炎症应激通过激活mTOR通路诱导THP-1巨噬细胞泡沫化的实验研究

Experiment of THP-1 macrophage foam-cell formation through mT OR signal pathway activation induced by inflammatory stress

目的研究脂多糖(LPS)诱导的炎症应激是否通过激活m TOR通路,增加SCAP/SREBP2表达,干扰SCAP/SREBP2负反馈调控,增加THP-1巨噬细胞对非修饰低密度脂蛋白胆固醇(LDL)摄取,导致泡沫细胞形成。方法诱导分化成功的THP-1源性巨噬细胞在无血清培养基中培养4 h后,分为对照组(5 mg·L^(-1)LDL)、炎症刺激组(5 mg·L^(-1)LDL+200μg·L^(-1)LPS)、炎症+雷帕霉素组(5 mg·L^(-1)LDL+200μg·L^(-1)LPS+10μg·L^(-1)Rapamycin)。以上各组细胞培养24 h后收获。油红O染色法检测各组细胞内脂质沉积情况,Real-time PCR法检测LDLr、SREBP2、SCAP、S6K1和m TOR mRNA水平,Western blot法检测LDLr、S6K1、mTOR蛋白表达,激光共聚焦法检测SCAP在内质网与高尔基体间的转位情况。结果油红O染色发现,炎症应激增加THP-1巨噬细胞内脂质沉积,雷帕霉素抑制炎症应激诱导的脂质聚积。Real-time PCR检测显示,炎症应激上调THP-1巨噬细胞LDLr、SREBP2、SCAP、S6K1和mTOR mRNA水平(P<0.05),雷帕霉素减少炎症应激诱导的LDLr、SREBP2、SCAP、S6K1和mTOR mRNA水平升高(P<0.05)。Western blot检测发现,炎症应激上调LDLr、S6K1和m TOR蛋白表达(P<0.05),雷帕霉素减少炎症应激诱导的LDLr、S6K1和mTOR蛋白表达水平升高(P<0.05)。激光共聚焦检测发现,炎症应激增加SCAP/SREBP2从内质网转位到高尔基体,雷帕霉素则抑制炎症应激诱导的SCAP/SREBP2复合物从内质网转位到高尔基体。结论炎症应激通过激活mTOR通路,上调SCAP/SREBP2表达,致SCAP/SREBP2复合体转位至高尔基体异常增加,LDLr表达上调,致使胆固醇聚积于细胞内,泡沫细胞形成。雷帕霉素能够逆转炎症应激诱导mTOR通路激活,减少细胞内胆固醇沉积,提示炎症应激状态下,mTOR可能是泡沫细胞形成的关键通路。

Aim To investigate if LPS increases the sterol regulatory element binding proteins （ SREBPs ） cleavage-activating protein （ SREBP ） -SREBP 2 expressionby activation of mTOR signal pathway in THP-1macrophages, upgrading LDLr level, causing foam-cellformation. Methods THP-1 macrophages were incubatedin serum free medium in the absence of 5mg·L^-1 LDL alone,or 5mg·L^-1 LDL plus 200μg·L^-1 LPS ,or 5mg·L^-1 LDL plus 200μg·L^-1 LPS plus 10μg·L^-1 rapamycin. Morphological examination ofmacrophages was performed with Oil Red O staining.Expression changes of LDLr, SREBP 2 , SREBP , S6 K 1and mTOR m R N A were detected by real time quantitativepolymerase chain reaction （ PCR ） . Western blotwas used to analyze protein expression changes of LDLr,S6K1 and mTOR . Translocation of SREBP -SREBP 2complex from the endoplasmic reticulum （ ER ） to theGolgi was determined by confocal microscopy. ResultsLPS enhanced transformation of THP-1 macrophagesinto foam cells by increased uptake of lipid as evidencedby Oil Red Oasay . LPS increased mRNA levelsof LDLr , SREBP 2 ,SREBP ,S6K 1 and mTOR （ P 〈0. 05）. Rapamycin reduced the mRNA levels of LDLr,SREBK ,SREBP ,S6K1 and mTOR induced by LPS （ P〈 0. 05 ） . Western blot demonstrated that LPS alsocaused over-expression of protein of LDLr, S6K1 andmTOR （ P 〈0. 05） . Rapamycin reduced the expressionof protein of LDLr,S6 K 1 and m TOR induced by LPS（P 〈 0. 05 ） . Confocal microscopy demonstrated LPScaused an escape of SREBP -SREBP 2 complex from theE Rto the Golgi. Rapamycin inhibited the translocationof SREBP -SREBP 2 complex from the ER to the Golgi.Conclusions Inflammatory stress increases SREBP /SREBP expression by activation of mTOR signal pathway, resulting in an escape of SREBP - SREBP complexfrom the ER to the Golgi,furthermore elevating LDLrexpression and causing foam-cell formation. Rapamycinreverses the activation of mTOR signal pathway anddecreases lipid deposition in THP-1 macrophages inducedby LPS .