异丙酚对缺氧/复氧诱导PC12细胞损伤的影响及其分子机制

Effects of propofol on hypoxia/reoxygenation induced PC12 cell damage and its molecular mechanism

目的探讨异丙酚对缺氧/复氧(H/R)诱导PC12细胞损伤的影响及其分子机制。方法体外培养PC12细胞,将其分为control组(正常条件21%O_(2)/5%CO_(2)/74%N_(2)培养)、H/R组(缺氧条件3%O_(2)/5%CO_(2)/92%N_(2)培养2 h,正常条件下培养12 h)、H/R+异丙酚组(50μmol/L异丙酚预处理1 h后用H/R处理)、H/R+anti-miR-NC组(转染anti-miR-NC后用H/R处理)、H/R+anti-miR-1246组(转染anti-miR-1246后用H/R处理)、H/R+异丙酚+miR-NC组(转染miR-NC,再经50μmol/L异丙酚和H/R处理)、H/R+异丙酚+miR-1246(转染miR-1246,再经50μmol/L异丙酚和H/R处理);MTT和流式细胞术分别用于检测细胞活力和凋亡;Western blot分析凋亡相关蛋白表达[B淋巴细胞瘤-2(Bcl-2)、B淋巴细胞瘤-2关联x蛋白(Bax)];酶联免疫吸附试验检测促炎性细胞因子和抗炎细胞因子表达水平;定量逆转录聚合酶链反应检测miR-1246的表达水平。结果采用50μmol/L异丙酚处理时细胞存活率最高,故后续实验,异丙酚浓度选择50μmol/L。与control组比较,H/R组细胞凋亡率、Bax蛋白、肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-1β和IL-18水平升高,Bcl-2蛋白和IL-10水平降低(P<0.05)。与H/R组比较,H/R+异丙酚组细胞凋亡率、Bax蛋白、TNF-α、IL-1β和IL-18水平降低,Bcl-2蛋白和IL-10水平升高(P<0.05)。与control组比较,H/R组中miR-1246表达升高(P<0.05)。与H/R组比较,H/R+异丙酚组中miR-1246表达降低(P<0.05)。H/R组与H/R+anti-miR-NC组miR-1246水平、细胞凋亡率、Bcl-2蛋白、Bax蛋白、TNF-α、IL-1β、IL-18、IL-10水平比较,差异无统计学意义(P>0.05)。与H/R+anti-miR-NC组比较,H/R+anti-miR-1246组miR-1246水平、细胞凋亡率、Bax蛋白、TNF-α、IL-1β和IL-18水平降低,Bcl-2蛋白和IL-10水平升高(P<0.05)。与H/R+异丙酚+miR-NC组比较,H/R+异丙酚+miR-1246组miR-1246表达水平、细胞凋亡率、Bax蛋白及TNF-α、IL-1β、IL-18水平升高,Bcl-2蛋白和IL-10水平降低(P<0.05)。结论异丙酚通过下调miR-1246缓解H/R诱导的PC12细胞损伤。

Objective To investigate the effect of propofol on hypoxia/reoxygenation(H/R)induced PC12 cell damage and its molecular mechanism.Methods PC12 cells were cultured in vitro and divided into control group(21%O_(2)/5%CO_(2)/74%N_(2) culture under normal conditions)and H/R group(3%O_(2)/5%CO_(2)/92%N_(2) culture under hypoxia condition for 2 h,cultured for 12 h under normal conditions),H/R+propofol group(50μmol/L propofol pretreatment for 1 h and then treated with H/R),H/R+anti-miR-NC group(transfected with anti-miR-NC and then treated with H/R),H/R+anti-miR-1246 group(transfected with anti-miR-1246 and then treated with H/R),H/R+propofol+miR-NC group(transfected miR-NC,and then treated with 50μmol/L propofol and H/R),H/R+propofol+miR-1246(transfected with miR-1246 and then treated with 50μmol/L propofol and H/R).MTT and flow cytometry were used to detect cell viability and apoptosis,respectively.Western blot analysis of apoptosis-related protein expression(B lymphoblastoma-2[Bcl-2],B lymphoblastoma-2 associated with x protein[Bax]).The expression levels of pro-inflammatory cytokines and anti-inflammatory cytokines were detected by enzyme-linked immunosorbent assay.The expression level of miR-1246 was detected by quantitative reverse transcription polymerase chain reaction.Results The survival rate of cells was the highest when 50μmol/L propofol was applied,so the concentration of propofol was 50μmol/L in subsequent experiments.Compared with control group,the apoptosis rate,levels of Bax protein,tumor necrosis factor-α(TNF-α),interleukin(IL)-1β,and IL-18 in H/R group were increased,while the levels of Bcl-2 protein and IL-10 were decreased(P<0.05).Compared with H/R group,the apoptosis rate,levels of Bax protein,TNF-α,IL-1β,and IL-18 were decreased in H/R+propofol group,while the levels of Bcl-2 protein and IL-10 were increased(P<0.05).Compared with control group,the expression of miR-1246 in H/R group was increased(P<0.05).Compared with H/R group,the expression of miR-1246 in H/R+propofol group was decreased(P<0.05).There were no significant differences in miR-1246 level,apoptosis rate,Bcl-2 protein,Bax protein,TNF-α,IL-1β,IL-18,and IL-10 between H/R group and H/R+anti-miR-NC group(P>0.05).Compared with H/R+anti-miR-NC group,the levels of miR-1246,apoptosis rate,Bax protein,TNF-α,IL-1β,and IL-18 were decreased in H/R+anti-miR-1246 group,while the levels of Bcl-2 protein and IL-10 were increased(P<0.05).Compared with H/R+propofol+miR-NC group,the expression level of miR-1246,apoptosis rate,Bax protein,TNF-α,IL-1β,and IL-18 levels were increased in H/R+propofol+miR-1246 group,while the levels of Bcl-2 protein and IL-10 were decreased(P<0.05).Conclusion Propofol can relieve H/R induced PC12 cell damage by down-regulating miR-1246.