本研究旨在探讨丹参的有效成分丹酚酸B(salvianolic acid B，Sal B)通过调控线粒体分裂融合对氧糖剥夺/再复(oxygen and glucose deprivation/reperfusion，OGD/R)损伤的H9C2心肌细胞的保护作用。通过建立OGD/R模型模拟心肌缺血再灌注损伤过程。细胞增殖及细胞毒性检测试剂盒(cell counting kit-8，CCK-8)检测细胞活力;试剂盒法检测细胞内活性氧(reactive oxygen species，ROS)、总谷胱甘肽(total glutamyl cysteinyl glycine，t-GSH)、一氧化氮(nitric oxide，NO)含量，蛋白质印迹法检测线粒体分裂融合、细胞凋亡相关蛋白表达水平，线粒体通透性转换孔(mitochondrial permeability transition pore，MPTP)检测试剂盒与 Hoechst 33342 荧光观察MPTP 开放水平，分子对接技术确定丹酚酸B分子靶点。结果表明，与对照组相比，OGD/R损伤降低H9C2细胞活力，ROS含量增加，t-GSH、NO含量减少，线粒体动力相关蛋白1(dynamin-related protein 1，Drp1)、线粒体融合蛋白有线粒体融合蛋白2(mitofusions 2，Mfn2)、Bcl-2相关X蛋白(Bcl-2 associated X protein，Bax)、半胱氨酸蛋白水解酶3(cysteinyl aspartate specific proteinase 3，caspase 3)的蛋白表达水平升高，Mfn1的蛋白表达水平降低，MPTP开放水平增加。与OGD/R组相比，丹酚酸B各组在6。25～100μmol·L-1具有保护作用，丹酚酸B降低ROS含量，增加t-GSH、NO含量，降低Drp1、Mfn2、Bax、caspase3的蛋白表达水平，增加Mfn1的蛋白表达水平，降低MPTP开放水平。综上所述，丹酚酸B可能通过调节氧化与抗氧化平衡、线粒体分裂融合平衡，抑制MPTP开放，减少细胞凋亡进而使H9C2细胞减少OGD/R损伤，可为丹酚酸B治疗心肌缺血再灌注损伤提供有价值的科学依据。
Mechanism of salvianolic acid B protecting H9C2 from OGD/R injury based on mitochondrial fission and fusion
This study aims to investigate the effect of salvianolic acid B(Sal B),the active ingredient of Salvia miltiorrhiza,on H9C2 cardiomyocytes injured by oxygen and glucose deprivation/reperfusion(OGD/R)through regulating mitochondrial fission and fusion.The process of myocardial ischemia-reperfusion injury was simulated by establishing OGD/R model.The cell proliferation and cytotoxicity detection kit(cell counting kit-8,CCK-8)was used to detect cell viability;the kit method was used to detect intracellular reactive oxygen species(ROS),total glutathione(t-GSH),nitric oxide(NO)content,protein expression levels of mitochondrial fission and fusion,apoptosis-related detection by Western blot.Mitochondrial permeability transition pore(MPTP)detection kit and Hoechst 33342 fluorescence was used to observe the opening level of MPTP,and molecular docking technology was used to determine the molecular target of Sal B.The results showed that relative to control group,OGD/R injury reduced cell viability,increased the content of ROS,decreased the content of t-GSH and NO.Furthermore,OGD/R injury increased the protein expression levels of dynamin-related protein 1(Drp1),mitofusions 2(Mfn2),Bcl-2 associated X protein(Bax)and cysteinyl aspartate specific proteinase 3(caspase 3),and decreased the protein expression levels of Mfnl,increased MPTP opening level.Compared with the OGD/R group,it was observed that Sal B had a protective effect at concentrations ranging from 6.25 to 100 μmol·L-1.Sal B decreased the content of ROS,increased the content of t-GSH and NO,and Western blot showed that Sal B decreased the protein expression levels of Drp1,Mfn2,Bax and caspase 3,increased the protein expression level of Mfn1,and decreased the opening level of MPTP.In summary,Sal B may inhibit the opening of MPTP,reduce cell apoptosis and reduce OGD/R damage in H9C2 cells by regulating the balance of oxidation and anti-oxidation,mitochondrial fission and fusion,thereby providing a scientific basis for the use of Sal B in the treatment of myocardial ischemia reperfusion injury.
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