Correlation Of ANGPTL4 And CD8~+T/M_? In Patients With IBD And Its Effect On The Pathogenesis Of Experimental Mouse Colitis

Inflammatory bowel disease(IBD)is a chronic recurrent inflammatory disease involving the entire digestive tract and multiple organ systems,including Crohn's disease(CD)and Ulcerative colitis(UC).The course of this disease is long and the condition is repeated,moreover there is no absolute standard of diagnosis and effective radical treatment.In recent years,the incidence rate of IBD has increased significantly in China,endangering national health.Its main pathological features were slow recurrent intestinal inflammation and epithelial damage,and large number of mononuclear cells infiltrated in the intestinal tissues during the active period.Angiopoietin-like protein 4(ANGPTL4)is a multifunctional signaling protein involved in the regulation of glycolipid metabolism,angiogenesis and leakage,tumor growth and metastasis.In recent years,its role in the regulation of inflammation has been discussed hotly.IBD is an immune disease,and the occurrence and development of inflammation is a key link in its pathogenesis.Previous studies have found that ANGPTL4 can be involved in the regulation of intestinal inflammation.However,whether ANGPTL4 can directly regulate the pathogenesis of IBD and which immune cells are mainly mediated is expected to be further studied.Objective:1.To analyze the expression of ANGPTL4 in intestinal tract and the concentration of plasma ANGPTL4 in IBD patients and experimental colitis mice,and to correlate with the infiltration and activity of CD8 T cells and macrophages(M?)respectively.2.To observe the sensitivity of ANGPTL4 knockout(ANGPTL4-/-)mice to experimental colitis and the changes of CD8 T/M?.3.To analysis the direct effect of ANGPTL4 recombinant protein on experimental colitis and CD8+T/M?.Methods:1.The concentration of ANGPTL4 and TG was tested by enzyme-linked immunosorbent assay(ELISA).Peripheral blood mononuclear cells(PBMCs)were used to detect the frequency of CD3+CD8+CXCR3+?CD8+IFN-y+?CD14+CD86+cells by flow cytometry(FCM).Immunohistochemistry(IHC)was used to observe the expression of ANGPTL4 in diseased intestinal tissues and the infiltration of CD8 T/M?.The expression of ANGPTL4 in intestinal lesions and adjacent normal tissues was detected by Western blot(WB).Then Spearman correlation analysis was used to analyze the correlation between ANGPTL4 and IBD disease degree and CD8 T/M?.2.Dextran sulfate sodium salt(DSS)was used to induce mice with different days of experimental colitis.The concentration of ANGPTL4 in serum was detected by ELISA.The frequency of CD8 T/M? in intestinal tissues was detected by FCM.The expression of ANGPTL4 in intestinal was observed and detected by IHC and WB.3.DSS was used to induce experimental colitis in ANGPTL4-/-and wild-type(WT)mice.At the same time the weight and disease index curves were recorded and plotted.HE staining was used to compare with the pathological changes of intestine.The frequency of CD8-T/M? in intestine was detected by FCM.4.Experimental colitis in mice was induced by DSS,at the same time mice were intraperitoneal injected with ANGPTL4 recombinant protein(rANGPTL4).The weight and disease index curves were recorded and plotted.The frequency of CD8 T/M? in intestinal tissues was detected by FCM and the infiltration of CD8 T/M? in intestinal tissues was observed and compared by IHC.RESULTS:1.The plasma ANGPTL4 concentration in CD/UC patients with mild/moderate active period was higher than that in healthy controls,and the increase in CD group was more significant.The expression of ANGPTL4 in intestinal lesions of patients with CD and UC was higher than that of normal tissues.Among them,the expression of ANGPTL4 was positively correlated with the severity of disease in CD patients,and the expression of ANGPTL4 in peripheral blood and intestinal tract was negatively correlated with the frequency of CD8 T cells.The expression of ANGPTL4 in the intestine was positively correlated with macrophage infiltration.In UC patients,the expression of ANGPTL4 in peripheral blood and intestine was both negatively correlated with CD8 T cell frequency.The correlation between the expression of ANGPTL and disease severity was not found,meanwhile the correlation between the expression of ANGPTL4 in peripheral blood and intestinal tissues and the frequency of macrophages was not found.2.In experimental colitis mice,both of serum ANGPTL4 concentration and intestinal ANGPTL4 expression peaked at 5 days,then decreased at 7 days the same with the frequency of CD8+T cells in intestinal tissues.The frequency of intestinal macrophages increased significantly to 3 days until to 7 days.3.The colitis in ANGPTL4-/-mice were significantly aggravated compared with WT.The frequency of CD8 T cells in intestinal tissues was significantly increased,and there was no difference in the frequency of M?.4.After intraperitoneal injection of rANGPTL4 in mice with colitis,symptoms of intestinal inflammation were alleviated.The frequency of CD8 T/M? cells in the intestine was decreased and the infiltration was decreased.The changes of CD8 T cells were more obvious.Conclusion:ANGPTL4 is expected to be a bio-marker reflecting the severity of CD activity and experimental colitis.Meanwhile,ANGPTL4 may attenuate the onset of IBD and experimental colitis by down-regulating the inflammatory activity of CD8+T cells.This research may provide a new direction for clinical treatment of IBD.