摘要
目的评价亚甲蓝对过氧化氢(H2O2)诱导小鼠巨噬细胞线粒体途径凋亡的影响。方法小鼠腹腔巨噬细胞株RAW264.7培养于含10%胎牛血清的DMEM培养基,采用随机数字表法分为6组(n=24):对照组(C组)、H2O2组、不同浓度亚甲蓝预防性给药组(MB1,2组)和不同浓度亚甲蓝治疗性给药组(MB3.4组)。H2O2组在培养基中加入50ixmol/L H2O2;MB1,2组于加入H2O2前30min时、MB3,4组于加入H2O2,后30rain时在培养基中加入亚甲蓝,终浓度分别为0.1μmol/L(MB,组和MB3组)和1.0μmol/L(MB2组和MB4组)。于各组培养或孵育24h时,采用噻唑蓝比色法测定细胞存活率,采用DHE荧光探针法测定细胞ROS活性,采用分光光度计测定上清液LDH活性,采用比色法测定细胞SOD活性,采用罗丹明123染色法测定线粒体膜电位(MMP),采用ATP生物发光法测定ATP含量,采用Western blot法检测caspase-3前体及剪切体P20蛋白和P18蛋白表达,采用流式细胞仪检测细胞凋亡率。结果与C组比较,其余5组细胞存活率、SOD活性、MMP和ATP含量降低,ROS活性和上清液LDH活性升高,caspase-3前体及剪切体P20蛋白和P18蛋白表达上调,早期凋亡率和晚期凋亡率升高(P<0.05)。与H2O2组比较,MB1-4组细胞存活率、SOD活性、MMP和ATP含量升高,ROS活性和上清液LDH活性降低,caspase-3前体及剪切体P20蛋白和P18蛋白表达下调,早期凋亡率和晚期凋亡率降低(P<0.05)。与MB1组比较,MB2组细胞存活率降低,caspase-3剪切体P18蛋白表达下调,MB3组细胞存活率和SOD活性降低,ROS活性增高(P<0.05)。与MB4组比较,MB2组caspase-3剪切体P18蛋白表达下调,早期凋亡率和晚期凋亡率降低,ROS活性升高,MB3组ROS活性升高(P<0.05)。结论亚甲蓝减轻H2O2诱导小鼠巨噬细胞氧化损伤的机制与抑制线粒体途径凋亡有关。
Objective To evaluate the effect of methylene blue (MB)on hydrogen peroxide (H2O2)-induced apoptosis in macrophages through mitochondria-dependent pathway in mice.Methods Mouse peritoneal macrophage line RAW264.7cells were cultured in DMEM culture medium containing 10%fetal bovine serum.Cells were divided into 6groups (n =24each)using a random number table method: control group (group C),H2O2group,prophylactic different concentrations of MB groups (MB1.2groups) and therapeutic different concentrations of MB groups (MB3,4groups).H2O2 50μmol/L was added to the culture medium in group H202.MB was added to the culture medium with the final concentrations of 0.1 μmol/L (in MB1 and MB3groups)and 1.0μmol/L (in MB2and MB4groups)at 30min before adding H2P2in MB1,2groups and 30min after adding H2P2in MB3.4groups.At 24h of culture or incubation in each group,the cell survival rate was measured by methyl thiazolyl tetrazolium assay,the activity of reactive oxygen species (ROS)in cells was determined with the fluorescent probe,the lactate dehydrogenase (LDH)activity in supernatant was detected by spectrophotometry,the activity of superoxide dismutase (SOD)in cells was detected by colorimetric method,mitochondrial membrane potential (MMP)was measured using rhodamine 123staining,the content of ATP was determined by an ATP bioluminescent method, the expression of pro-caspase-3and spliceosomes P20protein and P18protein was detected by Western blot, and cell apoptosis was detected using flow cytometry.Results Compared with group C,the cell survival rate,SOD actiyity and contents of MMP and ATP were significantly decreased,the ROS activity and activity of LDH in supernatant were increased,the expression of pro-caspase-3and spliceosomes P20protein and P18protein was up-regulated,and early and late apoptosis rates were increased in the other five groups (P<0.05),Compared with group H2O2,the cell survival rate,SOD activity and contents of MMP and ATP were significantly increased,the ROS activity and activity of LDH in supernatant w
作者
豆立冬
曾思
盛琼
苑佳佳
张晓彤
庞庆丰
Dou Lidong;Zeng Si;Sheng Qiong;Yuan Jiajia;Zhang Xiaotong;Pang Qingfeng(Department of Anesthesiology,Henan Province People's Hospital,Zhengzhou 450003,China;Department of Anesthesiology,Sichuan Provincial People's Hospital,Chengdu 610072,China;Department of Pathophysiology,Wuxi Medical College of Jiangnan University,Wuxi 214122,China)
出处
《中华麻醉学杂志》
CAS
CSCD
北大核心
2018年第6期723-727,共5页
Chinese Journal of Anesthesiology
基金
国家自然科学基金(81270126)
中央高校基本科研业务费专项资金(JUSRP51412B)
四川省卫生和计划生育委员会科研项目(150234).
关键词
亚甲蓝
过氧化氢
线粒体
细胞凋亡
巨噬细胞
Methylene blue
Hydrogen peroxide
Mitochondria
Apoptosis
Macrophages
