| Background:Ulcerative colitis（UC）is a chronic inflammation of the colon with recurrent characteristics,and its main symptoms include abdominal pain,diarrhea,and bloody stools.ulcerative colitis is a common type of idiopathic inflammatory bowel disease（IBD）worldwide.So far,its complex pathogenesis includes:genetic susceptibility,epithelial barrier defects,Intestinal microecology disorder,immune disorders and environmental factors[1],the incidence of UC is high in developed countries（>0.3%）and increases rapidly in newly industrialized countries（annual percentage change increased by 14.9%）[2,3].Studies have shown that the cancer rate of ulcerative colitis after 20 years of chronic disease is 7.2%[4].The principles of clinical treatment are to control acute attacks,maintain remission,reduce recurrence,and prevent complications.Most patients have a chronic course and poor quality of life.Therefore,if the pathogenesis of ulcerative colitis can be clarified,it is extremely important to take effective intervention measures early and timely to improve the quality of life of patients and provide guarantee for the prognosis.The concept of"inflammasome"was first proposed by Martinon in 2002[5],who found that Nod-like receptors,NLR protein,apoptosis-associated speck-like protein containing CARD,ASC,cysteine-containing aspartate protein hydrolase（Caspase）can activate caspase,and then promote the maturation and secretion of cytokines IL-1βand IL-18 to participate in inflammatory responses[6].A variety of inflammasomes have been identified,named NLRP1,NLRP3,NLRC4 and AIM2inflammasomes according to receptor proteins.Previous studies have found that NLRP3 inflammasome plays an important role in a variety of cardiovascular diseases,neurodegenerative diseases and tumorgenesis.For example,Samuel found that targeting NLRP3 inflammasome can treat cardiovascular fibrosis[7],and Heneka found that in mouse models of Alzheimer’s disease,activation of NLRP3 of inflammasome drives the accumulation of amyloid protein and exacerbates neuroinflammation[8].In the context of intestinal inflammation,Bauer et al found that NLRP3 knockout mice in a dextran sodium sulfate（DSS）-induced acute colitis model had milder colitis than wild-type（WT）mice and lower levels of pro-inflammatory cytokines in colon tissues.NLRP3 is associated with the severity of ulcerative colitis[9].In addition,the physical barrier composed of intestinal epithelium can isolate harmful substances in the intestinal lumen.When injury occurs,epithelial cells can restore the integrity of the intestinal barrier through a series of events such as proliferation,differentiation and migration[10].Zhao J et al found that MCT4 disrupts the normal epithelial barrier of colon in ulcerative colitis and exacerbates colonic inflammation by increasing the expression of NLRP3 and increasing the secretion of IL-1βinduced by NLRP3[11].In conclusion,NLRP3 is an important effector molecule that promotes the inflammatory response and plays an important role in ulcerative colitis.However,the regulated mechanism of NLRP3 has not been fully elucidated and needs further study.Studies have reported that the ubiquitin-like protein FAT10（the human HLA-F adjacent transcript 10,FAT10）gene plays an important role in the occurrence and development of various diseases in the body.As an ubiquitin-like protein,the encoded protein is degraded by covalently binding the targeted substrate protein to the 26S proteasome,and this process of targeting the substrate is called FAT10ylation[12].FAT10 participates in various basic biological functions of cells,including signal transduction,autophagy,DNA damage repair,and cell cycle regulation,through FAT10ylation of its substrate.For example,FAT10 promotes the invasion and metastasis of liver cancer cells by activating theβ-catenin/TCF4 signaling pathway[13].Furthermore,FAT10 protects the myocardium from ischemic injury by inhibiting SIRT1-mediated autophagy[14].In addition,when cells occurred with DNA damage,the highly expressed FAT10 can inhibit the process of DNA damage response（DRR）by binding proliferating cell nuclear antigen（PCNA）to promote its proteasomal degradation[15].However,in normal tissues,FAT10 is mainly expressed in the spleen and thymus[16],suggesting that it may play an important role in immunity.In summary,we speculate that FAT10 may be involved in the process of colonic inflammatory response,but the specific mechanism has not been fully elucidated and needs to be further explored.Objective:In view of the above research progress at home and abroad and the basis of our previous work,we speculate that NLRP3 is a downstream effector gene of FAT10aggravating ulcerative colitis in mice,and FAT10 promotes the maturation and secretion of IL-1βby stabilizing NLRP3.In order to prove this hypothesis,in this study,the mouse body weight,colon length,colon tissue HE staining and other techniques were used to confirm the successful establishment of ulcerative colitis in mice,q RT-PCR,Western blot,and immunohistochemistry were used to detect the ulcerative colitis in mice.Infiltration of macrophages in colon tissue in a colitis model.The mouse primary macrophages were further extracted to determine the expression of FAT10,NLRP3 and IL-1β.The recovery experiments and inhibitor experiments proved that FAT10 promotes the maturation and secretion of IL-1βby stabilizing NLRP3.Furthermore,various in vitro experiments such as GST-pulldown,IP,and ubiquitination experiments were used to demonstrate the complex molecular regulation mechanism between FAT10 and NLRP3.Finally,FAT10 knockout mice were used to construct an ulcerative colitis model to demonstrate that ulcers in FAT10knockout mice the inflammatory degree of colitis was milder than that of wild mice.The above results provide new targets and theoretical basis for the treatment of ulcerative colitis.Methods:1.A mouse model of acute colitis was induced by feeding with 3%dextran sodium sulfate（DSS）for one week.During the establishment of the model,the body weight,stool consistency and blood in the stool were measured to draw the disease activity index.After the model was successfully constructed,the mice were sacrificed and their colon tissues were taken,and fluorescence quantitative PCR,Western blot,HE staining,immunohistochemical staining,primary extraction and other experiments were used to detect the levels of FAT10,NLRP3 and FAT10,NLRP3 and m RNA in primary colon macrophages in the model group and control group.The expression of IL-1β,to clarify the expression changes of FAT10 in colitis macrophages.2.In the immortalized mouse macrophage leukemia cell RAW264.7,the changes of NLRP3 and IL-1βafter endogenous interference or overexpression of FAT10 were detected by q RT-PCR,Western blot,Elisa and other experiments.Next,exogenous addition of TNFαand IFNγstimulated the expression of FAT10,and the changes of NLRP3 and IL-1βwere observed by q RT-PCR,Western blot,Elisa and other experiments.In order to further prove the upstream and downstream regulatory relationship among FAT10,NLRP3 and IL-1β,we designed a reverse experiment:firstly,a stable transfected RAW264.7 was constructed using mouse-derived lentiviral plasmids overexpressing FAT10 and sh-FAT10 lentiviral plasmids cell lines（RAW264.7 over-FAT10 and RAW264.7 sh-FAT10）,interfered with NLRP3 in RAW264.7 over-FAT10,and detected the expression of FAT10,NLRP3 and IL-1βby Western blot,overexpress NLRP3 in sh-FAT10 RAW264.7 cells,the expression of FAT10,NLRP3 and IL-1βwas detected by WB.In addition,we also used the NLRP3specific inhibitor MCC950 to perform the recovery experiment,and Western blot and Elisa were used to detect the changes of FAT10,NLRP3 and IL-1β.Conclusions were drawn from the above experiments:In macrophages,FAT10 promoted the secretion of IL-1βby up-regulating the expression of NLRP3.3.It has been reported that FAT10 can activate the NF-κB signaling pathway,and the NF-κB signaling pathway can promote the transcription of NLRP3.We put forward the hypothesis that FAT10 can promote the transcription of NLRP3 by activating the NF-κB signaling pathway.In order to prove the above hypothesis,experiments such as dual luciferase reporter gene and cell fluorescence were used to prove that FAT10 can promote the nuclear translocation of p65 protein and increase the transcriptional activity of NF-κB signaling pathway.The cell transfection was further used for recovery experiments,q RT-PCR and dual-luciferase reporter gene were used to detect the transcriptional activity of NLRP3,and it was determined that FAT10 could promote the transcription of NLRP3 through the NF-κB signaling pathway.It has been reported in the literature that the binding region of NF-κB and the NLRP3 promoter is detected after mutation of the binding region above,and the changes in the transcription level of NLRP3 after changing FAT10.4.As a ubiquitin-like protein,FAT10 also regulates the level of post-translational modification of substrate proteins.The ubiquitin-proteasome degradation of NLRP3protein was demonstrated using the proteasome inhibitor MG132.To further clarify the specific molecular mechanism,the first step:IP,confocal experiments and GST pull-down experiments to prove whether FAT10 binds to NLRP3.Step 2:Prove that FAT10 is involved in NLRP3 protein degradation.The proteasome inhibitor MG132was added to observe the changes of NLRP3 protein expression after changing FAT10.Step 3:GST pulldown experiments proved that FAT10 competes with ub to bind NLRP3.Step 4:In vivo ubiquitination assay to detect changes in the ubiquitination level of NLRP3 and its protein level after changing FAT10.Using the FAT10-p62complex as a positive control,it was analyzed whether the FAT10-NLRP3 complex was degraded by the proteasome pathway.The only receptor of FAT10,Rpn10,was further changed,and the changes of FAT10-NLRP3 complex were analyzed.5.The FAT10 knockout mice and WT mice were used to construct ulcerative colitis.The comparison of the general state of the mice and the severity of local inflammation in the colon between the two groups of FAT10 mice and WT mice were observed,and primary macrophages were extracted from the colons of the two groups.Cell fluorescence experiments,q RT-PCR and WB experiments were performed to detect the expression intensity of NLRP3 and IL-1β.Results:1.The results of disease activity index（DAI）,colon length,and colon HE showed that the acute ulcerative colitis in mice was successfully constructed.Colon primary macrophages were extracted for RNA-seq detection.Compared with the colonic macrophages in the normal group,the expressions of NLRP3,IL-1β,FAT10,etc.in the cells of the model group were significantly increased.The protein expression of primary macrophages in the two groups was detected by q RT-PCR and Western blot,and it was found that the expression of NLRP3,IL-1βand FAT10 in the model group was higher than that in the normal group.2.Knockdown of FAT10 in macrophage RAW264.7 resulted in decreased expression of NLRP3 and IL-1β,whereas overexpression of FAT10 could upregulate the expression of NLRP3 and IL-1β.Elisa experiments demonstrated that IL-1βsecretion of RAW264.7 was decreased after interfering with FAT10,whereas IL-1βsecretion was increased when FAT10 was overexpressed.After stimulating the expression of endogenous FAT10 in macrophages with TNFα+IFNγ,it was found that after the increase of endogenous FAT10 expression,the m RNA and protein expressions of NLRP3 and IL-1βincreased,and the secretion of IL-1βincreased.The recovery experiment proved that the increase of IL-1βexpression caused by overexpression of FAT10 could be recovered by interference with NLRP3,while the decreased expression of IL-1βafter interference with FAT10 was increased by overexpression of NLRP3.When FAT10 was overexpressed and NLRP3 inhibitor MCC950 was added,the increase in IL-1βexpression was restored by MCC950.Elisa detection of IL-1βsecretion was consistent with Western blot results.3.The results of the dual-luciferase reporter gene assay showed that when FAT10 was overexpressed,the transcriptional activity of the NF-κB signaling pathway was increased,the downstream NLRP3 transcriptional activity was increased,and the m RNA expression was increased.The results of cytofluorescence assay showed that the nuclear translocation of p65 protein occurred.The results of the recovery experiments showed that overexpression of FAT10 also interfered with p65,and the transcriptional activity of downstream NLRP3 decreased,while interference with FAT10 and overexpression of p65 increased the transcriptional activity of NLRP3.These results demonstrate that FAT10 regulates NLRP3 transcription through the NF-κB signaling pathway.After transfection of the NLRP3 plasmid with a mutation in the promoter-binding region,FAT10 was overexpressed,but the transcriptional activity of NLRP3 did not change,suggesting that after the mutation of the binding region,FAT10 lost its role in promoting the transcription of NLRP3.4.Western blot results showed that the expression of NLRP3 protein increased in a time-dependent manner after adding the proteasome inhibitor MG132,suggesting that NLRP3 protein was degraded by the proteasome pathway.Confocal experiments demonstrated that NLRP3 was spatially consistent with FAT10,and IP experiments demonstrated that NLRP3 was bound to FAT10.FAT10 was changed with or without MG132.Western blot results showed that FAT10 could stabilize NLRP3 protein without adding MG132.After adding MG132,NLRP3 protein did not change after changing FAT10.It is proved that FAT10 is involved in the process of NLRP3 protein degradation.GST pull-down experiments showed that FAT10 was changed in dose,the FAT10-NLRP3 complex was gradually increased,and the ub-NLRP3 complex was gradually decreased,which proved that FAT10 competes with ub to bind NLRP3protein.In vivo ubiquitination experiments demonstrated that NLRP3 ubiquitination decreased when FAT10 was overexpressed,and increased NLRP3 ubiquitination when FAT10 was disturbed.The FAT10-p62 complex was further used as a positive control to prove that the FAT10-NLRP3 complex was not degraded by the proteasome pathway,and the FAT10-NLRP3 complex did not change when the receptor Rpn10,which is a substrate protein for FAT10 degradation,was changed.5.FAT10-/-knockout mice and WT wild mice were used to construct a colitis model,and it was found that FAT10-/-mice had less severe colonic inflammation and systemic state than wild mice,more complete colonic physiological structure,and less macrophage infiltration.Extraction of primary macrophages found that the expression of NLRP3 and IL-1βin macrophages in the knockout mouse group was decreased.Conclusions:1.Increased FAT10 expression in DSS-induced colitis model mouse macrophages.2.FAT10 promotes IL-1βsecretion by upregulating NLRP3 in macrophages.3.FAT10 promotes NLRP3 transcription through the NF-κB signaling pathway.4.FAT10 stabilizes NLRP3 expression through post-translational modification.5.Deficiency of FAT10 mouse relieved experimental colitis. |
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