α-酮戊二酸二甲酯预处理对犬脂肪间充质干细胞氧化应激损伤的保护作用

研究旨在探究α-酮戊二酸二甲酯(dimethyl α-ketoglutarate,DMKG)预处理对犬脂肪间充质干细胞(adipose-derived mesenchymal stem cells,ADSCs)抗氧化应激损伤的影响。以含0、0.2、0.4、0.6、0.8、1.0 mmol/L过氧化氢(H₂O₂)的DMEM/F12基础培养基处理犬ADSCs,1 h后采用CCK-8法检测细胞存活率,筛选最适浓度以建立犬ADSCs氧化应激模型。选取生长良好的P3代犬ADSCs分为3组,分别为空白对照组、模型组、DMKG组。空白对照组正常培养,不作处理;模型组使用最佳浓度的H₂O₂溶液处理犬ADSCs 1 h;DMKG组使用1 mmol/L DMKG预处理犬ADSCs 24 h后,使用最佳浓度的H₂O₂溶液处理犬ADSCs 1 h。培养结束后,采用CCK-8法检测各组的细胞存活率,可见分光光度法检测还原型谷胱甘肽含量,荧光探针DCFH-DA检测活性氧水平,Annexin V/PI双染法检测细胞凋亡率,实时荧光定量PCR检测超氧化物歧化酶(superoxide dismutase,SOD)、过氧化氢酶(catalase,CAT)、抗凋亡相关基因Bcl-2和促凋亡相关基因Bax、Cleaved-Caspase3的mRNA表达水平。结果显示,与模型组相比,DMKG预处理能有效减少细胞内活性氧的产生(P<0.05),增加细胞存活率(P<0.01),并抑制细胞凋亡(P<0.05);同时,DMKG预处理可增加还原型谷胱甘肽含量(P<0.01),上调相关抗氧化酶基因SOD1、SOD2(P<0.01)和CAT(P<0.05),下调凋亡基因Cleaved-Caspase3的表达水平(P<0.05),增加Bcl-2/Bax的比值(P<0.05)。综上所述,DMKG预处理可显著提高犬ADSCs抗氧化、抗凋亡的能力,从而增加犬ADSCs在氧化应激条件下的存活。

Protective Effect of Dimethyl α-Ketoglutarate Pretreatment on Oxidative Stress in Canine Adipose-derived Mesenchymal Stem Cells

The aim of the present study was to explore the effect of dimethyl α-ketoglutarate (DMKG) on antioxidative stress injury of canine adipose-derived mesenchymal stem cells(ADSCs).The canine ADSCs were treated with DMEM/F12 basic medium which containing hydrogen peroxide (H₂O₂) at concentrations of 0, 0.2, 0.4, 0.6, 0.8 and 1.0 mmol/L.After 1 h, the cell survival rate was detected by CCK-8 assay to screen the optimal concentration for building canine ADSCs oxidative stress model.In this experiment, the well-growing passage 3 canine ADSCs were divided into three groups: Blank control group, model group and DMKG group.The blank control group was cultured normally without treatment.In the model group, canine ADSCs were treated with the best concentration of H₂O₂ solution for 1 h. In DMKG group, canine ADSCs were pretreated with 1 mmol/L DMKG for 24 h, and then treated with the best concentration of H₂O₂ solution for 1 h.After the culture of each group, the cell survival rate of each group was detected by CCK-8 assay, the content of reduced glutathione was detected by visible spectrophotometry, the level of reactive oxygen was detected by DCFH-DA fluorescent probe, the apoptosis rate was detected by Annexin V/PI double staining, and the mRNA expression levels of superoxide dismutase(SOD), catalase (CAT), anti-apoptotic gene Bcl-2, pro-apoptotic genes Bax and Cleaved-Caspase3 were detected by Real-time quantitative PCR.The results showed that compared with the model group, DMKG pretreatment could effectively reduce the production of reactive oxygen species (P<0.05), increase the cell survival rate (P<0.01), and decrease the rate of cell apoptosis (P<0.05).Meanwhile, DMKG pretreatment could increase the content of reduced glutathione (P<0.01), up-regulate the expression levels of antioxidant enzyme-related genes SOD1, SOD2 (P<0.01)and CAT (P<0.05), down-regulate the expression levels of Cleaved-Caspase3 gene (P<0.05)and increase the rate of Bcl-2/Bax (P<0.05).In conclusion, DMKG pretreatment could improve the ability of anti-oxidation and anti-apoptosis of canine ADSCs, so as to increase the survival of canine ADSCs under oxidative stress.