Endothelin receptor B enhances liver injury and pro-inflammatory responses by increasing G-protein-coupled receptor kinase-2 expression in primary biliary cholangitis

Severe diseases like cirrhosis and liver failure can be developed from primary biliary cholangitis (PBC). Endothelin-2 (EDN2) and endothelin receptor B (EDNRB) are related to the pathogenesis of PBC. However, the roles of EDN2 and EDNRB in PBC-related liver injury and inflammation along with molecular mechanisms are poorly defined. In this study, histopathologic alterations of liver tissues were assessed through hematoxylin–eosin staining. Alanine transaminase (ALT), alkaline phosphatase (ALP), aspartate transaminase (AST), and γ-Glutamyltranspetidase (GGT) (4 liver function indexes) serum levels were detected with corresponding activity assay kits. Also, we determined the levels of M2 subtype anti-mitochondrial antibody (AMA-M2), interferon-gamma (IFN-γ), and tumor-necrosis factor alpha (TNFα) in serum with ELISA assay. Later, RT-qPCR assay was used to measure the expression of genes at mRNA levels, while western blotting and immunohistochemical techniques were used to detect protein levels of genes. Our results showed that the liver tissues of PBC patients and mice presented with severe hepatocyte injury and inflammatory cell infiltration as well as destruction of intrahepatic small bile ducts. ALP, AST, ALT, GGT, AMA-M2, IFN-γ, and TNF-α serum levels were higher in PBC patients and mice. Besides, EDN2 and EDNRB were highly expressed in serums and livers of PBC patients and mice. EDNRB potentiated PBC-related liver injury and pro-inflammatory responses, as evidenced by observation of serious liver pathologic injury and increased serum levels of ALP, AST, ALT, AMA-M2, IFN-γ, and TNF-α in PBC mice following EDNRB overexpression. EDNRB overexpression or activation via its agonist IRL-1620 TFA triggered liver injury and pro-inflammatory responses, increased GRK2 expression and induced NF-κB expression and activation in wild-type mice. EDNRB knockdown or inhibition by Bosentan alleviated liver damage and inflammation, reduced GRK2 expression, and inhibited NF-κB in PBC mice. These findings suggested EDNRB loss or inhibition weakened liver injury and pro-inflammatory responses by down-regulating GRK2 and inhibiting the NF-κB pathway in PBC mice.