Effects and mechanism of hydrogen peroxide pretreatment with low molarity on oxidative stress induced apoptosis of mouse bone marrow mesenchymal stem cells
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摘要:
目的 探讨低物质的量浓度过氧化氢预处理对小鼠骨髓间充质干细胞（BMSC）氧化应激性凋亡的作用及其机制。 方法 采用实验研究方法。通过全骨髓培养法从2只2周龄雄性BALB/c小鼠中分离培养BMSC，鉴定后取第3~7代对数生长期细胞进行实验。将细胞按随机数字表法（分组方法下同）分为用相应终物质的量浓度过氧化氢处理的0 μmol/L过氧化氢组（即不加入过氧化氢，下同）、25 μmol/L过氧化氢组、50 μmol/L过氧化氢组、100 μmol/L过氧化氢组、150 μmol/L过氧化氢组、200 μmol/L过氧化氢组、250 μmol/L过氧化氢组、300 μmol/L过氧化氢组，培养24 h后，用流式细胞术检测细胞凋亡率（样本数为4）。将细胞分为用相应终物质的量浓度过氧化氢处理的0 μmol/L过氧化氢组、25 µmol/L过氧化氢组、50 µmol/L过氧化氢组、100 µmol/L过氧化氢组，培养24 h后，用蛋白质印迹法检测B淋巴细胞瘤-2（Bcl-2）、Bcl-2相关X蛋白（Bax）的蛋白表达，计算Bcl-2/Bax比值（样本数为3）。将细胞分为用相应终物质的量浓度过氧化氢处理的0 μmol/L过氧化氢组、25 μmol/L过氧化氢组、50 μmol/L过氧化氢组、100 μmol/L过氧化氢组、200 μmol/L过氧化氢组、300 μmol/L过氧化氢组，培养24 h后，用蛋白质印迹法检测糖原合成酶激酶-3β（GSK-3β）及磷酸化GSK-3β（p-GSK-3β）的蛋白表达（样本数为3）。将细胞分为用相应终物质的量浓度过氧化氢处理的0 μmol/L过氧化氢组、50 μmol/L过氧化氢组、300 μmol/L过氧化氢组，以及预先加入50 μmol/L过氧化氢刺激12 h再加入300 μmol/L过氧化氢的过氧化氢预处理组，培养24 h后，用倒置荧光显微镜（非荧光条件）和免疫荧光法观察细胞形态和生长情况，用流式细胞术检测细胞凋亡率，用蛋白质印迹法检测Bcl-2、Bax、胱天蛋白酶3（caspase-3）、caspase-9、剪切型caspase-3、剪切型caspase-9、GSK-3β及p-GSK-3β蛋白表达，计算Bcl-2/Bax比值，样本数均为3。对数据进行单因素方差分析、Bonferroni检验。 结果 培养24 h后，与0 μmol/L过氧化氢组比较，25 μmol/L过氧化氢组、50 μmol/L过氧化氢组、100 μmol/L过氧化氢组细胞的凋亡率均无明显变化（P>0.05），150 μmol/L过氧化氢组、200 μmol/L过氧化氢组、250 μmol/L过氧化氢组、300 μmol/L过氧化氢组细胞凋亡率均明显上升（P<0.01）。培养24 h后，与0 μmol/L过氧化氢组比较，25 µmol/L过氧化氢组、50 μmol/L过氧化氢组细胞Bcl-2/Bax比值均明显增加（P<0.05或P<0.01），100 µmol/L过氧化氢组细胞Bcl-2/Bax比值显著减少（P<0.05）。培养24 h后，与0 μmol/L过氧化氢组比较，25 μmol/L过氧化氢组、50 μmol/L过氧化氢组细胞GSK-3β蛋白表达均无明显变化（P>0.05），p-GSK-3β蛋白表达均显著增加（P<0.01）；100 μmol/L过氧化氢组细胞GSK-3β、p-GSK-3β蛋白表达均无明显变化（P>0.05）；200 μmol/L过氧化氢组、300 μmol/L过氧化氢组细胞GSK-3β蛋白表达均明显增加（P<0.05），p-GSK-3β蛋白表达均显著减少（P<0.05）。培养24 h后，0 μmol/L过氧化氢组与50 μmol/L过氧化氢组细胞形态及生长情况相近，均正常，与之相比，300 µmol/L过氧化氢组细胞变小、变圆，细胞突起变短或消失，细胞核出现空化，细胞脱落明显增多；过氧化氢预处理组大部分细胞形态正常，细胞脱落少于300 µmol/L过氧化氢组，细胞核形态正常。培养24 h后，4组间细胞caspase-9蛋白表达相近（P>0.05）。与0 μmol/L过氧化氢组比较，50 μmol/L过氧化氢组细胞凋亡率以及剪切型caspase-9、caspase-3、剪切型caspase-3的蛋白表达均无明显变化（P>0.05），Bcl-2/Bax比值明显增加（P<0.05）；300 μmol/L过氧化氢组细胞凋亡率显著升高（P<0.01），剪切型caspase-9、caspase-3、剪切型caspase-3蛋白表达均明显增加（P<0.01），Bcl-2/Bax比值明显减少（P<0.05）。与300 μmol/L过氧化氢组比较，过氧化氢预处理组细胞凋亡率显著下降（P<0.01），剪切型caspase-9、caspase-3、剪切型caspase-3的蛋白表达均明显减少（P<0.05或P<0.01），Bcl-2/Bax比值显著增加（P<0.01）。培养24 h后，0 μmol/L过氧化氢组、50 μmol/L过氧化氢组、300 μmol/L 过氧化氢组、过氧化氢预处理组细胞GSK-3β、p-GSK-3β蛋白表达分别为1.09±0.14、0.62±0.17、1.35±0.21、0.74±0.34，0.68±0.03、0.85±0.08、0.38±0.10、0.54±0.09。与0 μmol/L过氧化氢组比较，50 μmol/L过氧化氢组细胞p-GSK-3β蛋白表达明显增加（P<0.05）；300 μmol/L过氧化氢组细胞GSK-3β蛋白表达明显增加（P<0.05），p-GSK-3β蛋白表达明显减少（P<0.01）。与300 μmol/L过氧化氢组比较，过氧化氢预处理组细胞GSK-3β蛋白表达明显减少（P<0.01），p-GSK-3β蛋白表达明显增加（P<0.01）。 结论 50 μmol/L可能是过氧化氢预处理小鼠BMSC的最佳物质的量浓度，50 μmol/L过氧化氢预处理通过抑制GSK-3β活性对抗线粒体途经的氧化应激性凋亡。 Abstract:Objective To investigate the effects and mechanism of hydrogen peroxide (HP) pretreatment with low molarity on oxidative stress induced apoptosis of mouse bone marrow mesenchymal stem cells (BMSCs). Methods The experimental research methods were used. BMSCs were isolated and cultured from two 2-week-old male BALB/c mice by the whole bone marrow culture method. The 3rd-7th passages of cells in logarithmic growth phase were used for the experiments after identification. According to the random number table (the same grouping method below), the cells were divided into 0 μmol/L HP group (without HP, the same below), 25 μmol/L HP group, 50 μmol/L HP group, 100 μmol/L HP group, 150 μmol/L HP group, 200 μmol/L HP group, 250 μmol/L HP group, and 300 μmol/L HP group in which cells were treated by the corresponding final molarity of HP, respectively. The apoptosis rate was detected by flow cytometry (n=4) after 24 hours of culture. The cells were divided into 0 μmol/L HP group, 25 μmol/L HP group, 50 μmol/L HP group, and 100 μmol/L HP group in which cells were treated by the corresponding final molarity of HP, respeclively. After 24 hours of culture, the protein expressions of B-lymphoma-2 (Bcl-2) and Bcl-2-related X protein (Bax) were detected by Western blotting, and the Bcl-2/Bax ratio was calculated (n=3). The cells were divided into 0 μmol/L HP group, 25 μmol/L HP group, 50 μmol/L HP group, 100 μmol/L HP group, 200 μmol/L HP group, and 300 μmol/L HP group in which cells were treated by the corresponding final molarity of HP, respectively. After 24 hours of culture, the protein expressions of glycogen synthase kinase-3β (GSK-3β) and phosphorylated GSK-3β (p-GSK-3β) were detected by Western blotting (n=3). The cells were divided into 0 μmol/L HP group, 50 μmol/L HP group, and 300 μmol/L HP group in which cells were treated by the corresponding final molarity of HP, respeclively, and HP pretreatment group with 50 μmol/L HP being added in advance for 12 h and then 300 μmol/L HP being added. After 24 hours of culture, the morphology and growth of cells were observed by inverted fluorescence microscopy (non-fluorescent condition) and immunofluorescence method, the apoptosis rate was detected by flow cytometry, the protein expressions of Bcl-2, Bax, cysteine aspartic acid specific protease-3 (caspase-3), caspase-9, cleavage caspase-3, cleavage caspase-9, GSK-3β, and p-GSK-3β were detected by Western blotting, and the Bcl-2/Bax ratio was calculated, with all the number of samples being 3. Data were statistically analyzed with one-way analysis of variance and Bonferroni test. Results After 24 hours of culture, compared with that in 0 μmol/L HP group, the apoptosis rate of cells did not change significantly in 25 μmol/L HP group, 50 μmol/L HP group, or 100 μmol/L HP group (P>0.05) but increased significantly in 150 μmol/L HP group, 200 μmol/L HP group, 250 μmol/L HP group, and 300 μmol/L HP group (P<0.01). After 24 hours of culture, compared with that in 0 μmol/L HP group, the Bcl-2/Bax ratio of cells increased significantly in 25 μmol/L HP group and 50 μmol/L HP group (P<0.05 or P<0.01) but decreased significantly in 100 µmol/L HP group (P<0.05). After 24 hours of culture, compared with those in 0 μmol/L HP group, the protein expression of GSK-3β in cells showed no significant change in 25 μmol/L HP group and 50 μmol/L HP group (P>0.05), the protein expressions of p-GSK-3β in cells significantly increased in 25 μmol/L HP group and 50 μmol/L HP group (P<0.01), the protein expressions of GSK-3β and p-GSK-3β in cells in 100 μmol/L HP group showed no significant change (P>0.05), the protein expressions of GSK-3β in cells in 200 μmol/L HP group and 300 μmol/L HP group were significantly increased (P<0.05). but the protein expression of p-GSK-3β in cells in 200 μmol/L HP group and 300 μmol/L HP group was significantly decreased (P<0.05). After 24 hours of culture, the morphology and growth of cells in 0 μmol/L HP group and 50 μmol/L HP group were similar and normal; in contrast, the cells in 300 µmol/L HP group became smaller and round, with the cell protrusions being shorter or disappeared, the nucleus being cavitated, and the cell abscission being increased significantly; the morphology of most cells in HP pretreatment group was normal, with the shedding of cells being less than that in 300 µmol/L HP group, and the morphology of nucleus being normal. After 24 hours of culture, the protein expression of caspase-9 was similar among the four groups (P>0.05). Compared with that in 0 μmol/L HP group, the apoptosis rate and the protein expressions of cleavage caspase-9, caspase-3, and cleavage caspase-3 of cells in 50 μmol/L HP group showed no significant changes (P>0.05), the Bcl-2/Bax ratio of cells in 50 μmol/L HP group increased significantly (P<0.05), the apoptosis rate and the protein expressions of cleavage caspase-9, caspase-3, and cleavage caspase-3 of cells in 300 μmol/L HP group were significantly increased (P<0.01), while the Bcl-2/Bax ratio of cells in 300 μmol/L HP group was significantly decreased (P<0.05). Compared with those in 300 μmol/L HP group, the apoptosis rate and the protein expressions of cleavage caspase-9, caspase-3, and cleavage caspase-3 of cells were significantly decreased in HP pretreatment group (P<0.05 or P<0.01), while the Bcl-2/Bax ratio of cells was significantly increased in HP pretreatment group (P<0.01). After 24 hours of culture, the protein expressions of GSK-3β and p-GSK-3β of cells in 0 μmol/L HP group, 50 μmol/L HP group, 300 μmol/L HP group, and HP pretreatment group were 1.09±0.14, 0.62±0.17, 1.35±0.21, 0.74±0.34, 0.68±0.03, 0.85±0.08, 0.38±0.10, and 0.54±0.09, respectively. Compared with those in 0 μmol/L HP group, the protein expression of p-GSK-3β of cells was significantly increased in 50 μmol/L HP group (P<0.05) but significantly decreased in 300 μmol/L HP group (P<0.01), while the protein expression of GSK-3β of cells was significantly increased in 300 μmol/L HP group (P<0.05). Compared with those in 300 μmol/L HP group, the protein expression of GSK-3β of cells was significantly decreased in HP pretreatment group (P<0.01), while the protein expression of p-GSK-3β of cells was significantly increased in HP pretreatment group (P<0.01). Conclusions The molarity of 50 μmol/L may be the optimal molarity of HP to pretreat mouse BMSCs, and 50 μmol/L HP pretreatment can antagonize mitochondrial pathway of oxidative stress induced apoptosis by inhibiting the activity of GSK-3β. -
参考文献
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1 8个物质的量浓度过氧化氢组小鼠骨髓间充质干细胞培养24 h后细胞凋亡率比较（样本数为4，
注：横坐标下1、2、3、4、5、6、7、8分别指0 μmol/L过氧化氢组、25 µmol/L过氧化氢组、50 µmol/L过氧化氢组、100 µmol/L过氧化氢组、150 µmol/L过氧化氢组、200 µmol/L 过氧化氢组、250 µmol/L过氧化氢组、300 µmol/L过氧化氢组；与0 μmol/L过氧化氢组比较，aP<0.01
2 蛋白质印迹法检测4个物质的量浓度过氧化氢组小鼠骨髓间充质干细胞培养24 h后Bcl-2与Bax蛋白表达。2A.条带图；2B.Bcl-2/Bax比值条图（样本数为3，
注：Bcl-2为B淋巴细胞瘤-2，Bax为Bcl-2相关X蛋白，条带图上方和条图横坐标下1、2、3、4均分别指0 μmol/L过氧化氢组、25 µmol/L过氧化氢组、50 µmol/L过氧化氢组、100 µmol/L过氧化氢组；与0 μmol/L过氧化氢组比较，aP<0.05，bP<0.01
3 蛋白质印迹法检测6个物质的量浓度过氧化氢组小鼠骨髓间充质干细胞培养24 h后GSK-3β与p-GSK-3β蛋白表达。3A.条带图；3B.条图（样本数为3，
注：p-GSK-3β为磷酸化糖原合成酶激酶-3β，GSK-3β为糖原合成酶激酶-3β，条带图上方和条图横坐标下1、2、3、4、5、6均分别指0 μmol/L过氧化氢组、25 µmol/L过氧化氢组、50 µmol/L过氧化氢组、100 µmol/L过氧化氢组、200 µmol/L过氧化氢组、300 µmol/L过氧化氢组；与0 μmol/L过氧化氢组比较，aP<0.01，bP<0.05
4 3个物质的量浓度过氧化氢组与过氧化氢预处理组小鼠骨髓间充质干细胞培养24 h后形态和生长 倒置荧光显微镜×400，图中标尺为20 µm。4A.0 μmol/L过氧化氢组，细胞呈短梭形或圆形；4B.50 µmol/L 过氧化氢组，细胞形态与图4A相近；4C.300 µmol/L过氧化氢组，细胞脱落明显，与图4A比较，细胞数量减少，细胞体积变小，细胞突起变短或消失；4D.过氧化氢预处理组，细胞脱落较图4C减少，大部分细胞形态正常；4E.0 μmol/L过氧化氢组，细胞核形态正常，大小均匀；4F.50 μmol/L过氧化氢组，细胞核形态与图4E相似；4G.300 μmol/L过氧化氢组，染色质从细胞核流入细胞质内使得细胞质蓝染，细胞核出现空化;4H.过氧化氢预处理组，细胞核与图4E相似，未见细胞质蓝染
注：过氧化氢预处理组细胞先加入终物质的量浓度50 μmol/L过氧化氢培养12 h后，再加入终物质的量浓度为300 μmol/L过氧化氢培养；图4A~4D为非荧光条件下观察；图4E~4H为荧光染色后观察，细胞核染色剂为4',6-二脒基-2-苯基吲哚
5 蛋白质印迹法检测3个物质的量浓度过氧化氢组与过氧化氢预处理组小鼠骨髓间充质干细胞培养24 h后凋亡相关蛋白的表达。5A.Bcl-2与Bax条带图；5B.Bcl-2/Bax比值条图（样本数为3，
注：过氧化氢预处理组细胞先加入终物质的量浓度50 μmol/L过氧化氢培养12 h后，再加入终物质的量浓度为300 μmol/L过氧化氢培养；Bcl-2为B淋巴细胞瘤-2，Bax为Bcl-2相关X蛋白，caspase为胱天蛋白酶，条带图的上方和条图横坐标下1、2、3、4均分别指0 μmol/L过氧化氢组、50 µmol/L 过氧化氢组、300 µmol/L过氧化氢组、过氧化氢预处理组；图5B中，与0 μmol/L过氧化氢组比较，aP<0.05；与300 μmol/L 过氧化氢组比较，bP<0.01；图5D中，与0 μmol/L过氧化氢组比较，aP<0.01；与300 μmol/L 过氧化氢组比较，bP<0.01,cP<0.05
6 蛋白质印迹法检测3个物质的量浓度过氧化氢组与过氧化氢预处理组小鼠骨髓间充质干细胞培养24 h后p-GSK-3β与GSK-3β蛋白表达。6A.条带图；6B.条图（样本数为3，
注：过氧化氢预处理组细胞先加入终物质的量浓度50 μmol/L过氧化氢培养12 h后，再加入终物质的量浓度为300 μmol/L过氧化氢培养；p-GSK-3β为磷酸化糖原合成酶激酶-3β，GSK-3β为糖原合成酶激酶-3β，条带图上方和条图横坐标下1、2、3、4均分别指0 μmol/L过氧化氢组、50 µmol/L 过氧化氢组、300 µmol/L过氧化氢组、过氧化氢预处理组；与0 μmol/L过氧化氢组比较，aP<0.05，bP<0.01；与300 μmol/L过氧化氢组比较，cP<0.01
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