| Objective Inflammatory bowel disease(IBD)is a chronic and inflammatory disease,which is prone to recurrence after clinical operation and seriously threatens the life and health of modern people.At present,the etiology of IBD is still unclear.In order to better explore the pathogenesis of IBD and develop natural clinical treatment methods,this topic intends to use IL-16-/-mice to establish an inflammatory bowel disease model,explore the role of IL-16 in the pathogenesis of inflammatory bowel disease in mice and the regulatory mechanism,so as to provide new research directions and therapeutic targets for the treatment of IBD.Methods 1.the IBD model was constructed using C57BL/6 mice,and the expression and localization of IL-16 were detected by RT-q PCR,ELISA and immunofluorescence.2.C57BL/6 and IL-16-/-mice were randomly divided into two groups: WT control group,WT DSS group,IL-16 KO control group,and IL-16 KO DSS group.The DSS model group was given 25 g/L DSS for 7 days to establish an IBD model,while the control group was given normal drinking water.The weight of mice was recorded during the modeling period and the body mass curve was drawn.After 7 days,the spleen and whole colon of mice were dissected and separated.HE was used to detect pathological damage of colon tissue,and apoptosis in colon tissue was detected by TUNEL.Flow cytometry was used to detect the number and polarization of macrophages in peritoneal cells(F4/80,CD86),immunohistochemical staining was used to detect the distribution of macrophages in colon tissue,RT-q PCR was used to detect the levels of IL-6?IL-12?TNF-? and IL-1? in spleen and colon tissue,and ELISA was used to detect the changes of IL-6 and IL-12 protein levels in whole blood and colon tissue.3.The effects of IL-16 on the polarization of macrophages was verified in vitro experiments.Bone marrow cells from C57BL/6 and IL-16-/-mouse were isolated in vitro.GM-CSF was induced to culture bone marrow-derived macrophages for 7 days.LPS and IFN-? were added to stimulate their activation to M1 macrophages.Flow cytometry was used to detect the polarization level of macrophages in cultured cells(CD86,CD11b/TNF-?,CD11b/IL-12p40).RT-q PCR was used to detect the levels of IL-6?IL-12?TNF-? and IL-1?,ELISA was used to detect the levels of IL-6?IL-12?TNF-? and IL-1? in the supernatant of cultured cells,and Western Blot was used to detect the phosphorylation levels of P38 MAPK and P65NF-?B.Results 1.The model of inflammatory bowel disease in mice was established.Compared with WT control group:(1)After 4 days,the body weight of mice in WT DSS group decreased significantly(P < 0.05),with spleen abnormal enlargement and colon redness atrophy(P < 0.01);(2)the level of IL-16 in spleen and colon tissue increased significantly(P < 0.01,P < 0.05),and the level of IL-16 protein in colon tissue was significantly higher(P < 0.01,P < 0.01);(3)Immunofluorescence showed high expression of IL-16 in the severely damaged colonic tissues(P < 0.01).2.Compared with WT DSS group:(1)After 4 days,the body weight of mice in IL-16 KO DSS group decreased slowly(P < 0.01);(2)HE staining showed that the glandular structure of colonic tissues in IL-16 KO DSS group was more complete,the arrangement of colonic tissues was orderly,and the infiltration of submucosal cells decreased significantly;(3)TUNEL results showed that apoptotic cells in colon tissue of IL-16 KO DSS group decreased significantly(P < 0.01);(4)Flow cytometry showed that the number and polarization of macrophages in peritoneal single cell suspension of mice in IL-16 KO DSS group decreased significantly(P < 0.01,P < 0.05);(5)Immunohistochemical staining showed that macrophages in colon tissue of IL-16 KO DSS group were severely damaged(P < 0.01,P < 0.05);(6)The expression of IL-6?IL-12 and TNF-? m RNA in spleen tissue of IL-16 KO DSS group decreased significantly(P < 0.05,P < 0.01,P < 0.05),while the expression of IL-6?IL-12?TNF-? and IL-1? in colon tissue of IL-16 KO DSS group decreased significantly(P < 0.05,P < 0.01,P < 0.05,P < 0.01);(7)The levels of IL-6 and IL-12 protein in colon tissue of mice in IL-16 KO DSS group were significantly decreased(P < 0.05,P < 0.001),while the levels of IL-6 and IL-12 protein in colon tissue of mice in IL-16 KO DSS group were significantly decreased(P < 0.05,P < 0.05).3.The macrophages derived from bone marrow of C57BL/6 and IL-16-/-mice were induced and cultured in vitro.Compared with WT BMDMs group.(1)Flow cytometry showed that the polarization level of macrophages in IL-16 KO BMDMs group decreased significantly(P < 0.05);(2)RTq PCR results showed that the levels of IL-6?IL-12?TNF-? and IL-1? m RNA were significantly lower in IL-16 KO BMDMs group;(3)The results showed that IL-6,IL-12 and TNF-? protein expression in the supernatant of macrophages cultured in IL-16 KO BMDMs group was significantly decreased;(4)Western Blot results showed that the phosphorylation levels of P65NF-?B proteins in macrophages cultured in IL-16 KO BMDMs group were decreased.Conclusion 1.The model of DSS-induced colitis in mice was successfully constructed.IL-16 was overexpressed in the colitis model and aggravated the pathological formation of colitis;2.IL-16 promotes the recruitment of macrophages to colon tissue,increases the polarization of macrophages to M1 subtype,and induces abnormal apoptosis of colon tissue cells in DSS-induced colitis model of mice;3.In vitro,IL-16 promotes the polarization of M1 macrophages by up-regulating phosphorylation of NF-?B proteins. |
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