Inhibition of the substance P/neurokinin-1 receptor (NK-1R) axis decreases liver fibrosis in the Mdr2(-/-) mouse model of primary sclerosing cholangitis (PSC) through changes in senescence in cholangiocytes and hepatic stellate cells

PSC is characterized by biliary damage, senescence and liver fibrosis. Senescence contributes to liver fibrosis in PSC. In cholangiopathies, proliferating cholangiocytes acquire neuroendocrine features and secrete sensory neurotransmitters such as substance P (SP). We have previously shown that: (i) SP (transcribed from the tachykinin, Tac1, gene) is deactivated by membrane metalloendopeptidase (MME) and stimulates biliary growth and liver fibrosis by interacting with NK-1R; and (ii) total knockdown of NK-1R decreases biliary hyperplasia in bile duct ligated mice. No information exists on the role of sensory innervation in PSC. We aim to determine the role of the SP/ NK-1R axis on biliary senescence and liver fibrosis in PSC. Methods: We performed studies in: (i) serum, total liver, liver sections and cholangiocytes and hepatic stellate cells (HSCs) isolated by laser capture microscopy (LCM) from normal WT mice and Mdr2-/- mice treated with saline or L-733,060 (NK-1R antagonist, 20 mg/kg BW by daily IP injections) for 2 wk; and (ii) serum and cDNA from early and late stage male PSC patients. We measured serum SP levels by ELISA and hepatic fibrosis and senescence by Sirius red and SA-β-gal staining, respectively, in liver sections. We evaluated by qPCR the mRNA expression of: (i) Tac1, MME and NK1R; (ii) fibronectin 1, alpha-SMA and collagen-1a (markers of fibrosis); and (iii) CCL2, p16, p21 and p18 (markers of senescence) in cholangiocytes and HSCs from mouse models and human PSC samples. In vitro, murine biliary and human hepatic stellate cell lines were treated with SP (25 μM) with/ without L-733,060 before measuring fibrosis and senescence gene expression. Results: There were increased serum SP levels and mRNA expression of Tac1 and NK-1R but reduced expression of MME in total liver from Mdr2-/- mice and PSC samples compared to controls. Overall, there was enhanced liver fibrosis and cellular senescence in PSC samples (by qPCR) as well as in Mdr2-/- mice (by Sirius red and SA-β-gal staining), which was reduced by L-733,060. Enhanced senescence (seen in Mdr2-/- mice) was limited to LCM-isolated cholangiocytes as there was reduced senescence in LCM-isolated stellate cells. In vitro, SP: (i) increased the expression of fibrosis and senescent genes in cholangiocytes; and (ii) increased fibrosis but reduced senescent gene expression in HSCs (expressing NK-1R); all effects were reversed by L-733,060. Conclusion: The increase in liver fibrosis observed in PSC is likely due to increase biliary senescence but reduced senescence of stellate cells. Modulation of senescence by the SP/ NK-1R axis may be a key approach for modulating liver fibrosis in PSC