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目的 构建恩曲替尼(ENT)小鼠心脏毒性模型并对自噬机制进行初步探索。方法 将C57BL/6小鼠随机分为ENT心脏毒性模型组和溶媒对照组,模型组小鼠每日灌胃给予ENT 60mg/kg,溶媒对照组同法灌胃等量的生理盐水。分别于给药后的第3周、5周、6周进行小鼠心脏超声检查。于第6周处死小鼠,取心脏称重,计算心重指数(CWI);超声心动图分析左心室射血分数(LVEF)、左室短轴缩短率(LVFS)、左室舒张末期内径(LVIDd)、左室收缩末期内径(LVIDs)、左室后壁舒张期厚度(LVPWd)和左室后壁收缩期厚度(LVPWs)等指标来评估小鼠的心功能;ELISA试剂盒检测血清乳酸脱氢酶(LDH)、肌酸激酶同工酶MB (CKMB)、N末端B型脑钠肽前体(NT-proBNP)和心肌肌钙蛋白Ⅰ(cTnⅠ)等心肌损伤标志物;HE和Masson染色评估心肌组织病理学,观察心肌纤维化的程度,并计算心肌胶原容积分数(CVF);免疫荧光染色技术标记心肌组织,观察小鼠心肌细胞变化;Western blot检测心肌组织Beclin-1、P62、LC3Ⅰ和LC3Ⅱ蛋白的表达;Real-time PCR检测Beclin-1、STX7、ATG5、LAMP1、LAMP2 mRNA的表达。结果 与溶媒对照组相比,ENT给药3周后小鼠心脏功能无明显变化;给药5周后有下降趋势;给药6周后小鼠出现心功能障碍,表现为LVEF和LVFS明显下降,LVIDd和LVIDs明显增大,LVPWs明显变薄(P<0.05或P<0.01),CWI明显增加(P<0.01),心肌损伤标志物LDH、CK-MB、NT-proBNP和cTnⅠ明显升高(P<0.05或P<0.01)。HE染色结果显示模型组心肌纤维间隙明显增大,心肌结构紊乱;Masson染色结果显示模型组心肌纤维破坏,CVF显著增加(P<0.01);免疫荧光染色结果显示模型组心肌细胞膜受损,心肌胶原含量增加;Western blot结果显示模型组小鼠心肌组织中Beclin-1、p62、LC3Ⅱ/Ⅰ比值蛋白表达均显著增加(P<0.01);Real-time PCR结果显示模型组小鼠心肌组织中Beclin-1、STX7、ATG5、LAMP1、LAMP2 mRNA的表达均显著增加(P<0.05或P<0.01)。结论 成功构建ENT小鼠心脏毒性模型,自噬过度激活可能参与其心脏毒性进程。
Abstract：Objective To establish a mouse model of entrectinib(ENT)-induced cardiotoxicity and determine its potential autophagy mechanism.Methods C57BL/6 mice were randomly divided into an ENT cardiotoxicity model group and a vehicle control group.Mice in the model group received ENT [60mg/(kg·d)] via oral gavage,while the control group received an equal volume of saline with the same administration method.Echocardiography was performed at weeks 3,5,and 6 post-administration.At week6,the mice were sacrificed for heart weight measurement and cardiac weight index(CWI) calculation.Echocardiographic parameters,namely the left ventricular ejection fraction(LVEF),the left ventricular fractional shortening(LVFS),the left ventricular internal diameter at end-diastole(LVIDd),left ventricular internal diameter at end-systole(LVIDs),the left ventricular posterior wall diastolic thickness(LVPWd),and the left ventricular posterior wall systolic thickness(LVPWs) were analyzed to assess the cardiac function.The serum levels of lactate dehydrogenase(LDH),creatine kinase-MB(CK-MB),N-terminal pro-brain natriuretic peptide(NT-proBNP),and cardiac troponin I(cTn I) were measured with ELISA kits.The myocardial histopathology was evaluated by HE and Masson staining to observe fibrosis degree and calculate collagen volume fraction(CVF).Immunofluorescence staining was used to label myocardial tissue and observe changes in cardiomyocytes.Western blot detected Beclin-1,P62,LC3,and LC3 Ⅱprotein expression in the myocardial tissues,while real-time PCR measured the mRNA expression of Beclin-1,STX7,ATG5,LAMP 1,and LAMP2.Results Compared with the vehicle control group,no significant changes in mouse cardiac function were observed after 3 weeks of ENT administration;after 5weeks of administration,a decreasing trend was observed;after 6 weeks of administration,mice developed cardiac dysfunction,manifested as decreased LVEF and LVFS,increased LVIDd and LVIDs,reduced LVPWs(P <0.05 or P <0.01),increased CWI(P <0.01),and elevated levels of the cardiac injury biomarkers LDH,CK-MB,NT-proBNP,and cTnI(P <0.05 or P <0.01).HE staining showed enlarged myocardial fiber interstitium and disorganized myocardial structure in the model group;Masson staining showed disrupted myocardial fibers and a significant increase in CVF(P <0.01) in the model group;immunofluorescence staining showed damaged cardiomyocyte membranes and increased myocardial collagen content in the model group;Western blot demonstrated remarkable upregulation of Beclin-1and p62 proteins,as well as increased LC3 Ⅱ/Ⅰ ratio in the myocardial tissue(P <0.01).Real-time PCR demonstrated that the mRNA expression levels of Beclin-1,STX7,ATG5,LAMP1,and LAMP2were greatly increased(P <0.05 or P <0.01).Conclusion The model of ENT mice cardiotoxicity is successfully established,and over-activation of autophagy participates in the process of cardiotoxicity.
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基本信息:
中图分类号:R965
引用信息:
[1]赵喜林,朱敏燕,房洪杰,等.恩曲替尼致小鼠心脏毒性模型的制备及自噬机制初探[J].中南药学,2026,24(06):15-21.
基金信息:
国家自然科学基金资助项目(No.82373951); 中华医学会临床药学分会临床药学科研基金项目(No.Z-2021-46-2101-2023); 吴阶平医学基金会临床科研专项(No.320.6750.2023-25-9); 苏州市卫生青年骨干人才“全国导师制”项目(No.Qngg2021002); 苏州大学研究生教育改革成果奖培育项目(No.202406)
2026-06-18
2026-06-18