A semi-synthetic derivative of artemisinin,artesunate inhibits prostaglandin E2 production in LPS/IFNγ-activated BV2 microglia

Artesunate is a semi-synthetic derivative of artemisinin used to treat malaria, and has been shown to possess anti-inflammatory activity. In this study, we have investigated the effect of artesunate on PGE₂ production/COX-2 protein expression in LPS + IFNγ-activated BV2 microglia. To further understand the mechanism of action of this compound, we investigated its interference with NF-κB and p38 MAPK signalling pathways. PGE₂ production was determined using EIA, while protein expressions of inflammatory targets like COX-2, mPGES-1, IκB, p38 and MAPKAPK2 were evaluated using western blot. An NF-κB-bearing luciferase reporter gene assay was used to test the effect of artesunate on NF-κB-mediated pro-inflammatory gene expression in HEK293 cells stimulated with TNFα (1 ng/ml). Artesunate (2 and 4 μM), significantly (p <0.01) suppressed PGE₂ production in LPS + IFNγ-activated BV2 microglia. This effect was found to be mediated via reduction in COX-2 and mPGES-1 proteins. Artesunate also produced significant inhibition of TNFα and IL-6 production in activated BV2 microglia. Further investigations showed that artesunate (0.5–4 μM) significantly (p <0.001) reduced NF-κB-driven luciferase expression, and inhibited IκB phosphorylation and degradation, through inhibition of IKK. Artesunate inhibited phosphorylation of p38 MAPK and its substrate MAPKAPK2 following stimulation of microglia with LPS + IFNγ. Taken together, we have shown that artesunate prevents neuroinflammation in BV2 microglia by interfering with NF-κB and p38 MAPK signalling.     

Dexamethasone enhances trichosanthin-induced apoptosis in the HepG2 hepatoma cell line

AimsTrichosanthin (TCS) is a type I ribosome-inactivating protein (RIP) with antitumor activities for various cancers. In this paper, we aimed to investigate whether dexamethasone, an important synthetic member of the glucocorticoid steroids, in combination with TCS can be a potential therapy in treating hepatoma.Main methodsCell viability was investigated using MTT assay, and apoptosis was evaluated with Hoechst 33258 staining. Western blot analysis was used to examine the changes in the expression levels of IκB-α, NF-κB p65 subunit and Cox-2. Additionally, we took advantage of dominant-negative IκB (IκB-DM) over-expression and chemical inhibitor PDTC to inhibit NF-κB activation.Key findingsOur results demonstrated that dexamethasone could enhance TCS-induced apoptosis in the hepatoma cell line HepG2, decreasing IC50 values from in excess of 200 μg/ml to 50 µg/ml. In addition, our results demonstrated that TCS could induce rapid degradation of IκB-α, nuclear translocation of NF-κB and decrease of COX-2 expression in HepG2 cells. Inhibition of NF-κB by biological (IκB-DM) or chemical inhibitor (PDTC) increased HepG2 cells' sensitivity to TCS, resulting in cell viability rate decreasing and apoptotic rate increasing. Simultaneously, dexamethasone increased the level of IκB-α protein and effectively inhibited TCS-induced degradation of IκB-α.SignificanceThese results suggest that dexamethasone could enhance trichosanthin-induced apoptosis in the HepG2, at least in part, by inhibiting the NF-κB signaling pathway and thus strengthening the antitumor effects of TCS, which highlights the possibility of combined drug application of TCS and dexamethasone in the clinical treatment of hepatoma.     

HDAC inhibitor-induced activation of NF-κB prevents apoptotic response of E1A + Ras-transformed cells to proapoptotic stimuli

HDAC inhibitors (HDACIs) are capable of suppressing the cell growth of tumour cells due to the induction of apoptosis and/or cell cycle arrest. This allows of considering HDACIs as promising agents for tumour therapy. The final outcome – apoptotic cell death or cell cycle arrest – depends on the type of tumour and cellular context. In this report, we addressed the issue by analysing effects produced in E1A + Ras-transformed MEF cells by HDAC inhibitors sodium butyrate (NaB), Trichostatin A (TSA) and some others. It has been shown that the HDACIs induced cell cycle arrest in E1A + Ras-transformed cells but not apoptosis. The antiapoptotic effect of HDACIs is likely to be a result of NF-κB-dependent signaling pathway activation. HDACI-induced activation of NF-κB takes place in spite of a deregulated PI3K/Akt pathway in E1A + Ras cells, suggesting an alternative mechanism for the activation of NF-κB based on acetylation. HDACI-dependent activation of NF-κB prevents the induction of apoptosis by cytostatic agent adriamycin and serum deprivation. Accordingly, suppression of NF-κB activity in HDACI-arrested cells by the chemical inhibitor CAPE or RelA-siRNA resulted in the induction of an apoptotic programme. Thus, our findings suggest that the activation of the NF-κB pathway in HDACI-treated E1A + Ras-transformed cells blocks apoptosis and may thereby play a role in triggering the programme of cell cycle arrest and cellular senescence.     

Quorum sensing modulators exhibit cytotoxicity in Hodgkin’s lymphoma cells and interfere with NF-κB signaling

In recent years it has become evident that bacteria can modulate signaling pathways in host cells through the secretion of small signaling molecules. We have evaluated the cytotoxic effects and NF-κB inhibitory activities of a panel of quorum sensing molecules and their reactive analogs on Hodgkin's lymphoma cells (L428). We found that several molecules inhibited NF-κB signaling in a dose dependent manner. Three inhibitors (ITC-12, ITC-Cl and Br-Furanone) showed 50% NF-κB inhibition at concentrations less than 10 µM (4.1 µM, 12.8 µM and 9.9 µM, respectively). Furthermore, all three molecules displayed cytotoxic effects against L428 cells with IC50 values of 12.4 µM, 18.3 µM and 3.1 µM respectively after 48 h incubation. They also showed inhibition of A549 adenocarcinoma cell migration at low concentrations 5.6 µM, 2.6 µM and 7.9 µM respectively. Further analysis showed that these molecules significantly decrease the degree of expression of proteins of NF-κB subunits p50, p65 and RelB both in cytosolic and nuclear fractions. This confirms that these compounds have the potential to modulate the NF-κB pathway by suppressing their subunits and thus exhibit cytotoxicity and inactivation of NF-κB signaling in Hodgkin's lymphoma cells.     

Dual in vitro effects of cortisol on cell turnover in the medaka esophagus via the glucocorticoid receptor

AimsCortisol is a glucocorticoid in mammals, but has both gluco- and mineralocorticoid activities in teleost fish. Our previous in vivo studies on osmoregulatory esophagi of euryhaline fish showed that epithelial apoptosis for the simple epithelium in seawater and cell proliferation for the stratified epithelium in fresh water are both induced by cortisol. The aim of the present study was to examine the mechanism of these dual cortisol effects on esophageal cell turnover.Main methodsWe developed a tissue culture method for the esophagus from euryhaline medaka (Oryzias latipes) and assessed cell proliferation and apoptosis in vitro in response to cortisol and 11-deoxycorticosterone (DOC), a recently identified agonist of the teleostean mineralocorticoid receptor.Key findingsEpithelial apoptosis, a well-established glucocorticoid function, was stimulated by treatment of the esophagus culture with 10 nM cortisol for 8 days, but no effects were seen at higher doses (100 and 1000 nM). In contrast, cell proliferation was induced by 1000 nM cortisol treatment for 8 days and this response was dose-dependent. Both effects were blocked by RU-486, a glucocorticoid receptor antagonist. DOC showed no significant effects at 10–1000 nM.SignificanceIn the esophageal epithelium in euryhaline fish, cortisol induces either apoptosis or cell proliferation via the glucocorticoid receptor, depending on the cortisol concentration. The glucocorticoid signaling may play a more important role than mineralocorticoid signaling in differentiation of the osmoregulatory esophagus in euryhaline fishes.     

The regulation of the UCH-L1 gene by transcription factor NF-κB in podocytes

In kidney, the ubiquitin carboxy-terminal hydrolase 1 (UCH-L1) is involved in podocyte injury and proteinuria but details of the mechanism underlying its regulation are not known. Activation of NF-κB is thought to be the predominant risk factor for kidney disease; therefore, it is postulated that UCH-L1 may be one of the NF-κB target genes. In this study, we investigated the involvement of NF-κB activation in the regulation of UCH-L1 expression and the function of murine podocytes. Stimulation of podocytes with the cytokines TNF-α and IL-1β up-regulated UCH-L1 expression rapidly at the mRNA and protein levels and the NF-κB-specific inhibitor pyrrolidine dithiocarbamate resulted in down-regulation. NF-κB up-regulates UCH-L1 via binding the ? 300 bp and ? 109 bp sites of its promoter, which was confirmed by the electrophoretic mobility shift assay of DNA–nuclear protein binding. In the renal biopsy from lupus nephritis patients, the expressions of NF-κB and UCH-L1 increased in immunohistochestry staining and were positively correlated. Activation of NF-κB up-regulates UCH-L1 expression following changing of other podocytes molecules, such as nephrin and snail. These results suggest that activation of the NF-κB signaling pathway could be the major pathogenesis to up-regulate UCH-L1 in podocyte injury, followed by the turnover of other molecules, which might result in morphological changes and dysfunction of podocytes. This work help us to understand the effect of NF-κB on specific target molecules of podocytes, and suggest that targeting the NF-κB–UCH-L1 interaction could be a novel therapeutic strategy for the treatment of podocyte lesions and proteinuria.     

Activation of cardiac hypertrophic signaling pathways in a transgenic mouse with the human PRKAG2 Thr400Asn mutation

Human mutations in PRKAG2, the gene encoding the γ2 subunit of AMP activated protein kinase (AMPK), cause a glycogen storage cardiomyopathy. In a transgenic mouse with cardiac specific expression of the Thr400Asn mutation in PRKAG2 (TG^T400N), we previously reported initial cardiac hypertrophy (ages 2–8 weeks) followed by dilation and failure (ages 12–20 weeks). We sought to elucidate the molecular mechanisms of cardiac hypertrophy. TG^T400N mice showed significantly increased cardiac mass/body mass ratios up to ～ 3-fold beginning at age 2 weeks. Cardiac expression of ANP and BNP were ～ 2- and ～ 5-fold higher, respectively, in TG^T400N relative to wildtype (WT) mice at age 2 weeks. NF-κB activity and nuclear translocation of the p50 subunit were increased ～ 2- to 3-fold in TG^T400N hearts relative to WT during the hypertrophic phase. Phosphorylated Akt and p70S6K were elevated ～ 2-fold as early as age 2 weeks. To ascertain whether these changes in TG^T400N mice were a consequence of increased AMPK activity, we crossbred TG^T400N with TG^α2DN mice, which express a dominant negative, kinase dead mutant of the AMPK α2 catalytic subunit and have low myocardial AMPK activity. Genetic reversal of AMPK overactivity led to a reduction in hypertrophy, nuclear translocation of NF-κB, phosphorylated Akt, and p70S6K. We conclude that inappropriate activation of AMPK secondary to the T400N PRKAG2 mutation is associated with the early activation of NF-κB and Akt signaling pathway, which mediates cardiac hypertrophy.     

Chronic inhibition of nuclear factor kappa B attenuates aldosterone/salt-induced renal injury

AimsRecent studies suggested that nuclear factor kappa B (NF-κB) plays a key role in the pathogenesis of renal injury. This study investigated whether NF-κB inhibition attenuates progressive renal damage in aldosterone/salt-induced renal injury and its mechanisms.Main methodsAdult male rats were uninephrectomized and treated with one of the following for 4 weeks: vehicle (0.5% ethanol, subcutaneously); vehicle/1% NaCl (1% NaCl in drinking solution); aldosterone/1% NaCl (1% NaCl in drinking solution and aldosterone, 0.75 μg/h, subcutaneously); or aldosterone/1%NaCl + pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-κB (100 mg/kg/day, by gavage). The activity of NF-κB was measured by EMSA and immunohistochemistry, CTGF and ICAM-1 were measured by Western blot and real-time PCR, and TGF-β and CTGF were measured by immunohistochemistry.Key findingsRats that received aldosterone/1% NaCl exhibited hypertension and severe renal injury. Renal cortical mRNA levels of CTGF, TGF-β, ICAM-1 and collagen IV, protein expression of CTGF and ICAM-1, and NF-κB–DNA binding activity were significantly upregulated in rats that received aldosterone/1% NaCl. Treatment with PDTC significantly decreased the percentage of cells positive for CTGF and TGF-β; mRNA levels of CTGF, TGF-β, ICAM-1 and collagen IV, and protein levels of CTGF and ICAM-1 were also inhibited by PDTC.SignificanceThese data suggest that the NF-κB signal pathway plays a role in the progression of aldosterone/salt-induced renal injury.     

Tetracyclines downregulate the production of LPS-induced cytokines and chemokines in THP-1 cells via ERK,p38, and nuclear factor-κB signaling pathways

Recent reports have shown that antibiotics such as macrolide, aminoglycoside, and tetracyclines have immunomodulatory effects in addition to essential antibiotic effects. These agents may have important effects on the regulation of cytokine and chemokine production. However, the precise mechanism is unknown. This time, we used Multi Plex to measure the production of cytokines and chemokines following tetracycline treatment of lipopolysaccharide (LPS)-induced THP-1 cells. The signaling pathways were investigated with Western blotting analysis. Three tetracyclines significantly suppressed the expression of cytokines and chemokines induced by LPS. Minocycline (50 μg/ml), tigecycline (50 μg/ml), or doxycycline (50 μg/ml) were added after treatment with LPS (10 μg/ml). Tumor necrosis factor-α was downregulated to 16%, 14%, and 8%, respectively, after 60 min compared to treatment with LPS without agents. Interleukin-8 was downregulated to 43%, 32%, and 26% at 60 min. Macrophage inflammatory protein (MIP)-1α was downregulated to 23%, 33%, and 16% at 120 min. MIP-1β was downregulated to 21%, 11%, and 2% at 120 min. Concerning about signaling pathways, the mechanisms of the three tetracyclines might not be the same. Although the three tetracyclines showed some differences in the time course, tetracyclines modulated phosphorylation of the NF-κB pathway, p38 and ERK1/2/MAPK pathways, resulting in inhibition of cytokine and chemokine production. In addition, SB203580 (p38 inhibitor) and U0126 (ERK1/2 inhibitor) significantly suppressed the expression of TNF-α and IL-8 in LPS-stimulated THP-1 cells. And further, the NF-κB inhibitor, BAY11-7082, almost completely suppressed LPS-induced these two cytokines production. Thus, more than one signaling pathway may be involved in tetracyclines downregulation of the expression of LPS-induced cytokines and chemokines in THP-1 cells. And among the three signaling pathways, NF-κB pathway might be the dominant pathway on tetracyclines modification the LPS-induced cytokines production in THP-1 cells. In general, minocycline and doxycycline suppressed the production of cytokines and chemokines in LPS-stimulated THP-1 cell lines via mainly the inhibition of phosphorylation of NF-κB pathways. Tigecycline has the same structure as the other tetracyclines, however, it showed the different properties of cytokine modulation in the experimental time course.     

Inhibitory and mechanistic investigations of oxo-lipids with human lipoxygenase isozymes

Oxo-lipids, a large family of oxidized human lipoxygenase (hLOX) products, are of increasing interest to researchers due to their involvement in different inflammatory responses in the cell. Oxo-lipids are unique because they contain electrophilic sites that can potentially form covalent bonds through a Michael addition mechanism with nucleophilic residues in protein active sites and thus increase inhibitor potency. Due to the resemblance of oxo-lipids to LOX substrates, the inhibitor potency of 4 different oxo-lipids; 5-oxo-6,8,11,14-(E,Z,Z,Z)-eicosatetraenoic acid (5-oxo-ETE), 15-oxo-5,8,11,13-(Z,Z,Z,E)-eicosatetraenoic acid (15-oxo-ETE), 12-oxo-5,8,10,14-(Z,Z,E,Z)-eicosatetraenoic acid (12-oxo-ETE), and 13-oxo-9,11-(Z,E)-octadecadienoic acid (13-oxo-ODE) were determined against a library of LOX isozymes; leukocyte 5-lipoxygenase (h5-LOX), human reticulocyte 15-lipoxygenase-1 (h15-LOX-1), human platelet 12-lipoxygenase (h12-LOX), human epithelial 15-lipoxygenase-2 (h15-LOX-2), soybean 15-lipoxygenase-1 (s15-LOX-1), and rabbit reticulocyte 15-LOX (r15-LOX). 15-Oxo-ETE exhibited the highest potency against h12-LOX, with an IC₅₀ = 1 ± 0.1 μM and was highly selective. Steady state inhibition kinetic experiments determined 15-oxo-ETE to be a mixed inhibitor against h12-LOX, with a K_ic value of 0.087 ± 0.008 μM and a K_iu value of 2.10 ± 0.8 μM. Time-dependent studies demonstrated irreversible inhibition with 12-oxo-ETE and h15-LOX-1, however, the concentration of 12-oxo-ETE required (K_i = 36.8 ± 13.2 μM) and the time frame (k₂ = 0.0019 ± 0.00032 s⁻¹) were not biologically relevant. These data are the first observations that oxo-lipids can inhibit LOX isozymes and may be another mechanism in which LOX products regulate LOX activity.     

Lipoic acid effects on established atherosclerosis

AimsAlpha-lipoic acid (LA) is a commonly used dietary supplement that exerts anti-oxidant and anti-inflammatory effects in vivo and in vitro. We investigated the mechanisms by which LA may confer protection in models of established atherosclerosis.Main methodsWatanabe heritable hyperlipidemic (WHHL) rabbits were fed with high cholesterol chow for 6 weeks and then randomized to receive either high cholesterol diet alone or combined with LA (20 mg/kg/day) for 12 weeks. Vascular function was analyzed by myography. The effects of LA on T cell migration to chemokine gradients was assessed by Boyden chamber. NF-κB activation was determined by measuring translocation and electrophoresis migration shift assay (EMSA).Key findingsLA decreased body weight by 15 ± 5% without alterations in lipid parameters. Magnetic Resonance Imaging (MRI) analysis demonstrated that LA reduced atherosclerotic plaques in the abdominal aorta, with morphological analysis revealing reduced lipid and inflammatory cell content. Consistent with its effect on atherosclerosis, LA improved vascular reactivity (decreased constriction to angiotensin II and increased relaxation to acetylcholine and insulin), inhibited NF-κB activation, and decreased oxidative stress and expression of key adhesion molecules in the vasculature. LA reduced T cell content in atherosclerotic plaque in conjunction with decreasing ICAM and CD62L (l-selectin) expression. These effects were confirmed by demonstration of a direct effect of LA in reducing T cell migration in response to CCL5 and SDF-1 and decreasing T cell adhesion to the endothelium by intra-vital microscopy.SignificanceThe present findings offer a mechanistic insight into the therapeutic effects of LA on atherosclerosis.     

Genistein attenuates D-galactose-induced oxidative damage through decreased reactive oxygen species and NF-κB binding activity in neuronal PC12 cells

AimsWe investigated the mechanism of D-galactose (DG)-induced oxidative damage and the neuroprotective action of genistein in PC12 cells.Main methodsPC12 cells were treated with 40 mM DG dissolved in medium containing 85% RPMI1640, 10% HBS and 5% FBS with or without genistein. We measured the protein expression of β-amyloid (Aβ), advanced glycation end products (AGEs), IκB-α and manganese-superoxide dismutase (MnSOD) by western blotting, intracellular reactive oxygen species (ROS) by 2, 7-dichlorofluorescin-diacetate, and the binding activity of nuclear factor kappa B (NF-κB) by electrophortic mobility shift assay.Key findingsDG (40 mM) completely retarded cell growth after incubation for 72 h, and this effect was not due to osmotic changes, as 40 mM mannitol had no effect. Mechanistically, we found that DG increased intracellular ROS starting at 4 h and increased Aβ and AGEs at 24 h. DG treatment for 24 h also increased the binding activity of NF-κB but strongly decreased the expression of IκB-α protein. Furthermore, DG treatment for 48 h increased MnSOD protein expression. All these effects of DG were effectively inhibited by genistein (0.5–10 μM).SignificanceThe present study indicates that the protection of genistein against DG-induced oxidative stress in PC12 cells, and the effect is likely mediated by decreased intracellular ROS and binding activity of NF-κB.     

Baicalein inhibits nuclear factor-κB and apoptosis via c-FLIP and MAPK in d-GalN/LPS induced acute liver failure in murine models

The hepatoprotective effects and molecular mechanisms of baicalein on acute liver failure induced by d-galactosamine (d-GalN)/lipopolysaccharides (LPS) were investigated in vivo. Mice were administered with different doses of baicalein (50, 100 or 150 mg/kg, p.o.) 1 h before injection of d-GalN (700 mg/kg)/LPS (10 μg/kg) and then sacrificed 6 h after treatment with d-GalN/LPS. Pretreatment with baicalein prevented d-GalN/LPS-induced liver damage by preventing associated increases of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) and by reducing serum tumor necrosis factor α (TNF-α), nitric oxide (NO) or inducible nitric oxide synthase (iNOS) expressions. The molecular mechanisms involved in baicalein-induced inhibition of d-GalN/LPS-caused apoptosis were associated with the protection of mitochondria, increasing the Bcl-2/Bax ratio, blocking the release of cytochrome c, and suppressing the phosphorylation of IκBα, ERK and JNK. Moreover, baicalein activated c-FLIP_L, XIAP and cIAP2 proteins, potentially blocking the recruitment of NF-κB signaling molecules. The results support the investigation of baicalein as a therapeutic candidate for acute liver apoptosis or injury and indicate that baicalein might inhibit liver apoptosis by mediating one or more of these pathways.     

Synthesis and cytotoxic potential of heterocyclic cyclohexanone analogues of curcumin

A series of 18 heterocyclic cyclohexanone analogues of curcumin have been synthesised and screened for their activity in both adherent and non-adherent cancer cell models. Cytotoxicity towards MBA-MB-231 breast cancer cells, as well as ability to inhibit NF-κB transactivation in non-adherent K562 leukemia cells were investigated. Three of these analogues 3,5-bis(pyridine-4-yl)-1-methylpiperidin-4-one B1, 3,5-bis(3,4,5-trimethoxybenzylidene)-1-methylpiperidin-4-one B10, and 8-methyl-2,4-bis((pyridine-4-yl)methylene)-8-aza-bicyclo[3.2.1]octan-3-one C1 showed potent cytotoxicity towards MBA-MB-231, MDA-MB-468, and SkBr3 cell lines with EC₅₀ values below 1 μM and inhibition of NF-κB activation below 7.5 μM. The lead drug candidate, B10, was also able to cause 43% of MDA-MB-231 cells to undergo apoptosis after 18 h. This level of activity warrants further investigation for the treatment of ER-negative breast cancer and/or chronic myelogenous leukemia as prototypical cellular models for solid and liquid tumors.     

Effects of carbon monoxide releasing molecule-liberated CO on severe acute pancreatitis in rats

Recent studies have suggested that exogenously administered carbon monoxide (CO) is beneficial for resolution of acute inflammation. Severe acute pancreatitis (SAP) is an inflammatory condition which leads to a systemic inflammatory response syndrome (SIRS). In this study, we investigated the role of CO liberated from carbon monoxide releasing molecule-2 (CORM-2) in rats with SAP. SAP was induced by retrograde infusion of 5% sodium taurocholate into the pancreatobiliary duct. Forty Wistar rats were randomly divided into four groups. Sham group was given normal saline after the sham operation. SAP group was treated with normal saline after the induction of SAP. CORM-2 group was injected with CORM-2 (8 mg/kg, i.v.) after the onset of SAP. iCORM-2 group was given iCORM-2 (an inactive compound used as negative control) after SAP induction. All animals were sacrificed at 12 h after the operation. Eighty rats (n = 20 for each group) were monitored for 7 days to observe their survival rates. In another set of experiments, the former three groups received the same treatment as mentioned above. The last group was given ZnPPIX (HO-1 inhibitor) by peritoneal injection at 1 h before the administration of CORM-2 (n = 10 for each group). Serum levels of amylase, tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), and interleukin 10 (IL-10) as well as myeloperoxidase (MPO) activity in pancreatic tissue were determined. Histological score, mRNA expression of these cytokines, heme oxygenase-1 (HO-1) expression, HO activity, and nuclear factor κB (NF-κB)-binding activity in the pancreas were also evaluated. Our results showed that compared with SAP group, CORM-2 treatment significantly reduced the serum levels of amylase, TNF-α, and IL-1β, suppressed pancreatic tissue mRNA expression of TNF-α and IL-1β, and decreased MPO activity in the pancreas. In contrast with the pro-inflammatory cytokines, the serum level and pancreatic tissue mRNA expression of IL-10 were markedly increased by the injection of CORM-2. The severity of pancreatic histology and survival rate were also significantly improved by the administration of CORM-2. Treatment with CORM-2 was associated with an increase in HO-1 expression at 12 h after SAP induction. Pretreatment with ZnPPIX had no effect on the production and mRNA expression of these cytokines at 12 h after the development of SAP with the treatment of CORM-2 as compared to CORM-2 group. Furthermore, CORM-2 treatment inhibited the activation of NF-κB in the pancreas. These results indicate that CORM-2-liberated CO exerts protective effects on SAP in rats, and the beneficial effects may be due to the suppression of NF-κB activation and subsequent regulation of NF-κB-dependent expression of cytokines.