目的 探究小檗堿及其體內代謝產物對高糖誘導大鼠H9c2心肌細胞損傷的保護作用。方法 將H9c2心肌細胞分成對照組、模型組、正常給藥組、模型給藥組，對照組用無血清DMEM培養(yǎng)，正常給藥組于無血清DMEM中加藥，模型組用含50 mmol·L^-1葡萄糖的無血清DMEM培養(yǎng)（造模劑量篩選實驗設置25、50、100、200 mmol·L^-1），模型給藥組于高糖無血清DMEM中加藥，加藥濃度分別為小檗堿1.25、2.50、5.00、10.00 μmol·L^-1，二氫小檗堿（DHB）0.5、1.0、2.0、4.0 μmol·L^-1，小檗紅堿1.25、2.50、5.00、10.00 μmol·L^-1，非洲防己堿（COL） 1.25、2.50、5.00、10.00 μmol·L^-1，巴馬汀3.125、6.250、12.500、25.000 μmol·L^-1，藥根堿6.25、12.50、25.00、50.00 μmol·L^-1，去亞甲基小檗堿（DEM） 6.25、12.50、25.00、50.00 μmol·L^-1，處理48 h后，通過細胞增殖計數(shù)（CCK-8）法檢測細胞存活率；通過實時熒光定量聚合酶鏈式反應（qRT-PCR）檢測經(jīng)典代謝調控通路沉默調節(jié)蛋白1（Sirt1）、過氧化物酶體增殖物激活受體-γ共激活因子1α（PGC1α）、過氧化物酶體增殖物激活受體α（PPARα）基因水平，葡萄糖代謝相關基因丙酮酸脫氫酶激酶4 （PDK4）、葡萄糖激酶（GCK）、己糖激酶（HK2）、葡萄糖轉運蛋白4（Glut4）表達水平，線粒體動力學相關基因線粒體融合蛋白2 （Mfn2）、視神經(jīng)萎縮蛋白1 （OPA1）、動力學相關蛋白1（Drp1）水平及細胞凋亡相關基因天冬氨酸特異性半胱氨酸蛋白酶3（Caspase-3）、Caspase-9、Bcl-2相關X蛋白（Bax）、B淋巴細胞瘤-2 （Bcl-2）水平；通過Western blotting實驗檢測PGC1α、Glut4、線粒體氧化磷酸化系統(tǒng)（OXPHOS）蛋白表達。結果 與高糖模型組相比，小檗堿2.5、5.0、10.0 μmol·L^-1，DHB 1、2 μmol·L^-1，COL 5、10 μmol·L^-1，巴馬汀12.5 μmol·L^-1，藥根堿25、50 μmol·L^-1，DEM 12.5、25.0 μmol·L^-1處理48 h的細胞存活率均顯著上升（P＜0.05、0.01、0.001）；小檗堿、DHB、藥根堿、DEM處理的心肌細胞中PDK4水平顯著增加（P＜0.05、0.001），小檗堿、DHB、DEM處理的心肌細胞中GCK水平顯著增加（P＜0.01、0.001），小檗堿、DHB、COL、巴馬汀、DEM處理的心肌細胞中HK2水平顯著增加（P＜0.05、0.01），DEM處理的心肌細胞中Glut4水平顯著增加（P＜0.001）；藥根堿、DEM處理的心肌細胞中OPA1水平顯著增加（P＜0.05、0.001），小檗堿、DHB、COL、巴馬汀、藥根堿、DEM處理的心肌細胞中Drp1水平顯著降低（P＜0.01、0.001），小檗堿、DHB、藥根堿、DEM處理的心肌細胞中Mfn2水平顯著增加（P＜0.05、0.001）；小檗堿、DHB、COL處理的心肌細胞中Caspase-3水平顯著降低（P＜0.01、0.001），小檗堿、小檗紅堿、COL、巴馬汀處理的心肌細胞中Caspase-9水平顯著降低（P＜0.01、0.001），小檗堿、DHB、小檗紅堿、COL、巴馬汀、藥根堿、DEM處理的心肌細胞中Bax水平顯著降低（P＜0.05、0.001），小檗堿、DHB、巴馬汀處理的心肌細胞中Bcl-2水平顯著增加（P＜0.01、0.001）；小檗堿及其代謝產物處理的心肌細胞中PGC1α、Glut4、OXPHOS表達均明顯增加。結論 小檗堿體內代謝產物可以緩解高糖造成的H9c2心肌細胞損傷，其作用機制與調控Sirt1/PGC1α/PPARα信號通路，促進H9c2心肌細胞葡萄糖代謝、改善線粒體功能、抑制細胞凋亡有關。

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

國家自然科學基金資助項目（82000825）；北京市中醫(yī)藥科技發(fā)展資金項目資助（JJ-2020-25）