| Deoxynivalenol（DON）,also called vomitoxin,which is mainly produced by Fusarium graminearum,is a member of the type B trichothecenes family.DON can contaminate different crops and its by-products,which not only affects the output and quality of crops,but also has a wide range of toxic effects on human and animals.In animals that ingest mouldy feed or foods contaminated with DON may suffer from a series of symptoms such as diarrhea,vomiting,gastrointestinal hemorrhage,anorexia and weight lose,which result from the intestinal inflammation induced by DON.Besides,some works report that DON can lead to peripheral and central inflammation.Inflammasome is an intracellular complex consists of multiple proteins,which plays an important regulatory role in the inherent immune responses.With recognition of various pathogens and danger signals,the cysteine protease pro-caspase-1 of inflammasome was activated by autocatalytic cleavage to process pro-IL-1β to the proinflammatory form,as well as leading to cell death termed pyroptosis.At present,several inflammasomes have been reported that include NLRP1,NLRP3,NLRP6,NLRP7,NLRP12,NLRC4 and AIM2,in which the NLRP3 inflammasome was best characterized in mutiple cell lines,and series of reports demonstrate that NLRP3 is implicated in many inflammatory diseases.Common mechanisms implicated in inflammasome activation induced by different stimuli include K+efflux,lysosomal damage with release and activation of cathepsin B and reactive oxygen species（ROS）.In addition,src-family kinase and P2X7R protein may be involved in the activation of inflammatory induced by mycotoxin.This study sought to characterize whether DON can activate the NLRP3 inflammasome in murine RAW 264.7 macrophages and identify how DON regulates IL-1βsecretion.Experiment Ⅰ.The Ⅲeffect of DON on activation of NLRP3 inflammasome in murine RAW264.7 macrophagesThis part of test is aimed at analyze whether DON can induce NLRP3 inflammasome activation and IL-1β secretion in murine RAW 264.7 macrophages,DON-stimulated murine RAW 264.7 macrophages were used as an inflammation model.At first,the MTT assay was performed to determine the viability of murine RAW 264.7 macrophages stimulated with a series of concentration of DON,as 0.01,0.025,0.05,0.1,0.25,0.5 and 1μg/mL doses included.The resuts shows that 0.25～1 μg/mL concentration of DON can significantly reduce cell viability.Therefore,0.5 and 1 μg/mL concentration of DON were used to stimulate cells for 4 hours,after which the total cellular RNA was extracted and quantitative real time RT-PCR assay was performed to analyze the expression of pro-caspase-1,NLRP3,ASC and pro-IL-1β mRNA,which result indicates that DON strongly activated transcription of the NLRP3 and pro-IL-1β gene（p<0.05）,whereas pro-caspase-1 and ASC mRNA expression was not modified（p>0.05）.Then the cells was cultured with 1 μg/mL dose of DON for 6 or 12 hours and western blotting was performed on cell lysates to detect the protein expression of NLRP3,procaspase-1,caspase-1,ASC and proIL-1β.The result shows all the protein expression increased（p<0.05）.The biologically active IL-1β released to supernatants was significantly induced by DON（1μg/mL）at 12 hours,16 hours or 24 hours that showed in enzyme-linked immunosorbent（ELISA）assay.Finally,to study whether the IL-1β activated and secreted by DON is NLRP3,small interfering RNA assay was performed.We can conclude from the result that DON can induce IL-1β secretion through NLRP3 inflammasome pathway in murine RAW264.7 macrophages.Experiment Ⅱ.Study on mechanism of NLRP3 inflammasome activation induced by DON in murine RAW264.7 macrophagesThis study is aimed at characterize the specific mechanism by which DON induces the NLRP3 inflammasome activation in murine RAW264.7 macrophages.The first step is to analyze whether K+efflux,ROS and cathepsin B are implicated in NLRP3 inflammasome activation in response to DON.Therefore,the cells were pretreated with antioxidant BHA（10 μmol/L）,CA-07-Me（10 μmol/L,cathepsin Binhibitor）and KCl（12.5,25 and 50 mmol/L）for 30min,and then stimulate the cells with DON at dose of 1 μg/mL for 24 hours.The secreted IL-1β was detected by ELISA assay and IL-1β secretion was notably decreased as seen（p<0.05）.Besides,to study if Src tyrosine kinase and P2X7R are associated with IL-1β release induced by NLRP3 inllammasome activation,we first detected the protein expression of P2X7R and phosphate-Src induced by DON,which result show that DON can significantly induce the protein expression of P2X7R and phosphate-Src after 2 h.BBG（20 μmol/L,P2X7Rinhibitor）and PP2（5 μmol/L,Src tyrosine kinase inhibitor）primed cells for 30min,and 1 μg/mL concentration of DON was added for 24 h.As the ELISA assay shows that the IL-1β in supernatants was abolished by inhibitor compared with DON-induced control.SiRNA assay was taken to verify the result.The conclusion is that K+ efflux,ROS generation,the release of cathepsin B and Src-P2X7R pathway take important role in the activation of NLRP3 inflammasome induced by DON in murine RAW264.7 macrophages. |
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