Epidermal growth factor and prostaglandin E(2) accelerate mucosal recovery from stress-induced gastric lesions via inhibition of apoptosis.

The repair of damaged gastric mucosa is a complex process involving prostaglandins (PG) and mucosal growth factors such as epidermal growth factor (EGF). Recently, we postulated that the increased occurrence of apoptosis in the gastric epithelium might be of pathophysiological importance in the development of stress lesions. The aim of the present study was to assess the effect of the pretreatment of rats, exposed to 3.5 h of water immersion and restraint stress (WRS), with EGF and PG (16,16 dmPGE(2)) on the number of stress lesions, recovery of gastric mucosa from stress and the expression of apoptosis related genes such as caspase-3 and antiapoptotic bcl-2. Rats were divided in following groups: (1) vehicle; (2) EGF 100 microg/kg i.p.; (3) 16,16 dm-PGE(2) (5 microg/kg i.g.) and caspase-1 inhibitor (ICE-I; 100 microg/kg i.p.). One hour later, the rats were exposed to 3.5 h of WRS and then sacrificed immediately (0 h) or at 6, 12, or 24 h after WRS. The number of acute gastric lesions was determined. Gastric epithelial apoptosis was assessed by TUNEL staining. In addition, mRNA expression of caspase-3, Bcl-2 and proinflammatory cytokines (IL-1 beta, TNFalpha) was assessed by RT-PCR. PGE(2) generation in gastric mucosa and luminal EGF were determined by RIA. Exposure to WRS resulted in the development of multiple acute stress erosions ( approximately 18) which almost completely healed during 24 h. The gastric blood flow was significantly reduced (approximately 70% of intact mucosa) immediately after WRS. The expression of mRNA for IL-1 beta and TNF alpha reached their peak at 12 h after stress exposure. The apoptosis rate was highest at 6 h after WRS and was accompanied by the highest caspase-3 expression. In rats pretreated with EGF or 16,16 dm-PGE(2), a significant decrease in caspase-3 mRNA and upregulation of bcl-2 mRNA as observed as compared to vehicle controls. Caspase-1 inhibitor significantly reduced the number of stress lesions. We conclude that EGF and PGE(2) accelerate healing of stress-induced lesions due to the attenuation of apoptosis via upregulation of bcl-2 in gastric mucosa. Inhibitors of apoptosis accelerate healing of stress lesions and may be potentially effective agents in the healing of damaged gastric mucosa.     

Apoptosis in gastric mucosa with stress-induced gastric ulcers.

The maintenance of gastric mucosal integrity depends upon the interplay between epithelial cell proliferation and apoptosis (programmed cell death). The Bcl-2 family of proteins plays a central role in the regulation of apoptotic cell death by suppressing the apoptosis while some others such as Bax proteins promote this process. Stress-induced gastric ulcerations are accompanied by the fall in gastric mucosal cell proliferation but little is known about the influence of the stress on the apoptosis in gastric mucosa. In the present study, the gastric epithelial apoptosis was determined by means of expression of Bax and Bcl-2 mRNA in the gastric mucosa following acute stress. Wistar rats were exposed to mild water immersion and restraint stress (WRS) for 3.5 h and then sacrificed at 0, 2, 4, 6, 12 and 24 h after the termination of WRS. At each time interval after WRS, the gastric blood flow (GBF) and the proliferating cell nuclear antigen (PCNA) labeling were determined. The apoptosis rate in the gastric mucosa was determined by the terminal deoxynucleotidyl transferase (TDT) mediated 2-deoxyuridine 5-triphosphate (dUTP)-biotin nick end-labeling (TUNEL) staining method and the expression of Bax and Bcl-2 mRNA was analyzed by RT-PCR and southern blot hybridization. WRS produced multiple erosions accompanied by the fall in GBF and PCNA index and by a dramatic enhancement in gastric epithelial apoptosis rate reaching maximum at 4 h after exposure to WRS. Following 6 and 12 h after the end of WRS the apoptotis declined but even 24 h after WRS it failed to reach the value recorded in intact gastric mucosa. The PCNA index was still significantly inhibited at 2 h after WRS but then showed significant rise at 6 and 12 h to reach at 24 h after WRS, the level similar to that measured in intact gastric mucosa. The expression of Bax mRNA was detected in intact gastric mucosa and gradually increased in first 4 h after WRS to decline at 24 h to the level not significantly different from that observed in the intact mucosa. In contrast, the expression of Bcl-2 mRNA was almost undetectable during first 4 h but showed strong signal at 6 and 12 h to decline to the control level 24 h after WRS. We conclude that: 1. Healing of WRS lesions involves an increase in GBF and mucosal cell proliferation and 2. The enhancement in gastric epithelial apoptosis accompanies the mucosal damage induced by stress and this appears to be triggered by the shift from the cell death effector Bax to the cell death repressor Bcl-2 protein.     

Expression of cyclooxygenase (COX)-1 and COX-2 in adaptive cytoprotection induced by mild stress.

Prostaglandins (PG) derived from COX-1 play an important role in the maintenance of mucosal integrity but the role of COX-2-derived products in mucosal defence mechanism has not been fully explained. Mild stress is known to prevent gastric mucosal lesions induced by severe stress via the phenomenon of adaptive cytoprotection but it remains unknown which COX is involved in this adaptation. In this study, the mucosal expression of COX-1 and COX-2 was examined and the inhibitors of these enzymes were used to determine the contribution of these enzymes in adaptive cytoprotection induced by mild stress. Male Wistar rats were exposed to mild water immersion and restraint stress (WRS) at various time intervals ranging from 5 min up to 2 h followed 1 h later by exposure to severe 3.5 h WRS with or without pretreatment with: 1) NS-398 (10 mg x kg(-1) i.g.), a selective COX-2 inhibitor; 2) resveratrol (5 mg x kg(-1) i.g.), a selective COX-1 inhibitor; 3) meloxicam (2 mg x kg(-1) i.g.), preferential COX-2 inhibitor; and 4) indomethacin (5 mg x kg(-1) i.p), non-selective inhibitor of COX. The number of WRS lesions was counted, gastric blood flow (GBF) was measured by H2-gas clearance technique, mucosal biopsy samples were taken for the assessment of PGE2 by radioimmunoassay, and the expression of COX-1 and COX-2 mRNA by RT-PCR. WRS for 3.5 h produced numerous gastric lesions, decreased GBF by 48% and inhibited formation of PGE2 by 68% as compared to intact mucosa. Exposure to mild WRS during 5-30 min by itself failed to affect mucosal integrity but significantly attenuated gastric lesions induced by exposure to severe 3.5 h stress; the maximal protective effect being achieved with mild WRS during 15 min. This protective effect was accompanied by the rise in GBF and the generation of PGE2 in the gastric mucosa. After extension of mild WRS from 15 min up to 1 or 2 h before more severe 3.5 h WRS, the loss of cytoprotective effect of mild WRS against severe stress accompanied by significant fall in the GBF were observed. Pretreatment with NS-398 (10 mg x kg(-1) i.g.) that failed to affect mucosal PGE2 generation, reduced significantly the protection and accompanying rise in GBF produced by mild WRS whereas resveratrol partly reduced the protection and the rise in GBF induced by mild WRS. Meloxicam or indomethacin significantly inhibited PGE2 generation and completely abolished the hyperemia and protection induced by mild WRS against more severe stress. The protective and hyperemic effects of mild WRS were completely restored by the addition of 16,16 dm PGE2 (5 microg x kg(-1) i.g.) to NS-398 or resveratrol, while the deleterious effects of meloxicam and indomethacin were significantly attenuated by the concomitant treatment with this PGE2 analogue. We conclude that PG derived from both, COX-1 and COX-2 appear to be involved in adaptive cytoprotection developed in response to mild stressors.     

Role of salivary glands and epidermal growth factor (EGF) in gastric secretion and mucosal integrity in rats exposed to stress

EGF, produced mainly by salivary glands, inhibits gastric acid secretion, stimulates the proliferation of gastric mucosal cells and protects the mucosa against various ulcerogens, but its role in the pathogenesis of stress ulcerations is unknown. In this study, rats with intact or resected salivary glands were exposed to water immersion and restraint stress (WRS) without and with pretreatment with exogenous EGF or dimethyl PGE2 (dmPGE2) at doses which were shown previously to protect the mucosa against topical irritants. During 1.5-12 h of WRS, the formation of gastric ulcerations increased progressively with the duration of stress reaching peak after 6 h of stress and being significantly higher in rats with removed salivary glands than in intact animals. Gastric acid secretion and DNA synthesis in oxyntic mucosa declined with the duration of WRS, but after sialoadenectomy a significant increase in gastric acid secretion and a further decline in DNA synthesis were observed after WRS. EGF contents in the gastric lumen and the gastric mucosa were several times higher in rats subjected to stress than in control unstressed animals, indicating that stress causes an extensive release of EGF. Both exogenous EGF (17 nmol/kg/h) and dmPGE2 (143 nmol/kg) prevented, in part, the formation of gastric lesions, while inhibiting gastric acid secretion both in rats with intact or resected salivary glands. We conclude that water immersion and restraint stress is accompanied by an excessive release of EGF, which appears to attenuate gastric secretion, enhances the DNA synthesis and may limit the formation of stress-induced gastric ulcerations.     

Endoplasmic reticulum stress response is involved in the pathogenesis of stress induced gastric lesions in rats

Stress gastric ulcer is a serious complication, but the mechanism involved is not fully clarified. It is well known that mucosal cell apoptosis plays a crucial role in the pathogenesis of gastric ulceration. Recent studies have shown that endoplasmic reticulum (ER) stress is an important pathway leading to cellular apoptosis. To investigate the role of ER stress in the pathogenesis of stress gastric ulcer, we studied the alteration in the expression of ER stress markers GRP78 (glucose-regulated protein 78) and caspase-12 (an ER stress-specific proapoptotic molecule) and their relations with gastric mucosal apoptosis during development of stress gastric lesions in the water-immersion and restraint stress (WRS) model in rats. Rats developed severe gastric lesions after 6 h of WRS. Typical apoptosis was observed at the edge cells of WRS induced gastric lesions. Western blot analysis showed that GRP78 and activated caspase-12 were over-expressed in the gastric tissues of WRS rats. Immunohistochemical analysis demonstrated that increased GRP78 and caspase-12 were distributed only under the lesions. In addition, dithiothreitol and tunicamycin (ER stress inducers), which increased the expression of GRP78 and activated caspase-12, caused gastric mucosal injury and mucosal cell apoptosis in vitro. These findings suggest that ER stress might be involved in the development of stress gastric ulcer through an apoptotic mechanism.     

Depletion of OLFM4 gene inhibits cell growth and increases sensitization to hydrogen peroxide and tumor necrosis factor-alpha induced-apoptosis in gastric cancer cells

Background

Human olfactomedin 4 (OLFM4) gene is a secreted glycoprotein more commonly known as the anti-apoptotic molecule GW112. OLFM4 is found to be frequently up-regulated in many types of human tumors including gastric cancer and it was believed to play significant role in the progression of gastric cancer. Although the function of OLFM4 has been indicated in many studies, recent evidence strongly suggests a cell or tissue type-dependent role of OLFM4 in cell growth and apoptosis. The aim of this study is to examine the role of gastric cancer-specific expression of OLFM4 in cell growth and apoptosis resistance.

Methods

OLFM4 expression was eliminated by RNA interference in SGC-7901 and MKN45 cells. Cell proliferation, anchorage-independent growth, cell cycle and apoptosis were characterized in vitro. Tumorigenicity was analyzed in vivo. The apoptosis and caspase-3 activation in response to hydrogen peroxide (H₂O₂) or tumor necrosis factor-alpha (TNF α) were assessed in the presence or absence of caspase inhibitor Z-VAD-fmk.

Results

The elimination of OLFM4 protein by RNA interference in SGC-7901 and MKN45 cells significantly inhibits tumorigenicity both in vitro and in vivo by induction of cell G1 arrest (all P < 0.01). OLFM4 knockdown did not trigger obvious cell apoptosis but increased H₂O₂ or TNF α-induced apoptosis and caspase-3 activity (all P < 0.01). Treatment of Z-VAD-fmk attenuated caspase-3 activity and significantly reversed the H₂O₂ or TNF α-induced apoptosis in OLFM4 knockdown cells (all P < 0.01).

Conclusion

Our study suggests that depletion of OLFM4 significantly inhibits tumorigenicity of the gastric cancer SGC-7901 and MKN45 cells. Blocking OLFM4 expression can sensitize gastric cancer cells to H₂O₂ or TNF α treatment by increasing caspase-3 dependent apoptosis. A combination strategy based on OLFM4 inhibition and anticancer drugs treatment may provide therapeutic potential in gastric cancer intervention.     

New satiety hormone nesfatin-1 protects gastric mucosa against stress-induced injury: Mechanistic roles of prostaglandins,nitric oxide,sensory nerves and vanilloid receptors

Nesfatin-1 belongs to a family of anorexigenic peptides, which are responsible for satiety and are identified in the neurons and endocrine cells within the gut. These peptides have been implicated in the control of food intake; however, very little is known concerning its contribution to gastric secretion and gastric mucosal integrity. In this study the effects of nesfatin-1 on gastric secretion and gastric lesions induced in rats by 3.5 h of water immersion and restraint stress (WRS) were determined. Exogenous nesfatin-1 (5–40 μg/kg i.p.) significantly decreased gastric acid secretion and attenuated gastric lesions induced by WRS, and this was accompanied by a significant rise in plasma NUCB2/nefatin-1 levels, the gastric mucosal blood flow (GBF), luminal NO concentration, generation of PGE₂ in the gastric mucosa, an overexpression of mRNA for NUBC2 and cNOS, as well as a suppression of iNOS and proinflammatory cytokine IL-1β and TNF-α mRNAs. Nesfatin-1-induced protection was attenuated by suppression of COX-1 and COX-2 activity, the inhibition of NOS with L-NNA, the deactivation of afferent nerves with neurotoxic doses of capsaicin, and the pretreatment with capsazepine to inhibit vanilloid VR1 receptors. This study shows for the first time that nesfatin-1 exerts a potent protective action in the stomach of rats exposed to WRS and these effects depend upon decrease in gastric secretion, hyperemia mediated by COX-PG and NOS-NO systems, the activation of vagal and sensory nerves and vanilloid receptors.     

Bioelectric,tissue, and molecular characteristics of the gastric mucosa at different times of ischemia

Gastrointestinal ischemia may be presented as a complication associated with late shock detection in patients in critical condition. Prolonged ischemia can cause mucosal integrity to lose its barrier function, triggering alterations that can induce organ dysfunction and lead to death. Electrical impedance spectroscopy has been proposed to identify early alteration in ischemia-induced gastric mucosa in this type of patients. This work analyzed changes in impedance parameters, and tissue and molecular alterations that allow us to identify the time of ischemia in which the gastric mucosa still maintains its barrier function. The animals were randomly distributed in four groups: Control, Ischemia 60, 90, and 120 min. Impedance parameters were measured and predictive values were determined to categorize the degree of injury using a receiver operating characteristic curve. Markers of inflammatory process and apoptosis (iNOS, TNFα, COX-2, and Caspase-3) were analyzed. The largest increase in impedance parameters occurred in the ischemia 90 and 120 min groups, with resistance at low frequencies (R_L) and reactance at high frequencies (X_H) being the most related to damage, allowing prediction of the occurrence of reversible and irreversible tissue damage. Histological analysis and apoptosis assay showed progressive mucosal deterioration with irreversible damage (p < 0.001) starting from 90 min of ischemia. Furthermore, a significant increase in the expression of iNOS, TNFα, and COX-2 was identified in addition to apoptosis in the gastric mucosa starting from 90 min of ischemia. Tissue damage generated by an ischemia time greater than 60 min induces loss of barrier function in the gastric mucosa.     

Gastroprotective Effects of Astragaloside IV against Acute Gastric Lesion in Rats

Background

Protection of the gastric mucosa from acute lesions induced by various irritants is a pertinent issue in the field of critical care medicine. In this study, we investigated the gastroprotective effects of astragaloside IV on acute gastric lesions in rats under stressful conditions.

Methods

Rats were randomized into six groups. Group 1 and 2 received 10% Tween 80 (vehicle). Group 3 received 20 mg/kg of omeprazole, a proton pump inhibitor. Groups 4, 5 and 6 received astragaloside IV at concentration of 1, 10, and 50 mg/kg, respectively. As a means to induce gastric lesions, Groups 2–6 were subjected to water immersion and restraint stress for 12 hours after treatment.

Results

Our present studies show that compared to rats in group 2, treatment with 1 to 50 mg/kg astragaloside IV significantly decreased the size of gastric lesions, MDA, TNFα and MCP1 levels, in addition to normalizing gastric pH, gastric mucus and SOD levels (P<0.05). Histomorphological examination confirmed that treatment with astragaloside IV elicited a dosage-dependent protective effect on the gastric mucosa. Furthermore, pretreatment with astragaloside IV resulted in significant elevations in HSP70 and reduction in Bax, along with over-expression of PLCγ response level, which was further confirmed via immunohistochemical analysis.

Conclusions

The acute gastric lesions induced are attenuated by pretreatment with astragaloside IV which is possibly due to the enhancing of the expression of HSP70 with concomitant antioxidant, anti-inflammatory and anti-apoptotic capacity.     

Prostaglandin stimulation of gastrointestinal transit in post-operative ileus rats

The prostaglandins PGF_2α, PGE₂ and 16,16-dimethyl PGE₂, when administered intravenously, orally, subcutaneously or intraduodenally to laparotomized rats, decreased gastric emptying, small intestinal transit and colonic transit as compared to unoperated controls. All three prostaglandins increased colonic transit above that found with unoperated controls. This activity was independent of small intestinal fluid accumulation (i.e., enteropooling) since ligating the ileal-cecal junction had no effect on colonic transit. Small intestinal transit was increased, but not normalized, by PGE₂ and 16,16-dimethyl PGE₂. 16,16-Dimethyl PGE₂ completely restored gastric emptying when given intravenously to laparotomized rats at doses greater than 5.0 μg/kg. This effect on gastric emptying lasted approximately 4 hrs. Thus, 16,16-dimethyl PGE₂, when given intravenously, normalized gastric emptying, significantly increased small intestinal transit, and made the colon hypermotile. Prostaglandins may be beneficial in the treatment of post-operative ileus and other conditions of sluggish gastrointestinal propulsion.     

Diosmin Protects against Ethanol-Induced Gastric Injury in Rats: Novel Anti-Ulcer Actions

Alcohol consumption has been commonly associated with gastric mucosal lesions including gastric ulcer. Diosmin (DIO) is a natural citrus flavone with remarkable antioxidant and anti-inflammatory features that underlay its protection against cardiac, hepatic and renal injuries. However, its impact on gastric ulcer has not yet been elucidated. Thus, the current study aimed to investigate the potential protective effects of DIO against ethanol-induced gastric injury in rats. Pretreatment with DIO (100 mg/kg p.o.) attenuated the severity of ethanol gastric mucosal damage as evidenced by lowering of ulcer index (UI) scores, area of gastric lesions, histopathologic aberrations and leukocyte invasion. These actions were analogous to those exerted by the reference antiulcer sucralfate. DIO suppressed gastric inflammation by curbing of myeloperoxidase (MPO) and tumor necrosis factor-α (TNF-α) levels along with nuclear factor kappa B (NF-κB) p65 expression. It also augmented the anti-inflammatory interleukin-10 (IL-10) levels. Meanwhile, DIO halted gastric oxidative stress via inhibition of lipid peroxides with concomitant enhancement of glutathione (GSH), glutathione peroxidase (GPx) and the total antioxidant capacity (TAC). With respect to gastric mucosal apoptosis, DIO suppressed caspase-3 activity and cytochrome C (Cyt C) with enhancement of the anti-apoptotic B cell lymphoma-2 (Bcl-2) in favor of cell survival. These favorable actions were associated with upregulation of the gastric cytoprotective prostaglandin E₂ (PGE₂) and nitric oxide (NO). Together, these findings accentuate the gastroprotective actions of DIO in ethanol gastric injury which were mediated via concerted multi-pronged actions, including suppression of gastric inflammation, oxidative stress and apoptosis besides boosting of the antioxidant and the cytoprotective defenses.     

Inhibition by 16, 16-dimethyl PGE2 of ethanol-induced gastric mucosal damage and leukotriene B4 and C4 formation

The effects of PGE₂ and its stable analogue, 16, 16 dimethyl PGE₂ (dmPGE₂) were investigated on ethanol-induced gastric mucosal haemorrhagic lesions and leukotriene formation in the rat. Exposure of the rat gastric mucosa to ethanol , produced a concentration-related increase in the mucosal formation of leukotriene B₄ (LTB₄) which was correlated with macroscopically-apparent haemorrhagic damage to the mucosa. Challenge with absolute ethanol likewise enhanced the mucosal formation of LTC₄ whereas the mucosal formation of 6-keto-PGF_1α was unaffected. Challenge of the rat gastric mucosa with ethanol induced a concentration-dependent increase in the formation of LTB₄ and LTC₄, but not 6-keto PGF_1α. Pretreatment with PGE₂ (200–500μg/kg p.o.) prevented the haemorrhagic mucosal damage induced by oral administration of absolute ethanol but not the increased formation of leukotrienes by the mucosa. In contrast, pretreatment with a high dose of dmPGE₂ (20μg/kg p.o.) prevented both the gastric mucosal lesions and the increase mucosal leukotriene formation. The differences in the effects of these prostaglandins may be related to the nature or degree of protection of the gastric mucosa. Thus, high doses of dmPGE₂ but not PGE₂ may protect the cells close the luminal surface of the mucosa and hence reduce the stimulation of leukotriene synthesis by these cells.     

Gastroprotective action of orexin-A against stress-induced gastric damage is mediated by endogenous prostaglandins, sensory afferent neuropeptides and nitric oxide

Orexin-A, identified in the neurons and endocrine cells in the gut, has been implicated in control of food intake and sleep behavior but little is known about its influence on gastric secretion and mucosal integrity. The effects of orexin-A on gastric secretion and gastric lesions induced in rats by 3.5 h of water immersion and restraint stress (WRS) or 75% ethanol were determined. Orexin-A (5-80 microg/kg i.p.) increased gastric acid secretion and attenuated gastric lesions induced by WRS and this was accompanied by the significant rise in plasma orexin-A, CGRP and gastrin levels, the gastric mucosal blood flow (GBF), luminal NO concentration and an increase in mRNA for CGRP and overexpression of COX-2 protein and the generation of PGE(2) in the gastric mucosa. Orexin-A-induced protection was abolished by selective OX-1 receptor antagonist, vagotomy and attenuated by suppression of COX-1 and COX-2, deactivation of afferent nerves with neurotoxic dose of capsaicin, pretreatment with CCK(2)/gastrin antagonist, CGRP(8-37) or capsazepine and by inhibition of NOS with L-NNA. This study shows for the first time that orexin-A exerts a potent protective action on the stomach of rats exposed to non-topical ulcerogens such as WRS or topical noxious agents such as ethanol and these effects depend upon hyperemia mediated by COX-PG and NOS-NO systems, activation of vagal nerves and sensory neuropeptides such as CGRP released from sensory nerves probably triggered by an increase in gastric acid secretion induced by this peptide.     

Endogenous Prostaglandins and Afferent Sensory Nerves in Gastroprotective Effect of Hydrogen Sulfide against Stress-Induced Gastric Lesions

Hydrogen sulfide (H₂S) plays an important role in human physiology, exerting vasodilatory, neuromodulatory and anti-inflammatory effects. H₂S has been implicated in the mechanism of gastrointestinal integrity but whether this gaseous mediator can affect hemorrhagic lesions induced by stress has been little elucidated. We studied the effect of the H₂S precursor L-cysteine, H₂S-donor NaHS, the H₂S synthesizing enzyme (CSE) activity inhibitor- D,L-propargylglycine (PAG) and the gastric H₂S production by CSE/CBS/3-MST activity in water immersion and restraint stress (WRS) ulcerogenesis and the accompanying changes in gastric blood flow (GBF). The role of endogenous prostaglandins (PGs) and sensory afferent nerves releasing calcitonin gene-related peptide (CGRP) in the mechanism of gastroprotection induced by H₂S was examined in capsaicin-denervated rats and those pretreated with capsazepine to inhibit activity of vanilloid receptors (VR-1). Rats were pretreated with vehicle, NaHS, the donor of H₂S and or L-cysteine, the H₂S precursor, with or without the concurrent treatment with 1) nonselective (indomethacin) and selective cyclooxygenase (COX)-1 (SC-560) or COX-2 (rofecoxib) inhibitors. The expression of mRNA and protein for COX-1 and COX-2 were analyzed in gastric mucosa pretreated with NaHS with or without PAG. Both NaHS and L-cysteine dose-dependently attenuated severity of WRS-induced gastric lesions and significantly increased GBF. These effects were significantly reduced by pretreatment with PAG and capsaicin denervation. NaHS increased gastric H₂S production via CSE/CBS but not 3-MST activity. Inhibition of COX-1 and COX-2 activity significantly diminished NaHS- and L-cysteine-induced protection and hyperemia. NaHS increased expression of COX-1, COX-2 mRNAs and proteins and raised CGRP mRNA expression. These effects of NaHS on COX-1 and COX-2 protein contents were reversed by PAG and capsaicin denervation. We conclude that H₂S exerts gastroprotection against WRS-induced gastric lesions by the mechanism involving enhancement in gastric microcirculation mediated by endogenous PGs, sensory afferent nerves releasing CGRP and the activation of VR-1 receptors.     

Glycogen Synthase Kinase-3β and Caspase-2 Mediate Ceramide- and Etoposide-Induced Apoptosis by Regulating the Lysosomal-Mitochondrial Axis

Glycogen synthase kinase-3β (GSK-3β) regulates the sequential activation of caspase-2 and caspase-8 before mitochondrial apoptosis. Here, we report the regulation of Mcl-1 destabilization and cathepsin D-regulated caspase-8 activation by GSK-3β and caspase-2. Treatment with either the ceramide analogue C₂-ceramide or the topoisomerase II inhibitor etoposide sequentially induced lysosomal membrane permeabilization (LMP), the reduction of mitochondrial transmembrane potential, and apoptosis. Following LMP, cathepsin D translocated from lysosomes to the cytoplasm, whereas inhibiting cathepsin D blocked mitochondrial apoptosis. Furthermore, cathepsin D caused the activation of caspase-8 but not caspase-2. Inhibiting GSK-3β and caspase-2 blocked Mcl-1 destabilization, LMP, cathepsin D re-localization, caspase-8 activation, and mitochondrial apoptosis. Expression of Mcl-1 was localized to the lysosomes, and forced expression of Mcl-1 prevented apoptotic signaling via the lysosomal-mitochondrial pathway. These results demonstrate the importance of GSK-3β and caspase-2 in ceramide- and etoposide-induced apoptosis through mechanisms involving Mcl-1 destabilization and the lysosomal-mitochondrial axis.     

Mitochondria-targeted Ogg1 and Aconitase-2 Prevent Oxidant-induced Mitochondrial DNA Damage in Alveolar Epithelial Cells

Mitochondria-targeted human 8-oxoguanine DNA glycosylase (mt-hOgg1) and aconitase-2 (Aco-2) each reduce oxidant-induced alveolar epithelial cell (AEC) apoptosis, but it is unclear whether protection occurs by preventing AEC mitochondrial DNA (mtDNA) damage. Using quantitative PCR-based measurements of mitochondrial and nuclear DNA damage, mtDNA damage was preferentially noted in AEC after exposure to oxidative stress (e.g. amosite asbestos (5–25 μg/cm²) or H₂O₂ (100–250 μm)) for 24 h. Overexpression of wild-type mt-hOgg1 or mt-long α/β 317–323 hOgg1 mutant incapable of DNA repair (mt-hOgg1-Mut) each blocked A549 cell oxidant-induced mtDNA damage, mitochondrial p53 translocation, and intrinsic apoptosis as assessed by DNA fragmentation and cleaved caspase-9. In contrast, compared with controls, knockdown of Ogg1 (using Ogg1 shRNA in A549 cells or primary alveolar type 2 cells from ogg1^−/− mice) augmented mtDNA lesions and intrinsic apoptosis at base line, and these effects were increased further after exposure to oxidative stress. Notably, overexpression of Aco-2 reduced oxidant-induced mtDNA lesions, mitochondrial p53 translocation, and apoptosis, whereas siRNA for Aco-2 (siAco-2) enhanced mtDNA damage, mitochondrial p53 translocation, and apoptosis. Finally, siAco-2 attenuated the protective effects of mt-hOgg1-Mut but not wild-type mt-hOgg1 against oxidant-induced mtDNA damage and apoptosis. Collectively, these data demonstrate a novel role for mt-hOgg1 and Aco-2 in preserving AEC mtDNA integrity, thereby preventing oxidant-induced mitochondrial dysfunction, p53 mitochondrial translocation, and intrinsic apoptosis. Furthermore, mt-hOgg1 chaperoning of Aco-2 in preventing oxidant-mediated mtDNA damage and apoptosis may afford an innovative target for the molecular events underlying oxidant-induced toxicity.     

IL-18 mediates the formation of stress-induced, histamine-dependent gastric lesions

A role of IL-18 in the induction of gastric lesions by water immersion and restraint stress (WRS) was investigated. When wild-type BALB/c mice were exposed to WRS, levels of IL-18 in the serum and stomach increased rapidly with the development of acute gastric lesions. In IL-18-deficient mice [IL-18 knockout (KO) mice] similarly exposed to WRS, no gastric lesions were observed, but the administration of IL-18 before exposure to WRS resulted in the induction of WRS-induced gastric lesions. WRS enhanced gastric histidine decarboxylase (HDC) activity with concomitant increases in gastric histamine content. In IL-18 KO mice, the WRS-induced elevation of gastric HDC activity and histamine levels was much less than that in wild-type mice, but it was augmented by prior administration of IL-18. Treatment of wild-type mice with cimetidine, a histamine H2 receptor antagonist, inhibited the formation of WRS-induced gastric lesions with no effect on the induction of gastric IL-18 by WRS. Levels of corticosterone, one of the stress indicators, were lower in IL-18 KO mice than in wild-type mice. The glucocorticoid receptor antagonist mifepristone had no effect on gastric IL-18 and histamine levels but aggravated the stress-induced gastric lesions, indicating that corticosterone was not involved in the IL-18-mediated formation of stress-induced gastric lesions. These results indicate that IL-18 is involved in the induction of gastric lesions by WRS through augmentation of HDC activity and production of histamine in the stomach.     

Hormesis in Cholestatic Liver Disease; Preconditioning with Low Bile Acid Concentrations Protects against Bile Acid-Induced Toxicity

Introduction

Cholestasis is characterized by accumulation of bile acids and inflammation, causing hepatocellular damage. Still, liver damage markers are highest in acute cholestasis and drop when this condition becomes chronic, indicating that hepatocytes adapt towards the hostile environment. This may be explained by a hormetic response in hepatocytes that limits cell death during cholestasis.

Aim

To investigate the mechanisms that underlie the hormetic response that protect hepatocytes against experimental cholestatic conditions.

Methods

HepG2.rNtcp cells were preconditioned (24 h) with sub-apoptotic concentrations (0.1–50 μM) of various bile acids, the superoxide donor menadione, TNF-α or the Farsenoid X Receptor agonist GW4064, followed by a challenge with the apoptosis-inducing bile acid glycochenodeoxycholic acid (GCDCA; 200 μM for 4 h), menadione (50 μM, 6 h) or cytokine mixture (CM; 6 h). Levels of apoptotic and necrotic cell death, mRNA expression of the bile salt export pump (ABCB11) and bile acid sensors, as well as intracellular GCDCA levels were analyzed.

Results

Preconditioning with the pro-apoptotic bile acids GCDCA, taurocholic acid, or the protective bile acids (tauro)ursodeoxycholic acid reduced GCDCA-induced caspase-3/7 activity in HepG2.rNtcp cells. Bile acid preconditioning did not induce significant levels of necrosis in GCDCA-challenged HepG2.rNtcp cells. In contrast, preconditioning with cholic acid, menadione or TNF-α potentiated GCDCA-induced apoptosis. GCDCA preconditioning specifically reduced GCDCA-induced cell death and not CM- or menadione-induced apoptosis. The hormetic effect of GCDCA preconditioning was concentration- and time-dependent. GCDCA-, CDCA- and GW4064- preconditioning enhanced ABCB11 mRNA levels, but in contrast to the bile acids, GW4064 did not significantly reduce GCDCA-induced caspase-3/7 activity. The GCDCA challenge strongly increased intracellular levels of this bile acid, which was not lowered by GCDCA-preconditioning.

Conclusions

Sub-toxic concentrations of bile acids in the range that occur under normal physiological conditions protect HepG2.rNtcp cells against GCDCA-induced apoptosis, which is independent of FXR-controlled changes in bile acid transport.     

Inhibition of 15-hydroxy prostaglandin dehydrogenase activity in rabbit gastric antral mucosa by 13-hydroperoxyoctadecadienoic acid

The effect of 13-hydroperoxyoctadecadienoic acid (13-HPODE), a hydroperoxy adduct of linoleic acid (LA), on the activities of prostaglandin (PG) synthesizing and catabolizing enzymes in rabbit gastric antral mucosa was examined. 13-HPODE had no effect on the synthesis of PGE₂, PGF_2α and PGD₂ from exogenous arachidonic acid in the microsomal fraction of the gastric mucosa at concentrations ranging from 5–20 μM. On the other hand, at 1–10 μM, it inhibited the activity of 15-hydroxy PG dehydrogenase (PGDH), which catalyzes the initial step of catabolism of PGs, in a dose-dependent manner. The concentration required for 50% inhibition was approximately 1 μM. Experiments utilizing LA, 13-hydroxyoctadecadienoic acid and Fe²⁺ indicated the requirement of the hydroperoxy moiety for the inhibitory effect of 13-HPODE on the PGDH activity. These results suggest that 13-HPODE has the potential to increase the levels of biologically active PGs in gastric mucosa by preventing their inactivation and may have functional effects within the stomach.     

瘦素对小鼠脑缺血/再灌注损伤神经元凋亡的影响

目的：研究Leptin在脑缺血性损伤神经元凋亡中的作用及其机制。方法：将75只雄性昆明小鼠完全随机分成3组,即假手术组、缺血/再灌注模型组、Leptin干预组;通过大脑中动脉栓塞（MCAO）复制小鼠局灶性脑缺血再灌注损伤模型,Leptin干预组在缺血0 min腹腔注射Leptin（1μg/g体重）,TUNEL染色检测神经元凋亡,RT-PCR检测凋亡相关基因bcl-2和caspase-3 mRNA表达,免疫组化凋亡相关基因bcl-2和caspase-3蛋白水平的表达。结果：模型组脑缺血中心区神经元以坏死为主,与假手术组相比,其半影区神经元凋亡数量显著增多、促凋亡基因cas-pase-3和抑凋亡基因bcl-2的mRNA和蛋白表达水平均显著升高（P<0.01）;与模型组比较,Leptin干预组半影区凋亡神经元数量显著减少、caspase-3 mRNA和蛋白表达水平显著降低（P<0.01）,抑凋亡基因bcl-2 mRNA和蛋白表达水平显著升高（P<0.01）。结论：Leptin能够通过上调抑凋亡基因bcl-2表达,下调促凋亡基因caspase-3表达抑制神经元凋亡,在脑缺血性损伤中发挥神经保护作用。