Design of a highly potent GLP-1R and GCGR dual-agonist for recovering hepatic fibrosis

Abstract

Currently, there is still no effective curative treatment for the development of late-stage liver fibrosis. Here, we have illustrated that TB001, a dual glucagon-like peptide-1 receptor/glucagon receptor (GLP-1R/GCGR) agonist with higher affinity towards GCGR, could retard the progression of liver fibrosis in various rodent models, with remarkable potency, selectivity, extended half-life and low toxicity. Four types of liver fibrosis animal models which were induced by CCl₄, α-naphthyl-isothiocyanate (ANIT), bile duct ligation (BDL) and Schistosoma japonicum were used in our study. We found that TB001 treatment dose-dependently significantly attenuated liver injury and collagen accumulation in these animal models. In addition to decreased levels of extracellular matrix (ECM) accumulation during hepatic injury, activation of hepatic stellate cells was also inhibited via suppression of TGF-β expression as well as downstream Smad signaling pathways particularly in CCl₄-and S. japonicum-induced liver fibrosis. Moreover, TB001 attenuated liver fibrosis through blocking downstream activation of pro-inflammatory nuclear factor kappa B/NF-kappa-B inhibitor alpha (NFκB/IKBα) pathways as well as c-Jun N-terminal kinase (JNK)-dependent induction of hepatocyte apoptosis. Furthermore, GLP-1R and/or GCGR knock-down results represented GCGR played an important role in ameliorating CCl₄-induced hepatic fibrosis. Therefore, TB001 can be used as a promising therapeutic candidate for the treatment of multiple causes of hepatic fibrosis demonstrated by our extensive pre-clinical evaluation of TB001.

Keywords: Apoptosis; Candidate peptides; GCGR; GLP-1R; Inflammation; Liver fibrosis.

Graphical abstract

Figure 1

Peptide design. (A) GLP-1 and GCG chimerae amino acid sequence and structure. ∗Represents the side chain modification position. (B) Ex-4 crystal structure (9–39) (PDB code 3C5T). and three-dimensional model of OXM. (C) The sequence information of TB001. (D) Helical wheel representation of TB001 showing residues 12–29. (E) Three-dimensional model of TB001/GLP-1R. (F) In vitro activity of TB001 in the GLP-1/GCG receptor-mediated CRE-Luciferase reporter assay. (G) The mean concentration vs. time profiles of TB001 following a single and multiple s.c. administration (n = 6).

Figure 2

TB001 inhibits the progress of CCl₄-induced rodent liver fibrosis. HE staining (A), Sirius Red staining, and IHC for α-SMA, and Col1α1 (B) of liver sections representative images of mice treated with CCl₄ or Corn Oil and liraglutide or TB001 therapy. (C) ALT and AST level from mice treated with CCl₄ or Corn Oil and liraglutide or TB001 therapy (n = 6). (D) H&E staining, Sirius Red staining and IHC for α-SMA of liver sections representative images of rat at a magnification of 4× or 20× and analysis of the positive area (n = 10). (E) Serum ALT and AST level from rat treated with CCl₄ or Corn Oil and liraglutide or TB001 therapy. (F) qPCR of genes related to ECM metabolism (Pdgf, and Timp1) in CCl₄-induced rat. Here and later, if not mentioned specifically, data were shown as the mean ± SEM, statistical significance of the differences between each group was determined by Student's two-tailed t-test. Triplicates were performed in each experiment.

Figure 3

TB001 inhibits the progress of ANIT–induced rat liver fibrosis. (A) HE staining, Sirius Red staining and IHC for α-SMA and CD68 of liver sections representative images at a magnification of 20× and analysis of the positive area (n = 10). (B) Serum ALT, AST, D-BIL and I-BIL level from rat in the indicated groups (n = 10). (C–D) qPCR of genes which were related to ECM metabolism (Fn, Pdgf, and Timp1) and inflammation (Tnf-α and F4/80) in ANIT-induced rat liver fibrosis (n = 5).

Figure 4

TB001 inhibits the progress of BDL-induced rat liver fibrosis. (A) HE staining, Sirius Red staining, and IHC for α-SMA and CD68 of liver sections representative images at a magnification of 20× and analysis of the positive area (n = 5). (B) Serum ALT, AST and ALP level from rat in the indicated groups were measured (n = 10).

Figure 5

TB001 inhibits the progress of liver fibrosis and restores the immune microenvironment homeostasis in schistosomiasis induced mice liver fibrosis. (A) H&E staining, Masson staining, and IHC for α-SMA of liver sections representative images at a magnification of 20× and analysis of the positive area (n = 6). (B) Serum ALT and AST level from mice in indicated group (n = 6). (C) qPCR analysis of genes related to Th1 cytokines (Il-1β, Il-6, Tnf-α) and Th2 cytokines (Il-4, Il-5, Il-13) (n = 6). (D) The number of F4/80+CD11b+ macrophage (n = 6) and CD25 + Foxp3 + Treg cells (n = 5) in each of mice liver were analyzed by flow cytometry. One-way ANOVA followed by Dunnett's multiple comparison test was used here for determining statistical significance.

Figure 6

TB001 ameliorates hepatic fibrosis via improving ECM metabolism, inflammation, and apoptosis. (A) Bulk mRNA sequencing analysis of fibrosis-, inflammation-, apoptosis- and oxidative stress-related genes (P < 0.05, fold change (FC) log₂ > 1). (B) Western blotting assay of α-SMA, Smad2/3, phosphorylated Smad2/3 and TGF-β measured in liver samples of mice in each group (n = 3). Here and later, unless specified, protein expression was normalized to GAPDH. (C) qPCR of genes related to fibrosis (α-Sma, Tgf-β, Mmp2, Mmp9, and Timp1) (n = 5). (D) IHC for CD68 of liver sections representative images from mice treated with CCl₄ or Corn Oil and TB001 therapy (n = 5). (E) The expression level of pIKBα/IKBα and pNFκB/NFκB were measured in liver samples from each group (n = 3). (F) qPCR analysis of inflammation related genes (n = 5). (G) TUNEL assay of liver sections representative images from mice treated with CCl₄ or Corn Oil and TB001 therapy (n = 5). (H) The expression levels of pJNK/JNK and Cle-caspase-3/caspase-3 were measured by Western blot (n = 3). (I) qPCR of genes related to apoptosis (Fas, Fasl, Bid, Bim, and Bcl2) (n = 5). (J) Oxygen consumption rates (OCR) of primary hepatocytes from each treated group mice. (K) The expression level of Col1a and NOX4 were measured by Western blot in primary hepatocytes from each treated group mice (n = 3).

Figure 7

TB001 ameliorates hepatic fibrosis induced by schistosomiasis via improving ECM metabolism, apoptosis, and ER stress. (A) Western blotting assay of Col1α, α-SMA, Smad2/3, phosphorylated Smad2/3 and TGF-β from indicated treatment group. (n = 4). (B) qPCR of genes related to fibrosis (Col1α, α-Sma and Tgf-β) from indicated treatment group mice liver (n = 5). (C) TUNEL assay of liver sections representative images from different treated mice (n = 5). (D, E) qPCR of genes involved in apoptosis (Caspase-1, Caspase-3, Capase-4, Bax, Bcl2) and ER stress (Grp78, Xbp-1, Perk) in liver samples from different treated mice (n = 5). (F) ROS assay of primary hepatocyte representative images from each group (n = 3). (G) MDA concentration in primary hepatocytes from different groups. (H and I) Inflammation- (Tnf-α, Il-33, Il-1β, Il-6) and apoptosis- (Caspase-1, Caspas-3, Caspase-4) related genes mRNA expression in primary hepatocytes from each group (n = 3). (J) The pIRE1α/IRE1α and GRP78 and XBP-1 expression level were measured by Western blot in primary hepatocytes stimulated with PBS, SEA or SEA plus TB001 therapy (n = 3).

Figure 8

TB001 ameliorate the progress of CCl₄-induced mouse liver fibrosis by targeting GLP-1R/GCGR. H&E staining (A) and Sirius Red staining (B) of mice liver sections representative images from each group. (C) Quantifications of each staining. (D) Col1α mRNA expression level in the liver of mice from different group (n = 3–6). (E) ALT, T-BIL and D-BIL activity (n = 6).

Figure 9

Scheme of GLP-1R/GCGR dual agonist TB001 in recovering liver fibrosis.