mitoTEMPO改善糖尿病小鼠脂肪干细胞的氧化损伤

目的:探讨线粒体靶向抗氧化剂mitoTEMPO对糖尿病小鼠脂肪干细胞(Adipose-derived stem cells,ADSCs)氧化损伤的影响。方法:采用60%高脂饮食喂养雄性C57小鼠(4周龄)连续8周,并在高脂喂养第2周,对小鼠进行连续5天腹腔注射低剂量链脲佐菌素(streptozotocin,STZ)(25 mg·kg-1)制备2型糖尿病小鼠模型。喂养2周后,检测小鼠血浆葡萄糖水平等指标符合2型糖尿病标准者纳入实验。分别从正常小鼠与STZ诱导的糖尿病小鼠的腹股沟处皮下脂肪组织分离培养脂肪干细胞(ADSCs),并将其各分为4组:DMEM培养的正常ADSCs组(nADSCs组),DMEM培养的糖尿病ADSCs组(dADSCs组),TEMPO治疗的糖尿病ADSCs组(T-dADSCs组),mitoTEMPO治疗的糖尿病ADSCs组(mitoT-dADSCs组)。采用细胞计数试剂盒-8(CCK-8)检测细胞存活能力;油红-O和茜素红染色分别检测成脂细胞分化与成骨细胞分化能力;划痕实验和Transwell试验分别测定细胞迁移能力;DCF和mito SOX染色荧光成像分别检测细胞内和线粒体中的活性氧簇(Reactive oxygen species, ROS)水平。结果:①与nADSCs组相比,d ADSCs组的细胞活力明显下降(P0.05)、成骨细胞分化与成脂细胞分化程度明显下降(P0.05)、脂肪干细胞迁移能力明显下降(P0.05)、细胞内和线粒体中ROS水平明显升高(P0.05)。②与dADSCs组相比,T-dADSCs和mitoT-dADSCs组的细胞内和线粒体中的ROS水平明显降低(P0.05);与dADSCs组相比,mitoT-dADSCs组的成骨细胞分化与成脂细胞分化能力明显提升(P0.05),基本达到nADSCs组的分化水平;与dADSCs组相比,mitoT-dADSCs治疗组的细胞迁移能力显著升高(P0.05)、T-dADSCs组的细胞迁移能力增长无明显差异。结论:mitoTEMPO可以减轻糖尿病时线粒体内活性氧簇蓄积造成的脂肪干细胞的氧化应激损伤与功能紊乱。

mitoTEMPO Improves Oxidative Damage in Adipose-derived Stem Cells of Diabetic Mice

ABSTRACT Objective: To investigate the effect of mitochondria-targeted antioxidant mitoTEMPO on the oxidative damage of adipose-derived stem cells (ADSCs) in diabetic mice. Methods: Male C57 mice (4-week-old) were fed 60 % high-fat diet for 8 weeks and intraperitoneally injected with low-dose streptozotocin (STZ) (25 mg?kg-1) for 5 consecutive days in the second week of high-fat diet to establish type 2 diabetes mouse model. After fed for 2 weeks, the plasma glucose levels and other test indicators of mice were determined and the ones complaint with the criteria for type 2 diabetes were involved in the experiment. Adipose-derived stem cells (ADSCs) were isolated from normal mouse and STZ-induced diabetic mouse respectively in the inguinal subcutaneous adipose tissue and divided into 4 groups: DMEM cultured normal ADSCs group (nADSCs group), DMEM cultured diabetic ADSCs group (dADSCs group), TEMPO-treated diabetic ADSCs group (T-dADSCs group), and mitoTEMPO-treated diabetic ADSCs group (mitoT-dADSCs group). Cell viability was determined by Cell Counting Kit-8 (CCK-8); oil red-O and alizarin red staining were adopted to assess adipogenic differentiation and osteoblast differentiation respectively; scratch assay and Transwell assay were used to determine cell migration ability; DCF and mitoSOX staining fluorescence imaging were performed to detect the intracellular and mitochondrial reactive oxygen species (ROS) levels respectively. Results: 1). Compared with nADSCs group, the cell viability was significantly decreased in dADSCs group (P<0.05), the degree of osteoblast differentiation and adipogenic differentiation was significantly declined in dADSCs group (P<0.05), the migration ability of adipose-derived stem cells was significantly decreased in dADSCs group (P<0.05), and the intracellular and mitochondrial ROS levels were significantly increased in dADSCs group (P<0.05) ; 2). Compared with dADSCs group, the intracellular and mitochondrial ROS levels were significantly declined in T-dADSCs and mitoT-dADSCs groups (P<0.05), the ability of osteoblast differentiation and adipogenic differentiation was significantly increased in mitoT-dADSCs group (P<0.05) which basically reached the differentiation level of nADSCs group, the cell migration ability was significantly increased in mitoT-dADSCs group (P<0.05), and the cell migration ability indicated no significant difference in T-dADSCs group. Conclusion: mitoTEMPO can alleviate the oxidative stress damage and dysfunction of adipose-derived stem cells caused by mitochondrial ROS accumulation in diabetic environment.