淋浆对内毒素休克大鼠的干预作用及其机制
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摘要
内毒素休克(endotoxic shock,ES)是临床常见的急危重症,是危重病患者的重要死亡原因之一,病死率逐年增高;革兰阴性菌产生的脂多糖(lipopolysaccharide,LPS)在ES的发病学中发挥重要的作用。尽管某些休克的治疗取得了可喜的进展,但ES的死亡率仍居高不下,本研究目的是探索ES治疗的新途径。实验采用LPS复制大鼠ES模型,观察正常淋浆对ES的干预作用。观察血压、肾、肝微区血流灌注量、及器官功能、结构损伤及存活时间,揭示淋浆对ES的干预效果,并通过检测肠系膜微循环变化(白细胞黏附、微血管口径、血液流态、淋巴管收缩性)和血浆及组织匀浆中的细胞间黏附分子-1(intercellularadhesion molecule-1,ICAM-1)、P-选择素含量、组织匀浆中的髓过氧化物酶(myeloperoxidase, MPO)和Na+-K+-ATPase活性,探讨淋浆干预ES的机制,为临床治疗感染性休克提供新思路。
SPF级Wistar雄性大鼠90只,随机均分模型组(Model group)、淋浆组(Lymph plasma group)、对照组(Control group),每组30只。以戊巴比妥钠(50 mg·kg-1·bw)肌注麻醉、肝素全身抗凝(420 U·kg-1·bw)后,行颈部手术,右颈总动脉插管,通过生物信号采集系统连续记录平均动脉压(mean arterial pressure,MAP),左颈浅静脉插管,备注药及取血用。三组大鼠稳定30min后,模型组和淋浆组经颈浅静脉注射LPS(15 mg·kg-1,Sigma)复制ES模型;对照组以等量生理盐水代替LPS;15 min后,淋浆组用ZCZ-50自动抽注机以0.5 ml·min-1的速度自颈静脉输入淋浆(占全血量1/15,全血量按体重的7.4%计算),模型组和对照组以生理盐水代替淋浆。
上述每组大鼠又分为3个亚组:亚组1每组10只大鼠用于微区血流灌注量的观察,通过Perimed激光多普勒血流灌注仪观察LPS攻击后360 min内右肾中下极和肝右中叶下部微区血流灌注量,实验结束后,取出光导纤维探头及插管,逐层缝合伤口,观察自注入LPS到死亡的存活时间,存活24 h以上者为长期存活;亚组2每组10只用于回肠下段肠系膜血液微循环观察,通过Olympus微循环显微电视录像系统连续观察LPS攻击后360 min内肠系膜微血管口径、白细胞黏附及血液流态改变;亚组3每组10只用于肠系膜淋巴微循环观察,观察注射LPS后180 min内微淋巴管收缩频率、最大收缩口径、最大舒张口径、静态口径及收缩参数的改变。观察肠系膜微循环的两亚组大鼠分别于注入LPS(或相应液体)后180 min及360 min,取血液及固定位置的肺、肝、肾组织,分别制备10%组织匀浆,360 min者做石蜡切片、HE染色,检测血浆中尿素氮(blood urea nitrogen,BUN)、肌酐(creatinine,Cre)、天门冬氨酸氨基转移酶(aspartate aminotransferase,AST)和丙氨酸氨基转移酶(alanine aminotransferase,ALT),血浆及组织匀浆中P选择素和ICAM-1含量,及组织匀浆中MPO及Na+-K+-ATPase活性的变化,以及肺、肝、肾组织的湿/干比(wet/dry,W/D),观察肺、肝、肾组织的形态学变化。
研究结果显示:实验前,各组大鼠各项观测指标均无组间差异(P>0.05),对照组大鼠在整个观察过程中, MAP、肾、肝的微区血流灌注量、肠系膜微血管的口径和流态,微淋巴管的最大收缩口径、最大舒张口径、静态口径、自主收缩频率以及IndexⅠ、IndexⅡ、LD-Index三个收缩性指数均维持恒定(P>0.05)。肠系膜细静脉无白细胞黏附现象,随着观察时间的延长,偶见少数白细胞黏附。
LPS攻击后15 min,模型组和淋浆组的MAP均下降,肾和肝的微区血流灌注量显著减少,肠系膜细动脉口径缩窄,微血流流态变慢,加权积分值增高,白细胞黏附于细静脉壁,肠系膜微淋巴管的最大收缩幅度减小,其IndexⅠ、IndexⅡ和LD-Index三个收缩性指数均降低,两组间未见统计学差异(P>0.05),均与对照组形成鲜明的对比(P<0.01,P<0.05)。但淋浆组给予小量正常淋浆后,各观察指标显著优于模型组。
尽管模型组和淋浆组在注射LPS MAP迅速下降后均有所回升,但模型组仍显著低于对照组(P<0.01,P<0.05),且自180 min后进行性下降;淋浆组的MAP在120~240 min维持正常水平,其后虽有降低,但自注射淋浆至90 min及自330 min起,显著优于模型组(P<0.05,P<0.01)。
在MAP出现回升波的同时,两组大鼠肾和肝的微区血流灌注量均出现回升,且肾灌注量均高于对照组(P<0.05,P<0.01),肝灌注量维持正常水平。模型组的肾灌注量在45~105 min降至正常水平,之后逐渐波动式下降,自195 min起血流灌注量持续进行性下降,肝灌注量自270 min开始持续进行性下降,肾脏出现持续性低灌注的时间显著早于肝脏(P<0.01)。淋浆组的肾灌注量高水平维持至75 min,其后至实验结束一直接近对照组水平(P>0.05),其肝灌注量除偶高于对照组外,均处于实验前及对照组的恒定水平(P>0.05),且自270 min起,其肾、肝微区血流灌注量均显著高于模型组(P<0.05,P<0.01)。
模型组大鼠的肠系膜细动脉口径在整个实验过程中均呈缩窄状态,显著低于实验前及对照组(P<0.01,P<0.05),而淋浆组在注射淋浆后,细动脉口径均恢复至实验前和对照组水平(P>0.05),至330 min时方出现口径缩窄。模型组的肠系膜细静脉自实验150 min起,细静脉口径进行性缩小,显著低于对照组和淋浆组(P<0.05,P<0.01),而淋浆组的细静脉口径一直维持正常的恒定水平。同时,模型组细静脉的血流状态由线粒流减慢为粒线流、粒流甚至粒缓流,出现红细胞聚集及出血,加权积分值显著高于对照组和淋浆组(P<0.01,P<0.05),而淋浆组自300 min起,方出现流态改变,积分值虽高于对照组,但仍显著优于模型组。注射LPS后,细动脉的流态变化晚于细静脉,淋浆组的血流状态显著优于模型组(P<0.01,P<0.05)。LPS攻击后,模型组肠系膜细静脉出现白细胞黏附,进行性加重,白细胞黏附数显著多于对照组和淋浆组(P<0.01,P<0.05)。而淋浆组则维持在注射淋浆前的低水平,自270 min后方缓慢增多,但仍显著少于模型组(P<0.01)。模型组和淋浆组的肠系膜微淋巴管自LPS攻击30 min起,降低的收缩性指数和最大收缩口径均恢复到实验前及对照组水平(P>0.05),尽管微淋巴管口径有所改变,除模型组的LD-Index偶见增高(P<0.05)外,在实验180 min内一直维持此正常水平。在实验180 min内,三组的微淋巴管自主收缩频率均未见显著变化。
LPS攻击后180min,模型组大鼠血浆、肺和肝组织匀浆中的P-选择素含量显著增高(P<0.05,P<0.01),淋浆组血浆中P-选择素含量虽高于对照组(P<0.05),但血浆及肺中的P-选择素均显著低于模型组(P<0.01),肾匀浆中的含量未见组间差异(P>0.05)。同时,模型组的肺和肝组织匀浆中ICAM-1含量显著高于对照组和淋浆组(P<0.01,P<0.05),其360 min的血浆和肺、肝、肾组织匀浆中ICAM-1含量均显著高于对照组(P<0.01,P<0.05);而淋浆组180 min时血浆及各组织匀浆中的含量均维持对照组的低水平,360 min时虽血浆及肺、肾匀浆中的含量高于对照组(P<0.01,P<0.05),但显著低于模型组(P<0.05)。
LPS攻击后180 min和360 min,模型组大鼠肺、肝、肾匀浆中的MPO活性均显著高于对照组(P<0.01,P<0.05),其Na+-K+-ATPase活性均显著低于对照组(P<0.01,P<0.05);淋浆组肾匀浆中MPO活性与对照组无统计学差异,其肺、肝匀浆的MPO活性在两个时间点虽高于对照组,但肺匀浆显著低于模型组(P<0.01,P<0.05),肝匀浆在180min时显著低于模型组。淋浆组除180 min时肺、肝匀浆的Na+-K+-ATPase活性与对照组无差异外,其余组织匀浆的Na+-K+-ATPase活性虽低于对照组,但仍显著高于模型组。
对血浆肾、肝功能生化指标检测表明,注射LPS 180 min和360min,模型组和淋浆组的BUN、Cre、AST和ALT含量均显著高于对照组(P<0.01);但淋浆组的BUN和Cre在360 min时均显著低于模型组(P<0.01,P<0.05)。其AST和ALT在180 min及360 min也均显著低于模型组(P<0.01,P<0.05)。病理形态学研究表明,模型组的肺、肾、肝组织损伤严重,淋浆组病变较轻,与两组上述器官的湿/干比值均高于对照组(P<0.01,P<0.05)相一致,但淋浆组肺组织的W/D比值显著低于模型组(P<0.05)。对照组大鼠长期存活,淋浆组大鼠存活时间为(11.80±2.67)h,显著长于模型组(7.21±1.33)h(P<0.01)。
上述结果表明,正常淋浆对LPS攻击引起的ES具有良好的干预作用,表现在改善低血压及组织低灌注;减轻脏器的功能障碍和形态损伤,延长存活时间。淋浆干预ES的机制与改善微循环障碍、抑制P-选择素及ICAM-1等黏附分子的合成、减少白细胞黏附于微血管和扣押到器官有关,从而优化微血流,恢复组织灌注,改善细胞代谢和膜泵功能,有利于逆转休克。以正常淋浆干预ES的策略,为临床治疗ES提出了新思路。
Endotoxic shock (ES) is akind of common serious diseases in clinic.It is one of the important causes of death and the mortality increases yearby year. Lipopolysaccharide (LPS) plays an essential role in itspathogenesis, which come from Gram-negative bacteria. Although theencouraging progress has been made in the treatment of some shock, thedeath rate of ES is still high. The aim of the study is to investigate the newmethod in ES therapy. The ES model of rats were duplicated byintravenous injection with LPS. The blood pressure, micro-local blood flowperfusion volume, functional and structural organs and survival time wereobeserved to reveal interference effects of lymph plasma on ES, andmoreover the changes of mesenteric microcirculation (adherent leukocytes,micro- vascular diameter, blood flow condition, lymphatic contractility)were observed and levels of intercellular adhesion molecule-1 (ICAM-1),P-selectin in plasma and homogenate, and the activities of myeloperoxidase(MPO) and Na+-K+-ATPase in homogenate were determined to explore theinterferernce mechanisms of lymph plasma on ES, which provided newideas for the clinic to treat septic shock.
90 SPF-class male Wistar rats were randomly divided into threegroups: control group, model group and lymph plasma group (n=30). Allrats were anesthetized with 1% sodium pentobarbital (50mg·kg-1·bw, im)and anticoagulated with venous injection of heparin (420 U·kg-1·bw). Thenthe above animals were operated on neck. The right common carotid arterywas cannulated to record mean arterial pressure (MAP) continuously withbiological signal acquisition system, the left jugular vein were cannulatedto perepare for infusing drugs and taking blood. After stabilizaion for 30minutes, the LPS solution was infused (15mg·kg-1, Sigma) via the left jugular vein into model group and lymph plasma groups, normal salinereplaced LPS in the same moment in control group. After 15 minutes,lymph plasm was infused in lymph plasma group with ZCZ-50 automaticpump and injection machine at the speed of 0.5 ml per minute. The amountof lymph plasma was one fifteenth of whole blood volume whichcalculated by 7.4% of body weight. In control and model groups, lymphplasmwas replaced by normal saline.
Each group above was divided into three subgroups: ten rats of thefirst subgroup were used for observing blood flow perfusion volume ofkidney and liver in 360 minutes after LPS challenge with laser Dopplerflowmetry of Perimed company. At the end of the experiment, thefiberoptic probe and canulas were removed, the wounds were sewed uplayer by layer, and than the survival time of rats were observed from themoment of infusion with LPS to death. If the survival time was more than24 hours, the rats were considered long-term life. Ten rats of the secondsubgroup were used for observing mesenteric blood microcirculation ofmesentery near the ileum lower by Olympus microcirculatory microscopeand video record system for 360 minutes after LPS challenge. The indexesobserved included the changes of mesenteric microvascular diameter,leukocyte adhesion and blood flow condition. Ten rats of the third subgroupwere used for observing mesenteric lymphatic microcirculation. Thechanges of lymphatic spontaneous contractile frequency, maximumcontractile diameter, maximum diastolic diameter, stillstand state diameterand contractile parameters were observed after administration with LPS. At180 minutes and 360 minutes after LPS (or corresponding liquid) infusionrespectively, blood and lung, kidney, liver tissues of fixed position weretaken from the two subgroups, which were used for observing mesentericmicrocirculation. The tissues were prepared for homogenate of 10 percentand made into paraffin section to HE staining, which tissues at 360 minutesafter LPS challenge. The blood urea nitrogen (BUN), creatinine (Cre), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) inplasma and the P-selectin, ICAM-1 in plasma and homogenate, and theactivities of MPO and Na+-K+-ATPase in homogenate were determined.The wet/dry (W/D) ratio of lung, kidney and liver were calculated, as wellas to observe the morphologic changes of lung, kidney and liver tissues.
The results showed that every index observed in experiment had nosignificant difference between three groups before administration (P>0.05).In control group, the MAP, micro-local blood flow perfusion volume ofkidney and liver, the mesenteric microvascular diameter and blood flowcondition maintained constant level throughout the experiment, so did themaximum contractile diameter, maximum diastolic diameter, stillstand statediameter, spontaneous contractile frequency and three contractileparameters of IndexⅠ, IndexⅡand LD-Index (P>0.05). The adherentleukocyte in mesenteric venule was no found and only a few of adherentleukocytes were observed along the time of observation in control group.
In model and lymph plasma groups, the MAP, kidney and livermicro-local blood flow perfusion volume significantly decreased and themesenteric arteriolar diameter contracted, the blood flow condition sloweddown and the number of leukocytes adherent to venular wall increased,maximum contractile range and three contractile parameters of mesentericmicro-lymphatic decreased after LPS challenge for 15 minutes. All indexeshad no significant difference between the two groups (P>0.05), but therewas significant difference compared with control group (P<0.01, P<0.05).The lymph plasma group was prevailed over model group on these indexesafter infusion with lymph plasma.
Although the MAP recovered after the degression at the moment ofadministration with LPS in model and lymph plasma groups, the modelgroup were still significantly lower than that of control group (P<0.01,P<0.05) and had been decreasing continually since 180 minutes after LPSinfusion. The MAP remained a normal level during 120~240 minutes in lymph plasma group. From infusion with lymph plasma to 90 minutes andsince 330 minutes to the end, that of lymph plasma group was obviouslyhigher than that of the model group (P<0.05, P<0.01).
With the MAPrecovering, the kidney and liver micro-local blood flowperfusion volume also recovered, and that of kidney in two groups werehigher than that of control group (P<0.05, P<0.01). Those of livermaintained normal level. In model group, blood flow perfusion volume ofkidney was reduced to a normal level from 45 to 105 minutes, and thenshowed fluctuant decreasing. Its blood flow perfusion volume had beengradually progressive decreased since 195 minutes in kidney anddecreasing since 270 minutes in liver. The time during which started tocontinuously decrease of kidney blood flow perfusion volume wassignificantly earlier than that of liver (P<0.01). In lymph plasma group,high level of blood flow perfusion volume of kidney maintained up to 75minutes and had been closed to control group since 75 minutes (P>0.05).The blood flow perfusion volume of liver was approached to the level ofpre-experiment and control group, except several times higher than that ofcontrol group by chance (P>0.05). Since 270 minutes, the kidney and livermicro-local blood flow perfusion volume was significantly higher than thatof model group (P<0.05, P<0.01).
In model group, mesenteric arteriole diameter remained contractilestate throughout the experiment and it was significantly narrowedcompared with pre-experiment and control group (P<0.01, P<0.05), but inlymph plasma group, arteriole diameter recovered to the level ofpre-experiment and control group after infusion with lymph plasma(P<0.05), and contracted starting from 330 minutes. The mesenteric venulediameter progressive contracted since 150 minutes in model group, whichwas significantly lower than that of control group and lymph plasma groups(P<0.05, P<0.01), but the venular diameter maintained normal levelconstantly in lymph plasma group. Meanwhile, blood flow condition in model group slowed down from line flow to granular-line flow, to granularflow even to slow granular flow which resulted in multiple score valueincreased significantly compred with control and lymph plasma groups(P<0.01, P<0.05), and there appeared erythrocyte aggregation and bleeding.But in lymph plasma group, there was no change of blood flow conditionuntil 300 minutes, and since then the multiple score value was higher thanthat of control group, but lower than that of model group. The changes ofarteriole blood flow condition were later than that of the venule in modelgroup and lymph plasma groups, and blood flow condition of lymphplasma group was significantly better than that of the model group (P<0.01,P<0.05). After infusion with LPS, there were progressively increasingleukocytes adherent to mesenteric venules in model group, and numbers ofadherent leukocytes were significantly increased compared with controland lymph plasma groups (P<0.01, P<0.05). The adherent numbers oflymph plasma group maintained a low level the same as those beforeinfusing with lymph plasma, although it had been increasing slowly since270 minutes, but still significantly less than that of model group (P<0.01).Since 30 minutes after LPS challenge in model and lymph plasma groups,the decreased contractile parameters and maximum contractile diameter ofmesenteric micro-lymphatic had recoverd to normal levels ofpre-experiment and control group (P>0.05), although the changes ofmicro-lymphatic diameter were displayed. The normal level of spontaneouscontractile parameters maintained up to 180 minutes, except that LD-Indexincreased occasionally in model group (P<0.05). The spontaneouscontractile frequency of micro-lymphatic had no significant change in threegroups within 180 minutes.
After LPS challenge for 180 minutes, the concentration of P-selectinwas significantly increased in plasma and homogenate of lung and liver inmodel group (P<0.05, P<0.01). In lymph plasma group, the concentrationof plasma P-selectin was higher than that of control group (P<0.05), but P-selectin of plasma and pulmonary homogenate was significantly lowerthan that of model group (P<0.01). There was no significant difference ofP-selectin in renal homogenate between the model and lymph plasmagroups (P>0.05). At the same time, the level of ICAM-1 in pulmonary andhepatic homogenate in model group was significantly higher than that oflymph plasma and control groups (P<0.01, P<0.05). After challenge LPSfor 360 minutes, the level of ICAM-1 in plasma and homogenate of lung,liver and kidney was significantly higher than that of control group (P<0.01,P<0.05), while the level of ICAM-1 in homogenates of lymph plasmagroup were all closed to control group after 180 minutes. Although theICAM-1 of plasma and homogenates of lung and kidney was significantlyhigher than that of control group (P<0.01, P<0.05), the level of ICAM-1was significantly lower than that of model group (P<0.05).
After LPS challenge for 180 and 360 minutes, the MPO activity inhomogenate of lung, liver and kidney in model group was significantlyhigher than that of control group (P<0.01, P<0.05), and the Na+-K+-ATPaseactivity was obviously lower than that of control group (P<0.01, P<0.05).The MPO activity in renal homogenate of lymph plasma group showed nostatistically significant difference with control group, and pulmonary andhepatic homogenates was higher than that of control group, but thepulmonary was lower than that of model group and MPO of liver waslower than that of model group in 180 minutes(P<0.01, P<0.05). Exceptthe activity showed no significant difference between lymph plasma andcontrol groups at 180 minutes, every one of Na+-K+-ATPase activity inhomogenates of lymph plasma group was significantly lower than that ofcontrol group, but those were obviously higher than model group.
The results of detecting biochemical indexes of kidney and liverfunction in plasma showed that the concentration of BUN, Cre, AST, andALT significantly increased in model and lymph plasma groups afterinfusion with LPS for 180 minutes and 360 minutes, which compared with control group (P<0.01). In lymph plasma group, the level of BUN and Cresignificantly decreased compared with model group at 360 minutes(P<0.05, P<0.01), the level of AST and ALT were all lower than that ofmodel group at 180 minutes and 360 minutes (P<0.05, P<0.01).Pathomorphologic study showed that the tissue structures were foundserious damage in lung, kidney, liver of model group, and only mild lesionscould be found in organs of lymph plasma group. The W/D ratio ofabove-mentioned organs in both groups was higher than that of controlgroup (P<0.01, P<0.05), which in accordance with the tissue morphologicdamage. The W/D ratio of lung in lymph plasma group was significantlylower than that of model group (P<0.05). The rats of control group werelong-term life. The survival time of lymph plasma was (11.80±2.67) hours,it was significantly than that of model group(P<0.01), which was(7.21±1.33) hours.
The results above suggests that normal lymph plasma plays a positiveinterference effect on endotoxic shock induced by LPS, including impro-ving hypotension and tissue hypoperfusion volume, depressing organsdysfunction and morphologic damage and prolonging survival time. Theinterference mechanisms of lymph plasma on ES may be concerned withthe improvement of microcirculatory dysfunction, alleviatingion of theproduction of cell adhesion molecules (such as P-selectin and ICAM-1) andreducing leukocytes adherent to microvascular wall and detained in organs.Thus protecting micro blood flow condition and recovering tissue bloodflow perfusion, improving cellular metabolism and membrane pump, all ofthese help to reverse shock. The tactics of normal lymph plasma to interfereendotoxic shock may present a new way to clinical treatment.
SPF级Wistar雄性大鼠90只,随机均分模型组(Model group)、淋浆组(Lymph plasma group)、对照组(Control group),每组30只。以戊巴比妥钠(50 mg·kg-1·bw)肌注麻醉、肝素全身抗凝(420 U·kg-1·bw)后,行颈部手术,右颈总动脉插管,通过生物信号采集系统连续记录平均动脉压(mean arterial pressure,MAP),左颈浅静脉插管,备注药及取血用。三组大鼠稳定30min后,模型组和淋浆组经颈浅静脉注射LPS(15 mg·kg-1,Sigma)复制ES模型;对照组以等量生理盐水代替LPS;15 min后,淋浆组用ZCZ-50自动抽注机以0.5 ml·min-1的速度自颈静脉输入淋浆(占全血量1/15,全血量按体重的7.4%计算),模型组和对照组以生理盐水代替淋浆。
上述每组大鼠又分为3个亚组:亚组1每组10只大鼠用于微区血流灌注量的观察,通过Perimed激光多普勒血流灌注仪观察LPS攻击后360 min内右肾中下极和肝右中叶下部微区血流灌注量,实验结束后,取出光导纤维探头及插管,逐层缝合伤口,观察自注入LPS到死亡的存活时间,存活24 h以上者为长期存活;亚组2每组10只用于回肠下段肠系膜血液微循环观察,通过Olympus微循环显微电视录像系统连续观察LPS攻击后360 min内肠系膜微血管口径、白细胞黏附及血液流态改变;亚组3每组10只用于肠系膜淋巴微循环观察,观察注射LPS后180 min内微淋巴管收缩频率、最大收缩口径、最大舒张口径、静态口径及收缩参数的改变。观察肠系膜微循环的两亚组大鼠分别于注入LPS(或相应液体)后180 min及360 min,取血液及固定位置的肺、肝、肾组织,分别制备10%组织匀浆,360 min者做石蜡切片、HE染色,检测血浆中尿素氮(blood urea nitrogen,BUN)、肌酐(creatinine,Cre)、天门冬氨酸氨基转移酶(aspartate aminotransferase,AST)和丙氨酸氨基转移酶(alanine aminotransferase,ALT),血浆及组织匀浆中P选择素和ICAM-1含量,及组织匀浆中MPO及Na+-K+-ATPase活性的变化,以及肺、肝、肾组织的湿/干比(wet/dry,W/D),观察肺、肝、肾组织的形态学变化。
研究结果显示:实验前,各组大鼠各项观测指标均无组间差异(P>0.05),对照组大鼠在整个观察过程中, MAP、肾、肝的微区血流灌注量、肠系膜微血管的口径和流态,微淋巴管的最大收缩口径、最大舒张口径、静态口径、自主收缩频率以及IndexⅠ、IndexⅡ、LD-Index三个收缩性指数均维持恒定(P>0.05)。肠系膜细静脉无白细胞黏附现象,随着观察时间的延长,偶见少数白细胞黏附。
LPS攻击后15 min,模型组和淋浆组的MAP均下降,肾和肝的微区血流灌注量显著减少,肠系膜细动脉口径缩窄,微血流流态变慢,加权积分值增高,白细胞黏附于细静脉壁,肠系膜微淋巴管的最大收缩幅度减小,其IndexⅠ、IndexⅡ和LD-Index三个收缩性指数均降低,两组间未见统计学差异(P>0.05),均与对照组形成鲜明的对比(P<0.01,P<0.05)。但淋浆组给予小量正常淋浆后,各观察指标显著优于模型组。
尽管模型组和淋浆组在注射LPS MAP迅速下降后均有所回升,但模型组仍显著低于对照组(P<0.01,P<0.05),且自180 min后进行性下降;淋浆组的MAP在120~240 min维持正常水平,其后虽有降低,但自注射淋浆至90 min及自330 min起,显著优于模型组(P<0.05,P<0.01)。
在MAP出现回升波的同时,两组大鼠肾和肝的微区血流灌注量均出现回升,且肾灌注量均高于对照组(P<0.05,P<0.01),肝灌注量维持正常水平。模型组的肾灌注量在45~105 min降至正常水平,之后逐渐波动式下降,自195 min起血流灌注量持续进行性下降,肝灌注量自270 min开始持续进行性下降,肾脏出现持续性低灌注的时间显著早于肝脏(P<0.01)。淋浆组的肾灌注量高水平维持至75 min,其后至实验结束一直接近对照组水平(P>0.05),其肝灌注量除偶高于对照组外,均处于实验前及对照组的恒定水平(P>0.05),且自270 min起,其肾、肝微区血流灌注量均显著高于模型组(P<0.05,P<0.01)。
模型组大鼠的肠系膜细动脉口径在整个实验过程中均呈缩窄状态,显著低于实验前及对照组(P<0.01,P<0.05),而淋浆组在注射淋浆后,细动脉口径均恢复至实验前和对照组水平(P>0.05),至330 min时方出现口径缩窄。模型组的肠系膜细静脉自实验150 min起,细静脉口径进行性缩小,显著低于对照组和淋浆组(P<0.05,P<0.01),而淋浆组的细静脉口径一直维持正常的恒定水平。同时,模型组细静脉的血流状态由线粒流减慢为粒线流、粒流甚至粒缓流,出现红细胞聚集及出血,加权积分值显著高于对照组和淋浆组(P<0.01,P<0.05),而淋浆组自300 min起,方出现流态改变,积分值虽高于对照组,但仍显著优于模型组。注射LPS后,细动脉的流态变化晚于细静脉,淋浆组的血流状态显著优于模型组(P<0.01,P<0.05)。LPS攻击后,模型组肠系膜细静脉出现白细胞黏附,进行性加重,白细胞黏附数显著多于对照组和淋浆组(P<0.01,P<0.05)。而淋浆组则维持在注射淋浆前的低水平,自270 min后方缓慢增多,但仍显著少于模型组(P<0.01)。模型组和淋浆组的肠系膜微淋巴管自LPS攻击30 min起,降低的收缩性指数和最大收缩口径均恢复到实验前及对照组水平(P>0.05),尽管微淋巴管口径有所改变,除模型组的LD-Index偶见增高(P<0.05)外,在实验180 min内一直维持此正常水平。在实验180 min内,三组的微淋巴管自主收缩频率均未见显著变化。
LPS攻击后180min,模型组大鼠血浆、肺和肝组织匀浆中的P-选择素含量显著增高(P<0.05,P<0.01),淋浆组血浆中P-选择素含量虽高于对照组(P<0.05),但血浆及肺中的P-选择素均显著低于模型组(P<0.01),肾匀浆中的含量未见组间差异(P>0.05)。同时,模型组的肺和肝组织匀浆中ICAM-1含量显著高于对照组和淋浆组(P<0.01,P<0.05),其360 min的血浆和肺、肝、肾组织匀浆中ICAM-1含量均显著高于对照组(P<0.01,P<0.05);而淋浆组180 min时血浆及各组织匀浆中的含量均维持对照组的低水平,360 min时虽血浆及肺、肾匀浆中的含量高于对照组(P<0.01,P<0.05),但显著低于模型组(P<0.05)。
LPS攻击后180 min和360 min,模型组大鼠肺、肝、肾匀浆中的MPO活性均显著高于对照组(P<0.01,P<0.05),其Na+-K+-ATPase活性均显著低于对照组(P<0.01,P<0.05);淋浆组肾匀浆中MPO活性与对照组无统计学差异,其肺、肝匀浆的MPO活性在两个时间点虽高于对照组,但肺匀浆显著低于模型组(P<0.01,P<0.05),肝匀浆在180min时显著低于模型组。淋浆组除180 min时肺、肝匀浆的Na+-K+-ATPase活性与对照组无差异外,其余组织匀浆的Na+-K+-ATPase活性虽低于对照组,但仍显著高于模型组。
对血浆肾、肝功能生化指标检测表明,注射LPS 180 min和360min,模型组和淋浆组的BUN、Cre、AST和ALT含量均显著高于对照组(P<0.01);但淋浆组的BUN和Cre在360 min时均显著低于模型组(P<0.01,P<0.05)。其AST和ALT在180 min及360 min也均显著低于模型组(P<0.01,P<0.05)。病理形态学研究表明,模型组的肺、肾、肝组织损伤严重,淋浆组病变较轻,与两组上述器官的湿/干比值均高于对照组(P<0.01,P<0.05)相一致,但淋浆组肺组织的W/D比值显著低于模型组(P<0.05)。对照组大鼠长期存活,淋浆组大鼠存活时间为(11.80±2.67)h,显著长于模型组(7.21±1.33)h(P<0.01)。
上述结果表明,正常淋浆对LPS攻击引起的ES具有良好的干预作用,表现在改善低血压及组织低灌注;减轻脏器的功能障碍和形态损伤,延长存活时间。淋浆干预ES的机制与改善微循环障碍、抑制P-选择素及ICAM-1等黏附分子的合成、减少白细胞黏附于微血管和扣押到器官有关,从而优化微血流,恢复组织灌注,改善细胞代谢和膜泵功能,有利于逆转休克。以正常淋浆干预ES的策略,为临床治疗ES提出了新思路。
Endotoxic shock (ES) is akind of common serious diseases in clinic.It is one of the important causes of death and the mortality increases yearby year. Lipopolysaccharide (LPS) plays an essential role in itspathogenesis, which come from Gram-negative bacteria. Although theencouraging progress has been made in the treatment of some shock, thedeath rate of ES is still high. The aim of the study is to investigate the newmethod in ES therapy. The ES model of rats were duplicated byintravenous injection with LPS. The blood pressure, micro-local blood flowperfusion volume, functional and structural organs and survival time wereobeserved to reveal interference effects of lymph plasma on ES, andmoreover the changes of mesenteric microcirculation (adherent leukocytes,micro- vascular diameter, blood flow condition, lymphatic contractility)were observed and levels of intercellular adhesion molecule-1 (ICAM-1),P-selectin in plasma and homogenate, and the activities of myeloperoxidase(MPO) and Na+-K+-ATPase in homogenate were determined to explore theinterferernce mechanisms of lymph plasma on ES, which provided newideas for the clinic to treat septic shock.
90 SPF-class male Wistar rats were randomly divided into threegroups: control group, model group and lymph plasma group (n=30). Allrats were anesthetized with 1% sodium pentobarbital (50mg·kg-1·bw, im)and anticoagulated with venous injection of heparin (420 U·kg-1·bw). Thenthe above animals were operated on neck. The right common carotid arterywas cannulated to record mean arterial pressure (MAP) continuously withbiological signal acquisition system, the left jugular vein were cannulatedto perepare for infusing drugs and taking blood. After stabilizaion for 30minutes, the LPS solution was infused (15mg·kg-1, Sigma) via the left jugular vein into model group and lymph plasma groups, normal salinereplaced LPS in the same moment in control group. After 15 minutes,lymph plasm was infused in lymph plasma group with ZCZ-50 automaticpump and injection machine at the speed of 0.5 ml per minute. The amountof lymph plasma was one fifteenth of whole blood volume whichcalculated by 7.4% of body weight. In control and model groups, lymphplasmwas replaced by normal saline.
Each group above was divided into three subgroups: ten rats of thefirst subgroup were used for observing blood flow perfusion volume ofkidney and liver in 360 minutes after LPS challenge with laser Dopplerflowmetry of Perimed company. At the end of the experiment, thefiberoptic probe and canulas were removed, the wounds were sewed uplayer by layer, and than the survival time of rats were observed from themoment of infusion with LPS to death. If the survival time was more than24 hours, the rats were considered long-term life. Ten rats of the secondsubgroup were used for observing mesenteric blood microcirculation ofmesentery near the ileum lower by Olympus microcirculatory microscopeand video record system for 360 minutes after LPS challenge. The indexesobserved included the changes of mesenteric microvascular diameter,leukocyte adhesion and blood flow condition. Ten rats of the third subgroupwere used for observing mesenteric lymphatic microcirculation. Thechanges of lymphatic spontaneous contractile frequency, maximumcontractile diameter, maximum diastolic diameter, stillstand state diameterand contractile parameters were observed after administration with LPS. At180 minutes and 360 minutes after LPS (or corresponding liquid) infusionrespectively, blood and lung, kidney, liver tissues of fixed position weretaken from the two subgroups, which were used for observing mesentericmicrocirculation. The tissues were prepared for homogenate of 10 percentand made into paraffin section to HE staining, which tissues at 360 minutesafter LPS challenge. The blood urea nitrogen (BUN), creatinine (Cre), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) inplasma and the P-selectin, ICAM-1 in plasma and homogenate, and theactivities of MPO and Na+-K+-ATPase in homogenate were determined.The wet/dry (W/D) ratio of lung, kidney and liver were calculated, as wellas to observe the morphologic changes of lung, kidney and liver tissues.
The results showed that every index observed in experiment had nosignificant difference between three groups before administration (P>0.05).In control group, the MAP, micro-local blood flow perfusion volume ofkidney and liver, the mesenteric microvascular diameter and blood flowcondition maintained constant level throughout the experiment, so did themaximum contractile diameter, maximum diastolic diameter, stillstand statediameter, spontaneous contractile frequency and three contractileparameters of IndexⅠ, IndexⅡand LD-Index (P>0.05). The adherentleukocyte in mesenteric venule was no found and only a few of adherentleukocytes were observed along the time of observation in control group.
In model and lymph plasma groups, the MAP, kidney and livermicro-local blood flow perfusion volume significantly decreased and themesenteric arteriolar diameter contracted, the blood flow condition sloweddown and the number of leukocytes adherent to venular wall increased,maximum contractile range and three contractile parameters of mesentericmicro-lymphatic decreased after LPS challenge for 15 minutes. All indexeshad no significant difference between the two groups (P>0.05), but therewas significant difference compared with control group (P<0.01, P<0.05).The lymph plasma group was prevailed over model group on these indexesafter infusion with lymph plasma.
Although the MAP recovered after the degression at the moment ofadministration with LPS in model and lymph plasma groups, the modelgroup were still significantly lower than that of control group (P<0.01,P<0.05) and had been decreasing continually since 180 minutes after LPSinfusion. The MAP remained a normal level during 120~240 minutes in lymph plasma group. From infusion with lymph plasma to 90 minutes andsince 330 minutes to the end, that of lymph plasma group was obviouslyhigher than that of the model group (P<0.05, P<0.01).
With the MAPrecovering, the kidney and liver micro-local blood flowperfusion volume also recovered, and that of kidney in two groups werehigher than that of control group (P<0.05, P<0.01). Those of livermaintained normal level. In model group, blood flow perfusion volume ofkidney was reduced to a normal level from 45 to 105 minutes, and thenshowed fluctuant decreasing. Its blood flow perfusion volume had beengradually progressive decreased since 195 minutes in kidney anddecreasing since 270 minutes in liver. The time during which started tocontinuously decrease of kidney blood flow perfusion volume wassignificantly earlier than that of liver (P<0.01). In lymph plasma group,high level of blood flow perfusion volume of kidney maintained up to 75minutes and had been closed to control group since 75 minutes (P>0.05).The blood flow perfusion volume of liver was approached to the level ofpre-experiment and control group, except several times higher than that ofcontrol group by chance (P>0.05). Since 270 minutes, the kidney and livermicro-local blood flow perfusion volume was significantly higher than thatof model group (P<0.05, P<0.01).
In model group, mesenteric arteriole diameter remained contractilestate throughout the experiment and it was significantly narrowedcompared with pre-experiment and control group (P<0.01, P<0.05), but inlymph plasma group, arteriole diameter recovered to the level ofpre-experiment and control group after infusion with lymph plasma(P<0.05), and contracted starting from 330 minutes. The mesenteric venulediameter progressive contracted since 150 minutes in model group, whichwas significantly lower than that of control group and lymph plasma groups(P<0.05, P<0.01), but the venular diameter maintained normal levelconstantly in lymph plasma group. Meanwhile, blood flow condition in model group slowed down from line flow to granular-line flow, to granularflow even to slow granular flow which resulted in multiple score valueincreased significantly compred with control and lymph plasma groups(P<0.01, P<0.05), and there appeared erythrocyte aggregation and bleeding.But in lymph plasma group, there was no change of blood flow conditionuntil 300 minutes, and since then the multiple score value was higher thanthat of control group, but lower than that of model group. The changes ofarteriole blood flow condition were later than that of the venule in modelgroup and lymph plasma groups, and blood flow condition of lymphplasma group was significantly better than that of the model group (P<0.01,P<0.05). After infusion with LPS, there were progressively increasingleukocytes adherent to mesenteric venules in model group, and numbers ofadherent leukocytes were significantly increased compared with controland lymph plasma groups (P<0.01, P<0.05). The adherent numbers oflymph plasma group maintained a low level the same as those beforeinfusing with lymph plasma, although it had been increasing slowly since270 minutes, but still significantly less than that of model group (P<0.01).Since 30 minutes after LPS challenge in model and lymph plasma groups,the decreased contractile parameters and maximum contractile diameter ofmesenteric micro-lymphatic had recoverd to normal levels ofpre-experiment and control group (P>0.05), although the changes ofmicro-lymphatic diameter were displayed. The normal level of spontaneouscontractile parameters maintained up to 180 minutes, except that LD-Indexincreased occasionally in model group (P<0.05). The spontaneouscontractile frequency of micro-lymphatic had no significant change in threegroups within 180 minutes.
After LPS challenge for 180 minutes, the concentration of P-selectinwas significantly increased in plasma and homogenate of lung and liver inmodel group (P<0.05, P<0.01). In lymph plasma group, the concentrationof plasma P-selectin was higher than that of control group (P<0.05), but P-selectin of plasma and pulmonary homogenate was significantly lowerthan that of model group (P<0.01). There was no significant difference ofP-selectin in renal homogenate between the model and lymph plasmagroups (P>0.05). At the same time, the level of ICAM-1 in pulmonary andhepatic homogenate in model group was significantly higher than that oflymph plasma and control groups (P<0.01, P<0.05). After challenge LPSfor 360 minutes, the level of ICAM-1 in plasma and homogenate of lung,liver and kidney was significantly higher than that of control group (P<0.01,P<0.05), while the level of ICAM-1 in homogenates of lymph plasmagroup were all closed to control group after 180 minutes. Although theICAM-1 of plasma and homogenates of lung and kidney was significantlyhigher than that of control group (P<0.01, P<0.05), the level of ICAM-1was significantly lower than that of model group (P<0.05).
After LPS challenge for 180 and 360 minutes, the MPO activity inhomogenate of lung, liver and kidney in model group was significantlyhigher than that of control group (P<0.01, P<0.05), and the Na+-K+-ATPaseactivity was obviously lower than that of control group (P<0.01, P<0.05).The MPO activity in renal homogenate of lymph plasma group showed nostatistically significant difference with control group, and pulmonary andhepatic homogenates was higher than that of control group, but thepulmonary was lower than that of model group and MPO of liver waslower than that of model group in 180 minutes(P<0.01, P<0.05). Exceptthe activity showed no significant difference between lymph plasma andcontrol groups at 180 minutes, every one of Na+-K+-ATPase activity inhomogenates of lymph plasma group was significantly lower than that ofcontrol group, but those were obviously higher than model group.
The results of detecting biochemical indexes of kidney and liverfunction in plasma showed that the concentration of BUN, Cre, AST, andALT significantly increased in model and lymph plasma groups afterinfusion with LPS for 180 minutes and 360 minutes, which compared with control group (P<0.01). In lymph plasma group, the level of BUN and Cresignificantly decreased compared with model group at 360 minutes(P<0.05, P<0.01), the level of AST and ALT were all lower than that ofmodel group at 180 minutes and 360 minutes (P<0.05, P<0.01).Pathomorphologic study showed that the tissue structures were foundserious damage in lung, kidney, liver of model group, and only mild lesionscould be found in organs of lymph plasma group. The W/D ratio ofabove-mentioned organs in both groups was higher than that of controlgroup (P<0.01, P<0.05), which in accordance with the tissue morphologicdamage. The W/D ratio of lung in lymph plasma group was significantlylower than that of model group (P<0.05). The rats of control group werelong-term life. The survival time of lymph plasma was (11.80±2.67) hours,it was significantly than that of model group(P<0.01), which was(7.21±1.33) hours.
The results above suggests that normal lymph plasma plays a positiveinterference effect on endotoxic shock induced by LPS, including impro-ving hypotension and tissue hypoperfusion volume, depressing organsdysfunction and morphologic damage and prolonging survival time. Theinterference mechanisms of lymph plasma on ES may be concerned withthe improvement of microcirculatory dysfunction, alleviatingion of theproduction of cell adhesion molecules (such as P-selectin and ICAM-1) andreducing leukocytes adherent to microvascular wall and detained in organs.Thus protecting micro blood flow condition and recovering tissue bloodflow perfusion, improving cellular metabolism and membrane pump, all ofthese help to reverse shock. The tactics of normal lymph plasma to interfereendotoxic shock may present a new way to clinical treatment.
引文
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