[Roles of the CXCR1/CXCL8 axis in abnormal proliferation of bile duct epithelial cells in primary biliary cholangitis]

by Selleckchem | Aug 17 2023

Objective: To investigate the role of the CXC chemokine receptor 1 (CXCR1)/CXC chemokine ligand 8 (CXCL8) axis in the abnormal proliferation of bile duct epithelial cells in primary biliary cholangitis (PBC).

Methods: 30 female C57BL/6 mice were randomly divided into the PBC model group (PBC group), reparixin intervention group (Rep group), and blank control group (Con group) in an in vivo experiment. PBC animal models were established after 12 weeks of intraperitoneal injection of 2-octanoic acid coupled to bovine serum albumin (2OA-BSA) combined with polyinosinic acid polycytidylic acid (polyI:C). After successful modelling, reparixin was injected subcutaneously into the Rep group (2.5 mg · kg(-1) · d(-1), 3 weeks). Hematoxylin-eosin staining was used to detect histological changes in the liver. An immunohistochemical method was used to detect the expression of cytokeratin 19 (CK-19). Tumor necrosis factor-α (TNF-α), γ-interferon (IFN-γ) and interleukin (IL)-6 mRNA expression were detected by qRT-PCR. Western blot was used to detect nuclear transcription factor-κB p65 (NF-κB p65), extracellularly regulated protein kinase 1/2 (ERK1/2), phosphorylated extracellularly regulated protein kinase 1/2 (p-ERK1/2), Bcl-2-related X protein (Bax), B lymphoma-2 (Bcl-2), and cysteine proteinase-3 (Caspase- 3) expression. Human intrahepatic bile duct epithelial cells were divided into an IL-8 intervention group (IL-8 group), an IL-8+Reparicin intervention group (Rep group), and a blank control group (Con group) in an in vitro experiment. The IL-8 group was cultured with 10 ng/ml human recombinant IL-8 protein, and the Rep group was cultured with 10 ng/ml human recombinant IL-8 protein, followed by 100 nmol/L Reparicin. Cell proliferation was detected by the EdU method. The expression of TNF-α, IFN-γ and IL-6 was detected by an enzyme-linked immunosorbent assay. The expression of CXCR1 mRNA was detected by qRT-PCR. The expression of NF-κB p65, ERK1/2 and p-ERK1/2 was detected by western blot. A one-way ANOVA was used for comparisons between data sets.

Results: The results of in vivo experiments revealed that the proliferation of cholangiocytes, the expression of NF-κB and ERK pathway-related proteins, and the expression of inflammatory cytokines were increased in the Con group compared with the PBC group. However, reparixin intervention reversed the aforementioned outcomes (P<0.05). In vitro experiments showed that the proliferation of human intrahepatic cholangiocyte epithelial cells, the expression of CXCR1 mRNA, the expression of NF-κB and ERK pathway-related proteins, and the expression of inflammatory cytokines were increased in the IL-8 group compared with the Con group. Compared with the IL-8 group, the proliferation of human intrahepatic cholangiocyte epithelial cells, NF-κB and ERK pathway-related proteins, and inflammatory indicators were significantly reduced in the Rep group (P < 0.05).

Conclusion: The CXCR1/CXCL8 axis can regulate the abnormal proliferation of bile duct epithelial cells in PBC, and its mechanism of action may be related to NF-κB and ERK pathways.

Comments:

The objective of this study was to investigate the role of the CXC chemokine receptor 1 (CXCR1)/CXC chemokine ligand 8 (CXCL8) axis in the abnormal proliferation of bile duct epithelial cells in primary biliary cholangitis (PBC). The study utilized both in vivo and in vitro experiments.

In the in vivo experiment, female C57BL/6 mice were divided into three groups: the PBC model group (PBC group), the reparixin intervention group (Rep group), and the blank control group (Con group). PBC animal models were established by injecting the mice with 2-octanoic acid coupled to bovine serum albumin (2OA-BSA) combined with polyinosinic acid polycytidylic acid (polyI:C). After successful modeling, reparixin (an inhibitor of CXCR1) was injected subcutaneously into the Rep group for three weeks. Hematoxylin-eosin staining and immunohistochemistry were performed to assess histological changes in the liver and the expression of cytokeratin 19 (CK-19), respectively. mRNA expression of tumor necrosis factor-α (TNF-α), γ-interferon (IFN-γ), and interleukin-6 (IL-6) was measured using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Western blot analysis was conducted to assess the expression of nuclear transcription factor-κB p65 (NF-κB p65), extracellularly regulated protein kinase 1/2 (ERK1/2), phosphorylated ERK1/2 (p-ERK1/2), Bcl-2-related X protein (Bax), B lymphoma-2 (Bcl-2), and cysteine proteinase-3 (Caspase-3).

The in vitro experiment used human intrahepatic bile duct epithelial cells, which were divided into three groups: the IL-8 intervention group (IL-8 group), the IL-8+Reparicin intervention group (Rep group), and the blank control group (Con group). The IL-8 group was cultured with human recombinant IL-8 protein, while the Rep group was cultured with IL-8 protein followed by Reparicin. Cell proliferation was assessed using the EdU method. The expression of TNF-α, IFN-γ, and IL-6 was measured using an enzyme-linked immunosorbent assay. CXCR1 mRNA expression was detected using qRT-PCR. The expression of NF-κB p65, ERK1/2, and p-ERK1/2 was assessed using western blot analysis.

The results of the in vivo experiment showed that the Con group exhibited increased proliferation of cholangiocytes, increased expression of NF-κB and ERK pathway-related proteins, and increased expression of inflammatory cytokines compared to the PBC group. However, the Rep group, which received reparixin intervention, showed a reversal of these outcomes.

In the in vitro experiment, the IL-8 group exhibited increased proliferation of human intrahepatic cholangiocyte epithelial cells, increased expression of CXCR1 mRNA, increased expression of NF-κB and ERK pathway-related proteins, and increased expression of inflammatory cytokines compared to the Con group. However, the Rep group showed significant reductions in cell proliferation, NF-κB and ERK pathway-related proteins, and inflammatory indicators compared to the IL-8 group.

Based on these findings, the study concluded that the CXCR1/CXCL8 axis can regulate the abnormal proliferation of bile duct epithelial cells in PBC, and this regulation may occur through the NF