| The necrosis of liver tissue and regional inflammatory response were involved in chronic liver inflammation induced by xenobiotics,leading to further liver fibrosis and cirrhosis,which has become one of the most important public health problems.Duo to the liver sinusoidal structure and abundant hepatic immune cells,xenobiotics promote the formation of the heptic regional immune microenvironment via crosstalk between hepatocytes and hepatic immune cells,to regulate inflammatory injury.Among them,liver resident Kupffer cells(KC)account for 80%-90%of tissue macrophages of the whole body,which respond to intrahepatic dangerous signals quickly.In response to endogenous or exogenous dangerous signals,NLRP3 inflammasomes,the cytoplasmic protein complex to produce pro-inflammatory cytokines,result in pyroptosis,an immungentic cell death.NLRP3 inflammasomes are closely related to liver inflammation via the release of damaged-associated molecular patterns and IL-1β,to mediate cellular communication and immune cascade respectively.Cyclooxygenase-2(COX-2)is induced by xenobiotics or pro-inflammatory cytokines.For example,aflatoxin B1(AFB1)upregulated the expression of COX-2 in liver tissue of rat.It has been reported that recruitment and assembly of NLRP3 on mitochondria were reduced with COX-2 intervention through decreasing mitochondrial oxidative damage in lipopolysaccharide-treated murine marcrophages.However,the mechanism that COX-2,an endoplasmic reticulum(ER)resident protein,translocates to mitochondria and regulates NLRP3 inflammasomes activation remains to be farther studied.Objectives:We established a AFB1-treated model of upregulated COX-2 and inflammatory hepatotoxicity in hepatocytes and liver,to demonstrate the ER quality control(ERQC)mechanism that regulated the expression,distribution,and degradation of COX-2 involved in NLRP3-dependent pyroptosis of hepatocytes;to explore the hepatic regional immune mechanism that DAMPs released from pyroptotic cells activate immune cascade.This study provided potential targets for the intervention of AFB1-induced liver inflammatory diseases.Methods:(1)GEO analysis:The mRNA levels of PTGS2 and caspase-related genes in HepaRG cells treated with AFB1 were obtained from GEO database(GSE25844).And the linear correlation of PTGS2 with indicated genes was analyzed.(2)In vivo:C57BL/6 mice were intragastrically administered with AFB1(1 mg/kg)or celecoxib(CELE,30 mg/kg),a selective inhibitor of COX-2,every other day for 4 weeks to establish an exposure model and an intervention model.The blood and liver tissue samples were performed as follows:①The levels of AST and ALT in serum or liver homogenate were detected.②The ratio of F4/80+ KC in liver non-parenchymal cells was analyzed by flow cytometry(FCM).③The pathological changes of liver tissues were observed by H&E staining.④The collagen deposition of liver tissues was determined by sirius red staining.⑤The mRNA levels of phase Ⅰ metabolic enzymes,phase Ⅱ metabolic enzymes,inflammatory factors,and Ptgs2 in liver tissues were analyzed by real-time quantitative PCR(qRT-PCR).⑥The levels of COX-2,NLRP3 inflammasomc-(p65,NLRP3,ASC)and pyroptosis-related(p10,IL-1β,GSDMD)proteins in liver tissues were detected by western blot(WB).⑦The expression and localization of COX-2 and caspase 1 in liver tissues were observed by immunohistochemistry(IHC).⑧IL-1β secretion in serum and liver homogenate were analyzed by enzyme-linked immunosorbent assay(ELISA).(3)Ex vivo:The murine primary hepatocytes and KC were isolated by two-step liver perfusion or immunomagnetic beads respectively.And cells were treated with AFB1(1 μM,24 h)or CY-09(1μM,24 h)after adherence for 48 hours.①The levels of COX-2,pro-inflammatory signaling-related proteins(AP-1,c-JUN,STAT3),NLRP3 inflammasome-and pyroptosis-related proteins were detected by WB.②IL-1β secretion in the supernatant was analyzed by ELISA.(4)In vitro:Differentiated human HepaRG cells were treated with AFB1(1 μM,24 h).①The mRNA levels of phase I metabolic enzymes were detected by qRT-PCR.②Pyroptosis-related indicators:pyroptotic bodies and plasma membrane rupture were observed by transmission electron microscopy(TEM).The fluorescence signal of GSDMD was detected by immunofluorescence(IF)with confocal microscopy.The levels of pyroptosis-related proteins were detected by WB.The ratio of pyroptotic cells of caspase 1 and PI double-positive was analyzed by FCM.The integrity of plasma membrane was detected by lactate dehydrogenase(LDH)release assay.And IL-1β secretion in the supernatant was detected by ELISA.③NLRP3 inflammasomc-related indicators:the levels of NLRP3 inflammasome-related proteins were detected by WB.And the fluorescence signal of NLRP3 and its co-localization with mitochondria were detected by IF.④ER reduction-oxidation(redox)-related indicators:the level of total ROS was analyzed by chemiluminescent assay.And the levels of ER-redox-related proteins(ERO1α,PDI)and unfolded protein response(UPRER)-related proteins were detected by WB.⑤The protein interaction between COX-2-NLRP3 and COX-2-B55δ were analyzed by Co-IP.⑥CY-09,a specific inhibitor of NLRP3,was used to establish an intervention model.And the indicators of NLRP3 inflammasome activation and pyroptosis were detected.⑦Small interfering RNA(siRNA)of COX-2(siPTGS2)was used to establish an intervention model.The expression of COX-2,and the indicators of NLRP3 inflammasome activation and pyroptosis were detected.⑧siRNA of ERO1α(siERO1A)and ERO1α-overexpressed human hepatic L02 cells(L02-ERO1A)were applied.The expression and distribution of ERO1α and COX-2,and the indicators of NLRP3 inflammasome activation and pyroptosis were detected.⑨siRNA of B55δ(siPPP2R2D)was used to establish an intervention model.The interaction and distribution of B55δ and COX-2,and the indicators of NLRP3 inflammasome activation and pyroptosis were detected.⑩Targeted at serine 582,583,584,586 and 601 of COX-2,L02 cells with mimic phosphorylation(D)or dephosphorylation(A)of COX-2-overexpression were applied.The expression and distribution of COX-2,and the indicators of NLRP3 inflammasome activation and pyroptosis were detected.Results:(1)GEO analysis:Compared with the control group,the mRNA levels of PTGS2 and CASP1 in AFB1-treated HepaRG cells were upregulated and positively correlated.(2)In vivo:Compared with the control group,in AFB1-treated group,①the levels of AST and ALT of liver homogenate were increased;②the ratio of F4/80+ KC was increased(5.4%to 16.57%,P<0.05);③the quantity of inflammatory foci in liver tissues was increased;④collagen deposition of liver tissues was observed;⑤the mRNA levels of phase I metabolic enzymes,inflammatory factors,and Ptgs2 in liver tissues were upregulated,and the mRNA levels of phase Ⅱ metabolic enzymes were decreased;⑥the levels of COX-2,NLRP3 inflammasome-and pyroptosis-related proteins in liver tissues were increased;⑦the positive staining of COX-2 and caspase 1 of liver tissues were enhanced;⑧IL-1β secretion in serum and liver homogenate were increased.Compared with AFB1-treated group,the indicated phenomena above were reversed in AFB1-treated combined with CELE group.(3)Ex vivo:①Compared with the control group,the levels of COX-2,NLRP3 inflammasome-and pyroptosis-related proteins were enhanced in primary hepatocytes and KC of AFB1-treated group,accompanied with increased secretion of IL-1β in the supernatant.②In primary hepatocyte-KC co-culture model,compared with the control group,AFB1 induced the indicated phenomena above;compared with the AFB1-treated group,CY-09 inhibited the indicated phenomena above.③In primary hepatocyte-KC co-culture model of un-treated mice,compared with monolayer culture,AFB1 induced the indicated phenomena above.(4)In vitro:Compared with the control group,in AFB1-treated HepaRG cells,①the mRNA levels of phase I metabolic enzymes were increased;②the formation of pyroptotic bodies and plasma membrane rupture were obsevered;fluorescence signal of GSDMD was increased;the ratio of caspase 1 and PI double-positive cells was increased(7.18%to 19.22%,P<0.05);the level of pyroptosis-related proteins were upregulated,and LDH release and IL-1βsecretion in the supernatant were increased.③the level of NLRP3 inflammasome-related proteins were upregulated;the mitochondrial translocation of NLRP3 and the co-localization of NLRP3 with ASC were enhanced.④the level of ROS was increased,and the level of ER-redox-and UPRER-related proteins were upregulated.⑤the protein interaction of COX-2-NLRP3 and COX-2-B55δ were enhanced.Compared with AFB1-treated HepaRG cells,⑤the ratio of caspase 1 and PI double-positive cells was decreased(19.22%to 10.67%,P<0.05);LDH release and IL-1β secretion in the supernatant were reduced with CY-09 intervention.⑦the expression of COX-2 and NLRP3,and the co-localization of NLRP3 with mitochondria were reduced;the level of NLRP3 inflammasome-and pyroptosis-related proteins were decreased,and LDH release and IL-1β secretion in the supernatant were reduced with COX-2 inhibition.⑧the nuclear translocation of ATF4 was reduced;the expression of NLRP3 and its co-localization with mitochondria were reduced;the level of ERO1α,COX-2,and NLRP3 inflammasome-and pyropotosis-related proteins were decreased,and LDH release and IL-1p secretion in the supernatant were decreased with ERO1α intervention.⑨the co-localization of NLRP3 with mitochondria were reduced;the level of B55δ,NLRP3 inflammasome-and pyroptosis-related proteins,and LDH release and IL-1β secretion in the supernatant were reduced;the co-localization and proteion interaction of COX-2-Derlin 1 were enhanced with B555 inhibition.⑩Compared with L02-pBabe cells,the levels of NLRP3 inflammasome-and pyropotosis-related proteins,and LDH release and IL-1β secretion in the supernatant were enhanced in L02-COX-2S601A cells.To compare phosphorylated-with dephosphorylated-COX-2-overexpressing cells at 5 serine sites respectively,the indicated phenomena of L02-COX-2S601D cells were decreased in comparision with L02-COX-2S601A cells.Conclusions:The study suggested the mechanism that dcphosphorylation of COX-2Ser601 activated NLRP3-dependent pyroptosis of hepatocytes,to mediate liver inflammatory injury via intercellular communication between hepatocytes and KC.The results revealed a novel model of dephosphorylated COX-2 on AFB1-induced hepatotoxicity,and provided a theoretical basis for COX-2 as an intervention target in liver inflammatory injury induced by xenobiotics. |
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