硫化氢在大鼠阿霉素心肌病发病中的意义

The role of hydrogen sulfide in adriamycin-induced dilated cardiomyopathy in rats

目的探讨新型气体信号分子硫化氢(hydrogen sul-fide,H2S)对阿霉素(adriamycin,ADR)心肌病大鼠心肌细胞凋亡的影响。方法♂性Wistar大鼠66只,随机分为6组①阿霉素组(ADR组,n=12):腹腔注射ADR,每次2.5mg.kg-1,1次/周,共用药10wk;②ADR+小剂量NaHS组(n=12):ADR用药方法同ADR组,同时经腹腔注射H2S供体NaHS,2.8μmol.kg-1.d-1,连续用药10wk;③ADR+大剂量NaHS组(n=12):ADR用药方法同ADR组,同时经腹腔注射NaHS,14μmol.kg-1.d-1,连续用药10wk;④ADR+PPG组(12只):ADR用药方法同ADR组,同时经腹腔注射PPG30mg.kg-1.d-1,连续用药10wk;⑤对照组(n=9):用相同容量的生理盐水代替ADR,给药方法同ADR组;⑥NaHS组(n=9):经腹腔注射NaHS,14μmol.kg-1.d-1,连续用药10wk。于第10周检测大鼠心功能和血流动力学指标,处死大鼠,取心肌组织制作病理切片,分别于光镜及透射电镜下观察其显微以及超微结构,用TdT介导dUTP缺口末端标记法(TUNEL)方法检测心肌细胞凋亡及用免疫组化测定心肌细胞中Fas和Bcl-2蛋白的表达,同时用敏感硫电极法测定血浆及心肌组织里的H2S含量。结果阿霉素组大鼠左室内压最大上升速率(+dp/dtmax)及左室内压最大下降速率(-dp/dtmax)明显降低(P均<0.01),心肌病理结果显示阿霉素组心肌细胞变性、线粒体和肌浆网水肿、细胞内空泡增多,呈心肌病样改变;心肌细胞凋亡数明显增加,心肌细胞中的Fas蛋白表达明显增加,Bcl-2蛋白的表达明显降低(P均<0.01);同时大鼠血浆及心肌组织H2S含量均明显低于对照组(P<0.01)。经外源性补充H2S供体NaHS后,大鼠心功能较前明显改善,光镜及电镜显示ADR+小剂量NaHS组及ADR+大剂量NaHS组心肌组织病理损害程度明显较阿霉素组减轻;心肌细胞凋亡率明显降低,Fas蛋白表达明显降低,Bcl-2蛋白表达明显增加(P均<0.01)。ADR+PPG组与ADR组相比较,差异无显著性,NaHS组和对照组相比较,差异无显著性(P>0.05)。结论H2S参与大鼠阿霉素心肌病的发病过程,外源性补充H2S供体可以改善阿霉素心肌病大鼠心功能,减轻心肌损伤,减少细胞凋亡,对其发病起到调节作用。

Aim To investigate the effect of hydrogen sulfide（H2S） on apoptosis of myocardial tissues of adriamycin-induced dilated cardiomyopathy in rats and to explore the underlying mechanisms.Methods Weight-matched male Wistar rats were randomly divided into 5 groups as follows：（1） ADR group（n=12）,2.5 mg·kg^-1 of adriamycin was injected intraperitoneally once a week for 10 weeks（total dose of 25 mg·kg^-1）;（2） ADR＋small-dose-NaHS group（n=12）,the dosage and use of adriamycin were as mentioned above,while NaHS solution was injected to rats at a dosage of 2.8 μmol·kg-1·d-1 at the same time;（3） ADR＋large-dose-NaHS group（n=12）,the dosage and use of adriamycin were as mentioned above,while NaHS solution was injected to rats at a dosage of 14 μmol·kg-1·d-1 at the same time;（4） ADR＋PPG group（n=12）,in which the dosage and use of adriamycin were as mentioned above,while PPG solution was injected to rats at a dosage of 30 mg·kg^-1d-1;（5） control group（n=9）,an equivalent volume of physiological saline was administered weekly for 10 weeks.（6） NaHS group（n= 9）,14 μmol·kg-1 of NaHS solution was injected to rats intraperitoneally for 10 weeks.Hemodynamic and echocardiographic measurements were obtained 10 weeks after treatment.Histological examination was implemented at the 10th week after sacrificed.And apoptotic index,Fas and Bcl-2 expressions in myocardiocytes were analyzed semiquantitatively by TUNEL or immunohistochemical analysis.Meanwhile,H2S concentrations in serum and myocardial tissues were evaluated by a modified sulfide electrode-based method.Results Left ventricular systolic pressure（LVSP）,difference of left ventricular pressure（△LVP=left ventricular systolic pressure minus left ventricular diastolic pressure） and ventricular peak rate of contraction（＋LV dp/dtmax）,peak rate of relaxation（-LV dp/dtmax） were significantly reduced in the group of ADR rats（P〈0.01）.Left ventricular diastolic pressure（LVDP）,the ratio of LVW/BW and HW/BW were significantly increased in ADR group（P〈0.01）.Pathological examination demonstrated that characteristics consistent with cardiomyopathy were found in ADR group,such as cardiac muscle cell degeneration,mitochondrial and sarcoplasmic reticular edema,and numerous intracellular vacuoles.Compared with those in control group,apoptotic cells and Fas expression were obviously seen in the ADR group,but Bcl-2 expression obviously decreased（all P〈0.01）.The plasma levels of H2S and H2S in myocardial tissues of the rats in ADR group were both significantly decreased compared with those in the control group（Both P〈0.01）.Exogenous administration of the H2S donor NaHS improved the cardiac function,and histological sections showed that myocardiocytic necrosis and myocardial injury were reduced.And aopoptotic cells and Fas expression were observed weakened,meanwhile Bcl-2 expression increased significantly as compared with ADR group（all P〈0.01）.After the administration with NaHS,the hemodynamic and echocardiographic parameters,ultra-and micro-structure of myocardial tissues were improved markedly with the elevation of H2S concentration in serum and myocardial tissues and at the same time apoptotic index of myocardiocytes as well as Fas expressions were significantly lessened but Bcl-2 expression strengthened as compared with the ADR group（P〈0.01）.The ADR＋PPG group did not differ from ADR group and the NaHS group did not differ from control group（P〉0.05）.Conclusion H2S might play an important role in the development of adriamycin-induced dilated cardiomyopathy.Administration of exogenous H2S could protect the cardiac function and myocardial pathological lesions via the down-regulation of augmented myocardiocyte apoptosis.