【作者】 王艳；

【导师】 柴伟； 姚立农；

【作者基本信息】 第四军医大学 ， 麻醉学， 2010， 硕士

【摘要】 创伤失血性休克(traumatic hemorrhagic shock, THS)是指机体遭受严重创伤后引起的以微循环障碍为主要特征的急性循环功能不全的休克类型,常伴随多器官损伤。创伤失血性休克早期,由于血管内有效血容量不足,组织缺血缺氧性坏死、细胞凋亡,引起大量伤害性化学介质释放;后期液体复苏时缺血再灌注(ischemia-reperfusion, I/R)损伤又增加耗氧,增强氧化应激反应,激发炎症因子爆发,血管被阻塞出现“无复流”现象,导致细胞摄取氧功能障碍,进一步造成器官再灌注损伤,降低复苏后存活率。因此,如何维持组织细胞“氧供-氧耗”平衡,减少缺血再灌注损伤成为救治创伤失血性休克的关键。硫化氢(hydrogen sulfide,H2S)作为第三类气体信号分子,已被证实广泛参与了神经系统、心血管系统、呼吸系统、消化系统、氧化应激、细胞代谢及炎症反应等众多病理生理调节过程。研究发现,H2S具有保护重要器官抵御缺血再灌注损伤、提高机体抗氧化应激性损伤能力、减轻炎症反应的作用,并具有明显的代谢抑制效应。但H2S对于创伤失血性休克是否同样具有保护作用至今仍未见报道。本研究利用大鼠创伤失血性休克模型,通过复苏初期给予外源性硫化氢后,观察模型血流动力学、血气分析指标、重要器官功能及观察病理形态学改变和复苏成功率及存活率的变化,以评价外源性H2S对创伤失血性休克的作用并初步探讨其可能的机制,为创伤失血休克救治提供新思路和方法。目的利用大鼠创伤失血性休克模型观察外源性硫化氢对创伤失血性休克损伤的保护作用并初步探讨其可能的作用机理,为创伤失血性休克的救治提供新思路。方法1、选择健康清洁级成年SD大鼠48只,雄性,220g-250g,随机分为三组,每组16只:假手术组(Sham组),对照组(Vehicle组),NaHS处理组(NaHS组)。采用创伤失血性休克模型,经3%戊巴比妥麻醉后(40 mg/kg ip),于大鼠腹正中切口5cm然后缝合造成软组织创伤;经右侧股动脉插入聚乙烯导管以备放血,分离右侧股静脉插管以备输液,分离左侧股动脉插管连接压力转换器测量平均动脉压(mean arterial pressure, MAP)及心率(heart rate, HR)。所有切口用利多卡因浸润阻滞,除Sham组外,各组均在10min内迅速放血使MAP降至35-40mmHg;若血压不能维持需补充Ringer’s液,此时为最大放血量(maximal bleed-out, MBO),放血量为大鼠总体血容量的60%,(大鼠总血容量为体重的6%)。MBO后,回输40%MBO容量的Ringer’s液以维持血压在35-40mmHg约90mi(n出血后开始),然后以4倍MBO容量的Ringer’s液在60min内进行复苏,复苏后缝合所有伤口。Sham组完成所有手术操作,但不进行放血和复苏;Vehicle组放血后Ringer’s液复苏前,腹腔注射与NaHS组等容量的生理盐水;NaHS组在复苏前给与腹腔注射28μmol/kg NaHS。复苏后的大鼠拔除所有插管,结扎血管,丝线缝合伤口后放回笼子自由进食饮水观察。2、术中持续监测各组平均动脉压MAP、HR,复苏后2h检测左室压(left ventricular pressure,LVP)、左心室压力最大上升速率(positive first derivatives of pressure.+dp/dtmax)和左心室压力最大下降速率(negative first derivatives of pressure ,-dp/dtmax),动脉血气分析等指标变化。3、检测各组大鼠肝功转氨酶及肾功能指标变化。4、检测肺系数与肺湿/干重比等指标变化,并观察肺组织损伤(IQA)的程度。5、监测各组大鼠即刻复苏成活率、复苏后6h、24h存活率的变化。6、测定血浆中H2S、MDA(malondialdehyde)、SOD(superoxide dismutase)、MPO(myeloperoxidase)的变化,观察硫化氢对氧化应激及炎症反应作用。7、制备大鼠心、肺、肝和肾组织切片行HE染色,观察创伤失血性休克复苏后器官病理形态学的变化。结果1、与Sham组相比,Vehicle组复苏后MAP、HR、LVP、+dp/dtmax、-dp/dtmax均明显降低(P<0.05),pH、PaO2值及BE值降低(P<0.05)。与Vehicle组相比,NaHS组MAP、LVP、+dp/dtmax、-dp/dtmax有明显升高(P<0.05),血气指标pH、PaO2值及BE值升高(P<0.05)。2、Vehicle组在复苏后,肝肾功能指标AST(aspartate aminotransferase)、ALT(alanine aminotransferase)、BUN(blood urea nitrogen)、SCr(serum creatinine)升高(P<0.05)。给予NaHS处理后,上述肝肾功能指标有所降低(P<0.05)。3、复苏后,Vehicle组及NaHS组肺系数、肺组织湿/干重比及肺损伤指数(IQA)升高(P<0.05),但NaHS组较Vehicle组肺系数、肺湿/干重比减少,肺损伤指数(IQA)降低(P<0.05),提示肺组织水肿减轻、损伤减小。4、较Vehicle组,NaHS组复苏后24h存活率明显提高(P<0.05)。5、Vehicle组复苏后,血浆中MDA、MPO增加,SOD活性及血浆H2S浓度降低(P<0.05);与Vehicle组相比,NaHS组血浆中MDA、MPO明显降低,SOD活性及血浆H2S浓度增加(P<0.05)。6、较Vehicle组,NaHS组在光镜下心、肝、肾组织病理学形态有明显改善。结论1、外源性硫化氢对创伤失血性休克大鼠具有保护作用,能够改善复苏后血流动力学参数、增强心脏舒缩功能,改善酸中毒状态,提高液体复苏后24h存活率。2、外源性硫化氢能够减轻创伤失血性休克复苏后肺组织水肿,提高肝肾功能,改善心、肺、肝、肾组织病理形态。3、外源性硫化氢对创伤失血性休克大鼠保护作用的机制可能与硫化氢具有的提高机体抗氧化应激反应能力有关。更多还原

【Abstract】 Traumatic hemorrhagic shock (THS) is a type of shock caused by severe trauma with acute circulatory insufficiency. THS is accompanied with the hypovolemia, hypoxemia,apotosis and microcirculatory disturbance, releasing abundant toxic chemical mediators. Resuscitation will also cause ischemia- reperfusion (I/R) damages which will increase the consumption of oxygen,enhance lipid oxidative stress response, stimulate inflammatory cytokines, resulting in dysfunction of cellular oxygen uptake and aggravating tissue damage. It has been reported that treament of reducing oxidative stress damage and inhibiting the outbreak of inflammatory cytokines make sense in the remedy of trauma-hemorrhagic shock.H2S is proved to be extensively involved in many pathophysiological processes of the nervous system, cardiovascular system, respiratory system, digestive system, oxidative stress, cell metabolism, and inflammatory responses. Studies found that H2S can protect vital organs against ischemia-reperfusion injuries, improve the defense against oxidative stress injury, reduce the activation of inflammation, and has significant inhibitory effect on metabolism. However, the effect of H2S on traumatic hemorrhagic shock is still not reported yet.In this study, traumatic hemorrhagic shock model of rats was used to observe the alterations in hemodynamics, cardiac function, tissue injuries, blood gas analysis parameters, and survive rates when giving the exogenous hydrogen sulfide at the beginning of resuscitation. By evaluating the clinical effects of exogenous H2S on traumatic-hemorrhagic shock, we explored the possible mechanisms and expected to provide a new way for the treatments of traumatic hemorrhagic shock.The protective effects of exogenous hydrogen sulfide against trauma-hemorrhagical shock in ratsObjectTo invest the protective effects of exogenous hydrogen sulfide on traumatic hemorrhagic shock in rats.Methods1. Forty-eight male Sprague-Dawely rats were divided into 3 groups randomly as follows: (1) Sham group (n=16); (2) Vehicle group (n=16); (3) NaHS group (n=16). Sham-operated rats underwent all the surgical procedures, except neither hemorrhage nor resuscitation. The rats were given the same volume of vehicle or NaHS solution (28μmol/kg) intraperitoneally at the onset of of the resuscitation in group (2) and (3) respectively. After overnight fast with a free access to water, rats were anesthetized with 3% pentobarbital sodium intraperitoneally (40mg/kg). Trauma hemorrhage and resuscitation model was carried out. Briefly, a 5-cm midline laparotomy was performed to make a soft tissue trauma. The abdominal wound was then closed in two layers with sutures. Polyethylene catheters were put in both femoral arteries and the right femoral vein. The incision sites were then closed. The hemodynamic parameters were measured via one of the arteries using a blood pressure analyzer.Then blood was then withdrawn rapidly to a mean arterial pressure (MAP) of 35–40 mmHg in 10 min through the other artery until the animals could no longer maintain MAP of 35–40 mmHg unless some Ringer lactate solution (RL) was administered. This time and volume were recorded as maximal bleed out (MBO). After the MBO, hypotension was maintained between 35 and 40 mmHg by giving 40% of the MBO volume of RL (about 90 min since the beginning of bleeding). The blood withdrawn was about 60% the same as the total volume (6% of the body weight). The animals were then resuscitated with four times the volume of MBO with RL over 60 minutes in vein. After resuscitation, all the catheters were removed and all wounds were closed with sutures. All the incisions were flushed with lidocaine in order to inhibit pain. At the end of whole procedure, rats was then put in a solo cage with food and water ad libitum.2. Rats were sacrificed 2 hours after resuscitation, and monitored artery mean blood pressure (MAP), heart rate(HR), left ventricular pressure (LVP), positive (+dp/dtmax) and negative (-dp/dtmax) first derivatives of left ventricle pressure. Meanwhile, blood samples were taken to determine the blood gas analysis.3. The serum samples were then analyzed for aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN) and serum creatinine (SCr). 4. W/D ratio and IQA of lung tissue were messured to estimate the edema as well as the injury dgree of pulmonary alveoli.5. The changes of H2S, malondialdehyde (MDA), myeloperoxidase (MPO) level and superoxide dismutase (SOD) activity were determined after taking serum samples.6 .The rats were also monitored the variances of survive rates immediaterly and 6h, 24h after resuscitation.7. Histopathological changes of heart, lung, liver and kidney were observed under light microscope at 2 hours after resuscitation among groups.Results1. Compared with sham group, MAP, HR, LVP, +dp/dtmax and -dp/dtmax decreased largely in vehicle group (P<0.05). Compared with vehicle group, NaHS displayed a rise in MAP, LVP, +dP/dtmax and -dP/dtmax (P<0.05).2. Compared with sham group, pH、PaO2 and BE decreased in vehicle group(P<0.05). Compared with vehicle group, NaHS displayed a rise in pH、PaO2 and BE (P<0.05).3. NaHS showed a reverse against the raising AST, ALT, BUN and SC(rserum creatinine)compared with the vehicle group after T-H(P<0.05).4. In the vehicle group, the W/D ratio of lung tissue and IQA increased compared with sham group (P<0.05).But compared with vehicle group, the W/D ratio and IQA in NaHS group decreased largely (P<0.05).5. NaHS also significantly reversed the high mortality caused by T-H after 24 hours.6. The NaHS-treated group showed a remarkable decreasing in MDA and MPO levels in plasma as well as a rise in SOD activity compared with those in vehicle-treated group.7. The histopathological analysis indicated conservation of morphous in heart, liver and kidney in NaHS.group.Conclusions1. NaHS attenuated the depression of cardiac function, promoted the stability of hemodynamic parameters and reversed the acidosis, enhancing survival rate 24 hours after trauma hemorrhage and resuscitation.2. NaHS decreases edema of lung tissue and pulmonary alveoli. NaHS postconditioning also promoted the restoration of hepatic and renal functions.3. Mechanisms for protective effects of NaHS may be associated with the antioxidative damage of hydrogen sulfide on T-H and resuscitation.更多还原