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AIM:To investigate the role and mechanism of insulinlike 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 mea-sured 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,andα-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 ofα-SMA and p-Smad2/3,and AdshSmad3 inhibited IGFBPrP1-stimulated p-Smad2/3 activation and the expression ofα-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%vs10.24%±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 attenuatedα-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.
AIM: To investigate the role and mechanism of insulinlike 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 mea-sured 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. Shshmad3-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? -smooth muscle actin (a-SMA) was determined by Western blot analysis and immunohistochemistry. Hepatatocyte apoptosis was assessed us ing TUNEL assay.RESULTS: IGFBPrP1 overexpression induced collagen deposition and up-regulated the expression ofα-SMA and p-Smad2 / 3, and AdshSmad3 inhibited IGFBPrP1-stimulated p-Smad2 / 3 activation and the expression ofα-SMA, collagen? SMA positive stained cells (29.84% ± 1.36% vs 5.83% ± 1.47%, P <0.05) and fibronectin in HSC-T6cells.Similarly, increased hepatocyte apoptosis (38.56% ± 3.42% vs0.24% ± 0.03%, P < (28.87% ± 2.73% vs 8.23% ± 0.98%, P <0.05), and increased numbers of Smad3 (35.88% ± 2.15% vs10.24% ± 1.31%, 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 < , 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.