Alterations Of Mucin Expression In Graft Bile Ducts After Liver Transplantation And Their Relationships With Bile Duct Injury

BackgroudOrthotopic liver transplantation is the most effective therapeutic option for patients with end-stage liver diseases. Nonanastomotic biliary strictures (NAS) are considered to represent the most troublesome complications after OLT as their resistance to therapy. NAS, with the incidence 5%-15%, are defined as strictures, dilatations and irregularity of nonanastomotic graft bile ducts and are reported to more easily involve larger bile ducts at first presentation. The occurrence of NAS has emerged as an important source of graft failure and retransplantation.The exact mechanism underlying the formation of NAS has not yet been determined. Cold ischemia-reperfusion (I/R) injury aggravates bile duct injury through different pathways and is considered to be a major cause of NAS. The microenvironment of biliary epithelial cells is filled with damage elements under physiologic condition, and there are also many protective measures to antagonize the damage elements to maintain the integrity of epithelia barrier. Previous studies showed that cold I/R injury not only directly damages the biliary epithelial cells, but also breaks such balance between damage elements and protective measures. Increased bile salts/phospholipids (BS/PL) ratio by cold I/R injury aggravates graft bile duct injury and is significantly associated with NAS.Mucins are a family of large molecular weight glycoproteins which expressed by various epithelial cells and glands lining the respiratory, gastrointestinal, genitourinary tracts. The putative functions of mucins are to protect epithelia from various damage elements. Mucin expression shows tissue- and disease-specificity. Alterations of mucin expression have been noted in conditions such as gastritis, peptic ulcer disease, Crohn's disease, ulcerative colitis, asthma, cystic fibrosis, chronic obstructive pulmonary disease and some epithelial malignancies. Mucins are also expressed in bile ducts and alterations of mucins have been studied in pathogenesis of hepatolithiasis, cholangiocarcinoma and many other biliary diseases. While whether mucin expression such a self-protective measure of biliary epithelial cells changed after liver transplantation has not been well established.ObjectiveIn present study, by adopting rat arterialized orthotropic liver transplantation model, we investigated the alterations of mucin expression in graft bile ducts and their Potential roles after OLT.Methods and Results1. To evaluate the alterations of mucin expression after liver transplantation. The rats were divided into four groups: (1) Normal group (n=10); (2) Sham-operated group (n=20), only received external biliary drainage and jejunal fistula; OLT-1h group (n=20), arterialized OLT with continuous external biliary drainage and jejunal fistula, and graft cold ischemic time is 1 hour in University of Wisconsin (UW) solution at 4℃; and (4) OLT-12h group (n=20), same operation as OLT-1h, just the graft cold ischemic time is 12 hour. In last three groups, four time points were predetermined as postoperative 1, 3, 7 and 14 days. Five animals were killed at each time point for histologic and biochemical analysis. Liver and bile duct samples were collected. Five normal bile duct samples were divided into larger and smaller ducts from the end of third order branches as NAS are reported to more easily involve larger bile ducts. mRNA and protein levels of Muc1, Muc2, Muc3A, Muc4, Muc5AC and Muc6 were detected by Real-Time PCR, Immunohistochemistry and Western blotting analysis.Normal larger bile ducts expressed mRNA for Muc1, Muc2, Muc3A, Muc4 and Muc6, while smaller ducts expressed Muc1, Muc2, Muc3A, and Muc4 mRNA, the levels of which, especially the Muc3A mRNA, were all remarkably lower than in larger bile ducts by Real-Time PCR analysis. Muc6 mRNA is absent in smaller bile ducts. Muc5AC mRNA is absent in both two parts. Immunohistochemistry examination showed that both Muc1 and Muc4 proteins were expressed on the apical membrane of biliary epithelial cells. Western blotting analysis showed that both Muc1 and Muc4 protein levels in larger bile ducts were significantly higher than in smaller bile ducts which were consistent with the levels of mRNA. The results showed that mucins were mainly expressed in larger bile ducts where NAS more easily involved. After liver transplantation, mRNA levels of Muc1, Muc3A and Muc4 decreased significantly during the early period compared with those in Sham, and then recovered gradually. It was noticeable that the longer the donor cold ischemic time, the lower mRNA levels of Muc1, Muc3A and Muc4, the more slowly to recover. Decrease of Muc3A mRNA after transplantation was seemed more dramatic. The mRNA levels of Muc2 and Muc6 after transplantation were almost the same as those of Sham at most time points. Muc5AC mRNA could not be detected in graft bile ducts after liver transplantation. The profiles of Muc1 and Muc4 protein expression were similar with their mRNA expression by western blotting analysis. What a pity that Muc3A protein was not detected as the lack of commercial antibody.2. To evaluate the correlations between downregulations of mucin levels and bile duct injury. Biliary ALP andγ-GT levels were detected. Larger and smaller Bile duct injury was semi-quantified by calculating a larger or smaller bile duct injury severity score (BDISS) respectively.By Pearson Correlation-tests, No obvious correlations were seen between the mucin levels and either ALP orγ-GT levels. By Spearman Correlation-tests, we found that mRNA levels of Muc1 and Muc3A in OLT-12h group were both negatively associated with larger BDISS(r=-0.62, P<0.01; r=-0.66, P<0.01, respectively). Muc1 protein levels in OLT-12h group was also negatively associated with larger BDISS (r=-0.52, P=0.02). There were no significant correlations between other mucins and larger BDISS. In addition, no obvious correlations were seen between mucin levels and smaller BDISS.3. To seek an ideal in vitro model for further study. Larger and smaller bile ducts were cultured in rat tail collagen, respectively. The experiment included three groups: I: control group, n=5; II: Stored for 1 hour at 4℃in UW-solution, n=5; III: Stored for 12 hours at 4℃in UW-solution, n=5. Use Real-Time PCR and Western blot to describe the expression of Muc1,Muc3A and Muc4 mucins.In Cold preservation-12h group, Muc1,Muc3A and Muc4 mucins were remarkably decreased in larger bile ducts compared with those of control group.Conlutions1. Muc1, Muc3A and Muc4 mucin levels deceased significantly early after rat liver transplantation, the longer graft cold ischemic time, the lower levels of Muc1,Muc3A and Muc4 mucins, the more slowly to recover.2. Downregulations of Muc1, Muc3A levels by prolonged graft cold ischemic time were significantly associated with histopathologic signs of larger bile duct injury. On the basis of these results, we speculated that downregulations of Muc1 and Muc3A mucins by prolonged graft cold ischemic time played a role in pathogenesis of larger bile duct injury after OLT.3. Bile duct culture in rat tail collagen was an ideal in vitro model for mechanism researches of downregulations of Muc1 and Muc3A in bile duct by prolonged cold ischemic time.