免疫因素在大鼠肺缺血再灌注损伤中的作用

目的探讨免疫因素在大鼠肺缺血再灌注(I/R)损伤中的作用。方法选取90只健康雄性Wistar大鼠,随机分为3组,即为I/R组(左肺I/R组),Sham组(假手术组),C组(对照组),均于缺血30 min、再灌注60、120 min时采集标本,检测肺组织的湿/干重比(W/D),支气管肺泡灌洗液的蛋白总量(TP),白细胞数目(WBC),中性粒细胞(PMN)的百分比及肺组织匀浆肿瘤坏死因子(TNF)-α、IL-1、IL-6及IL-8含量。结果在各组的对应时间点,I/R组中再灌注60、120 min时,W/D比值、WBC、PMN比例及TP含量明显高于C组及Sham组(P<0.05);I/R组中再灌注120 min,W/D比值、WBC、PMN比例及TP含量明显高于I/R组中再灌注60 min(P<0.05);I/R组中缺血30 min,肺组织匀浆TNF-α含量明显高于C组及Sham组(P<0.05);I/R组中再灌注60 min及再灌注120 min时,肺组织匀浆TNF-α、IL-1、IL-6及IL-8含量明显高于C组及Sham组(P<0.05);I/R组中再灌注120 min时,肺组织匀浆TNF-α、IL-1、IL-6及IL-8含量明显高于I/R组再灌注60 min时(P<0.05)。结论免疫因素在大鼠肺I/R损伤发挥重要作用。     

吸入一氧化氮对犬肺血栓栓塞症再灌注后血气及血流动力学的影响

目的 建立血栓栓塞1周的犬肺栓塞(PTE)模型,观察吸入20 ppm一氧化氮(NO)对取栓再灌注后血气及血流动力学的影响并探讨其可能的机制.方法 对该PTE犬模型行取栓术,观察再灌注0、2、4、6 h后生命体征、血气变化,通过漂浮导管监测肺血流动力学的变化,再灌注6 h后肺泡灌洗液(BALF)中的白蛋白含量(g/L)、肺组织湿干重比值(W/D)、肺泡腔多核中性粒细胞(PMN)数.结果再灌注2 h后与再灌注前比较(下同)平均肺动脉压(MPAP)升高[(3.20±0.53)kPa对(2.27±0.67)kPa,F=63,P=0.02],再灌注6 h后,心率增快最明显[(175±8)次/min对(155±5)次/min,F=38.72,P=0.01],氧合指数下降(41.70±8.04对54.71±3.78,F=48.36,P=0.03);再灌注后2 h,吸入NO组与再灌注组(下同)比较MPAP降低((2.53±0.40)kPa对(3.20±0.53)kPa,F=55,P=0.04],4 h,氧合指数有升高趋势(49.17±7.37对39.71±7.31,F=2.36,P=0.11),再灌注后2、4、6 h,吸入NO组肺血管阻力较再灌注组也有减小趋势(F=1.49,P=0.26),6 h后吸入NO组肺泡腔PMN数低于再灌注组[(19±6)个/10 HPF对(31±11)个/10 HPF,F=98,P=0.01]. 结论 血栓栓塞1周的PTE犬模型再灌注后导致了肺再灌注损伤,再灌注不同时间点对肺组织损伤程度不一.吸入20 ppm NO可降低PTE再灌注损伤导致的升高的肺动脉压,减少PMN向肺组织的迁移而减轻再灌注损伤,可能减轻肺微血管渗漏.     

N-乙酰半胱氨酸对大鼠肺缺血再灌注损伤的保护作用

目的探讨N-乙酰半胱氨酸(NAC)对大鼠在体肺缺血再灌注(I/R)损伤的保护作用。方法建立大鼠在体肺缺血再灌注模型,将30只SD大鼠随机分成假手术对照组,缺血再灌注组(I/R组)和N-乙酰半胱氨酸组(NAC组),NAC组缺血前1 h给予腹腔注射N-乙酰半胱氨酸200 mg/kg。再灌注2 h后摘取左肺,分别对各组进行以下检测:肺湿/干比(W/D)、超氧化物歧化酶(SOD)活力、髓过氧化物酶(MPO)活性、丙二醛(MDA)含量并进行病理学检查及肺组织损伤定量评价(IQA)。结果I/R组肺W/D和IQA显著高于假手术组(P0.01),NAC组上述指标明显降低(P0.01)。病理学结果显示三组动物肺组织结构基本正常,假手术组无充血;与NAC组比较,I/R组肺组织充血明显、白细胞浸润更严重及肺间质高度淤血水肿。I/R组MDA含量和MPO活性较假手术组明显升高(P0.01),SOD活性显著下降(P0.01)。NAC能明显减少MDA含量和降低MPO活性,提高SOD活性(P0.01)。结论N-乙酰半胱氨酸对肺缺血再灌注损伤具有保护作用,可能与其抗氧化作用和抑制中性粒细胞激活有关。     

移植胰腺再灌注后细胞凋亡及Fas表达的实验研究

目的 探讨胰腺移植再灌注后胰腺细胞凋亡发生的过程及凋亡相关基因Fas的表达。方法 将60只SD大鼠随机分成7组:假手术组(5只),冷缺血2小时组(I组,5只),冷缺血2小时再灌注1、3、6、9、12小时组(I/R1组、I/R3组、I/R6组、I/R9组、I/R12组,各10只)。HE染色后光镜及电镜观察各组胰腺组织的病理变化;流式细胞仪检测各组胰腺细胞的凋亡百分比;采用免疫组织化学方法检测各组胰腺组织中Fas蛋白的表达。结果胰腺移植后早期即可观察到细胞凋亡的典型改变,胰腺冷缺血再灌注后发生凋亡的高峰期为再灌注后3小时[凋亡率(AR)为(12.61％±2.94％),P<0.01],再灌注6小时较3小时细胞凋亡有所减少[AR为(6.35％±2.19％),P<0.01),再灌注9小时及12小时细胞凋亡进一步减少[AR分别为(3.67％±1.44％)及(3.33％±1.03％),P<0.05]。假手术组与I/R3组均未见Fas蛋白表达,I/R1组Fas(+);至I/R3组Fas表达增强为(++),以后各组表达有所下降。结论 细胞凋亡是胰腺移植后的早期事件,移植胰腺冷缺血再灌注后的凋亡高峰发生在再灌注后3小时;移植胰腺的细胞凋亡受基因调控,Fas基因可促进胰腺细胞的凋亡。     

犬急性肺缺血再灌注损伤的病理学改变及中药814的干预作用

目的 :探讨肺缺血再灌注损伤的病理学改变及中药 814的干预作用。方法 :7只杂种犬随机分两组 :①急性肺缺血再灌注组 (3只 ) :结扎左主肺动脉 2 4h后恢复血流 4h ;②中药 814干预组 (4只 ) :再灌注前予中药 814腹腔注射 ,应用光镜和电镜等病理检测方法观察缺血期、再灌注期、中药 814干预后肺组织病理形态学改变 ,计算受损肺泡百分率。结果 :①急性肺缺血再灌注组 :缺血期 ,左肺散在出血 ,部分肺泡上皮细胞及肺血管内皮细胞胞质肿胀、线粒体空泡化、内质网扩张 ;同例对照右肺基本正常。再灌注期 ,左肺出血加重 ,肺间质及广泛肺泡腔内有水肿液 ,受损肺泡百分率显著增加 ,肺泡上皮细胞和肺血管内皮细胞肿胀、线粒体空泡化及嵴溶解加重 ,可见细胞坏死 ;同例对照右肺大致正常或散在小灶性肺泡腔内有水肿和出血 ,少数肺泡上皮细胞和肺血管内皮细胞轻度线粒体肿胀和空泡化。②中药 814干预组 :再灌注期左肺受损肺泡百分率显著减少 ,少数肺泡上皮细胞和肺血管内皮细胞线粒体轻度肿胀和空泡化。结论 :急性缺血后再灌注肺组织病理学形态显示比较明显的损伤 ,中药 814对再灌注肺损伤有一定的实验防治作用     

缺血后处理对心肌缺血再灌注损伤过程中内质网应激的影响研究

目的 探讨缺血后处理(IPostC)对心肌缺血再灌注损伤(MIRI)中细胞凋亡的影响,以及特异性内质网应激(ERS)损伤相关蛋白Grp78(葡萄糖调节蛋白78)和Caspase 12(半胱氨酸蛋白酶12)的蛋白表达水平的变化和意义。方法 Wistar 大鼠24只随机分为假手术组、I/R组(缺血再灌注组)、IPostC组(缺血后处理组)3组,每组8只。分别测定各组心肌缺血和梗死面积、细胞凋亡指数和Caspase12、 Grp78蛋白表达检测。结果 假手术组未见梗死心肌和缺血心肌,IPostC组心肌缺血区面积(46.46±2.13)%和梗死区面积(41.02±2.93)%均明显小于I/R组(53.31±3.87, 52.19±3.44)%,P值均<0.01。假手术组心肌细胞凋亡指数(6.70±2.25)%明显低于I/R组(26.92±1.91)% 、IPostC组(20.54±3.05)%,P值均<0.001。心肌组织Caspase12蛋白表达水平在假手术组(0.11±0.01)明显低于I/R组(0.41±0.06)和IPostC组(0.35±0.06),并且IPostC组低于I/R组,P值均<0.01;心肌组织Grp78蛋白水平在假手术组(0.13±0.03)亦明显低于I/R组(1.04±0.16)和IPostC组(1.17±0.14),并且IPostC组高于I/R组,P值均<0.01。结论 本研究从动物实验证实IPostC能减轻心肌细胞凋亡,而ERS激活参与了大鼠MIRI过程,推测IPostC在大鼠MIRI过程中可能通过调节ERS途径抑制细胞凋亡,改善MIRI。     

针对Fas的RNA抑制对缺血再灌注神经细胞的保护作用

目的探讨Fas-RNAi对缺血再灌注(I/R)损伤后神经细胞凋亡的保护作用。方法细胞随机分四组对照组、模型组、GFPsiRNA组、FassiRNA组。对照组用完全培养液培养,其他三组细胞制作体外(I/R)模型,GFPsiRNA组、FassiRNA于转染24h后造模,造模后6h进行Caspase-3、Fas表达检测,24h进行细胞凋亡相关检测。结果(1)Fas、Caspase-3阳性率模型组模型组的Fas阳性率(37.25±8.37)%、caspase-3阳性率(28.79±6.23)%明显高于对照组(7.21±3.28,8.01±2.31)%,差异有统计学意义(P<0.01)。GFPsiRNA组Fas阳性率(14.35±7.31)%、caspase-3阳性率(16.33±4.15)%明显低于模型组,差异有统计学意义(P<0.01)。(2)凋亡率模型组细胞(39.13±10.63)明显高于对照组(2.85±0.43),差异有显著性(P<0.01)。FassiRNA组(1.70±0.31)明显低于模型组(P<0.01)。结论针对Fas的siRNA能有效地抑制I/R损伤后神经细胞凋亡。对神经细胞I/R损伤有一定的保护作用。     

肺缺血-再灌注氧化应激反应对超微结构的影响及川芎嗪的作用

目的 探讨川芎嗪对肺缺血-再灌注(IR)损伤超微结构的影响及其机制.方法 采用在体兔单侧肺左肺门持续性阻断1 h,再灌注1,3,5 h IR损伤的动物模型.90只兔随机分为3组:(1)对照组:对兔进行假手术,不发生缺血;(2)IR组:缺血1 h,分别再灌注1,3,5 h.(3)TMP组:缺血前1 h静脉滴注射川芎嗪60 mg/kg,其余同IR组.测定各组血浆1,3,5 h末血浆超氧化物歧化酶(SOD)、丙二醛(MDA)的含量.常规电镜制片、染色,观察肺组织超微结构的变化.结果 与对照组相比IR组各时相血浆MDA含量显著升高(P＜0.01),SOD显著降低(P＜0.05);与IR组相比川芎嗪能减轻MDA的升高(P＜0.05,P＜0.01),而使SOD升高(P＜0.01).电镜下见对照组:肺微小动脉内皮结构、毛细血管内皮细胞及肺泡上皮细胞结构正常;IR组肺微小动脉内皮细胞损伤明显,线粒体水肿、空泡变性.肺毛细血管腔内多形核白细胞(PMN)堵塞,肺泡Ⅱ型上皮微绒毛脱失,细胞内板层小体疏松肿大;TMP组肺微小动脉内皮结构基本正常,个别线粒体轻度水肿,无空泡变性,毛细血管腔内PMN少,堵塞相对较轻,肺泡Ⅱ型上皮微绒毛少许脱落.结论 川芎嗪通过抑制氧自由基的作用,明显改善肺IR肺组织超微结构的改变,从而对IR肺组织有保护作用.     

缺氧诱导因子-1α在鼠肺缺血再灌注损伤中的表达与意义

目的:探讨缺氧诱导因子-1α(hypoxia inducible factor-1 alpha,HIF-1α)对鼠肺缺血再灌注损伤是否具有保护作用及可能的机制。方法:选择体重250～350 g的健康雄性SD大鼠72只,随机分为3组,缺血再灌注组、二甲基乙二酰基甘氨酸(DMOG)组、假手术组,每组24只。缺血再灌注组:建立左肺缺血和再灌注动物模型:在肺充气状态下用无损伤动脉钳夹闭左侧肺门,阻断45 min后松开动脉夹,形成再灌注;DMOG组:在缺血和再灌注动物模型建立前24 h进行腹腔内注射DMOG 20μg/g;假手术组:仅行左侧开胸,不行缺血再灌注处理。缺血再灌注组与DMOG组于再灌注1 h、3 h、6 h、12h后颈动脉放血处死大鼠,假手术组于开胸后1 h、3 h、6 h、12 h颈动脉放血处死大鼠,取左肺上半部制成匀浆,硫代巴比妥酸比色法检测丙二醛含量、黄嘌呤氧化酶法检测超氧化物歧化酶活力,酶联免疫吸附法测定匀浆内白细胞介素-8含量,下半部通过免疫组化法行HIF-1α蛋白表达的观察与光镜下苏木素伊红(HE)染色后肺组织病理学改变的观察。结果:HIF-1α蛋白主要表达于肺泡上皮细胞的胞核或胞浆,与假手术组比较,缺血再灌注组、DMOG组各时间点HIF-1α蛋白的表达增强,DMOG组较缺血再灌注组各时间点HIF-1α蛋白的表达增强;缺血再灌注组、DMOG组在再灌注后3 h、6 h、12 h(缺血再灌注组12 h除外)较1 h HIF-1α蛋白的表达增强,差异有统计学意义(P<0.05或P<0.01)。与假手术组比较,缺血再灌注组、DMOG组各时间点白细胞介素-8(除DMOG组1 h外)、丙二醛含量均升高,而超氧化物歧化酶活力降低;DMOG组较缺血再灌注组各时间点白细胞介素-8、丙二醛含量均降低,而超氧化物歧化酶活力升高;缺血再灌注组、DMOG组在再灌注后3 h、6 h、12 h较1 h:白细胞介素-8含量(除DMOG组3 h外)升高,丙二醛含量(除缺血再灌注组、DMOG组12 h外)升高,超氧化物歧化酶活力降低,差异均有统计学意义(P<0.05或P<0.01)。光镜下DMOG组肺组织炎性渗出较缺血再灌注组减轻。结论:HIF-1α通过降低肺组织中丙二醛、白细胞介素-8含量,增强肺组织中超氧化物歧化酶活力,减轻肺内毛细血管充血及炎性细胞浸润,对大鼠肺缺血与再灌注损伤发挥保护作用。     

吸入高浓度氢气对大鼠肺缺血再灌注损伤的影响

目的 探究高浓度氢气对大鼠肺缺血再灌注损伤的影响。方法 雄性SD大鼠30只,体重220～260 g,随机分为3组(n=10):假手术组(Sham组)、缺血再灌注组(I/R组)和氢气组(H2组)。采用阻断左侧肺门30min,再灌注1h的方法制备大鼠肺缺血再灌注模型。Sham组,只开胸,不建立缺血再灌注模型,I/R组和H2组建立缺血再灌注模型,H2组在再灌注期间吸入67%H2。再灌注1h后,采用ELISA法检测肺组织中白细胞介素-6(IL-6)、肿瘤坏死因子α(TNF-α)的水平,采用黄嘌呤氧化酶法和硫代巴比妥酸法检测肺组织中超氧化物歧化酶(SOD)的活性和丙二醛(MDA)的水平,并且用Western blot方法测定凋亡相关蛋白caspase-3和caspase-9的含量。结果 与Sham组比较,I/R组IL-6、TNF-α和MDA水平显著升高,SOD的活性降低,caspase-3和caspase-9的含量明显升高(P<0.05)。与I/R组比较,H2组IL-6、TNF-α和MDA水平显著降低,SOD的活性升高,caspase-3和caspase-9的含量明显降低(P<0.0...     

Protective effect of L-arginine preconditioning on ischemia and reperfusion injury associated with rat small bowel transplantation

AIM: To investigate the protective effect and possible mechanism of L-arginine preconditioning on ischemia and reperfusion injury associated with small bowel transplantation (SBT). METHODS: Male inbred Wistar rats weighting between 180 and 250 g were used as donors and recipients in the study. Heterotopic rat SBT was performed according to the techniques of Li and Wu. During the experiment, intestinal grafts were preserved in 4℃ Ringer's solution for 8 h before being transplanted. Animals were divided into three groups. In group 1, donors received intravenous L-arginine (50 mg/kg, 1 mL) injection 90 min before graft harvesting. However, donors in control group were given normal saline (NS) instead. In group 3, six rats were used as sham-operated control. Specimens were taken from intestinal grafts 15 min after reperfusion. Histological grading, tissue malondialdehyde (MDA) and myeloperoxidase (MPO) levels were assessed. The graft survival of each group was monitored daily until 14 d after transplantation. RESULTS: Levels of MDA and MPO in intestine of sham-operated rats were 2.0±0.22 mmol/g and 0.66±0.105 U/g. Eight hours of cold preservation followed by 15 min of reperfusion resulted in significant increases in tissue MDA and MPO levels. Pretreatment with L-arginine before graft harvesting resulted in lower enhancement of tissue levels of MDA and MPO and the differences were significant (4.71±1.02 mmol/g vs 8.02±3.49 mmol/g, 1.03±0.095 U/g vs 1.53±0.068 U/g, P<0.05). Besides, animals in L-arginine pretreated group had better histological structures and higher 2-wk graft survival rates comparing with that in NS treated group (3.3±0.52 vs 6±0.1, 0/6 vs 6/6, P<0.05 or 0.01). CONCLUSION: L-arginine preconditioning attenuates ischemia and reperfusion injury in the rat SBT model, which was due to antioxidant activities partially.     

Nuclear factor-kappaB decoy oligodeoxynucleotides attenuates ischemia/reperfusion injury in rat liver graft

AIM: To evaluate the protective effect of NF-kappaB decoy oligodeoxynucleotides (ODNs) on ischemia/reperfusion (I/R) injury in rat liver graft. METHODS: Orthotopic syngeneic rat liver transplantation was performed with 3 h of cold preservation of liver graft in University of Wisconsin solution containing phosphorothioated double-stranded NF-kappaB decoy ODNs or scrambled ODNs. NF-kappaB decoy ODNs or scrambled ODNs were injected intravenously into donor and recipient rats 6 and 1 h before operation, respectively. Recipients were killed 0 to 16 h after liver graft reperfusion. NF-kappaB activity in the liver graft was analyzed by electrophoretic mobility shift assay (EMSA). Hepatic mRNA expression of TNF-alpha, IFN-gamma and intercellular adhesion molecule-1 (ICAM-1) were determined by semiquantitative RT-PCR. Serum levels of TNF-alpha and IFN-gamma were measured by enzyme-linked immunosorbent assays (ELISA). Serum level of alanine transaminase (ALT) was measured using a diagnostic kit. Liver graft myeloperoxidase (MPO) content was assessed. RESULTS: NF-kappaB activation in liver graft was induced in a time-dependent manner, and NF-kappaB remained activated for 16 h after graft reperfusion. NF-kappaB activation in liver graft was significant at 2 to 8 h and slightly decreased at 16 h after graft reperfusion. Administration of NF-kappaB decoy ODNs significantly suppressed NF-kappaB activation as well as mRNA expression of TNF-alpha, IFN-gamma and ICAM-1 in the liver graft. The hepatic NF-kappaB DNA binding activity [presented as integral optical density (IOD) value] in the NF-kappaB decoy ODNs treatment group rat was significantly lower than that of the I/R group rat (2.16+/-0.78 vs 36.78+/-6.35 and 3.06+/-0.84 vs 47.62+/- 8.71 for IOD value after 4 and 8 h of reperfusion, respectively, P<0.001). The hepatic mRNA expression level of TNF-alpha, IFN-gamma and ICAM-1 [presented as percent of beta-actin mRNA (%)] in the NF-kappaB decoy ODNs treatment group rat was significantly lower than that of the I/R group rat (8.31+/-3.48 vs 46.37+/-10.65 and 7.46+/- 3.72 vs 74.82+/-12.25 for hepatic TNF-alpha mRNA, 5.58+/-2.16 vs 50.46+/-9.35 and 6.47+/-2.53 vs 69.72+/-13.41 for hepatic IFN-gamma mRNA, 6.79+/-2.83 vs 46.23+/-8.74 and 5.28+/-2.46 vs 67.44+/-10.12 for hepatic ICAM-1 mRNA expression after 4 and 8 h of reperfusion, respectively, P<0.001). Administration of NF-kappaB decoy ODNs almost completely abolished the increase of serum level of TNF-alpha and IFN-gamma induced by hepatic ischemia/reperfusion, the serum level (pg/mL) of TNF-alpha and IFN-gamma in the NF-kappaB decoy ODNs treatment group rat was significantly lower than that of the I/R group rat (42.7+/-13.6 vs 176.7+/-15.8 and 48.4+/-15.1 vs 216.8+/-17.6 for TNF-alpha level, 31.5+/-12.1 vs 102.1+/-14.5 and 40.2+/-13.5 vs 118.6+/-16.7 for IFN-gamma level after 4 and 8 h of reperfusion, respectively, P<0.001). Liver graft neutrophil recruitment indicated by MPO content and hepatocellular injury indicated by serum ALT level were significantly reduced by NF-kappaB decoy ODNs, the hepatic MPO content (A655) and serum ALT level (IU/L) in the NF-kappaB decoy ODNs treatment group rat was significantly lower than that of the I/R group rat (0.17+/-0.07 vs 1.12+/-0.25 and 0.46+/-0.17 vs 1.46+/-0.32 for hepatic MPO content, 71.7+/-33.2 vs 286.1+/-49.6 and 84.3+/-39.7 vs 467.8+/-62.3 for ALT level after 4 and 8 h of reperfusion, respectively, P<0.001). CONCLUSION: The data suggest that NF-kappaB decoy ODNs protects against I/R injury in liver graft by suppressing NF-kappaB activation and subsequent expression of proinflammatory mediators.     

Effects of ischemic preconditioning on cyclinD1 expression during early ischemic reperfusion in rats

AIM: To observe the effect of ischemic preconditioning on cyclinD1 expression in rat liver cells during early ischemic reperfusion. METHODS: Fifty-four SD rats were randomly divided into ischemic preconditioning group (IP), ischemia/ reperfusion group (IR) and sham operation group (SO). The IP and IR groups were further divided into four sub-groups (n - 6). Sham operation group (SO) served as the control group (n = 6). A model of partial liver ischemia/reperfusion was used, in which rats were subjected to liver ischemia for 60 min prior to reperfusion. The animals in the IP group underwent ischemic preconditioning twice for 5 min each time prior to the ischemia/reperfusion challenge. After 0, 1, 2, and 4 h of reperfusion, serum and liver tissue in each group were collected to detect the level of serum ALT, liver histopathology and expression of cyclinDi mRNA and protein. Flow cytometry was used to detect cell cycle as the quantity indicator of cell regeneration. RESULTS: Compared with IR group, IP group showed a significantly lower ALT level in 1 h to 4 h sub-groups (P < 0.05). Proliferation index(PI) indicated by the S-phase and G2/M-phase ratio [(S G2/M)/(G0/G1 S G2/M)] was significantly increased in IP group at 0 and 1 h (26.44±7.60% vs 18.56±6.40%,41.87±7.27% vs 20.25±6.70%, P < 0.05). Meanwhile, cyclinDi protein expression could be detected in IP group. But in IR group, cyclinDi protein expression occurred 2 h after reperfusion. The expression of cyclinDi mRNA increased significantly in IP group at 0 and 1 h (0.568±0.112 vs 0.274±0.069, 0.762±0.164 vs 0.348±0.093, P < 0.05). CONCLUSION: Ischemic preconditioning can protect liver cells against ischemia/reperfusion injury, which may be related to cell proliferation and expression of cyclinD1 during early ischemic reperfusion.     

Acid fibroblast growth factor reduces rat intestinal mucosal damage caused by ischemia-reperfusion insult

AIM: To detect the effects of acid fibroblast growth factor (aFGF) on apoptosis and proliferation of intestinal epithelial cells in differentiation or proliferation status to explore the protective mechanisms of aFGF. METHODS: Wistar rats were randomly divided into sham-operated control group (C, n= 6), intestinal ischemia group (I,n = 6), aFGF treatment group (A, n= 48) and intestinal ischemia-reperfusion group (R,n= 48). Apoptosis of intestinal mucosal cells was determined with terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) technique. Proliferating cell nuclear antigen (PCNA) protein expression and distribution were detected with immunohistochemical method. Plasma levels of D-lactate were determined with modified Brandts method. RESULTS: In A group, administration of exogenous aFGF could improve intestinal histological structure and decrease plasma D-lactate levels at 2-12 h after the reperfusion compared with R group. The apoptotic rates and PCNA protein expressions were not increased until 2 h after reperfusion and were maximal at 12 h. After reperfusion for 2-12 h, the apoptotic rates were gradually augmented along the length of jejunal crypt-villus units. Administration of aFGF could significantly reduce the apoptotic response at 2-12 h after reperfusion (P<0.05). Apoptosis rates in villus and crypt epithelial cells in A group at 12 h after reperfusion were (62.5±5.5)% and (73.2±18.6)% of those in R group, respectively. Treatment of aFGF could apparently induce protein expression of PCNA in intestinal mucosal cells of A group compared with R group during 2-12 h after reperfusion (P<0.05). There were approximately 1.3- and 1.5-times increments of PCNA expression levels in villus and crypt cells in A group at 12 h after reperfusion compared with R group, respectively. CONCLUSION: Intestinal I/R insult could lead to histological structure change and apoptotic rate increment. The protective effects of aFGF against ischemia/reperfusion in rat intestinal mucosa might be partially due to its ability to inhibit ischemia/reperfusion-induced apoptosis and to promote cell proliferation of crypt cells and villus epithelial cells.     

Direct hemoperfusion with a polymyxin B-immobilized cartridge in intestinal warm ischemia reperfusion

AIM： To investigate the effectiveness of direct hemoperfusion with polymyxin B-immobilized fibers （DHPPMX therapy） on warm ischemia-reperfusion （I/R） injury of the small intestine.
METHODS： The proximal jejunum and distal ileum of mongrel dogs were resected. Warm ischemia was performed by clamping the superior mesenteric artery （SMA） and vein （SMV） for 2 h. Blood flow to the proximal small intestine was restored 1 h after reperfusion, and the distal small intestine was used as a stoma. The experiment was discontinued 6 h after reperfusion. The dogs were divided into two groups： the DHP-PMX group （n = 6, DHP-PMX was performed for 180 min; from 10 min prior to reperfusion to 170 rain after reperfusion） and the control group （n = 5）. The rate pressure product （RPP）, SMA blood flow, mucosal tissue blood flow, and intramucosal pH （pHi） were compared between the two groups. The serum interleukin （IL）-10 levels measured 170 min after reperfusion were also compared.
RESULTS： The RPP at 6 h after reperfusion was significantly higher in the PMX group than in the control group （12174 ± 1832 mmHg/min vs 8929 ± 1797 mmHg/min, P 〈 0.05）. The recovery rates of the SMA blood flow at I and 6 h after reperfusion were significantly better in the PMX group than in the control group （61%±7% vs 44% ±4%, P 〈 0.05, and 59%±5% vs 35%±5%, P 〈 0.05, respectively）. The recovery rate of the mucosal tissue blood flow and the pHi levels at 6 h after reperfusion were significantly higher in the PMX group （61%±8% vs 31%±3%, P 〈 0.05 and 7.91±0.06 vs 7.69±0.08, P 〈 0.05, respectively）. In addition, the serum IL-IO levels just before DHP-PMX removal were significantly higher in the PMX group than in the control group （1 569 ± 253 pg/mL vs 211± 40 pg/mL, P 〈 0.05）.
CONCLUSION： DHP-PMX therapy reduced warm I/R injury of the small intestine. IL-10 may play a role in inhibiting I/R injury during DHP-PMX therapy.     

Beneficial effects of supplemental nitric oxide donor given during reperfusion period in reperfusion-induced lung injury

BACKGROUND: Reperfusion injury is a perplexing cause of early graft failure after lung transplantation and today we know that reperfusion may be more harmful to tissues than the preceding ischemia. We hypothesized that administration of the nitric oxide donor nitroglycerin (NTG) during flush perfusion and reperfusion periods would ameliorate reperfusion-induced lung injury. METHODS: Using an IN SITU normothermic ischemic lung rabbit model, three groups were studied (n = 7/group): (1) NTG given during flush perfusion (ischemia group); (2) NTG given in the flush perfusion and the reperfusion period (reperfusion group); and (3) no NTG (control group). All groups were flushed with low potassium dextran glucose solution. Blood gas analysis, tissue nitrite (nitric oxide metabolite) level analysis, bronchoalveolar lavage (BAL) fluid examination and morphological examinations were performed. RESULTS: Compared with the ischemia group, the reperfusion group had significantly improved arterial oxygenation (318 +/- 31.4 mmHg vs. 180 +/- 14.7 mmHg, P < 0.05), decreased BAL fluid neutrophil percentage (21 +/- 1.9 % vs. 30 +/- 5.6 %, P < 0.05), increased tissue nitrite level (32.55 +/- 4.12 nmol/g vs. 27.81 +/- 1.05 nmol/g, P < 0.05), and decreased tissue histopathological lesion scores (0.42 +/- 0.53 vs. 1.14 +/- 0.37, P < 0.05). CONCLUSIONS: This study suggests that nitric oxide donors supplemented during flush perfusion and reperfusion have more beneficial effects on lung functions against reperfusion injury than any other treatment modalities during IN SITU normothermic ischemic lung model.     

Reperfusion induces myocardial apoptotic cell death

OBJECTIVE: The purpose of the present study was to investigate whether apoptosis is triggered during ischemia (I) and reperfusion (R) and whether I/R-induced apoptosis is correlated with changes in expression of Bcl-2 and Bax. METHODS: Anesthetized open-chest dogs were divided into two groups. Group I: 7 h of permanent I without R (PI, n = 7); Group II: 60 min I followed by 6 h R (I/R, n = 8). Apoptosis was identified as "DNA ladder" by agarose gel electrophoresis or confirmed histologically using the terminal transferase UTP nick end labeling (TUNEL) assay. RESULTS: Collateral myocardial coronary blood flow during I, confirmed by colored microspheres was comparable in both groups. Although PI caused 72 +/- 5% infarct size, very few TUNEL-positive cells were detected in the necrotic area (0.2 +/- 0.1% of total normal nuclei), consistent with an absence of DNA laddering. In contrast, the appearance of TUNEL-positive cells was significantly displayed after 6 h R in the necrotic area in I/R group (26 +/- 4%, P < 0.001 vs. PI group), and DNA ladder occurred in all experimental animals, suggesting that myocardial apoptosis is primarily elicited by R. Densitometrically, Western blot analysis showed significant reduction in expression of Bcl-2 (16 +/- 1%) and increase in Bax (29 +/- 8%) after 6 h R in the necrotic area compared with normal tissue while expression of these two proteins was not changed in the PI group. Polymorphonuclear neutrophil (PMN) accumulation in the necrotic area determined either by immunohistochemistry with anti-CD18 antibody or by myeloperoxidase activity was significantly increased in the I/R group compared to the PI group (358 +/- 24 vs. 24 +/- 2, mm2 myocardium, P < 0.01) and (2.9 +/- 0.3 vs. 0.4 +/- 0.1, U/100 mg tissue, P < 0.01). There was a significant linear relationship between CD18-positive PMNs and TUNEL-positive cells (P < 0.05) in the I/R group. CONCLUSIONS: These results indicate that (1) PI without R did not induce apoptotic cell death, while two types of cell death, necrosis and apoptosis were found after I/R, (2) the Bcl-2 family may participate in early R-induced myocardial apoptosis, (3) PMN accumulation may play a role in the development of apoptosis.     

Preconditioning the human myocardium by simulated ischemia: studies on the early and delayed protection

BACKGROUND: There are data supporting the existence of ischemic preconditioning in man. This study investigated the most effective preconditioning protocol for the human myocardium and whether the second window of ischemic preconditioning (24 h) is as protective as the first window (< or = 2 h). METHODS AND RESULTS: Right atrial appendages (n = 6/group) obtained during coronary bypass surgery were prepared and superfused with normoxic and normothermic Krebs-Henseleit solution. After 30 min stabilisation, muscles were subjected to various preconditioning protocols followed by 90 min ischemia and 120 min reperfusion. At the end of each protocol, the leakage of creatinine kinase (CK, U/g wet wt) and the reduction of MTT to insoluble formazan dye (OD/mg wet wt), an index of cell viability, were measured. In study 1, preconditioning was induced by 2, 3, 5 and 10 min of ischemia followed by 5 min reperfusion. In study 2, 1-4 cycles of 2 or 5 min ischemia-5 min reperfusion were applied. In study 3, preconditioning was induced by 5 min ischemia-5 min reperfusion followed by 1, 2, 3 or 4 h reperfusion before the subsequent 90 min ischemia. In study 4, preconditioning with 5 min ischemia followed by 5 min reperfusion either immediately preceded 30 or 90 min ischemia/120 min reperfusion or was applied 24 h before. In study 1 and 2, optimal protection was achieved with 5 min or two cycles of 2 min preconditioning ischemia (CK = 3.06 +/- 0.31 and 2.89 +/- 0.02; MTT = 0.56 +/- 0.05 and 0.47 +/- 0.09, respectively vs. CK = 5.56 +/- 0.52 and MTT = 0.18 +/- 0.04 in ischemia alone group; P < 0.05). In study 3, protection was observed 2 h after preconditioning (CK = 3.43 +/- 0.22 and MTT = 0.46 +/- 0.09; P < 0.01 vs. ischemia alone group) but it was lost beyond 2 h (CK = 6.30 +/- 0.56 and MTT = 0.16 +/- 0.02 after 3 h; P = NS vs. ischemia alone group). In study 4, protection was observed 24 h following preconditioning when the atrial specimens were exposed to 30 min ischemia (CK = 2.96 +/- 0.38 and MTT = 0.61 +/- 0.01 vs. CK = 4.56 +/- 0.26 and MTT = 0.43 +/- 0.02 in ischemia alone group, P < 0.05); however, when the period of ischemia was extended to 90 min the beneficial effect of preconditioning was lost (CK = 10.28 +/- 0.05 and MTT = 0.11 +/- 0.05 vs. CK = 9.56 +/- 0.62 and MTT = 0.104 +/- 0.05 in ischemia alone group, P = NS). CONCLUSIONS: In the isolated human myocardium maximal protection induced by preconditioning is achieved by a total 4-5 min ischemic stimulus, an effect that is lost beyond 2 h of its application. Two windows of protection were identified, the first (< or = 2 h) being more potent than the second (24 h).     

Role of nitric oxide and peroxynitrite anion in lung injury induced by intestinal ischemia－reperfusion in rats

AIM: To evaluate effects of nitric oxide (NO) and peroxynitrite anion (ONOO-) on lung injury following intestinal ischemia-reperfusion (IR) in rats. METHODS: A rat model of intestinal ischemia was made by clamping superior mesenteric artery and lung injury was resulted from reperfusion. The animals were randomly divided into 3 groups: sham operation (Sham), 2 h ischemia followed by 2 h reperfusion (IR) and IR pretreated with aminoguanidine (AG) - an inhibitor of inducible NO synthase (iNOS) 15 minutes before reperfusion (IR+AG). The lung malondialdehyde (MDA) and nitrate/nitrite (NO2/NO3)contents and morphological changes were examined.Western blot was used to detect the iNOS protein expression.Immunohistochemical staining was used to determine the change of nitrotyrosine (NT)- a specific "footprint" of ONOO-. RESULTS: The morphology revealed evidence for lung edema, hemorrhage and polymorphonuclear sequestration after intestinal IR. Compared with sham group, lung contents of MDA and NO2-/NO3- in IR group were significantly increased (12.00±2.18 vs23.44±1.25 and 76.39±6.08 vs140.40±4.34,P＜0.01) and the positive signals of iNOS and NT were also increased in the lung. Compared with IR group, the contents of MDA and NO2/NO3 in IR+AG group were significantly decreased (23.44±1.25 vs14.66±1.66 and 140.40±4.34 vs 80.00±8.56, P＜0.01) and NT staining was also decreased. CONCLUSION: Intestinal IR increases NO and ONOO production in the lung, which may be involved in intestinal IR-mediated lung injury.     

重组人可溶性补体受体1型SCR15-18片段对心肌缺血再灌注损伤的保护作用

目的探讨重组人可溶性补体受体1型SCR15-18片段（sCR1-SCR15-18）对心肌缺血再灌注的保护作用。方法36只SD大鼠随机分为假手术（SO）组,缺血再灌注（I／R）组和sCR1-SCR15-18（sCR1）保护组。建立急性心肌缺血再灌注模型,结扎冠状动脉前立即注射磷酸盐缓冲液（0.1mL／100g）或sCR1-SCR15-18蛋白（15mg／kg）。测定心肌梗塞面积,血清中乳酸脱氢酶（LDH）和肌酸激酶（CK）,心肌组织髓过氧化物酶（MPO）活性,HE染色观察心肌病理改变和免疫组织化学法检测C3c。结果（1）心肌梗死面积：I／R组为（22．9±3．0）％,sCR1保护组为（16．1±3．3）％（P〈0．05）。（2）血清心肌酶CK（U／L）：I／R组为3400．9±534．9,sCR1保护组为2532．5±597．1（P〈0．05）。LDH（U／L）：I／R组为6572．0±476．3,sCR1保护组为5436．2±611．3（P〈0．05）。（3）心肌组织MPO活性（U／g）：I／R组为1．12±0．13,sCR1保护组为0．81±0．14（P〈0．05）。（4）心肌病理改变：I／R组心肌有断裂、坏死,间质肿胀,出血及中性粒细胞浸润,sCR1保护组心肌的以上病理变化明显较I／R组减轻。（5）与I／R组比sCR1保护组梗死区心肌组织C3c的沉积减少。结论sCR1-SCR15-18蛋白对大鼠急性心肌缺血再灌注损伤具有保护作用。