Protective effect and mechanism of clemastine fumarate on acute lung injury in mice with intestinal ischemia-reperfusion

Objective:To evaluate the protective effect and mechanism of clemastine fumarate(CLE)on acute lung injury(ALI)in intestinal ischemia-reperfusion(I/R)mice.Methods:Twenty-four SPF Balb/c mice were randomly divided into sham operation group(sham group),ischemia-reperfusion group(I/R group),and clemastine fumarate pretreatment group(I/R+C group).In the I/R group,an intestinal ischemia-reperfusion model was established(ischemia for 40 minutes,reperfusion for 2 hours).In the I/R+C group,CLE 5 mg/kg was intraperitoneally injected before the operation.Lung tissue morphology was observed and scored by HE staining;and the ratios of wet weight to dry weight(W/D)were recorded.the levels of MDA,SOD,GSH-px,NF-κB and TNF-αin lung tissue of each group were determined by ELISA;Western blot method was used to determine the expression of TLR4 protein in lung tissue.Results:Compared with the Sham group,the I/R group had significantly higher lung tissue injury score and wet/dry ratio(P<0.05),increased lung tissue MDA level(P<0.05),decreased SOD and GSH-px levels(P<0.05),and increased NF-κB and TNF-αlevels,the expression of TLR4 protein in lung tissue increased(P<0.05);compared with the I/R group,the lung tissue injury score and wet/dry ratio of the I/R+C group decreased(P<0.05),the level of MDA in lung tissue decreased(P<0.05),the levels of SOD and GSH-px increased(P<0.05),and the levels of NF-κB and TNF-毩decreased(P<0.05),the expression of TLR4 protein in lung tissue decreased(P<0.05).Conclusion:Clemastine fumarate can alleviate acute lung injury after intestinal ischemia-reperfusion in mice,and the mechanism may be related to the inhibition of oxidative stress and inflammatory response in lung tissue.

Effect of intestinal ischemia-reperfusion on expressions of endogenous basic fibroblast growth factor and transforming growth factor β in lung and its relation with lung repair

AIM To study the changes of endogenoustransforming growth factor β(TGFβ)and basicfibroblast growth factor(bFGF)in lung followingintestinal ischemia and reperfusion injury andtheir effects on lung injury and repair.METHODS Sixty Wistar rats were divided intofive groups,which underwent sham-operation,ischemia(45 minutes),and reperfusion(6,24and 48 hours,respectively)after ischemia(45minutes).Immunohistochemical method wasused to observe the localization and amounts ofboth growth factors.RESULTS Positive signals of both growthfactors could be found in normal lung,mainly inalveolar cells and endothelial cells of vein.Afterischemia and reperfusion insult,expressions ofboth growth factors were increased and theiramounts at 6 hours were larger than those ofnormal control or of 24 and 48 hours after insult.CONCLUSION The endogenous bFGF and TGF βexpression appears to be up-regulated in thelung following intestinal ischemia andreperfusion,suggesting that both growth factorsmay be involved in the process of lung injury andrepair.

Bone-marrow mesenchymal stem cells reduce rat intestinal ischemia-reperfusion injury, ZO-1 downregulation and tight junction disruption via a TNF-α-regulated mechanism

AIM: To investigate the effect of bone-marrow mesenchymal stem cells (BM MSCs) on the intestinal mucosa barrier in ischemia/reperfusion (I/R) injury. METHODS: BM MSCs were isolated from male Sprague-Dawley rats by density gradient centrifugation, cultured, and analyzed by flow cytometry. I/R injury was induced by occlusion of the superior mesenteric artery for 30 min. Rats were treated with saline, BM MSCs (via intramucosal injection) or tumor necrosis factor (TNF)-α blocking antibodies (via the tail vein). I/R injury was assessed using transmission electron microscopy, hematoxylin and eosin (HE) staining, immunohistochemistry, western blotting and enzyme linked immunosorbent assay.RESULTS: Intestinal permeability increased, tight junctions (TJs) were disrupted, and zona occludens 1 (ZO-1) was downregulated after I/R injury. BM MSCs reduced intestinal mucosal barrier destruction, ZO-1 downregulation, and TJ disruption. The morphological abnormalities after intestinal I/R injury positively correlated with serum TNF-α levels. Administration of anti-TNF-α IgG or anti-TNF-α receptor 1 antibodies attenuated the intestinal ultrastructural changes, ZO-1 downregulation, and TJ disruption. CONCLUSION: Altered serum TNF-α levels play an important role in the ability of BM MSCs to protect against intestinal I/R injury.

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 PREOPERATIVE GLUTAMINE ADMINISTRATION ON ICAM-1 EXPRESSION IN RAT LUNG INDUCED BY INTESTINAL ISCHEMIA-REPERFUSION

Objective To evaluate the effect of preoperative glutamine administration on intracellular adhesion molecule-1 （1CAM-l） expression in rat lung induced by intestinal ischemia-reperfusion（ I/R）. Methods Sprague-Dawley rats （n = 25） were randomly divided into 5 groups： sham group （sham surgery）, glutamine groups （three different doses） and control group. All groups except sham were subjected to intestinal 1/R injury, and superior mesenteric artery （SMA） occluded for 60 min followed by 90 min of reperfusion. Lung injury was evaluated with Evans blue dye concentration and histopathologic examination. The immunohistochemical expression and mRNA expression of 1CAM-1 were measured with immunohistochemical staining and RT-PCR method respectively. The level of myeloperoxidase （MPO） was also measured with biochemistry method. Results Intestinal 1/R resulted in lung injury characterized by an increase in Evans blue dye concentration, neutrophil sequestration, and obvious staining for expression of pulmonary 1CAM-l, compared with sham group. The expression of 1CAM-1 and the level of MPO in rat lung were lower in glutamine groups compared with control group. Conclusion 1-R injury increases the expression of 1CAM-1 within the lung. This may contribute to the migration, accumulation and activation of polymorphonuclear neutrophils （PAINs） after such injury. Preoperative glutamine administration attenuates rat lung injury induced by intestinal I-R, and inhibiting 1CAM-1 expression maybe one of the potential mechanisms.

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 nuclear factor kappa B on intercellular adhesion molecule-1 expression and neutrophil infiltration in lung injury induced by intestinal ischemia/reperfusion in rats

AIM： To investigate the role of nuclear factor kappa B （NF-κB） in the pathogenesis of lung injury induced by intestinal ischemia/reperfusion （I/R）, and its effect on intercellular adhesion molecule-1 （ICAM-1） expression and neutrophil infiltration. METHODS： Twenty-four Wistar rats were divided randomly into control, I/R and pyrrolidine dithiocarbamate （PDTC） treatment groups, n = 8 in each. I/R group and PDTC treatment group received superior mysenteric artery （SMA） occluding for 1 h and reperfusion for 2 h. PDTC group was administrated with intraperitoneal injection of 2% 100 mg/kg PDTC 1 h before surgery. Lung histology and bronchia alveolus lung fluid （BALF） protein were assayed. Serum IL-6, lung malondialdehyde （MDA） and myeloperoxidase （MPO） as well as the expression level of NF-κB and ICAM-1 were measured.RESULTS： Lung injury induced by intestinal I/R, was characterized by edema, hemorrhage and neutrophil infiltration as well as by the significant rising of BALF protein. Compared to control group, the levels of serum IL-6 and lung MDA and MPO increased significantly in I/R group （P=0.001）. Strong positive expression of NF-κB p65 and ICAM-1 was observed. After the administration of PDTC, the level of serum IL-6, lung MDA and MPO as well as NF-κB and ICAM-1 decreased significantly （P〈 0.05） when compared to I/R group.CONCLUSION： The activation of NF-κB plays an important role in the pathogenesis of lung injury induced by intestinal I/R through upregulating the neutrophil infiltration and lung ICAM-1 expression. PDTC as an inhibitor of NF-κB can prevent lung injury induced by intestinal I/R through inhibiting the activity of NF-κB.

N-acetylcysteine inhibits activation of toll-like receptor 2 and 4 gene expression in the liver and lung after partial hepatic ischemia-reperfusion injury in mice

BACKGROUND: Toll-like receptor 2 and 4 (TLR2/4) may play important roles in ischemia-reperfusion (I/R) injury, and N-acetylcysteine (NAC) can prevent the generation of reactive oxygen species (ROS) induced by I/R injury. This study aimed to investigate the changes in TLR2/4 gene expression in the liver and lung after I/R injury with or without NAC pretreatment. METHODS: BALB/c mice were used in a model of partial hepatic I/R injury and randomly assigned to a sham-operated control group (SH), a hepatic ischemia/reperfusion group (I/R) or a NAC pretreated, hepatic I/R group (I/R-NAC). The levels of TNF-alpha in the portal vein and plasma alanine aminotransferase (ALT) were measured at 1 and 3 hours after reperfusion. The lung wet-to-dry ratio was measured, and the expression of TLR2/4 mRNA and protein in the liver and lung were assessed with RT-PCR and Western blotting at the same time points. RESULTS: Compared with the I/R group, the expression of TLR2/4 mRNA and protein in the liver and lung in the I/R-NAC group was decreased at the same time point (P<0.05). The levels of portal vein TNF-a and plasma ALT increased continuously in the l/R group at I and 3 hours of reperfusion compared with the SH group; however, they declined significantly in the group pretreated with NAC (P<0.05). The extent of lung edema was relieved in the I/R-NAC group compared with the I/R group (P<0.05). CONCLUSIONS: TLR2/4 was activated in the liver and lung in the process of partial hepatic I/R injury. NAC inhibited the activation of TLR2/4 and the induction of TNF-alpha resulting from I/R injury via modulating the redox state, thus it may mitigate liver and lung injury following partial hepatic I/R in mice.

Ginkgo biloba extract(EGb 761) attenuates lung injury induced by intestinal ischemia/reperfusion in rats:Roles of oxidative stress and nitric oxide

AIM： To investigate the effect of ginkgo biloba extract （EGb 761） on lung injury induced by intestinal ischemia/ reperfusion （ Ⅱ/R）. METHODS： The rat model of Ⅱ/R injury was produced by damping the superior mesenteric artery for 60 min followed by reperfusion for 180 min. The rats were randomly allocated into sham, Ⅱ/R, and EGb ＋Ⅱ/R groups. In EGb ＋Ⅱ/R group, EGb 761 （100 mg/kg per day） was given via a gastric tube for 7 consecutive days prior to surgery. Rats in Ⅱ/R and sham groups were treated with equal volumes of the vehicle of EGb 761. Lung injury was assessed by light microscopy, wet-todry lung weight ratio （W/D） and pulmonary permeability index （PPT）. The levels of malondialdehyde （MDA） and nitrite/nitrate （NO2/NO3）, as well as the activities of superoxide dismutase （SOD） and myeloperoxidase （MPO） were examined. Western blot was used to determine the expression of inducible nitric oxide synthase （iNOS）. RESULTS： EGb 761 markedly improved mean arterial pressure and attenuated lung injury, manifested by the improvement of histological changes and significant decreases of pulmonary W/D and PPT （P 〈 0.05 or 0.01）.Moreover, EGb 761 markedly increased SOD activity, reduced MDA levels and MPO activity, and suppressed NO generation accompanied by down-regulation of iNOS expression （P 〈 0.05 or 0.01）. CONCLUSION： The results indicate that EGb 761 has a protective effect on lung injury induced by Ⅱ /R, which may be related to its antioxidant property and suppressions of neutrophil accumulation and iNOS- induced NO generation. EGb 761 seems to be an effective therapeutic agent for critically ill patients with respiratory failure related to Ⅱ/R.

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.

Gene Expression Profile of Pulmonary Tissues in Different Phases of Lung Ischemia-reperfusion Injury in Rats

In order to provide us new clues to induce some endogenous protective molecular mechanisms, the changes in gene expression profile induced by ischemia-reperfusion in pulmonary tissues of rats were investigated and the dynamic mechanism of pulmonary ischemia-reperfusion injury was elucidated. Thirty male Wistar rats were randomly divided into 6 groups： 5 ischemia-reperfusion （I/R） groups （I/R 0-h, I/R 1-h, I/R 3-h, I/R 6-h, I/R 24-h） and control group （n=5 in each）. An in situ ischemia-reperfusion lung injury rat model was established by occluded hilus of lung. The RatRef-12 Expression Beadchip （22 226 gene probes per array） was used to analyze the pattern of gene expression in all groups. The results showed that 648, 340, 711, 1279 and 641 genes were differentially expressed in I/R 0-, 1-, 3-, 6- and 24-h groups respectively. The differentially expressed genes were classified as following 7 functional categories： cytokine, adhesion molecule, growth factor and apoptosis-related factor, oxidation and antioxidation molecule, metabolic enzyme, ion channel and aquaporin, signal transduction molecule. It was suggested that gene chip technology was an effective and quick method for screening differentially expressed genes. Many differentially expressed genes with different functions interacted each other to result in pulmonary ischemia-reperfusion injury.

Benefit of prophylactic bronchodilator withβ2 adrenergic agonist in ischemia-reperfusion-induced lung injury

Primary lung graft dysfunction could significantly attribute to ischemia-reperfusion lung injury(IRLI)during transplantation surgery.β2-adrenergic agonists were one of the bronchodilators that had been well-established in the management of asthma and chronic obstructive pulmonary disease(COPD)with anti-inflammatory potency.By applying the model of isolated rat lung,we evaluated the efficacy of short-actingβ2-agonist inhalation to ameliorate ischemia-reperfusion damage.The experiment protocol was 180 min of global ischemia and then reperfusion for 60 min.In theβ2-agonist inhalation group,aerosolized albuterol was administrated prior ischemia procedure.Increased weight ratios of wet to dry lung and microvascular permeability were characterized in the IRLI group.In contrast,pre-inhaledβ2-agonist significantly mitigated the severity of pulmonary edema.Bronchoalveolar lavage from theβ2-agonist group presented decreased leukocyte counts and cytokines production,including interleukin-1β(IL-1β),tumor necrosis factor-α(TNF-α),and macrophage inflammatory protein 2(MIP-2).Devastating oxidative stress was widely recognized during the ischemia-reperfusion process,whileβ2-agonist pretreatment revealed subsided H2O2,myeloperoxidase(MPO),and the cleavage of caspase-3.Western blotting from lung homogenates identified the blockade of NF-κB and MAPK activation in theβ2-agonist inhalation group.Currently,there was no specific pharmacotherapy in IRLI management.Our results elucidated the protective effect ofβ2-agonist bronchodilator against ischemia-reperfusion induced oxidative stress,inflammation reaction,and pulmonary edema.

Effect of Dachengqi Decoction on NF-κB p65 Expression in Lung of Rats with Partial Intestinal Obstruction and the Underlying Mechanism

To investigate the effect of Dachengqi decoction on NF-κB p65 expression in lung of rats with partial intestinal obstruction and the underlying mechanism, 30 SD rats were randomly divided into three groups： sham-operation group, model group and Dachengqi decoction treatment group （Dachengqi group）, with 10 animals in each group. The models were made by partially ligating their large intestines outside the body. The pathological changes were analyzed by HE staining. The expression of NF-κB p65 in rats lung were measured by using real-time polymerase chain reaction and immunohistochemistry respectively. Moreover, the expression of caveolin-1 in rats lung was also measured to. Increased edema, interstitial thickening, hemorrhage, and infiltration of inflammatory cells were found in the model group. In contrast, this change was significantly reduced in Dachengqi group as compared with model group. In addition, the up-regulated caveolin-1 and NF-κB p65 were also suppressed by Dachengqi decoction in lung of rats with partial intestinal obstruction. We are led to concluded that the caveolin-l-NF-κB pathway plays an important role in the development of lung injury of rats with partial intestinal obstruction and Dachengqi decoction could down-regulate the expression of caveolin-1 and NF-κB p65 in lung of rats with partial intestinal obstruction.

Relationship between plasma D(-)-lactate and intestinal damage after severe injuries in rats

AIM To explore the kinetic changes in plasma D(-)- lactate and lipopolyssccharide(LPS)levels,and investigate whether D(-)-lactate could be used as a marker of intestinal injury in rats following gut ischemia/ reperfusion,burn,and acute necrotizing pancreatitis (ANP). METHODS Three models were developed in rats:① gut ischemia/ reperfusion obtained by one hour of superior mesenteric artery occlusion followed by reperfusion;② severe burn injury created by 30% of total body surface area(TBSA)full-thickness scald burn;and ③ ANP induced by continuous inverse infusion of sodium taurocholate and trypsin into main pancreatic duct. Plasma levels of D(-)-lactate in systemic circulation and LPS in portal circulation were measured by enzymatic- spectrophotometric method and limulus amebocyte lysate (LAL)test kit,respectively.Tissue samples of intestine were taken for histological analysis. RESULTS One hour gut ischemia followed by reperfusion injuries resulted in a significant elevation in plasma D(-)- lactate and LPS levels,and there was a significant correlation between the plasma D(-)-lactate and LPS(r =0.719,P<0.05).The plasma concentrations of D(-)- lactate and LPS increased significantly at 6h postburn, and there was also a remarkable correlation between them (r = 0.877,P < 0.01).D(-)-lactate and LPS levels elevated significantly at 2h after ANP,with a similar significant correlation between the two levels(r = 0.798, P < 0.01 ).The desquamation of intestine villi and infiltration of inflammatory cells in the lamina propria were observed in all groups. CONCLUSION The changes of plasma D(-)-lactate levels in systemic blood paralleled with LPS levels in the portal vein blood.The measurement of plasma D(-)-lactate level may be a useful marker to assess the intestinal injury and to monitor an increase of intestinal permeability and endotoxemia following severe injuries in early stage.

Inhibition of p38 mitogen-activated protein kinase may decrease intestinal epithelial cell apoptosis and improve intestinal epithelial barrier function after ischemia- reperf usion injury

AIM: To investigate the role of p38 mitogen-activated protein kinase in rat small intestine after ischemia-reperfusion (I/R)insult and the relationship between activation of p38 MAPK and apoptotic cell death of intestine.METHODS: Ninety Wistar rats were divided randomly into three groups, namely sham-operated group (C), I/R vehicle group (R) and SB203580 pre-treated group(S).In groups R and S, the superior mesenteric artery(SMA)was separated and occluded for 45 min, then released for reperfusion for0.25, 0.5, 1, 2, 6, 12 and 24 h. In group C, SMA was separated without occlusion. Plasma D-lactate levels were examined and histological changes were observed under a light microscope. The activity of p38 MAPK was determined by Western immunoblotting and apoptotic cells were detected by the terminal deoxynucleotidyl transferase (TdT)-mediated dUDP-biotin nick end labeling (TUNEL).RESULTS: Intestinal ischemia followed by reperfusion activated p38 MAPK, and the maximal level of activation (7.3-fold vs sham-operated group) was reached 30 min after I/R. Treatment with SB 203580, a p38 MAPK inhibitor,reduced intestinal apoptosis (26.72±3.39% vs62.50±3.08%in I/R vehicle, P＜0.01) and decreased plasma D-lactate level (0.78±0.15 mmol/L in I/R vehicle vs0.42±0.17 mmol/L in SB-treated group) and improved post-ischemic intestinal histological damage.CONCLUSION: p38 MAPK plays a crucial role in the signal transduction pathway mediating post-ischemic intestinal apoptosis, and inhibition of p38 MAPK may attenuate ischemia-reperfusion injury.

TLR4-HMGB1-, MyD88- and TRIF-dependent signaling in mouse intestinal ischemia/reperfusion injury

AIM: To characterize high-mobility group protein 1-toll-like receptor 4(HMGB1-TLR4) and downstream signaling pathways in intestinal ischemia/reperfusion(I/R) injury.METHODS: Forty specific-pathogen-free male C57BL/6 mice were randomly divided into five groups(n = 8 per group): sham, control, anti-HMGB1, anti-myeloid differentiation gene 88(My D88), and anti-translocatingchain-associating membrane protein(TRIF) antibody groups. Vehicle with the control Ig G antibody, antiHMGB1, anti-My D88, or anti-TRIF antibodies(all 1 mg/kg, 0.025%) were injected via the caudal vein 30 min prior to ischemia. After anesthetization, the abdominal wall was opened and the superior mesenteric artery was exposed, followed by 60 min mesenteric ischemia and then 60 min reperfusion. For the sham group, the abdominal wall was opened for 120 min without I/R. Levels of serum nuclear factor(NF)-κB p65, interleukin(IL)-6, and tumor necrosis factor(TNF)-α were measured, along with myeloperoxidase activity in the lung and liver. Inaddition,morphologic changes that occurred in the lung and intestinal tissues were evaluated. Levels of m RNA transcripts encoding HMGB1 and NF-κB were measured by real-time quantitative PCR, and levels of HMGB1 and NF-κB protein were measured by Western blot. Results were analyzed using one-way analysis of variance.RESULTS: Blocking HMGB 1, MyD 8 8, and TRIF expression by injecting anti-HMGB1, anti-My D88, or anti-TRIF antibodies prior to ischemia reduced the levels of inflammatory cytokines in serum; NF-κB p65: 104.64 ± 11.89, 228.53 ± 24.85, 145.00 ± 33.63, 191.12 ± 13.22, and 183.73 ± 10.81(P < 0.05); IL-6: 50.02 ± 6.33, 104.91 ± 31.18, 62.28 ± 6.73, 85.90 ± 17.37, and 78.14 ± 7.38(P < 0.05); TNF-α, 43.79 ± 4.18, 70.81 ± 6.97, 52.76 ± 5.71, 63.19 ± 5.47, and 59.70 ± 4.63(P < 0.05) for the sham, control, anti-HMGB1, anti-My D88, and anti-TRIF groups, respectively(all in pg/m L).Antibodies also alleviated tissue injury in the lung and small intestine compared with the control group in the mouse intestinal I/R model. The administration of antiHMGB1, anti-My D88, and anti-TRIF antibodies markedly reduced damage caused by I/R, for which anti-HMGB1 antibody had the most obvious effect.CONCLUSION: HMGB1 and its downstream signaling pathway play important roles in the mouse intestinal I/R injury, and the effect of the TRIF-dependent pathway is slightly greater.

Intestinal injury can be reduced by intra-arterial postischemic perfusion with hypertonic saline

AIM:To investigate the effect of local intestinal perfusion with hypertonic saline(HTS) on intestinal ischemia-reperfusion injury(IRI) in bothex vivo andin vivo rat models.METHODS:All experiments were performed on male Wistar rats anesthetized with pentobarbital sodium given intraperitoneally at a dose of 60 mg/kg.Ex vivo vascularly perfused rat intestine was subjected to 60-min ischemia and either 30-min reperfusion with isotonic buffer(controls),or 5 min with HTS of 365 or 415 mOsm/L osmolarity(HTS 365mOsm or HTS 415mOsm,respectively) followed by 25-min reperfusion with isotonic buffer.The vascular intestinal perfusate flow(IPF) rate was determined by collection of the effluent from the portal vein in a calibrated tube.Spontaneous intestinal contraction rate was monitored throughout.Irreversible intestinal injury or area of necrosis(AN) was evaluated histochemically using 2.3.5-triphenyltetrazolium chloride staining.In vivo,30-min ischemia was followed by either 30-min blood perfusion or 5-min reperfusion with HTS 365mOsm through the superior mesenteric artery(SMA) followed by 25-min blood perfusion.Arterial blood pressure(BP) was measured in the common carotid artery using a miniature pressure transducer.Histological injury was evaluated in both preparations using the Chui score.RESULTS:Ex vivo,intestinal IRI resulted in a reduction in the IPF rate during reperfusion(P < 0.05 vs sham).The postischemic recovery of the IPF rate did not differ between the controls and the HTS 365mOsm group.In the HTS 415mOsm group,postischemic IPF rates were lower than in the controls and the HTS 365mOsm group(P < 0.05).The intestinal contraction rate was similar at baseline in all groups.An increase in this parameter was observed during the first 10 min of reperfusion in the control group as compared to the sham-treated group,but no such increase was seen in the HTS 365mOsm group.In controls,AN averaged 14.8% ± 5.07% of the total tissue volume.Administration of HTS 365mOsm for 5 min after 60-min ischemia resulted in decrease in AN(5.1% ± 1.20% vs controls,P < 0.01).However,perfusion of the intestine with the HTS of greater osmolarity(HTS 415mOsm) failed to protect the intestine from irreversible injury.The Chiu score was lower in the HTS 365mOsm group in comparison with controls(2.4 ± 0.54 vs 3.2 ± 0.44,P = 0.042),while intestinal perfusion with HTS 415mOsm failed to improve the Chiu score.Intestinal reperfusion with HTS 365mOsm in the in vivo series secured rapid recovery of BP after its transient fall,whereas in the controls no recovery was seen.The Chiu score was lower in the HTS 365mOsm group vs controls(3.1 ± 0.26 and 3.8 ± 0.22,P = 0.0079 respectively,),although the magnitude of the effect was lower than in the ex vivo series.CONCLUSION:Brief intestinal postischemic perfusion with HTS 365mOsm through the SMA followed by blood flow restoration is a protective procedure that could be used for the prevention of intestinal IRI.

Enteral glutamine pretreatment does not decrease plasma endotoxin level induced by ischemia-reperfusion injury in rats

AIM:To investigate whether oral glutamine pretreatment prevents impairment of intestinal mucosal integrity during ischemia-reperfusion (I/R) in rats. METHODS: The study was performed as two series with 40 rats in each. Each series of animals was divided into four groups. The first group was used as a control. Animals in the second group were only pretreated with oral glutamine, 1 g/kg for 4 d. The third group received a normal diet, and underwent intestinal I/R, while the fourth group was pretreated with oral glutamine in the same way, and underwent intestinal I/R. Intestinal mucosal permeability to 51Cr-labeled EDTA was measured in urine in the first series of animals. In the second series, histopathological changes in intestinal tissue and plasma endotoxin levels were evaluated. RESULTS: Intestinal I/R produced a significant increase in intestinal permeability, plasma endotoxin level and worsened histopathological alterations. After intestinal I/R, permeability was significantly lower in glutamine- treated rats compared to those which received a normal diet. However, no significant change was observed in plasma endotoxin levels or histopathological findings. CONCLUSION: Although glutamine pretreatment seems to be protective of intestinal integrity, upon I/R injury, such an effect was not observable in the histopathological changes or plasma endotoxin level.

Influence of Intestinal Lymphatic Ligation on Pulmonary Injury in Rats with Severe Acute Pancreatitis

Objective:The present study explored the mechanisms involved in intestinal lymphatic ligation in rats with severe acute pancreatitis(SAP).Methods:Male Sprague Dawley rats were randomly divided into 4 groups:saline group,saline+ligation group,SAP group,and SAP+ligation group.Rats in the SAP group were administered sodium taurocholate solution.Isolated mesenteric lymph duct ligation was administered to the saline+ligation and SAP+ligation groups.Endotoxin(ET),nitric oxide(NO),tumor necrosis factor-α(TNF-α),interleukin-1(IL-1),myeloperoxidase(MPO),and superoxide dismutase(SOD)were detected.Nuclear factor-KB(NF-κB)and intercellular cell adhesion molecule-1(ICAM-1)proteins were observed.The mRNA of inducible nitric oxide synthase(iNOS)and Toll-like receptor 4(TLR4)was detected by PCR.Results:Pathomorphological analysis showed that necrosis was present in the lung of rats in the SAP group,but only mild lesions in the SAP+ligation group.ET,NO,TNF-α,and IL-1 in the serum and lung tissue were significantly decreased and MPO was increased in the SAP+ligation group as compared with the SAP group.However,MPO was increased.The expression of NF-κB and ICAM-1,either iNOS or TLR4,was upregulated in the SAP group,but downregulated in the SAP+ligation group.Intestinal lymph duct ligation prevented ET translocation,the release of inflammation factors,and inflammation injury.Conclusion:The intestinal lymph duct ligation could alleviate SAP-induced pulmonary injury by suppressing NF-κB activation in rats.

Reperfusion injury after critical intestinal ischemia and its correction with perfluorochemical emulsion "perftoran"

AIM： To investigate the anti-ischemic properties of perfluorochemical emulsion ＂perftoran＂ in mesenteric region. METHODS： Experiments were conducted on 146 nonlinear white male rats weighing 200-350 g. Partial critical intestinal ischemia was induced by thorough atraumatic strangulation of 5-6 cm jejunal loop with its mesentery for 90 rain. Global critical intestinal ischemia was made by atraumatic occlusion of the cranial mesenteric artery （CMA） for 90 rain also. Perftoran （PF, 0.8-1.0 mL per 100 g） in experimental groups or 0.9% sodium chloride in control groups was injected at 75 rain of ischemic period. Mean systemic arterial blood pressure （BPM） registration, intravital microscopy and morphological examination of ischemic intestine and its mesentery were performed in both groups. RESULTS： During 90 min of reperfusion, BPM progressively decreased to 27.3±7.4% after PF administration vs 38.6±8.0% in the control group of rats with partial intestinal ischemia （NS） and to 50.3±6.9% vs 53.1±5.8% in rats after global ischemia （NS）. During the reperfusion period, full restoration of microcirculation was never registered; parts with restored blood flow had leukocyte and erythrocyte stasis and intra-vascular clotting, a typical ＂non-reflow＂phenomenon. The reduction of mesenteric 50-400 μm feeding artery diameter was significantly less in the PF group than in the control group （24±5.5% vs 45.2±3.6%, P〈0.05） 5 min after partial intestinal ischemia. This decrease progressed but differences between groups minimized at the 90th min of reperfusion （41.5±4.2% and 50.3±2.8%, respectively）. In reperfusion of rat＇s intestine, a significant mucosal alteration was registered. Villous height decreased 2.5-3 times and the quantity of crypts decreased more than twice. In the group of rats administered PF, intestinal mucosal layer was protected from irreversible post-ischemic derangement during reperfusion. Saved cryptal epithelial cells were the source of regeneration of the epithelium, which began to cover renewing intestinal villi after 24 h of blood flow restoration. View of morphological alterations was more heterogeneous in CMA groups. CONCLUSION： Systemic administration of perftoran promotes earlier and more complete structural regeneration during reperfusion in rats after partial and global critical intestinal ischemia.