Mechanism Of Methamphetamine Inducing Intestinal Inflammatory Injury Via NLRP3 Inflammasome Overexpression

Background and Objectives:With the rapid development of economy and the acceleration of information globalization,the global illicit drug abuse problem keeps spreading and the number of drug abbusers keeps rising around the world.Resently,the number of people abbusing ICE surpassing the traditional drug-Heroin,which maked ICE the "King Drug".Chronic ICE abusing will cause injury to different kinds of organs and systems,but for lacking in ICE related injury mechanisms,it makes IEC abuser treatments less reasonable and efficient and calls for systemetic research of its related injury mechanisms about different kinds of organs and systems urgently.Methamphetamine(METH)is the main composed ingredient in ICE,which can quickly pass through the blood-brain barrier to the central nervous system,mainly causes hallucinations,delusions,paranoia,and even suicidal adverse physiological and psychological reaction.Besided,Methamphetamine,which can also reach several organs and systems such as Peripheral nervous system,Cardiovascular systems et.al,causes acute or chronic toxic efect and serious injury.Although Methamphetamine has been reported to cause inflammatory lesions such as ulcers and perforations in the digestive tract in several clinical cases,it still lacked in the injury mechanism in Digestive system,especially intestine.So we designed this experiment to find the intestinal inj ury mechanism of Methamphetamine.Pattern Recognition Receptors(PRRs)can recognize related harmful pathogens to lead to a series of inflammatory responses by activating Caspase.Nod-like Receptors(NLRs)are the intracellular pattern recognition Receptors,which are one of the main members of Pattern Recognition Receptors.NLRP3 receptor,the most popular Nod-like Receptor,can combine apoptosis with CARD chain protein(apoptosis-associated speck-like protein containing a CARD,ASC)and Caspase to generate a kind of a special polyprotein,NLRP3 inflammasome,which can activate mature Caspase-1 to generate the pro-inflammatory factor IL-1? and IL-18 resulted in several inflammatory signaling pathways activations.It has been verified that NLRP3 expresses widely in intestinal epithelial cells and NLRP3 inflammasomes can cause intestinal inflammatory inj ury.The activating of NLRP3 inflammasome needs 2 steps:Priming and Activating,in which NF-?b can participate in Priming step by increase the level of NLRP3.Toll-like receptor 4(TLR4),which widely expressed in intestinal cells,can be phosphorylated activing downstream Myeloid Differentiation Factor8 8(MyD88),IL-1 receptor associated kinase 1(IRAK1)and IL-1 receptor associated kinase4(IRAK4).TGF-Activated Kinase 1(TAK1)is Activated via ubiquitination in the classical pathway of NF-?b production(NF-?b regulatory dependence pathway,NF-?b essential modulator(NEMO)-dependent pathway).Therefore,intestinal pathogens can increase NF-?b through TLR4/NF-?b pathway,activate NLRP3 inflammasome and cause intestinal injury.In this study,Methamphetamine IEC-6 cells and C57BL/6 mice models were established.According to the obvious inflammatory injury in cells and intestinal tissues,it verified that Methamphetamine can cause intestinal inflammatory injury.After the addition of the NLRP3 inflamosome inhibitor MCC950,according that the intestinal inflammatory injury aggravated with NLRP3 inflammasome increasement and remitted with NLRP3 inflammasome decreasement after MCC950,it verified that Methamphetamine induced intestinal inflammatory injuryvia NLRP3 inflamosome overexpression.After the addition of the NF-?b inhibitor DHMEQ,according to the fact that the expression of NLRP3 inflammasome and related pro-inflammatory factors(IL-1 and IL-18)decreased with NF-?b decreasement after DHMEQ,it verified that Methamphetamine can induce NLRP3 inflammasome activation though TLR4/NF-?b pathway.Methods:After the establishment of Methamphetamine related IEC-6 cells and C57BL/6 mice intestinal inflammatory injury model,in the Methamphetamine IEC-6 cells modles,the number and morphology of the cells were observed,the changes in the Transepithelial Electrical Rsistance(TEER)of the cells were detected,and the expression of inflammatory factors(IL-6,IFN-?,TNF-?and NF-?b)in the cells were detected;in the Methamphetamine animal model,the changes of ileum tissue injury were observed through HE staining pathological sections,and the expressions of inflammatory factors(IL-6,IFN-?,TNF-?and NF-?b)in ileum tissues were detected.After adding MCC950 in Methamphetamine cell and animal model,whose inhibitory effect of NLRP3 inflammatory were confirmed though detecting NLRP3 inflammasome(NLRP3,ASC and Caspase-1)and its related inflammatory factors(IL-1? and IL-18)by Western Blot and q-PCR,we compared the changes of Transepithelial Electrical Rsistance(TEER),the percentage of apoptosis,the necroptosis related proteins RIPK1 and RIPK-3 with Western Blot and q-PCR in the Methamphetamine related IEC-6 cells modle and the expressions of inflammatory factors(IL-6,IFN-?,TNF-?and NF-?b)in the Methamphetamine related IEC-6 cells and mice modle.After adding DHMEQ in Methamphetamine cell and animal model,whose inhibitory effect of NF-?b were confirmed though detecting NF-?b by Western Blot and q-PCR,we compared the expression of TLR4,NLRP3 inflammasome(NLRP3,ASC and caspase-1)and its related pro-inflammatory cytokines(IL-1?and IL-18)in the Methamphetamine related IEC-6 cells and mice modle.by Western Blot and q-PCR.Results:Methamphetamine can cause intestinal inflammatory injury.1.The number of IEC-6 cells decreased and the morphology changed due to Methamphetamine.2.Methamphetamine decreased the tranepithelial resistance(TEER)of IEC-6 cells.3.Methamphetamine induced tissue damage in mice's ileum.4.Methamphetamine increased inflammatory factors(IL-6,IFN-?,TNF-?and NF-?b)in IEC-6 cells and ileum tissues of mice.Methamphetamine can cause intestinal inflammatory injury via NLRP3 inflammasome overexpression.1.Methamphetamine induced necoptosis via NLRP3 inflammarosome overexpression in cell modle,in which the percentage of apoptosis and the expression of necropoptosis-related proteins RIPK-1 and RIPK-3 increased depended on NLRP3 inflammarosome overexpression.2.Methamphetamine induced intestinal inflammatory injury via NLRP3 inflammarosome overexpression in cell and animal modle,in which,TEER decrease,inflammatory factors(IL-6,IFN-?,TNF-?and NF-?b)increased depended on NLRP3 inflammarosome overexpression.3.The NLRP3 inflammasome inhibitor,MCC950,could not inhibit ASC expression.4.The NLRP3 inflammasome inhibitor,MCC950,could not inhibit NF-?b expression.Methamphetamine can cause NLRP3 inflammasome overexpression through TLR4/NF-Kb pathway.1.Methamphetamine promoted the expression of TLR4 and NF-?b.2.The NF-?b inhibitor,DHMEQ,inhibited NF-?b expression without affecting TLR4 expression.3.The NF-?b inhibitor,DHMEQ,inhibited the expression of NLRP3 inflammasome and its related pro-inflammatory cytokines(IL-1? and IL-18).Conclusions:Methamphetamine induced inflammatory injury in IEC-6 cells and C57BL/6 mice ileum tissues,which led to cell number decreasement,morphological changes,TEER decreasement,inflammatory lesions in HE staining pathological sections and increased inflammatory factors(IL-6,IFN-?,TNF-?and NF-?b)in Methamphetamine cell and mice modle.Methamphetamine induced intestinal inflammatory injury via NLRP3 inflammarosome overexpression in cell an animal modle,in which the number of apoptosis decreased,necroptosis related proteins RIPK-1 and RIPK-3 increased,TEER increased,inflammation factor(IL-6,IFN-?,TNF-?)increased cells and the ileum tissue inflammation factor(IL-6,IFN-?,TNF-?)in Methamphetamine related cell and mice modle depended on NLRP3 inflammaosome overexpression and alleviated by NLRP3 inflammasome inhibitor MCC950;Methamphetamine promoted NLRP3 inflammasome overexpression through TLR4/NF-?b pathway,in which NLRP3 inflammasomes(NLRP3,ASC and caspase-1)and its related pro-inflammatory cytokines(IL-1? and IL-18)increased depended on NF-?b and alleviated by NF-?b inhibitor DHMEQ.Hence,Methamphetamine can induce intestinal inflammatory injury via NLRP3 inflammasome overexpression through TLR4/NF-?b pathway.