| Background:Hepatic fibrosis is the common result of liver injury from diverse origins. The sequence of response to injury is local inflammation followed by excessive deposition of extracellular matrix. Hepatic fibrosis is reversible and common pathological consequence of various chronic liver diseases which could eventually lead to hepatic cirrhosis. Up to date, hepatic stellate cells as the main matrix-producing cells and play a pivotal role in the process of liver fibrosis.Hepatic fibrosis is a complex pathological process in which participate a variety of cytokines and multiple signaling pathways. The main signal transduction pathway of hepatic fibrosis have: Smad, JAK/STAT, NF-κB and MAPK signal transduction pathway. The Smad signaling pathway is the relatively clear signal transduction pathway. It has showed that hepatic fibrosis is often accompanied by progressive increasing HSC and Smad3 mRNA expression, suggesting the Smad signaling pathway plays the key role in the development of hepatic fibrosis.RNAi is a widespread conservative defense response and an important post-transcriptional gene silencing approach. The double-stranded RNA induced the mRNA degradation by blocking specific gene expression to achieve "gene silencing" effectiveness.Insulin-like growth factor binding protein related protein 1 is a secretory protein with a molecular mass of approximately 31 kDa. Professor Lixin Liu discovered that IGFBPrPl is probably a new pathogenic factor in the formation of liver fibrosis. In human livers with fibrosis and cirrhosis, the expression of IGFBPrPl has positive correlation with TGFβ1 and Collagen I. Recombinant-IGFBPrPl can activate HSC in vitro and can increase the synthesis of extracelluar matrix. Anti-IGFBPrPl antibody can reduce the deposition of extracellular matrix in liver tissue of mice with liver fibrosis and the expression of extracelluar matrix has positive correlation with Smad3. In order to identify whether IGFBPrP1 effect on the secretion of extracellular matrix in hepatic stellate cells through Smad3 signaling pathway and to explore the effect of IGFBPrP1 on the expression of TGFβ1 in activated hepatic stellate cells, we designed this study.Part I The effect of siRNA Smad3 on Smad3 gene expression of hepatic stellate cellsObjective:To investigate the effect of small interfering RNA targeting Smad3 gene on the expression of Smad3 in rat hepatic stellate cells.Methods:1. Nonspecific siRNA with fluorescence were transfected into cultured HSC-T6 cells by the RNA interfering technology. We observed the transfection efficiency using fluorescence microscopy. 2. Screening the effective siRNA. Groups in experiment:negative contol group, two pairs of Smad3 siRNA groups and blank group. The inhibition rate of siRNA on Smad3 expression of HSC-T6 was determined by Real time PCR and Western blot.Results:1. Under the fluorescence microscope, the green fluorescence with the cells could be seen after transfection, suggesting the transfection was successful. The transfection efficiency was 70%.2. After 48 hours of transfecting siRNA 1 and siRNA2 into HSC-T6 cells, compared with negative control, the result of RT-PCR showed that the Smad3 mRNA inhibition rate was 32%and 41%, respectively. Western blot analysis revealed that the expression of Smad3 were down-regulated by 7.5%and 61.5%, respectively(P<0.01). siRNA2 inhibited Smad3 gene expression stronger than another siRNA.Conclusions: siRNA against Smad3 could effectively inhibit the expression of Smad3 and the protein level of Smad3 on HSC-T6 cell line.Part II The effect of IGFBPrPl with siRNA-mediated Smad3 silence on the secretion of extracellular matrix in activated hepatic stellate cellsObjective:To identify whether IGFBPrP1 effect on the secretion of extracellular matrix in hepatic stellate cells through Smad3 signaling pathway.Methods:1. HSC-T6 cells were cultured in vitro and established respectively the groups treated with IGFBPrP1 30μg/L and the blank control group (incubated with equal phosphate buffer saline(PBS)), the supernate was collected after 48 hours. The expression of Collagen I and FN in HSC was confirmed by Western blot.2. HSC-T6 cells were divided into four groups:Negative control group (transfection negative control siRNA), siRNA-Smad3 transfection group (transfection Smad3 siRNA2), siRNA-Smad3+IGFBPrP1 group (transfection Smad3 siRNA224 hours, adding IGFBPrP1 stimulating 48 hours), IGFBPrP1 group (adding IGFBPrP1 stimulating 48 hours). The expression of Smad3, Collagen I and FN were determined by Western blot.Results:1. The results of Western blot analysis:The expression of Collagen I and FN were significantly upregulated than that in the blank control group(0.74±0.09 vs 0.53±0.07; 0.63±0.07 vs 0.46±0.07) (P<0.01)2. After transfection of siRNA2 Smad3, the protein expression of Smad3 significantly decreased compared with the negative control group(0.14±0.02 vs 0.33±0.06) (P<0.01). The protein expression of fibronectin and Collagen I in IGFBPrP1 stimulating HSC treated with siRNA2 Smad3 were significantly decreased compared to that in IGFBPrP1 stimulating HSC without siRNA2 Smad3(0.42±0.05 vs 0.74±0.09; 0.34±0.06 vs 0.63±0.07) (P<0.01).Conclusions: The secretion of both Collagen I and FN which are the principal component of ECM wereincreasing by IGFBPrP1, the one of the mechanisms of which is through Smad3 signalingpathway.Part III The effect of IGFBPrPl on the expression of TGFβ1 in activated stellate cellsObjective:To observe the effect of IGFBPrP1 on the expression of TGFβ1 in hepatic stellate cells, and investigate whether IGFBPrP1 can be the one of the causes of TGFβ1.Methods:HSC-T6 was cultured in vitro and established respectively the groups treated with IGFBPrPl 10μg/L,20μg/L,30μg/L and the blank control group (incubated with equal phosphate buffer saline(PBS)), cell-coated dishes were attained after 24 hours, then the expression of TGFβ1 were detected by immunocytochemical staining and analyzed by Image-Pro image analysis system. Simultaneously, the supernatant were collected and the content of TGFp1 was measured by enzyme-linked immunosorbent assay (ELISA). The synthesis of TGFβ1 in HSC was confirmed by Western blot again.Results:The results of immunocyochemical staining:Each group had positive staining in cytoplasm showing some buffy or brown particles. The results of image analysis showed that the positive staining of TGFβ1 in IGFBPrPl treatment with 10μg/L group (10.90±0.35),20μg/L group(12.09±0.54),30μg/L group(11.89±0.32)were significantly higher than that in the blank control group (7.71±0.63) (P<0.05). The results of ELISA:The content of TGFβ1 in IGFBPrP1 treatment with 20μg/L group (169.59±24.87),30μg/L group (188.34±32.24) were significantly higher than that in the blank control group (136.16±19.52) (P<0.05). The results of Western blot analysis:The expression of TGFβ1 in IGFBPrPl group(0.46±0.05) were significantly upregulated than that in the blank control group(0.30±0.02) (P<0.05)Conclusions:The synthesis and secretion of TGFβ1 were increasing by IGFBPrP1 and the level of TGFβ1 gradually enhanced in a certain dose range of IGFBPrPl.
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