Study On The Mechanism Of Ischemic-type Biliary Lesion After Liver Transplantation

Objectives: To explore the possible mechanism of gut microbiota and DNA methylation in ischemic-type biliary lesions(ITBL)after liver transplantation.Methods: This study is composed of two parts.Part Ⅰ Study on the mechanism of bile acid metabolism in ITBL mediated by gut microbiota.(1)We collected the serum,liver biopsy and stool samples of patients with ITBL and without ITBL.Patients with ITBL and without ITBL were compared on intestinal flora distribution using 16 sRNA gene sequencing technology.Serum liver function was detected by a biochemical analyzer and serum deoxycholic acid(DCA)and cholic acid(CA)concentrations were measured by Elisa kits;(2)HIBEC were stimulated with DMSO,CA and DCA for 6h,respectively.CCK8 assay was used to detect cell viability,and flow cytometry and TUNEL staining were used to detect cell apoptosis.Through real-time fluorescent quantitative polymerase chain reaction(RT-PCR)and western blots,the BAX and cleaved-caspase3 expression levels were identified.We used JC-1 fluorescent probe to detect mitochondrial membrane potential changes,while the mitochondrial green fluorescent probe was used to detect mitochondrial number changes.Each group was stained with reactive oxygen species(ROS)fluorescence staining to detect their levels.The changes in mitochondria in cells were observed by transmission electron microscope.The BAX and FXR genes were knocked down with siRNAs in HIBEC cells,respectively,to detect the DCA-induced apoptosis level of the cells;(3)C57/BL mice were randomly divided into three groups and fed with DCA/CA diet at a ratio of 3:1,1:1 and 1:3,respectively.The mice’s liver and blood were obtained at 3 weeks.mouse serum liver function was measured to assess bile duct injury.Hematoxylin-eosin(HE)staining and cytokeratin(CK19)immunohistochemistry were performed on the mice’s liver tissue.At the same time,TUNEL and CK19 immunofluorescence staining was performed on the patients’ and mice’s liver tissue to evaluate the degree of cholangiocyte apoptosis.Part Ⅱ(1)The liver biopsy tissues of ITBL patients and non-ITBL patients after liver transplantation were collected.The liver tissue DNA was extracted and detected by Methylation EPIC(850K)Bead Chip.The differentially methylated sites and regions were analyzed,and the genes of differentially methylated sites and regions were analyzed by GO and KEGG analysis.Then genes with the most significant differences were screened;(2)HIBEC were treated with Co Cl2 to induce hypoxia in vitro.The cell apoptosis rate and cell viability of hypoxic HIBEC and normal cultured HIBEC were detected using flow cytometry and CCK8 respectively.The most significant difference gene was screened in the differential methylation sites.Both groups of HIBEC were tested for mRNA and protein expression levels using RT-PCR and Western blot analysis.Results:Part Ⅰ(1)Bifidobacteria,Lactobacilli,and Clostridia at the class level were significantly more prevalent in the ITBL group than in the Control group.Patients in the ITBL group had a higher serum DCA concentration and DCA/CA ratio,as well as impaired serum liver function;(2)HIBEC subjected to DCA showed a higher apoptosis rate,a higher proportion of TUNEL-positive cells,lower cell viability,and higher levels of BAX,ROS,and cleavedcaspase3,and lower mitochondrial membrane potential than CA at the same concentration.Under the electron microscope,we found that DCA changed the morphology of mitochondria.After the BAX gene was knocked down in HIBEC cells,the DCA-induced apoptosis rate was significantly reduced,the expression of BAX mRNA and BAX/BCL2 ratio,BAX and Cleaved-caspase3 protein were significantly decreased.Interestingly,DCAinduced apoptosis rate was significantly reduced when FXR or BAX gene was knocked down;(3)The liver function of mice gradually worsened with the increase of the DCA /CA ratio.In the DCA/CA 3:1 group,CK19 immunohistochemistry and HE staining showed the proliferation of small bile ducts in the portal area,and immunofluorescence staining showed that the proportion of TUNEL-positive cholangiocytes increased.The proportion of TUNEL-positive cholangiocytes in patients with ITBL was significantly higher than in those without ITBL.Part Ⅱ(1)Compared with the control group,a total of 8152 different DNA methylation sites in the ITBL group were identified,including 3023 hypermethylation sites and 5129 demethylation sites.The FASTK gene was the most significant difference gene between the two groups.Unsupervised hierarchical clustering analysis found significant differences in methylation sites between the two groups.GO enrichment analysis found the top10 GO entries in molecular function,biological process and cell component,and KEGG analysis found the Top30 KEGG pathway entries;(2)Compared with the control group,the mRNA and protein expression of FASTK decreased significantly after HIBEC hypoxia for 24 h,and the DNA methyltransferase 3b(DNMT3B)increased significantly,indicating that FASTK might be hypermethylated.Conclusions: Changes in gut microbiota may increase the proportion of hydrophobic bile acids(DCA)and aggravate bile duct injury through the FXR-mitochondrial apoptosis signaling pathway.Liver tissue DNA methylation is altered in patients with ITBL.FASTK gene as the most significant difference gene is hypermethylated and its expression is decreased,which may be involved in the pathogenesis of ITBL.This study provided new ideas for the treatment of ITBL after liver transplantation.