6％HES130/0.4对失血性休克大鼠液体复苏后继发肺损伤的影响及机制研究
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摘要
目的:失血性休克(hemorrhagic shock, HS)及液体复苏可引起全身炎症反应综合征(systemic inflammatory response syndrome, SIRS),SIRS发展失控必然导致多器官功能障碍综合征(multiple organ dysfunction syndrome, MODS);SIRS在肺部表现为急性肺损伤(acute lung injury, ALI)。SIRS→ALI→ARDS→MODS,体现着多米诺骨牌效应。因此,明确HS时ALI/ARDS的发病机制,早期预防和治疗ALI/ARDS,对降低HS的死亡率及改善疾病的预后具有重要意义。
HS导致肠源性菌血症和内毒素血症,是诱发ALI/ARDS的重要因素。继发的中性粒细胞(polymorphonuclear neutrophils, PMNs)过度活化,肺内炎症反应失控,肺血管内皮细胞的坏死和凋亡等是构成HS液体复苏后ALI/ARDS的主要作用机制。在ALI/ARDS的发病进程中,炎症反应导致肺通透性增高,其在内源性ARDS主要表现在肺微血管内皮;外源性ARDS则主要开始于肺泡上皮。炎症介质的释放、中性粒细胞与内皮细胞的相互作用以及细胞骨架的变化是导致肺微血管内皮通透性增高的主要原因。
HS后,尤其是在血源匮乏的情况下,复苏液体的选择要优先考虑在有效恢复血流动力学的前提下,改善机体的病理生理状况,提高生存率。羟乙基淀粉是临床上最常用的血浆容量扩张剂,其扩容强度和维持时间,决定于它们的浓度和相对分子质量以及克分子取代程度和取代方式。6%HES130/0.4(6%hydroxyethyl starch 130/0.4, Voluven)是最新一代羟乙基淀粉,克分子量为130 000道尔顿,取代级为0.4,C2/C6羟乙基化的比率为9︰1;与其他HES溶液和右旋糖苷比较,容量扩容效果相同,但副作用更少。6%HES130/0.4除具有良好的扩容作用外,还可减少白细胞迁移,减轻炎性反应,可减轻大鼠内毒素性ALI,但其对HS液体复苏后继发肺损伤的作用及其机制尚无定论,故本研究欲加以探讨。
本课题选用实验外科技术,采用颈总动脉放血法造成大鼠失血性休克,应用有创动脉血压监测技术对不同休克维持时间的大鼠行液体复苏,建立SD大鼠HS液体复苏后继发ALI模型,并在此基础上用HES130/0.4早期干预;应用血气分析、病理形态学观察、透射电镜技术、流式细胞术、酶联免疫技术等检测方法,观察肺功能变化、肺脏病理形态及超微结构变化、肺组织匀浆中TNF-α、IL-1β、IL-10含量的变化以及动脉血中性粒细胞CD11b、CD18表达的变化,探讨肺血管内皮细胞及中性粒细胞在HS液体复苏后继发ALI病程进展中的作用及机制,以及HES130/0.4对HS液体复苏后继发ALI早期干预的作用及机制;并在动物实验基础上,进一步观察了HES130/0.4对体外培养的肺微血管内皮细胞损伤的作用及相关机理,为HS液体复苏致ALI的早期干预提供新的治疗策略及理论依据。
方法:
第一部分:失血性休克大鼠液体复苏后继发肺损伤模型的建立
48只SD大鼠,按随机数字表法分为45min休克组(H45组)及45min对照组(C45组)、60min休克组(H60组)及60min对照组(C60组)和90min休克组(H90组)及90min对照组(C90组),每组8只。对照组仅麻醉及行动静脉穿刺置管,不放血及液体复苏。不同休克时间组按休克维持时间分为H45组、H60组以及H90组。颈总动脉放血法造成大鼠失血性休克,按照实验设计分别维持不同休克时段后,通过左侧股静脉输注三倍最大放血量的林格氏液进行复苏。计算各组的失血程度;记录不同时间点MAP;分别于休克前、液体复苏前即刻以及复苏后2、3h时行血气分析,并计算PO2/FiO2的比值;复苏3h后采用颈总动脉放血法快速处死动物,大体观察各组肺脏充血、水肿、出血情况,光镜下观察肺组织病理学变化,并计算各组肺组织的病理评分,测定支气管肺泡灌洗液(BALF)蛋白浓度和肺湿/干重比(W/D)。
第二部分:6%HES130/0.4对失血性休克大鼠液体复苏后肺组织TNF-α、IL-1β和IL-10的影响
成年雄性SD大鼠24只,按随机数字表法随机分为对照组(control组)、林格氏液组(RS组)、33ml/kg复苏组(H1组)、50ml/kg-1复苏组(H2组),每组6只。对照组(control组)仅麻醉及进行动静脉穿刺,不放血;林格氏液组(RS组)用三倍最大放血量的林格氏液复苏;H1组、H2组分别用33ml/kg、50ml/kg的羟乙基淀粉和林格氏液复苏。为保证理论上复苏效果的相同,H1、H2组复苏所用林格氏液的量为3倍最大的放血量减去相应剂量的羟乙基淀粉。
计算各休克组的最大失血程度;记录不同时间点MAP;分别于休克前(T0),休克后复苏前(T1),复苏后2(T4)、3(T5)h行血气分析,并计算PaO2/FiO2;于复苏后3h处死动物,测定BALF蛋白浓度及肺W/D;测定肺组织肿瘤坏死因子(TNF-α)、白细胞介素-1β(IL-1β)和白细胞介素-10(IL-10)的含量;大体观察肺脏充血、水肿、出血情况;光镜下观察肺组织病理学变化并计算肺组织的病理评分;透射电镜下观察肺超微组织结构变化。
第三部分:6%HES130/0.4对失血性休克大鼠液体复苏后肺组织MDA、SOD和MPO的影响
动物选择与分组同第二部分。
休克组分别于液体复后3h、control组于相应时间点处死动物,取右肺下叶肺组织测定MDA含量、SOD和MPO活性。
第四部分:6%HES130/0.4对失血性休克大鼠液体复苏后血中性粒细胞CD11b和CD18表达的影响
动物的选择和分组、HS模型的复制同第二部分;
分别于休克前(T0),复苏前即刻(T1),复苏后2h(T4)、3h(T5)行血气分析;流式细胞术(Flow Cytometry,FCM)检测动脉中性粒细胞CD11b和CD18的表达水平;复苏后大体观察肺脏充血、水肿、出血情况;光镜下观察肺组织病理学变化并计算肺组织的病理评分;透射电镜下观察肺超微组织结构变化。
第五部分:6%HES130/0.4对大鼠肺微血管内皮细胞凋亡率的影响
1用改良组织块法进行肺微血管内皮细胞(pulmonary microvascular endothelial cell, PMVECs)原代培养,倒置相差显微镜下观察细胞形态,扫描电镜、透射电镜下观察细胞形态及超微结构,进行细胞鉴定;2-3代培养的细胞用于下一步实验。
2将所得的PMVECs悬液浓度调整到1×109个/ml, 24孔细胞培养板中每孔加入1ml细胞悬液,共15孔,随机分为3组,空白对照组(C组)、LPS处理组(L组)和HES干预组(H组),每组5孔。L组加入1μg/ml LPS和等体积PBS液,H组加入1μg/ml LPS、等体积PBS液以及6% HES130/0.430mg/ml;C组加入等体积PBS液。将细胞培养板置于CO2细胞培养箱中,分别孵育3h后取出培养板后收集细胞。透射电镜、扫描电镜下观察细胞形态及超微结构;流式细胞仪检测PMVECs凋亡率和细胞增殖情况
结果:
第一部分:失血性休克大鼠液体复苏后继发肺损伤模型的建立
1各休克组大鼠失血程度、MAP组间差异无统计学意义;
2与各对照组比较,各休克组于T1时间点PaO2/FiO2升高,PaCO2降低(P<0.05);H45组、H90组于T5时间点PaO2/FiO2降低(P<0.05)。与H45组比较, H60组、H90组于T4时间点PaO2/FiO2、PaCO2降低(P<0.05)。与H60组比较,H90组于T5时间点PaO2/FiO2降低,于T4、T5时间点PH升高(P<0.05);
3.1肺大体观察结果:各对照组无明显改变;各休克组肺脏可见不同程度的缺血、充血和水肿,以H90组最为明显。
3.2各对照组肺泡结构未见明显异常;H45组、H60组肺间质增宽,炎性细胞浸润,H60组小支气管见炎性渗出和浸润;H90组肺泡腔内见红细胞及炎性细胞,肺间质增宽,炎性细胞浸润,间质小血管扩张充血;与各自对照组比较,H45组、H60组和H90肺组织病理评分增高(P<0.05);与H45或者H60组比较,H90肺组织病理评分增高(P<0.05);
3.3各对照组肺组织超微结构未见明显异常;H45组肺泡Ⅱ型细胞基本正常,板层颗粒正常,线粒体膜缺损;H60组核周间隙扩张,粗面内质网轻度扩张;H90组血管内皮基底膜断裂,气血屏障结构模糊,水肿增厚;
3.4与对照组比较,各休克组BALF蛋白浓度、肺W/D不同程度升高(P<0.05或0.01);与H45或H60比较,H90组BALF蛋白浓度、肺W/D显著升高(P<0.05或0.01);
第二部分6%HES130/0.4对失血性休克大鼠液体复苏后肺组织TNF-α、IL-1β和IL-10的影响
1各液体复苏组最大失血程度、不同时间点MAP组间比较差异无统计学意义;
2与control组比较,RS组、H1、H2组于T1时间点PaO2/FiO2升高,PaCO2降低(P<0.05);RS组于T4、T5时间点,H2组于T5时间点PaO2/FiO2(P<0.05)。与RS组比较, H1组T4、T5时间点,H2组于T4时间点PaO2/FiO2升高,H1、H2组于T4时间点PaCO2升高(P<0.05);与H1组比较,H2组于T5时间点PaO2/FiO2降低(P<0.05);
3与control组比较,RS组、H1、H2组肺组织匀浆TNF-α、IL-1β和IL-10含量、BALF蛋白浓度、W/D升高(P<0.05);与RS组比较,H1、H2组肺组织匀浆TNF-α、IL-1β含量降低,IL-10含量升高,BALF蛋白浓度、肺W/D降低(P<0.05);
4.1肺大体观察结果:control组无明显改变;各液体复苏组肺脏可见不同程度的缺血、充血和水肿,以RS组最为明显;
4.2 control组肺泡结构正常;RS组肺泡腔内见红细胞及炎性细胞,肺间质增宽,炎性细胞浸润,间质小血管扩张充血;H1组和H2组肺间质增宽,炎性细胞浸润,小支气管见炎性渗出和浸润;H1组、H2组肺组织损伤较RS组轻,其中H1组损伤最轻。与control组比较,各液体复苏组肺组织病理评分增高(P<0.05);与RS组比较,H1组和H2组肺组织病理评分降低(P<0.05);与H1组比较,H2组肺组织病理评分增高(P<0.05);
4.3 control组肺超微组织形态结构未见异常;RS组肺组织超微结构受损,血管内皮基底膜断裂,Ⅱ型上皮细胞微绒毛明显减少,粗面内质网扩张,可见颗粒融合现象;H1组除线粒体结构轻微改变外基本接近正常; H2组核周间隙轻度扩张,粗面内质网轻度扩张,有脱颗粒;
第三部分:6%HES130/0.4对失血性休克大鼠液体复苏后肺组织MDA、SOD和MPO的影响
1与control组比较,RS组、H1组和H2组MDA含量升高(P<0.05);与RS组比较,H1组和H2组MDA含量降低(P<0.05);与H1组比较,H2组MDA含量升高(P<0.05);
2与control组比较,RS组、H1组和H2组SOD活性降低(P<0.05);与RS组比较,H1组和H2组SOD活性升高(P<0.05);与H1组比较,H2组SOD活性减低(P<0.05);
3与control组比较,RS组、H1组和H2组MPO活性升高(P<0.05);与RS组比较,H1组和H2组MPO活性降低(P<0.05);
第四部分:6%HES130/0.4对失血性休克大鼠液体复苏后血中性粒细胞CD11b和CD18表达的影响
1复苏后各时点MAP组间比较差异无统计学意义。
2与control组比较,各液体复苏组于T1时间点PaO2升高,T1~5 PaCO2均降低(P<0.05);与T0比较,各液体复苏组PaO2于T1时间点、H1组T1~5时间点、H2组T5时间点升高;PaCO2各液体复苏组T1~5时间点降低;除H1组外,各液体复苏组PH于T4、5时间点降低(P<0.05);
3与control组比较,各液体复苏组于T1、T4、T5时间点CD11b和CD18的表达增强(P<0.05);与RS组比较,H1、H2组于T4、T5时间点CD11b和CD18的表达降低(P<0.05);与H1组比较,H2组于T4、T5时间点CD11b和CD18的表达增强(P<0.05);
4.1肺大体观察结果:control组无明显改变;各液体复苏组肺脏可见不同程度的缺血、充血和水肿,以RS组最为明显;
4.2 control组肺泡结构正常;RS组肺泡腔内见红细胞及炎性细胞,肺间质增宽,炎性细胞浸润,间质小血管扩张充血;H1组和H2组肺间质增宽,炎性细胞浸润,小支气管见炎性渗出和浸润;H1组、H2组肺组织损伤较RS组轻,其中H1组损伤最轻。与control组比较,各液体复苏组肺组织病理评分增高(P<0.05);与RS组比较,H1组和H2组肺组织病理评分降低(P<0.05);与H1组比较,H2组肺组织病理评分增高(P<0.05);
4.3 control组肺超微组织形态结构未见异常;RS组肺组织超微结构受损,血管内皮基底膜断裂,Ⅱ型上皮细胞微绒毛明显减少,粗面内质网扩张,可见颗粒融合现象;H1组除线粒体结构轻微改变外基本接近正常; H2组核周间隙轻度扩张,粗面内质网轻度扩张,有脱颗粒;
第五部分:6%HES130/0.4对大鼠肺微血管内皮细胞凋亡率的影响
1倒置显微镜下观察,培养的细胞呈铺路石样排列;扫描电子显微镜观察到培养的细胞表面存在微绒毛,细胞表面存在窗孔;透射电镜下内皮细胞表面有胞浆突起,核仁大且明显;胞质内有高尔基复合体,粗面内质网和滑面内质网发达;
2扫描电镜结果:C组细胞细胞形态无明显变化,呈梭形或多角形;L组、H组部分细胞形态变圆,细胞边缘突起减少,未见凋亡小体;
3 HES130/0.4对LPS诱导活化的PMVECs细胞增殖指数及凋亡率的影响:与C组比较,L组、H组PI无统计学差异(P>0.05);与L组比较,H组PI无统计学差异(P>0.05)。与C组比较,L组、H组凋亡率增加(P<0.05);与L组比较,H组凋亡率降低(P<0.05)。
结论
第一部分:失血性休克大鼠液体复苏后继发肺损伤模型的建立失血性休克大鼠在休克时间维持在90min后林格氏液复苏后3h肺功能、肺形态学出现典型的损伤性的变化,是稳定、可靠的失血性休克液体复苏后继发肺损伤的动物模型;
第二部分:6%HES130/0.4对失血性休克大鼠液体复苏后肺组织TNF-α、IL-1β和IL-10的影响
1与林格氏液比较,6%HES130/0.4可明显降低HS液体复苏后肺脏组织炎性介质TNF-α、IL-1和IL-10的含量,减轻失血性休克液体复苏后继发肺组织病理学损伤,从而改善肺的呼吸功能;
2 6%HES130/0.4 33ml/kg和50ml/kg HES液体复苏能不同程度减轻肺脏炎性反应,且33ml/kg剂量复合林格氏液作用明显;
第三部分:6%HES130/0.4对失血性休克大鼠液体复苏后肺组织MDA、SOD和MPO的影响
与林格氏液复苏比较,6%HES130/0.4可减轻HS液体复苏后肺组织MDA、SOD和MPO的变化,进而减轻继发肺组织损伤,且33ml/kg 6% HES130/0.4复苏较50ml/kg剂量作用明显;
第四部分:6%HES130/0.4对失血性休克大鼠液体复苏后血中性粒细胞CD11b和CD18表达的影响
与林格氏液相比,6%HES130/0.4可使血中性粒细胞CD11b和CD18的表达显著降低,减轻肺组织的病理性损伤,从而改善肺功能;
第五部分:6%HES130/0.4对大鼠肺微血管内皮细胞凋亡率的影响
1 HES130/0.4处理后通过降低活化PMVECs凋亡率影响细胞的凋亡;
2 HES130/0.4处理后所引起的细胞凋亡改变与细胞增殖周期的变化无明显关系。
Objectives:
Hemorrhagic shock(HS) might cause systemic inflammatory response syndrome (SIRS). It would certainly result in multiple organ dysfunction syndrome (MODS) if the development of SIRS was out of control. SIRS in lungs was manifested by acute lung injury (ALI). Obviously, the process, SIRS→ALI→ARDS→MODS, reflected the dormino effects. Therefore, the determination the pathogenesis of ALI/ARD, and the early prevention and treatment for ALI/ARD, were significant for reducing mortality rate and impoving the prognosis of HS.
Intestinal bacteriemia and endotoxemia induced by HS, was an important causative factor of ALI/ARDS. And the main mechanisms of ALI/ARDS after HS, were the resulting over activation of polymorphonuclear neutrophils (PMNs), the incontrollable inflammation in lungs, and the necrosis or apoptosis of pulmonary vascular endothelial cells. During the process of ALI/ARDS, the inflammation response resulted in the increased permeability of lungs. The endogenous ARDS usually appeared on pulmonary microvascular endothelium, and the exogenous ARDS on alveolar epithelium. The main reasons that pulmonary microvascular endothelium permeability increases were the release of inflammatory mediator, the interaction between polymorphonuclear neutrophils and endothelial cells, and the changes of cytoskeletons.
A prior consideration should be taken, that was to improve the pathology state and survival rate on the premise of regaining the hemodynamics, when chosing resuscitation fluid after a hemorrhagic shock, especially in the shortage of blood. Hydroxyl starch (HES) was the most common plasma volume expander on clinical application, whose expansion capability and maintenance was determined by its concentration, relative molecular mass, and the degree or patterns of gram molecule substitution. 6%HES130/0.4(hydroxyethyl starch 130/0.4, Voluven)was a new generation of HES, with a gram molecular weight of 130,000 dalton, substitution degree of 0.4, and C2/C6 hydroxyethylated rate of 9:1. Compared with other HES fluids and daxtran, it had the same effect of volume expansion, but less side effects. Besides a good expansion effect, the 6%HES130/0.4 could also reduce the leucocytes migration, the inflammatory reaction and endotoxin-induced ALI in rats. But it had no fixed conclusion about the effects and mechanisms for the damage of non-infectious pulmonary inflammatory.
This study was carried out with experimental surgery technique. Models with hemorrhagic shock were made in rats by blood loss from carotid. Then invasive monitoring of arterial pressure method was used to resuscitate rats in HS with different shock-time duration, and estabilished the SD rat ALI models. Based on these, HES130/0.4 was used for early intervention. To observe the changes of pulmonary function, pathomorphology and ultra-microstructure, TNF-α、IL-10 in lung tissue homogenates and CD11b\CD18 of pulmonary polymorphonuclear neutrophils were made; to study the effects or the mechanism of pulmonary vascular endothelial cell and polymorphonuclear neutrophils in the pocess of ALI induced by HS fluid resuscitation, and the effects and mechanisms of HES130/0.4 on the early intervention with ALI induced by HS fluid resuscitation, several detection technologies were used here, such as fully auto blood gas analysis, pathomorphological observation, transmission electronic microscope, flow cytometry, enzyme-linked immunosorbent assay, and so on. On the basis of animal experiment, the effects and mechanism of HES130/0.4 on the damage of pulmonary microvascular endothelial cells had been futher observed. So, new treatment strategies and theories were provided for early intervation with ALI induced by hemorrhagic shock and fluid resuscitation.
Methods:
Part I: Establishing models of acute lung injury induced by hemorrhagic shock and resuscitation in rats
Fourty-eight SD rats were divided into 6 groups (45min hemorrhagic group,60min hemorrhagic group,and 90min hemorrhagic group and their corresponding control groups) by means of random number table. The control operation group was only anesthetized and dealt with venipuncture indwelling catheter,without removal of blood or fluid resuscitation. Different shock duration groups were divided into group H45, group H60, group H90 by time. The models of hemorrhagic shock in rats established with the method of blood in carotid atery were resuscitated by infusing Ringer's lactate of 3 times the maximum volume of the removed blood in femoral veins after shock. Then, the hemorrhagic level of different groups were calculated; MAP at different time points were recorded; blood gas analysis were taken before shock, before fluid resuscitation and during the 3 hours resuscitation separately, and the PO2/FiO2 ratios were calculated. These animals were killed quickly by the method of removal blood in carotid artery after 3 hours resuscitation. Then the lung congestion, edema, hemorrhage were generally observed in each group. The pulmonary pathological changes at light microscope observed and the lung pathological score were calculated in the different groups. And the ultrastructure in lung tissue was observed by transmission electron microscope (TEM). The brochoalveolar lavage fluid (BALF) protein concentration and the wet/dry weight (W/D) ratio of lung were determined.
Part II: Effect of 6% HES 130/0.4 resuscitation on lung TNF-α, IL-1βand IL-10 in hemorrhagic shock rats
Twenty-four adult male SD rats were divided into 4 groups: control operation group (group control) , Ringer's lactate group (group RS), resuscitation group of 33ml/kg (group H1), resuscitation group of 50ml/kg (group H2) by the means of random number table. The rats in control group was anesthetized and dealt with only venipuncture,without blood removal. After shock, Ringer's solution of 3 times the maximum volume of the removed blood was infused in femoral veins to make resuscitation in group RS. 33ml/kg of 6% HES130/0.4 were infused to make resuscitation in group H1 and 50ml/kg in H2 group. To ensure the same effect of theoretical resuscitation, the Ringer's solution equalled 3 times the maximum volume of the removed blood minus corresponding amount of hydroxyethyl starch in H1and H2 were infused.
The largest hemorrhagic level of different shock groups were calculated; MAP at different time points were recorded; Blood gas analysis were made before shock (T0), before resuscitation (T1), after 2 hrs (T4) and 3 hrs (T5) of resuscitation; PO2/FiO2 ratio were calculated. Rats were killed after 3 hrs of resuscitation. BALF protein concentration and the lung wet/dry weight (W/D) ratios were determined. The concentrations of tumor necrosis factor-α(TNF-α), interleukin-1 (IL-1β) and interleukin-10 (IL-10) in lung issue were determined. Generally the lung congestion, edema, hemorrhage in each group were observed. The pulmonary pathological changes were observed at light microscope and the lung pathological score were calculated in the different groups. The ultrastructural organization were observed in lung tissue by TEM.
Part III: Effect of 6% HES 130/0.4 resuscitation on lung MDA, SOD and MPO in hemorrhagic shock rats
Rats was selected and classified as in the second part.The rats were killed at 3h after fluid resuscitation in shock groups, and at the same time in to obtain the lower lobe lung issue in right lung. And the amount of MDA, activity of SOD and MPO were determined.
Part IV: Effect of 6% HES 130/0.4 resuscitation on the expression of CD11b and CD18 in arterial PMNs in hemorrhagic shock rats
Rats’selection, group and establishing models of hemorrhagic shock were the same as described in second part. To reduce the differences in different groups during the test with flow cytometry (FCM), this part of the experimental rats were divided into batches, each batches of four, divided into control operation group (group S),Ringer's lactate group (group RS) , resuscitation group of 33ml/kg (group H1) , resuscitation group of 50ml/kg (group H2)
Blood gas analysis were made before shock (T0), before resuscitation (T1), 2 hours (T4) and 3 hours (T5) after resuscitation separately; Flow Cytometry (FCM) were detected for the expression level of polymorphonuclear neutrophils (PMNs) of CD11b and CD18 in artery blood.
Part V: Exprimental study of the effect of 6% HES 130/0.4 on the apoptosis rate of injuried pulmonary microvascular endothelial cell induced by LPS
1. Modified tissue block pasted culture method was used in primary culture of pulmonary microvascular endothelial cell (PMVECs). The tissue block were inverted under the phase contrast microscope and the cellular morphology were observed. The cellular morphology and ultrastructure were observed with scanning electron microscope (SEM) and TEM, and the cells were identified. 2-3 generation of cells cultured were used in next experiments.
2. Suspension concentration of the first part of PMVECs was adjusted to 1×109/ml. 1ml of cell suspension was added into each hole of a 24 holes’cell culture plate. A total of 15 holes were randomly divided into 3 groups: control group (group C)、LPS treatment group (group L) and HES intervention group (group H). Group L was added with 1μg/ml LPS and other fluid PBS with the same volume. H group was added with 1μg/ml LPS and other fluid PBS with the same volume and HES130/0.4 30mg/ml. Group C was added with the fluid PBS with the same volume. The cell culture plates were put into cell incubators, removed after 3 hours and the cells were collected. The cellular morphology and ultrastructure were observed by SEM and TEM. Apoptosis rate and cell proliferation of PMVECs were detected by flow cytometer. Results:
Part I: Establishing models of acute lung injury induced by hemorrhagic shock and resuscitation in rats
1. Each group of rats were alive till the experiments. It was meaningless in statistics in the groups of shock about the blood loss level and the interblock diference of MAP.
2. Compared with the value in group control, PaO2/FiO2 rised at time point T1, PaCO2 were decreased (P<0.05) in three shock groups; in group H45 and H90, PaO2/FiO2 decreased at time point T4 (P<0.05). Comapared with the value in group H45 and H60, PaO2/FiO2 rised at time point T5, and PaCO2 decreased (P<0.0) in group H90. Compared with the value in group H60, PaO2/FiO2 decreased at time point T5, and PH rised at time point T4 and T5 in group H90 (P<0.05).
3.1 The general observing results of lung tissue: no obvious changes were observed in the group control. Ischemia, congestion and oedema in various degrees could be found in each group of shock rats, espessially in group H90.
3.2 The structure of alveolus was not found obvious abnormity. In the groups H45 and H60, the lung interstitial substance was broadened, and inflammatory cell infiltration could be found. In group H60, inflammatory exudation and lammatory infiltration in small bronchus could be found. Hematid and inflammatory cell in the intracavity of alveolus could be found, the broadened lung interstitium, inflammatory cell infiltration, expansion and congestion interstitium minute vessel could be found in group H90. Compared with the values in group control and H45, group H60 and H90, pathology grade of lung tissue rised (P<0.05). Compared with the value in group H60, pathology grade of lung tissue in H90 rised (P<0.05).
3.3 In group control, there was no obvious abnormity found in ultrastructure of lung tissue. In group H45, alveolitoidⅡcell was basically nomal, lamellated granule was nomal, but mitochodrial membrane was impairment.In group H60, the interstistial substance of lung was broaden, inflammatory cell intitration, inflammatory exudation and lammatory infiltration cound be found in small bronchus. In group H90, hematid and inflammatory cell in the intracavity of alveolus, the broadened interstitial substance of lung, inflammatory cell infiltration, expansion and congestion of interstritial substance of minute vessel could be found.
3.4 Compared with the value in group control, viscosity albuminose of BALF and W/D of lung rised in each shock group (P<0.05 or 0.01); Compared with the value in group H45 and H60, lung viscosity albuminose of BALF and W/D rised (P<0.05or 0.01) in group H90.
Part II: Effect of 6% HES 130/0.4 resuscitation on lung TNF-α, IL-1βand IL-10 in hemorrhagic shock rats
1. There was no difference statisticly of MAP at different time points between fluid resuscitation groups.
2. Compared with the value in group control, PaO2/FiO2 increased and PaCO2 decreased (P<0.05) at time point T1 in group H1, H2 and RS. In group RS, PaO2/FiO2 increased at time point T4 and T5. PaO2/FiO2 in group H2 increased at time point T5 (P<0.05). Compared with the value in group RS,, PaO2/FiO2 increased at time point T4 and T5 in group H1. PaO2/FiO2 increased at time point T4 in group H2. In the group H1 and H2, PaCO2 increaesd at time point T4 (P<0.05). Compared with the value in group H1, PaO2/FiO2 decreased at time point T5 (P<0.05) in group H2.
3. Compared with the value in group control, IL-10 and protein in BALF and lung W/D increased in group RS, H1 and H2 (P<0.05); compared with the value in group RS, the concentration of TNF-α, IL-1β, the protein in BALF and lung W/D in group H1 and H2 decreased, while the concentration of IL-10 increased (P<0.05).
4.1 The observed results of lung tissue: there was no obviously change in group control; Ischemia, congestion and edema in various degrees could be found in each fluid resucitation group, especially in group RS.
4.2 The stucture of alveolus was nomal in group control. Hematid and inflammatory cell in the intracavity of alveolus, the broadened interstitial substance of lung, inflammatory cell infiltration, expansion an congestion of interstitial substance of minute vessel could be found in group RS. The broadened interstitial substitial of lung, inflammatory cell infiltration, and inflammatory exudationg and lammatory infiltration in interstitial substance of minute vessel could be found in group H1 and H2. The damage of lung tissue in group H1 and H2 were less than the group RS, the least damage of lung tissue was in group H1. Compared with the value in group control, the pathology grade of lung tissue increased in each of fluid resuscitation group (P<0.05). Compared with the value in group RS, the pathology grade of lung tissue was decreased in group H1 and H2 (P<0.05). Compared with the value in group H1, the lung tissue pathology grade increased in the group H2 (P<0.05).
4.3 In group control, the ultrastructure of lung tissue was obviously nomal, while the ultrastructure of lung tissue was damaged in group RS. Basement membrane of blood vessel was reptured, epithelial microvillus was obviously reduced, rough surfaced endoplasmic reticulum was expanded, and the phenomenon of kermel integration could be found; Those in group H1 were basically nomal except slight structural modificationg of mitochondria; Perinucleat space was low-grade expanded, rough surfaced endoplasmic reticulum was low-grade expanded and there was phenomenon of degranlation in group H2.
Part III: Effect of 6% HES 130/0.4 resuscitation on lung MDA, SOD and MPO in hemorrhagic shock rats
1. Compared with the value in group control, the concentration of MDA increased in group RS, H1 and H2 (P<0.05); Compared with the value in group RS, the concentration of MDA decreased in group H1 and H2 (P <0.05); Compared with group H1, the concentration of MDA increased in group H2 (P <0.05);
2. Compared with the value in group control, the concentration of SOD decreased in group RS, H1 and H2 (P<0.05); Compared with the value in group RS, the concentration of SOD increased in group H1 and H2 (P <0.05); Compared with the value in group H1, the concentration of SOD decreased in group H2 (P <0.05);
3. Compared with the value in group control, the concentration of MPO increased in group RS, H1 and H2 (P<0.05); Compared with the value in group RS, the concentration of MPO decreased in group H1 and H2 (P <0.05); There was no difference statisticly of MPO beteween group H1 and H2 (P >0.05);
Part IV: Effect of 6% HES 130/0.4 resuscitation on the expression of CD11b and CD18 in arterial PMNs in hemorrhagic shock rats
1.There was no statistical significance of MAP at each time point between groups (P >0.05);
2.Compared with the value in group control, PaO2 increased at T1, while PaCO2 decreased at T1~5 in every fluid resuscitation group (P<0.05); compared with the value at T0, PaO2 in every fluid resuscitation group at T1, group H1 at T1~5 and group H2 at T5 increased; PaCO2 in every fluid resuscitation group decreased at T1~5; except group H1, PH in every fluid resuscitation group was lower at T4, 5 (P<0.05);
3.Compared with the value in group control, the expression of CD11b and CD18 in every fluid resuscitation group increased at T1, T4, T5 (P<0.05); compared with the value in group RS, the expression of CD11b and CD18 in both group H1 and H2 decreased at T4, T5 (P<0.05); compared with the value in group H1, the expression of CD11b and CD18 decreased at T4, T5 time in group H2 (P<0.05).
Part V: Exprimental study of the effect of 6% HES 130/0.4 on the apoptosis rate of injuried pulmonary microvascular endothelial cell induced by LPS
1 Under inverted microscope, cultured cells were cobblestone arrangement; microvilli and fenestrae on the surface of cultured cell could be observed by SEM. Protuberances on the surface of endothelial cells were observed; large and clear nucleolus, Golgi complex in the cytoplasm and rough endoplasmic reticulum as well as well developed smooth endoplasmic reticulum were observed by TEM.
2 In group C, cell morphology with no significant changes were spindle or polygona by SEM; in both group L and group H, part of the cells became round shape, with decreased protrudings in cell edges but no apoptosis bodies were observed;
3 Effects on the proliferation index and apoptosis rates of LPS-induced PMVECs HES130/0.4: compared with group C, PI of group L and group H was no statistical significance (P>0.05); compared with group L, PI of group H was no statistical significance (P>0.05); compared with group C, the apoptosis rate in group L and H increased (P>0.05); compared with group L, the apoptosis rate decreased in group H (P>0.05). Conclusions:
Part I: Establishing models of acute lung injury induced by hemorrhagic shock and resuscitation in rats
As hemorrhagic shock lasts for 45, 60 and 90 minutes while using Ringers solution to resuscitate for 2 or 3 hours may cause lung tissue changes functionally and morphologically, and the 90min is the most obvious one; and one reason of lung damage in hemorrhagic shock using fluid resuscitation during 45, 60 and 90 min is increasing of pulmonary capillary permeability, and the 90min was the most obvious one.
Part II: Effect of 6% HES 130/0.4 resuscitation on lung TNF-α, IL-1βand IL-10 in hemorrhagic shock rats
1. Lungs may have pathomorphological damage by using Ringers solution to resuscitation HS for 3 hours, and with 6%HES130/0.4 33ml/kg or 50ml/kg HES resuscitation can mitigate the pathomorphological change differently;
2. The 33ml/kgHES make much more effort than the 50ml/kg one to mitigate the lung’s pathomorphological change and caused by HS Part III: Effect of 6% HES 130/0.4 resuscitation on lung MDA, SOD and MPO in hemorrhagic shock rats
By using 6%HES130/0.4 for HS fluid resuscitation can bring decreased the organic oxidation reaction conspicuously, and the 33ml/kg 6%HES130/0.4 is much more obviously than the 50ml/kg one.
Part IV: Effect of 6% HES 130/0.4 resuscitation on the expression of CD11b and CD18 in arterial PMNs in hemorrhagic shock rats
The fluid resuscitation and HS itself may induce the activation of neutrophils, 6%HES130/0.4 can reduce the activation of neutrophils; and 33ml/kg 6%HES130/0.4 is much more effective than the 50ml/kg one for HS fluid resuscitation.
Part V: Exprimental study of the effect of 6% hydroxyethyl starch 130/0.4 on the apoptosis rate of injuried pulmonary microvascular endothelial cell induced by LPS
1. When the damaged PMVECs induced by LPS are treated with HES130/0.4, the apoptosis rates increased.
2. There is no connection between the cell cycle and apoptosis percentage of damaged PMVECs induced by LPS when treated with HES130/0.4.
HS导致肠源性菌血症和内毒素血症,是诱发ALI/ARDS的重要因素。继发的中性粒细胞(polymorphonuclear neutrophils, PMNs)过度活化,肺内炎症反应失控,肺血管内皮细胞的坏死和凋亡等是构成HS液体复苏后ALI/ARDS的主要作用机制。在ALI/ARDS的发病进程中,炎症反应导致肺通透性增高,其在内源性ARDS主要表现在肺微血管内皮;外源性ARDS则主要开始于肺泡上皮。炎症介质的释放、中性粒细胞与内皮细胞的相互作用以及细胞骨架的变化是导致肺微血管内皮通透性增高的主要原因。
HS后,尤其是在血源匮乏的情况下,复苏液体的选择要优先考虑在有效恢复血流动力学的前提下,改善机体的病理生理状况,提高生存率。羟乙基淀粉是临床上最常用的血浆容量扩张剂,其扩容强度和维持时间,决定于它们的浓度和相对分子质量以及克分子取代程度和取代方式。6%HES130/0.4(6%hydroxyethyl starch 130/0.4, Voluven)是最新一代羟乙基淀粉,克分子量为130 000道尔顿,取代级为0.4,C2/C6羟乙基化的比率为9︰1;与其他HES溶液和右旋糖苷比较,容量扩容效果相同,但副作用更少。6%HES130/0.4除具有良好的扩容作用外,还可减少白细胞迁移,减轻炎性反应,可减轻大鼠内毒素性ALI,但其对HS液体复苏后继发肺损伤的作用及其机制尚无定论,故本研究欲加以探讨。
本课题选用实验外科技术,采用颈总动脉放血法造成大鼠失血性休克,应用有创动脉血压监测技术对不同休克维持时间的大鼠行液体复苏,建立SD大鼠HS液体复苏后继发ALI模型,并在此基础上用HES130/0.4早期干预;应用血气分析、病理形态学观察、透射电镜技术、流式细胞术、酶联免疫技术等检测方法,观察肺功能变化、肺脏病理形态及超微结构变化、肺组织匀浆中TNF-α、IL-1β、IL-10含量的变化以及动脉血中性粒细胞CD11b、CD18表达的变化,探讨肺血管内皮细胞及中性粒细胞在HS液体复苏后继发ALI病程进展中的作用及机制,以及HES130/0.4对HS液体复苏后继发ALI早期干预的作用及机制;并在动物实验基础上,进一步观察了HES130/0.4对体外培养的肺微血管内皮细胞损伤的作用及相关机理,为HS液体复苏致ALI的早期干预提供新的治疗策略及理论依据。
方法:
第一部分:失血性休克大鼠液体复苏后继发肺损伤模型的建立
48只SD大鼠,按随机数字表法分为45min休克组(H45组)及45min对照组(C45组)、60min休克组(H60组)及60min对照组(C60组)和90min休克组(H90组)及90min对照组(C90组),每组8只。对照组仅麻醉及行动静脉穿刺置管,不放血及液体复苏。不同休克时间组按休克维持时间分为H45组、H60组以及H90组。颈总动脉放血法造成大鼠失血性休克,按照实验设计分别维持不同休克时段后,通过左侧股静脉输注三倍最大放血量的林格氏液进行复苏。计算各组的失血程度;记录不同时间点MAP;分别于休克前、液体复苏前即刻以及复苏后2、3h时行血气分析,并计算PO2/FiO2的比值;复苏3h后采用颈总动脉放血法快速处死动物,大体观察各组肺脏充血、水肿、出血情况,光镜下观察肺组织病理学变化,并计算各组肺组织的病理评分,测定支气管肺泡灌洗液(BALF)蛋白浓度和肺湿/干重比(W/D)。
第二部分:6%HES130/0.4对失血性休克大鼠液体复苏后肺组织TNF-α、IL-1β和IL-10的影响
成年雄性SD大鼠24只,按随机数字表法随机分为对照组(control组)、林格氏液组(RS组)、33ml/kg复苏组(H1组)、50ml/kg-1复苏组(H2组),每组6只。对照组(control组)仅麻醉及进行动静脉穿刺,不放血;林格氏液组(RS组)用三倍最大放血量的林格氏液复苏;H1组、H2组分别用33ml/kg、50ml/kg的羟乙基淀粉和林格氏液复苏。为保证理论上复苏效果的相同,H1、H2组复苏所用林格氏液的量为3倍最大的放血量减去相应剂量的羟乙基淀粉。
计算各休克组的最大失血程度;记录不同时间点MAP;分别于休克前(T0),休克后复苏前(T1),复苏后2(T4)、3(T5)h行血气分析,并计算PaO2/FiO2;于复苏后3h处死动物,测定BALF蛋白浓度及肺W/D;测定肺组织肿瘤坏死因子(TNF-α)、白细胞介素-1β(IL-1β)和白细胞介素-10(IL-10)的含量;大体观察肺脏充血、水肿、出血情况;光镜下观察肺组织病理学变化并计算肺组织的病理评分;透射电镜下观察肺超微组织结构变化。
第三部分:6%HES130/0.4对失血性休克大鼠液体复苏后肺组织MDA、SOD和MPO的影响
动物选择与分组同第二部分。
休克组分别于液体复后3h、control组于相应时间点处死动物,取右肺下叶肺组织测定MDA含量、SOD和MPO活性。
第四部分:6%HES130/0.4对失血性休克大鼠液体复苏后血中性粒细胞CD11b和CD18表达的影响
动物的选择和分组、HS模型的复制同第二部分;
分别于休克前(T0),复苏前即刻(T1),复苏后2h(T4)、3h(T5)行血气分析;流式细胞术(Flow Cytometry,FCM)检测动脉中性粒细胞CD11b和CD18的表达水平;复苏后大体观察肺脏充血、水肿、出血情况;光镜下观察肺组织病理学变化并计算肺组织的病理评分;透射电镜下观察肺超微组织结构变化。
第五部分:6%HES130/0.4对大鼠肺微血管内皮细胞凋亡率的影响
1用改良组织块法进行肺微血管内皮细胞(pulmonary microvascular endothelial cell, PMVECs)原代培养,倒置相差显微镜下观察细胞形态,扫描电镜、透射电镜下观察细胞形态及超微结构,进行细胞鉴定;2-3代培养的细胞用于下一步实验。
2将所得的PMVECs悬液浓度调整到1×109个/ml, 24孔细胞培养板中每孔加入1ml细胞悬液,共15孔,随机分为3组,空白对照组(C组)、LPS处理组(L组)和HES干预组(H组),每组5孔。L组加入1μg/ml LPS和等体积PBS液,H组加入1μg/ml LPS、等体积PBS液以及6% HES130/0.430mg/ml;C组加入等体积PBS液。将细胞培养板置于CO2细胞培养箱中,分别孵育3h后取出培养板后收集细胞。透射电镜、扫描电镜下观察细胞形态及超微结构;流式细胞仪检测PMVECs凋亡率和细胞增殖情况
结果:
第一部分:失血性休克大鼠液体复苏后继发肺损伤模型的建立
1各休克组大鼠失血程度、MAP组间差异无统计学意义;
2与各对照组比较,各休克组于T1时间点PaO2/FiO2升高,PaCO2降低(P<0.05);H45组、H90组于T5时间点PaO2/FiO2降低(P<0.05)。与H45组比较, H60组、H90组于T4时间点PaO2/FiO2、PaCO2降低(P<0.05)。与H60组比较,H90组于T5时间点PaO2/FiO2降低,于T4、T5时间点PH升高(P<0.05);
3.1肺大体观察结果:各对照组无明显改变;各休克组肺脏可见不同程度的缺血、充血和水肿,以H90组最为明显。
3.2各对照组肺泡结构未见明显异常;H45组、H60组肺间质增宽,炎性细胞浸润,H60组小支气管见炎性渗出和浸润;H90组肺泡腔内见红细胞及炎性细胞,肺间质增宽,炎性细胞浸润,间质小血管扩张充血;与各自对照组比较,H45组、H60组和H90肺组织病理评分增高(P<0.05);与H45或者H60组比较,H90肺组织病理评分增高(P<0.05);
3.3各对照组肺组织超微结构未见明显异常;H45组肺泡Ⅱ型细胞基本正常,板层颗粒正常,线粒体膜缺损;H60组核周间隙扩张,粗面内质网轻度扩张;H90组血管内皮基底膜断裂,气血屏障结构模糊,水肿增厚;
3.4与对照组比较,各休克组BALF蛋白浓度、肺W/D不同程度升高(P<0.05或0.01);与H45或H60比较,H90组BALF蛋白浓度、肺W/D显著升高(P<0.05或0.01);
第二部分6%HES130/0.4对失血性休克大鼠液体复苏后肺组织TNF-α、IL-1β和IL-10的影响
1各液体复苏组最大失血程度、不同时间点MAP组间比较差异无统计学意义;
2与control组比较,RS组、H1、H2组于T1时间点PaO2/FiO2升高,PaCO2降低(P<0.05);RS组于T4、T5时间点,H2组于T5时间点PaO2/FiO2(P<0.05)。与RS组比较, H1组T4、T5时间点,H2组于T4时间点PaO2/FiO2升高,H1、H2组于T4时间点PaCO2升高(P<0.05);与H1组比较,H2组于T5时间点PaO2/FiO2降低(P<0.05);
3与control组比较,RS组、H1、H2组肺组织匀浆TNF-α、IL-1β和IL-10含量、BALF蛋白浓度、W/D升高(P<0.05);与RS组比较,H1、H2组肺组织匀浆TNF-α、IL-1β含量降低,IL-10含量升高,BALF蛋白浓度、肺W/D降低(P<0.05);
4.1肺大体观察结果:control组无明显改变;各液体复苏组肺脏可见不同程度的缺血、充血和水肿,以RS组最为明显;
4.2 control组肺泡结构正常;RS组肺泡腔内见红细胞及炎性细胞,肺间质增宽,炎性细胞浸润,间质小血管扩张充血;H1组和H2组肺间质增宽,炎性细胞浸润,小支气管见炎性渗出和浸润;H1组、H2组肺组织损伤较RS组轻,其中H1组损伤最轻。与control组比较,各液体复苏组肺组织病理评分增高(P<0.05);与RS组比较,H1组和H2组肺组织病理评分降低(P<0.05);与H1组比较,H2组肺组织病理评分增高(P<0.05);
4.3 control组肺超微组织形态结构未见异常;RS组肺组织超微结构受损,血管内皮基底膜断裂,Ⅱ型上皮细胞微绒毛明显减少,粗面内质网扩张,可见颗粒融合现象;H1组除线粒体结构轻微改变外基本接近正常; H2组核周间隙轻度扩张,粗面内质网轻度扩张,有脱颗粒;
第三部分:6%HES130/0.4对失血性休克大鼠液体复苏后肺组织MDA、SOD和MPO的影响
1与control组比较,RS组、H1组和H2组MDA含量升高(P<0.05);与RS组比较,H1组和H2组MDA含量降低(P<0.05);与H1组比较,H2组MDA含量升高(P<0.05);
2与control组比较,RS组、H1组和H2组SOD活性降低(P<0.05);与RS组比较,H1组和H2组SOD活性升高(P<0.05);与H1组比较,H2组SOD活性减低(P<0.05);
3与control组比较,RS组、H1组和H2组MPO活性升高(P<0.05);与RS组比较,H1组和H2组MPO活性降低(P<0.05);
第四部分:6%HES130/0.4对失血性休克大鼠液体复苏后血中性粒细胞CD11b和CD18表达的影响
1复苏后各时点MAP组间比较差异无统计学意义。
2与control组比较,各液体复苏组于T1时间点PaO2升高,T1~5 PaCO2均降低(P<0.05);与T0比较,各液体复苏组PaO2于T1时间点、H1组T1~5时间点、H2组T5时间点升高;PaCO2各液体复苏组T1~5时间点降低;除H1组外,各液体复苏组PH于T4、5时间点降低(P<0.05);
3与control组比较,各液体复苏组于T1、T4、T5时间点CD11b和CD18的表达增强(P<0.05);与RS组比较,H1、H2组于T4、T5时间点CD11b和CD18的表达降低(P<0.05);与H1组比较,H2组于T4、T5时间点CD11b和CD18的表达增强(P<0.05);
4.1肺大体观察结果:control组无明显改变;各液体复苏组肺脏可见不同程度的缺血、充血和水肿,以RS组最为明显;
4.2 control组肺泡结构正常;RS组肺泡腔内见红细胞及炎性细胞,肺间质增宽,炎性细胞浸润,间质小血管扩张充血;H1组和H2组肺间质增宽,炎性细胞浸润,小支气管见炎性渗出和浸润;H1组、H2组肺组织损伤较RS组轻,其中H1组损伤最轻。与control组比较,各液体复苏组肺组织病理评分增高(P<0.05);与RS组比较,H1组和H2组肺组织病理评分降低(P<0.05);与H1组比较,H2组肺组织病理评分增高(P<0.05);
4.3 control组肺超微组织形态结构未见异常;RS组肺组织超微结构受损,血管内皮基底膜断裂,Ⅱ型上皮细胞微绒毛明显减少,粗面内质网扩张,可见颗粒融合现象;H1组除线粒体结构轻微改变外基本接近正常; H2组核周间隙轻度扩张,粗面内质网轻度扩张,有脱颗粒;
第五部分:6%HES130/0.4对大鼠肺微血管内皮细胞凋亡率的影响
1倒置显微镜下观察,培养的细胞呈铺路石样排列;扫描电子显微镜观察到培养的细胞表面存在微绒毛,细胞表面存在窗孔;透射电镜下内皮细胞表面有胞浆突起,核仁大且明显;胞质内有高尔基复合体,粗面内质网和滑面内质网发达;
2扫描电镜结果:C组细胞细胞形态无明显变化,呈梭形或多角形;L组、H组部分细胞形态变圆,细胞边缘突起减少,未见凋亡小体;
3 HES130/0.4对LPS诱导活化的PMVECs细胞增殖指数及凋亡率的影响:与C组比较,L组、H组PI无统计学差异(P>0.05);与L组比较,H组PI无统计学差异(P>0.05)。与C组比较,L组、H组凋亡率增加(P<0.05);与L组比较,H组凋亡率降低(P<0.05)。
结论
第一部分:失血性休克大鼠液体复苏后继发肺损伤模型的建立失血性休克大鼠在休克时间维持在90min后林格氏液复苏后3h肺功能、肺形态学出现典型的损伤性的变化,是稳定、可靠的失血性休克液体复苏后继发肺损伤的动物模型;
第二部分:6%HES130/0.4对失血性休克大鼠液体复苏后肺组织TNF-α、IL-1β和IL-10的影响
1与林格氏液比较,6%HES130/0.4可明显降低HS液体复苏后肺脏组织炎性介质TNF-α、IL-1和IL-10的含量,减轻失血性休克液体复苏后继发肺组织病理学损伤,从而改善肺的呼吸功能;
2 6%HES130/0.4 33ml/kg和50ml/kg HES液体复苏能不同程度减轻肺脏炎性反应,且33ml/kg剂量复合林格氏液作用明显;
第三部分:6%HES130/0.4对失血性休克大鼠液体复苏后肺组织MDA、SOD和MPO的影响
与林格氏液复苏比较,6%HES130/0.4可减轻HS液体复苏后肺组织MDA、SOD和MPO的变化,进而减轻继发肺组织损伤,且33ml/kg 6% HES130/0.4复苏较50ml/kg剂量作用明显;
第四部分:6%HES130/0.4对失血性休克大鼠液体复苏后血中性粒细胞CD11b和CD18表达的影响
与林格氏液相比,6%HES130/0.4可使血中性粒细胞CD11b和CD18的表达显著降低,减轻肺组织的病理性损伤,从而改善肺功能;
第五部分:6%HES130/0.4对大鼠肺微血管内皮细胞凋亡率的影响
1 HES130/0.4处理后通过降低活化PMVECs凋亡率影响细胞的凋亡;
2 HES130/0.4处理后所引起的细胞凋亡改变与细胞增殖周期的变化无明显关系。
Objectives:
Hemorrhagic shock(HS) might cause systemic inflammatory response syndrome (SIRS). It would certainly result in multiple organ dysfunction syndrome (MODS) if the development of SIRS was out of control. SIRS in lungs was manifested by acute lung injury (ALI). Obviously, the process, SIRS→ALI→ARDS→MODS, reflected the dormino effects. Therefore, the determination the pathogenesis of ALI/ARD, and the early prevention and treatment for ALI/ARD, were significant for reducing mortality rate and impoving the prognosis of HS.
Intestinal bacteriemia and endotoxemia induced by HS, was an important causative factor of ALI/ARDS. And the main mechanisms of ALI/ARDS after HS, were the resulting over activation of polymorphonuclear neutrophils (PMNs), the incontrollable inflammation in lungs, and the necrosis or apoptosis of pulmonary vascular endothelial cells. During the process of ALI/ARDS, the inflammation response resulted in the increased permeability of lungs. The endogenous ARDS usually appeared on pulmonary microvascular endothelium, and the exogenous ARDS on alveolar epithelium. The main reasons that pulmonary microvascular endothelium permeability increases were the release of inflammatory mediator, the interaction between polymorphonuclear neutrophils and endothelial cells, and the changes of cytoskeletons.
A prior consideration should be taken, that was to improve the pathology state and survival rate on the premise of regaining the hemodynamics, when chosing resuscitation fluid after a hemorrhagic shock, especially in the shortage of blood. Hydroxyl starch (HES) was the most common plasma volume expander on clinical application, whose expansion capability and maintenance was determined by its concentration, relative molecular mass, and the degree or patterns of gram molecule substitution. 6%HES130/0.4(hydroxyethyl starch 130/0.4, Voluven)was a new generation of HES, with a gram molecular weight of 130,000 dalton, substitution degree of 0.4, and C2/C6 hydroxyethylated rate of 9:1. Compared with other HES fluids and daxtran, it had the same effect of volume expansion, but less side effects. Besides a good expansion effect, the 6%HES130/0.4 could also reduce the leucocytes migration, the inflammatory reaction and endotoxin-induced ALI in rats. But it had no fixed conclusion about the effects and mechanisms for the damage of non-infectious pulmonary inflammatory.
This study was carried out with experimental surgery technique. Models with hemorrhagic shock were made in rats by blood loss from carotid. Then invasive monitoring of arterial pressure method was used to resuscitate rats in HS with different shock-time duration, and estabilished the SD rat ALI models. Based on these, HES130/0.4 was used for early intervention. To observe the changes of pulmonary function, pathomorphology and ultra-microstructure, TNF-α、IL-10 in lung tissue homogenates and CD11b\CD18 of pulmonary polymorphonuclear neutrophils were made; to study the effects or the mechanism of pulmonary vascular endothelial cell and polymorphonuclear neutrophils in the pocess of ALI induced by HS fluid resuscitation, and the effects and mechanisms of HES130/0.4 on the early intervention with ALI induced by HS fluid resuscitation, several detection technologies were used here, such as fully auto blood gas analysis, pathomorphological observation, transmission electronic microscope, flow cytometry, enzyme-linked immunosorbent assay, and so on. On the basis of animal experiment, the effects and mechanism of HES130/0.4 on the damage of pulmonary microvascular endothelial cells had been futher observed. So, new treatment strategies and theories were provided for early intervation with ALI induced by hemorrhagic shock and fluid resuscitation.
Methods:
Part I: Establishing models of acute lung injury induced by hemorrhagic shock and resuscitation in rats
Fourty-eight SD rats were divided into 6 groups (45min hemorrhagic group,60min hemorrhagic group,and 90min hemorrhagic group and their corresponding control groups) by means of random number table. The control operation group was only anesthetized and dealt with venipuncture indwelling catheter,without removal of blood or fluid resuscitation. Different shock duration groups were divided into group H45, group H60, group H90 by time. The models of hemorrhagic shock in rats established with the method of blood in carotid atery were resuscitated by infusing Ringer's lactate of 3 times the maximum volume of the removed blood in femoral veins after shock. Then, the hemorrhagic level of different groups were calculated; MAP at different time points were recorded; blood gas analysis were taken before shock, before fluid resuscitation and during the 3 hours resuscitation separately, and the PO2/FiO2 ratios were calculated. These animals were killed quickly by the method of removal blood in carotid artery after 3 hours resuscitation. Then the lung congestion, edema, hemorrhage were generally observed in each group. The pulmonary pathological changes at light microscope observed and the lung pathological score were calculated in the different groups. And the ultrastructure in lung tissue was observed by transmission electron microscope (TEM). The brochoalveolar lavage fluid (BALF) protein concentration and the wet/dry weight (W/D) ratio of lung were determined.
Part II: Effect of 6% HES 130/0.4 resuscitation on lung TNF-α, IL-1βand IL-10 in hemorrhagic shock rats
Twenty-four adult male SD rats were divided into 4 groups: control operation group (group control) , Ringer's lactate group (group RS), resuscitation group of 33ml/kg (group H1), resuscitation group of 50ml/kg (group H2) by the means of random number table. The rats in control group was anesthetized and dealt with only venipuncture,without blood removal. After shock, Ringer's solution of 3 times the maximum volume of the removed blood was infused in femoral veins to make resuscitation in group RS. 33ml/kg of 6% HES130/0.4 were infused to make resuscitation in group H1 and 50ml/kg in H2 group. To ensure the same effect of theoretical resuscitation, the Ringer's solution equalled 3 times the maximum volume of the removed blood minus corresponding amount of hydroxyethyl starch in H1and H2 were infused.
The largest hemorrhagic level of different shock groups were calculated; MAP at different time points were recorded; Blood gas analysis were made before shock (T0), before resuscitation (T1), after 2 hrs (T4) and 3 hrs (T5) of resuscitation; PO2/FiO2 ratio were calculated. Rats were killed after 3 hrs of resuscitation. BALF protein concentration and the lung wet/dry weight (W/D) ratios were determined. The concentrations of tumor necrosis factor-α(TNF-α), interleukin-1 (IL-1β) and interleukin-10 (IL-10) in lung issue were determined. Generally the lung congestion, edema, hemorrhage in each group were observed. The pulmonary pathological changes were observed at light microscope and the lung pathological score were calculated in the different groups. The ultrastructural organization were observed in lung tissue by TEM.
Part III: Effect of 6% HES 130/0.4 resuscitation on lung MDA, SOD and MPO in hemorrhagic shock rats
Rats was selected and classified as in the second part.The rats were killed at 3h after fluid resuscitation in shock groups, and at the same time in to obtain the lower lobe lung issue in right lung. And the amount of MDA, activity of SOD and MPO were determined.
Part IV: Effect of 6% HES 130/0.4 resuscitation on the expression of CD11b and CD18 in arterial PMNs in hemorrhagic shock rats
Rats’selection, group and establishing models of hemorrhagic shock were the same as described in second part. To reduce the differences in different groups during the test with flow cytometry (FCM), this part of the experimental rats were divided into batches, each batches of four, divided into control operation group (group S),Ringer's lactate group (group RS) , resuscitation group of 33ml/kg (group H1) , resuscitation group of 50ml/kg (group H2)
Blood gas analysis were made before shock (T0), before resuscitation (T1), 2 hours (T4) and 3 hours (T5) after resuscitation separately; Flow Cytometry (FCM) were detected for the expression level of polymorphonuclear neutrophils (PMNs) of CD11b and CD18 in artery blood.
Part V: Exprimental study of the effect of 6% HES 130/0.4 on the apoptosis rate of injuried pulmonary microvascular endothelial cell induced by LPS
1. Modified tissue block pasted culture method was used in primary culture of pulmonary microvascular endothelial cell (PMVECs). The tissue block were inverted under the phase contrast microscope and the cellular morphology were observed. The cellular morphology and ultrastructure were observed with scanning electron microscope (SEM) and TEM, and the cells were identified. 2-3 generation of cells cultured were used in next experiments.
2. Suspension concentration of the first part of PMVECs was adjusted to 1×109/ml. 1ml of cell suspension was added into each hole of a 24 holes’cell culture plate. A total of 15 holes were randomly divided into 3 groups: control group (group C)、LPS treatment group (group L) and HES intervention group (group H). Group L was added with 1μg/ml LPS and other fluid PBS with the same volume. H group was added with 1μg/ml LPS and other fluid PBS with the same volume and HES130/0.4 30mg/ml. Group C was added with the fluid PBS with the same volume. The cell culture plates were put into cell incubators, removed after 3 hours and the cells were collected. The cellular morphology and ultrastructure were observed by SEM and TEM. Apoptosis rate and cell proliferation of PMVECs were detected by flow cytometer. Results:
Part I: Establishing models of acute lung injury induced by hemorrhagic shock and resuscitation in rats
1. Each group of rats were alive till the experiments. It was meaningless in statistics in the groups of shock about the blood loss level and the interblock diference of MAP.
2. Compared with the value in group control, PaO2/FiO2 rised at time point T1, PaCO2 were decreased (P<0.05) in three shock groups; in group H45 and H90, PaO2/FiO2 decreased at time point T4 (P<0.05). Comapared with the value in group H45 and H60, PaO2/FiO2 rised at time point T5, and PaCO2 decreased (P<0.0) in group H90. Compared with the value in group H60, PaO2/FiO2 decreased at time point T5, and PH rised at time point T4 and T5 in group H90 (P<0.05).
3.1 The general observing results of lung tissue: no obvious changes were observed in the group control. Ischemia, congestion and oedema in various degrees could be found in each group of shock rats, espessially in group H90.
3.2 The structure of alveolus was not found obvious abnormity. In the groups H45 and H60, the lung interstitial substance was broadened, and inflammatory cell infiltration could be found. In group H60, inflammatory exudation and lammatory infiltration in small bronchus could be found. Hematid and inflammatory cell in the intracavity of alveolus could be found, the broadened lung interstitium, inflammatory cell infiltration, expansion and congestion interstitium minute vessel could be found in group H90. Compared with the values in group control and H45, group H60 and H90, pathology grade of lung tissue rised (P<0.05). Compared with the value in group H60, pathology grade of lung tissue in H90 rised (P<0.05).
3.3 In group control, there was no obvious abnormity found in ultrastructure of lung tissue. In group H45, alveolitoidⅡcell was basically nomal, lamellated granule was nomal, but mitochodrial membrane was impairment.In group H60, the interstistial substance of lung was broaden, inflammatory cell intitration, inflammatory exudation and lammatory infiltration cound be found in small bronchus. In group H90, hematid and inflammatory cell in the intracavity of alveolus, the broadened interstitial substance of lung, inflammatory cell infiltration, expansion and congestion of interstritial substance of minute vessel could be found.
3.4 Compared with the value in group control, viscosity albuminose of BALF and W/D of lung rised in each shock group (P<0.05 or 0.01); Compared with the value in group H45 and H60, lung viscosity albuminose of BALF and W/D rised (P<0.05or 0.01) in group H90.
Part II: Effect of 6% HES 130/0.4 resuscitation on lung TNF-α, IL-1βand IL-10 in hemorrhagic shock rats
1. There was no difference statisticly of MAP at different time points between fluid resuscitation groups.
2. Compared with the value in group control, PaO2/FiO2 increased and PaCO2 decreased (P<0.05) at time point T1 in group H1, H2 and RS. In group RS, PaO2/FiO2 increased at time point T4 and T5. PaO2/FiO2 in group H2 increased at time point T5 (P<0.05). Compared with the value in group RS,, PaO2/FiO2 increased at time point T4 and T5 in group H1. PaO2/FiO2 increased at time point T4 in group H2. In the group H1 and H2, PaCO2 increaesd at time point T4 (P<0.05). Compared with the value in group H1, PaO2/FiO2 decreased at time point T5 (P<0.05) in group H2.
3. Compared with the value in group control, IL-10 and protein in BALF and lung W/D increased in group RS, H1 and H2 (P<0.05); compared with the value in group RS, the concentration of TNF-α, IL-1β, the protein in BALF and lung W/D in group H1 and H2 decreased, while the concentration of IL-10 increased (P<0.05).
4.1 The observed results of lung tissue: there was no obviously change in group control; Ischemia, congestion and edema in various degrees could be found in each fluid resucitation group, especially in group RS.
4.2 The stucture of alveolus was nomal in group control. Hematid and inflammatory cell in the intracavity of alveolus, the broadened interstitial substance of lung, inflammatory cell infiltration, expansion an congestion of interstitial substance of minute vessel could be found in group RS. The broadened interstitial substitial of lung, inflammatory cell infiltration, and inflammatory exudationg and lammatory infiltration in interstitial substance of minute vessel could be found in group H1 and H2. The damage of lung tissue in group H1 and H2 were less than the group RS, the least damage of lung tissue was in group H1. Compared with the value in group control, the pathology grade of lung tissue increased in each of fluid resuscitation group (P<0.05). Compared with the value in group RS, the pathology grade of lung tissue was decreased in group H1 and H2 (P<0.05). Compared with the value in group H1, the lung tissue pathology grade increased in the group H2 (P<0.05).
4.3 In group control, the ultrastructure of lung tissue was obviously nomal, while the ultrastructure of lung tissue was damaged in group RS. Basement membrane of blood vessel was reptured, epithelial microvillus was obviously reduced, rough surfaced endoplasmic reticulum was expanded, and the phenomenon of kermel integration could be found; Those in group H1 were basically nomal except slight structural modificationg of mitochondria; Perinucleat space was low-grade expanded, rough surfaced endoplasmic reticulum was low-grade expanded and there was phenomenon of degranlation in group H2.
Part III: Effect of 6% HES 130/0.4 resuscitation on lung MDA, SOD and MPO in hemorrhagic shock rats
1. Compared with the value in group control, the concentration of MDA increased in group RS, H1 and H2 (P<0.05); Compared with the value in group RS, the concentration of MDA decreased in group H1 and H2 (P <0.05); Compared with group H1, the concentration of MDA increased in group H2 (P <0.05);
2. Compared with the value in group control, the concentration of SOD decreased in group RS, H1 and H2 (P<0.05); Compared with the value in group RS, the concentration of SOD increased in group H1 and H2 (P <0.05); Compared with the value in group H1, the concentration of SOD decreased in group H2 (P <0.05);
3. Compared with the value in group control, the concentration of MPO increased in group RS, H1 and H2 (P<0.05); Compared with the value in group RS, the concentration of MPO decreased in group H1 and H2 (P <0.05); There was no difference statisticly of MPO beteween group H1 and H2 (P >0.05);
Part IV: Effect of 6% HES 130/0.4 resuscitation on the expression of CD11b and CD18 in arterial PMNs in hemorrhagic shock rats
1.There was no statistical significance of MAP at each time point between groups (P >0.05);
2.Compared with the value in group control, PaO2 increased at T1, while PaCO2 decreased at T1~5 in every fluid resuscitation group (P<0.05); compared with the value at T0, PaO2 in every fluid resuscitation group at T1, group H1 at T1~5 and group H2 at T5 increased; PaCO2 in every fluid resuscitation group decreased at T1~5; except group H1, PH in every fluid resuscitation group was lower at T4, 5 (P<0.05);
3.Compared with the value in group control, the expression of CD11b and CD18 in every fluid resuscitation group increased at T1, T4, T5 (P<0.05); compared with the value in group RS, the expression of CD11b and CD18 in both group H1 and H2 decreased at T4, T5 (P<0.05); compared with the value in group H1, the expression of CD11b and CD18 decreased at T4, T5 time in group H2 (P<0.05).
Part V: Exprimental study of the effect of 6% HES 130/0.4 on the apoptosis rate of injuried pulmonary microvascular endothelial cell induced by LPS
1 Under inverted microscope, cultured cells were cobblestone arrangement; microvilli and fenestrae on the surface of cultured cell could be observed by SEM. Protuberances on the surface of endothelial cells were observed; large and clear nucleolus, Golgi complex in the cytoplasm and rough endoplasmic reticulum as well as well developed smooth endoplasmic reticulum were observed by TEM.
2 In group C, cell morphology with no significant changes were spindle or polygona by SEM; in both group L and group H, part of the cells became round shape, with decreased protrudings in cell edges but no apoptosis bodies were observed;
3 Effects on the proliferation index and apoptosis rates of LPS-induced PMVECs HES130/0.4: compared with group C, PI of group L and group H was no statistical significance (P>0.05); compared with group L, PI of group H was no statistical significance (P>0.05); compared with group C, the apoptosis rate in group L and H increased (P>0.05); compared with group L, the apoptosis rate decreased in group H (P>0.05). Conclusions:
Part I: Establishing models of acute lung injury induced by hemorrhagic shock and resuscitation in rats
As hemorrhagic shock lasts for 45, 60 and 90 minutes while using Ringers solution to resuscitate for 2 or 3 hours may cause lung tissue changes functionally and morphologically, and the 90min is the most obvious one; and one reason of lung damage in hemorrhagic shock using fluid resuscitation during 45, 60 and 90 min is increasing of pulmonary capillary permeability, and the 90min was the most obvious one.
Part II: Effect of 6% HES 130/0.4 resuscitation on lung TNF-α, IL-1βand IL-10 in hemorrhagic shock rats
1. Lungs may have pathomorphological damage by using Ringers solution to resuscitation HS for 3 hours, and with 6%HES130/0.4 33ml/kg or 50ml/kg HES resuscitation can mitigate the pathomorphological change differently;
2. The 33ml/kgHES make much more effort than the 50ml/kg one to mitigate the lung’s pathomorphological change and caused by HS Part III: Effect of 6% HES 130/0.4 resuscitation on lung MDA, SOD and MPO in hemorrhagic shock rats
By using 6%HES130/0.4 for HS fluid resuscitation can bring decreased the organic oxidation reaction conspicuously, and the 33ml/kg 6%HES130/0.4 is much more obviously than the 50ml/kg one.
Part IV: Effect of 6% HES 130/0.4 resuscitation on the expression of CD11b and CD18 in arterial PMNs in hemorrhagic shock rats
The fluid resuscitation and HS itself may induce the activation of neutrophils, 6%HES130/0.4 can reduce the activation of neutrophils; and 33ml/kg 6%HES130/0.4 is much more effective than the 50ml/kg one for HS fluid resuscitation.
Part V: Exprimental study of the effect of 6% hydroxyethyl starch 130/0.4 on the apoptosis rate of injuried pulmonary microvascular endothelial cell induced by LPS
1. When the damaged PMVECs induced by LPS are treated with HES130/0.4, the apoptosis rates increased.
2. There is no connection between the cell cycle and apoptosis percentage of damaged PMVECs induced by LPS when treated with HES130/0.4.
引文
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