瑞芬太尼对BV⁃2小胶质细胞沉默调节蛋白2及炎症因子的影响

Effects of remifentanil on silent information regulator 2 protein and inflammatory factors in BV‑2 microglia

目的探讨瑞芬太尼对BV⁃2小胶质细胞沉默调节蛋白2(silent information regulator 2,SIRT2)及炎症因子的影响。方法将培养的BV⁃2小胶质细胞分为4组(每组1孔):空白对照组(C组)、瑞芬太尼10μmol/L组(R1组)、瑞芬太尼50μmol/L组(R2组)、瑞芬太尼100μmol/L组(R3组),待细胞基本贴满板底后,R1组、R2组、R3组分别将培养液更换为单纯DMEM/F12培养液配置的瑞芬太尼2 ml(R1组瑞芬太尼10μmol/L、R2组瑞芬太尼50μmol/L、R3组瑞芬太尼100μmol/L),孵育2 h。另取培养的BV⁃2小胶质细胞分为4组(每组1孔):空白对照2组(C2组)、15 min组(T1组)、1 h组(T2组)、2 h组(T3组),待细胞基本贴满板底后,T1组、T2组、T3组将培养液更换为单纯DMEM/F12培养液配置的100μmol/L瑞芬太尼2 ml(T1组刺激15 min、T2组刺激1 h、T3组刺激2 h)。Western blot法检测各组细胞SIRT2、离子钙接头蛋白1(ionized calcium binding adaptor molecule 1,Iba1)水平,ELISA法检测各组细胞TNF⁃α、IL⁃1β水平。结果与C组比较:R1组、R2组、R3组Iba1、IL⁃1β、TNF⁃α增加(P<0.05),SIRT2水平降低(P<0.05)。与R1组比较:R2组、R3组Iba1、IL⁃1β水平增加(P<0.05),R3组SIRT2水平降低、TNF⁃α水平增加(P<0.05)。与R2组比较:R3组SIRT2水平降低(P<0.05)。与C2组比较:T1组、T2组、T3组Iba1、IL⁃1β、TNF⁃α水平增加(P<0.05),SIRT2水平降低(P<0.05)。与T1组比较:T2组、T3组Iba1、IL⁃1β、TNF⁃α水平增加(P<0.05),SIRT2水平降低(P<0.05)。与T2组比较:T3组SIRT2水平降低(P<0.05),IL⁃1β、TNF⁃α水平增加(P<0.05)。结论瑞芬太尼可通过剂量依赖性和时间依赖性方式使BV⁃2小胶质细胞中的Iba1和炎症因子的表达显著增加,并显著抑制SIRT2表达,从而激活小胶质细胞。

Objective To investigate the effect of remifentanil on the expression of silent information regulator 2(SIRT2)and inflammatory factors in BV⁃2 microglia cells.Methods The cultured BV⁃2 microglia cells were divided into four groups(one well per group):a blank control group(group C),a remifentanil 10μmol/L group(group R1),a remifentanil 50μmol/L group(group R2)and a remifentanil 100μmol/L group(group R3).After the cells were grown to 70%-80%confluence,the culture media of group R1,group R2 and group R3 were replaced with 2 ml of remifentanil with DMEM/F12 culture medium(containing 10,50,100μmol/L of remifentanil,respectively),before incubation for 2 h.In addition,cultured BV⁃2 microglia cells were divided into four groups(one well per group):a blank control groups 2(group C2),a 15 min group(group T1),a 1 h group(group T2)and a 2 h group(group T3).After the cells were grown to 70%-80%confluence,the culture media of group T1,group T2 and group T3 was replaced with 2 ml of remifentanil at 100μmol/L with DMEM/F12 culture medium,followed by treatment for 15 min,1 h and 2 h,respectively.The levels of SIRT2 and ionized calcium binding adaptor molecule 1(Iba1)in each group were determined by Western blot,and the levels of tumor necrosis factor⁃α(TNF⁃α)and interleukin(IL)⁃1βwere determined by enzyme⁃linked immunosorbent assay(ELISA).Results Compared with group C,groups R1,R2 and R3 showed increased levels of Iba1,IL⁃1β,TNF⁃αincreased and decreased SIRT2 levels(P<0.05).Compared with group R1,the levels of Iba1 and IL⁃1βincreased in group R2 and group R3(P<0.05),while decreased SIRT2 levels and increased TNF⁃αlevels were seen in group R3(P<0.05).Compared with group R2,the levels of SIRT2 decreased in group R3(P<0.05).Compared with group C2,group T1,group T2 and group T3 showed increased levels of Iba1,IL⁃1βand TNF⁃α(P<0.05),and decreased SIRT2 levels(P<0.05).Compared with group T1,group T2 and group T3 presented increases in Iba1,IL⁃1βand TNF⁃αlevels(P<0.05),and decreases in SIRT2 levels(P<0.05).Compared with group T2,group T3 showed decreased levels of SIRT2(P<0.05),as well as increased levels of IL⁃1βand TNF⁃α(P<0.05).Conclusions Remifentanil can activate BV⁃2 microglia through significantly increasing the expression of Iba1 and inflammatory cytokines and inhibiting the expression of SIRT2 in a dose⁃dependent and time⁃dependent manner.