龙牙楤木总皂苷及楤木皂苷A对缺氧/复氧诱导的AC16心肌细胞铁死亡的影响

Effects of Aralosides and Araloside A on hypoxia/reoxygenation-induced ferroptosis in AC16 cells

目的探讨龙牙楤木总皂苷及其组分楤木皂苷A能否减轻缺氧/复氧诱导的AC16心肌细胞铁死亡。方法将AC16细胞随机分为正常组、模型组、龙牙楤木总皂苷组、楤木皂苷A组。通过缺氧24小时、复氧12小时建立AC16细胞缺氧/复氧模型,药物组缺氧/复氧损伤前6小时给予药物处理。CCK-8检测细胞存活率,乳酸脱氢酶检测试剂盒检测细胞受损程度,透射电子显微镜观察AC16细胞形态学变化,比色法检测Fe^(2+)水平,酶联免疫试剂盒检测丙二醛(malondialdehyde,MDA)和谷胱甘肽(glutathione,GSH)水平,DHE荧光探针检测活性氧(reactive oxygen species,ROS)水平,蛋白免疫印迹法检测铁死亡相关蛋白p53、SLC7A11、SAT1、GLS2的表达。结果与正常组相比,模型组AC16细胞存活率明显下降,细胞受损程度明显升高,细胞内的线粒体皱缩、内嵴增厚、膜电子密度增高,Fe^(2+)、ROS、MDA水平明显上升,GSH活力明显下降,铁死亡相关蛋白p53、SAT1、GLS2蛋白表达明显升高,SLC7A11蛋白表达明显降低,以上结果差异均具有统计学意义(P<0.05);与模型组相比,龙牙楤木总皂苷组及楤木皂苷A组AC16细胞存活率明显上升,细胞受损程度明显降低,细胞内的线粒体形态趋于正常状态,Fe^(2+)、ROS、MDA水平下降,GSH活力升高,铁死亡相关蛋白p53、SAT1、GLS2蛋白表达下调,SLC7A11蛋白表达上调,以上结果差异均具有统计学意义(P<0.05)。结论龙牙楤木总皂苷及其组分楤木皂苷A能减轻缺氧/复氧诱导的AC16心肌细胞铁死亡,其机制可能与抑制p53、SAT1、GLS2蛋白表达,上调SLC7A11蛋白表达有关。

Objective To research the influence of Aralosides(As)and its components Araloside A on the ferroptosis of hypoxia/reoxygenation(H/R)injure AC16 myocardial cell.Methods AC16 myocardial cells was cultured and randomly divided into 4 groups:normal group,the H/R group,As group(H/R+As),the As group A(H/R+As A).The AC16 cell hypoxia/reoxygenation injury model was established by 24 hours of hypoxia and 12 hours of reoxygenation.Drug group cells were given drug treatment 6 hours before hypoxia/reoxygenation injury.CCK-8 method was used to detect the AC16 cells viability.LDH assay kit was used to detect the degree of AC16 cells damage.The electron microscope was used to observe the cells morphology changes.the Fe^(2+)level was detected by colorimetry.The levels of malondialdehyde(MDA)and glutathione(GSH)were detected by ELISA kit,and the levels of reactive oxygen species(ROS)were detected by DHE fluorescent probe Western blot was used to detect the proteins expression of p53,SLC7A11,SAT1,GLS2.Results Compared with control group,the survival rate of AC16 cells was decreased and the degree of cell damage was significantly increased,mitochondria were shrunkenand the ridge were thickened,membrane electron density were increased,the levels of Fe^(2+),MDA and ROS were significantly accumulated,and GSH were decreased,the protein expression of ferroptosis-related proteins p53,SAT1,GLS2 were increased and SLC7A11 was decreased and the differences in the above results were statistically significant(P<0.05).After intervention with As or As A,the survival rate of AC16 cells was increased and the degree of cell damage was decreased,the mitochondrial morphology in the cells tended to be normal,and the levels of Fe^(2+),ROS,and MDA decreased,the levels of GSH were increased,the levels of p53,SAT1,GLS2 were decreased and SLC7A11 was increased after H/R(P<0.05).Conclusion Ferroptosis exists in the H/R-injured AC16 cell model,while As and As A protect the cells against ferroptosis.The mechanism may be related to the inhibition of p53,SAT1,GLS2 protein expression and the up-regulation of SLC7A11 protein expression.