A Study On The Pathogenesis Of Inflammatory Bowel Disease Based On Clinicopathological Characteristics

Inflammatory bowel disease(IBD)is a chronic inflammatory disease of the gastrointestinal tract.With the progression of IBD,it leads to dysplasia and eventually develop into small bowel cancer or colorectal cancer.Early identification and diagnosis of IBD-related dysplasia is beneficial for early clinical intervention and follow-up of patients,as well as reducing the mortality rate of colitis-related colorectal cancer.Therefore,the systematic analysis of the clinical and pathological features of IBDrelated dysplasia is particularly important for accurately identifying dysplasia.The pathogenesis of IBD is unclear.At present,it is believed that environmental factors,genetic background and infection are involved in the pathogenesis of IBD.Among them,the inflammatory response caused by abnormal intestinal mucosal immune system plays an important role in the initiation and progression of IBD.However,due to the differences in genetic background and environmental factors between the East and the West,IBD-related susceptibility genes are also significantly different in various ethnic IBD patients.Based on current research states,exploring the role of susceptibility genes in Chinese patients with IBD will help to clarify the pathogenesis of IBD and provide a theoretical basis for related drug target research.LRRK2 has been widely studied in Parkinson’s disease.GWAS studies have shown that LRRK2 is also closely associated with some inflammatory diseases such as Crohn’s disease,leprosy and tuberculosis besides Parkinson’s disease,and LRRK2 has been identified as a CD susceptibility gene.In Japanese CD patients,LRRK2 is involved in the sorting and secretion of lysozyme in Paneth cells,while the Paneth cell defect in North American CD is irrelevant to LRRK2,indicating that LRRK2 has different effects in CD patients of different races.Considering the key role of intestinal mucosal immune system in IBD,we have taken intestinal innate immune response as a crucial point to study the effect and molecular mechanism of LRRK2 involved in intestinal immune regulation.This research is divided into three parts.PART Ⅰ The expression of LRRK2 in IBD patients.Objective The expression verification and localization of LRRK2 were studied by in vitro and in vivo experiments,and the related signaling pathways and interacting proteins of LRRK2 were analyzed by bioinformatics.Methods The expression of LRRK2 in different diseases was verified by WB,immunohistochemistry and q PCR.Immunofluorescence technology was used to explore the localization of LRRK2 in intestinal tissue,and the expression was further verified in vitro experiments.GSEA was used to analyze LRRK2-related signaling pathways,and the interaction between LRRK2 and key proteins of signaling pathways was further analyzed.Resullts LRRK2 is positively correlated with key proteins in these pathways.In vivo experiments,it was confirmed that LRRK2 expression was up-regulated in colon mucosa of CD patients,and there was no difference in other intestinal diseases.Immunofluorescence results confirmed that LRRK2 was mainly expressed in intestinal macrophages.In vitro experiments,LRRK2 was confirmed to be highly expressed in macrophages as the target gene of IFN-γ.GSEA analysis showed that LRRK2 was mainly related to the inflammatory signal pathways such as apoptosis signaling pathway,chemokine signaling pathway,JAK-STAT signaling pathway,leukocyte trans-endothelial migration pathway,NOD-like receptor signaling pathway,Toll-like receptor signaling pathway,MAPK signal pathway,and so on.Conclusion LRRK2 is abundantly expressed in colon mucosa of CD patients and is mainly expressed in macrophages,indicating that LRRK2 plays an important role in immune regulation in CD.PART Ⅱ Study on the function and mechanism of LRRK2 in colitis mouse model.Objective To explore the effect and mechanism of LRRK2 in CD by inhibiting or interfering the expression of LRRK2 in acute colitis mouse model and in vitro inflammation model.Methods The mouse model of acute colitis was established by enema with TNBS solution,and LRRK2 inhibitor was given for drug intervention to explore the effect of LRRK2 on experimental colitis;in vitro inflammation model stimulated by IFN-γ,lentivirus was used to interfere with the expression of LRRK2 in macrophages to detect the changes of related signal pathway proteins and the transcription level of inflammatory factors,and to study the specific mechanism of LRRK2.Resullts After treatment with LRRK2 inhibitors,the diarrhea symptoms of colitis mice were alleviated,promoting fecal granule formation,reducing mucosal thickening and weight loss,reducing colon shortening,reducing mucosal inflammatory cell infiltration and intestinal epithelial injury,and the transcription level of inflammatory factors in colon tissue was reduced,including IL-1β,IL-6,TNF-α,and MCP-1.In vitro,interfering with the expression of LRRK2 inhibited the transcription of inflammatory cytokines induced by IFN-γ in macrophages and decreased the phosphorylation level of P38,ERK and JNK in MAPK signaling pathway.Conclusion Inhibition of LRRK2 expression can effectively alleviate the clinical and pathological changes of TNBS-induced acute colitis mice,significantly reduce the secretion of many inflammatory factors in colon tissue,and down-regulate the transcription levels of macrophage inflammatory factors(IL-1β,IL-6,TNF-α,MCP-1).This effect is regulated by reducing the phosphorylation level of P38,ERK and JNK proteins in MAPK signaling pathway.PART Ⅲ The clinical and pathological characteristics of IBD-related dysplasiaObjective Through the retrospective analysis of the clinical and pathological data of patients with IBD in the past five years,the clinicopathological features of IBDrelated dysplasia in Hubei were systematically analyzed and summarized.Methods The clinical and pathological data of IBD patients treated in Zhongnan hospital of Wuhan University from January 2017 to December 2021 were collected.All cases were reviewed,and all suspected IBD-related dysplasia were screened out and further diagnosed by p53 detection.Resullts 497 patients with IBD has been involved,including 333 males(67%)and 164 females(33%),the age range from 10 to 87 years old,with an average age of 37 years old.There were 281 patients with CD(56.5%),200 patients with UC(40.2%)and 16 patients with indeterminate IBD(3.3%).Under the endoscope,most lesions were flat(n=422 cases,84.9%),and a few lesions were polypoid or raised(n=75,15.1%).According to morphological evidence,71 patients(14.3%)had suspected IBDrelated dysplasia among 497 patients with IBD,and 11 cases of IBD-related dysplasia were further diagnosed by p53 staining,including 2 cases of IBD-related small intestinal cancer,and the average course of dysplasia was 4.5 years.Histologically,the features were crowed crypt cells,deep nuclear staining,enlarged and elongated nucleus,increased nuclear-to-cytoplasmic(N:C)ratio,lack of surface maturation,and mutant p53 staining.Conclusion We found that the course of dysplasia in Chinese patients with IBD is shorter than that in Europe and America,which may be due to the diagnosis level of IBD,genetic factors,environmental factors and so on.P53 staining results and expression patterns have good specificity and application value in distinguishing IBDrelated dysplasia from mucosal reparative changes.the expression of p53 can help clinicians to identify and diagnose IBD-related dysplasia in the early stage without a unified and strict morphological standard of IBD-related dysplasia.