截肢创伤应激后心肌及线粒体损伤的机制及保护

发布时间：2018-06-26 05:10

本文选题：创伤 + 心肌损伤　；参考：《中国人民解放军军医进修学院》2008年硕士论文

【摘要】： 背景与目的:应激是机体对有害刺激(应激原)所做出的适应性综合反应。机体在受到外界刺激如烧伤、缺血再灌注以及手术等创伤应激后,可以引起一系列神经内分泌的激活、氧化应激以及细胞内的Ca~(2+)超载,诱导体内产生大量的血管紧张素Ⅱ(AngⅡ)、醛固酮(Ald)、内皮素(ET-1)以及各种细胞因子和活性氧自由基,从而引起应激性高血压、应激性心律失常、应激性心肌缺血、心功能障碍等。肢体创伤也是一种应激状态,心血管系统是多种应激因素作用的重要靶器官。本实验用手术的方法建立大鼠的左后肢截肢创伤应激模型,动态检测血浆H_2S、NO、髓过氧化物酶(myeloperoxidase,MPO)、丙二醛(MDA)、血管紧张素Ⅱ(angiotensinⅡ,AngⅡ)、醛固酮(aldosterone,ALD)的变化,检测心肌CSE、MPO活性及MDA、ALD含量,并用RT-PCR的方法检测心肌CaN mRNA的表达,观察心肌组织CaN Aβ基因表达的变化,同时光镜下观察心肌的病理损伤,电镜观察心肌线粒体形态和结构的变化。并应用H_2S供体NaHS、CSE抑制剂PPG、CaN抑制剂FK506及醛固酮拮抗剂螺内酯进行干预,观察上述指标及线粒体呼吸功能、膜电位及线粒体总ATP酶的变化,进一步研究H_2S、CaN及醛固酮在肢体创伤应激后心肌损伤中的信号传导机制及各信号通路之间的相互作用,同时研究H_2S、CaN抑制剂FK506及醛固酮受体拮抗剂螺内酯对心血管系统的保护作用。本研究分为三部分:一、截肢创伤应激后心肌H_2S/CSE体系和CaN的动态变化及干预措施:二、截肢创伤应激后心肌损伤相关信号转导机制的研究;三、截肢创伤应激后心肌损伤的线粒体机制。方法:本实验用手术的方法建立大鼠的左后肢截肢创伤应激模型。第一部分实验分组:雄性Wistar大鼠63只,随机分为9组,每组7只:正常对照组;截肢后1h组;2h组;4h组;6h组;12h组;24h组;48h组;72h组。第二部分实验分组:雄性Wistar大鼠42只,分为6组,每组7只:正常对照组;创伤对照组;NaHS组:截肢后立即给予腹腔注射NaHS(28μmol/kg);PPG组:截肢后立即给予腹腔注射PPG(50mg/kg);FK506组:截肢后立即给予腹腔注射FK506(0.1mg/kg);螺内酯组:正常大鼠螺内酯每天灌胃(20mg/kg),6天后截肢。观察各组大鼠心肌病理、血浆H_2S、NO、MPO、MDA、AngⅡ、ALD的变化,同时检测各组大鼠心肌CSE活性、MPO、MDA、ALD的含量;并用RT-PCR的方法检测心肌CaN mRNA的表达,电镜观察心肌线粒体形态结构的变化。第三部分实验动物为48只雄性SD大鼠,随机分为6组,每组8只:分组及处理同第二部分。观察心肌线粒体呼吸功能和膜电位的变化,测定线粒体总ATP酶。结果:第一部分结果:与正常对照组比较,血浆MPO、MDA在截肢后1h即有明显升高,至截肢后6h达高峰,其后虽有所下降,但仍维持在较高水平。心肌MPO及MDA较血浆变化稍晚,于截肢创伤应激后2h开始升高,心肌MPO于12h最高,MDA于截肢后6h最高。血浆H_2S及NO均于创伤应激后4h开始明显下降,6h最低,至创伤后24h逐渐恢复。而心肌CSE酶活性的变化稍晚于血浆H_2S,于创伤后12h最低,72h后才逐渐恢复。第二部分结果:与创伤对照组比,NaHS组及FK506组血浆及心肌MDA、ALD、H_2S、NO含量增加,PPG组血浆MDA、ALD、H_2S、NO及心肌CSE酶活性均下降,螺内酯组血浆H_2S、NO含量增加而心肌CSE酶活性变化不明显。第三部分结果:与正常对照组比,创伤组线粒体呼吸功能下降,RCR及P/O明显降低,ATP酶含量及膜电位下降。与创伤组比较,NaHS组及FK506组心肌线粒体呼吸功能明显改善,RCR、P/O及ATP酶含量增加,膜电位升高,而PPG组RCR、P/O及ATP酶含量减少。与创伤对照组比,螺内酯组线粒体RCR、ATP及膜电位增加。结论:截肢创伤应激后可以造成远隔心肌及线粒体损伤,截肢创伤应激后4～6小时,心肌损伤最重。炎细胞及炎性细胞因子的活化、过度的氧化应激、CaN信号通路及内源性血浆H_2S/心肌CSE体系均参与了截肢创伤应激后心肌损伤。外源性H_2S补充及CaN抑制剂FK506可以通过降低炎细胞浸润及炎性细胞因子激活、直接清除氧自由基、增加心肌线粒体呼吸功能、提高膜电位及线粒体ATP酶产量,对心肌损伤发挥保护作用。CaN信号通路及气体信号通路相互作用共同调节截肢创伤应激后的心肌及线粒体损伤。
[Abstract]:Background and purpose: stress is an adaptive comprehensive response to harmful stimuli (Ying Jiyuan). The body can induce a series of neuroendocrine activation, oxidative stress, and intracellular Ca~ (2+) overload after external stimuli such as burns, ischemia reperfusion and surgery, and induce a large number of blood vessels in the body. Tension II (Ang II), aldosterone (Ald), endothelin (ET-1) as well as various cytokines and reactive oxygen radicals can cause stress hypertension, stress arrhythmia, stress myocardial ischemia, cardiac dysfunction, and so on. Limb trauma is also a stress state, and cardiovascular system is an important target organ for various stress factors.
In this experiment, the rat's left hind limb amputation stress model was established by operation, and the changes of plasma H_2S, NO, myeloperoxidase (MPO), malondialdehyde (MDA), angiotensin II (angiotensin II, Ang II), aldosterone (aldosterone, ALD), and CSE, MPO activity and MDA, content of the myocardium were detected. The expression of CaN mRNA in myocardium was detected and the changes of CaN A beta gene expression in myocardial tissue were observed. At the same time, the pathological damage of myocardium was observed under light microscope. The changes of the morphology and structure of myocardial mitochondria were observed by electron microscope. The intervention of H_2S donor NaHS, CSE inhibitor PPG, CaN inhibitor FK506 and aldosterone antagonist spironolactone were used to observe the above indexes and grain lines. The changes of body respiration, membrane potential and mitochondrial ATP enzyme are further studied. The signal transduction mechanism of H_2S, CaN and aldosterone in the myocardial injury after trauma stress and the interaction of various signal pathways are further studied, and the protective effects of H_2S, CaN inhibitor FK506 and aldosterone receptor antagonist spironolactone on cardiovascular system are also studied.
This study is divided into three parts: first, the dynamic changes of H_2S/CSE system and CaN after the amputation of the amputation stress and the intervention measures: two, the study of the signal transduction mechanism of myocardial injury after the amputation stress; three, the mitochondrial mechanism of myocardial injury after the amputation stress.
Methods: the first part of the experiment was to establish the left hind limb amputation trauma stress model in rats. 63 male Wistar rats were divided into 9 groups randomly, 7 rats in each group: normal control group, group 1h after amputation; group 2H; group 4H; 6h group; 12h group; 24h group; 48H group; 72h group. 42 male Wistar rats were divided into 6 groups. 7 rats in each group: normal control group, trauma control group and group NaHS: immediately after amputation, NaHS (28 mol/kg) was given by intraperitoneal injection; group PPG: PPG (50mg/kg) was given immediately after amputation; group FK506: FK506 (0.1mg/kg) immediately after amputation; spironolactone group: normal rat spironolactone was intragastric daily (20mg/kg), and 6 days after amputation. Observe groups large The changes in plasma H_2S, NO, MPO, MDA, Ang II, ALD were observed in rat myocardium, and the content of CSE activity, MPO, MDA and ALD were detected in each group, and the expression of myocardial CaN was detected by RT-PCR method and changes in the morphological structure of myocardial mitochondria were observed by electron microscope. The third experimental animals were randomly divided into 6 groups, 8 rats in each group. Group and treatment of the same second parts. The changes of respiratory function and membrane potential of mitochondria were observed, and the total ATP enzyme of mitochondria was determined.
Results: the first part results: compared with the normal control group, the plasma MPO and MDA increased significantly after the amputation, and the 6h reached the peak after amputation. After the amputation, the 6h reached a high level, but still maintained at a higher level. The myocardial MPO and MDA were slightly later than the plasma changes, and the 2H began to rise after the amputation stress, and the heart MPO was the highest in 12h, and MDA was the highest after the amputation. Plasma 6h was the highest after amputation. Both H_2S and NO began to decrease obviously after the trauma stress, and the lowest 6h, and the 24h gradually recovered after the trauma. The change of myocardial CSE activity was slightly later than that of plasma H_2S, and 12h was lowest after trauma, and gradually recovered after 72h. The second part: compared with the trauma control group, the plasma and FK506 group of NaHS and FK506 groups were increased. The activity of ALD, H_2S, NO and myocardial CSE decreased, the plasma H_2S, NO content of spironolactone group increased and the myocardial CSE enzyme activity was not significantly changed. Third results: compared with the normal control group, the mitochondrial respiratory function decreased, RCR and P/O decreased significantly, the ATP enzyme content and membrane potential decreased. Compared with the trauma group, NaHS group and FK506 group myocardial mitochondria granules were compared. The volume of body respiration was obviously improved, the content of RCR, P/O and ATP increased, the membrane potential was increased, while the content of RCR, P/O and ATP in group PPG decreased. Compared with the control group, the mitochondrial RCR, ATP and membrane potential of the spironolactone group were increased.
Conclusion: the injury of the amputation injury can cause the injury of the distant myocardium and mitochondria, the most serious injury of the myocardium is 4~6 hours after the amputation stress. The activation of the inflammatory cells and inflammatory cytokines, the excessive oxidative stress, the CaN signaling pathway and the endogenous plasma H_2S/ myocardial CSE system all participate in the myocardial injury after the amputation stress. Exogenous H_2S Supplementation and CaN inhibitor FK506 can directly remove oxygen free radicals, increase myocardial mitochondrial respiratory function, increase membrane potential and mitochondrial ATP enzyme production by reducing infiltration of inflammatory cells and inflammatory cytokines activation, and improve the membrane potential and mitochondrial ATP enzyme production. The protective effect of.CaN signaling pathway and gas signal pathway interaction to regulate amputation stress stress on myocardial injury Injury of the posterior myocardium and mitochondria.
【学位授予单位】：中国人民解放军军医进修学院
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R363

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