The Potential Mechanism Of TRPA1 And Substance P In Acute Gastric Mucosal Lesions

BackgroundAcute gastric mucosal lesions(AGML)is a weakened protective effect of gastric mucosal barrier with different degrees edema,ulcer,erosion and hemorrhage in the gastric mucosa,which is a common clinical problem.Although there are many drugs for the treatment of acute gastric mucosal lesions,gastric mucosal lesions often relapse and the number of patients afflicted with this entity has remained stable.Excessive and/or persistent nociceptive stress is a key pathogenic factor of acute gastric mucosal lesions.The stomach is one of the most sensitive organs under stress.Existing studies have shown that many kinds of difficult and complicated surgery,shock,severe infection,sepsis,multiple organ dysfunction syndrome or failure,and severe burn trauma can all cause acute gastric mucosal lesions.Especially the low blood perfusion caused by shock,can reduce the blood flow of gastric wall and damage the gastric mucosal barrier,which affect the function of gastric mucosal epithelial cells and develop stress gastric mucosal edema,ulcer or even erosive bleeding.The gastric mucosal lesions associated with stress can affect more than 75%of acute critically ill patients,and three to four percent of them had severe gastric bleeding,which may aggravate or worsen the original disease and increase the mortality rate.The incidence of gastric hemorrhage associated with stress in ICU was 0.6%-6%.Many studies have shown that stress ulcers have little to do with increased gastric acid secretion,and mainly dependent on gastric mucosal blood flow reduction,hypoxia and ischemia-reperfusion injury.Any factors affecting gastric wall blood flow will affect the function of gastric mucosal epithelial cells and weaken the gastric mucosal protective barrier.An increase in gastric acid obviously can increase the load on the gastric mucosal defense system,but gastric acid is not generally increased or even is reduced in stress ulcers.Nevertheless,the role of H~+in the pathogenesis of stress gastric ulcer cannot be denied.Although the concentration of H~+is not high in stomach,it can still spread retrogradely and cause gastric intramural acidification due to gastric mucosal barrier damage,which can cause acute gastric mucosal lesions.In addition,some inflammatory mediators and metabolites also affect the function of gastric mucosa.Although some of these factors have been identified to affect gastric mucosal function,the pathophysiological mechanism of acute gastric mucosal lesions induced by stress is still entirely unclear.Considering the multiple factors and multichannel regulation of acute gastric mucosal lesions,it is urgent to find new signaling molecules and signal transduction pathways related to the pathogenesis of acute gastric mucosal lesions,which can provide new potential targets for treatment AGML.At present,it is believed that the occurrence of AGML is related to many factors,such as nerve and body fluids.Transient receptor potential anchor protein 1(TRPA1)and substance P(SP)expressed abundantly and also co-expressed in rat's gastric tissue and dorsal root ganglion(DRG,T8-11)innervating the corresponding stages of gastric tissue,so there may be some interaction between them.It has been reported that TRPA1 was involved in the regulation of visceral hyperalgesia in rats.Kondo T reported that intrathecal injection and intraperitoneal injection HC-030031,a specific antagonist of TRPA1,or TRPA1knockout,can reduce effectively the noxious stimuli induced by gastric distention in rats.Giving the neurokinin-1 receptor(NK1R)of SP antagonist SR140333 and CP99994 can respectively reduce C57/BL6J mice gastric injury induced by alcohol and SD rats bladder injury induced by water immersion restraint stress.Some studies reported that TRPA1 and SP can cause neurogenic inflammation and non-neurogenic inflammation.Although TRPA1can regulate the SP release in colitis model induced by TNBS in mice,their role in gastric mucosal lesions is not very clear.ObjectiveWater immersion restraint stress(WIRS)is widely used as an experimental model of stress-induced AGML based on its reproducibility and clinical relevance.Therefore,we preliminarily discussed the following issues by using WIRS induced acute gastric mucosal lesions rat models.1.WIRS affected the expression of TRPA1 and SP in DRG(T8-11)innervating the corresponding stages of gastric tissue,gastric tissue,and gastric mucosa in rats.The effect of TRPA1 and SP on AGML was observed and the possible mechanisms of them in AGML were explored.2.In order to further observe the role of TRPA1and SP on AGML and explore the possible mechanisms of them in AGML,and the influence of TRPA1 on the SP release,rats were injected intrathecally or intraperitoneally TRPA1 specific antagonist HC-030031respectively and taken orally TRPA1 agonist AITC.MethodsWistar rats were randomly divided into 6 groups in this study.The rats in the control group did not undergo any procedures.In the WIRS group,the rats were immobilized in plastic restraint devices and were immersed up to the depth of the xiphoid process in a water bath at 16±1°C for 6 h to induce AGML formation.In the ITIH group,intrathecal injection of 10?l of HC-030031(10?g/?l,dissolved in 100%dimethyl sulfoxide)was administered at30min before WIRS.In the IPIH group,intraperitoneal injection of HC-030031(150 mg/kg,dissolved in 0.5%methyl cellulose)was administered at 60 min before WIRS.DRG(T8-11),gastric tissue and gastric mucosa were isolated from rats after WIRS 6h.In the AITC group,intragastric administration of TRPA1 agonists AITC(17 mg/kg,diluted in saline containing3%ethanol and 10%Tween 80)was administered.In the negative control group,saline containing 3%ethanol and 10%Tween 80 vehicle was administered.DRG(T8-11),gastric tissue and gastric mucosa were isolated from rats after oral AITC 2h.After gastric tissue were stained with hematoxylin and eosin(HE)or fixed with glutaraldehyde and osmium tetroxide and a series of treatments,gastric mucosal lesions and gastric mucosal cellular damage in six rats groups were observed by optical microscope or transmission electron microscope respectively.TRPA1 and SP mRNA and protein expression in DRG and gastric tissue was detected by RT-PCR(Reverse transcription-polymerase chain reaction),Western blotting and immunohistochemistry(IHC),respectively.SP concentration in gastric mucosa was detected by ELISA(Enzyme-linked immunosorbent assay).Results1.We identified gastric mucosal lesions stained by HE histopathology in each group rats by OM.In control group,the normal gastric mucosal structure,the clear layer,and the intact epithelium were observed.The epithelial cells were arranged closely and regularly,without necrosis and abscission.No edema,erythrocyte exudation and inflammatory cell infiltration in the stroma were observed.The glands in the lamina propria were arranged neatly and densely,without atrophy or reduction;There was no swelling,the tendency of capillary hemorrhage and stasis dilatation in the submucosa.In WIRS group,flake shedding of gastric mucosa,multiple superficial ulcer,occasional crater-like tremendous ulcer,and a large number of acute erosive hemorrhagic necrosis foci were observed.The extensive epithelial cells showed diffuse coagulation necrosis and abscission,accompanied by extensive erythrocyte exudation and inflammatory cell infiltration,capillary hemorrhage and stasis dilatation in the mucosal layer.The lamina propria gland was disordered and obvious atrophy,coupled to the reduction or even lack of the gland quantity.The intercellular substance became large and edema.The submucosal structure was loose,edema and vascular congestion and dilatation.Compared with the control group,the gastric mucosal lesions scores were significantly increased(P<0.001).In ITIH group or IPIH group,the integrity of gastric mucosa was better.The damaged mucosa was superficial and local,together with mild hyperemia and edema,occasional inflammatory cell infiltration.The local surface epithelium was necrotic and exfoliated,and local gland structure was disorder.Compared with WIRS group,gastric mucosal lesions scores were significantly decreased(P<0.001).In AITC group,gastric mucosa is damaged,accompanied by mucosal epithelial cell necrosis and exfoliation.Mucosal edema,erythrocyte exudation and capillary hemorrhage were observed.Occasionally,submucosa is loose,edema and vascular congestion and dilatation.Compared with the control group,the gastric mucosal lesions scores were significantly increased(P<0.001).2.TEM was used to observe the gastric mucosal cellular damage in each group rats.In control group,gastric epithelial cells were intact.Meanwhile,the structure of mitochondria,granular endoplasmic reticulum(GER)and nuclear were normal.The WIRS group exhibited gastric epithelial cell degeneration,perinuclear space with nuclear damage in chief cells and/or parietal cells,mitochondrial damage seriously with completely lost the normal structure,contour blur and reduction or loss of mitochondrial cristae,dilatation and even vacuoles in GER,loose tight junctions,and extreme secretory tubular dilation.Compared with the control group,the gastric mucosal cellular damage scores were significantly increased(P<0.001).In ITIH group or IPIH group,we observed local mitochondrial damage with mitochondrial cristae and contour profile,local dilatation in the GER and vacuoles disappearance,normal tight connection structure,and no secretory tubules dilated.Compared with WIRS group,gastric cellular damage scores were significantly decreased(P<0.001).In AITC group,perinuclear space with nuclear damage in the chief cells and/or parietal cells in the local gastric tissue were observed.Mitochondria were damaged,coupled to the decrease or disappearance of mitochondrial cristae,but the contour existed.GER was locally dilated and occasional vacuoles.Secretory tubules were dilated occasionally.Compared with the control group,the gastric cellular damage scores were significantly increased(P<0.001).3.Compared with the control group,TRPA1 and SP mRNA expression and protein expression were up-regulated in DRG(T8-11)and stomach,and SP release in gastric mucosa was significantly increased in WIRS group(P<0.001).4.Intrathecal injection and intraperitoneal injection of TRPA1 antagonists HC-030031significantly alleviated WIRS-induced acute gastric mucosal lesions and strongly inhibited SP release in DRG(T8-11),stomach and gastric mucosa in rats,compared with the WIRS group(P<0.001).5.Compared with the control group,oral administration TRPA1 agonist AITC induced moderate gastric mucosal lesions and increased SP release in DRG(T8-11),stomach and gastric mucosa in rats(P<0.001).Conclusions1.WIRS induced AGML and significantly increased the expression of TRPA1 and SP in DRG(T8-11)and stomach,and SP release in gastric mucosa.AGML induced by stress was closely related to the activation of TRPA1 and SP.This is because TRPA1 and SP can cause neuronal and non-neurogenic inflammation and promote visceral hyperalgesia.2.TRPA1 antagonist HC-030031 can obviously inhibit the AGML induced by stress in rats.The mechanisms may be that HC-030031 may significantly inhibit the expression of TRPA1 and SP release in DRG,stomach and gastric mucosa,which alleviated neurogenic and non-neurogenic inflammation in stomach and visceral hyperalgesia.3.TRPA1 agonist AITC induced moderate gastric mucosal lesions and increased SP release in DRG(T8-11),stomach and gastric mucosa in rats4.In the rat model of AGML induced by stress,SP release is correlated with TRPA1activity.