Research On The Pathogenesis Of Inflammatory Bowel Disease And The Effect Of Alcohol Based On The Lymphatic System

BackgroundInflammatory bowel disease(IBD)is chronic,non-specificity inflammatory diseases of intestine due to unknown causes.IBD is one of the independent risk factors of colorectal cancer.For the study of IBD,the initial focus was only on the damage of intestinal epithelial cells in patients with IBD.However,in recent years,some scholars found that IBD is related to the damage of lymphatic system,and speculated that the essence of IBD is lymphedema caused by intestinal lymphatic abnormalities,and the damage of lymphatic endothelial cells may also be one of the pathogenesis of IBD.The onset of IBD is insidious and difficult to detect,so it is not easy to distinguish IBD from other intestinal diseases,such as radiation enteritis and intestinal tuberculosis,and there is no accurate method for the diagnosis of IBD at home and abroad,therefore,often leads to misdiagnosis.At present,the lymphatic system is examined by indocyanine green(ICG)near-infrared imaging.However,due to its low lymphatic targeting and rapid metabolism,indocyanine green can only be used for subcutaneous superficial lymphatic system imaging,and cannot be used for examination of the visceral lymphatic system.In this case,a new type of high-efficiency imaging material for targeting intestinal lymphatic system is eagerly needed,and it will have a significant role in promoting the diagnosis of IBD.The research on the role of alcohol in IBD process is not sufficient,and the research results are also controversial.Studies have shown that alcohol can cause intestinal lymphedema and inflammation by destroying lymphatic vessels.Therefore,we speculate that alcohol should play an important role in the pathogenesis of IBD.Recent studies have found that lysosomes can participate in the activities of lymphatic endothelial cells.Therefore,we believe that alcohol can also affect the lysosome abnormalities of lymphatic endothelial cells,thereby causing damage to lymphatic endothelial cells and aggravating inflammation.At present,for the pathogenesis of IBD,genomics,proteomics and bioinformatics technologies have become new research technologies.For the prevention and treatment of IBD.These new technologies can extensively and comprehensively search for undiscovered IBD-related genes in the whole gene bank,and accurately search for pathways that may be related to this gene and IBD,which provides reliable data for subsequent research on the mechanism of IBD,thereby greatly improving research efficiency and obtaining sufficient evidence.Based on this status,we have carried out research on the mechanism of alcohol-induced intestinal injury through transcriptomics and bioinformatics,so as to lay a solid foundation for the subsequent prevention and treatment of IBD and the development of new drugs.ObjectiveUnder the existing conditions,we studied the fluorescent nano-imaging materials targeting the lymphatic system of IBD rats,and the pathogenesis of IBD based on lymphoid system and the role of alcohol in the process of IBD.We aim to develop an efficient and specific intestinal imaging material for IBD rats,so as to explore a new pathogenesis of IBD,and to clarify the role of alcohol in the process of IBD.This work not only provides great significance to the prevention and treatment of IBD,but also provides a new idea for the research of IBD.Materials and methods1.Based on the indocyanine green(ICG),a new nano-fluorescent material Tp Pa-1@ICG-HG was synthesized by chemical reaction.The stability,innocuity and specificity to lymphatic endothelial cells were verified by structure analysis,cytotoxicity test and immunofluorescence.180-220 g male SD rats were selected to establish the IBD rat model by drinking DSS solution daily.Tp Pa-1@ICG-HG was given to rats by intragastric administration,and the lymphatic system targeting and metabolic rate of ICG were compared by immunofluorescence co-localization.2.180-220 g male SD rats were selected to establish the IBD rat.The optimal daily DSS intake of rats was 0.5L / kg.Subsequently,the IBD rat model was constructed by quantitative daily free drinking of DSS solution and 2.5g / kg once daily,The experiment groups are as below: DSS + alcohol group,DSS group,alcohol group and control group.Seven days after the establishment of the IBD model,applied to a synthetic novel fluorescent nanomaterial,Tp Pa-1 @ICG-HG,the ileums,colons and mesenteries of dissected rats were stained with HE staining and immunohistochemical staining to determine the effect of alcohol on the lymphatic vessels of IBD rats.We also measured lenght of intestine and leakage of intestinal lymphatic vessels.At the same time,q PCR and western blot experiments were used to detect the molecular changes of related genes in rat lymphatic endothelial cells to verify its molecular mechanism.3.The above rats were selected and given DSS and alcohol according to the above method,and the experiments were divided into: DSS+ alcohol group,DSS group,alcohol group and control group.After 7 days,their mesenteries were taken.The whole transcriptome analysis was done by the second generation sequencing method,and the bioinformatics analysis was done by the enrichment method of GO and KEEG database pathway in order to explore the specific genes and signal pathways in the process of alcohol-induced lymphatic system injury in IBD rats.Results1.According to structural analysis,cytotoxicity experiments,and immunofluorescence,the physical and chemical structure stability and fluorescence intensity of Tp Pa-1@ICG-HG are better than those of ICG,and Tp Pa-1@ICG-HG has no biological toxicity.2.Verified by cell experiments and IBD rat models,Tp Pa-1@ICG-HG has lymphatic system targeting,and the specificity and stability of binding to lymphatic endothelial cells are better than ICG,and its metabolic half-life in vivo is longer than ICG.3.Compared with other groups,the DSS+Alcohol group rats had the highest DAI score,the lowest body weight,and the shortest total intestinal length,with significant differences.The HE staining showed that the intestinal structure destruction and inflammation were the most severe in the DSS+Alcohol group.Immunohistochemistry showed that in the DSS+Alcohol group,lymphatic vessels were obviously destroyed,lymphatic endothelial cells proliferated disorderly,and intestinal lymphedema was obvious.The colorimetric method showed that the lymph fluid of the rats in the DSS+Alcohol group leaked severely.The results of PCR and Western Blot showed that the expression of lymphatic endothelial cell-related marker Lyve-1 was significantly increased.4.Transcriptomics showed that there was a significant difference in the expression of 61 genes in DSS+ alcohol group compared with DSS and control group.Among them,Lyve-1,Acacb,Fmo3,Glul,Grin2 d,Alox5ap,Hmmr,Prodh1,S100a8 and other genes had the most significant differences.In addition,the expression of VEGF-D,a product associated with lymphatic endothelial cells,was also significantly increased.5.The results of.GO enrichment showed that among the functions involved in the above genes,the functions of organic acid binding,carboxylic acid binding and hyaluronic acid binding were significantly changed.The results of KEGG enrichment showed that in the above genes,calcium ion signaling pathway was significantly changed,and Lyve-1,Grin2 d,Hmmr and VEGF-D genes were involved in these processes.Conclusion1.Tp Pa-1@ICG-HG is a stable,harmless dye,with targeting to the lymphatic system and a long metabolic half-life.Tp Pa-1@ICG-HG can accurately,efficiently and sustainably locate the intestinal lymphatic vessels of IBD rats.2.Alcohol can damage the original structure of lymphatic vessels in the lymphatic system,by damaging the lymphatic endothelial cells,leading to the leakage of lymphatic fluid in the lymphatic cavity,and it can lead to the disordered and compensatory proliferation of lymphatic endothelial cells blocking the lymphatic vessels,resulting in downstream intestinal lymphedema,and further aggravate IBD.3.Lyve-1 is the key gene that alcohol can aggravate IBD patients by destroying the lymphatic system.The key mechanism is carboxylic acid binding,organic acid binding,hyaluronic acid binding function,calcium ion signaling pathway and Epstein-Barr virus infection pathway.The key genes involved in these functions and pathways are Lyve-1,Grin2 d,Hmmr and VEGF-D genes.