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Objective:To investigate the protective effect of carbon monoxide releasing molecule 2 (CORM-2) on intestinal barrier by regulating the differentiation of T lymphocytes in rats with hemorrhagic shock.Methods:Healthy male Sprague-Dawley rats ( n=56) were randomly (random number) divided into the sham operation group, shock group, dimethyl sulfoxide (DMSO) control group, inactivated carbon monoxide release molecule-2 (iCORM-2) pretreatment group and three pretreatment CORM-2 with the doses of 2, 4 and 6 mg/kg separately. The hemorrhagic shock was induced with the use of a modified Wiggers model. Rats in the CORM-2 group and iCORM-2 group were intraperitoneally injected with CORM-2 with the doses of 2, 4 and 6 mg/kg and 6 mg/kg iCORM-2 instantly before shock induction. Rats in the DMSO group received intraperitoneal administration of 2% DMSO with the same volume of iCORM-2. Rats in the shock group and sham operation group were not pretreated before inducing shock. Mean arterial pressure of each rat was recorded at different phases after catheterization or shock. Twenty-three hours after shock induction, the permeability of intestinal barrier was measured by FITC-dextran flux, and then ileum tissues were harvested to observe histopathologic changes. Immunohistochemistry was used to detect the expression of transcription factors T-bet and Foxp3 of intestinal mucosa in rats, and the expression of interferon-γ (IFN-γ), interleukin-10 (IL-10) and transforming growth factor-β (TGF-β) in intestinal mucosa was measured by Western blot. One-way analysis of variance or Kruskal Wallis rank sum test was used to compare the difference among groups for normal or non-normal distributed data. Results:Compared with the sham operation group, serum concentrations of FITC-dextran were significantly increased in the shock group, DMSO group and iCORM-2 group (all P<0.05). The concentrations of FITC-dextran in serum of three CORM-2 pretreatment groups pretreatment were significantly decreased compared with other groups undergoing shock (all P<0.05). Rats in the shock group, DMSO group and iCORM-2 group showed severe ileum injury. CORM-2 intervention resulted in alleviation of intestinal mucosal injury in rats with shock, and rats in groups pretreatment CORM-2 at the doses of 4 and 6 mg/kg exhibited integrity of anatomic ileac structure. Compared with the sham operation group, T-bet levels of intestinal intraepithelial lymphocytes were increased in shock group and DMSO group (all P<0.05). Compared with the shock group, levels of T-bet were decreased in intestinal epithelium of three groups pretreatment with CORM-2 at the doses of 2, 4 and 6 mg/kg (all P<0.05). Foxp3 levels in intestinal intraepithelial lymphocytes of the iCORM-2 group and two groups pretreatment with CORM-2 at the doses of 4 and 6 mg/kg were increased compared with the shock group and DMSO group (all P<0.05), but there was no significant difference among the shock group, DMSO group and group pretreatment with CORM-2 at 6 mg/kg (all P>0.05). The shock group showed increased expression of IFN-γ (all P<0.05), but unchangeable expression of IL-10 and TGF-β (all P>0.05) compared with the sham operation group. Compared with the shock group, the expression levels of IL-10 in three groups pretreatment with CORM-2 at the doses of 2, 4 and 6 mg/kg were significantly increased (all P<0.05), and the expression levels of TGF-β were increased in two groups pretreatment with CORM-2 at the doses of 4 and 6 mg/kg (all P<0.05). The expression of IFN-γ in group pretreatment with CORM-2 at 6 mg/kg was significantly decreased compared with the shock group ( P<0.05). Conclusions:CORM-2 inhibited the activation of type 1 helper T cells to decrease the expression of proinflammatory factors and upregulated the expression of anti-inflammatory factors. Thus, CORM-2 reduced gut inflammation and protected the intestinal barrier.
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Objective@#To investigate the protective effect of carbon monoxide releasing molecule 2 (CORM-2) on post-resuscitation myocardial dysfunction in rats.@*Methods@#Forty male SD rats which were healthy were randomly divided into 5 groups: sham operated group(sham group), cardiopulmonary resuscitation(PCR) group, DMSO group, inactivated CORM-2(iCORM-2) group and CORM-2 group (n=8 each). Established the model of post-cardiac arrest myocardial dysfunction by intravenous potassium chloride (4 ℃) injection combined with asphyxiation for 4 minutes and then followed by artificial chest compression for 3 minutes. Sham group: rats were instrumented with catheter without inducing cardiac arrest and resuscitation, and intraperitoneal injection of 0.9% normal saline (4 ml/kg) was performed 12 hours before catheterization. CPR group: rats were instrumented with catheters and underwent CPR, and intraperitoneal injection of 0.9% normal saline (4 ml/kg) was performed 12 hours before surgery.CORM-2 group: rats were instrumented with catheters and underwent CPR, intraperitoneally injected the prepared CORM-2 solution (4 mg/kg) at 12 hours before surgery. DMSO group: rats were instrumented with catheters and underwent CPR, intraperitoneally injected the prepared DMSO solution (4 ml/kg) at 12 hours before surgery. iCORM-2 group: rats were instrumented with catheters and underwent CPR, iCORM-2 solution (4 mg/kg) was intraperitoneally injected at 12 hours before surgery. Hemodynamic data (MAP, +dp/dtmax, -dp/dt) were continuously monitored and recorded for 4 hours after resuscitation (or catheterization) in each group. Myocardial tissue specimen and blood samples were taken after resuscitation (or catheterization). The myocardial ultrastructure was observed by transmission electron microscope. Lactate dehydrogenase (LDH) activity was measured by lactate-pyruvate method. Serum creatine kinase isoenzyme (CK-MB) concentration was measured by ELISA. Western blot was used to detect the levels of Caspase-3, Caspase-9 and Cyt-C protein in myocardial tissue.@*Results@#MAP, +dp/dtmax and -dp/dt at 0.5, 1, 2, 3 and 4 hours post resuscitation were significantly lower than those immediately after catheterization in CRP, DMSO, iCORM-2 groups (all P<0.05). MAP at 0.5, 1, 2, 3, and 4 hours post resuscitation were significantly lower in CRP, DMSO and iCORM-2 groups than those at respective time points in sham group (all P<0.05), while MAP was similar between CORM-2 group and Sham group at these time points (all P>0.05). +dp/dtmax and -dp/dt values at 0.5, 1, 2, 3, and 4 hours post resuscitation were lower than those at respective time points in sham group and significance was found at 0.5, 1 and 2 hours post resuscitation (both P<0.05), while +dp/dtmax and -dp/dt values were similar between CORM-2 group and sham group at various time points (all P>0.05). Myocardial ultrastructure, especially mitochondrial structural integrity was better preserved in the CORM-2 group than those in the other resuscitation groups at 4 hours after resuscitation. Serum LDH activity and CK-MB concentration were significantly elevated at 4 hours after resuscitation in the CPR group, DMSO group and iCORM-2 group than those in sham group (all P<0.01); CK-MB concentration was also higher in CORM-2 group than that in sham group,and LDH level was similar between CORM-2 group and sham group (P>0.05). Serum LDH activity and CK-MB concentrations were significantly lower in the CORM-2 group than those in the other resuscitation groups (all P<0.01). The myocardial expressions of Caspase-3, Caspase-9 and Cyt-C at 4 hours after resuscitation were significantly higher in the CPR group, DMSO group and iCORM-2 group than those in sham group; the myocardial expressions of Caspase-3 and Caspase-9 were significantly higher in CORM-2 group than those in sham group (both P<0.05), while Cyt-C expression was similar between CORM-2 group and sham group. The expressions of the above 3 proteins were significantly lower in the CORM-2 group than those in the other resuscitation groups (all P<0.05).@*Conclusions@#CORM-2 can effectively alleviate post-resuscitation myocardial injury in rats with cardiopulmonary resuscitation and improve cardiac function. Protecting myocardial mitochondria and inhibiting mitochondrial apoptosis pathway may serve as the protective mechanisms in this model.
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AIM:To investigate the effects of carbon monoxide-releasing molecule-2 (CORM-2) on intestinal barrier injury in cardiopulmonary resuscitation (CPR) rats.METHODS:The model of intestinal ischemia-reperfusion injury after cardiac arrest (CA) -CPR was established by intravenous injection of potassium chloride (4℃) combined with asphyxiation for 4 min, followed by artificial chest compression for 3 min.The SD rats (n=32) were randomly divided into 4 groups:sham group, CPR group, CORM-2 group and inactivated CORM-2 (i CORM-2) group, with 8 rats in each group.The rats in sham group were instrumented with catheter without experiencing cardiac arrest or CPR.The rats in other groups were inserted with the catheters and underwent CA-CPR.In CORM-2 group and i CORM-2 group, the rats were given CORM-2 at 4 mg/kg and i CORM-2 at 4 mg/kg by intraperitoneal injection at 12 h before CA-CPR.In sham group and CPR group, the rats were administrated with an equivalent volume of vehicle.The hemodynamic data were monitored for 4 h after resuscitation (or catheterization) in each group.The ileum tissues and blood samples were harvested at 4 h after resuscitation (or catheterization).Histopathological changes and ultrastructure of the ileum were observed by HE staining and transmission electron microscopy.The levels of intestinal fatty acid binding protein (I-FABP) in the blood was measured by ELISA.The protein expression of claudin-3, occludin, ZO-1 and proinflammatory factor tumor necrosis factor-α (TNF-α) in the ileum tissues was determined by Western blot.RESULTS:No significant difference of mean arterial pressure (MAP) immediately after catheterization between sham group and resuscitation groups was observed.In CPR group, CORM-2 group and i CORM-2 group, the MAP at each time point after resuscitation was obviously lower than that immediately after catheterization and this phenomenon continued until 4 h after resuscitation (P<0.05).The MAP at each time point after resuscitation in all resuscitation groups was prominently lower than that at the same time points in sham group (P<0.05).The MAP at each time point after resuscitation in CORM-2 group was higher than that in CPR group and i CORM-2 group (P<0.05).The histopathological changes and ultrastructural damage of ileum mucosa in CPR group were obvious.In CORM-2 group, the histopathological changes and ultrastructural integrity of ileum mucosa were significantly better than those in other resuscitation groups.Compared with sham group, the level of I-FABP in CPR group and i CORM-2 group was significantly increased (P<0.05).The level of I-FABP in CORM-2 group showed no statistically significant difference as compared with sham group.The level of I-FABP in CORM-2 group was notably lower than that in CPR group and i CORM-2 group (P<0.05).Compared with sham group, the expression of claudin-3, occludin and ZO-1 in ileum tissues was decreased in CPR group and i CORM-2 group (P<0.05) , but in CORM-2 group, only the expression of claudin-3 and ZO-1 were decreased (P<0.05).The levels of the above proteins in CORM-2 group were higher than those in CPR group and i CORM-2 group (P<0.05).Compared with sham group, the expression of TNF-αin ileum tissues was increased in CPR group and i CORM-2 group (P<0.05) , and the level of TNF-αin CORM-2 group showed no statistically significant difference as compared with sham group.The protein level of TNF-αin CORM-2 group was lower than that in CPR group and i CORM-2 group (P<0.05).CONCLUSION:Cardiac arrest-cardiopulmonary resuscitation causes severe damage of intestinal mechanical barrier in rats.CORM-2 may protect the intestinal barrier integrity in cardiopulmonary resuscitation rats by up-regulating the expression of tight junction proteins in the intestinal epithelium.
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Objective To investigate the protective effect of carbon monoxide release molecule-2 (CORM-2) on intestinal barrier injury in shock rats. Methods Thirty-two rats were equally and randomly divided into 4 groups: the sham operated group, shock group, carbon monoxide releasing molecule-2 (CORM-2) group and inactivated CORM-2 (iCORM-2) group. A modified Wiggers model was used in inducing hemorrhagic shock. In the CORM-2 group and iCORM-2 group, the animals were intraperitoneally injected CORM-2 (5 mg/kg) or iCORM-2 (5 mg/kg) 1 h before inducing shock. The ileum tissues were harvested 24 h after operation or 24 h after inducing shock, and the histopathologic changes and ultrastructure of the ileum were observed. The expressions of ZO-1, Occludin, and Claudin3 protein in ileum intestinal mucosa were determined by Western blot. TNF-α and D-lactic acid levels in the blood were measured by enzyme-linked immunosorbent assay (ELISA). Data of multi-groups were analyzed by one way variance (ANOVA) and inter-group comparisons were made by the least significant difference (LSD)-t tests. Kruskal Wallis rank sum test was used when homogeneity of variance were not met. The value of P<0.05 was considered statistically significant. Results (1) The pathological changes and ultrastructural damage of ileum mucosa in the shock group were obvious. CORM-2 can significantly improve the pathological morphology and ultrastructural integrity of intestinal mucosa in shock rats. (2) Compared with the sham operated group, the expressions of Claudin3, Occludin, and ZO-1 of ileum intestinal mucosa were significantly decreased in the shock group and iCORM-2 group(all P<0.05), but in the CORM-2 group, only the expression of Claudin3 and ZO-1 were decreased(P<0.05). The expression of these proteins in the CORM-2 group was higher than that in the shock group and iCORM-2 group. (3) Compared with the sham operated group, the levels of D-lactic acid and TNF-α in the shock group and iCORM-2 group were significantly higher[D-lactic acid(ng/mL): 139.49 ± 28.83, 135.75 ± 25.19 vs 65.09 ± 33.16; TNF-α(pg/mL):358.60(314.18, 395.73),312.25(275.75, 345.40) vs 65.85(47.82, 84.32); all P<0.05], TNF-α value and D-lactic acid level showed increased trend in CORM-2 group, but it's not statistically significant[ D-lactic acid(ng/mL): 75.65±34.14 vs 65.09±33.16; TNF-α(pg/mL): 104.00(93.10, 131.95) vs 65.85(47.82, 84.32); all P>0.05]. The levels of D-lactic acid and TNF-αin the CORM-2 group were lower than those in the shock group and iCORM-2 group (all P<0.05). Conclusions CORM-2 may reduce the intestinal barrier damage by anti-inflammatory effects and up-regulate the expression of tight junction proteins in the intestinal epithelium.
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Objective To investigate the effects and mechanisms of carbon monoxide-releasing molecule-2 (CORM-2) on LPS induced barrier injure of Caco-2 cells.Methods The model of Caco-2 monolayer cells damage induced by LPS was established by using 50 μg/ml LPS for 24 hours.After preconditioning with different concentrations (10 μmol/L,50 μmol/L,and 100 μ mol/L) of CORM-2 for 1 hour,the cultured well-grown Caco-2 monolayer cells were stimulated with 50 μ g/ml lipopolysaccharides (LPS) for 24 hour.The 100 μmol/L CORM-2 was put into 37℃ and 5％ CO2 incubator for 18 hours until the CO has been fully released,and it became an inactive CORM-2(iCORM-2).The cultured Caco-2 monolayer cells were divided into six groups:the control group,the LPS group,the LC1(10 μmol /L CORM-2 preconditioning) group,the LC2(50 μmol/L CORM-2 preconditioning) group,the LC3(100 μmol/L CORM-2 preconditioning) group,and the LC4 (iCORM-2 preconditioning) group.The apoptosis rates of different groups of Caco-2 monolayer cells were detected using flow cytometry.Cytokines (TNF-α,IL-1β and HMGB-1) levels of different groups were detected using ELISA kits.The levels of tight junction proteins (occludin ZO-1,claudin-1 and claudin-4) of every group were detected using Western blotting with specific antibodies.The structural changes of tight junction proteins were visualized by immunofluorescence technique.Results Compared with control group,cell apoptosis rate and release of inflammatory cytokines such as TNF-α,IL-1β and HMGB-1 in LPS group were significantly higher,and the levels of tight junction proteins were apparently decreased (P＜0.05) in LPS group.Compared with LPS group,cell apoptosis rate and release of inflammatory cytokines such as TNF-α,IL-1β and HMGB-1 decreased,and the levels of tight junction proteins were attenuated obviously,P＜0.05,in CORM-2 preconditioning groups.And the higher the concentration of CORM-2,the more obvious the protective effects.Conclusions This study demonstrates that CORM-2,as one of exogenous CO-releasing molecules,has the capacity to protect the barrier damage of LPS-stimulated Caco-2 monolayer cells in a concentration dependent manner.The higher the concentration of CORM-2 was,the stronger the protective effects were.The protective effects of CORM-2 include reducing Caco-2 monolayer cells apoptosis rate,inhibiting inflammatory cytokines production and release,and restoring distribution and levels of tight junction proteins.
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Objective To investigate the suppressive effect of exogenous carbon monoxide (CO) on abnormal platelet exocytosis and its possible molecular mechanism. Methods Venous blood was collected from healthy volunteers. Platelet-rich plasma (PRP) was isolated from the blood by differential centrifugation. The PRP was randomly divided into five groups by random number table, namely normal control group, lipopolysaccharide (LPS) group (challenged with 10 mg/L LPS), inactively exogenous carbon monoxide releasing molecule 2 (iCORM-2) group (given 10 mg/L LPS + 50 μmol/L iCORM-2 for intervention), exogenous carbon monoxide releasing molecule 2 (CORM-2) 10 μmol/L and 50 μmol/L groups (given 10 mg/L LPS + CORM-2 10 μmol/L or 50 μmol/L for intervention). After 30 minutes, enzyme linked immunosorbent assay (ELISA) was used to determine the platelet-derived growth factor BB (PDGF-BB) and matrix metalloproteinase 2 (MMP-2). Chemical fluorescein method was used to determine the platelet adenosine triphosphate (ATP). Flow cytometer was used to determine the expression of P-selectin. The expressions of Toll-like receptor 4 (TLR4), phosphorylation of protein kinase Cθ (PKCθ) and syntaxin binding protein 1 (STXBP-1) were determined by Western Bolt. The soluble N-ethylmaleimide-sensitive factor-attachment protein receptors (SNAREs) complex formation [syntaxin 2-synaptosomal-associated protein 23-vesicle associated membrane protein 8 (STX2-SNAP23-VAMP8)] mediated by STXBP-1 was determined by immunoprecipitation. Results ① Compared with normal control group, the platelet release of PDGF-BB, MMP-2 and ATP was significantly increased after LPS challenge, and the P-selectin expression of platelet was also obviously up-regulated [PDGF-BB (μg/L): 127.53±1.78 vs. 94.35±5.84, MMP-2 (ng/L): 51.87±9.20 vs. 35.83±3.17, ATP (μmol/L): 1.288±0.056 vs. 0.975±0.010, P-selectin: (3.93±0.19)% vs. (0.44±0.10)%, all P < 0.05]. The increases in platelet release of PDGF-BB, MMP-2 and ATP were suppressed by 10 μmol/L or 50 μmol/L CORM-2 administration, as well as high-expression of P-selectin in a dose-dependent manner [PDGF-BB (μg/L): 114.68±1.35, 97.08±6.14 vs. 127.53±1.78, MMP-2 (ng/L): 32.67±8.00, 24.63±1.63 vs. 51.87±9.20, ATP (μmol/L): 0.999±0.015, 0.965±0.008 vs. 1.288±0.056, P-selectin: (1.95±0.27)%, (0.94±0.11)% vs. (3.93±0.19)%, all P < 0.05]. ② Compared with normal control group, LPS challenge resulted in a significant increase in the expression of TLR4 and the phosphorylation of PKCθ and STXBP-1 [TLR4 (gray value): 1.21±0.38 vs. 0.67±0.06, p-PKCθ (gray value): 1.36±0.20 vs. 0.44±0.03, p-STXBP-1 (gray value): 1.13±0.06 vs. 0.59±0.04, all P < 0.05]. The increases in above parameters were suppressed by 10 μmol/L or 50 μmol/L CORM-2 administration in a dose-dependent manner [TLR4 (gray value): 0.76±0.05, 0.65±0.04 vs. 1.21±0.38; p-PKCθ (gray value): 0.71±0.07, 0.47±0.10 vs. 1.36±0.20; p-STXBP-1 (gray value): 0.56±0.02, 0.48±0.01 vs. 1.13±0.06, all P < 0.05]. ③ Compared with normal control group, the SNAREs proteins in platelet that combined with STXBP-1, including STX2, SNAP23 and VAMP8, were obviously increased after LPS challenge [STX2 (gray value): 1.35±0.06 vs. 0.57±0.04, SNAP23 (gray value): 0.97±0.04 vs. 0.30±0.12, VAMP8 (gray value): 1.37±0.12 vs. 0.77±0.10, all P < 0.05]. The increases in SNAREs complex formation were suppressed by 10 μmol/L or 50 μmol/L CORM-2 administration in a dose-dependent manner [STX2 (gray value): 0.77±0.02, 0.39±0.03 vs. 1.35±0.06, SNAP23 (gray value): 0.41±0.03, 0.22±0.08 vs. 0.97±0.04, VAMP8 (gray value): 0.85±0.07, 0.66±0.07 vs. 1.37±0.12, all P < 0.05]. There was no significant difference in the above mentioned parameters between iCORM-2 group and LPS group. Conclusions LPS-induced abnormal secretion of platelet was suppressed by CORM-2 administration. The mechanism may involve the TLR4/PKCθ/STXBP-1 signaling pathway activation and the SNAREs complex formation.