阿霉素诱导扩张型心肌病模型小鼠心脏淋巴管生成障碍及扩心方干预作用的研究

Study on Myocardial Lymphangiogenesis Disorder in Dilated Cardiomyopathy Mice Induced by Doxorubicin and the Protective Mechanism of Kuoxin Decoction

目的观察阿霉素(Doxorubicin,DOX)诱导扩张型心肌病(Dilated cardiomyopathy,DCM)模型小鼠心脏淋巴管生成的动态变化,并研究中药扩心方(KXD)对心脏淋巴管生成的干预作用。方法应用每周腹腔注射DOX方式构建DCM小鼠模型,并每周进行动态观察。在此基础上,60只C57BL/6小鼠随机分为6组(n=10):空白组,模型组,KXD低、中、高剂量组及卡托普利组。造模成功后应用KXD及阳性对照药卡托普利连续灌胃28天。采用心脏超声检测小鼠心功能,HE染色、Masson染色观察心脏病理形态学改变,Whole-mount免疫荧光染色检测心外膜淋巴管LYVE-1、Podoplanin表达,Western blot检测VEGFR-3蛋白表达,qPCR检测VEGFR-3 mRNA表达。结果DOX诱导DCM小鼠自造模第3周(DOX:15 mg·kg^(-1))心功能降低明显,第5周(DOX:25 mg·kg^(-1))心肌纤维化程度明显(P<0.05);VEGFR-3蛋白自第3周(DOX:15 mg·kg^(-1))表达下降明显(P<0.01),心外膜淋巴管密度自第四周(DOX:20 mg·kg^(-1))降低明显(P<0.01),以上指标均随造模时间延长、剂量的累积而不断加重;应用KXD干预后发现,DCM小鼠心功能改善,心肌纤维化程度减轻,心外膜淋巴管密度升高,VEGFR-3蛋白及mRNA表达水平呈剂量依赖性上升(P<0.01),且KXD效果优于卡托普利。结论DOX诱导DCM小鼠心脏淋巴管生成不足,心肌纤维化严重,心脏功能减弱,且随造模时间延长、剂量的累积而逐渐加重;中药扩心方可促进DOX诱导DCM小鼠心脏淋巴管生成,改善心功能,其机制可能与上调VEGFR-3的表达有关。

Objective To observe the dynamic changes of cardiac lymphangiogenesis in Doxorubicin(DOX)-induced dilated cardiomyopathy(DCM)model mice,and to study the the protective mechanism of Kuoxin Decoction.Methods The DCM mouse model was established by intraperitoneal injection of DOX,and the dynamic observation was performed every week.On this basis,60 C57BL/6 mice were randomly divided into 6 groups(n=10):control group,Model group,LKXD,M-KXD and H-KXD groups and Captopril group.After successful modeling,the KXD and the positive control drug Captopril were administered continuously for 28 days.Echocardiography was used to detect cardiac function in mice,HE staining and Masson staining were used to observe pathological and morphological changes of the heart,Whole-mount immunofluorescent staining was used to detect the expression of LYVE-1 and Podoplanin in epicardial lymphatic vessels,Western blot was used to detect the expression of VEGFR-3 protein,and qPCR was used to detect the expression of VEGFR-3 mRNA.Results DCM mice induced by DOX showed significant cardiac function decline from the third week(DOX:15 mg·kg^(-1),P<0.05),and significant ventricular remodeling at the fifth week(DOX:15 mg·kg^(-1),P<0.01);The lymphatic vessel area of the mouse heart decreased significantly from the fourth week(DOX:20 mg·kg^(-1),P<0.0001),and the expression of VEGFR-3 decreased significantly from the third week(DOX:15 mg·kg^(-1),P<0.01).Conclusion KXD can improve ventricular remodeling and cardiac function in DOX-induced DCM mice,promote cardiac lymphangiogenesis,and upregulate the expression of VEGFR-3 at protein and mRNA levels,with a better effect than captopril.DOX-induced cardiac lymphangiogenesis in DCM mice leads to severe myocardial fibrosis and weakened cardiac function,which gradually worsens with the accumulation of modeling time and dose.KXD can promote cardiac lymphangiogenesis and improve cardiac function in DOX-induced DCM mice.The mechanism may be related to the upregulation of VEGFR-3 expression.