Research Of Platelet Microparticles Participating In Inflammation Of Inflammatory Bowel Disease Through Inducing The Formation Of Neutrophils Extracellular Traps

Backgroud and Objectives Intestinal mucosal immune dysfunction is considered to be an important link in the pathogenesis of inflammatory bowel disease(IBD).Among them,the mechanism of injury-related molecular patterns(DAMPs)that stimulates intestinal immune inflammation damage has received widespread attention.Platelet microparticles(PMPs),as an important product after platelet activation,can express many vital DAMPs signal.Studies have shown that PMPs have the ability to induce neutrophils to produce neutrophil extracellular traps(NETs)in systemic sclerosis,and whether it is involved in the mucosal immune injury mechanism of IBD is still unclear.The purpose of this study was to investigate the expression of PMPs in IBD and the effect of PMPs on the permeability of the intestinal mucosa,and to analyze the effect of PMPs on intestinal mucosal permeability of DSS-induced colitis.Method1.A total of 118 patients with IBD and 35 healthy controls(HCs)were recruited and divided into ulcerative colitis group(UC group,n = 54),Crohn’s disease group(CD group,n = 64),and healthy controls group(HCs group,n= 35).Specimens were collected,and the expression level of PMPs was detected by flow cytometry to analyze its correlation with disease activity by statistical methods.2.Intestinal mucosa samples from IBD patients were collected at the site of severe intestinal lesions and normal sites,labeled with immunofluorescence method,and the expression and distribution of PMPs and NETs in intestinal mucosa were observed under laser confocal scanning microscope(LCSM).3.After separating and purifying peripheral blood neutrophils,we co-cultured neutrophils with PMPs for 6 hours,use immunofluorescence staining to observe the formation capacity of NETs under LCSM,and detected the content of each component of NETs by fluorescence DNA quantitative method or ELISA.Grouping:(1)HC-PMN group(n = 6):only peripheral blood neutrophils derived from healthy controls were cultured;(2)IBD-PMN group(n = 6): only peripheral blood neutrophils derived from IBD group were cultured;(3)HC-PMN + IBD-PMPs group(n = 12): neutrophils from healthy controls and PMPs from IBD group were co-cultured;(4)PMN + HC-PMPs group(n = 6):Neutrophils and PMPs derived from healthy controls were co-cultured.4.We established the experimental colitis model in mice induced by 5% DSS for 1 week,at the same time,PMPs were injected daily through the abdominal cavity.We recorded the disease activity index(DAI)score.Immunofluorescence method was used to stain intestinal mucosa samples to observed the expression and distribution of PMPs under LCSM.The histopathological score(HI)of mice were calculated to assess the severity of intestinal inflammation.The expression levels of MPO,NE and DNA in colonic homogenate were detected by ELISA.The intestinal epithelial tissue structure was observed by transmission electron microscopy and the FITC-Dextran content in the serum of mice was detected to evaluate the changes in intestinal mucosal permeability.Grouping:(1)NCs group(n=10): drank water and injected with normal saline;(2)PMPs group(n=10): daink water and injected with PMPs;(3)DSS group(n=10): drank DSS and injected with normal saline;(4)experimental group(n=15):drank DSS and injected with PMPs.Results:The expressions of PMPs in UC group,CD group and normal control group were2724.00±2590.34 /μL,2733.02±2460.61 /μL and 810.94±439.44 /μL.The expression level of PMPs in the plasma of patients with active IBD was significantly higher than those in the healthy control group,and there was no significant difference in the elevated levels between the UC group and the CD group(P> 0.05).The expression level of PMPs was positively correlated with the degree of disease activity(P<0.05).By immunofluorescence staining the colon of IBD patients,CD61 mark PMPs,the results showed that: compared with normal control group,CD61 expressed in intestinal mucosa epithelial cells lesion group increased significantly.In the intestinal mucosal lesions of both CD group and UC group,epithelial lesions can be seen,glands are fused or infiltrated by a large number of inflammatory cells,intestinal mucosal full-thickness have CD61 expression,CD61 in inflammatory cells infiltration area especially colonic crypt abscesses in UC group gathered themselves together,and expression.1.In the absence of any stimulation,there was no significant difference in the level of NETosis induced in the healthy control group(2.00±0.63%)compared with the IBD group(1.67±0.82%)(P>0.05).Under the stimulation of PMPs,PMPs can induce NETs formation in both HCs’(5.83±2.86%)and IBD patients’(14.67±5.35%)peripheral blood neutrophils,the amount of NETs generated by IBD group was significantly increased compared with HCs group,and the difference was statistically significant,P<0.05.2.In the study of PMPs in mice with colitis,compared with normal control group,the NETs components(MPO,NE)levels in colonic homogenate of PMPs group were increased,and the levels of DNA were decreased,P<0.05,DAI score have no significant change(P>0.05),HI score was slightly elevated(P<0.05),FITC-D levels in plasma increased significantly(P<0.05).The microscopic substructure of epithelial tissue was slightly changed and the degree of intercellular connectivity was slightly reduced.NETs components(MPO,NE and free DNA)levels in colonic homogenate in DSS group mice were elevated(P > 0.05),DAI grade and HI abnormally were elevated(P < 0.05),FITC-D levels in plasma were increased significantly(P < 0.05).The destruction of the epithelial structure and the blurring and looseness of the intercellular connections were observed under electron microscopy,indicating an increase in intestinal mucosal permeability.Compared to the DSS model group,the NETs components(MPO,NE)levels in colonic homogenate of experimental group were significantly increased,and the levels of DNA were decreased,P<0.05,,DAI score and HI score were moderately elevated(P > 0.05),FITC-D content in plasma was increased(P < 0.05).Under the electron microscope,obvious epithelial damage was observed,the changes of intercellular junction structure and tightness were increased,and bacterial invasion was observed locally,indicating increased intestinal mucosal permeability.It suggested that the increased expression of NETs might be the cause of the aggravation of intestinal inflammation.Conclusion:1.The PMPs expression level in plasma of IBD patients was significantly higher than that of healthy patients,and the difference between UC patients and CD patients was not significant.The PMPs expression level in plasma of active IBD patients may be related to the degree of disease activity.2.The expression of PMPs in the intestinal mucosa of the patients with IBD increased compared with the normal site,and there were abnormal concentrations in the infiltration site of inflammatory cells,suggesting that there might be a correlation with inflammation.In some patients with UC,NETs formation has been observed in lesions in the colon.3.PMPs have the ability to induce neutrophils to release NETs,and PMPs from IBD patients can induce the formation of NETs more easily than PMPs from normal people.4.PMPs can induce the formation of NETs in mice,promote the generation of colon inflammation in mice,increase the intestinal mucosal permeability of DSS colitis mice,and aggravate the intestinal inflammation in colitis mice,suggesting that PMPs may affect the occurrence and development of intestinal inflammation by inducing the release of NETs.