| BackgroundInflammatory bowel disease(IBD)is a chronic intestinal disease closely related to human immunity,including ulcerative colitis and Crohn’s disease.The common clinical manifestations are persistent or recurrent abdominal pain,mucous pus and bleeding stools,weight loss,etc.,and may be accompanied by extrenteral manifestations such as skin,eyes,liver and gallbladder.In the past 20 years,the number of patients with IBD has been increasing rapidly in China.The etiology and pathogenesis of IBD are unknown,and many factors such as heredity,environment and immune disorders are involved in its pathogenesis.More and more studies have confirmed that inflammatory cytokines and immune cells are involved in its pathogenesis.Th17 cells are currently considered to be the major pro-inflammatory cells in a variety of autoimmune diseases,including IBD.Increased Stat3 expression induces the formation of ROR-γt,which is required for differentiation of juvenile CD4~+T cells into Th17 cells.Mouse colitis model confirmed the high expression of TLR4,My D88 and NF-κB in the inflammatory site of colonic mucosa.Studies have shown that TLR4/My D88/NF-κB signaling pathway is involved in the pathogenesis of IBD.TLR is an important part of innate immune recognition receptors and plays an important role in the recognition of pathogenic microorganisms.Studies have found that TLR4 signal transduction pathway is involved in the pathogenesis of UC,which initiates signal transduction by non-specific binding with pathogen-related molecules and triggers the release of intestinal inflammatory mediators.My D88 is a adaptor protein containing the TLR domain,which plays a key role in the TLR signaling pathway as a downstream signaling factor.TLR4 can transduce the signaling pathway through the My D88 pathway.As a highly conserved transcription factor,NF-κB is widely found in various tissues and plays an important role in immune and inflammatory responses.NF-κB,as the convergence point of multiple signal transduction pathways,plays a crucial role in regulating the expression of genes related to inflammatory response.More and more studies have confirmed the involvement of TLR4/My D88/NF-κB signaling pathway in the pathogenesis of UC.However,whether this pathway is also involved in the pathogenesis of CD remains to be further confirmed.ObjectiveIn this study,the expressions of TLR4,My D88,NF-κB,ROR-γ,p-STAT3 and STAT3 in endoscopic colon tissues of patients with CD were detected by immunohistochemical staining,and the differences between the expressions and normal colon tissues were analyzed,and the correlation between the expressions and the degree of disease was further analyzed according to the Best CDAI score.At the same time,ESR,CRP,albumin and hematocellular volume of patients were collected to analyze the correlation between them and the above indexes.To investigate whether STAT3/ROR-γpathway and TLR4/My D88/NF-κB pathway are involved in the pathogenesis of CD.Methods1.Patient tissue samples collection:collection in December 2019 to December2020 in hospital or outpatient treatment of tissue samples,a total of 17 patients with CD,including 6 cases remission,6 cases of moderate and severe in 5 cases,in addition to collect 5 cases at the same time line hospitalized patients with colorectal cancer surgery cut edge negative tissue samples from cancer and confirmed by a pathologist.2.Gender,age,course of disease and other information of patients were collected through admission medical records;symptoms(abdominal pain,frequency of stool,etc.),physical signs and previous history of patients were recorded at admission;first blood routine,liver function,ESR,CRP,colonoscopy reports were collected after admission.The Best CDAI marking condition assessment in patients with CD and grouped,there are number of stools,abdominal pain degree,general situation,parenteral manifestations and complications,opioid antidiarrheal,abdominal mass,hematocrit value reduction,100 x(1-weight/body weight),a total of eight indicators,calculated the index score,multiplied by the relative weight after scoring,<150 was classified as remission stage,≥150 as active stage,150-220 as mild,221-450 as moderate,>450 is severe.3.Histochemical score was counted after HE staining of colon tissue;The expressions of TLR4,My D88,NF-κB,ROR-γ,p-STAT3 and STAT3 in colon tissues were detected by immunohistochemistry.4.Analysis of process data with SPSS 22.0 software,counting analysis data using mean±standard deviation,between groups than by chi-square test,each data after f or normal inspection,two or more groups to compare using single factor analysis of variance of S-N-K multiple comparison or rank and inspection,correlation analysis uses Spearman correlation analysis,inspection standards of alpha=0.05,P<0.05 difference is statistically significant.Results1.Hematoxylin-eosin staining of colon tissue of patients with CDColonic epithelial tissue of patients in CD group showed varying degrees of damage,accompanied by intestinal mucosal layer and submucosal gland structure disorder,disappearance in severe cases,and infiltration of different inflammatory cells in the whole layer,such as lymphocytes and plasma cells,to varying degrees.The infiltration of neutrophils in the crypt and beadlike proliferation of lymphatic follicles were observed.These changes were related to the activity of CD.2.Immunohistochemical resultsCompared with the control group,the average level of TLR4 expression in the colon tissue of patients was higher in the CD group at remission stage,mild and moderate severe stage than in the CD group,and the differences were statistically significant,while there was no statistically significant difference between the CD groups.The positive grade score of My D88 expression showed that the expression of moderate and severe CD group and mild CD group was higher than that of CD group in remission and control group,and the differences were statistically significant.There was no statistical difference between mild CD group and moderate and severe CD group as well as between the remission group and control group.The positive grade of NF-κB expression was higher in the CD group than in the control group,and higher in the moderate and severe group than in the remission group,with statistical significance.There was no statistical difference between the remission group and the mild CD group.Compared with the control group,the positive level of ROR-γexpression was increased in the CD group,with statistical significance.Compared with the remission group,the expression of ROR-γexpression was increased in the mild and moderate severe CD group,with statistical difference.There was no statistical difference in the positive grade score of p-STAT3 and STAT3 expression in colon tissue of patients,as well as in moderate and severe CD group,mild CD group,remission CD group and control group.3.Analysis of test indexes and their correlationThe results of correlation analysis showed that TLR4,My D88,NF-κB were correlated with CD activity compared with clinical inflammatory markers.Correlation analysis showed that TLR4,My D88,NF-κB,ROR-γand p-STAT3 were positively correlated with each other to different degrees,and there was a significant strong correlation between TLR4 and My D88,while there was also a significant strong correlation between My D88 and NF-κB.There was no direct correlation between STAT3 and ROR-γexpression,but there was a significant moderate positive correlation between p-STAT3 and ROR-γexpression.Conclusions1.The expression levels of TLR4,My D88,NF-κB and ROR-γin colon tissue of CD were higher than those in normal colon tissue,and TLR4/My D88/NF-κB pathway was involved in the pathogenesis of CD.The expression levels of p-STAT3 and STAT3 were not different from those in normal colon tissues.2.The expression levels of TLR4,My D88 and NF-κB were significantly correlated with various inflammatory markers. |
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