槐角苷对多柔比星引起的心肌细胞损伤保护作用的机制研究

Mechanism of Protective Effect of Sophoricoside against Doxorubicin-Induced Myocardial Ce Injury

目的:探讨槐角苷对多柔比星引起的H9c2心肌细胞损伤的保护作用及机制。方法:采用不同浓度的槐角苷预处理H9c2大鼠心肌细胞6 h后,加入终浓度为1μmol/L的多柔比星处理24 h。应用MTT法测定H9c2心肌细胞的活力;采用Hoechst 33342染色及流式细胞术检测H9c2心肌细胞的凋亡情况;采用二氯荧光黄双乙酸盐(DCFH-DA)染色荧光显微镜检测活性氧(ROS)水平;蛋白免疫印迹(Western Blot)法测定细胞凋亡蛋白BCL2-相关蛋白X(BAX)、B淋巴瘤细胞-2(BCL-2)、活化半胱氨酸蛋白酶-3(Cleaved Caspase-3)及相关信号通路蛋白P38与细胞外信号调节激酶(ERK)、c-Jun氨基末端激酶(JNK)、核因子κ-B(NF-κB)蛋白表达,并使用P38激动剂P79350进一步验证槐角苷与P38/MAPK/NF-κB信号通路的关系。结果:与空白对照组相比,模型对照组H9c2心肌细胞活力显著降低(P<0.01),凋亡率明显增加,H9c2心肌细胞内ROS生成增加(P<0.05),凋亡相关蛋白BAX、Cleaved Caspase-3表达上调,BCL-2表达下调,P38、ERK、JNK磷酸化蛋白表达上调,NF-κB表达上调(P<0.01);与模型对照组相比,槐角苷预8.65、34.59μg/mL处理后,H9c2心肌细胞活力显著升高(P<0.01),凋亡率明显降低(P<0.05),细胞内ROS生成降低(P<0.05),BAX、Cleaved Caspase-3表达下调(P<0.01),BCL-2表达上调(P<0.01),P38、ERK、JNK磷酸化蛋白表达下调(P<0.05),NF-κB表达下调(P<0.05);加入P38激动剂P79350后,与槐角苷34.59μg/mL组相比,槐角苷减轻多柔比星H9c2心肌细胞损伤的作用被明显逆转(P<0.05)。结论:槐角苷通过下调P38/MAPK/NF-κB信号通路相关蛋白的表达从而减少ROS生成,抑制氧化应激,减轻多柔比星引起的心肌细胞损伤。

Objective:To investigate the protective effect and mechanism of sophoricoside against doxorubicin-induced H9c2 myocardial cell injury.Methods:H9c2 myocardial cells were pretreated with different concentrations of sophoricoside for 6 hours,followed by treatment with a final concentration of 1μmol/L doxorubicin for 24 hours.Cell viability of H9c2 myocardial cells was determined using the MTT assay.Apoptosis of H9c2 myocardial cells was detected using Hoechst 33342 staining and flow cytometry.The level of reactive oxygen species(ROS)was measured using dichlorodihydrofluorescein diacetate(DCFH-DA)and fluorescence microscopy.Western blot analysis was performed to determine the expression of apoptosis-related proteins B-cell lymphoma 2(Bcl-2),Bcl-2-associated X protein(Bax),cleaved Caspase-3,and signaling pathway proteins P38,extracellular signal-regulated kinase(ERK),c-Jun N-terminal kinase(JNK),and nuclear factor kB(NF-kB).The relationship between sophoricoside and the P38/MAPK/NF-kB signaling pathway was further validated using the P38 agonist P79350.Results:Compared with the blank control group,the model control group showed reduced cell viability(P<0.O1),increased apoptosis rate(P<0.05),increased R0S generation(P<0.05),up-regulated expression of Bax and cleaved Caspase-3(P<0.01),down-regulated expression of Bcl-2(P<0.01),up-regulated phosphorylation of P38,ERK,and JNK(P<0.01),and up-regulated expression of NF-kB(P<0.01).Compared with the model control group,sophoricoside pretreatment resulted in increased cell viability(P<0.01),decreased apoptosis rate(P<0.05),decreased ROS generation(P<0.05),down-regulated expression of Bax and cleaved Caspase-3(P<0.01),up-regulated expression of Bcl-2(P<0.01),down-regulated phosphorylation of P38,ERK,and JNK(P<0.05),and down-regulated expression of NF-kB(P<0.05).However,when the P38 agonist P79350 was added,the protective effect of sophoricoside against doxorubicin-induced H9c2 myocardial cell injury was reversed compared with the sophoricoside group at 34.59μg/mL(P<0.05).Conclusion:Sophoricoside reduces ROS generation,inhibits oxidative stress,and alleviates doxorubicin-induced myocardial cell injury by down-regulating the expression of proteins related to the P38/MAPK/NF-kB signaling pathway.