Inhibiting Caspase-1 Activation Of NLRP3 Inflammasome Attenuates Acute Gastric Ulcer In Mice

BackgroundAcute gastric ulcer is usually a superficial and diffuse mucosal lesion of stomach, it frequently occurs as a result of alcohol consumption, ingestion of nonsteroidal anti-inflammatory drugs and major stressful events such as severe burns, shock, surgery and trauma. Acute gastric ulcer may result in severe upper gastrointestinal bleeding with high morbidity and mortality. The pathogenesis of acute gastric ulcer is still obscure, but acute phase of inflammation is considered as one of major etiological factors maintaining and regulating of the severity of acute gastric ulcers.Inflammasomes are multi-protein oligomers of the innate immune system and play an important role in the pathogenesis of various diseases including gout, lung injury, diabetes mellitus and dengue hemorrhagic fever. NLRP3 inflammasome may be the most extensively studied one, which contains NLRP(Nucleotide-binding oligomerization domain, Leucine rich Repeat and Pyrin domain containing) protein, adaptor protein ASC(Apoptosis-associated Speck-like protein containing a Caspase recruitment domain) and Caspase-1. NLRP3 inflammasome can recognize certain microbial and danger components and serve as a platform for Caspase-1 activation and pro-inflammatory cytokine IL-1β maturation. Dinarello CA et al referred that proper regulation of inflammasome can affect the balance between pro- and anti-inflammatory cytokines production. And Rozza AL et al considered that cytokines such as TNF-α, IL-6 and IL-10 play important roles in the regulation of acute gastric ulcers.This is our first study to evaluate the protective effect of inhibiting Caspase-1 activation in NLRP3 inflammasome on experimental acute gastric injury in mice. As Caspase-1 is a key enzyme of NLRP3 inflammasome, which is a critical pro-inflammatory mediator that modulates host response to various stress conditions. Therefore, we used Caspase-1 inhibitor AC-YVAD-CMK to silence Caspase-1 expression of NLRP3 inflammasome, and investigated its protective effects using cold-restraint stress and ethanol-induced mouse acute gastric injury models. In the present study, we found that inhibiting Caspase-1 activation of NLRP3 inflammasome could attenuate mouse acute gastric injury and which was involved with alleviating inflammatory response and apoptosis, with underlying mechanism associated with NF-κB and P38 MAPK signal pathways.Part One. Mouse models of acute gastric injuryObjective: To select and make models of acute gastric ulcer in mice.Methods: ① The model of mouse cold-restraint stress-induced acute gastric injury was performed as follows: after fasting for 24 hours, mice were restrained in the devices and immersed to the depth of the xiphoid process in water at 20°C for 8 hours.② Another model of ethanol-induced gastric injury was also performed, after fasting for 24 hours, 100% ethanol(0.1ml/kg) was given to mice by intragastric administration, while mice in the Control group received PBS water instead. After 4 hours, the model of ethanol-induced gastric injury has been done.Results: One simple plastic device has been designed by our department for the model of mouse cold-restraint stress-induced acute gastric injury, which was evaluated as Chinese Patent(ZL 201420111867.X). There were obvious multiple hemorrhagic erosions in both of the two acute gastric injury models.Conclusions: Both models of cold-restraint stress and ethanol-induced acute gastric injury in mice were performed successfuly, the gastric mucosa histopathological changes were evaluated according to photographs and H&E staining.Part Two. Inhibiting Caspase-1 activation of NLRP3 inflammasome attenuates acute gastric injury in miceObjective: To evaluate the protective effect of Inhibiting Caspase-1 activation in NLRP3 inflammasome on mouse acute gastric injury.Methods: We established two models of acute gastic injury in mice, mice of both models were randomly divided into the following four groups(1) Control group,(2) Injury group,(3) Injury + AC-YVAD-CMK group,(4) Injury + Omeprazole group. AC-YVAD-CMK, Omeprazole or vehicle were given to mice half an hour before the injurys were induced. After the experiment, mice were killed and samples were stored at-80 °C for biochemical determinations. For histological analysis, we usd general photographs, H&E staining, PH values of gastric mucosal and index of gastric ulcer. Meanwhile, in order to test the acute phase of inflammation after mouse cold-restraint stress-induced gastric injury, serum pro--inflammatory cytokines of TNF-α and IL-6 were measured with commercial ELISA, macrophage infiltration was tested with Immunohistochemistry, protein expression of IL-18 and IL-1β were measured with Western blot. Futhermore, we tested of cleaved-Caspase-3, Caspase-3 and Bax protein expression with Western blot and cell apoptosis with TUNEL staining. In addition, on another model of ethanol-induced mouse acute gastric injury, the general anatomy and histological results also showed protective effects of Caspase-1 silencing in NLRP3 inflammasome on acute gastric injury in mice.Results: General photographs and H&E staining showed that cold-restraint stress-induced gastric damage induced multiple hemorrhagic erosions and ulcers, and which were prevented significantly by pretreatment with AC-YVAD-CMK. Quantitatively, index of gastric ulcer was reduced from 18.83±2.32 of the Injury group to 8.67±1.21 of the Injury+ AC-YVAD-CMK group(P<0.01), but there was no statistically difference between groups of mice treated with AC-YVAD-CMK and Omeprazole(P>0.05). Gastric mucosal PH was 3.33±0.82 in Injury group, and which was 4.83±0.75 in mice pretreated with Omeprazole, indicating that gastric acid production was effectively inhibited by Omeprazole, but mucosal PH of Injury+AC-YVAD-CMK was 4.167±0.41, there was no statistical difference compared with that in Injury group(P>0.05). Pro-inflammatory cytokines, IL-1β and IL-18 were dramatically increased after cold-restraint stresss-induced injury, and which were markedly reduced by pretreatment with AC-YVAD-CMK(P<0.05), but levels of IL-1β and IL-18 in mice pretreated with Omeprazole were not reduced significantly compared with those in Injury group(P>0.05). Furthermore, the immunohistochemistry results showed that number of F4/80 positive cells in gastric tissues was significantly reduced by AC-YVAD-CMK treatment compared with that in Injury group. ELISA results exhibited that mice pretreated with AC-YVAD-CMK had lower levels of TNF-α and IL-6 compare with those in Injury group(P<0.01 and P<0.05). Pretreatment with AC-YVAD-CMK significantly reduced high levels of cleaved-Caspase-3 in Injury group(P<0.05). Moreover, expression of Bax was high in both Injury group and Injury+Omeprazole group, and which was reduced markedly by pretreatment with AC-YVAD-CMK(P<0.05). The TUNEL results exhibited that a large number of apoptotic cells located at the top of mucous epidermis were observed in the Injury group, and which was reduced obviously with AC-YVAD-CMK pretreatment. In addition, the results of general photographs and H&E staining showed that ethanol-induced gastric damage induced multiple hemorrhagic erosions and ulcers, which were alleviated significantly by pretreatment with AC-YVAD-CMK.Conclusions: Our results proved that Inhibiting Caspase-1 activation of NLRP3 inflammasome had a protective effect on cold-restraint stress and ethanol-induced acute gastric injury in mice. The protective effect might be involved in attenuating inflammatory response and apoptosis.Part Three. The protective mechanism of inhibiting Caspase-1 activation in NLRP3 inflammasome on acute gastric injury in miceObjective: To discuss the protective mechanism of inhibiting Caspase-1 activation in NLRP3 inflammasome on mouse acute gastric injury.Methods: In order to evaluate the mechanismof inhibiting Caspase-1 activation in NLRP3 inflammasome on acute gastric injury in mice, we performed Western blot assays to measure the protein expression of P38, P-P38 and P-IκB.Results: MAPK/NF-κB signal pathways play quite an important role in acute gastric injury in mice. We found that with the pretreatment of AC-YVAD-CMK, protein expression of P-P38 was significantly lower than that in Injury group and Injury+Omeprazole group(P<0.05). Furthermore, AC-YVAD-CMK pretreatment also decreased over-expression of P-IκB induced by cold-restraint stress compared with that in Injury group and Injury+Omeprazole group(P<0.05).Conclusions: Our results proved that inhibiting Caspase-1 activation of NLRP3 inflammasome had a protective effect on cold-restraint stress and ethanol-induced acute gastric injury in mice. Its underlying mechanism might be associated with NF-κB and P38 MAPK signal pathways.