三七总皂苷联合淫羊藿总黄酮改善D-gal致H9c2大鼠心肌细胞衰老的保护作用

Protective effect of total saponins of Panax notoginseng combined with total flavonoids of epimedium on D-galactose-incuced senescence of H9c2 cell

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 investigate the protective effect of Panax notoginseng saponins combined with total flavonoids of epimedium on D-galinduced senescence of H9c2 cells and explore its underlying mechanisms. The 50 mol·L^（-1）D-gal was used to induce H9c2 cells senescence. Different concentrations of TPNS,TFE,and TPNS combined with TFE were used for 4 hours for pre-treatment. D-gal was used to stimulate H9c2 cardiac muscle cells for 24 h. Then in order to determine the best combined scheme,MTT was used to detect cell viability. Cell senescence was identified by β-galactosidase staining. Levels of reactive oxygen species（ ROS） was observed by DCFH-DA detection. The changes of mitochondrial membrane potential were identified by JC-1 detection. Protein levels of silentmating type information regulation 2 Homolog^（-1）（ SIRT1）,peroxisomal proliferator-activated receptor-coactivator 1α（ PGC-1α） and silentmating type information regulation 2 Homolog-3（ SIRT3） were detected by western blot analysis. The results showed that TPNS（ 5 mg·L^（-1）） combined with TFE（ 5 mg·L^（-1）） had significant synergistic effect on H9c2 myocardial cell proliferation（ Q = 1. 154）,so 5 mg·L^（-1）TPNS combined with 5 mg·L^（-1）TFE was determined as the best scheme. The quantity of β-galactosidase staining and the fluorescence intensity of ROS were apparently decreased in 5 mg· L^（-1）TPNS combined with 5 mg · L^（-1）TFE scheme. Meanwhile,it markedly increased the florescence intensity of mitochondrial membrane potential and enhanced the protein expression of SIRT1,PGC-1 α and SIRT3. TPNS combined with TFE could protect H9 c2 cells from D-gal-induced senescence. The mechanism might be related to adjusting the signal pathways of SIRT1/PGC-1 α,SIRT3,adjusting the structure and function of mitochondria and reducing oxidative stress injury.