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Background: We previously showed that insulin-like growth factor binding protein-related protein 1 (IGFBPrP1) is a novel mediator in liver fibrosis. Transforming growth factor beta 1 (TGFβ1) is known as the strongest effector of liver fibrosis. Therefore, we aimed to investigate the detailed interaction between IGFBPrP1 and TGFβ1 in primary hepatic stellate cells (HSCs)., Methods: We overexpressed TGFβ1 or IGFBPrP1 and inhibited TGFβ1 expression in primary HSCs for 6, 12, 24, 48, 72, and 96 hours to investigate their interaction and observe the accompanying expressions of α-smooth muscle actin (α-SMA), collagen I, fibronectin, and phosphorylated-mothers against decapentaplegic homolog 2/3 (p-Smad2/3)., Results: We found that the adenovirus vector encoding the TGFβ1 gene (AdTGFβ1) induced IGFBPrP1 expression while that of α-SMA, collagen I, fibronectin, and TGFβ1 increased gradually. Concomitantly, AdIGFBPrP1 upregulated TGFβ1, α-SMA, collagen I, fibronectin, and p-Smad2/3 in a time-dependent manner while IGFBPrP1 expression was decreased at 96 hours. Inhibition of TGFβ1 expression reduced the IGFBPrP1-stimulated expression of α-SMA, collagen I, fibronectin, and p-Smad2/3., Conclusions: These findings for the first time suggest the existence of a possible mutually regulation between IGFBPrP1 and TGFβ1, which likely accelerates liver fibrosis progression. Furthermore, IGFBPrP1 likely participates in liver fibrosis in a TGFβ1-depedent manner, and may act as an upstream regulatory factor of TGFβ1 in the Smad pathway., (Copyright © 2017 The Editorial Board of Hepatobiliary & Pancreatic Diseases International. Published by Elsevier B.V. All rights reserved.)

Aim: To investigate the role and mechanism of insulin-like growth factor binding protein-related protein 1 (IGFBPrP1) in the development of liver fibrosis., Methods: An in vitro model using hepatic stellate cell (HSC)-T6 cells and an in vivo model of rat liver overexpressing IGFBPrP1 were established using an IGFBPrP1-expressing recombinant adenovirus. The expression of IGFBPrP1 was examined by immunofluorescence, and the expression of collagen I and fibronectin was measured by real-time reverse transcription-polymerase chain reaction and Western blot analysis. The expression of Smad2/3 and p-Smad2/3 was examined by Western blot and immunohistochemistry. A shSmad3-expressing recombinant adenovirus (AdshSmad3) was designed and used to knockdown the Smad3 gene in HSC-T6 cells and rat liver fibrosis transfected with IGFBPrP1. The expression of collagen I, fibronectin, and α-smooth muscle actin (α-SMA) was determined by Western blot analysis and immunohistochemistry. Hepatocyte apoptosis was assessed using TUNEL assay., Results: IGFBPrP1 overexpression induced collagen deposition and up-regulated the expression of α-SMA and p-Smad2/3, and AdshSmad3 inhibited IGFBPrP1-stimulated p-Smad2/3 activation and the expression of α-SMA, collagen I and fibronectin in HSC-T6 cells. Similarly, increased hepatocyte apoptosis (38.56% ± 3.42% vs 0.24% ± 0.03%, P < 0.05), α-SMA positive stained cells (29.84% ± 1.36% vs 5.83% ± 1.47%, P < 0.05), and increased numbers of Smad3 (35.88% ± 2.15% vs 10.24% ± 1.31%, P < 0.05) and p-Smad2/3 positive cells (28.87% ± 2.73% vs 8.23% ± 0.98%, P < 0.05) were detected in the livers of IGFBPrP1-overexpressing rats compared with the control group. Moreover, AdshSmad3 reduced IGFBPrP1-stimulated Smad3 expression and attenuated α-SMA expression (29.84% ± 1.36% vs 8.23% ± 1.28%, P < 0.05), hepatocyte apoptosis (38.56% ± 3.42% vs 6.75% ± 0.52%, P < 0.05), and both collagen I and fibronectin deposition in the livers of AdIGFBPrP1-treated rats., Conclusion: IGFBPrP1 induces liver fibrosis by mediating the activation of hepatic stellate cells and hepatocyte apoptosis in a Smad3-dependent mechanism.

Objective: The aim of this study was to investigate the role of insulin-like growth factor binding protein-related protein 1 (IGFBP-rP1) in the development of hepatic fibrogenesis in experimental disease models and human liver samples., Methods: Cellular distribution patterns of IGFBP-rP1 were assessed by immunohistochemistry in fibrotic and cirrhotic human liver specimens. Gene silencing of IGFBP-rP1 was performed on cultured hepatic stellate cells (HSCs) by small interfering RNA (siRNA), and the silencing effect was determined by quantitative real-time polymerase chain reaction (PCR) and Western blot. We also determined the effects of siRNA-mediated gene silencing of IGFBP-rP1 on the production of extracellular matrix (ECM) components by Western blot. The expression of ECM components and transforming growth factor (TGF)-β1 was studied by immunohistochemistry and Western blot in C57BL/6 wild-type mice treated with recombinant IGFBP-rP1 (rIGFBP-rP1)., Results: Expression of IGFBP-rP1 was significantly elevated in fibrotic and cirrhotic human liver specimens, and this increase was positively correlated with the number of collagen fibers observed. siRNA-mediated gene silencing of IGFBP-rP1 resulted in significantly decreased levels of collagen I and fibronectin in HSCs. Moreover, IGFBP-rP1 overexpression significantly increased the production of collagen, fibronectin and TGF-β1 in rIGFBP-rP1-treated mice., Conclusions: IGFBP-rP1 contributes to the development of liver fibrosis and may be a novel molecule involved in the progression of hepatic fibrogenesis., (© 2013 Chinese Medical Association Shanghai Branch, Chinese Society of Gastroenterology, Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd.)

Background: Insulin-like growth factor binding protein-related protein 1 (IGFBPrP1) can activate hepatic stellate cells and increase extracellular matrix (ECM) in vitro. However, the effects of IGFBPrP1 in mice with hepatic fibrosis, and the mechanisms of these effects, are currently unknown. We aim to address these issues in this study., Methods: Intraperitoneal injection of thioacetamide (TAA) is a classic method for establishing a mouse model of hepatic fibrosis. Using this model, we administered anti-IGFBPrP1 antibody, again via intraperitoneal injection. The morphological changes of liver fibrosis were observed with both HE and Masson stainning. The immunohistochemical assays and Western blotting were used to measure changes in IGFBPrP1, α-smooth muscle actin (α-SMA) and ECM in liver tissues, and the expression of transforming growth factor-β1 (TGF-β1) and Smad3. Data were statistically analyzed using one-way analysis of variance (ANOVA), the SNK-q test for inter-group differences., Results: The Masson staining analysis showed that compared with normal control group, content of collagen fiber in TAA5w group was significantly increased (P < 0.01), and it was significantly decreased in TAA5w/aIGFBPrP1 group compared with in TAA5w group (P < 0.01). The expression of hepatic IGFBPrP1, α-SMA, TGF-β1, Smad3, collagen I and fibronectin (FN) was significantly up-regulated in the TAA5w group (P < 0.01). Anti-IGFBPrP1 treatment reversed these changes (P < 0.01)., Conclusions: IGFBPrP1 plays an important role in the development of hepatic fibrosis. Anti-IGFBPrP1 prevents fibrosis in mice by suppressing the activation of hepatic stellate cells, inhibiting the synthesis of major components of the ECM (namely, collagen I and FN). The mechanism for this suppression of fibrosis is associated with the TGF-β1/Smad3 signaling pathways.