Clinical Features And Pathogenesis Of Primary Sclerosing Cholangitis And Inflammatory Bowel Disease

Part 1 Clinical features of primary sclerosing cholangitis and inflammatory bowel diseaseObjectives:To explore disease characteristics of primary sclerosing cholangitis(PSC)and inflammatory bowel disease(IBD)and to compare the differences between PSC-IBD and PSC without IBD.Methods:Forty-two PSC patients admitted from January 2000 to January 2021 were included.The data were collected through the electronic medical record system.We analyzed their demographic characteristics,clinical manifestations,concomitant diseases,auxiliary examination,and treatment.Results:The age at diagnosis of 42 PSC patients was 43.2 ±17.5 years old.The incidence rate of PSC-IBD was 33.3%,and the age at diagnosis of PSC-IBD was 41.9 ±17.1 years old.In PSC-IBD patients,the incidence of jaundice and fatigue was lower,and the incidence of diarrhea was higher(P<0.05).The levels of ALT,TBil,DBil,TBA and CA19-9 were higher(P<0.05)in PSC patients without IBD.The positive rates of ANA and fecal occult blood were higher(P<0.05)in PSC-IBD patients.Patients with PSC complicated with ulcerative colitis(UC)mainly suffered from extensive colonic involvement.The proportion of 5-aminosalicylic acid and glucocorticoid application in PSC-IBD patients significantly increased(P=0.025).Conclusions:We found that the incidence of PSC-IBD in Peking Union Medical College Hospital is lower than that in Western countries.Colonoscopy screening for PSC patients with diarrhea and fecal occult blood positive may be beneficial to the early detection and diagnosis of IBD.Part 2 The characteristics of gut microbiota and bile acid metabolism in a murine model of primary sclerosing cholangitis and ulcerative colitisObjectives:To construct a novel murine model of sclerosing cholangitis and acute colitis and to explore the characteristics of intestinal flora and bile acid metabolism.Methods:10-12-week-old male C57BL/6 mice were selected as subjects and were randomly divided into normal control group,DSS induced colits group,DDC induced sclerosing cholangitis group and DDC combined with DSS treated group.For sclerosing cholangitis mice model,mice were fed with standard chow containing 0.1%(w/w)DDC for 28 consecutive days.For colitis mice model,mice were treated with 2.5%(w/v)DSS dissolved in distilled water ad libitum for 5 days.To establish a murine model simultaneously possess the characteristics of sclerosing cholangitis and intestinal inflammation,mice were fed a 0.1%DDC-supplemented diet for 4 weeks to cause chronic liver damage and were administered 2.5%DSS dissolved in water for 5 days to induce colitis.Fresh mice fecal pellets were collected on the morning of the 29th day.The weight loss,mental state,and stool consistency change were recorded,and the colon length was counted after sacrifice.The colon and liver were histologically evaluated,and biochemical indicators such as ALT,ALP,TBil,and TBA were detected.After the evaluation of the murine model of sclerosing cholangitis complicated with acute colitis,16S rDNA sequencing and fecal bile acid metabolomics were performed.Immunohistochemistry was performed to detect farnesoid X receptor(FXR)location and expression.Results:The significantly higher disease activity index(P<0.05)and lower colon length(P<0.05)were observed in DDC+DSS treated group compared with DSS treated group and there was no significant difference in relative body weight loss(P=0.495)and histological scores for colits(P=0.862)between DDC+DSS treated group and DSS group.The levels of ALT,ALP and TBil in the DDC+DSS treated group were lower than those in the DDC group(all P<0.05).Porphyrin plugs in the bile ductules,obvious inflammatory cell infiltration and excess collagen deposition around the bile ducts were observed by HE staining in DDC+DSS treated group and DDC group,indicating bile duct inflammation.Evaluation of alpha diversity of fecal samples based on the OTU levels showed that the Simpson index,Shannon index,and Ace index in DDC+DSS treated group were significantly lower than those in the control group(all P<0.05).Principal co-ordinates analysis(PCoA)showed significant differences of the microbial community between two groups.Bacteroides,Akkermansia,Prevotella,Anaerostipes,Bacteroides acidogens,Bacteroides vulgaris,and Paraprevotella_clara were significantly enriched in DDC+DSS group by LEfSe.Fecal bile acid metabolomics showed that fecal CA and TUDCA concentrations increased(all P<0.05),GCA,UDCA,isoLCA,12_ketoLCA,LCA,βUDCA,α-MCA,HDCA,alloLCA,and DC A concentrations decreased significantly(all P<0.05)in DDC+DSS treated group.The fecal total bile acid level of DDC+DSS group was significantly decreased(P=0.0005).Orthogonal partial least squares-discriminant analysis showed that fecal bile acids in the normal control group and DDC+DSS treated group had a clear trend of separation.According to variable importance in projection value and the P value of significance test,HDCA,GCA,UDCA,DCA,12_ketoLCA,pUDCA,LCA,alloLCA,CA,α-MCA,and isoLCA were identified as the differential bile acids.Bacteroides,Prevotella,and Anaerostipes were negatively correlated with most differential bile acids.Immunohistochemistry revealed FXR was mainly distributed in the nucleus and cytoplasm of intestinal epithelial cells and FXR was downregulated in the DDC+DSS treated group compared with the normal control group.Conclusions:We built a novel murine model to study the characteristics of sclerosing cholangitis and acute colitis.We found that alpha diversity of fecal samples in DDC+DSS group were significantly lower than those in the control group.PCoA showed significant differences of the microbial community between two groups.The fecal bile acid spectrum of DDC+DSS group changed significantly,and the total fecal bile acid pool was significantly reduced.Bacteroides,Prevotella,and Anaerostipes were negatively correlated with most differential bile acids.The expression of bile acid receptor FXR is downregulated in the colon tissues of DDC and DSS treated mice,which may play an important role in the pathogenesis of primary sclerosing cholangitis and ulcerative colitis.Part 3 The role of bile acid receptor FXR in the pathogenesis of primary sclerosing cholangitis and ulcerative colitisObjectives:To explore the role of FXR in sclerosing cholangitis and acute colitis by constructing Fxr knockout mice.Methods:Male Fxr+/+ and Fxr-/-mice were selected as subjects.To establish a murine model simultaneously possess the characteristics of sclerosing cholangitis and intestinal inflammation,mice were fed a 0.1%DDC-supplemented diet for 4 weeks to cause sclerosing cholangitis and were administered 2.5%DSS dissolved in water for 5 days to induce colitis.The weight loss,mental state,and stool consistency change were recorded,and the colon length was counted after sacrifice.The colon was histologically evaluated.Real time quantitative polymerase chain was performed to detect the relative mRNA expression of colonic pro-inflammatory cytokines Il-1β,Il-6 and Tnf-α.RNA sequencing was performed to find differential expression genes and potential FXRmediated downstream signaling pathways.Immunohistochemistry was used to detect the expression of colonic autophagy related protein LC3,and Western blot was used to detect the expression of colonic mechanical barrier proteins E-Cadherin and Cluadin 1,as well as the expression of colonic autophagy related proteins LC3 and Beclin-1.For vitro experiment,HT29 cells were stimulated with 100 ng/ml rhTNF-α for 12 hours to establish a cellular inflammation model.Small interfering RNA was used to knock down FXR.The cells were divided into control siRNA group,control siRNA+rhTNF-α group,FXR siRNA group,and FXR siRNA group+rhTNF-α group.Western blot was used to detect the expression of LC3 and Beclin-1,and cell immunofluorescence was used to detect the aggregation points of LC3.The potential target genes of FXR to regulate autophagy were analyzed by bioinformatics.The binding of FXR to target genes was further verified by CUT&Tag.Results:Compare with Fxr+/+ DDC+DSS treated group,the significant decreased relative body weight(P=0.001),increased disease activity index(P=0.0008)and shortened colon length(P=0.0364)were observed in Fxr-/-DDC+DSS treated group.There was no significant difference in histological scores(P=0.916).The relative colonic mRNA expression of Il-1β and Il-6 increased,while the relative colonic mRNA expression of Tnf-α decreased(Il-1β,P=0.0339;il-6,P=0.410;Tnf-α,P=0.106).RNA sequencing found that the expression of colonic genes was changed after Fxr knockout in DDC+DSS treated mice.The differential expression genes were involved in multiple processes,including ferroptosis,tight junction,and autophagy.Immunohistochemical staining of colon tissue showed that LC3 expression in Fxr-/-group increased in intestinal epithelial cells compared with Fxr+/+ group.Western blot showed that the expression of colonic epithelial mechanical barrier proteins E-Cadherin and Claudin 1 in Fxr-/-group were lower than those in Fxr+/+ group(P<0.01)and autophagy related proteins LC3 Ⅱ/Ⅰratio,Beclin-1 were significantly higher in Fxr-/-group(P<0.05).The effect of FXR siRNA-transfected HT29 cells on the autophagy activity was detected by Western blot.In the absence of rhTNF-α stimulation,the ratio of LC3-Ⅱ/Ⅰincreased in FXR siRNA-transfected HT29 cells,but there was no statistical difference(P=0.298).The change of Beclin-1 protein expression was not observed.While under the stimulation of rhTNF-α,LC3 cleavage was significantly up-regulated(P=0.0329),and Beclin-1 protein expression was also up-regulated(P=0.0 759).Cell immunofluorescence staining demonstrated that under the stimulation of rhTNF-α,the LC3 aggregation points increased in FXR siRNA-transfected HT29 cells compared with the control group.Bioinformatics analysis suggested that Mras,Atg9b,Camkk2,Dapk2,Dapk1,Gabarapl1,Map1lc3a may be potential target genes of FXR to regulate autophagy in sclerosing cholangitis and colits mice model.CUT&Tag demonstrated that FXR could enrich in the upstream promoter region of ATG9B.Conclusions:Aggravated colonic inflammation and increased expression of autophagy related proteins LC3-Ⅱ and Beclin-1 were found in Fxr-/-DDC+DSS treated group.Transcriptomic data analysis found that after knocking out Fxr,many signaling pathways in colon tissue,including ferroptosis,tight junction,and autophagy,were altered.Knocking down FXR in HT29 cells increased the conversion of LC3-Ⅰ to LC3-Ⅱunder the stimulation of rhTNF-α.Bioinformatics analysis suggested that Mras,Atg9b,Camkk2,Dapk2,Dapk1,Gabarapl1,Map1lc3a may be potential target genes of FXR to regulate autophagy signaling.