Exploring Potential Therapeutic Targets Of Crohn’s Disease Based On Proteomics And Effects And Mechanism Of Loureirin B On The Treatment Of Crohn’s Disease

Background:Crohn’s disease(CD)is a chronic and refractory intestinal inflammatory disease with a dramatic increase in incidence in our country.At present,the etiology and pathogenesis of CD are still unclear.Although biologic agents targeting TNF-α,IL-12/IL-23,or integrin α4β7 are effective in ameliorating CD,only a subset of patients benefit from them.Exploring new therapeutic targets is an important direction for CD research.Proteomics has been used to explore therapeutic targets for CD.However,most studies are still in the initial discovery stage and lack of functional and mechanistic verification of a target protein.In addition to target therapy,more and more CD patients seek complementary therapies,such as herbal medicine and acupuncture.In the theoretical system of traditional Chinese medicine,blood stasis is an important pathogenic factor of CD.The method of promoting blood circulation and removing blood stasis can effectively relieve CD luminal inflammation.Previously,the research group used the Chinese patent medicine of DaemonoropsdracoBl in the treatment of CD and achieved ideal curative effect.Nevertheless,its mechanism of action is still unclear.Purpose:(1)To discover a potential therapeutic target for CD by using proteomics,and then to verify its function and mechanism through a cell experiment.(2)To reveal the effect and mechanism of Loureirin B(LB),the main pharmacodynamic substance of DaemonoropsdracoBl,in the treatment of CD rat model.Methods:The plasma protein profiles of 20 CD patients and 20 matched healthy people were detected by proteomics.Differential expression proteins were screened based on a fold change greater than 1.5 or less than 2/3 and P values less than 0.05.The MCODE plugin in Cytoscape software was used to screen hub genes.A hub gene with immunoregulatory function was selected for functional verification and mechanism study.The expression levels of this hub gene in the intestinal mucosa of additional 20 CD patients and 20 matched healthy people were determined by immunohistochemistry.Subsequently,plasmid or siRNA of the selected hub gene was used to treat a CD cell model induced by lipopolysaccharide.The concentrations of pro-inflammatory cytokines were detected by ELISA.qRT-PCR and Western blot were used to respectively detect the gene levels and the protein expressions of classical NF-κB signaling pathway to determine the effect of the selected hub gene on regulating intestinal inflammation.After LB treated the CD rat model induced by 2,4,6-trinitrobenzenesulfonic acid induction,the general condition,disease activity index scores and histopathological scores of rats were observed.The concentrations of inflammatory cytokines and indexes of oxidative stress were measured by ELISA.TUNEL staining and Masson staining were used to respectively observe the apoptosis of intestinal epithelial cells and intestinal fibrosis.Western blot was used to detect the expressions of indexes related to apoptosis and epithelial-to-mesenchymal transition.qRT-PCR and Western blot were used to respectively detect the mRNA and protein expressions of fibrinogen-like protein 1(FGL1)/NF-κB/STAT3 positive feedback loop.Results:A total of 35 differential expression proteins were found by proteomics.Among them,22 hub genes were identified.FGL1,one hub gene with an immunomodulatory function and involvement in coagulation and thrombosis,became the target gene for further research.The expression level of FGL1 in peripheral blood of CD patients is 1.722-fold higher than that of healthy subjects.FGL1 expressions in lamina propria and epithelial layer of intestinal mucosa were also significantly higher in CD patients than in healthy individuals.In vitro,overexpression of FGL1 increased the concentrations of pro-inflammatory cytokines IL-1β,IL-6,IL-17 and TNF-α,while siRNA silencing of FGL1 decreased the concentrations of these pro-inflammatory cytokines.FGL1 overexpression also upregulated the leves of NF-κB mRNA and protein expressions of IKKα,IKKβ,p-IKKα/β,p-IκBα,and p-p65.Knockdown of FGL1 can reverse the gene level of NF-κB and the protein expressions of its classical signaling pathway.LB could reduce disease activity index and histopathological scores of CD rats,decrease the concentrations of pro-inflammatory cytokines IL-1,IL-1β,IL-6 and TNF-α,and increase the concentration of anti-inflammatory cytokine IL-10 to relieve intestinal inflammation.LB might inhibit oxidative stress and lipid peroxidation by reducing the concentrations of iNOS,MPO and MDA.LB was able to inhibit apoptosis of intestinal epithelial cells via upregulating the expression of Bcl-2 and downregulating the expressions of Bax,cleaved caspase-3,caspase-3,cleaved caspase-9 and caspase-9.LB also displayed a function to improve intestinal fibrosis via reducing the expressions of Snail,N-cadherin and Vimentin and increasing E-cadherin expression to inhibit epithelial-to-mesenchymal transition.In addition,the mRNA expressions of IL-6,NF-κB,STAT3 and protein levels of FGL1,p-p65 and p-STAT3 in the colon of CD rats were significantly decreased after LB intervention.Conclusions:FGL1 could induce inflammatory response in intestinal epithelial cells through activation of IKK/NF-κB signaling pathway,which suggested that FGL1 might be a potential therapeutic target and biomarker for CD.LB might reduce apoptosis of intestinal epithelial cells and pro-inflammatory cytokine secretion to ameliorate colitis of CD rats,and inhibit epithelial-to-mesenchymal transition to improve intestinal fibrosis by blocking FGL1/NF-κB/STAT3 positive feedback loop.Results of this research preliminarily revealed the mechanism of the method of promoting blood circulation and removing blood stasis in the treatment of CD,and might provide a novel therapeutic target for CD.