目的 观察姜黄素对过氧化氢(H2O2)诱导少突胶质细胞(OL)前体细胞损伤作用的保护作用,并探讨其作用机制.方法 (1)体外原代培养OL前体细胞并加入0、100、250、500μmol/L H2O2作用30 min,Hoechst33342/PI双染观察细胞凋亡和死亡情况;(2)将细胞分为正常对照组、模型组及5、10、20 μmol/L姜黄素组,后3组细胞分别加入5、10、20 μmol/L姜黄素孵育1h,后4组细胞均加入100 μmol/L H2O2作用30 min后,MTT法检测各组细胞存活率;(3)将细胞分为正常对照组、模型组及10 μmol/L姜黄素组,进行相应处理后用流式细胞术检测细胞凋亡,分光光度法测定氧化/抗氧化物质含量,Western blotting法检测B细胞淋巴瘤基因-2、Bcl-2相关X蛋白和半胱天冬蛋白酶(caspase)-3、caspase-9蛋白的表达.结果 (1)与正常对照组比较,100、250、500μmol/L H2O2组正常细胞减少,凋亡和坏死细胞均增多,差异有统计学意义(P＜0.05).100μmol/LH2O2组凋亡细胞多于坏死细胞,250、500 μmol/LH2O2组坏死细胞多于凋亡细胞;(2)MTT检测显示5、10和20 μmol/L姜黄素组细胞存活率高于模型组,且10和20μmol/L姜黄素组高于5μmol/L姜黄素组,差异均有统计学意义(P＜0.05);(3)与模型组比较,10 μmol/L姜黄素组凋亡和坏死细胞明显减少,且细胞总超氧化物歧化酶、谷胱甘肽过氧化物酶、过氧化氢酶的活性增加,谷胱甘肽含量升高、丙二醛含量降低,Bcl-2表达升高、Bax、caspase-3、9的表达降低,差异均有统计学意义(P＜0.05).结论 姜黄素对氧化应激损伤的OL前体细胞具有保护作用,其机制可能与减轻OL前体的脂质过氧化、调节Bcl-2/Bax的表达以及抑制caspase-3、9的活化有关.%Objective To study the protective effect of curcumin on precurosor oligodendrocytes (preOLs) damage induced by hydrogen peroxide (H2O2),and explore its mechanism.Methods (1) In vitro primary culture ofpreOLs was performed; and 0 (normal controls),100,250 and 500 μmol/L H2O2 were added for 30 min; the apoptosis and death of preOLs were observed by Hoechst33342/PI double staining.(2) The preOLs were divided into normal control group,model group,and 5,10 and 20 μmol/L curcumin treatment groups; cells the later three groups were given 5,10 and 20 μmol/L curcumin for one h,and the later four groups were given 100 μmol/L H2O2 for 30 min; MTT assay was,then,employed to detect the cell viability.(3) The preOLs were divided into normal control group,model group,and 10 μmol/L curcumin treatment group; the apoptosis ofpreOLs was detected by Annexin V/FITC flow cytometry; Western blotting was used to detect the protein expressions of B cell lymphoma/leukemia-2 (Bcl2),Bcl-2 associated X protein (Bax) and cleaved caspase-3 and caspase-9; activities of total superoxide dismutase (T-SOD),glutathione peroxidase (GPx) and catalase (CAT) and malondialdehyde (MDA) were detected by spectrophotometry.Results (1) As compared with those in the normal control group,significantly decreased number of normal healthy cells and statistically increased apoptotic and necrotic cells in the 100,250 and 500 μmol/L H2O2 treatment groups were noted (P＜0.05); 100 μmol/L H2O2 treatment group had larger number of apoptotic cells than that of necrotic cells,while 250 and 500 μmol/L H2O2 treatment groups had larger number of necrotic cells than that of apoptotic cells.(2) Cells from 5,10 and 20 μmol/L curcumin treatment groups had significantly higher preOLs viability than those from model group,and 10 and 20 μmol/L curcumin treatment groups had significantly higher preOLs viability than those from 5 μmol/L curcumin treatment group (P＜0.05).(3) As compared with the model group,the 10 μmol/L curcumin treatment group had obviously decreased apoptotic and necrotic cells,increased activities of T-SOD,GPx and CAT,increased GSH level and decreased MDA concentration,up-regulated Bcl-2 expression,and inhibited Bax,caspase-3 and caspase-9 expressions,with significant differences (P＜0.05).Conclusion Curcumin has protective effect on preOLs against oxidative injury through effectively reducing lipid peroxidation,regulating Bcl-2/Bax expression and suppressing caspase-3 and caspase-9 activation.
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