| Background and PurposeDespite nearly70years of research, acute gastric mucosal lesion (AGML) is still one of the major challenges in the field of critical care medicine.As a non-specific stress response, the pathogenesis of AGML is results of multifactorial, multi-level interaction involving three anatomical levels characterized as the gastrointestinal tissue, the sensory afferent nerves and the central nervous system. Noxious stimuli can act on these anatomical structures via nervous, endocrine, immune and so on, thus causing AGML. Research showed that some of the ion channels and receptors as acid receptors、nociceptors. And increasing attention is being paied in these receptors because of there regulation of noxious stimuli towards the gastrointestinal tract.Among these nociceptors, acid sensing ion channel3(ASIC3)is one of the hotspots currently.Previous studies had shown that, ASIC3as acid receptor, nociceptor played an important role in the information processing in the gastrointestinal tract.In rats,82%of the DRG neurons projected to the stomach express ASIC3. And in the human gastrointestinal tract, An increase expression of ASIC3in the intestinal mucosa was detected in inflammatory bowel disease. In the crowd of gastrointestinal disorders caused by allergic purpura,high expression of ASIC3was also showed in the gastric mucosa. But so far, no studies in rats have confirmed the ASIC3expression in the gastrointestinal tissue, no studies have systematically described the spatial and temporal distribution patterns of ASIC3in WIRS induced AGML rat model in the three anatomical structures of the gastrointestinal tissue, the sensory afferent nerves and the central nervous system.Yet, no research have clarify the mechanism of ASIC3in AGML models by giving a specific antagonist,APETx2. Therefore, our study intends to establish AGML models caused by WIRS, focusing on analysis the spatial and temporal distribution of ASIC3in three anatomical structures of the gastrointestinal tissue, the sensory afferent nerves and the central nervous system. By giving a specific antagonist,APETx2, we intends to explore the mechanism of ASIC3in AGML.Furthermore,with pretreatment of sufentanil,we want to explore how an opioid receptor analgesics can protect AGML induced by WIRS.Above all,This project will clarify the regulative role and mechanism of ASIC3in AGML regeneration so as to provide more sufficient scientific evidence for the establishment of ASIC3as a new target for prevention and treatment of stress induced AGML.MethodsThis study was approved by the Ethics Committee of Guangzhou General Hospital of Guangzhou Military Command, following the National Institutes of Health guidelines for the care and use of laboratory animals. All efforts were made to reduce both the suffering and number of animals used.Pathogen-free male Wistar rats (weight170-220g and7-8weeks old) were provided and housed at24℃with a12h light/12h dark cycle, and the rats had free access to water and regular chow. Before the experiments, each animal was housed in a single cage that had wire-net bottoms to avoid coprophagy. All animals were starved for36h before the experiments. The rats were divided randomly into6groups:(1) normal control group(NC group or WIRS Oh group):rats were not exposed to any stress.(2) water immersion restraint stress2h group(WIRS2h group):rats were immersed up to the depth of the xiphoid process in water bath at18℃for2h as previously described.(3) WIRS4h group:rats were immersed for4h.(4) WIRS6h group:rats were immersed for6h.(5) Sufentanil pretreatment group(Sufen group):Sufentanil was dissolved in saline at5μg/ml. The rats received intraperitoneal (IP) injection of Sufentanil (25μg/kg body weight) at the time just before the WIRS procedure.(6)APETx2pretreatment group(AT group):APETx2was dissolved in saline at5μg/ml. The rats received intraperitoneal (IP) injection of APETx2(25μg/kg body weight) at the time just before the WIRS procedure.At each time point of the experiments, rats were anesthetized.The stomachs were removed.Intragastric pH, total activity of MDA and SOD in the stomach tissues were measured; HE stain used for detection of gastric histopathology by microscopy; Immunohistochemical and Westemblot methods used for detection of expression of ASIC3in three anatomical structures of the gastric tissue, the DRG neurons and the hypothalamic paraventricular nucleus(PVN)neurons. Real-time quantitative PCR assay used for detection of expression of ASIC3mRNA in the gastric tissues, DRG neurons and the hypothalamus.Results1. Value of intragastric pH in different groupsthe value of intragastric pH was significantly lower in WIRS2h group、WIRS4h group and WIRS6h group than control group(P<0.05). Compared among each WIRS group, the value of intragastric pH showed no significant(P>0.05). 2.Histopathological analysis of gastric injury in different groupsNo inflammatory changes or gastric mucosal lesions were observed in NC group However, in WIRS2h group、WIRS4h group and WIRS6h group,we found gastric bleeding erosions from Mild、moderate and serious, as indicated by mucosal hemorrhage, mucosal erosive lesion, and higher scores of ulcer index. The hemorrhage was observed mainly in the corpus mucosa and antral mucosa, and the mucosa was disrupted. Massive hemorrhagic necrosis and the exfoliation of surface mucosal cells into gastric lumen were observed in WIRS6h group. Notably, these features of mucosal lesions in WIRS6h group were significantly attenuated in Sufen group and AT group, manifested by less mucosal hemorrhage, less inflammatory cell infiltration, and lower scores of ulcer index.3. Total activity of SOD and MDA in the stomach tissuesCompared with WIRS Oh group (NC group), values of SOD in the rest group were significantly decreased (P<0.05), and MDA values were significantly higher (P<0.05); With the prolonged WIRS time, downward trend of SOD values and upward trend of MDA values were significantly significant between the groups (P<0.05). Compared with WIRS6h group, SOD values of Sufen group and AT group were significantly increased (P<0.05), and MDA values were significantly decreased (P<0.05); compared with Sufen group, SOD and MDA values of AT group were no significant (P>0.05).4.Immunohistochemical expression and localization of ASIC3Expression and localization of ASIC3in the stomach sections were described below.WIRS Oh group (NC group):Only weakly positive expression, occasionally in the epithelial cell layer; WIRS2h group:Brown strong expression over full-thickness gastric mucosa,mainly distributed in the epithelial cell layer, gastric primary cells and gastric parietal cells; WIRS4h group:Brown strong expression can be seeing over full thickness gastric mucosa,submucosal plexus was also positive expression; WIRS6h group:The epithelial layer was significantly damage, Brown strongly expressed mainly located in the main cells and gastric wall cells, submucosal plexus and stomach muscular plexus. Sufen group:Also had positive expression,mainly in the epithelial cell layer、primary cells、gastric parietal cells、 submucosal plexus and stomach muscular plexus; AT group:Weakly positive expression only in the mucosal layer.Expression and localization of ASIC3in the DRG neurons were described below:ASIC3immunohistochemical staining localized on neurons. WIRS Oh group (NC group):Only weakly positive expression.WIRS2h,4h and6h group:Brown strongly expressed and the WIRS4h group was the most obvious.Expression and localization of ASIC3in the PVN neurons were described below:In each group ASIC3protein immunohistochemical localized in the neuronal membrane and cytoplasm. NC group showed the most brown staining; WIRS2h group、WIRS4h group and WIRS6h group showed only a small numbers of neurons with yellow staining;, Sufen group and the AT group showed brown positive cells increased significantly.5.Expression results of ASIC3protein by Western blotWestern blot results of ASIC3protein in the gastric tissues were described below.Compared with WIRS Oh group (NC group),the rest group showed significantly higher gray values of ASIC3prote (P<0.05);Compared with WIRS2h group, gray values of ASIC3protein in the WIRS4h group and WIRS6h group were significantly increased (P<0.05); Compared with WIRS4h group, gray values of ASIC3protein in the WIRS6h group showed a downward trend, but not statistically significant (P>0.05). Compared with WIRS6h group, gray values of ASIC3protein in the AT group decreased significantly (P<0.05), gray values of ASIC3protein in the Sufen group declined slightly, but the difference was not statistically significant (P>0.05).Western blot results of ASIC3protein in the DRG neurons were described below. Compared with WIRS Oh group (NC group),the rest group showed significantly higher gray values of ASIC3protein (P<0.05);Compared with WIRS2h group, gray values of ASIC3protein in the WIRS4h group were significantly increased (P<0.05); Compared with WIRS4h group, gray values of ASIC3protein in the WIRS6h group showed a downward trend, and this was statistically significant (P<0.05).6. qPCR results of ASIC3mRNARelative mRNA level of ASIC3in the gastric tissues were described below.Compared with WIRS Oh group (NC group),the rest group showed significantly higher relative mRNA level of ASIC3.(P<0.05);Compared with WIRS2h group, Relative mRNA level of ASIC3in the WIRS4h group and WIRS6h group were significantly increased (P<0.05); Compared with WIRS4h group, relative mRNA level of ASIC3in the WIRS6h group showed a downward trend, and this was statistically significant (P<0.05). Compared with WIRS6h group, relative mRNA level of ASIC3in the AT group decreased significantly (P<0.05), relative mRNA level of ASIC3in the Sufen group declined slightly, but the difference was not statistically significant (P>0.05).Relative mRNA level of ASIC3in the DRG neurons were described below. Compared with WIRS Oh group (NC group),the rest group showed significantly higher relative mRNA level of ASIC3(P<0.05);Compared with WIRS2h group, relative mRNA level of ASIC3in the WIRS4h group were significantly increased (P<0.05); Compared with WIRS4h group, relative mRNA level of ASIC3in the WIRS6h group showed a downward trend, and this was statistically significant (P<0.05).Relative mRNA level of ASIC3in the hypothalamus neurons were described below. Compared with NC group, relative mRNA levels of ASIC3in rest groups were significantly lower (P<0.05); Compared with WIRS2h group, ASIC3mRNA expressions in the WIRS4h group、WIRS6h group、Sufen group and the AT group were significantly increased (P<0.05); Compared with WIRS4h group, WIRS6h group, Sufen group and the AT group ASIC3mRNA expression were significantly increased (P<0.05); Compared with WIRS6h group, Sufen group and the AT group ASIC3mRNA expression were significantly increased (P<0.05); compared with Sufen group, ASIC3mRNA expression level in the AT group was no significant change (P>0.05).7.Numbers of c-fos immunoreactivity neurons in the PVNImmunohistochemical staining of c-fos proteins in the PVN neurons in each group located in the nucleus,with c-fos immunoreactive nuclei dark brown and c-fos immune negative nuclei blue. NC group showed very small amount of brown nuclear staining; WIRS2h group showed a lot of dark brown nuclear staining; WIRS4h group and WIRS6h group were seen in the amount of neuronal nuclei stained dark brown; Sufen group and the AT group showed a small amount of nerve yuan dark brown nuclear staining.8. Correlation analysis between the various indicatorsASIC3expression and intragastric PH value and stomach tissue SOD values in the gastric tissue and DRG neurons showed a significant negative correlation was significantly positively correlated with MDA in gastric tissue; stomach tissue ASIC3expression level of DRG neurons ASIC3expression levels were significantly positive correlation.ASIC3mRNA expression in the hypothalamus and PVN ASIC3immunoreactive cells in the PVN c-fos protein immunoreactive cells was significantly negatively correlated. Conclusions1. The WIRS time closely associated with gastric mucosal injury, showed as WIRS prolonged,gastric mucosal injury increased, gastric acidification continued, gastric tissue oxidative damage increased.Decreased antioxidant capacity is an important factor in AGML progressive. To copy the severe hemorrhagic necrotizing acute gastric mucosal injury model in rats, at least6hours of water immersion restraint stress should be given.2. To our knowledge, for the first time we investigated ASIC3expression in the stomach tissue:ASIC3weak positive expression in the stomach tissue of rats, WIRS2h can make ASIC3in full-thickness gastric mucosa were positive; WIRS4h ASIC3not only visible in the whole strong expression of gastric mucosal layer, submucosal plexus was also positive expression; WIRS6h ASIC3buffy strong positive expression was mainly localized in the gastric parietal cells and primary cells, submucosal plexus and stomach muscular plexus.3. This study described the expression of ASIC3in rat DRG neurons projecting stomach:in the NC group,weakly positive expression ASIC3can be presented on the neurons,WIRS make these DRG neurons showed high expression of ASIC3.With4h of WIRS, the strong expression of ASIC3was the most obvious. When compared WIRS4h group,ASIC3expression in the WIRS6h group has decreased significantly, but still significantly higher than normal.4. In this study,we described expression of ASIC3in the paraventricular nucleus neurons and its effect to neuronal activity:ASIC3highly expressed in normal rat hypothalamus, ASIC3immunoreactive cells are widely distributed in the PVN. Rats can reduce flooding bondage ASIC3mRNA expression levels in the hypothalamus, inhibiting ASIC3protein expression in PVN neurons, and its trends and c-fos protein expression was significantly negatively correlated, suggesting that changes in WIRS hypothalamic ASIC3may be related to neuronal activity5. To our knowledge, for the first time we confirmed pretreatment with APETx2can downregulate ASIC3expressed in the stomach tissue and play a mucosal protective role by free radical antioxidant effect. Thus confirmed that ASIC3is a key stress factors in control of gastric tissue damage. As the role and mechanisms of ASIC3in the hypothalamus are not yet clear,further studies are needed to explore how ASIC3act on the regulation of WIRS in the CNS.6. Intraperitoneally administered sufentanil through pretreatment, while clearly sufentanil pretreatmentdid not reduce the expression of ASIC3stomach tissue, suggesting sufentanil in the stomach anti-nociceptive effect of organizational level stress may exist other mechanisms. And at central level, sufentanil showed inhibition of hypothalamic neuronal activity, relieve inhibition of expression of hypothalamic ASIC3WIRS,it may be central mechanism of the protective effect of sufentanil for AGMLinduced by WIRS. This also worth of further exploration. |
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