CAJ | 학술논문

目的探讨钙激活中性蛋白酶(calpain)在脓毒症小鼠心肌半胱氨酸蛋白酶-3(caspase-3)活化中的作用及其机制.方法 (1)体内实验:腹腔注射脂多糖(LPS,4 mg/kg)建立脓毒症小鼠模型.Western blot检测心肌组织中calpain、caspase-3活性和calpain-1、calpain-2、calpain特异性抑制蛋白calpastatin水平以及凋亡相关蛋白Bcl-2、Bid水平及剪切片段,TUNEL法检测心肌细胞凋亡情况,Langendorff灌注装置评价小鼠心脏的收缩和舒张功能.(2)体外实验:成年大鼠心肌细胞给予LPS(1μg/ml)处理4 h,或同时予以calpain抑制剂calpain inhibitor-Ⅲ(10 μmol/L)干预后,检测心肌细胞calpain和caspase-3活性,Bcl-2、Bid蛋白水平以及心肌细胞凋亡情况.结果 (1)体内实验:在脓毒症小鼠心肌组织中,calpain活性增高2.7倍,caspase-3活性增高1.8倍,给予calpain-inhibitor-Ⅲ或PD150606,均可抑制caspase-3活性的增高.脓毒症小鼠心肌组织calpain-1、calpain-2、calpastatin以及Bcl-2、Bid蛋白水平未见改变,亦未检测到Bcl-2、Bid剪切片段.Calpain inhibitor-Ⅲ则可使脓毒症小鼠心室最快压力上升速率和心室最快压力下降速率分别增加34.5%和34.6%,从而改善脓毒症小鼠心功能障碍.(2)体外实验:LPS可诱导成年大鼠心肌细胞calpain和caspase-3活性增高,给予calpain inhibitor-Ⅲ则可抑制caspase-3活性的增高,心肌细胞Bcl-2、Bid蛋白水平未见改变.体内外实验均未发现LPS可诱导心肌细胞凋亡的增加.结论脓毒症小鼠心肌calpain活性增高,可活化心肌caspase-3,但未导致心肌细胞凋亡,其机制与凋亡蛋白Bcl-2和Bid无关.
목적 탐토개격활중성단백매(calpain)재농독증소서심기반광안산단백매-3(caspase-3)활화중적작용급기궤제.방법 (1)체내실험:복강주사지다당(LPS,4 mg/kg)건립농독증소서모형.Western blot검측심기조직중calpain、caspase-3활성화calpain-1、calpain-2、calpain특이성억제단백calpastatin수평이급조망상관단백Bcl-2、Bid수평급전절편단,TUNEL법검측심기세포조망정황,Langendorff관주장치평개소서심장적수축화서장공능.(2)체외실험:성년대서심기세포급여LPS(1μg/ml)처리4 h,혹동시여이calpain억제제calpain inhibitor-Ⅲ(10 μmol/L)간예후,검측심기세포calpain화caspase-3활성,Bcl-2、Bid단백수평이급심기세포조망정황.결과 (1)체내실험:재농독증소서심기조직중,calpain활성증고2.7배,caspase-3활성증고1.8배,급여calpain-inhibitor-Ⅲ혹PD150606,균가억제caspase-3활성적증고.농독증소서심기조직calpain-1、calpain-2、calpastatin이급Bcl-2、Bid단백수평미견개변,역미검측도Bcl-2、Bid전절편단.Calpain inhibitor-Ⅲ칙가사농독증소서심실최쾌압력상승속솔화심실최쾌압력하강속솔분별증가34.5%화34.6%,종이개선농독증소서심공능장애.(2)체외실험:LPS가유도성년대서심기세포calpain화caspase-3활성증고,급여calpain inhibitor-Ⅲ칙가억제caspase-3활성적증고,심기세포Bcl-2、Bid단백수평미견개변.체내외실험균미발현LPS가유도심기세포조망적증가.결론 농독증소서심기calpain활성증고,가활화심기caspase-3,단미도치심기세포조망,기궤제여조망단백Bcl-2화Bid무관.
Objective In septic mice, myocardial calpain was activated and induced caspase-3 activation, the association between calpain activation and apoptosis was explored in this experiment. Methods In in vivo model, adult C57 mice were injected with lipopolysaccharide (LPS, 4rg/kg, i. p. ) to induce sepsis. Myocardial calpain and caspase-3 activities, protein levels of calpain-1,calpain-2, calpastatin, Bcl-2 and Bid were detected by Western blot analysis and myocardial apoptosis was detected by TUNEL, myocardiac function was evaluated by Langendorff system. In in vitro model, adult rat cardiomyocytes were incubated with LPS (1μg/ml) or co-incubated with calpain inhibitor-Ⅲ (10μmol/L), calpain activity, caspase-3 activity, protein levels of Bcl-2 and Bid, and cardiomyocyte apoptosis were detected. Results In septic mice, myocardial calpain and caspase-3 activity were increased up to 2. 7-and 1.8-folds, respectively. Both calpain inhibitor-Ⅲ and PD150606 significantly attenuated the increase of caspase-3 activity. Myocardial protein levels of calpain-1, calpain-2, calpastatin, Bcl-2 and Bid were similar between control and septic mice, and no cleavage of both Bcl-2 and Bid was found in septic mice. Calpain inhibitor-Ⅲ significantly improved myocardial function in septic mice. In in vitro model, calpain and caspase-3 activities were increased after 4 h LPS treatment, co-treatment with calpain inhibitor-Ⅲ prevented caspase-3 activity increase, protein Bcl-2 and Bid were similar between normal cardiomyocytes and LPS-treated cardiomyocytes. Cardiomyocyte apoptosis was similar in in vivo and in vitro septic models. Conclusion Myocardial calpain activity is increased in LPS induced septic mice, subsequent caspase-3 activation may contribute to myocardial dysfunction in septic mice without aggravating myocardial apoptosis and Bcl-2 and Bid are not involved on calpain induced caspase-3 activation in our model.