目的 探讨沉默信息调控因子1(SIRTl)在激活型小胶质细胞介导PC12细胞损伤中所起的作用及相关机制. 方法 体外常规培养BV-2小胶质细胞和PC12细胞,ELISA检测1μg/mL脂多糖(LPS)作用BV-2细胞6、12、24 h后上清肿瘤坏死因子-α(TNF-α)、白介素-6(IL-6)的水平;MTT检测LPS与BV-2细胞共培养上清对PC12细胞存活率的影响,同时设对照组、LPS组、单纯BV-2上清液组;MTT检测SIRT1激活剂藜芦醇(5、10、25、50、100 μmol/L)、SIRT1抑制剂尼克酰胺(5、10、25、50 mmol/L)对PC12细胞存活率的影响,以筛选二者的干预浓度.实验分对照组、LPS+BV-2细胞共培养上清液组、白藜芦醇干预组、尼克酰胺干预组,分别加入培养基、LPS+BV-2细胞共培养上清液、50 μmol/L白藜芦醇、25 mmol/L尼克酰胺,培养18h后MTT检测4组细胞存活率,Western blotting检测细胞内SIRT1、乙酰化p53的表达水平. 结果 LPS作用BV-2细胞6、12、24 h后IL-6、TNF-α分泌量均依次增高,差异有统计学意义(P<0.05);MTT比色显示与对照组、LPS组、单纯BV-2上清液组比较,LPS+BV-2细胞共培养上清组PC12细胞存活率下降,差异有统计学意义(P<0.05);1 00μmol/L白藜芦醇组、50 μmol/L尼克酰胺组细胞存活率较对照组降低,差异有统计学意义(P<0.05),因此选择50 μmol/L白藜芦醇、25 μmol/L尼克酰胺进行实验;与对照组比较,LPS+BV-2细胞共培养上清组细胞存活率降低,SIRT1的表达下降,乙酰化p53的表达上升,差异有统计学意义(P<0.05),与LPS与BV-2共培养上清组比较,白藜芦醇干预组细胞活性上升,SIRT1的表达水平较高,乙酰化p53的表达量较低,尼克酰胺干预组结果相反,细胞活性下降,SIRT1的表达较低,乙酰化p53的表达较高,差异有统计学意义(P<0.05). 结论 在LPS与小胶质细胞共培养上清损伤多巴胺(DA)能神经元过程中,SIRT1具有保护作用,其机制与乙酰化p53受抑制有关.
Objective To observe the effects of silent information regulator 1 (SIRT1) on toxicity of activated BV-2 to PC12 cells and the possible mechanisms.Methods BV-2 microglial cells and PC12 cells were routinely cultured in vitro; ELISA was used to measure to the levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) stimulated by lipopolysaccharide (LPS,1 μg/mL) in BV-2 cells.MTT assay was employed to identify the cell viability of PC12 cells injured by culture medium of activated BV-2 and determined the suitable concentrations of resveratrol (a potent SIRT1 activator,5,10,25,50 and 100 μmol/L) and nicotinamide (a known SIRT1 inhibitor,5,10,25 and 50 mmol/L).PC12 cells were divided into groups as follows:control group Ⅱ,LPS+BV-2 co-cultured group,resveratrol treatment group and nicotinamide treatment group (pretreated with resveratrol or nicotinamide for 2 h,and then subjected to culture medium of activated BV-2 cells,in the presence of resveratrol or sirtinol for 18 h); the cell viability was measured by OD value in MTT assay,and the expressions of SIRT1 and acetyl-p53 were detected by Western blotting.Results TNF-α and IL-6 secretions increased gradually at 6,12 and 24 h after LPS being induced BV-2,with significant difference between each two time points (P<0.05).PC12 cell viability decreased in the LPS+BV-2 co-cultured group as compared with that in the control group Ⅰ,LPS treatment group and BV-2 supernate group (P<0.05).The cell viability of cells in the 100 μmol/L resveratrol treatment group and 50 μmol/L niacinamide treatment group decreased as compared with that in the control group Ⅱ (P<0.05),therefore,50 μmol/L resveratrol and 25 μmol/L nicotinamide were chosen in the next experiment.As compared with those in the control group Ⅲ,the cell viability and SIRT1 expression significantly decreased,and acetyl-p53expression significantly increased in the LPS+BV-2 co-cultured group (P<0.05); as compared with those in the in the LPS+BV-2 co-cultured group,the cell viability and SIRT1 expression significantly increased,and acetyl-p53 expression significantly decreased in the 50 μmol/L resveratrol treatment group,and opposite results were noted in the 25 μmol/L nicotinamide treatment group (P<0.05).Conclusion SIRT1 can inhibit toxicity of activated BV-2 to PC12 cells,the mechanism of which is partly via p53 activation.