一氧化氮合酶抑制剂治疗实验性自身免疫性心肌炎的研究

Study of L-NAME treatment on experimental autoimmune myocarditis

目的:观察一氧化氮合酶(NOS)抑制剂NG-硝基-L-精氨酸甲酯(L-NAME)对小鼠实验性自身免疫性心肌炎(EAM)的治疗效果并且探讨与凋亡相关的治疗机制。方法:30只Balb/C雄性小鼠,随机分成3组(n=10):正常对照组、模型对照组和实验组。模型对照组和实验组分别在第0天和第7天在小鼠双侧腹股沟和腋窝皮下给予猪心肌肌凝蛋白注射,腹腔注射百日咳毒素制作EAM模型,实验组于第1～21天给予L-NAME(5 mg/(kg.day))腹腔注射,模型对照组给予等剂量生理盐水。第21天眼球取血后断髓处死小鼠,计算心脏重量/体重(HW/BW);HE染色检测各组心肌炎症积分;原位末端缺口标记技术(TUNEL)检测各组心肌细胞凋亡指数;化学发光法检测各组血清一氧化氮(NO)的含量和分光光度法检测血清诱导型一氧化氮合酶(iNOS)的活性;实时定量PCR检测心脏中半胱氨酸天冬氨酸蛋白酶3、半胱氨酸天冬氨酸蛋白酶8和半胱氨酸天冬氨酸蛋白酶9(Caspase-3、Caspase-8和Caspase-9)和iNOSmRNA表达水平;免疫组织化学检测Caspase-3、Caspase-8和Caspase-9蛋白表达水平。结果:模型对照组的HW/BW、心肌炎症积分、NO含量、iNOS活性和mRNA表达、Caspase-3、Caspase-8和Caspase-9 mRNA和蛋白表达以及心肌细胞凋亡指数明显高于正常对照组(P<0.01);与模型对照组相比,实验组除HW/BW(P<0.05)降低外,心肌炎症积分、血清NO含量、血清iNOS活性和心脏iNOS的mRNA表达、Caspase-3、Caspase-8和Caspase-9的mRNA和蛋白表达以及心肌细胞凋亡指数均显著降低(P<0.01)。结论:EAM小鼠心肌组织内高表达的iNOS催化产生的NO促进了心肌细胞的凋亡和EAM病程进展,初期给予L-NAME治疗能通过抑制iNOS活性,减少NO生成,降低对心肌的损伤程度,作用机制可能与其抑制心肌细胞凋亡有关。

Objective： To observe the therapeutic effect of N-nitro-L-arginine methyl ester （L-NAME）, an inhibitor of NOS, on experimental autoimmune myocarditis （EAM） in Balb/C mice and discuss the therapeutic mechanism induced by apoptosis. Methods： Thirty male Balb/C mice were divided into normal control group, model control group and experimental group randomly （ n = 10）. Model control group and experimental group were created into EAM by injection of porcine cardiac myosin subcutaneously in double groin and axilla and pertussis toxin intraperitoneally on day 0 and 7 respectively. Model control group was intraperitoneally administered 5 mg/（kg.day ） of physiological saline af- ter injective myosin and pertussis toxin. Experimental group was intraperitoneally given 5 mg/（kg.day） of L-NAME on day 1-21. The hearts and blood were processed after sacrificed on day 21. Cardiac inflammation score was measured by HE staining. Heart weight / body weight （HW/BW）, serum nitric oxide （NO） level, activity of induced nitric oxide synthase （iNOS） and mRNA expression of iNOS in heart were measured in each group. Degree of heart apoptosis were evaluated by cardiac apoptotic index through TUNEL, examination and real time PCR of Caspase-3. Caspase-8 and Caspase-9. Results： Compared with normal control group, cardiac inflammation score, HW/BW level of NO and activity of iNOS, mR_NA expression of iNOS, the levels of mRNA and protein of Caspase-3, Caspase-8 and Caspase- 9 and cardiac apoptotie index were significantly higher （ P 〈 0.01 ） in model control group, and those of model control group were higher than those of experimental group （ P 〈 0.01 ）. HW/BW was only a little elevation in model control group compared with that in the experiment group （P 〈0.05）. Conclusion： The development of EAM is related with the NO catalyzed by iNOS. L-NAME protects cardiac myocyte v/a suppressing the activity of iNOS and further decreased production of NO in EAM. The mechanism might be that L-NAME alleviated myocardial inflammation thwugh inhibited the apoptosis of eardiac myocyte.