| Objective:Inflammatory bowel disease is a kind of chronic nonspecific intestinal inflammatory disease with unclear etiology.Recently,it has been found that neutrophils can form the extracellular network structure called neutrophil extracellular traps(NETs)under the activation of some special stimuli,which plays an important role in the anti-infection of the body,limiting the spread of pathogenic microorganisms through adhesion and fixation,and then killing pathogens by its own secreted immune effector molecules.Concomitantly,NETs can degrade some immune effector molecules to limit the inflammatory reaction.However,excessive production or untimely degradation of NETs could promote "inflammatory storm" and cause damage to normal cells and tissues.The formation time,the expression patterns and its relationship with intestinal mucosal injury of NETs have not been reported.On this basis,we investigated the roles of NETs in the model of colitis,made clear the relationship of NETs and the degree of disease activity,and preliminarily discussed the relationship between NETs and the impairment of intestinal epithelial barrier function,and laid a theoretical and experimental foundation for developing new effective drugs to maintain and repair intestinal mucosal barrier.Methods:1.Collecting clinical cases:Collecting blood samples from patients with ulcerative colitis(UC)(n=17)and Crohn’s disease(CD)(n=29)who were treated in Department of Pediatric Gastroenterology,Shengjing Hospital affiliated to China Medical University from March 2017 to September 2018,and healthy children(n=8)for physical examination in the same period as the blank control group.Serum free DNA(cf-DNA)level was detected by fluorescence microplate reader.2.Animal experiment:(1)A total of 48 BALB/c mice aged 6-8 weeks were randomly divided into control group and colitis group,and each group was divided into Dayl,Day3 and Day5 groups according to different observation time after administration.To induce ulcerative colitis,mice received 3%Dextran sulfate sodium salt(DSS)in drinking water for 7 days,while Ctrl group drank distilled water.The peripheral blood and colon tissues of two groups of mice were collected at different time points(1st,3rd and 5th day)after the establishment of colitis.(2)Twenty six BALB/c mice aged 6-8 weeks were randomly divided into Ctrl group(n=8)、DSS group(n=10)and DNasel group(n=8).Mice were fed distilled water containing 3%DSS for 7 days.Thus DNaseI group was injected subcutaneously with DNaseI(10mg/kg)on the 1st,3rd and 5th day during drinking DSS,and DSS group was injected subcutaneously with the same volume of PBS.After the establishment of model,the peripheral blood and colon tissues of mice were collected.The animal tissues to be stained with paraffin sections were soaked in 4%paraformaldehyde,fixed and embedded in paraffin,and the animal specimens prepared for Western Blot were stored at-80°℃.The level of serum free cf-DNA and histone were detected by fluorescence microplate reader.The levels of TNF-α,IL-1β,IL-10,IL-17 and IGF-1 in peripheral blood of mice were detected by Luminex multi-factor detection kit.The expression and localization of CitH3 and MPO in colonic tissue were observed by immunofluorescence staining,the expression of Claudin-1 in colon was observed by immunohistochemistry staining,and the apoptosis of colon tissue was observed by TUNEL staining.The expressions of CitH3,Claudin-1 and Cleaved Caspase-3 in colonic tissue were detected by Western Blot.3.Cell experiment:(1)PLB-985 cells were induced to differentiate into neutrophil-like cells by Dimethyl formamide(DMF),and different doses of Phorbol myristate acetate(PMA)(0nM group,20nM group,50nM group and 100nM group)were used to stimulate the cells to produce NETs.Cell supernatants were collected at 0,30,60,120,180,and 240min after administration,and the level of cf-DNA was detected by fluorescence microplate reader to select the best concentration and reaction timze.(2)Differentiated PLB-985(d-PLB-985)cells were inoculated into the well plate and divided into the following groups:control group,PMA group,DnaseI group,Cl-amidine group,PMA+DNasel group and PMA+Cl-amidine group.After 4 hours of administration,some cells were collected together with culture medium(NETs)for the following experiments.The cell supernatant were collected to detect cf-DNA level.After centrifugation,the cells were divided into two parts:one part was used to observe the production of NETs by immunofluorescence staining,and the other part was used to extract protein and detect the expression of CitH3 by Western Blot.(3)Adjust the status of Caco-2 cells to logarithmic growth stage,and divide cells into the following groups:Ctrl group,PLB-985 group,PMA group,NETs group,NETs+DNasel group,PLB-985+DnaseI group.After 4 hours,collect cells and detect the transepithelial electrical resistance(TEER)and FD-4 concerntration to evaluate monolayer permeability、the scratch test and Transwell test to evaluate the effect NETs on wound healing of monolayer cells,the expression of Claudin-1 by immunofluorescence staining and Western Blot.(4)Divide cells into the following groups:Ctrl group,NETs group,NETs+IGF-1 group and IGF-1 group.After 4 hours,collect cells to measure the test as described in part(3),and detect the expression of Claudin-1,PI3K,Akt and p-Akt by Western Blot.Results:1.The level of serum cf-DNA in IBD patients was significantly higher than that in control group(P<0.01),the level of serum cf-DNA in IBD active group was significantly higher than that in remission group(P<0.0001),and that in UC active group was significantly higher than that in CD active group(P<0.0001).2.Under normal physiological conditions,the expression of NETs in mice was very low.On the first day after the establishment of colitis model,the expression of cf-DNA and histone level in peripheral blood increased significantly(P<0.0001),and gradually decreased with time,and it was still significantly higher than that in the control group on the fifth day(P<0.01).CitH3 was hardly expressed in colon tissue under normal physiological conditions,and the expression level of CitH3 increased significantly on the first day after DSS model was established,and decreased gradually on the fifth day(P<0.05).3.Compared with PBS group,the degree of disease activity index in DNaseI group was reduced(P<0.01),and the degree of colon tissue injury was reduced.The levels of cf-DNA and histone in peripheral blood were reduced(P<0.05),the expression of CitH3 and MPO in colonic tissue of DNaseI group decreased significantly,and the same trend was found in the semi-quantitative detection of CitH3 protein(P<0.0001).Compared with PBS group,Claudin-1 protein expression in colon tissue of DNaseI group increased(P<0.05),and TUNEL staining showed that apoptotic cells and Cleaved Caspase-3 protein expression decreased(P<0.01).4.D-PLB-985 cells were stimulated by PMA,and the level of cf-DNA in the supernatant of 100nM group was the highest at 240min(P<0.0001).Immunofluorescence staining showed that CitH3 and MPO were expressed in large quantities outside the cells.The level of cf-DNA in supernatant reduced by the intervention of DNaseI and Cl-amidine(P<0.0001),but there is no significant difference in inhibitory effect between them.After intervention of DNaseI and Cl-amidine,the expression of CitH3 and MPO decreased by immunofluorescence staining,and the expression of CitH3 protein decreased significantly by WB(P<0.05).Co-culture experiments showed that NETs reduced the TEER but increased the permeability of monolayer cells,reduced cell migrateon,and inhibited the expression of Claudin-1 protein in Caco-2 cells(P<0.01),while adding IGF-1 could improve the barrier function of Caco-2 cell.Compared with Ctrl group,the expression of p-Akt in NETs group was significantly increased(P<0.05),and the expression of p-Akt protein in NETs+IGF-1 group was further increased(P<0.05),but there was no significant difference in PI3K and Akt protein among the groups.Conclusions:1.The expression of NETs in IBD patients is positively correlated with the degree of disease activity,and the expression of NETs in UC patients is more than that in CD patients.2.The expression of NETs is obvious in active stage of colitis,and decreases with the remission of the disease.Inhibition of NETs expression can alleviate the degree of disease activity,inhibit apoptosis of tissue cells,improve the barrier function of epithelial cells and inhibit the expression of inflammatory factors.3.NETs can damage the barrier function of intestinal epithelial cells by regulating PI3K/Akt pathway,and IGF-1 synergistically regulates Akt phosphorylation to improve the barrier function of intestinal epithelial cells. |
PDF Full Text Request