Liver fibrosis and biliary proliferation are enhanced in the rat model of inflammatory bowel disease

Abstract

BackgroundPatients who suffer from Inflammatory Bowel Disease (IBD) often experience concurrent conditions such as lymphatic dysfunction and bone loss. Additionally, liver and biliary abnormalities are frequently observed, including primary sclerosing cholangitis (PSC), autoimmune hepatitis, and cholelithiasis. The pathogenesis is unknown but is presumed to be secondary to chronic inflammation, which drives intrahepatic inflammation and fibrogenesis. The lymphatic system, via transport of inflammatory stimuli, is a likely route by which the regional inflammatory changes in the bowel effect distal organs such as the liver. Therefore, the aim of this study was to evaluate changes in biliary damage and senescence, ductular reaction (DR), and liver inflammation and fibrosis in an experimental rat model of inflammatory bowel disease.MethodsStudies were performed in male rats receiving a 2,4,6‐trinitrobenzene sulfonic acid (TNBS) enemas at 30mg/kg of body weight, once a week, for 4 weeks. Lymph, livers, isolated cholangiocytes, and hepatic stellate cells were collected. Ductular reaction was evaluated by immunohistochemistry (IHC) for CK19 in paraffin embedded liver sections and cellular senescence was determined by SA‐β‐gal staining. Gene expression was measured using qPCR for p16, CCL2 and p21 in total liver and laser‐captured microscopy (LCM) isolated cholangiocytes. Liver fibrosis was assessed via Sirius Red staining and qPCR for the following genes; α‐SMA, TGFβ‐1, FN‐1, and collagen1a1 in total liver, HSCs, and pure cholangiocytes. To determine the level of inflammation, IHC for CD68 and MHCII was performed, while gene expression was measured using qPCR for inflammatory/senescence markers (IL‐1β, IL‐6, IL‐33 and TNF‐α) in total liver and LCM isolated cholangiocytes. Immunofluorescence was used to visualize the expression of TLR4 in the intact bile ducts of total liver sections. Lymph was used for proteomic analysis post‐instillation at peak inflammation, to identify potential inflammatory signals that may precipitate the changes that were noted.ResultsRats treated with TNBS had increased DR, collagen deposition, and fibrotic gene expression in both cholangiocytes and HSCs; enhanced liver inflammation with increased CD68 positive macrophages in liver sections; increased biliary senescence and biliary expression of senescence markers; and increased biliary expression of TLR4. There was a strong correlation between the increase in inflammatory pathways in the lymph from IBD animals, with marked increases in pathways associated with bacterial translocation and lipid metabolism.ConclusionWe observed increased biliary ductular reaction that was associated with increased liver inflammation/fibrosis similar to the co‐morbidity of PSC reported in IBD patients.Support or Funding InformationNIH RO1 DK110035