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目的研究附子有效成分次乌头碱和甘草有效成分甘草次酸不同比例配伍对H9c2心肌细胞缺氧缺糖损伤的影响及可能作用机制。方法取长成致密单层的H9c2心肌细胞分成正常组、模型组、地高辛组(80μmol/L)、次乌头碱组(120μmol/L)及1∶0.5组、1∶1组、1∶2组(次乌头碱为120μmol/L,甘草次酸分别为60、120、240μmol/L),每组6个复孔。正常组加入完全培养基1 ml,模型组加入无糖Earle’s液1 ml,地高辛组、次乌头碱组和各配伍组分别加相应药时用无糖Earle’s液稀释成相应浓度,加入1 ml,除正常组外其余各组加药后构建缺氧缺糖模型培养4 h。观察各组H9c2心肌细胞的形态学变化,检测细胞培养上清液中肿瘤坏死因子α(TNF-α)、白细胞介素1β(IL-1β),肌酸肌酶(CK)的含量及Caspase-3、Fas-L、Fas蛋白表达。结果正常组心肌细胞呈贴壁生长,多为梭形,形成不规则的星形,并形成呈放射状排列的细胞簇;模型组心肌细胞胞体折光性下降,胞浆皱缩,可见折光性强的脱壁死亡细胞;各配伍组的细胞形态较模型组均有明显改善,并以配伍组1∶1组最明显。与正常组比较,模型组TNF-α、IL-1β、CK含量及Caspase-3、Fas-L、Fas的蛋白表达均升高(P<0.01)。与模型组比较,次乌头碱组各组指标均无明显改变(P>0.05);而地高辛组和各配伍组不同程度降低TNF-α、IL-1β、CK含量及Caspase-3、Fas-L、Fas蛋白表达,并且以1∶1组效果最好(P<0.05或P<0.01)。结论次乌头碱配伍甘草次酸对心肌细胞缺氧缺糖损伤有保护作用,并以1∶1配伍最佳,其作用机制可能与抗炎及抑制细胞凋亡有关。
Objective To study the effect of different concentrations of glycyrrhizin, the active ingredient of aconitine and licorice, on the injury of hypoxia-glucose deprivation in H9c2 cardiomyocytes and its possible mechanism. Methods H9c2 cardiomyocytes were divided into normal group, model group, digoxin group (80μmol / L), hypaconitine group (120μmol / L), 1: 0.5 group, 1: : 2 groups (hypaconitine was 120μmol / L, glycyrrhetinic acid were 60,120,240μmol / L), each group 6 complex wells. Normal group was added 1 ml of complete medium, the model group added 1 ml of sugar-free Earle’s solution, digoxin group, hypaconitine group and the corresponding drug groups were added with the corresponding drug-free Earle’s solution diluted to the appropriate concentration, adding 1 ml, in addition to the normal group, the rest of the group after dosing to build oxygen and glucose deprivation model cultured 4 h. The morphological changes of H9c2 cardiomyocytes in each group were observed. The levels of TNF-α, IL-1β, CK and Caspase-3 , Fas-L, Fas protein expression. Results In normal group, myocardial cells grew adherently, mostly in the form of spindles, forming irregular stars and forming clusters of cells arranged radially; in model group, the refolding of cell bodies of cardiomyocytes decreased and the cytoplasm collapsed, showing strong refraction The cells in detached wall were significantly more than those in the untreated group. Compared with the normal group, the levels of TNF-α, IL-1β, CK and the protein expressions of Caspase-3, Fas-L and Fas in model group were significantly increased (P <0.01). Compared with the model group, there was no significant change in each index of hypaconitine group (P> 0.05), while the levels of TNF-α, IL-1β and CK and Caspase-3, Fas-L, Fas protein expression, and 1: 1 group the best (P <0.05 or P <0.01). Conclusions Hypaconitine and glycyrrhetinic acid can protect cardiomyocytes against hypoxia and hypoglycemia, and have the best compatibility with 1: 1. The mechanism may be related to anti-inflammation and inhibition of apoptosis.