目的 探究小檗碱及其体内代谢产物对高糖诱导大鼠H9c2心肌细胞损伤的保护作用。方法 将H9c2心肌细胞分成对照组、模型组、正常给药组、模型给药组，对照组用无血清DMEM培养，正常给药组于无血清DMEM中加药，模型组用含50 mmol·L^-1葡萄糖的无血清DMEM培养（造模剂量筛选实验设置25、50、100、200 mmol·L^-1），模型给药组于高糖无血清DMEM中加药，加药浓度分别为小檗碱1.25、2.50、5.00、10.00 μmol·L^-1，二氢小檗碱（DHB）0.5、1.0、2.0、4.0 μmol·L^-1，小檗红碱1.25、2.50、5.00、10.00 μmol·L^-1，非洲防己碱（COL） 1.25、2.50、5.00、10.00 μmol·L^-1，巴马汀3.125、6.250、12.500、25.000 μmol·L^-1，药根碱6.25、12.50、25.00、50.00 μmol·L^-1，去亚甲基小檗碱（DEM） 6.25、12.50、25.00、50.00 μmol·L^-1，处理48 h后，通过细胞增殖计数（CCK-8）法检测细胞存活率；通过实时荧光定量聚合酶链式反应（qRT-PCR）检测经典代谢调控通路沉默调节蛋白1（Sirt1）、过氧化物酶体增殖物激活受体-γ共激活因子1α（PGC1α）、过氧化物酶体增殖物激活受体α（PPARα）基因水平，葡萄糖代谢相关基因丙酮酸脱氢酶激酶4 （PDK4）、葡萄糖激酶（GCK）、己糖激酶（HK2）、葡萄糖转运蛋白4（Glut4）表达水平，线粒体动力学相关基因线粒体融合蛋白2 （Mfn2）、视神经萎缩蛋白1 （OPA1）、动力学相关蛋白1（Drp1）水平及细胞凋亡相关基因天冬氨酸特异性半胱氨酸蛋白酶3（Caspase-3）、Caspase-9、Bcl-2相关X蛋白（Bax）、B淋巴细胞瘤-2 （Bcl-2）水平；通过Western blotting实验检测PGC1α、Glut4、线粒体氧化磷酸化系统（OXPHOS）蛋白表达。结果 与高糖模型组相比，小檗碱2.5、5.0、10.0 μmol·L^-1，DHB 1、2 μmol·L^-1，COL 5、10 μmol·L^-1，巴马汀12.5 μmol·L^-1，药根碱25、50 μmol·L^-1，DEM 12.5、25.0 μmol·L^-1处理48 h的细胞存活率均显著上升（P＜0.05、0.01、0.001）；小檗碱、DHB、药根碱、DEM处理的心肌细胞中PDK4水平显著增加（P＜0.05、0.001），小檗碱、DHB、DEM处理的心肌细胞中GCK水平显著增加（P＜0.01、0.001），小檗碱、DHB、COL、巴马汀、DEM处理的心肌细胞中HK2水平显著增加（P＜0.05、0.01），DEM处理的心肌细胞中Glut4水平显著增加（P＜0.001）；药根碱、DEM处理的心肌细胞中OPA1水平显著增加（P＜0.05、0.001），小檗碱、DHB、COL、巴马汀、药根碱、DEM处理的心肌细胞中Drp1水平显著降低（P＜0.01、0.001），小檗碱、DHB、药根碱、DEM处理的心肌细胞中Mfn2水平显著增加（P＜0.05、0.001）；小檗碱、DHB、COL处理的心肌细胞中Caspase-3水平显著降低（P＜0.01、0.001），小檗碱、小檗红碱、COL、巴马汀处理的心肌细胞中Caspase-9水平显著降低（P＜0.01、0.001），小檗碱、DHB、小檗红碱、COL、巴马汀、药根碱、DEM处理的心肌细胞中Bax水平显著降低（P＜0.05、0.001），小檗碱、DHB、巴马汀处理的心肌细胞中Bcl-2水平显著增加（P＜0.01、0.001）；小檗碱及其代谢产物处理的心肌细胞中PGC1α、Glut4、OXPHOS表达均明显增加。结论 小檗碱体内代谢产物可以缓解高糖造成的H9c2心肌细胞损伤，其作用机制与调控Sirt1/PGC1α/PPARα信号通路，促进H9c2心肌细胞葡萄糖代谢、改善线粒体功能、抑制细胞凋亡有关。

Objective To investigate the protective effect of berberine metabolites on H9c2 cardiomyocytes injury induced by high glucose. Methods H9c2 cardiomyocytes were divided into control group, model group, berberine metabolites-treated group and high glucose together with berberine metabolites-treated group. The control group was cultured in serum-free DMEM, the normal treatment group was treated with medication in serum-free DMEM, and the model group was cultured in serum-free DMEM containing 50 mmol·L^-1 glucose (modeling dose screening experiments were set at 25, 50, 100, and 200 mmol·L^-1). The model treatment group was treated in high glucose serum-free DMEM with concentrations of berberine (BBR) 1.25, 2.50, 5.00, and 10.00 μmol·L^-1, dihydroberberine (DHB) 0.5, 1.0, 2.0, and 4.0 μmol·L^-1, berberine (BRB) 1.25, 2.50, 5.00, and 10.00 μmol·L^-1, columbamine (COL) 1.25, 2.50, 5.00, and 10.00 μmol·L^-1, palmatine (PAL) 3.125, 6.250, 12.500, and 25.000 μmol·L^-1, jatrorrhizine （JAT) 6.25, 12.50, 25.00, and 50.00 μmol·L^-1, demethyleneberberine (DEM) 6.25, 12.50, 25.00, and 50.00 μmol·L^-1. After 48 hours of treatment, the cell survival rate was measured by cell proliferation count (CCK-8) method. Detection of classical metabolic regulatory pathway silencing regulatory protein 1 (Sirt1) and peroxisome proliferator activated receptors-γ Co-activation factor 1 α (PGC1α), peroxisome proliferator activated receptor α (PPARα) Gene level, expression levels of glucose metabolism related genes pyruvate dehydrogenase kinase 4 (PDK4), glucokinase (GCK), hexokinase (HK2), glucose transporter 4 (Glut4), and mitochondrial dynamics related genes mitochondrial fusion protein 2 (Mfn2), optic atrophy protein 1 (OPA1), dynamics related protein 1 (Drp1) and apoptosis related genes Caspase-3, Caspase-9, Bcl-2 related X protein (Bax), and B lymphomatoma-2 (Bcl-2) by real-time fluorescence quantitative polymerase chain reaction (qRT-PCR). Detection of PGC1α, Glut4, mitochondrial oxidative phosphorylation system (OXPHOS) protein expression through Western blotting experiment. Results Compared with the high glucose model group, the cell survival rate significantly increased in BBR 2.5, 5.0, 10.0 μmol·L^-1, DHB 1, 2 μmol·L^-1, COL 5, 10 μmol·L^-1, PAL 12.5 μmol·L^-1, JAT 25, 50 μmol·L^-1, and DEM 12.5, 25.0 μmol·L^-1 group after 48 hours of treatment (P＜0.05, 0.01, 0.001). The levels of PDK4 were significantly increased in cardiomyocytes treated with BBR, DHB, JAT, and DEM (P＜0.05, 0.001), GCK levels were significantly increased in cardiomyocytes treated with BBR, DHB, and DEM (P＜0.01, 0.001), HK2 levels were significantly increased in cardiomyocytes treated with BBR, DHB, COL, PAL, and DEM (P＜0.05, 0.01), and Glut4 levels were significantly increased in cardiomyocytes treated with DEM (P＜0.001). The levels of OPA1 were significantly increased in cardiomyocytes treated with JAT and DEM (P＜0.05, 0.001), while the levels of Drp1 were significantly reduced in cardiomyocytes treated with BBR, DHB, COL, PAL, JAT, and DEM (P＜0.01, 0.001). The levels of Mfn2 were significantly increased in cardiomyocytes treated with BBR, DHB, JAT, and DEM (P＜0.05, 0.001). The levels of Caspase-3 in cardiomyocytes treated with BBR, DHB, and COL were significantly reduced (P＜0.01, 0.001), while the levels of Caspase-9 in cardiomyocytes treated with BBR , BRB, COL, and PAL were significantly reduced (P＜0.01, 0.001). The levels of Bax in cardiomyocytes treated with BBR, DHB, BRB, COL, PAL, JAT, and DEM were significantly reduced (P＜0.05, 0.001), while the levels of Bcl-2 in cardiomyocytes treated with BBR, DHB, and PAL were significantly increased (P＜0.01, 0.001). The expression of PGC1α, Glut4 and OXPHOS in cardiomyocytes treated with BBR and its metabolites was significantly increased. Conclusion BBR metabolites can ameliorate high glucose induced H9c2 cardiomyocyte injury, and its mechanism may be through regulating Sirt1/PGC1 α/PPAR α signaling pathway, promoting glucose metabolism of H9c2 cardiomyocytes, improving mitochondrial function, and inhibiting apoptosis.

R285.5

国家自然科学基金资助项目（82000825）；北京市中医药科技发展资金项目资助（JJ-2020-25）