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.

Protective effect of clodronate-containing liposomes on intestinal mucosal injury in rats with severe acute pancreatitis

BACKGROUND: Severe acute pancreatitis (SAP) can result in intestinal mucosal injury. This study aimed to demonstrate the protective effect of clodronate-containing liposomes on intestinal mucosal injury in rats with SAP. METHODS: Liposomes containing clodronate or phosphate buffered saline (PBS) were prepared by the thin-film method SAP models were prepared by a uniform injection of sodium taurocholate (2 mL/kg body weight) into the subcapsular space of the pancreas. Sprague-Dawley rats were randomly divided into a control group (C group), a SAP plus PBS-containing liposomes group (P group) and a SAP plus clodronate-containing liposomes group (T group). At 2 and 6 hours after the establishment of SAP models, 2 mL blood samples were taken from the superior mesenteric vein to measure the contents of serum TNF-α and IL-12. Pathological changes in the intestine and pancreas were observed using hematoxylin and eosin staining, while apoptosis was detected using TUNEL staining. In addition, the macrophage markers cluster of differentiation 68 (CD68) in the intestinal tissue was assessed with immunohistochemistry. RESULTS: At the two time points, the levels of TNF-α and IL-12 in the P group were higher than those in the C group (P<0.05) Compared with the P group, the levels of TNF-α and IL-12 decreased in the T group (P<0.05). The pathological scores of the intestinal mucosa and pancreas in the T group were lower than those of the P group. In the T group, large numbers of TUNEL-positive cells were observed, but none or few in the C and P groups. The number of CD68-positive macrophages decreased in the T group.CONCLUSIONS: Clodronate-containing liposomes have prote- ctive effects against intestinal mucosal injury in rats with SAP. The blockade of macrophages may provide a novel therapeutic strategy in SAP.

Preventive effect of tetramethylpyrazine on intestinal mucosal injury in rats with acute necrotizing pancreatitis

AIM： To evaluate the role of microcirculatory disorder （MCD） and the therapeutic effectiveness ;of tetramethylpyrazine （TMP） on intestinal mucosa injury in rats with acute necrotizing pancreatitis （ANP）.METHODS： A total of 192 Sprague-Dawley rats were randomly divided into three groups： normal control group （C group）, ANP group not treated with TMP （P group）, ANP group treated with TMP （T group）. An ANP model was induced by injection of 50 g/L sodium taurocholate under the pancreatic membrane （4 mL/kg）. C group received isovolumetric injection of 9 g/L physiological saline solution using the same method. T group received injection of TMP （10 mL/kg） via portal vein. Radioactive biomicrosphere technique was used to measure the blood flow at 0.5, 2, 6 and 12 h after the induction of ANP. Samples of pancreas, distal ileum were collected to observe pathological changes using a validated histology score. Intestinal tissues were also used for examination of myeloperoxidase （MPO） expressed intraceUularly in azurophilic granules of neutrophils.RESULTS： The blood flow was significantly lower in P group than in C group （P 〈 0.01）. The pathological changes were aggravated significantly in P group. The longer the time, the severer the pathological changes. The intestinal MPO activities were significantly higher in P group than in C group （P 〈 0.01）. The blood flow of intestine was significantly higher in T group than in P group after 2 h （P 〈 0.01）. The pathological changes were alleviated significantly in T group. MPO activities were significantly lower in T group than in P group （P 〈 0.01 or P 〈 0.05）. There was a negative correlation between intestinal blood flow and MPO activity （r = -0.981, P 〈 0.01） as well as between intestinal blood flow and pathologic scores （r = -0.922, P 〈 0.05）.CONCLUSION： MCD is an important factor for intestinal injury in ANP. TMP can ameliorate the condition of MCD and the damage to pancreas and intestine.

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.

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.

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.

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.

Over-starvation aggravates intestinal injury and promotes bacterial and endotoxin translocation under high-altitude hypoxic environment

AIM:To study whether over-starvation aggravates intestinal mucosal injury and promotes bacterial and endotoxin translocation in a high-altitude hypoxic environment.METHODS:Sprague-Dawley rats were exposed to hy-pobaric hypoxia at a simulated altitude of 7000 m for 72 h.Lanthanum nitrate was used as a tracer to detect intestinal injury.Epithelial apoptosis was observed with terminal deoxynucleotidyl transferase dUTP nick end labeling staining.Serum levels of diamino oxidase(DAO),malondialdehyde(MDA),glutamine(Gln),superoxide dismutase(SOD) and endotoxin were measured in intestinal mucosa.Bacterial translocation was detected in blood culture and intestinal homogenates.In addition,rats were given Gln intragastrically to observe its protective effect on intestinal injury.RESULTS:Apoptotic epithelial cells,exfoliated villi and inflammatory cells in intestine were increased with edema in the lamina propria accompanying effusion of red blood cells.Lanthanum particles were found in the intercellular space and intracellular compartment.Bacterial translocation to mesenteric lymph nodes(MLN) and spleen was evident.The serum endotoxin,DAO and MDA levels were significantly higher while the serum SOD,DAO and Gln levels were lower in intestine(P< 0.05).The bacterial translocation number was lower in the high altitude hypoxic group than in the high altitude starvation group(0.47±0.83 vs 2.38±1.45,P<0.05).The bacterial translocation was found in each organ,especially in MLN and spleen but not in peripheral blood.The bacterial and endotoxin translocations were both markedly improved in rats after treatment with Gln.CONCLUSION:High-altitude hypoxia and starvation cause severe intestinal mucosal injury and increase bacterial and endotoxin translocation,which can be treated with Gln.

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.

Dynamic changes of IL-2/IL-10, sFas and expression of Fas in intestinal mucosa in rats with acute necrotizing pancreatitis

AIM:To investigate dynamic changes of serum IL-2, IL-10, IL-2/IL-10 and sFas in rats with acute necrotizing pancreatitis. To explore the expression of Fas in intestinal mucosa of rats with acute necrotizing pancreatitis (ANP). METHODS:A total of 64 Sprague-Dawley (SD) rats were randomly divided into two groups:normal control group (C group), ANP group (P group). An ANP model was induced by injection of 50 g/L sodium taurocholate under the pancreatic membrane. Normal control group received isovolumetric injection of 9 g/L physiological saline solution using the same method. The blood samples of the rats in each group were obtained via superior mesenteric vein to measure levels of IL-2, IL-10, sFas and calculate the value of IL-2/IL-10. The levels of IL-2, IL-10 and sFas were determined by ELISA. The severity of intestinal mucosal injury was evaluated by pathologic score. The expression of Fas in intestinal mucosal tissue was determined by immunohistochemistry staining. RESULTS:Levels of serum IL-2 were significantly higher in P group than those of C group (2.79 ± 0.51 vs 3.53 ± 0.62, 2.93 ± 0.89 vs 4.35 ± 1.11, 4.81 ± 1.23 vs 6.94 ± 1.55 and 3.41 ± 0.72 vs 4.80 ± 1.10, respectively, P < 0.01, for all) and its reached peak at 6 h. Levels of serum IL-10 were significantly higher in P group than those of C group at 6 h and 12 h (54.61 ± 15.81 vs 47.34 ± 14.62, 141.15 ± 40.21 vs 156.12 ± 43.10, 89.18 ± 32.52 vs 494.98 ± 11.23 and 77.15 ± 22.60 vs 93.28 ± 25.81, respectively, P < 0.01, for all). The values of IL-2/IL-10 were higher significantly in P group than those of C group at 0.5 h and 2 h (0.05 ± 0.01 vs 0.07 ± 0.02 and 0.02 ± 0.01 vs 0.03 ± 0.01, respectively, P < 0.01, for all), and it were significantly lower than those of C group at 6 h (0.05 ± 0.02 vs 0.01 ± 0.01, P < 0.01) and returned to the control level at 12 h (0.04 ± 0.01 vs 0.05 ± 0.02, P > 0.05). In sFas assay, there was no significant difference between P group and C group (3.16 ± 0.75 vs 3.31 ± 0.80, 4.05 ± 1.08 vs 4.32 ± 1.11, 5.93 ± 1.52 vs 5.41 ± 1.47 and 4.62 ± 1.23 vs 4.44 ± 1.16, respectively, P > 0.05, for all). Comparison of P group and C group, the pathological changes were aggravated significantly in P group. Immunohistochemistry staining show the expression of Fas was absent in normal intestinal tissues, however, it gradually increased after induction of pancreatitis in intestinal tissue, then reached their peaks at 12 h.CONCLUSION:Fas were involved in the pathogenesis of pancreatitis associated intestinal injury. The mechanisms of Fas may be associated to Fas mediated T helper cell apoptosis.

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.

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.

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.

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.

Protection effect of Emodin pretreatment on intestinal I-RI damage of intestinal mucosa in ratsa

Objective:To linvestigate the protective effect and mechanism of emodin pretreatment on intestinal mucosa of rats with intestinal ischemia-reperfusion injury.Methods:A total of 50SD rats were randomly divided into control group,model group,emodin groups of low,medium and high dose,with 10 in each group.Ischemia-reperfusion injury(I-RI)mode was established by using noninvasive clamp on superior mesentericartery(SMA).Control group and model group were pretreated with 0.5%sodium carboxymethyl cellulose solution lavage 2 h before operation,emodin groups of low,medium and high dose were given emodin lavage with 20,40,60 mg/kg pretreatment,femoral venous blood before the lavage pretreatment(TO)and 1 h ischemia(Tl),and inferior vena venous blood after 1 h of reperfusion(T2)were extracted from each group of rats for detection of serun level of intestinal fatty acid binding protein(I-FABP),tumor necrosis factor(TNF-α),endotoxin,interleukin 6(IL-6),and die content of diamine oxidase(DAO);Mter model establishment,the rats were sacrificed,intestine homogenate was prepared by using blind intestinal tissue to detect intestinal tissue myeloperoxidase(MPO],malondialdehyde(MDA)and superoxide dismutase(SOD)levels.And upper small intestine tissue was retrieved,followed by fixation and conventional HE staining to observe intestinal tissue morphology under light microscopy.Results:In emodin groups of low,medium and high dose at T1 and T2,I-FABP,TNF-α,endotoxin.IL,-6 and DAO level were significandy lower than that of model group(P<0.05);in emodin group of low,medium and high dose,MPO and MDA content in intestinal tissue homogenate was significantly lower than that in model group(P<0.05),SOD level was significantly higher than that of model group(P<0.05).Intestinal damage of emodin low,medium and high dose groups were significandy lighter than model group.Conclusions:Emodin pretreatment has certain protective effect on intestinal mucosa in ischemia reperfusion injury.

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.

Melatonin protects liver from intestine ischemia reperfusion injury in rats

AIM: To investigate the protective effect of melatonin on liver after intestinal ischemia-reperfusion injury in rats. METHODS: One hundred and fifty male Wistar rats, weighing 190-210 g, aged 7 wk, were randomly divided into melatonin exposure group, alcohol solvent control group and normal saline control group. Rats in the melatonin exposure group received intraperitoneal (IP) melatonin (20 mg/kg) 30 min before intestinal ischemia-reperfusion (IR), rats in the alcohol solvent control group received the same concentration and volume of alcohol, and rats in the normal saline control group received the same volume of normal saline. Serum samples were collected from each group 0.5, 1, 6, 12, and 24 h after intestinal IR. Levels of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured with an auto-biochemical analyzer. Serum TNF-α was tested by enzyme-linked immunosorbent assay (ELISA). Malondialdehyde (MDA) in liver was detected by colorimetric assay. Pathological changes in liver and immunohistochemical straining of ICAM-1 were observed under an optical microscope. RESULTS: The levels of ALT measured at various time points after intestinal IR in the melatonin exposure group were significantly lower than those in the other two control groups (P < 0.05). The serum AST levels 12 and 24 h after intestinal IR and the ICAM-1 levels (%) 6, 12 and 24 h after intestinal IR in the melatonin exposure group were also significantly lower than those in the other two control groups (P < 0.05). CONCLUSION: Exotic melatonin can inhibit the activity of ALT, AST and TNF-α, decrease the accumulation of MDA, and depress the expression of ICAM-1 in liver after intestinal IR injury, thus improving the liver function.

Pretreatment of cromolyn sodium prior to reperfusion attenuates early reperfusion injury after the small intestine ischemia in rats

AIM: To investigate the effects of Cromolyn Sodium (CS) pretreated prior to reperfusion on the activity of intestinal mucosal mast cells (IMMC) and mucous membrane of the small intestine in ischemia-reperfusion (IR) injury of rats. METHODS: Thirty-two Sprague-Dawley (SD) rats were randomly divided into four groups: sham group (group S), model group (group M), high and low dosage of CS groups, (treated with CS 50 mg/kg or 25 mg/kg, group C1 and C2). Intestinal IR damage was induced by clamping the superior mesenteric artery for 45 min followed by reperfusion for 60 min. CS was intravenouly administrated 15 min before reperfusion. Ultrastructure and counts of IMMC, intestinal structure, the expression of tryptase, levels of malondisldehyde (MDA), TNF-α, histamine and superoxide dismutase (SOD) activity of the small intestine were detected at the end of experiment. RESULTS: The degranulation of IMMC was seen in group M and was attenuated by CS treatment. Chiu’s score of group M was higher than the other groups. CS could attenuate the up-regulation of the Chiu’s score, the levels of MDA, TNF-α, and expression of tryptase and the down-regulation of SOD activity and histamine concentration. The Chiu’s score and MDA content were negatively correlated, while SOD activity was positively correlated to the histamine concentration respectively in the IR groups. CONCLUSION: Pretreated of CS prior to reperfusion protects the small intestine mucous from ischemia- reperfusion damage, the mechanism is inhibited IMMC from degranulation.

Cystic fibrosis transmembrane conductance regulator prevents ischemia/reperfusion induced intestinal apoptosis via inhibiting PI3K/AKT/NF-κB pathway

BACKGROUND Intestinal ischemia/reperfusion(I/R)injury is a fatal syndrome that occurs under many clinical scenarios.The apoptosis of intestinal cells caused by ischemia can cause cell damage and provoke systemic dysfunction during reperfusion.However,the mechanism of I/R-induced apoptosis remains unclear.Cystic fibrosis transmembrane conductance regulator(CFTR)is a cAMP-activated chloride channel.Few researchers have paid attention to its role in intestinal I/R injury,or the relationship between CFTR and intestinal apoptosis induced by hypoxia/reoxygenation(H/R).AIM To investigate the effects of CFTR on I/R-induced intestinal apoptosis and its underlying molecular mechanisms.METHODS An intestinal I/R injury model was established in mice with superior mesenteric artery occlusion, and Caco2 cells were subjected to H/R for the simulation of I/R in vivo.RESULTSThe results suggested that CFTR overexpression significantly increased the Caco2 cell viability anddecreased cell apoptosis induced by the H/R. Interestingly, we found that the translocation of p65,an NF-κB member, from the cytoplasm to the nucleus after H/R treatment can be reversed by theoverexpression of CFTR, the NF-κB P65 would return from the nucleus to the cytoplasm asdetermined by immunostaining. We also discovered that CFTR inhibited cell apoptosis in theH/R-treated cells, and this effect was significantly curbed by the NF-κB activator BA, AKTinhibitor GSK690693 and the PI3K inhibitor LY294002. Moreover, we demonstrated that CFTRoverexpression could reverse the decreased PI3K/AKT expression induced by the I/R treatment invivo or H/R treatment in vitro.CONCLUSIONThe results of the present study indicate that the overexpression of CFTR protects Caco2 cells fromH/R-induced apoptosis;furthermore, it also inhibits H/R-induced apoptosis through thePI3K/AKT/NF-κB signaling pathway in H/R-treated Caco2 cells and intestinal tissues.

Hydrogen gas and preservation of intestinal stem cells in mesenteric ischemia and reperfusion

BACKGROUND Patients with mesenteric ischemia frequently suffer from bowel necrosis even after revascularization.Hydrogen gas has showed promising effects for ischemiareperfusion injury by reducing reactive oxygen species in various animal and clinical studies.We examined intestinal tissue injury by ischemia and reperfusion under continuous initiation of 3%hydrogen gas.AIM To clarify the treatment effects and target cells of hydrogen gas for mesenteric ischemia.METHODS Three rat groups underwent 60-min mesenteric artery occlusion(ischemia),60-min reperfusion following 60-min occlusion(reperfusion),or ischemiareperfusion with the same duration under continuous 3%hydrogen gas inhalation(hydrogen).The distal ileum was harvested.Immunofluorescence staining with caspase-3 and leucine-rich repeat-containing G-protein-coupled 5(LGR5),a specific marker of intestinal stem cell,was conducted to evaluate the injury location and cell types protected by hydrogen.mRNA expressions of LGR5,olfactomedin 4(OLFM4),hairy and enhancer of split 1,Jagged 2,and Neurogenic locus notch homolog protein 1 were measured by quantitative polymerase chain reaction.Tissue oxidative stress was analyzed with immunostaining for 8-hydroxy-2'-deoxyguanosine(8-OHdG).Systemic oxidative stress was evaluated by plasma 8-OHdG.RESULTS Ischemia damaged the epithelial layer at the tip of the villi,whereas reperfusion induced extensive apoptosis of the cells at the crypt base,which were identified as intestinal stem cells with double immunofluorescence stain.Hydrogen mitigated such apoptosis at the crypt base,and the LGR5 expression of the tissues was higher in the hydrogen group than in the reperfusion group.OLFM4 was also relatively higher in the hydrogen group,whereas other measured RNAs were comparable between the groups.8-OHdG concentration was high in the reperfusion group,which was reduced by hydrogen,particularly at the crypt base.Serum 8-OHdG concentrations were relatively higher in both reperfusion and hydrogen groups without significance.CONCLUSION This study demonstrated that hydrogen gas inhalation preserves intestinal stem cells and mitigates oxidative stress caused by mesenteric ischemia and reperfusion.