Mechanism Of Gastric Inflammation Induced By Nonylphenol Via NF-κB/NLRP3 Signaling Pathway

Chronic gastritis is an chronic disease with high incidence rate and complex process of migration,which makes it hard to be cured.More than half of people in the world are suffering from this disease.Epidemiological investigation showed that the intake of environmental endocrine disruptors was positively correlated with the incidence of gastric cancer.Nonylphenol(NP),a kind of typical environmental endocrine disruptor,exists widely.It enters the human body through the digestive system commonly.Studies have shown that NP is difficult to remove from the body and exists in the gastrointestinal tract for a long time.However,whether long-term exposure to NP can lead to gastric inflammation is rarely reported,the activation of NF-κB/NLRP3 signaling pathway is an important mechanism to regulate the inflammatory response.Therefore,this study intends to find it out through animal experiments and cell experiments.For the first part,we observed the pathological changes of gastric tissue and detected the expression of inflammatory factors such as tumor necrosis factor-α(TNF-α)and interleukin-18(IL-18)in the stomach of male SD rats after chronic NP exposure after 33 weeks.Meanwhile,the related proteins and genes expression of NF-κB/NLRP3 pathway were detected;In the second part,we used normal gastric epithelial GES-1 cells as the model.At first we injected NP to verify the results of the first part,and then Pyrrolidinedithiocarbamate ammonium(PDTC)was given to inhibit NF-κB pathway,in the final part we detected the content of inflammatory factors and the expression of NLRP3 inflammasome related proteins.Above processes were used to confirm whether NP can lead to gastric inflammation and what role of NF-κB/NLRP3 signaling pathway plays.Part Ⅰ Effects of nonylphenol on gastric mucosal inflammation and NF-κB/NLRP3 signaling pathway in male ratsObjective: To explore the effects of NP ingestion through digestive tract on gastric mucosal inflammation and NF-κB/NLRP3 signaling pathway in ratsMethods: Forty eight male Sprague Dawley rats were randomly divided into four groups:control group(corn oil),low dose group(0.4 mg/kg NP),middle dose group(4 mg/kg NP)and high dose group(40 mg/kg NP),with 12 rats in each group.After 33 weeks of gavage exposure,the content of NP in gastric tissue was detected by HPLC,The pathological changes of gastric mucosa were observed by hematoxylin eosin staining.The levels of TNF-α and IL-18 were detected by ELISA;mRNA expression of NF-κB,NLRP3 were detected by RT-PCR,Caspase-1 and IL-1β;Western blot was used to detect gastric mucosal injury factors TFF1 and the expression of NF-κB/NLRP3 pathway related proteins(NF-κB p65,IκB,p-NF-κB p65,p-IκB,NLRP3,Caspase-1,Pro-caspase-1,IL-1β,Pro-IL-1β).Results : With the increase of NP concentration,the content of NP in gastric tissue increased gradually.The content of NP in the middle and high dose NP group was significantly higher than that in the control group and the low dose NP group(P < 0.05),and the content of NP in the high dose NP group was higher than that in the middle dose NP group(P < 0.05);the epithelial cells of gastric mucosa in NP middle and high dose groups were necrotic and exfoliated,and the glands were disordered,the gland cavity was expanded obviously,the chief cells reduced,the inflammatory cells were infiltrated,and the red blood cells appeared in the mucosa;The content of TNF-α in NP high-dose group was significantly higher than that in the control group,NP low dose group and NP middle dose group(P < 0.05),and the level of IL-18 in NP middle dose group and NP high dose group was significantly higher than that in the control group and NP low dose group(P <0.05);Compared with the control group,the expression of NLRP3 mRNA in the low,middle and high dose NP groups increased significantly(P < 0.01).The expression of NF-κB mRNA and IL-1β mRNA in each NP exposure group increased significantly(P <0.001).Compared with the control group and the low dose NP group,the expression of Caspase-1 mRNA in the middle and high dose NP groups increased significantly(P <0.001);Compared with the control group,the expression of TFF1 protein decreased in the middle and high dose groups(P < 0.05);Compared with the control group,the expression of phosphorylated protein p-NF-κB p65 and p-IκB of NF-κB signaling pathway increased in the middle and high dose NP groups(P < 0.05);compared with the control group,the expression of NLRP3 inflammasome related protein NLRP3,Pro-il-1β,Caspase-1 and Pro-caspase-1 increased significantly in the middle and high dose NP groups(P < 0.05)The expression of IL-1β protein in NP high-dose group was significantly higher than that in the control group,low-dose and middle dose NP groups(P < 0.05).Conclusion: Long term NP exposure can induce gastric mucosal inflammation in rats,and NP can activate NF-κ B/NLRP3 signaling pathway in gastric tissuePart Ⅱ Study on the mechanism of inflammatory response induced by nonylphenol in GES-1 cells based on NF-κB/NLRP3 signaling pathwayObjective: To explore the mechanism of NP-induced inflammatory response in GES-1cells based on the NF-κB/NLRP3 pathway by using model of gastric epithelial GES-1cells.Methods: 1.After GES-1 cells were treated with different concentrations of NP for 24 hours,CCK8 was used to detect the cell viability and determine the working concentration of NP: Control group(0 μmol/L NP),low dose group(2.5 μmol/L NP),middle dose group(40 μmol/L NP)and high dose group(60 μmol/L NP)were set,The levels of TNF-α and IL-18 were detected by ELISA 24 hours after NP treatment;The expression of NLRP3 AND NF-κB p65 protein was detected by immunofluorescence;The protein expressions of NF-κB p65,IκB,p-NF-κB p65,p-IκB,NLRP3,Caspase-1 and IL-1β were detected by Western blot.2.The experiment was divided into control group(0 μmol/L NP),PDTC group(15μmol/L),PDTC + NP group(15 μmol/L PDTC + 40 μmol/L NP)and NP group(40 μmol/L NP).In PDTC + NP group,PDTC was pretreated for 1 hour and then co-treated with NP for 24 hours.The levels of TNF-α and IL-18 were detected by ELISA.The expression of NLRP3 was detected by immunofluorescence.The protein expressions of NF-κB p65,p-NF-κB p65,NLRP3,Pro-caspase-1,Caspase-1,Pro-il-1β and IL-1β were detected by Western blot.Results: 1.With increase of NP concentration,the cell viability decreased;compared with the control group,the contents of TNF-α and IL-18 in NP middle and high dose groups increased(P < 0.05);the protein expressions of NF-κB p65,p-iκb and IκB in NP middle and high dose groups increased(P < 0.05);Compared with the control group,the protein expression of NLRP3,Caspase-1,IL-1β and pro-il-1β increased in NP exposed group,and the protein content of NLRP3 increased in NP exposure groups(P < 0.05).2.The protein expressions of NF-κB p65 and p-NF-κB p65 in NP group were significantly higher than those in other groups(P < 0.05),but there was no significant difference between PDTC group and PDTC + NP group(P > 0.05);the contents of TNF-α and IL-18 in NP group were significantly higher than those in PDTC + NP group(P < 0.05);compared with control group,PDTC group and PDTC + NP group,the protein expressions of NLRP3,pro-caspase-1 and IL-1β in NP group were increased(P < 0.05);The protein expression of pro-il-1β and Caspase-1 in NP group was higher than that in control group and PDTC group(P < 0.05).Conclusion: NP could induce inflammation in GES-1 cells,which is related to NF-κB/NLRP3 signaling pathway.