吡咯并喹啉醌对氧化应激条件下大鼠骨髓间充质干细胞线粒体功能和细胞存活的影响及机制

Effects and mechanism of pyrroloquinoline quinine on mitochondrial function and cell survival of rat bone marrow mesenchymal stem cells under oxidative stress

目的:观察吡咯并喹啉醌(PQQ)在氧化应激条件下对大鼠骨髓间充质干细胞(BMSC)线粒体功能和细胞存活的影响,并探讨其作用机制。方法:体外用含体积分数10%胎牛血清的DMEM/F12培养基(下称正常培养基)培养大鼠BMSC,选取对数生长期的第3~5代细胞进行实验。(1)取细胞,分为正常对照组、正常对照+PQQ组、单纯过氧化氢组、过氧化氢+PQQ组。正常对照组细胞用正常培养基培养24 h;正常对照+PQQ组细胞用含终物质的量浓度为100μmol/L PQQ的正常培养基培养24 h;单纯过氧化氢组细胞用含有终物质的量浓度为200μmol/L过氧化氢的正常培养基培养24 h;过氧化氢+PQQ组细胞用含有终物质的量浓度为100μmol/L PQQ的正常培养基培养2 h后,加入终物质的量浓度为200μmol/L的过氧化氢培养24 h。于倒置相差显微镜下观察各组细胞形态,采用细胞计数试剂盒8法检测细胞存活率。(2)取5个批次细胞,均分为正常对照组、单纯过氧化氢组、过氧化氢+PQQ组,各组细胞处理同实验(1)中相应各组。培养24 h后,采用流式细胞术对1个批次细胞进行细胞凋亡检测,计算细胞凋亡率;采用JC-1线粒体膜电位检测试剂盒和倒置相差荧光显微镜对1个批次细胞分别进行线粒体膜电位检测和JC-1荧光染色观察;采用透射电镜观察1个批次细胞线粒体形态;采用过氧化氢酶(CAT)和超氧化物歧化酶(SOD)活性测定试剂盒对1个批次细胞分别进行CAT和SOD活性检测;采用蛋白质印迹法对1个批次细胞环磷酸腺苷活化交换蛋白1(Epac1)、腺苷酸活化蛋白激酶(AMPK)、磷酸化AMPK、剪切型半胱氨酸天冬氨酸蛋白酶3(caspase-3)、caspase-3蛋白表达进行检测,计算AMPK磷酸化水平、剪切型caspase-3/caspase-3比值。除形态观察外,其余各指标每组样本数均为6。对数据行单因素方差分析、独立样本等方差t检验。结果:(1)培养24 h后,单纯过氧化氢组细胞出现空泡且数量较正常对照组减少,细胞存活率为(74.3±2.9)%,较正常对照组的100.0%明显降低(t=6.39,P<0.01);与单纯过氧化氢组比较,过氧化氢+PQQ组细胞形态明显改善,细胞存活率明显升高至(116.9±4.2)%(t=6.92,P<0.01);正常对照+PQQ组细胞存活率为(101.2±1.1)%,与正常对照组相近(t=1.06,P>0.05)。(2)培养24 h后,与正常对照组的(13.6±1.0)%比较,单纯过氧化氢组细胞凋亡率明显增加[(37.1±2.0)%,t=10.57,P<0.01];与单纯过氧化氢组比较,过氧化氢+PQQ组细胞凋亡率明显降低[(17.0±0.7)%,t=9.49,P<0.01]。(3)培养24 h后,与正常对照组比较,单纯过氧化氢组细胞线粒体膜电位出现去极化,JC-1荧光染料主要以单体形式存在于细胞质中,发出绿色荧光,表现为膜电位明显降低(t=4.18,P<0.01);与单纯过氧化氢组比较,过氧化氢+PQQ组细胞线粒体膜电位升高至正常水平(t=4.43,P<0.01),JC-1荧光染料顺着极化线粒体膜电位进入线粒体聚集,发出红色荧光。(4)培养24 h后,与正常对照组的规则形态比较,单纯过氧化氢组细胞线粒体结构紊乱,线粒体嵴消失,线粒体基质密度降低;与单纯过氧化氢组比较,过氧化氢+PQQ组细胞线粒体结构规则完整,线粒体嵴清晰可见,线粒体基质密度增加。(5)培养24 h后,与正常对照组比较,单纯过氧化氢组细胞CAT活性明显升高(t=4.54,P<0.05),SOD活性明显降低(t=3.93,P<0.05);与单纯过氧化氢组比较,过氧化氢+PQQ组细胞CAT活性明显上升(t=8.65,P<0.01),SOD活性无明显变化(t=0.72,P>0.05)。(6)培养24 h后,与正常对照组比较,单纯过氧化氢组细胞Epac1蛋白表达明显降低(t=4.67,P<0.01),AMPK磷酸化水平、剪切型caspase-3/caspase-3比值明显升高(t=7.88、3.62,P<0.01);与单纯过氧化氢组比较,过氧化氢+PQQ组细胞Epac1蛋白表达、AMPK磷酸化水平均明显升高(t=4.34、16.37,P<0.01),剪切型caspase-3/caspase-3比值明显降低(t=3.17,P<0.05)。结论:PQQ预处理能够改善氧化应激条件下大鼠BMSC的线粒体功能,降低细胞凋亡率,提高细胞存活率,且可能与Epac1蛋白表达上调、AMPK信号通路激活和剪切型caspase-3蛋白水平降低有关。

Objective To observe the effects of pyrroloquinoline quinine(PQQ)on the mitochondrial function and cell survival of rat bone marrow mesenchymal stem cells(BMSCs)under oxidative stress,and to explore its mechanism.Methods BMSCs of rats were cultured in vitro with Dulbecco′s minimum essential medium/F12 medium containing fetal bovine serum in the volume fraction of 10%(hereinafter referred to as normal medium).The rat BMSCs of third to fifth passages in logarithmic growth phase were selected for the following experiments.(1)The cells were divided into normal control group,normal control+PQQ group,hydrogen peroxide(H2O2)alone group,and H2O2+PQQ group.The cells in normal control group were cultured in normal medium for 24 hours;the cells in normal control+PQQ group were cultured in normal medium containing 100μmol/L PQQ for 24 hours;the cells in H2O2 alone group were cultured in normal medium containing 200μmol/L H2O2 for 24 hours;the cells in H2O2+PQQ group were pre-incubated with normal medium containing 100μmol/L PQQ for 2 hours,and then with H2O2 added to the concentration of 200μmol/L and cultured for 24 hours.The cell morphology of each group was observed under the inverted phase contrast microscope,and the cell survival rate was detected by cell count kit 8 method.(2)Five batches of cells were collected,and the cells of each batch were divided into normal control group,H2O2 alone group,and H2O2+PQQ group.The cells in each group received the same treatment as that in the corresponding group of experiment(1).After 24 hours of culture,one batch of cells was collected for apoptosis detection by flow cytometry,and the apoptosis rate was calculated.One batch of cells was subjected to mitochondrial membrane potential assay and JC-1 fluorescent staining observation using the JC-1 mitochondrial membrane potential detection kit and the inverted phase contrast fluorescence microscope,respectively.One batch of cells was collected for mitochondrial morphology observation under the transmission electron microscope.One batch of cells was subjected to catalase(CAT)and superoxide dismutase(SOD)activity assay by CAT activity assay kit and SOD activity assay kit,respectively.One batch of cells was subjected to Western blotting for determination of protein level of Epac1,adenine monophosphate activated protein kinase(AMPK),phosphorylated AMPK,cysteinyl aspartate-specific proteinase 3(caspase-3),and cleaved caspase-3,and the phosphorylation level of AMPK and cleaved caspase-3/caspase-3 ratio were calculated.Six replicates were measured in each group for each index except for morphological observation.Data were statistically analyzed with one-way analysis of variance and independent sample equal variance t test.Results(1)After 24 hours of culture,compared with those in normal control group(the cell survival rate was set to 100.0%),there was an increase in cell vacuole and a decrease in cell number in H2O2 alone group,and the cell survival rate was significantly reduced to(74.3±2.9)%(t=6.39,P<0.01).Compared with those in H2O2 alone group,the cell morphology of H2O2+PQQ group was significantly improved,and the cell survival rate was significantly increased to(116.9±4.2)%(t=6.92,P<0.01);the cell survival rate in normal control+PQQ group was(101.2±1.1)%,close to that of control group(t=1.06,P>0.05).(2)After 24 hours of culture,compared with(13.6±1.0)%in normal control group,the apoptosis rate of cells in H2O2 alone group was significantly increased to(37.1±2.0)%(t=10.57,P<0.01).Compared with that in H2O2 alone group,the apoptosis rate of cells in H2O2+PQQ group was significantly declined to(17.0±0.7)%(t=9.49,P<0.01).(3)After 24 hours of culture,compared with those in normal control group,the mitochondrial membrane potential of cells in H2O2 alone group was depolarized,the JC-1 fluorescent dye mainly existed in the cytoplasm in the form of monomer,which emitted green fluorescence,and a significant decrease in mitochondrial membrane potential was shown(t=4.18,P<0.01).Compared with those in H2O2 alone group,the mitochondrial membrane potential of cells in H2O2+PQQ group was increased to normal level(t=4.43,P<0.01),and the JC-1 fluorescent dye accumulated in mitochondria following the polarized mitochondrial membrane potential and emitted red fluorescence.(4)After 24 hours of culture,compared with that in normal control group,the mitochondrial structure of cells in H2O2 alone group was disordered,with disappeared mitochondrial cristae and decreased mitochondrial matrix density.Compared with that in H2O2 alone group,the mitochondrial structure of cells in H2O2+PQQ group was regular and intact,with clearly visible mitochondrial cristae and increased mitochondrial matrix density.(5)After 24 hours of culture,compared with those in normal control group,the CAT activity of cells in H2O2 alone group was significantly increased(t=4.54,P<0.05),and the SOD activity was significantly decreased(t=3.93,P<0.05).Compared with those in H2O2 alone group,the CAT activity of cells in H2O2+PQQ group was obviously increased(t=8.65,P<0.01),while there was no significant change in the SOD activity(t=0.72,P>0.05).(6)After 24 hours of culture,compared with those in normal control group,the protein expression of Epac1 of cells in H2O2 alone group was significantly decreased(t=4.67,P<0.01),while the AMPK phosphorylation level and the cleaved caspase-3/caspase-3 ratio were significantly increased(t=7.88,3.62,P<0.01).Compared with those in H2O2 alone group,the protein expression of Epac1 and the AMPK phosphorylation level of cells in H2O2+PQQ group were both significantly increased(t=4.34,16.37,P<0.01),while the cleaved caspase-3/caspase-3 ratio was significantly declined(t=3.17,P<0.05).Conclusions Pretreatment with PQQ can improve the mitochondrial function,reduce cell apoptosis rate,and enhance cell survival rate of rat BMSCs under oxidative stress,which may be related to the up-regulation of Epac1 protein expression,activation of AMPK signaling pathway,and down-regulation of cleaved caspase-3 protein level.