基于线粒体动力学失衡探讨参芪复方含药血清对高糖诱导的骨骼肌细胞线粒体损伤的改善作用

Study on the Ameliorative Effect of Drug Serum of Shenqi Prescription Based on Mitochondrial Kinetic Imbalance on Mitochondrial Injury Induced by High Glucose in Skeletal Muscle Cells

目的:观察参芪复方含药血清对高糖环境下L6细胞(大鼠成肌细胞)内线粒体形态与功能的影响,并基于线粒体动力学探讨其作用机制。方法:采用SD大鼠制备空白血清及参芪复方含药血清;将以DMEM低糖培养基(含5.56 mmol/L Glu)培养的细胞设为空白对照组,并予10%空白血清进行干预;将以DMEM高糖培养基(含25 mmol/L Glu)培养的细胞设为模型组(10%空白血清)、罗格列酮(300 ng/mL)阳性对照组及参芪复方含药血清低(5%)、中(10%)、高(15%)浓度组。各组均加入20 mIU/L胰岛素。采用电镜观察L6细胞内线粒体形态,流式细胞术检测L6细胞内线粒体膜电位,RT-qPCR法及Western Blot法检测L6细胞内线粒体动力学相关因子Opa1、Mfn1、Mfn2、Drp1、Fis1、Mff mRNA及蛋白表达。结果:与模型组比较,参芪复方含药血清各浓度组L6细胞线粒体的形态明显改善,线粒体肿胀程度减轻,线粒体膜电位显著升高,Fis1、Mff mRNA表达显著降低,Mfn2蛋白表达显著升高(P<0.05);参芪复方含药血清高浓度组Mfn1、Mfn2 mRNA及Mfn1蛋白表达显著升高,Mff、Drp1蛋白表达显著降低(P<0.05);参芪复方含药血清低浓度组Fis1蛋白表达显著降低(P<0.05)。结论:参芪复方可改善高糖环境下骨骼肌细胞的形态与功能障碍,其作用机制与参芪复方对线粒体动力学相关因子的调控有关。

Objective:To observe the effects of drug serum of Shenqi prescription on the morphology and function of mitochondria in L6 cells(rat myogenic cells) under high glucose environment,and to explore the mechanism of action based on mitochondrial dynamics.Methods:SD rats were used to prepare blank serum and drug serum of Shenqi prescription.The cells cultured in DMEM low glucose medium(containing 5.56 mmol/L Glu) were set up as blank group and intervened with 10% blank serum,cells cultured in DMEM high glucose medium(containing 25 mmol/L Glu) were set up as model group(10% blank serum),rosiglitazone(300 ng/mL) positive control group and drug serum of Shenqi prescription low(5%),medium(10%),high(15%) concentration groups.All groups were added with 20 mIU/L insulin.The morphology of mitochondria in L6 cells was observed by electron microscopy.The mitochondrial membrane potential in L6 cells was detected by flow cytometry.The mRNA and protein expressions of mitochondrial dynamics related factors Opa1,Mfn1,Mfn2,Drp1,Fis1 and Mff in L6 cells were detected by RT-qPCR and Western Blot.Results:Compared with the model group,the morphology of mitochondria in L6 cells were significantly improved,the degree of mitochondrial swelling was alleviated,the mitochondrial membrane potential was significantly increased,the mRNA expressions of Fis1 and Mff were significantly decreased,and the protein expression of Mfn2 was significantly increased in each drug serum of Shenqi prescription group(P0.05).The mRNA expressions of Mfn1 and Mfn2 and protein expression of Mfn1 in drug serum of Shenqi prescription high concentration group were significantly increased,the Mff,Drp1 protein expressions were significantly decreased.The protein expression of Fis1 in drug serum of Shenqi prescription low concentration group was significantly decreased(P0.05).Conclusion:Shenqi prescription can improve the morphology and dysfunction of skeletal muscle cells under high glucose environment,and its mechanism is related to the regulation of mitochondrial dynamics related factors by Shenqi prescription.