SIRT1/p66Shc介导小檗碱对阿霉素诱导心肌毒性拮抗作用研究  

Study on the SIRT1/p66Shc-Mediated Protective Effect of Berberine against Doxorubicin-induced Cardiotoxicity

作　　者：武彦昭[1] 张兰[1] 武子笑 单菊彤 时萍 刘妍[1] 杨菲[1] 熊晨[3] WU Yan-Zhao;ZHANG Lan;WU Zi-Xiao;SHAN Ju-Tong;SHI Ping;LIU Yan;YANG Fei;XIONG Chen(Dept.of Otolaryngology-Head and Neck Surgery,The Fourth Hospital of Hebei Medical University,Shijiazhuang 050011 Hebei,China;School of Preclinical Medicine,Chengde Medical University,Chengde 067000 Hebei,China;Dept.of Pharmacology,Hebei Medical University,Shijiazhuang 050017 Hebei,China)

机构地区：[1]河北医科大学第四医院耳鼻咽喉及头颈外科,河北石家庄050011 [2]承德医学院基础医学院,河北承德067000 [3]河北医科大学药理教研室,河北石家庄050017

出　　处：《广州中医药大学学报》2022年第6期1374-1382,共9页Journal of Guangzhou University of Traditional Chinese Medicine

基　　金：河北省自然科学基金资助项目(编号:H2020206273、H2021206012)

摘　　要：【目的】基于细胞水平观察小檗碱对阿霉素诱导心肌细胞沉默信息调节因子2同源蛋白1(SIRT1)/p66Shc通路的影响,探讨小檗碱抗阿霉素所致心肌毒性作用的机制。【方法】应用阿霉素(1μmol/L)处理人心肌细胞系AC16建立阿霉素心肌毒性模型。四甲基偶氮唑盐(MTT)比色法测定细胞存活率,二氯荧光黄双乙酸盐(DCFH-DA)染色荧光显微镜照像测定细胞活性氧簇(ROS)水平,罗丹明123(Rh123)染色荧光显微镜照像测定线粒体膜电位(MMP),荧光染料Rhod2-AM(分子探针)测定线粒体Ca^(2+)浓度([Ca^(2+)]m),蛋白免疫印迹(Western Blot)法测定细胞SIRT1、p66Shc及凋亡蛋白Bax的表达水平。【结果】用1μmol/L小檗碱预处理可明显抑制1μmol/L阿霉素引起的AC16心肌细胞毒性作用,使细胞存活率升高,表现为抑制阿霉素引起的细胞内ROS生成增多、抑制阿霉素致细胞凋亡(使凋亡蛋白Bax表达下调)和减轻线粒体损伤。阿霉素处理的AC16心肌细胞中p66Shc表达增强且SIRT1蛋白表达下降;预先加用小檗碱组中,阿霉素上调p66Shc及下调SIRT1表达的效应显著减弱;而1μmol/L EX527(SIRT1抑制剂)预处理则加重阿霉素引起的AC16细胞损伤,这种细胞损伤未能被小檗碱预处理所改善。【结论】小檗碱可通过SIRT1介导的p66Shc抑制保护AC16心肌细胞对抗阿霉素诱导的心肌毒性。Objective To observe the effects of berberine(Ber)against silencing information regulator 2homologous protein 1(SIRT1)/p66Shc pathway in doxorubicin(DOX)-induced cardiomyocytes,and to explore the mechanism of Ber in resisting DOX-induced cardiotoxicity.Methods DOX(1μmol/L)was applied to treat human cardiomyocyte line AC16 to establish a DOX cardiotoxicity model.Cell viability was detected by colorimetric assay with tetramethylazole salt(MTT),cellular reactive oxygen species(ROS)level was detected by fluorescence microscopy with diclofluorescein(DCFH-DA)staining,mitochondrial membrane potential(MMP)was detected by fluorescence microscopy with rhodamine 123(Rh123)staining,mitochondrial Ca^(2+)([Ca^(2+)]m)concentration was detected by fluorescent dye Rhod2-AM(molecular probe),the expression levels of SIRT1,p66Shc and apoptotic protein Bax were detected by Western Blot method.Results Pretreatment with 1μmol/L of Ber significantly inhibited the toxic effect of 1μmol/L of DOX on AC16 cardiomyocytes and increased cell survival,as evidenced by the inhibition of DOX-induced increase in intracellular ROS production,inhibition of DOX apoptosis(down-regulation of apoptotic protein Bax expression),and alleviation of mitochondrial damage.The p66Shc expression was increased and SIRT1 protein expression was decreased in AC16 cardiomyocytes treated with DOX;the up-regulation of p66Shc and down-regulation of SIRT1 expression by DOX was significantly reduced in the pre-treatment with Ber;while 1μmol/L of EX527(SIRT1 inhibitor)pre-treatment aggravated the DOX-induced damage in AC16 cells,which was not improved by pre-treatment with Ber.Conclusion Ber protects AC16 cardiomyocytes against DOX-induced cardiotoxicity through SIRT1-mediated inhibition of p66Shc.

关 键 词：小檗碱 阿霉素 心肌毒性 SIRT1/p66Shc通路 线粒体损伤 氧化损伤 钙超载 细胞凋亡 AC16细胞 

分 类 号：R285[医药卫生—中药学]