Dynamic Changes Of Both IGFBPrP1 And Transforming Growth Factorβ1/Smad On Liver Tissue With Fibrosis In Mice

Background:Liver fibrosis is the common pathological basis for diverse liver injuries. It is characterized by an excessive deposition of extra-cellular matrix (ECM). During liver injury, hepatic stellate cells (HSCs), are activated and transdifferentiated to become myofibroblasts (MFBs). MFBs multiply and manifest many fibrosis functions such as inducing ECM deposition,α-smooth muscle actin (α-SMA) expression and synthesis and secretion of type I collagen. Transforming growth factorβ1 (TGFβ1) is recognized as the strongest factor in activating and promoting this process. TGFβ1 classically transmits intracellular signaling via intracellular TGFβ/Smad signaling pathway, especially Smad3.Willem Boers [16] use the technique of serial analysis of gene expression (SAGE) to identify genes involved in the transformation of HSC into myofi-broblasts.And discovered that: IGFBPrP1 showed the highest expression in the midphase of the transdifferentiation process. LiXin Liu [17, 18, 19] discovered that: In liver tissue of patients with liver fibrosis and cirrhosis, the expression of IGFBPrP1 was high, which have positive correlation with high expression of TGFβ1. The expression of IGFBPrP1 induced by TGFβ1; Anti-IGFBPrP1 antibody can reverse over-production of collagen I induced by TGFβ1. HSC could be activated by IGFBPrP1 in vitro; The synthesis and secretion of both collagen I and FN was increasing by IGFBPrP1.Based on above review, we hypothesized that IGFBPrP1 was involved in the formation and development of hepatic fibrosis; moreover, it plays key roles in the emergence and development of liver fibrosis. Meanwhile, this function of IGFBPrP1 probably relates to promote activation of HSC ; promote the synthesis and secretion of both collagen I and FN which are the principal component of ECM; TGFβ1/Smad3 pathway.Objective:To investigate dynamic expressions of IGFBPrP1, TGFβ1 and Smad3 of mice with thioacetamide (TAA) - induced hepatic fibrosis.Methods:Liver fibrosis model of mice was made by intraperitoneal injecting with 5% TAA, 200mg/kg, three times per week, totally for 6 weeks. In the normally control group, mice were treated by saline with the same means. Animals were sacrificed 4, 5, 6 weeks after starting TAA injection, liver tissues were harvested. Liver pathological observation was detected by HE staining. Collagen accumulation in liver tissues was detected by Masson staining.Distribution and dynamic expression of IGFBPrP1,α-SMA, CollagenⅠ, FN, TGFβ1, Smad3 in the different groups were detected by immunohistochemistry staining. Meanwhile, the expression of IGFBPrP1,α-SMA, FN and Smad3 were examined by Western blot. The relationships of IGFBPrP1 andα-SMA, CollagenⅠ, FN, TGFβ1, Smad3 were analyzed.Results:1.Image analysis of immunohistochemical staining: during the progression of hepatic fibrosis, the expressions of Collagen, IGFBPrP1,α-SMA, CollagenⅠ, FN, TGFβ1, Smad3 were gradually increased. The intensity of expression in the model group was greater than those in the control group 1.06±0.05, 2.03±0.08, 5.03±0.09 vs 0.21±0.03, F=1783.141, P<0.05; 3.48±0.12, 7.93±0.15, 10.09±0.18 vs 0.11±0.04, F=998.200, P<0.05; 4.08±0.12, 7.99±0.15, 11.01±0.16 vs 0.22±0.01, F=886.715, P<0.05; 4.00±0.14, 10.18±0.15, 19.81±1.62 vs 0.31±0.09, F=935.242, P<0.05; 2.80±0.15, 3.81±0.17, 5.97±0.19 vs 0.49±0.02, F=931.241, P<0.05; 0.74±0.03, 1.24±0.05, 2.03±0.07 vs 0.22±0.03, F=697.118, P<0.05), and there was a significant difference among model group (P<0.05).2.Correlation analysis of immunohistochemical staining: during liver fibrosis developing phases, IGFBPrP1 was significantly positively correlated withα-SMA, CollagenⅠ, FN , TGFβ1, Smad3(r=0.906, 0.927, 0.988, 0.947, 0.977, P<0.01).3.The results of Western blot analysis: Molecular weight ofβ-actin, IGFBPrP1,α-SMA, FN, Smad3were 43, 31, 45, 220 and 58kD. The contents of IGFBPrP1,α-SMA, FN, Smad3 were significantly increased in model group compared with control group, which were gradually increased with process of fibrosis. After corrected byβ-actin showed that (0.41±0.05, 0.61±0.05, 0.92±0.07 vs 0.23±0.01, F=57.316, P<0.05; 0.64±0.03, 0.90±0.03, 1.39±0.03 vs 0.36±0.02, F=201.214, P<0.05; 0.06±0.02, 0.09±0.01, 0.12±0.02 vs 0.03±0.01, F=103.871, P<0.05; 0.20±0.02, 0.31±0.04, 0.56±0.04 vs0.09±0.01, F=72.966, P<0.05) and there was a significant difference among model group (P<0.05).4.Correlation analysis of Western blot analysis: There was a positive correlation between the expression of IGFBPrP1and the expression ofα-SMA, FN, Smad3 in hepatic tissue(r=0.982, 0.924, 0.965, P<0.001).Conclusion:1.The expression of IGFBPrP1 is positively correlated with that ofα-SMA, CollagenⅠ, FN, TGFβ1, Smad3, suggesting that IGFBPrP1 was involved in the formation and development of hepatic fibrosis ; moreover, it plays key roles in the emergence and development of liver fibrosis.2. This function of IGFBPrP1 probably relates to promote activation of HSC; promote the synthesis and secretion of both collagenI and FN which are the principal component of ECM.3. This function of IGFBPrP1 probably relates to affect TGFβ1/Smad3 pathway.