姜黄素调节PI3K/AKT/mTOR信号通路对LPS诱导的角膜基质细胞自噬的影响

Influence of curcumin on LPS-induced autophagy in corneal stromal cells by regulating PI 3 K/AKT/mTOR signaling pathway

目的:探讨姜黄素调节磷脂酰肌醇3-激酶(PI3K)/丝氨酸-苏氨酸激酶(AKT)/哺乳动物雷帕霉素靶蛋白(mTOR)信号通路对LPS诱导的角膜基质细胞自噬的影响。方法:小鼠角膜基质细胞分为对照组、LPS组(1μg/μl LPS)、L-Cur组(15μmol/L姜黄素)、M-Cur组(30μmol/L姜黄素)、H-Cur组(50μmol/L姜黄素)、H-Cur+740Y-P组(50μmol/L姜黄素+10μmol/L PI3K激活剂740Y-P);MTT检测姜黄素对角膜基质细胞毒性和细胞活力的影响;ELISA检测角膜基质细胞炎症因子水平;免疫荧光染色检测小鼠角膜基质细胞Beclin1、LC3水平;Western blot检测角膜基质细胞自噬相关蛋白及通路相关蛋白表达。结果:0~90μmol/L姜黄素对角膜基质细胞无明显毒性。与对照组相比,LPS组A570nm、Beclin1、LC3Ⅱ/Ⅰ水平显著降低(P<0.05),IL-6、IL-8水平、p-PI3K/PI3K、p-AKT/AKT、p-mTOR/mTOR水平显著升高(P<0.05);与LPS组相比,L-Cur组、M-Cur组、H-Cur组A570 nm、Beclin1、LC3Ⅱ/Ⅰ水平依次显著升高(P<0.05),IL-6、IL-8水平、p-PI3K/PI3K、p-AKT/AKT、p-mTOR/mTOR水平依次显著降低(P<0.05);H-Cur+740Y-P消除了姜黄素对角膜基质细胞的有利作用。结论:姜黄素可能通过抑制PI3K/AKT/mTOR信号通路促进LPS诱导的角膜基质细胞自噬。

Objective:To investigate influences of curcumin on LPS-induced autophagy in corneal stromal cells by regulating phosphatidylinositol 3-kinase(PI3K)/serine-threonine kinase(AKT)/mammalian target of rapamycin(mTOR)signaling pathway.Methods:Mouse corneal stromal cells were grouped into control group,LPS group(1μg/μl LPS),L-Cur group(15μmol/L curcumin),M-Cur group(30μmol/L curcumin),H-Cur group(50μmol/L curcumin),and H-Cur+740Y-P group(50μmol/L curcumin+10μmol/L PI3K activator 740Y-P).MTT was applied to detect effect of curcumin on corneal stromal cytotoxicity and cell viability;ELISA was applied to detect levels of inflammatory factors in corneal stromal cells;immunofluorescence staining was applied to detect levels of Beclin1 and LC3 in mouse corneal stromal cells;Western blot was applied to detect expressions of autophagy-related proteins and pathway-related proteins in corneal stromal cells.Results:0~90μmol/L curcumin had no obvious toxicity to corneal stromal cells.Compared with control group,A570 nm,levels of Beclin1 and LC3Ⅱ/Ⅰin LPS group were obviously decreased(P<0.05),levels of IL-6,IL-8,p-PI3K/PI3K,p-AKT/AKT and p-mTOR/mTOR were obviously increased(P<0.05);compared with LPS group,A570 nm,levels of Beclin1 and LC3Ⅱ/Ⅰin L-Cur group,M-Cur group,H-Cur group were obviously increased in turn(P<0.05),levels of IL-6,IL-8,p-PI3K/PI3K,p-AKT/AKT and p-mTOR/mTOR were obviously decreased in turn(P<0.05);H-Cur+740Y-P eliminated beneficial effect of curcumin on corneal stromal cells.Conclusion:Curcumin may promote LPS-induced autophagy in corneal stromal cells by down-regulating PI3K/AKT/mTOR signaling pathway.