P>0.05).ConclusionUnder high Pi condition,sodium phosphate transporter Pit1mediates the entry of phosphorus ions i

关 键 词：巨噬细胞 高磷酸盐血症 胆固醇贮积病 动脉粥样硬化 

分 类 号：R392.32[医药卫生—免疫学] R364.2[医药卫生—基础医学]