Human Intra-Hepatic Biliary Epithelial Cells

Apelin-APJ Induces Cholangiocyte Proliferation Via Nox4/ROS/ERK Signaling Pathway During Cholestasis

Apelin, a ligand for the G-protein coupled receptor APJ, is shown to have elevated serum levels in various human liver diseases. The apelin-APJ signaling axis is well-documented in influencing organ fibrosis, but its specific role remains debated. It has been observed to reduce fibrosis in renal and myocardial tissues but appears to promote liver fibrosis. Previous studies have correlated high serum apelin levels with the severity of liver conditions and increased APJ expression in cirrhotic livers.

Chen et al. aimed to evaluate the role of apelin-APJ signaling in regulating ductular reaction and liver fibrosis during cholestasis. They found that Apelin regulates Nox4 expression levels and stimulates the formation of reactive oxygen species (ROS) that triggers vascular smooth muscle cell proliferation via ERK. Therefore, they postulated that apelin-APJ promotes cholangiocyte proliferation through the Nox4/ROS/ERK signaling pathway in cholestasis. The findings demonstrated elevated NOX4 mRNA in human primary sclerosing cholangitis (hPSCL) cholangiocytes and BDL mice cholangiocytes compared to human intra-hepatic biliary epithelial cells (HIBEpiCs) and WT, respectively, with reductions observed in treatment by ML221 (Fig. 1A and B). Apelin increased NOX4 mRNA in HIBEpiCs in vitro, with ML221 pretreatment reversing this effect (Fig. 1C). ROS levels in cholangiocytes from BDL mice were higher compared to WT mice but were lowered by ML221 and apelin treatment (BDL+apelin) (Fig. 1D). In vitro, apelin upregulated ROS in HIBEpiCs, which was mitigated by ML221, DPI, or NAC pretreatment (Fig. 1E and F).

Elevated ERK phosphorylation in BDL mouse cholangiocytes was reduced by ML221 (Fig. 2A and B). Apelin-treated BDL mice showed decreased ERK phosphorylation compared to untreated BDL mice (Fig. 2C). In vitro, apelin enhanced ERK phosphorylation in HIBEpiCs, which was diminished by ML221, DPI, NAC, or PD98059 (Fig. 2D). Apelin induced HIBEpiC proliferation, with ML221, DPI, NAC, or PD98059 pretreatment inhibiting this proliferation. PCNA and KI67 expression were also increased by apelin in HIBEpiCs, but reversed by pretreatment of the aforementioned inhibitors (Fig. 2F).

Fig. 1. Detection of Nox gene expression levels (A-C). Detection of ROS expression levels (D-E). (F) Confocal microscopy assay for intracellular ROS regeneration in HIBEpiCs (Chen L, Zhou T, et al., 2021).

Fig. 2. The expression of p-ERK in mouse cholangiocytes, frozen liver sections and HIBEpiCs (A-D). (E) The measurement of proliferation in apelin treated HIBEpiCs. (F) The mRNA expression of PCNA and KI67 in apelin treated HIBEpiCs (Chen L, Zhou T, et al., 2021).

Sirt6 Ameliorated the GCDC‑Induced HiBEC Apoptosis

Cholestatic liver diseases expose biliary epithelial cells (BECs) and hepatocytes to toxic bile acids, leading to apoptosis and mitochondrial dysfunction. The underlying molecular mechanisms remain unclear. Research has shown that Sirt6 regulates liver injury and metabolism, yet its impact on human biliary epithelial cells (HiBECs) in cholestatic conditions is scarcely studied. Therefore, Li et al. aimed to explore the molecular mechanisms involved in Sirt6 protection against the apoptosis of HiBECs induced by the bile acid glycochenodeoxycholate (GCDC).

Li et al. discovered that Sirt6 ameliorates GCDC-induced apoptosis in human intrahepatic biliary epithelial cells (HiBECs) by upregulating the expression of PGC-1α through the AMPK pathway and its deacetylation activity. Given their observation that GCDC can downregulate Sirt6 expression in HiBECs, they subsequently aimed to determine the effect of Sirt6 on GCDC-induced cellular damage in HiBECs. HiBEC were transfected with a Sirt6 expression vector or a vector control for 48 hours, followed by treatment with 1 mM GCDC for 8 hours. Sirt6 transfection significantly increased Sirt6 mRNA and protein levels (Fig. 3a), enhanced cell viability (Fig. 3b), and reduced apoptosis (Fig. 3c). Sirt6 knockdown via si-Sirt6 transfection decreased cell viability and increased apoptosis (Fig. 3d-f). Western blot analysis indicated that Sirt6 overexpression decreased cleaved caspase-3 and Bax levels while increasing Bcl-2 levels, confirming its anti-apoptotic role. Conversely, Sirt6 knockdown had the opposite effects. Additionally, Sirt6 overexpression reduced cytochrome c release from mitochondria, while its knockdown increased it, implicating the involvement of Bax, Bak, and MPTP (Fig. 3g and h).

Fig. 3. Sirt6 ameliorated GCDC-induced HiBEC apoptosis (Li J, Yu D, et al., 2020).