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研究三七总皂苷(total saponins of Panax notoginseng,TPNS)联合淫羊藿总黄酮(total flavonoids of epimedium,TFE)改善D-半乳糖(D-galactose,D-gal)致H9c2大鼠心肌细胞衰老的保护作用及可能机制。采用50 mol·L~(-1)D-gal处理H9c2大鼠心肌细胞,以不同浓度TPNS单药、TFE单药、TPNS联合TFE预处理细胞4 h,D-gal刺激H9c2心肌细胞24 h,MTT检测细胞活率进而确定协同效果最佳的联合方案;β-半乳糖苷酶染色法鉴定细胞衰老程度;DCFH-DA探针检测细胞内活性氧(reactive oxgen species,ROS)水平;线粒体膜电位法(JC-1)检测线粒体膜电位变化情况;Western blot检测沉默信息调节因子2相关酶类1(silentmating type information regulation 2 Homolog~(-1),SIRT1),过氧化物酶体激活物受体γ辅激活因子1α(peroxisomal proliferator-activated receptor-coactivator 1α,PGC-1α),沉默信息调节因子2相关酶类3(silentmating type information regulation 2 homolog-3,SIRT3)蛋白表达情况。研究表明TPNS(5 mg·L~(-1))联合TFE(5 mg·L~(-1))对H9c2心肌细胞增殖具有显著的协同作用(Q=1.154),因此确定其为协同作用最佳的联合方案;TPNS(5 mg·L~(-1))联合TFE(5 mg·L~(-1))显著降低β-半乳糖苷酶染色阳性细胞数目和ROS荧光强度,显著提高线粒体膜电位和SIRT1,PGC-1α,SIRT3蛋白的表达量。TPNS(5 mg·L~(-1))联合TFE(5 mg·L~(-1))可产生改善D-gal致H9c2大鼠心肌细胞衰老的保护作用,机制可能与调节SIRT1/PGC-1α和SIRT3信号通路,进而调控线粒体的功能和减少氧化应激损伤有关。
To study the effects of total saponins of Panax notoginseng (TPNS) combined with total flavonoids of epimedium (TFE) on myocardial cell senescence induced by D-galactose (D-gal) in H9c2 rats Protection and possible mechanisms. The H9c2 cardiomyocytes were treated with 50 mol·L -1 D-gal, and the cells were pretreated with different concentrations of TPNS, TFE alone, TPNS and TFE for 4 h, D-gal stimulation of H9c2 cardiomyocytes for 24 h, Cell viability was determined by MTT to determine the optimal synergistic regimen; β-galactosidase staining was used to identify cell senescence; DCFH-DA probe was used to detect intracellular reactive oxygen species (ROS) levels; mitochondrial membrane potential (JC-1) was used to detect the changes of mitochondrial membrane potential. Western blot was used to detect the expressions of silent mastiff type 2 (SIRT1), silentmating type information regulation 2 (HIRT1), peroxisome proliferator-activated receptor (PGC-1α) and silentmating type information regulation 2 homolog-3 (SIRT3) protein expression were detected by Western blot. The results showed that TPNS (5 mg · L -1) combined with TFE (5 mg · L -1) had a synergistic effect on the proliferation of H9c2 cardiomyocytes (Q = 1.154) TPNS (5 mg · L -1) combined with TFE (5 mg · L -1) significantly decreased the number of β-galactosidase positive cells and ROS fluorescence intensity, and significantly increased mitochondria Membrane potential and SIRT1, PGC-1α, SIRT3 protein expression. TPNS (5 mg · L -1) combined with TFE (5 mg · L -1) could improve the cardiomyocyte senescence induced by D-gal in H9c2 rats, which may be related to the regulation of SIRT1 / PGC- 1α and SIRT3 signaling pathways, which in turn regulate the function of mitochondria and reduce oxidative stress injury.