Substance P Increases Liver Fibrosis by Differential Changes in Senescence of Cholangiocytes and Hepatic Stellate Cells

Substance P (SP) is involved in the proliferation of cholangiocytes in bile duct-ligated (BDL) mice and human cholangiocarcinoma growth by interacting with the neurokinin-1 receptor (NK-1R). To identify whether SP regulates liver fibrosis during cholestasis, wild-type or NK-1R knockout (NK-1R(-/-)) mice that received BDL or sham surgery and multidrug resistance protein 2 knockout (Mdr2(-/-)) mice treated with either an NK-1R antagonist (L-733,060) or saline were used. Additionally, wild-type mice were treated with SP or saline intraperitoneally. In vivo, there was increased expression of tachykinin precursor 1 (coding SP) and NK-1R in both BDL and Mdr2(-/-) mice compared to wild-type mice. Expression of tachykinin precursor 1 and NK-1R was significantly higher in liver samples from primary sclerosing cholangitis patients compared to healthy controls. Knockout of NK-1R decreased BDL-induced liver fibrosis, and treatment with L-733,060 resulted in decreased liver fibrosis in Mdr2(-/-) mice, which was shown by decreased sirius red staining, fibrosis gene and protein expression, and reduced transforming growth factor-beta 1 levels in serum and cholangiocyte supernatants. Furthermore, we observed that reduced liver fibrosis in NK-1R(-/-) mice with BDL surgery or Mdr2(-/-) mice treated with L-733,060 was associated with enhanced cellular senescence of hepatic stellate cells and decreased senescence of cholangiocytes. In vitro, L-733,060 inhibited SP-induced expression of fibrotic genes in hepatic stellate cells and cholangiocytes; treatment with L-733,060 partially reversed the SP-induced decrease of senescence gene expression in cultured hepatic stellate cells and the SP-induced increase of senescencerelated gene expression in cultured cholangiocytes. Conclusion: Collectively, our results demonstrate the regulatory effects of the SP/NK-1R axis on liver fibrosis through changes in cellular senescence during cholestatic liver injury.