褪黑素调控SIRT1/FOXO1通路延缓椎间盘髓核细胞退变的实验研究

An Experimental Study of Melatonin Regulating SIRT1/FOXO1 Pathway to Delay Degeneration of Intervertebral Disc Nucleus Pulposus Cells

目的探讨褪黑素调控沉默信息调节因子1(SIRT1)/叉头框转录因子O1(FOXO1)通路对椎间盘髓核细胞(NPCs)退变的影响。方法分离并培养NPCs,CCK-8法确定过氧化氢和褪黑素浓度;将过氧化氢处理的NPCs随机分为过氧化氢组(200μmol/L过氧化氢)、褪黑素组(200μmol/L过氧化氢、1μmol/L褪黑素)、褪黑素+siRNA NC组(细胞转染siRNA NC、200μmol/L过氧化氢、1μmol/L褪黑素)、褪黑素+siRNA SIRT1组(细胞转染siRNA SIRT1、200μmol/L过氧化氢、1μmol/L褪黑素),另取不使用过氧化氢处理的非转染NPCs为对照组。检测各组细胞中SIRT1 mRNA表达水平、细胞凋亡率、肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)含量,聚集蛋白聚糖(Aggrecan)、Ⅱ型胶原蛋白(collagenⅡ)、SIRT1蛋白表达和FOXO1乙酰化(acely-FOXO1)水平。结果与对照组比较,过氧化氢组NPCs中SIRT1 mRNA表达水平、Aggrecan、collagenⅡ、SIRT1蛋白表达水平显著降低,TNF-α和IL-1β含量、细胞凋亡率以及acely-FOXO1蛋白水平显著升高(P<0.05)。与过氧化氢组相比,褪黑素组NPCs中SIRT1 mRNA表达水平、Aggrecan、collagenⅡ、SIRT1蛋白表达水平显著升高,TNF-α和IL-1β含量、细胞凋亡率及acely-FOXO1蛋白水平显著降低(P<0.05);与褪黑素组和褪黑素+siRNA NC组比较,褪黑素+siRNA SIRT1组NPCs中SIRT1 mRNA表达水平、Aggrecan、collagenⅡ、SIRT1蛋白表达水平显著降低,TNF-α和IL-1β含量、细胞凋亡率以及acely-FOXO1蛋白水平显著升高(P<0.05)。结论褪黑素能够通过上调SIRT1表达,抑制FOXO1乙酰化,减轻炎症反应,减少NPCs凋亡,延缓椎间盘NPCs退化。

Objective To investigate the effect of melatonin on degeneration of intervertebral disc nucleus pulposus cells(NPCs)by regulating silent information regulator 3(SIRT1)/forkhead box transcription factor O1(FOXO1)pathway.Methods NPCs were isolated and cultured,and the concentrations of hydrogen peroxide and melatonin were determined by CCK-8.NPCs treated by hydrogen peroxide were randomly divided into hydrogen peroxide group(200μmol/L hydrogen peroxide),melatonin group(200μmol/L hydrogen peroxide,1μmol/L melatonin),melatonin+siRNA NC group(cells were transfected with siRNA NC,200μmol/L hydrogen peroxide,1μmol/L melatonin)and melatonin+siRNA SIRT1 group(cells were transfected with siRNA SIRT1,200μmol/L hydrogen peroxide,1μmol/L melatonin).Non-transfected NPCs without treatment by hydrogen peroxide were selected as control group.SIRT1 mRNA expressions,apoptosis rate,contents of tumor necrosis factor-α(TNF-α)and interleukin-1β(IL-1β),protein expressions of Aggrecan,collagen II and SIRT1,and levels of acetylation FOXO1(acely-FOXO1)were detected among all groups.Results Compared with those in control group,SIRT1 mRNA expression,and protein expressions of Aggrecan,collagenⅡand SIRT1 of NPCs were significantly lower,while contents of TNF-αand IL-1β,apoptosis rate and acely-FOXO1 protein level were significantly higher in hydrogen peroxide group(P<0.05).Compared with those in hydrogen peroxide group,SIRT1 mRNA expression,and protein expressions of Aggrecan,collagenⅡand SIRT1 of NPCs were significantly higher,while contents of TNF-αand IL-1β,apoptosis rate and acely-FOXO1 protein level were significantly lower in melatonin group(P<0.05).Compared with those in melatonin and melatonin+siRNA NC groups,SIRT1 mRNA expression,and protein expressions of Aggrecan,collagenⅡand SIRT1 were significantly lower,while contents of TNF-αand IL-1β,apoptosis rate and acely-FOXO1 protein level were significantly higher in melatonin+siRNA SIRT1 group(P<0.05).Conclusion Melatonin may inhibit FOXO1 acetylation,ameliorate inflammatory reactions,inhibit apoptosis of NPCs and delay degeneration of intervertebral disc NPCs by up-regulating SIRT1 expression.