硫化氢调控c-Jun氨基端激酶/活化蛋白-1信号通路对大鼠小肠缺血/再灌注损伤起保护作用

目的探讨硫化氢(H2S)是否通过调控c-Jun氨基端激酶/活化蛋白-1(JNK/AP-1)信号通路对大鼠小肠缺血/再灌注(I/R)损伤起保护作用。方法按随机数字表法将30只雄性Wistar大鼠分为假手术组(Sham组)、I/R组、H2S供体硫氢化钠干预组(I/R+NaHS组),每组10只。用无损伤血管夹阻断肠系膜上动脉法制备I/R损伤模型(缺血60 min、再灌注120 min)。I/R+NaHS组再灌注前10 min经大鼠尾静脉注射100μmol/kg的NaHS,随后按1.07 mmol·kg-1·h-1输注直到再灌注120 min。采用敏感硫电极法测定血浆H2S浓度。采用分光光度法检测小肠组织丙二醛(MDA)、超氧化物歧化酶(SOD)水平。对小肠病理组织切片行苏木素-伊红(HE)染色并进行Chiu评分以评价肠黏膜损伤程度;采用蛋白质免疫印迹试验(Western blotting)检测小肠组织磷酸化JNK(p-JNK)、JNK、AP-1、BCL-2蛋白表达。结果与Sham组比较,I/R组固有层破坏,出血、溃疡,Chiu评分明显升高(分:4.80±0.63比0.70±0.09,P<0.01);血浆H2S浓度和回肠组织SOD活性明显降低〔H2S(μmol/L):17.29±1.40比34.62±1.48,SOD(kU/g):5.38±0.93比20.56±1.85,均P<0.01〕,回肠组织MDA含量明显升高(μmol/g:16.06±1.71比4.80±0.92,P<0.01),回肠组织BCL-2蛋白表达明显降低(BCL-2/β-actin:0.32±0.06比0.79±0.05,P<0.01),p-JNK、AP-1蛋白表达明显升高(p-JNK/β-actin:0.69±0.03比0.10±0.03,AP-1/β-actin:0.82±0.02比0.22±0.02,均P<0.01)。与I/R组比较,I/R+NaHS组上皮层与固有层中度分离,部分绒毛顶端破损,Chiu评分明显下降(分:2.90±0.56比4.80±0.63,P<0.01);血浆H2S浓度和回肠组织SOD活性明显升高〔H2S(μmol/L):24.48±1.84比17.29±1.40,SOD(kU/g):10.29±1.26比5.38±0.93,均P<0.01〕,回肠组织MDA含量明显降低(μmol/g:7.88±1.01比16.06±1.71,P<0.01),回肠组织BCL-2蛋白表达明显升高(BCL-2/β-actin:0.44±0.06比0.32±0.06,P<0.01),p-JNK、AP-1蛋白表达明显降低(p-JNK/β-actin:0.54±0.05比0.69±0.03,AP-1/β-actin:0.66±0.04比0.82±0.02,均P<0.01)。Sham组、I/R组、I/R+NaHS组间回肠组织JNK表达差异无统计学意义(JNK/β-actin:0.63±0.02、0.66±0.02、0.64±0.02,P>0.05)。结论H2S通过下调JNK/AP-1信号通路表达减轻氧化应激水平,对大鼠小肠I/R损伤起保护作用。

中性粒细胞在肠缺血／再灌注损伤诱发多器官功能障碍综合征中的作用

脓毒症和多器官功能障碍综合征（MODS）是创伤及感染后最严重的并发症，发病率逐年增高，目前其病死率居外科重症加强治疗病房（SICI）中的首位。大量临床和实验研究的结果表明，肠缺血／再灌注（I／R）损伤是严重创伤、烧伤后全身炎症反应综合征（SIRS）、脓毒症及MODS发生发展的重要诱因。其中中性粒细胞（PMN）在肠内的激活、趋化、聚集、释放炎症介质和细胞因子等系列反应，

H2S对小肠缺血/再灌注损伤大鼠PI3K/Akt信号通路表达的影响

目的探讨硫化氢(H2S)对小肠缺血/再灌注损伤(IRI)大鼠磷脂酰肌醇3-激酶/蛋白激酶B(PI3K/Akt)信号通路表达的影响。方法将30只雄性Wistar大鼠按随机数字表法分为假手术组(Sham组)、IRI组、H2S供体硫氢化钠(NaHS)干预组(IRI+NaHS组),每组10只。采用无损伤血管夹夹闭肠系膜上动脉(SMA)60 min、再灌注120 min的方法建立大鼠肠IRI模型;Sham组仅分离SMA后关腹。恢复SMA血流前10 min,IRI+NaHS组经尾静脉注入100μmol/kg NaHS后以1.07 mmol·kg^-1·h^-1的速度维持输注至再灌注120 min;Sham组和IRI组则给予等体积生理盐水。实验结束后取下腔静脉血,采用敏感硫电极法测定血浆H2S浓度。取血后处死大鼠取回肠组织,采用苏木素-伊红(HE)染色观察组织病理学改变并进行Chiu评分;用蛋白质免疫印迹试验(Western Blot)检测磷酸化Akt(p-Akt)、Akt、PI3K、活化的天冬氨酸特异性半胱氨酸蛋白酶9(caspase-9)、哺乳动物雷帕霉素靶蛋白(mTOR)蛋白表达。结果与Sham组比较,IRI组肠黏膜组织结构紊乱、水肿,绒毛断裂、脱落,病理评分明显升高(分:4.21±0.15比0.15±0.03,P<0.01),血浆H2S水平明显降低(μmol/L:26.72±3.17比38.34±5.24,P<0.01),回肠组织p-Akt、PI3K、caspase-9、mTOR蛋白表达明显升高(p-Akt/GAPDH:2.67±0.12比0.24±0.05,PI3K/GAPDH:1.42±0.07比0.57±0.08,caspase-9/GAPDH:4.23±0.61比0.13±0.02,mTOR/GAPDH:2.17±0.23比0.23±0.02,均P<0.01)。与IRI组比较,IRI+NaHS组肠黏膜病理改变减轻,病理评分明显下降(分:1.56±0.02比4.21±0.15,P<0.01),血浆H2S水平明显升高(μmol/L:32.36±2.45比26.72±3.17,P<0.01),回肠组织p-Akt、PI3K蛋白表达进一步升高(p-Akt/GAPDH:5.12±0.08比2.67±0.12,PI3K/GAPDH:3.14±0.05比1.42±0.07,均P<0.01),而caspase-9、mTOR蛋白表达明显降低(caspase-9/GAPDH:2.12±0.24比4.23±0.61,mTOR/GAPDH:1.37±0.28比2.17±0.23,均P<0.01)。结论H2S通过上调PI3K/Akt信号通路表达,下调caspase-9、mTOR表达,从而减轻IRI大鼠肠损伤。

肠缺血／再灌注时卡巴胆碱对肠上皮细胞凋亡的影响

目的研究肠缺血／再灌注（I／R）时卡巴胆碱对肠上皮细胞凋亡的影响及可能机制。方法雄性Wistar大鼠，戊巴比妥钠腹腔麻醉，结扎肠管造成长约8cm肠袋，采用夹闭肠系膜上动脉（SMA）阻断血流45min后恢复血流的方法，制备肠I／R损伤模型。将动物按随机数字表法分为假手术组（n=40）、I／R模型组（n=40）和卡巴胆碱组（n=40）。卡巴胆碱组在阻断血流的同时向肠袋内注射卡巴胆碱（0．1mg／kg），I／R模型组给予等量生理盐水。分别在再灌注0、30、60、120和240min时，采用病理学方法按Chiu’s评分观察肠上皮细胞损伤；原位末端缺刻标记法（TUNEL）检测肠上皮细胞凋亡指数的变化；免疫组化法检测肠上皮细胞凋亡相关半胱氨酸天冬氨酸特异性蛋白酶（caspase-3）和Bcl-2蛋白表达的变化。结果与I／R模型组比较，卡巴胆碱组肠上皮细胞病理学变化较轻，凋亡指数明显降低，caspase-3阳性肠上皮细胞数明显减少，而Bcl-2阳性肠上皮细胞数明显增加（P均〈0．01）。结论卡巴胆碱能抑制I／R时肠上皮细胞caspase-3表达，增加Bcl-2表达，减少肠上皮细胞的凋亡。

犬肠缺血/再灌注时小肠对早期肠内营养耐受能力的实验研究

目的研究肠道低灌注状态下实施早期肠内营养(EEN)时小肠功能指标及耐受能力的变化。方法健康雄性杂种犬24只,夹闭肠系膜上动脉(SM A)1 h后恢复灌注造成肠缺血/再灌注(I/R)损伤,复流后4 h实施EEN,将瑞代营养液(4 m l.k-g 1.-h 1)经肠黏膜张力计管持续滴注3 h,如果动物出现肠道不能耐受症状(如呕吐、腹泻等)则停止,间隔6 h后再次实施EEN,直至动物肠道能够耐受为止。按随机数字表法将动物分成单纯EEN组、单纯I/R组、I/R后EEN组(I/R+EEN组),每组8只。检测小肠黏膜二氧化碳分压(P iCO2)、D木糖吸收量、小肠腔内压力判定小肠灌注和功能变化。结果肠I/R+EEN组肠道不能耐受发生率(87.5%)和严重程度均显著高于单纯EEN组(0)和单纯I/R组(12.5%)。实施EEN后,与单纯I/R组和单纯EEN组相比,I/R+EEN组小肠腔内压力及肠P iCO2均显著升高,而血浆D木糖吸收量显著降低(P均<0.01)。结论肠I/R能显著降低小肠对EEN的耐受能力,肠I/R后过早(<12 h)实施EEN能加重小肠I/R损伤,并进一步抑制小肠吸收和运动功能。

参附注射液对大鼠肠缺血/再灌注期间肾保护作用机制的研究

目的观察参附注射液(SFI)对大鼠肠缺血/再灌注损伤(IRI)期间肾组织内血红素加氧酶-1(HO-1)、诱生型一氧化氮合酶(iNOS)表达的影响,探讨其肾保护作用机制。方法采用钳闭大鼠肠系膜上动脉(SMA)诱导IRI模型。将36只雄性Wistar大鼠随机分为IRI模型组、SFI预处理组和假手术组。SFI预处理组:缺血前30 min静脉恒速泵入SFI 10 ml/kg,阻断SMA造成肠缺血1 h后再开放;IRI模型组:在缺血前30 min用微量泵持续注入等量生理盐水。应用免疫组化方法和图像分析系统检测肾组织中HO-1和iNOS的表达和分布,观察各组血清肌酐(SCr)、尿素氮(BUN),同时光镜下观察肾组织病理学改变。结果与假手术组比较,IRI模型组HO-1和iNOS表达均显著增强(P均<0.01),SCr、BUN明显增加(P均<0.01);与IRI模型组比较,SFI预处理组HO-1表达明显升高,而iNOS表达及SCr、BUN明显降低(P均<0.05)。病理学检查显示,SFI能明显减轻肠IRI导致的肾组织病理损害。结论SFI能明显减轻肠IRI所致的肾组织损伤,其分子机制为诱导肠IRI后肾组织中HO-1的表达,同时抑制iNOS的表达。

电针“足三里”穴激活多巴胺机制减轻肠缺血-再灌注大鼠心肌损伤

目的:探讨电针"足三里"对大鼠肠缺血/再灌注(I/R)引起的急性心肌损伤的保护作用。方法:雄性SD大鼠30只,随机分为假手术组、模型1h组和4h组、电针1h组和4h组,每组6只。采用夹闭大鼠肠系膜上动脉30min,恢复灌流1h和4h,制备肠I/R模型。电针组于肠缺血后即刻行电针双侧"足三里"穴30min。再灌注1h和4h后,腹主动脉取血,酶联免疫吸附法检测血浆肿瘤坏死因子-α(TNF-α)和多巴胺(DA)水平,并使用全自动生化分析仪测定血浆肌酸激酶同工酶(CK-MB)、乳酸脱氢酶(LDH);取心肌组织,酶联免疫吸附法检测髓过氧化物酶(MPO)活性和丙二醛(MDA)含量;HE染色观察左心尖组织病理变化。结果:与假手术组比较,模型1h组和4h组血浆DA水平下降,而TNF-α、CK-MB、LDH水平及心肌组织MDA含量、MPO活性升高(均P<0.05),心肌组织病理损伤明显。与相应时段模型组相比,电针"足三里"穴治疗后血浆DA水平升高,血浆TNF-α、CK-MB、LDH水平及心肌组织MDA含量、MPO活性降低(除CK-MB 4h、MPO 4h和MDA 1h外,余均P<0.05),心肌组织损伤减轻。I/R损伤1h时电针抑制心肌MDA含量、MPO活性的百分比分别是9%、13%,4h时分别是30%、15%。结论:电针"足三里"穴能显著减轻大鼠肠I/R后心肌损伤,其保护机制可能与电针"足三里"穴升高血浆DA,降低血浆TNF-α和心肌组织MPO、MDA,清除氧自由基,减轻炎性反应有关。

大鼠失血性休克肠粘膜形态学与功能变化

目的 探讨失血性休克肠缺血/再灌注损伤后,粘膜屏障功能与形态学的变化。方法 制作大鼠失血性休克模型,以休克复苏后0h、1h、3h、6h、12h、24h时间段取回肠组织标本,通过光镜和电镜下进行肠粘膜的形态学改变的观察,包括组织学检查、肠粘膜及绒毛厚度测量、粘膜损伤指数评定;同时在肝门静脉血作内毒素测定及复苏后1h、3h、6h的尿液作乳果糖/甘露醇比值检测以分析肠粘膜屏障功能的改变。结果 肠粘膜损伤主要表现出凋亡、坏死的两种细胞死亡形式。复苏0h组粘膜上皮就发生明显损伤改变,1h进一步加重,3h组出现修复现象,6h部分空肠及回肠绒毛已修复,12h时大部分肠粘膜结构基本恢复正常;内毒素和乳果糖/甘露醇比值在6h组达到高峰,仅24h组才恢复正常。结论 失血性休克缺血-再灌注后,肠粘膜屏障早期受累,表现为回肠粘膜细胞凋亡及坏死的两种损伤形式;肠粘膜具有强大的修复潜能;肠屏障功能的恢复滞后于形态学修复。

灵芝多糖对失血性休克兔肠黏膜屏障功能及肿瘤坏死因子-α的影响

目的：观察灵芝多糖（GLP）对失血性休克兔肠黏膜屏障功能及肿瘤坏死因子-α（TNF-α）的影响。方法将30只成年健康日本大耳白兔按随机数字表法分为假手术组、生理盐水复苏再灌注组（NS组）和GLP复苏再灌注组（GLP组）。股动脉放血10 min内使平均动脉血压（MAP）降至30～40 mmHg（1 mmHg＝0.133 kPa）制备失血性休克动物模型，维持40 min后给予抗休克治疗，NS组快速回输自体血及静脉滴注（静滴）2倍失血量的生理盐水，GLP组除用GLP代替生理盐水外，其他处理同NS组。假手术组仅给予插管并维持血压测定状态，不进行放血及复苏过程。3组分别于休克前（S0）、休克40 min（S40）及复苏再灌注40 min（R40）和90 min（R90）观察血中TNF-α含量及血液细菌培养阳性数。于R90时检测血清内毒素及肠黏膜TNF-α含量；光镜下观察肠黏膜病理学改变并进行肠黏膜损伤评分（Chiu）。结果①3组动物S0时血液细菌培养均为阴性；随时间延长，NS组和GLP组R40和R90时血液细菌培养阳性动物数逐渐增加，说明细菌移位增加，但GLP组R40和R90时血液细菌培养阳性动物数明显少于同期NS组水平（只：R40时2比4，R90时4比6，均P＜0.05）；血液中细菌以大肠杆菌最为多见，其余依次为肠球菌、嗜酸乳杆菌、葡萄球菌等。R90时，与假手术组比较，NS组和GLP组血浆内毒水平均明显升高（kU/L：1.823±0.963、1.361±0.529比0.064±0.036，均P＜0.05）。②光镜下，NS组肠绒毛水肿，上皮层下间隙增宽，有中性粒细胞和淋巴细胞浸润；GLP组肠黏膜损伤较NS组明显减轻；GLP组和NS组Chiu评分较假手术组显著升高，而GLP组较NS组明显降低（分：1.44±0.64比3.79±1.23，P＜0.05）。③NS组和GLP组血中TNF-α含量随缺血/再灌注（I/R）时间延长逐渐升高，于R90时达峰值，且均明显高于同期假手术组，而GLP组较NS组明显降低〔吸光度（A）值：33.350±7.950比85.080±4.330，P＜0.05〕；与假手术组比较，NS组和GLP组肠黏膜TNF-α含量较假手术组明显升高，GLP组含量明显低于NS组（A值：96.38±8.59比167.73±12.32，P＜0.05）。结论 GLP对失血性休克及复苏救治过程中肠黏膜屏障功能有保护作用，且可能与拮抗TNF-α释放有关。

Apoptosis induction with polo-like kinase-1 antisense phosph-orothioate oligodeoxynucleotide of colon cancer cell line SW480

AIM： To investigate the effects of polo-like kinase-1 （PLK1） antisense phosphorothioate oligodeoxynucleotide （ASODN） on apoptosis and cell cycle of human colon cancer cell line SW480. METHODS： After SW480 colon cancer cells were transfected with PLK1 ASODN, Northern and Western blot analyses were used to examine PLK1 gene expression in cancer cells. We studied apoptosis using terminal uridine deoxynucleotidyl nick end labeling. Apoptosis and cell cycle of SW480 cells were examined by fluorescence-activated cell sorter scan. RESULTS： The levels of PLK1 mRNA and protein were greatly inhibited by PLK1 ASODN in SW480 cancer cells transfected with PLK1 ASODN. Apoptosis index （AI） induced PLK1 ASODN in a time- and dose-dependent manner. Results from FLM showed that sub-2N DNA content of transfected cancer cells was significantly increased and arrested at G2/M compared with control groups. CONCLUSION： PLK1 ASODN can induce apoptosis of human colon cancer cell line SW480.

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 transferasemediated 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 2-22 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 ischernia/reperfusion in rat intestinal rnucosa might be partially due to its ability to inhibit ischernia/reperfusioninduced apoptosis and to promote cell proliferation of crypt cells and villus epithelial cells.

Rosuvastatin reduces rat intestinal ischemia-reperfusion injury associated with the preservation of endothelial nitric oxide synthase protein

AIM： To investigate the protective effect of rosuvastatin on ischemia-reperfusion （I-R）-induced small intestinal injury and inflammation in rats, and to determine the effect of this agent on the expression of endothelial nitric oxide synthase （eNOS） protein. METHODS： Intestinal damage was induced in male Sprague-Dawley rats by clamping both the superior mesenteric artery and the celiac trunk for 30 min, followed by reperfusion for 60 min. Rosuvastatin dissolved in physiological saline was administered intraperitoneally 60 min before ischemia. The severity of the intestinal mucosal injury and inflammation were evaluated by several biochemical markers, as well as by histological findings. The protein levels of eNOS were determined by Western blot. RESULTS： The levels of both intraluminal hemoglobin and protein, as indices of mucosal damage, were significantly increased in the I-R group compared with those in the sham-operated group. These increases, however, were significantly inhibited by treatment with rosuvastatin in a dose-dependent manner. The protective effects of rosuvastatin were also confirmed by histological findings. Exposure of the small intestine to I-R resulted in mucosal inflammation characterized by significant increases in thiobarbituric acid-reactive substances, tissueassociated myeloperoxidase activity, and the mucosal contents of rat cytokine-induced neutrophil chemoattracrant-1 （CINC-1） and tumor necrosis factor-α（TNF-α）. These increases in inflammatory parameters after I-R were significantly inhibited by pretreatment with rosuvo astatin at a dose of 10 mg/kg. Furthermore, mRNA expression of CINC-1 and TNF-α was increased after I-R, and this increase was also inhibited by rosuvastatin. The mucosal protein levels of eNOS decreased during I-R, but were preserved in rats treated with rosuvastatin. CONCLUSION： Rosuvastatin inhibits rat intestinal injury and inflammation induced by I-R, and its protection is associated with the preservation of eNOS protein.

Effect of shenfu injection on gastrointestinal microcirculation in rabbits after myocardial ischemia-reperfusion injury

AIM： To investigate the effect of shenfu injection on gastrointestinal microcirculation after myocardial ischemic-reperfusion （IR） injury in rabbits and probe into the mechanism. METHODS： Forty healthy flap-eared white rabbits were randomly divided into 4 groups： IR injury control group （group Ⅰ ）, shenfu injection 5 mL/kg per h group （group Ⅱ）, shenfu injection 10 mL/kg per h group （group Ⅲ） and shenfu injection 20 mL/kg per h group （group Ⅳ）. The four groups were treated with Lactated Ringer＇s solution, shenfu injection 5, 10, and 20 mL/ kg per h were infused intravenously 30 min before experiment respectively. The values of hemodynamics [mean arterial pressure （MAP）, heart rate （HR）, gastric intramucosal partial pressure of carbon dioxide （PCO2）, blood gas analysis and pH] were measured and compared with those before myocardial ischemia, 60 min after myocardial ischemia and 60, 90, and 180 rain after reperfusion. RESULTS： The MAP, HR and gastric intramucosal pH were （70.50 ± 4.50） kPa, （165 ± 14） beats per rain, 7.032 ± 0.024 in group Ⅰ 60 min after myocardial ischemia, which were significantly decreased compared with those before myocardial ischemia （88.50 ± 9.75 kPa, 217 ± 18 beats per rain, 7.112 ± 0.035, P 〈 0.05）. The MAP, HR and gastric intramucosal pH were significantly decreased in group Ⅰ 60, 90, and 180 min after reperfusion （61.50 ± 5.25 kPa, 133 ± 31 beats per rain, 6.997 ± 0.025） compared with those before reperfusion respectively （P 〈 0.05）, whereas the values were insignificantly different in groups Ⅱ, Ⅲ or Ⅳ after reperfusion, compared with those before reperfusion, and there were no significant differences between groups Ⅱ, Ⅲ, and Ⅳ after reperfusion. CONCLUSION： Pre-infusion of shenfu injection has a protective effect on gastrointestinal microcirculation after myocardial IR injury in rabbits, in a dose independent manner.

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.

Ketamine anesthesia reduces intestinal ischemia/reperfusion injury in rats

AIM: To investigate the effects of ketamine anesthesia on the motility alterations and tissue injury caused by ischemia/reperfusion in rats. METHODS: Thirty male Wistar rats weighing 200-250 g were used. Ischemia was induced by ob-structing blood flow in 25% of the total small intesti-nal length (ileum) with a vascular clamp for 45 min, after which either 60 min or 24 h of reperfusion was allowed. Rats were either anesthetized with pento-barbital sodium (50 mg/kg) or ketamine (100 mg/kg). Control groups received sham surgery. After 60 min of reperfusion, the intestine was examined for mor-phological alterations, and after 24 h intestinal basic electrical rhythm (BER) frequency was calculated, and intestinal transit determined in all groups. RESULTS: The intestinal mucosa in rats that were anesthetized with ketamine showed moderate altera-tions such as epithelial lifting, while ulceration and hemorrhage was observed in rats that received pento-barbital sodium after 60 min of reperfusion. Quantita-tive analysis of structural damage using the Chiu scaleshowed significantly less injury in rats that received ketamine than in rats that did not (2.35 ± 1.14 vs 4.58 ± 0.50, P < 0.0001). The distance traveled by a mark-er, expressed as percentage of total intestinal length, in rats that received pentobarbital sodium was 20% ± 2% in comparison with 25.9% ± 1.64% in rats that re-ceived ketamine (P = 0.017). BER was not statistically different between groups. CONCLUSION: Our results show that ketamine anesthesia is associated with diminished intestinal injury and abolishes the intestinal transit delay induced by ischemia/reperfusion.

Effects of penehyclidine hydrochloride in small intestinal damage caused by limb ischemia-reperfusion

AIM:To investigate the protective effect of penehyclidine hydrochloride post-conditioning in the damage to the barrier function of the small intestinal mucosa caused by limb ischemia-reperfusion(LIR) injury. METHODS:Male Wistar rats were randomly divided into three groups(36 rats each) :the sham-operation group(group S) ,lower limb ischemia-reperfusion group(group LIR) ,and penehyclidine hydrochloride postconditioning group(group PHC) .Each group was divided into subgroups(n=6 in each group) according to ischemic-reperfusion time,i.e.immediately 0 h(T1) ,1 h(T2) ,3 h(T3) ,6 h(T4) ,12 h(T5) ,and 24 h(T6) .Bilateral hind-limb ischemia was induced by rubber band application proximal to the level of the greater trochanter for 3 h.In group PHC,0.15 mg/kg of penehyclidine hydrochloride was injected into the tail vein immediately after 3 h of bilateral hind-limb ischemia.The designated rats were sacrificed at different time-points of reperfusion;diamine oxidase(DAO) ,superoxide dismutase(SOD) activity,myeloperoxidase(MPO) of small intestinal tissue,plasma endotoxin,DAO,tumor necrosis factor-α(TNF-α) ,and interleukin(IL) -10 in serum were detected in the rats. RESULTS:The pathological changes in the small intestine were observed under light microscope.The levels of MPO,endotoxin,serum DAO,and IL-10 at T1-T6,and TNF-αlevel at T1-T4 increased in groups LIR and PHC(P<0.05) compared with those in group S,but tissue DAO and SOD activity at T1-T6 decreased(P<0.05) .In group PHC,the tissue DAO and SOD activity at T2-T6,and IL-10 at T2-T5 increased to higher levels than those in group LIR(P<0.05) ;however,the levels of MPO,endotoxin,and DAO in the blood at T2-T6,and TNF-αat T2 and T4 decreased(P<0.05) . CONCLUSION:Penehyclidine hydrochloride post-conditioning may reduce the permeability of the small intestines after LIR.Its protection mechanisms may be related to inhibiting oxygen free radicals and inflammatory cytokines for organ damage.

Effect of intestinal ischemia-reperfusion injury on protein levels of leptin and orexin-A in peripheral blood and central secretory tissues

AIM: To explore the effect of intestinal ischemia-reperfusion injury on protein levels of leptin and orexin-A in peripheral blood and their central secretory tissues and to find out the role leptin and orexin-A play in acute inflammatory responses.METHODS: An intestinal ischemia-reperfusion (I/R)injury model of rats was established and rats were divided randomly into six groups: sham-operation group, 60 min ischemia/30 min reperfusion group (I60'R30'), I60'R90',I60'R150', I60'R240' and I60'R360', 9 rats each group.Two highly-sensitive radioimmunoassays for leptin and orexin-A were established and used to check the change of their concentrations in peripheral blood and central secretory tissues before and after intestinal I/R injury.RESULTS: Compared with the serum leptin level before injury, it decreased significantly in I60'R30' group and increased significantly in I60'R360' group; compared to sham-operation group after injury, serum leptin level increased significantly in I60'R360' group; compared to sham-operation group after injury, adipose leptin levels decreased significantly in I60'R30' and I60'R90' groups,while increased significantly in I60'R360' group. There was no significant difference between the expression levels of orexin-A before and after I/R injury.CONCLUSION: Leptin has a time-dependent response and orexin-A has a delayed response to acute inflammatory stimuli such as intestinal I/R injury and they may participate in metabolic disorders in injury as inflammatory cytokines.

Pyrrolidine dithiocarbamate reduces ischemia-reperfusion injury of the small intestine

AIM： To evaluate whether pyrrolidine dithiocarbamate （PDTC）, an enhancer of HO production, attenuates intestinal IR injury. METHODS： Eighteen male rats were randomly allocated into three groups： （a） sham; （b） IR, consisting of 30 min of intestinal ischemia, followed by 2-h period of reperfusion; and （c） PDTC treatment before IR. Intestinal microvascular perfusion （IMP） was monitored continuously by laser Doppler flowmetry. At the end of the reperfusion, serum samples for lactate dehydrogenase （LDH） levels and biopsies of ileum were obtained. HO activity in the ileum was assessed at the end of the reperfusion period. RESULTS： At the end of the reperfusion in the IR group, IMP recovered partially to 42.5% of baseline （P〈0.05 vs sham）, whereas PDTC improved IMP to 67.3% of baseline （P〈0.01 vs IR）. There was a twofold increase in HO activity in PDTC group （2 062.66±106.11） as compared to IR （842.3±85.12） （P〈0.001）. LDH was significantly reduced （P〈0.001） in PDTC group （585.6±102.4） as compared to IR group （1 973.8±306.5）. Histological examination showed that the ileal mucosa was significantly less injured in PDTC group as compared with IR group. CONCLUSION： Our study demonstrates that PDTC improves the IMP and attenuates IR injury of the intestine possibly via HO production. Additional studies are warranted to evaluate the clinical efficacy of PDTC in the prevention of IR injury of the small intestine.

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 thestudy. 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 shamoperated 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 vs8.02±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 vs6±0.1, 0/6 vs6/6, P＜0.05or 0.01).CONCLUSION: L-arginine preconditioning attenuates ischemia and reperfusion injury in the rat SBT model,which was due to antioxidant activities partially.

Intestinal ischemia and reperfusion induced second impairment of the rat heart

Objective: To study the changes of oxygen free radical, expression of apoptotic gene, ultrastructure of myocardial cell and second injury of the heart following the intestinal ischemia and reperfusion. Methods: Making the models of ischemia and reperfusion by clamping superior mesenteric artery, the concentration of NO and SOD in the blood was examined before clamping the artery and reperfusion for 0, 30, 60 min, 1, 3, and 7 d. The expression of Bax, Bal-2, and p53 in myocardial cell was studied by means of immunohistochemical SP method and the microstructure damage was observed under electron microscopy. Results: After clamping the superior mesenteric artery and reperfusion the concentration of NO increased continuously and reached a peak for reperfusion 7 d (P<0.01) but that of SOD decreased from 30 min to 7 d. The expression of Bax, p53 and Bcl-2 also increased obviously especially for reperfusion 30 min and 7 d following ischemia and reperfusion. After reperfusion for 30 min the positive cell rate of Bax, p53 and Bcl-2 was 53.6%, 45.9% and 67.9%, for reperfusion 7 d it was 52.4%, 43.4% and 31.9% respectively, but the positive cell rate of Bax and p53 was higher than that of Bcl-2. The result of electron microscopy observation showed mfofiliments breaked, dissolved and chromatin condensed. Conclusion: Intestinal ischemia and reperfusion of rat can induced the changes of oxygen free radical and the expression of apoptotic gene and second injury of myocardial cells.