| Objective:Intestinal fibrosis is a common complication of inflammatory bowel disease(IBD),which can occur in ulcerative colitis(UC)and Crohn’s disease(CD).Repeated intestinal inflammation in IBD promotes the activation of interstitial cells and produces a large number of extracellular matrix(ECM),leading to the formation of intestinal fibrosis.In recent years,biological agents have provided efficient and accurate targeted treatment for patients with IBD.However,due to the hidden symptoms and long course of disease in the early stage,patients are still inevitably suffering from intestinal fibrosis and intestinal stenosis in the process of injury and healing,which seriously reduces the quality of life of patients.The mechanism of fibrosis is complex and dynamic,involving a variety of cell types,related cell events and a large number of soluble factors.Due to the destruction of the epithelial barrier in inflammatory bowel disease,the bacterial products in the lumen enter the stroma,and induce the immune response by activating immune cells and non-immune cells.Finally,the composition of the extracellular matrix of the intestine has changed significantly to form fibrosis.The Dynamic equilibrium of production and degradation of the extracellular matrix plays an important role in tissue remodeling.The expression levels of matrix metalloproteinase(MMPs)and tissue inhibitors of metalloproteinases(TIMPs)in intestine tissue of IBD patients are unbalanced,which is one of the important mechanisms of the occurrence and development of IBD.At present,anti-inflammatory therapies used for IBD cannot prevent or reverse fibrosis.The traditional view is that intestinal fibrosis is an inevitable and irreversible process.With the improvement of understanding of the cellular and molecular mechanisms of its pathological occurrence,this view has gradually changed.Understanding the mechanisms of intestinal fibrosis may pave the way for the development of anti-fibrosis drugs and new treatment methods for IBD.Interleukin-33(IL-33)and its receptor ST2 combine to promote the secretion of type 2 cytokines and the survival and proliferation of ST2 receptor positive immune cells through downstream signal transduction.IL-33 is passively released from necrotic cells or secreted by stimulated leukocytes and epithelia when tissue damage is caused by various reasons,and its expression will also increase in inflammatory bowel disease.IL-33 has been reported to be involved in the process of organ fibrosis in many studies,and the mechanism of the IL-33/ST2 axis in intestinal fibrosis needs further research..The main physiological function of macrophages is phagocytosis and defensive secretion.Studies have reported that patients with CD have macrophage function defects,indicating that they may be involved in the occurrence and development of IBD.In addition,macrophages are also important participants in the process of fibrosis.They can differentiate into M1 and M2 subtypes in different cytokine environments.They have different biological functions and marker expression.M1 type macrophages highly express iNOS and TNFαafter activation,while M2 type macrophages can highly express Arg1,CD206,etc.It was found that M2 macrophages have anti-inflammatory,repair and fibrosis promoting tendencies,and the IL-33/ST2 axis can enhance M2 macrophages’phenotype.These results suggest that IL-33/ST2 may regulate the intestinal fibrosis process by mediating macrophage polarization.TNFαis a cytokine mainly secreted by monocyte macrophages,which plays an important role in the occurrence and development of IBD.The damage mechanism of TNFαto the intestinal tract is mainly that it damages the intestinal epithelial barrier,induces cell necrosis or apoptosis.At the same time,TNFαdoes not only play a one-way proinflammatory role,but also plays a beneficial regulatory role as a participant of the complex network.It stimulates the generation of downstream inflammatory factors and chemokines,and it also mediates the production of anti-inflammatory factors.TNFαtargeted biological agents are the first commercially available biological agents for the treatment of IBD.They bind to the soluble or transmembrane TNFαwith high affinity,which inhibits TNFαto bind to its receptors,and thus they cause TNFαto lose its biological activity.Their clinical efficacy in rapidly improving intestinal inflammation and promoting mucosal healing has been unanimously recognized by specialized doctors.The classic NF-κB pathway is one of the main pathways for TNFαto exert biological functions and participate in the regulation of intestinal inflammation.After activation,NF-κB can induce downstream genes to transcribe,transforming the transcription program of immune cells from a static state to a proliferative phenotype that can resist pathogens,thus playing a crucial role in the body’s immunity.In addition to its role as a therapeutic target,TNFαmay help to predict the response of IBD patients to anti-TNFαtreatment.Many studies have shown that the increased expression of transmembrane TNFα(m TNFα)of immune cells in patients’intestines and the increased expression of TNFαin peripheral blood monocytes indicate a better response to TNFαmonoclonal antibody in IBD patients.CD14~+mononuclear macrophages are the main source of producing the above TNFα,so the multidirectional function of TNFαin IBD needs further discussion.In summary,considering the potential clinical application of IL-33/ST2 axis in the treatment of fibrosis,this study will explore the regulatory effect and mechanism of IL-33/ST2 axis on intestinal fibrosis.Methods:1.Establishment of animal model of intestinal fibrosis and grouping of experimental animals:The intestinal fibrosis model of mice was established with 2,4,6-trinitrobenzene sulfonic acid(TNBS)of gradient concentration.The animal experiment cycle was 8weeks in total.After adaptive feeding and skin pre-sensitization,a 6-week chemical enema was started.The overall experiment set up three groups:control group,experimental modeling group and treatment group.From the third week of enema,mice in the treatment group were injected with ST2 monoclonal antibody(1.5μg/g)for 4weeks in total,and all mice were killed and sampled on the second day after the last enema.2.Evaluation of fibrosis degree,cytokine expression and macrophage phenotype in intestinal specimens of experimental mice.Firstly,we compared the general condition and intestinal morphology of mice;and then Type Ⅰ collagen(COL Ⅰ),Type Ⅲ collagen(COL Ⅲ),fibronectin(FN)andα-Smooth muscle Actin(α-SMA)were used to evaluate the differential expression of fibrosis with RT-qPCR,Western Blot,Masson trichrome staining,and immunohistochemical staining methods.Simultaneously we detected the expression of cytokines such as IL-33,TNFα,IL-1βand IL-6 in the intestinal tissue of mice in each group at the mRNA and protein levels.F4/80+CD206 double labeling is a classic method to identify M2 macrophages.The intestinal tissue sections of experimental animals in each group were stained with F4/80 and CD206 double immunohistochemical staining to evaluate the polarization of macrophage polarization in vivo.The proportion of M2 macrophages in the total number of macrophages in the intestinal tissue was calculated by double immunohistochemical staining.Arg1 is mainly expressed by M2 type macrophages.RT-qPCR and immunohistochemical methods were used to determine the expression level of Arg1 in intestinal tissue to evaluate the polarization degree of M2 type macrophages.3.In order to evaluate the mechanism of IL-33 in the pathogenesis of intestinal fibrosis,the gradient concentration of IL-33 recombinant protein was applied to human intestinal fibroblasts to evaluate the expression of Col I,Col Ⅲ,FN andαSMA through RT-qPCR,and Western Blot methods.4.Lipopolysaccharide and IL-4 were used to induce mouse macrophages to differentiate into M1 type or M2 type macrophages respectively,and then the model of macrophage polarization was established.Add IL-33 recombinant protein and lipopolysaccharide or IL-4 into mouse macrophages at the same time to evaluate the effect of IL-33 on the differentiated macrophage polarization markers iNOS,TNFα,IL1β,IL6,Arg1 and TGFβ1 expression.5.Correlation analysis of mRNA expression of TNFαwith COL Ⅰ,COL Ⅲ,FN andαSMA in intestinal tissue of mice was done;Western blot was used to evaluate the expression of NF-κB pathway protein components in intestinal tissue of mice in each group after treatment with ST2 monoclonal antibody.6.Apply TNFαrecombinant protein to stimulate human intestinal fibroblasts,and the expression of fibrosis indicators was evaluated by RT-qPCR and Western Blot;TNFαis a NF-κB pathway agonist,and in order to study whether this pathway is involved in the intestinal fibrosis,we add NF-κB pathway inhibitor to observe the changes of fibrosis index as well as MMP9 and TIMP1 expression.Results:1.After blocking the IL-33/ST2 axis with ST2 monoclonal antibody in mice,the scores of large intestine morphology in the experimental group and the treatment group were higher than those in the control group(P<0.01 or 0.05),but there was no statistical difference between the experimental group and the treatment group;The intestinal weight of mice in the treatment group and the experimental group was significantly higher than that in the control group(P<0.05),and the body weight was significantly lower than that in the control group after experiment(P<0.05).There was no statistical difference between the treatment group and the experimental group in intestinal weight and body weight of mice,and there was no statistical difference between the three groups in intestinal length of mice.2.The mRNA expression of COL Ⅰ,COL Ⅲ,FN andαSMA was detected.The mRNA expression of the four fibrosis indicators in the experimental group and the treatment group was higher than that in the control group(P<0.01),and the mRNA expression of the four fibrosis indicators in the treatment group was significantly lower than that in the experimental group(P<0.01 or 0.05).The protein expression of COL Ⅰ,COL Ⅲ,FN andα-SMA in the experimental group was higher than that in the control group(P<0.01 or0.05).Exceptα-SMA,the protein expression of the other three fibrosis indicators in the treatment group was significantly lower than that of the experimental group(P<0.05).Masson trichromatic staining is a classic method of connective tissue staining,in which the blue staining area shows the location of collagen fibers.The intestinal sections of mice in each group were stained with HE and Masson.HE staining showed that the infiltration of inflammatory cells in the epithelial layer and submucosa in the experimental group and the treatment group increased,and the mucosal muscular layer and submucosa were thickened,and the glands were irregularly arranged.Masson staining results showed that the blue area increased significantly in the experimental group,and the treatment group showed smaller region in blue area than the experimental group(P<0.05).Immunohistochemical method was used to detect COL Ⅰ,COL Ⅲ,FN andα-SMA staining showed that the first three were mainly distributed in the interglandular region of the epithelial lamina propria layer and submucosal layer;α-SMA is mainly expressed in smooth muscle cells and myofibroblasts which are mainly distributed in the mucosal muscle layer and muscle layer.During the experiment,it was found that when the fibrosis is aggravated,it can also show positive color between the glands in the epithelial lamina propria.According to statistics,the range and degree of four fibrosis indicators in the experimental group were higher than those in the control group,and the degree of fibrosis in the treatment group was lower than that in the experimental group(P<0.01 or 0.05).In intestinal tissues of mice at mRNA and protein levels,the expression of IL-33,TNF-α,IL1βAnd IL-6 were significantly increased in the experimental group(P<0.01),and the expression of IL-33、IL-1βand IL-6significantly decreased in the treatment group(P<0.01 or 0.05),while the expression of TNF-αin the treatment group was higher than that in the experimental group(P<0.05).3.Immunohistochemical F4/80 and CD206 double staining were performed on the intestinal tissue sections of mice,and F4/80(+)+CD206(+)double positive cells and F4/80(+)single positive cells were counted respectively.The ratio of M2 type macrophages to the total macrophages was evaluated by the ratio of the two.After statistics,the ratio of double positive cells to single positive cells in the treatment group was significantly lower than that in the experimental group(P<0.01).The expression of Arg1 in the intestinal tissue of mice in each group was determined by RT-qPCR and immunohistochemistry.The expression of Arg1 in the experimental group was significantly higher than that in the control group,and the expression of Arg1 was significantly decreased after treatment with ST2 monoclonal antibody.4.The recombinant IL-33 protein acts on human intestinal fibroblasts,and it is concluded that some concentrations of IL-33 promote the mRNA expression of COL3α1 increasing to about 1.2 times(P<0.01 or 0.05)at the highest.Similarly,100ng/ml IL-33significantly increased the protein expression of COL Ⅲ,1.6 times to that of the control group(P<0.05).However,IL-33 did not significantly change the mRNA and protein expression of other fibrosis indicators.5.The undifferentiated mouse macrophages were induced to transform into M1 type macrophages after applying lipopolysaccharide with the concentration of 1μg/ml and M2type macrophages by IL-4 with the concentration of 10ng/ml.The gradient concentration of mouse recombinant IL-33 protein and lipopolysaccharide or IL-4 were added to macrophages at the same time to evaluate the effect of IL-33 on the phenotype of differentiated macrophages.After M1 macrophages were stimulated by recombinant IL-33 at gradient concentration for 24 hours,their markers were detected by RT-qPCR.The mRNA level of iNOS and TNF-αdecreased(P<0.05),but IL-6 and IL-1βmRNA level increased including the increase of IL-1βwas statistically significant(P<0.05).Western blot was used to detect the protein expression level of the above four M1 type macrophage markers,and semi-quantitative analysis was made between the IL-33treatment groups and the LPS single drug group,respectively.IL-1βAnd IL-6 protein levels are increased in different degrees(P<0.05),while the level of TNF-αprotein expression decreased(P<0.05).M2 macrophages were stimulated by recombinant IL-33at gradient concentration for 24 hours,and their markers Arg-1 and TGFβ1 were detected by RT-qPCR.The results showed that the mRNA expression of Arg-1 showing an upward trend,and some concentrations of IL-33 significantly increased Arg-1 expression(P<0.05);Although the expression of TGFβ1 mRNA increased with the increase of drug concentration,it was not statistically significant.Western blot was used to detect the protein expression levels of the two M2 macrophage biomarkers mentioned above.It was found that at some concentrations of IL-33,the protein levels of both increased(P<0.05),while at other drug concentrations,although the expression also increased,it was not statistically significant.6.Correlation analysis was conducted on the relative expression of TNFαmRNA in intestinal tissue of experimental animals with the four fibrosis indicators,among which the relative expression of TNFαmRNA was significantly negatively correlated withα-SMA mRNA(P<0.05).Western blot detection of NF-κB pathway protein was carried out on the intestinal tissues of the three group mice.After semi-quantitative analysis,it was found that blocking the IL-33/ST2 axis caused IκBαprotein degradation and NF-κB p65phosphorylation increased(P<0.05).7.Gradient concentration TNF-αrecombinant protein adding to human intestinal fibroblasts can significantly reduce COL3lα(P<0.01or<0.05)andα-SMA mRNA expression(P<0.01).There was no effect on the mRNA expression of COL11αand FN.Western blot results showed that TNFαcan reduce the protein expression of FN andαSMA with statistical differences compared to the control group(P<0.01 or<0.05).8.To clarify whether TNF-αregulates the expression of fibrosis indicators of intestinal fibroblaststs through NF-κB pathway,NF-κB pathway inhibitor is selected.First,the concentration of NF-κB pathway inhibitor was screened.The cell survival rate was observed under the microscope,and the phosphorylation degree of pathway component protein was determined by western blot,and finally the concentration was choosen as 5μM.Follow-up experiment was carried out by adding the inhibitor into the cells for 2hours ahead of TNF-α.Human intestinal fibroblasts were divided into three groups:control group,TNF-α(30ng/ml)group,and TNF-α+NF-κB group.Western blot showed FN andα-SMA protein expression increased after inhibiting NF-κB pathway(P<0.05).The results of RT-qPCR and Western blot showed that TNF-αcould significantly increase the mRNA and protein expression of MMP9(P<0.05),and could be reduced by NF-κB pathway inhibitor(P<0.05);The protein expression of TIMP1 under TNFαwas significantly higher than that of the control group(P<0.05).The addition of pathway inhibitor had no effect on the protein expression of TIMP1,and the mRNA expression level of TIMP1 had no difference among the three groups.MMP9 and TIMP1 were confirmed to be 1:1 binding according to the protein spatial configuration.The ratio of the relative expression of MMP9 and TIMP1 mRNA and protein was calculated,and then compared between groups.It was found that there were differences among the three groups at the mRNA level ratio,TNF-αgroup>TNF-α+NF-κB pathway inhibitor group>control group(P<0.01 or P<0.05),and the ratio of MMP9 and TIMP1 in TNF-αgroup at the protein level was significantly higher than that in the TNF-α+NF-κB pathway inhibitor group(P<0.05).The above suggested that TNF-αcould activate the NF-κB pathway to improve the expression of MMP9 and increase MMP9/TIMP1 ratio.Conclusion:1.In animal experiments,blocking the IL-33/ST2 axis can alleviate intestinal fibrosis,reduce the expression of IL-1βand IL-6,increase TNFαexpression,and reduce the proportion of M2 macrophages in intestinal tissue.2.IL-33 can promote the expression of collagen type Ⅲ in intestinal fibroblasts and regulate the polarization of differentiated macrophage:it can reduce the expression of TNFαin M1 macrophages and promote the expression of Arg1 and TGFβ1 in M2macrophages.3.There is a significant negative correlation between TNFαandαSMA mRNA expression in the intestinal tissue of mice,and blocking the IL-33/ST2 axis can activate NF-κB.4.TNFαcan inhibit the expression of fibrosis markers in human intestinal fibroblasts;TNFαreduces FN andαSMA expression,and it increases MMP9 expression and MMP9/TIMP1 ratio through NF-κB pathway. |
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