d-amino acid modification protects N-Acetyl-seryl-aspartyl-lysyl-proline from physiological hydroxylation and increases its antifibrotic effects on hepatic fibrosis

Abstract

N-Acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is a critical negative regulator of fibrosis development in the liver. However, its extremely short half-life in vivo greatly compromises its potential applications. Here, we report an Ac-SDKP analog peptide with d-amino acid replacement (Ac-SDDKDP). The stability of Ac-SDDKDP and its prevention of liver fibrosis were investigated in vitro and in vivo. The stabilities of Ac-SDKP and Ac-SDDKDP exposed to angiotensin-1-converting enzyme (ACE) and their half-lives in rats and human sera were determined by high-performance liquid chromatography. The inhibitory effects of Ac-SDKP and Ac-SDDKDP on the proliferation and activation of hepatic stellate cells (HSC-T6) were evaluated using the Cell Counting Kit-8, Western blotting, reverse transcription quantitative polymerase chain reaction, and immunofluorescence assays. Finally, the protective effects of Ac-SDKP and Ac-SDDKDP on carbon tetrachloride (CCl4)-induced liver fibrosis in rats were compared. d-Amino acid replacement significantly enhanced the stability of the peptide to ACE and prolonged the half-life of Ac-SDKP in rats and human sera. The Ac-SDKP-mediated inhibition of HSC-T6 cell proliferation was well preserved, and Ac-SDDKDP exerted inhibitory effects comparable to Ac-SDKP on α-smooth muscle actin (α-SMA), collagen I and III expression, and phosphorylated-Smad-2 expression. After intraperitoneal (i.p.) administration, Ac-SDDKDP exhibited significantly greater protection than Ac-SDKP against CCl4-induced liver fibrosis in rats. The serum alanine aminotransferase, aspartate aminotransferase, albumin, and total protein levels of the Ac-SDDKDP-treated rats were significantly lower than those of the Ac-SDKP-treated rats. α-SMA, CD45, and collagen I and III expression, as well as Smad-2 phosphorylation were significantly attenuated in the livers of the Ac-SDDKDP-treated rats compared to those of the Ac-SDKP-treated rats. Furthermore, we showed that the Ac-SDDKDP concentration in the rat liver increased to a physiological level of 60 min after i.p. administration, although i.p. administration of Ac-SDKP failed to enhance the peptide concentration in the rat liver. Our findings indicate that d-amino acid replacement is a simple and effective method to enhance the stability of Ac-SDKP. Ac-SDDKDP represents potential application of Ac-SDKP in fibrosis treatment and provides a new potential treatment strategy for liver fibrosis. © 2019 IUBMB Life, 71(9):1302–1312, 2019.