Surgical stress during operation for gastrointestinal cancer increases plasma thioredoxin levels and decreases mitochondrial membrane potential in peripheral blood lymphocytes

Surgical stress is difficult to evaluate quantitatively. It has been reported that mitochondrial membrane potential (delta psi(m)) in the peripheral blood lymphocytes (PBLs) is decreased by surgical stress. Thioredoxin (TRX), a small protein with redox-active dithiol/disulfide in the active site, is induced by a variety of oxidative stresses and secreted from the cells. Accumulating evidence shows that plasma levels of TRX are elevated in oxidative stress-associated disorders. In the present study, we examined plasma levels of TRX in cases undergoing operations for gastrointestinal cancer. Plasma levels of TRX were significantly elevated on the first postoperative day compared with the pre-operative levels. The changes in the plasma TRX levels tended to show an inverse relationship with the changes in delta psi(m) in PBLs, which shows a significant decrease caused by surgical stress. Plasma TRX levels as well as delta psi(m) in PBLs are valuable markers to evaluate surgical stress.     

Thioredoxin-1 attenuates indomethacin-induced gastric mucosal injury in mice

Indomethacin is one of non-steroidal anti-inflammatory drugs that are commonly used clinically and often cause gastric mucosal injury as a side effect. Generation of reactive oxygen species (ROS) and activation of apoptotic signaling are involved in the pathogenesis of indomethacin-induced gastric mucosal injury. Thioredoxin-1 (Trx-1) is a small redox-active protein with anti-oxidative activity and redox-regulating functions. The aim of this study was to investigate the protective effect of Trx-1 against indomethacin-induced gastric mucosal injury. Trx-1 transgenic mice displayed less gastric mucosal damage than wild type (WT) C57BL/6 mice after intraperitoneal administration of indomethacin. Administration of recombinant human Trx-1 (rhTrx-1) or transfection of the Trx-1 gene reduced indomethacin-induced cytotoxicity in rat gastric epithelial RGM-1 cells. Pretreatment with rhTrx-1 suppressed indomethacininduced ROS production and downregulation of phosphorylated Akt in RGM-1 cells. Survivin, a member of inhibitors of apoptosis proteins family, was downregulated by indomethacin, which was suppressed in Trx-1 transgenic mice or by administration of rhTrx-1 in RGM-1 cells. Trx-1 inhibits indomethacin-induced apoptotic signaling and gastric ulcer formation, suggesting that it may have a preventive and therapeutic potential against indomethacin-induced gastric injury.     

Surgical stress during operation for gastrointestinal cancer increases plasma thioredoxin levels and decreases mitochondrial membrane potential in peripheral blood lymphocytes

Abstract

Surgical stress is difficult to evaluate quantitatively. It has been reported that mitochondrial membrane potential (Δψ_m) in the peripheral blood lymphocytes (PBLs) is decreased by surgical stress. Thioredoxin (TRX), a small protein with redox-active dithiol/disulfide in the active site, is induced by a variety of oxidative stresses and secreted from the cells. Accumulating evidence shows that plasma levels of TRX are elevated in oxidative stress-associated disorders. In the present study, we examined plasma levels of TRX in cases undergoing operations for gastrointestinal cancer. Plasma levels of TRX were significantly elevated on the first postoperative day compared with the pre-operative levels. The changes in the plasma TRX levels tended to show an inverse relationship with the changes in Δψ_m in PBLs, which shows a significant decrease caused by surgical stress. Plasma TRX levels as well as Δψ_m in PBLs are valuable markers to evaluate surgical stress.     

Mechanism of the antiulcerogenic effect of Ganoderma lucidum polysaccharides on indomethacin-induced lesions in the rat

Many cytokines, in particular tumor necrosis factor (TNF)-alpha have been known to play an important role in the pathogenesis of gastric mucosal lesions caused by various factors such as drugs and Helicobacter pylori infection. Our previous studies have shown that the polysaccharide fractions isolated from the fruiting bodies of Ganoderma lucidum (GLPS) prevented indomethacin- and acetic acid-induced gastric mucosal lesions in the rat. However, the mechanisms remain unclear. This study aimed to investigate whether GLPS had a direct mucosal healing effect in the indomethacin-treated rat, and to explore the possible mechanisms by determining the gastric mucosal mRNA and protein levels of TNF-alpha and ornithine decarboxylase (ODC) activity. In addition, the effects of GLPS on the cellular proliferation, ODC and c-Myc protein expression and mucus synthesis in the rat gastric cell culture (RGM-1) were examined. The present study demonstrated that GLPS at 250 and 500 mg/kg by intragastric input caused ulcer-healing effect in the rat; this was accompanied with a significant suppression of TNF-alpha gene expression, but with an increased ODC activity. In RGM-1 cells, GLPS at 0.05, 0.25 and 1.0 mg/ml significantly enhanced [3H]thymidine incorporation and ODC activity in a concentration-dependent manner. However, these effects were abrogated by the addition of the ODC inhibitor, DL-alpha-difluoromethyl-ornithine (DFMO). GLPS at 0.25-1.0 mg/ml also increased mucus synthesis, as indicated by the increased D-[6-3H]glucosamine incorporation in RGM-1 cells. Furthermore, GLPS at 0.05-1.0 mg/ml increased the c-Myc protein expression. These findings indicated that GLPS produced a mucosal healing effect in the rat model, perhaps due partly to the suppression of TNF-alpha and induction of c-myc and ODC gene.     

Catecholamines Inhibit Gastric Epithelial [RGM-1] Cell Proliferation via Beta Adrenoceptors

Catecholamines have been implicated in the modulation of normal cell growth, exerting inhibitory or excitatory control depending on the cell type. However, there is a dearth of information on the role of adrenergic mediators in gastric cell proliferation. In the present study, the effects of adrenaline (ADR) and noradrenaline (NOR) on mucosal cell growth and the cell cycle were evaluated in vitro using a normal rat gastric mucosal cell line RGM-1. Cell proliferation was assessed using [3H]-thymidine incorporation and cell cycle patterns were determined by DNA labeling with propidium iodide and flow cytometric quantification. The expressions of adrenoceptors in RGM-1 were determined by Western blot. ADR (0.01 - 10μM) and NOR (0.01 - 10μM) inhibited the growth of RGM-1 cells in a concentration-dependent manner. Pre-treatment of cells with ADR and NOR also inhibited the proliferation stimulated by epidermal growth factor (EGF). Neither phentolamine (non-selective α-adrenergic blocker), methoxamine (α1-selective agonist) nor clonidine (α2-selective agonist) significantly affected the inhibition of cell proliferation produced by ADR and NOR. Propranolol (non-selective β-adrenergic blocker) and butoxamine (selective β2-adrenergic blocker) significantly (but not totally) reversed the inhibitory action of ADR on cell proliferation. Furthermore, procaterol (selective beta-2 agonist) but not dobutamine (selective beta-1 agonist) had effects similar to those produced by ADR and NOR. Exposure of RGM-1 cells to both ADR and NOR caused significant inhibition of the G1 - S cycle progression as evidenced by the higher percentage of the G0/G1 phase and a decreased S- phase. This effect was blocked by pre-treatment with propranolol but not phentolamine These results indicate that catecholamines inhibit the proliferation of RGM-1 cells probably partly through beta-2 receptors.     

Adenoviral transfection of hepatocytes with the thioredoxin gene confers protection against apoptosis and necrosis

A recombinant adenovirus vector containing the human thioredoxin (TRX) gene was constructed using the Cre-loxP recombination system and used to transfect rat hepatocytes with very high efficiency. The TRX gene was expressed in a dose-dependent manner and significantly modulated rat cellular functions. The TRX gene conferred resistance to oxidative stress, such as hydrogen peroxide treatment, on the host hepatocytes. FACS analysis of DNA fragmentation showed that the TRX gene suppressed hepatocyte apoptosis. It also significantly extended the life span of hepatocytes cultured conventionally on polystyrene plates. Liver-specific functions were maintained in the viability-modulated hepatocytes. Moreover, TRX expression did not affect hepatocyte spheroid formation and it extensively suppressed necrosis in the internal cells. Thus, the transfection of hepatocytes with the TRX gene successfully confers global maintenance of liver functions. These findings provide important information for the development of bioartificial liver support systems and gene therapy for liver diseases.     

Protective effects of oral administration of yeast thioredoxin against gastric mucosal injury

Thioredoxin (TRX) is a redox regulating protein which has protective effects against oxidative stress-induced damage to cells and tissues. In this study, we investigated the effects of orally administered TRX derived from edible yeast, Saccharomyces cerevisiae, on gastric mucosa. First, we examined the digestibility of orally administered yeast TRX in mice, and detected yeast TRX in the stomach for 4?h after administration. Next, we investigated the mitigation of gastric mucosal injury after the oral administration of yeast TRX in water-immersion restraint stress and HCl/ethanol-induced gastric ulcer models. Furthermore, we conducted DNA microarray analysis, using the HCl/ethanol-induced model, which revealed that several groups of genes related to tissue repair were upregulated in ulcer regions in the stomachs of rats administered with yeast TRX. These results demonstrated the viability of the use of oral administrations of yeast TRX to protect the gastric mucosa.     

Oxygen sensing and redox signaling: the role of thioredoxin in embryonic development and cardiac diseases

It is important to regulate the oxygen concentration and scavenge oxygen radicals throughout the life of animals. In mammalian embryos, proper oxygen concentration gradually increases in utero and excessive oxygen is rather toxic during early embryonic development. Reactive oxygen species (ROS) are generated as by-products in the respiratory system and increased under inflammatory conditions. In the pathogenesis of a variety of adult human diseases such as cancer and cardiovascular disorders, ROS cause an enhancement of tissue injuries. ROS promote not only the development of atherosclerosis but also tissue injury during the reperfusion process. The thioredoxin (TRX) system is one of the most important mechanisms for regulating the redox balance. TRX is a small redox active protein distributed ubiquitously in various mammalian tissues and cells. TRX acts as not only an antioxidant but also an anti-inflammatory and an antiapoptotic protein. TRX is induced by oxidative stress and released from cells in response to oxidative stress. In various human diseases, the serum/plasma level of TRX is a well-recognized biomarker of oxidative stress. Here we discuss the roles of TRX on oxygen stress and redox regulation from different perspectives, in embryogenesis and in adult diseases focusing on cardiac disorders.     

Tumor necrosis factor-alpha stimulates gastric epithelial cell proliferation

TNF-alpha is a cytokine produced during gastric mucosal injury. We examined whether TNF-alpha could promote mucosal repair by stimulation of epithelial cell proliferation and explored further the underlying mechanisms in a rat gastric mucosal epithelial cell line (RGM-1). TNF-alpha treatment (1-10 ng/ml) for 12 or 24 h significantly increased cell proliferation but did not induce apoptosis in RGM-1 cells. TNF-alpha treatment significantly increased cytosolic phospholipase A(2) and cyclooxygenase-2 (COX-2) protein expression and PGE(2) level but did not affect the protein levels of EGF, basic fibroblast growth factor, and COX-1 in RGM-1 cells. The mRNA of TNF receptor (TNF-R) 2 but not of TNF-R1 was also increased. Dexamethasone dose dependently inhibited the stimulatory effect of TNF-alpha on cell proliferation, which was associated with a significant decrease in cellular COX-2 expression and PGE(2) level. A selective COX-2 inhibitor 3-(3-fluorophenyl)-4-[4-(methylsulfonyl)phenyl]-5,5-dimethyl-(5)H-furan-2-one (DFU) by itself had no effect on basal cell proliferation but significantly reduced the stimulatory effect of TNF-alpha on RMG-1 cells. Combination of dexamethasone and DFU did not produce an additive effect. PGE(2) significantly reversed the depressive action of dexamethasone on cell proliferation. These results suggest that TNF-alpha plays a regulatory role in epithelial cell repair in the gastric mucosa via the TNF-alpha receptor and activation of the arachidonic acid/PG pathway.     

Sofalcone, a gastric mucosa protective agent, increases vascular endothelial growth factor via the Nrf2-heme-oxygenase-1 dependent pathway in gastric epithelial cells

Sofalcone, 2′-carboxymethoxy-4,4-bis(3-methyl-2-butenyloxy)chalcone, is an anti-ulcer agent that is classified as a gastric mucosa protective agent. Recent studies indicate heat shock proteins such as HSP32, also known as heme-oxygenase-1(HO-1), play important roles in protecting gastrointestinal tissues from several stresses. We have previously reported that sofalcone increases the expression of HO-1 in adipocytes and pre-adipocytes, although the effect of sofalcone on HO-1 induction in gastrointestinal tissues is not clear. In the current study, we investigated the effects of sofalcone on the expression of HO-1 and its functional role in rat gastric epithelial (RGM-1) cells. We found that sofalcone increased HO-1 expression in RGM-1 cells in both time- and concentration-dependent manners. The HO-1 induction was associated with the nuclear translocation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) in RGM-1 cells. We also observed that sofalcone increased vascular endothelial growth factor (VEGF) production in the culture medium. Treatment of RGM-1 cells with an HO-1 inhibitor (tin-protoporphyrin), or HO-1 siRNA inhibited sofalcone-induced VEGF production, suggesting that the effect of sofalcone on VEGF expression is mediated by the HO-1 pathway. These results suggest that the gastroprotective effects of sofalcone are partly exerted via Nrf2-HO-1 activation followed by VEGF production.     

Inhibition of lysine-specific demethylase 1 by the acyclic diterpenoid geranylgeranoic acid and its derivatives

Lysine-specific demethylase 1 (LSD1) is upregulated in many cancers, especially neuroblastoma. We set out to explore whether geranylgeranoic acid (GGA) inhibits LSD1 activity by using recombinant human LSD1. GGA inhibited LSD1 activity with IC₅₀ similar to that of the clinically used drug tranylcypromine. In human neuroblastoma SH-SY5Y cells, GGA induced NTRK2 gene expression alongside upregulation of histone H3 with dimethylated lysine-4 in the regulatory regions of the NTRK2 gene. Dihydrogenation of GGA reinforced the LSD1-inhibitory effect in a position-dependent manner. The inhibitory effects of dihydro-derivatives of GGA on recombinant LSD1 strongly correlated with the induction of NTRK2 gene expression in SH-SY5Y cells. These data demonstrate for the first time the efficient LSD1-inhibitor activity of GGA and its derivatives, providing a novel prospect of preventing cancer onset by using GGA to regulate epigenetic modification.     

Thioredoxin inhibits NMDA-induced neurotoxicity in the rat retina

Thioredoxin (TRX) plays a variety of redox-related roles in organisms. To investigate its function as an endogenous redox regulator in NMDA-induced retinal neurotoxicity, we injected NMDA with TRX, mutant TRX or saline into the vitreous cavity of rat eyes. Retinal ganglion cells were rescued by TRX, compared with saline, when evaluated by retrograde labeling analysis at 7 days after NMDA injection. TRX, but not its mutant form, prevented NMDA-induced apoptosis in the retina, as measured by terminal deoxynucleotidyl transferase-mediated UTP nick-end labeling. The induction of caspase 3 and 9, but not caspase 8, by NMDA was significantly lower in TRX-treated eyes than in saline-treated eyes. NMDA-induced activation of the MAPKs, p38 kinase and c-Jun N-terminal kinase after 6 h and of the MAPK kinases (MKKs) MKK3/6 and MKK4 after 3 h was markedly suppressed in retinal ganglion cells by TRX but not by the mutant form. NMDA-induced increases in protein carbonylation, nitrosylation and lipid peroxidation were also suppressed in TRX-treated eyes. We concluded that the intravitreous injection of TRX effectively attenuated NMDA-induced retinal cell damage and that suppression of oxidative stress and inhibition of apoptotic signaling pathways were involved in this neuroprotection.     

Glucocorticoid receptors in human peripheral blood mononuclear cells in relation to explosive performance in elite handball players

A crude extract from Angelica sinensis (ASCE), which mainly consists of polysaccharides, prevents ethanol- or indomethacin-induced gastric mucosal damage and promotes ulcer healing. The aim of this study was to test the hypothesis that ASCE has a direct stimulating effect on gastric epithelial cells for wound healing. We found that ASCE significantly promoted the migration of epithelial cells over an artificial wound on the surface of an RGM-1 monolayer. The extract also stimulated DNA synthesis in a dose-dependent manner and concomitantly increased EGF mRNA expression. Co-incubation of ASCE with anti-EGF antibody reduced the speed of migration and the DNA synthesis, which however were still higher than the control without ASCE. These results strongly suggest that ASCE has a direct wound healing effect on gastric mucosa, and this is acting partially through an EGF-mediated pathway.     

Effect of fish oil on offensive and defensive factors in gastric ulceration in rats

The effect of fish oil (FO) derived from Scomberoides commersonianus containing omega-3 polyunsaturated fatty acids was studied on gastric ulcers and as well as on offensive and defensive factors in gastric mucosal damage, following experimental gastric ulceration. FO significantly reduced the severity of ulceration in gastric ulcers induced by aspirin, cold-restraint stress (CRS), alcohol, and pylorus ligation. The results also indicated the potentiality of FO in maintaining the integrity of gastric mucosa by virtue of its effect on both offensive and defensive gastric mucosal factors. It decreased the offensive acid-pepsin secretion and augmented the defensive factors like mucin secretion, cellular mucus and life span of mucosal cells following pylorus ligation. FO significantly increased activity of anti-oxidant enzymes (catalase and glutathione peroxidase) and decreased lipid peroxidation in gastric mucosa of CRS rats. The study indicates the beneficial role of FO in gastric ulceration by inhibition of offensive mucosal factors and oxidative stress, and augmentation of defensive mucosal factors.     

Over-expression of 70-kDa heat shock protein confers protection against monochloramine-induced gastric mucosal cell injury

The major heat shock protein, HSP70, is known to be involved in cytoprotection against environmental stresses mediated by their function as a "molecular chaperone". Monochloramine (NH(2)Cl) is a potent cytotoxic oxidant generated by neutrophil-derived hypochlorous acid and Helicobacter pylori urease-induced ammonia. In this study, to evaluate the cytoprotective effect of HSP70 against NH(2)Cl-induced gastric mucosal cell injury, rat gastric mucosal cells (RGM-1) were stably transfected with pBK-CMV containing the human HSP70 gene (7018-RGM-1) or pBK-CMV alone (pBK-CMV-12) as control cells. These cells were treated with various concentrations of NH(2)Cl. Cell Viability was determined by MTT assay and the direct plasma membrane damage was analyzed by lactate dehydrogenase (LDH) release assay. Apoptosis was determined by DNA fragmentation analysis. NH(2)Cl caused injury to pBK-CMV-12 cells in a concentration-dependent manner. NH(2)Cl-induced gastric cell injury was significantly diminished in HSP70 over-expressing cell line (7018-RGM-1) both necrosis and apoptosis compared to the control cell line (pBK-CMV-12) transfected with CMV vector alone. These result suggest that overexpression of HSP70 plays an important role in protecting gastric cells against NH(2)Cl-induced injury.     

Nonsteroidal anti-inflammatory drug induces proinflammatory damage in gastric mucosa through NF-κB activation and neutrophil infiltration: Anti-inflammatory role of heme oxygenase-1 against nonsteroidal anti-inflammatory drug

Nonsteroidal anti-inflammatory drug (NSAID)-induced mitochondrial oxidative stress (MOS) is an important prostaglandin (PG)-independent pathway of the induction of gastric mucosal injury. However, the molecular mechanism behind MOS-mediated gastric pathology is still obscure. In various pathological conditions of tissue injury oxidative stress is often linked with inflammation. Here we report that MOS induced by indomethacin (an NSAID) induces gastric mucosal inflammation leading to proinflammatory damage. Indomethacin, time dependently stimulated the expression of proinflammatory molecules such as intercellular adhesion molecule 1(ICAM-1), vascular cell adhesion molecule 1(VCAM-1), interleukin1β (IL-1β), and monocyte chemotactic protein-1 (MCP-1) in gastric mucosa in parallel with the increase of neutrophil infiltration and injury of gastric mucosa in rat. Western immunoblotting and confocal microscopic studies revealed that indomethacin induced nuclear translocation of nuclear factor kappa-B (NF-κB) in gastric mucosal cells, which resulted in proinflammatory signaling. The prevention of MOS by antioxidant tryptamine-gallic acid hybrid (SEGA) inhibited indomethacin-induced expression of ICAM-1, VCAM-1, IL-1β, and MCP-1. SEGA also prevented indomethacin-induced NF-κB activation and neutrophil infiltration as documented by chromatin immunoprecipitation studies and neutrophil migration assay, respectively. Heme oxygenase-1 (HO-1), a cytoprotective enzyme associated with tissue repair mechanisms is stimulated in response to oxidative stress. We have investigated the role of HO-1 against MOS and MOS-mediated inflammation in recovering from gastropathy. Indomethacin stimulated the expression of HO-1 and indomethacin-stimulated HO-1 expression was reduced by SEGA, an antioxidant, which could prevent MOS. Thus, the data suggested that the induction of HO-1 was a protective response against MOS developed by indomethacin. Moreover, the induction of HO-1 by cobalt protoporphyrin inhibited inflammation and chemical silencing of HO-1 by zinc protoporphyrin aggravated the inflammation by indomethacin. Thus, NSAID by promoting MOS-induced proinflammatory response damaged gastric mucosa and HO-1 protected NSAID-induced gastric mucosal damage by preventing NF-κB activation and proinflammatory activity.