Effect Of Estrogen Receptor On Zearalenone Induced Intestinal Inflammatory Response And Its Regulatory Mechanism

Zearelenone(ZEA)is a kind of mycotoxin produced by fusarium strains,which are widely found in maize,grain,wheat,barley,oat,and other crops,and seriously threatens the health of human beings,poultry and domestic animals.Cinsidering the estrogen-like action of ZEA,many studies on toxicity of ZEA are focus on its reproductive toxicity,but the enterotoxicity and immunotoxicity researches are scarce.The gut is an important part of the body for the digestion and absorption of nutrients and the first barrier against pathogens,food pollutants,chemical drugs and natural toxins.The animal intestinal tract has established a sophisticated immune system.In this immune system,cytokines secreted by epithelial cells can stimulate and regulate the function of immune cells,therefore it plays an important role in intestinal immunity.Because the ZEA exists widely in the feed,whether it will activate the intestinal inflammatory response,damage the intestinal barrier function,and the regulatory mechanism are still unclear.In this study,the oxidative stress and inflammatory response induced by ZEA in IPEC-J2 cells were first investigated.Then the ZEA-induced intestinal inflammation in mice was used to explore its regulatory mechanism.Based on the above results,the effect of ZEA on intestinal inflammation induced by 3%dextran sodium sulfate(3%DSS)in mice were further studied.Interestingly,the results showed that ZEA could alleviate 3%DSS-induced intestinal inflammation and inhibited the expression of NLRP3.Furthermore,the expression of ER subtype was changed from ERa to ERβ when high concentration ZEA was used.Therefore,we speculated that ZEA could relieve the inflammatory reaction in intestine by changing ER subtype expression.Then chromatin immunoprecipitation assay(Chip-qPCR),electrophoretic mobility shifts assay(EMSA)and dual luciferase reporter assay were used to demonstrate that ERa could directly regulate the transcription of NLRP3 gene.And ER specific agonist could promote the co-localization and assembly of NLRP3 inflammasome(NLRP3,ASC,Procaspase-1)with western blot and protein immunofluorescence analysis.The main research contents and results are as follows.1.Effect of ZEA on oxidative stress and inflammatory response in IPEC-J2 cellsIn this study,the IC50 of ZEA in IPEC-J2 cells was determined as13.59 ± 0.044 μg/mL.With the increase of ZEA concentration,the antioxidant enzymes(CAT,T-SOD,GSH-PX)activity decreased significantly and lipid peroxide malondialdehyde(MDA)increased significantly(P<0.05).Therefore,the imbalance of intracellular oxidant and antioxidant action will lead to the accumulation of reactive oxygen species(ROS),and further induce seriously damage to the mitochondrial membrane structure,especially to the mitochondrial inner membrane,which will eventually result in the decrease of mitochondrial membrane potential(MMP),increasing of mitochondrial permeability,and inducing mitochondrial dysfunction.In this study,fluorescence probes DCFH-DA and JC-1 were used to detect intracellular ROS and MMP,respectively.The results showed that ROS was significantly increased(P<0.05)with high dose ZEA(8 μg/mL)incubation in 2 hours,and the cells wrinkled.The change in the low-dose group was not significant compared to the control group.The MMP decreased with the increase of ZEA concentration,and the high concentration ZEA significantly reduced the MMP(P<0.05).The mitochondria of IEPC-J2 cells treated with ZEA appear abnormally enlarged or swollen,with an obvious loss of cristae and densematrix inclusions.Current studies have shown that increased ROS and damaged mitochondria could activate NLRP3 inflammasome,mature Caspase-1,regulate release of inflammatory cytokines such as interleukin-1β(IL-1β)and interleukin-18(IL-18).The results of western blot and ELISA showed that high concentration of ZEA could significantly activate the expression of NLRP3(NLRP3,ASC,Caspase-1)in IPEC-J2 cells(P<0.05),and promote the release of IL-1β and IL-18(P<0.05).Our experimental results indicate that ZEA can disrupt the oxidative balance of IPEC-J2 cells,cause oxidative stress and mitochondrial damage,further activate the NLRP3 inflammasome and promote the maturation and secretion of inflammatory cytokines.2.Effects of ZEA on intestinal inflammation in miceIn the study,the effect of high concentration of ZEA(4.5 mg/kg)on mice intestinal inflammation was studied,and 3%DSS induced colitis in mice was used as positive model.The results showed that the disease activity index(DAI)of ZEA-treated mice was significantly increased(P<0.05)and was lower than that of 3%DSS treated mice.The length of colon from ZEA-treated mice was obviously shortened(P<0.05).Histological analysis showed obvious inflammatory cell infiltration and tissue damage in the treated colon.The levels of IL-1β and IL-18 and neutrophil markers MPO were also significantly increased(P<0.05),and the expression of NLRP3 inflammasome(NLRP3,ASC,Caspase-1)were also significantly increased(P<0.05).In summary,high concentration ZEA could cause obvious inflammatory response in the intestinal tissue,which is mediated by the NLRP3 inflammasome signaling pathway.To verify the mechanism of NLRP3 inflammasome activation in the inflammation of mice intestinal tissue,abdominal macrophages from mice were isolated.The changes of ZEA treated abdominal macrophages were detected.The results showed that the concentration of IL-1β and IL-18 were significantly increased(P<0.05),and the expressions of NLRP3 inflammasome(NLRP3,ASC,Caspase-1)were also significantly increased(P<0.05).Studies on oxidative stress of mouse peritoneal macrophages showed that antioxidant enzyme activity was declined,MDA content and ROS levels were consistent with the results of IPEC-J2 cells.High concentration ZEA caused mitochondrial damage in mouse peritoneal macrophages.When antioxidant(NAC)was added to the cells,the contents of IL-1β and IL-18,the expressions of NLRP3 inflammasome(NLRP3,ASC,Caspase-1)were significantly decreased(P<0.05).In summary,the results of in vivo and in vitro experiments jointly identified that ZEA could cause oxidative stress and mitochondrial damage,further activate NLRP3 inflammasome,promote the maturation and release of inflammatory cytokines,and eventually lead to intestinal inflammation.These results support that the noncommunicable persistent diarrhea in farms may be associated with ZEA contamination in feed.3.Effect of ZEA on intestinal inflammatory response induced by 3%DSSThe intestinal inflammatory response model with the combined use of ZEA and 3%DSS in IPEC-J2 cells and mice were established.Interestingly,the DAI of ZEA+3%DSS treated mice was significantly lower than that of 3%DSS treated separately(P<0.05).The colon length of ZEA+3%DSS treated mice was also significantly longer than that of 3%DSS used separately(P<0.05).Histological analysis showed that the colon damage and inflammatory cell infiltration were alleviated in mice treated with ZEA+3%DSS.The contents of inflammatory cytokines IL-1β,IL-18 and neutrophil markers MPO were also significantly lower than those in the single treatment group(P<0.05).Additionally,the NLRP3 expression was significantly inhibited(P<0.05),while the expressions of ASC and Caspase-1 were not changed with ZEA+3%DSS treatment.In conclusion,ZEA had a certain alleviating effect on intestinal inflammation caused by 3%DSS.The relief of intestinal inflammation was due to the inhibition of NLRP3 expression.ZEA differs from other mycotoxins in that it has estrogen-like effects.Therefore,the expression of ER in different groups were further tested.Our results showed that the expression of ERa was observed in the control group,low concentration ZEA group,and 3%DSS group,while the expression of ERβ was observed in the high concentration ZEA group and ZEA+3%DSS group.That is the expression of ER changes from ERa to ERβ when high concentration ZEA treated.4.The regulation of ER on NLRP3 inflammasome signaling pathwayIn this experiment,the result suggests that the promoter sequence of NLRP3 contains the recognition motif of ER,which indicates NLRP3 may be the target gene of ER.The further study demonstrated that ER can directly regulate the transcription of NLRP3 gene through ChIP-qPCR,EMSA and dual luciferase reporter gene assay.The result showed ERa has significant transcriptional regulation effect on NLRP3 gene.Western blot and protein immunofluorescence assay confirmed that ERa specific agonists can promote the co-localization and assembly of NLRP3 inflammasome(NLRP3,ASC,Procaspase-1).The preliminary studies proved that ERa is a transcriptional regulator of NLRP3 and can directly regulate the transcription of NLRP3.In addition,ERa could promote the expression and assembly of NLRP3 inflammasome and further regulate the inflammatory response.To verify the experimental conclusion,we treated mice with specific ERa antagonist(AZD9496)and found that ERα-selective antagonist could significantly alleviate the intestinal inflammation caused by 3%DSS.All in all,this study proved that ZEA can activate NLRP3 inflammasome and cause intestinal inflammation by inducing oxidative stress and mitochondrial damage.When ZEA increases the inflammatory response,the expression of ER subtype will be changed.The effect of ERa on transcriptional regulation of NLRP3 gene and assembly of NLRP3 inflammasome were preliminarily elucidated.This study illustrated the intestinal immunotoxicity of ZEA and the regulation mechanism of ER on intestinal inflammatory response.This research provided theoretical basis for clinical studies on ZEA toxicity and rational drug use of IBD.