| BACKGROUND AND OBJECTIVEGastric cancer is the second leading cause of cancer death worldwide,accounting for about 10%of all aggressive cancer.Although the pathogenesis of gastric cancer has not yet been fully understood,it is worth noting that the positive correlation between gastric cancer and environmental pollution has been confirmed,and the total number of cases and deaths of gastric cancer increases with a wide range of population changes and increasing environmental pollution in China.Perfluorodecanoic acid(PFDA)is a highly toxic perfluorinated fatty acids(PFCA),a persistent environmental pollutants.PFDA presents in water,food and air,,especially in China,we detected 139ng/L of PFDA in Beijing’s surrounding snow.PFDA intake is mainly from food and drinking water,and PFDA can accumulate in human blood and organs,the serum clearance half-life for several years.It has been reported that PFDA treatment can cause severe weight loss,bradycardia,hypothermia,and reduced serum thyroid hormone levels in rats.However,so far,little is known about the toxic effects of PFDA on the human body and its role in promoting malignant tumors.Our aim is to investigate the effect of PFDA on the activation of inflammasome and inflammatory regulation in gastric cancer cells AGS,and to study the molecular mechanism of secretion of IL-1β and IL-18 by activation of inflammasome NLRP3 in gastric cancer cells after PFDA treatment.We hope to further study the important role of chronic inflammation in the development of gastric malignancies and the impact of environmental pollutants on human health and life.METHODSWe selected gastric cancer cells AGS In good condition,,and cultivated them in bacteria free six-well cell culture clusters.AGS cells in six well plates were treated with specific concentrations of PFDA(75μM)for use as a next step.1.RT-qPCRThe total RNA of AGS cells treated with PFDA was extracted by Trizol,and the mRNA expression of related genes was measured by RT-PCR after reverse transcription by reverse transcription kit.2.Enzyme-linked immunosorbent assay(ELISA)The human gastric cancer cells AGS were treated in sterile six-well plates and treated with PFDA.The supernatant of the cell culture supernatant was collected after 72h,and the concentration was determined by ELISA kit immediately after centrifugation.3.Western blotThe total protein was extracted from RIPA(containing PMSF),and the protein concentration was determined by BCA method.Then we performed the Western blot experiment.4.siRNA interferenceThe siRNA of cIAP2 and control siRNA were transfected into gastric cancer cell AGS by siRNA transfection reagent.The rest of the procedure was the same as RT-PCR.5.Hematoxylin and eosin(H&E)stainingPFDA was added to the drinking water of C57BL/6 mice at a dose of 25 mg/kg/d.In the control group,the same amount of DMSO was added to the drinking water.After two weeks,the gastric tissue was removed and stained with HE.RESULTSCompared to control cells,PFDA significantly stimulated the secretion of IL-18 and IL-1β,and increased the expression of mRNA levels.By RT-PCR and Western blot,we found NLRP3’s upregulation promoted the secretion of IL-1β and IL-18.The expression of cIAP1/2,c-Rel and p52 was analyzed,and the results showed that the enhancement of inflammatory mediators caused by PFDA resulted in increased mRNA expression in all test genes.The use of cIAP2 siRNA and NFκB reporter assays provided additional evidence that these genes were involved in PFDA-induced assembly of inflammatory mediators.In addition,the secretion of IL-1β and IL-18 was increased in the gastric tissue of PFDA-treated mice,and the disordered arrangement of epithelial cells and inflammatory cell infiltration were also observed in PFDA-treated gastric tissues.CONCLUSION1.PFDA can induce the activation of inflammasome NLRP3 in gastric cancer cell AGS.2.After treated with PFDA,AGS activates caspase-1 through inflammasome NLRP3,and the expression of IL-1β and IL-18 increased and the secretion increased.3.PFDA regulates inflammasome assembly and inflammation in gastric cancer cell AGS. |
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