| Purpose: Primary hepatolithiasis (HL), a condition marked by the presence of calculus in intrahepatic ducts, is a common disease in China and is especially prevalent in the Eastern and Southern regions of China. Although HL is a benign disease, it is intractable and usually requires operative interventions due to frequent stone recurrences. Most importantly, this disease has a high association with cholangiocarcinoma. Clinical data and experimental studies indicate that hepatolithiasis is associated with chronic inflammation of bile ducts, biliary fibrosis, biliary stricture, and bacterial infections. However, the cellular and molecular etiology of hepatolithiasis has remained elusive. Many previous studies have focused on the formation of stones, while little attention has been paid to the role of bile duct epithelial cells (BEC). Epithelial-mesenchymal transition (EMT) is, by definition, a process whereby epithelial cells convert to migratory mesenchymal cells. This process is characterized by a loss of cell polarity and acquired expression of mesenchymal components. EMT results in a dramatic remodeling of the cytoskeleton, which includes reduced expression of cell adhesion molecules (i.e., E-cadherin) and the transition of keratin-based cytoskeleton into vimentin-based cytoskeleton, thus leading to changes in cell morphology. It has been reported that EMT contributes to both renal fibrogenesis and the fibrogenetic processes that occur in chronic liver diseases such as hepatocirrhosis.Members of the transforming growth factor-β(TGF-β) family are pleiotropic cytokines involved in multiple physiological and pathological processes, including cellular proliferation, adhesion, apoptosis, differentiation and immunoregulatory activities. TGF-β1 activates Smad signaling via its two cell surface receptors—TGF-βtype I receptor (TβRI) and TGF-βtype II receptor (TβRII)—leading to Smad-mediated transcriptional regulation of target genes.Therefore, we assumed that primary hepatolithiasis could be caused by EMT, wherein BECs lose differentiated functions and develop a fibroblast phenotype. This study explored whether intrahepatic biliary epithelial cells undergo EMT during hepatolithiasis by detecting changes in epithelial and mesenchymal markers via immunohistochemical assays. Results:1.Expressions of the epithelial markers E-cadherin andα-catenin were frequently lost in HL samples (32.3% and 25.9%, respectively), while the mesenchymal marker vimentin was found to be present in normal amounts in 35.5% of hepatolithiasis samples;α-SMA was also found to be present in 29.0% of samples. The positive rates of TGF-β1 and Smad 2/3 in the samples paralleled those of the mesenchymal markers, but was comparable between the HLBECs and the control cells (p>0.05).2.Twenty-four hours after the administration of LPS, marked TGF-β1 mRNA was detected. The expression of TGF-β1 mRNA reached the peak at 48 h, but the mRNA level decreased after 72 h. These results suggest that the expression of TGF-β1 was induced by LPS and reached a peak value at 48 h . Therefore, we detected the expression of proteins involved in the EMT process at 48 h and 72 h in the following experiments.3.Along with the exposure to LPS, the mRNA of epithelial markers E-cadherin decreased gradually, whereas the mesenchymal markers (S100A andα-SMA) increased significantly . However, the expression of markers S100A4 andα-SMA at different time points did not show any difference (p >0.05). Consistent with the results of :westblotting, the protein level of E-cadherin decreased , and S100A andα-SMA increased .4.Administration of paclitaxel alone did not change the expression of TGF-β1 mRNA. However, pretreatment with paclitaxel before administration of LPS significantly down-regulated the mRNA of TGF-β1, Smad2/3, especially at 48 h, when the mRNA of TGF-β1, Smad2 /3 decreased by 60%, 36% and 28%, respectively. These results indicated that PT was not a specific antagonist of Smad2/3 because the blockage of Smad2/3 by PT was not as efficient as that of TGF-β1 . We assumed that there were other proteins besides Smad 2/3 involved in the reduced transcription of TGF-β1 that resulted from treatment with PT.5.It was observed that 48 hours after transfection with siRNA Smad2/3, the fluorescence intensity reached its peak, but the fluorescence intensity gradually weakened after 72 hours. The transfection efficiency of HIBEpiCs was 78% (data not shown). The results of real-time PCR showed that the levels of Smad 2/3 mRNA in transfected cells were significantly decreased by even more than 90% at both 48 h and 72 h and that the mRNA of epithelial marker E-cadherin was significantly up-regulated. Both at 48 h and at 72 h, E-cadherin mRNA increased by 64% and 20%, respectively. Expression of mesenchymal markers S100A4 andα-SMA was significantly reduced . It was confirmed that Smad 2/3 played a key role in the TGF-β/Smad signaling pathway. Forty-eight to seventy-two hours after the transfection of siRNA to Smad2/3, the E-cadherin protein in HIBEpiCs increased by 40% and 20%, respectively . The level of S100A4 protein decreased by 21 % and 41%, andα-SMA protein decreased by 35 % and 44% . Inhibition of S100A4 andα-SMA at 72 h was more efficient than that at 48 h (p <0.01), but the expression of E-cadherin showed no difference between the two time points (p >0.05).6.In rat model, the mesenchymal marker vimentin was found to be present, while expressions of the epithelial markers E-cadherin andα-catenin were frequently lost in bile duct epithelial cells of rat with LPS administration, suggesting the development of EMT in bile duct epithelial cells.Conclusions:1.in BECs from patients with primary HL, epithelial markers were lost, the TGF-β1/Smad pathway was activated, and mesenchymal markers were upregulated. These data indicate that TGF-β1-mediated EMT plays a role in the formation of hepatolithiasis.2.LPS can induce the expression of TGF-?1 and a subsequent EMT in HIBEpiCs, and the inhibition of TGF-?1 or Smad 2/3 could reverse this EMT, suggesting that the TGF–?/Smad pathway may be a potential target for the prevention and treatment of biliary fibrosis.3.LPS could also induce the EMT of bile duct epithelial cells in rat model... |
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