Screening And Validation Of Hepatic Fibrosis-related Signaling Pathway Induced By IGFBPrP1

Insulin-like growth factor binding protein related protein1(IGFBPrP1), alsoknown as IGFBP7, is a secreted protein belonging to the insulin-like growth factorbinding proteins (IGFBPs) family. IGFBPrP1exhibits a low affinity for insulin-like growth factor (IGF), however, its binding affinity for insulin is considerably higher. Itserves to regulate cell proliferation, differentiation, adhesion, senescence, apoptosisand angiogenesis.Our group have previously reported that IGFBPrP1is a novel molecule ofhepatic fibrogenesis. However, the exact mechanism by which IGFBPrP1exerts thenovel fibrotic effects has not yet been identified.Hepatic fibrosis-related signaling pathways firmly established include TGFβpathway, Jak-Stat pathway, Rho-ROCK pathway, NF-κB pathway, Wnt pathway,Hedgehog (Hh) pathway, leptin pathway, PPAR-mediated pathway and AngⅡreceptor-mediated pathway. There are Smad-dependent and Smad-independentsignaling pathways in TGFβ pathway. TGFβ/Smad pathway is classical signalingpathway responsible for hepatic fibrosis. MAPK and PI3K/AKT pathways areincluded in Smad-independent signaling pathways.Our group have previously explored the role of TGFβ pathway in hepaticfibrosis and have found that IGFBPrP1contributes to hepatic fibrosis via TGFβ/Smadpathway. Hepatic fibrosis induced by IGFBPrP1may be associated with ERK/MAPK.However, it has not yet been identified whether IGFBPrP1contributes to hepaticfibrosis via other signaling pathways. Signaling pathways play important role indifferent stages of hepatic fibrosis. TGFβ/Smad pathway provides quantitativeregulation of the signaling pathway, and serve as nodes for crosstalk with other majorsignaling pathways. Therefore, IGFBPrP1may contribute to hepatic fibrosis viaSmad-independent signaling pathway or other signaling pathways.PCR Array is the most reliable and accurate tool for analyzing the expression ofgenes concerned with research objects. It is comprised of a96-or384-well platecontaining qRT-PCR primer assays. The PCR Array offers a complete solution toexamine gene expression profiles for any pathway of interest and is a powerful new tool for research on signal transduction.In the present study, differential expression genes of hepatic fibrosis-relatedsignaling pathway induced by IGFBPrP1were screened using PCR Array and theexpression changes of some differential genes were validated in rat fibrotic livers.The mechanism regulating IGFBPrP1-induced hepatic fibrosis may in part bedelineated and new thinking may be provided for the treatment of hepatic fibrosis.There are six parts in the present study.Part1Adenovirus-mediated IGFBPrP1genetransfer to liver tissue in ratsObjective: To investigate whether IGFBPrP1expressed in rat liver tissue afterreceiving injections of an adenovirus carrying IGFBPrP1gene through tail veins.Methods: A total of98health SD rats weighing120-140g were randomly dividedinto Ad-IGFBPrP1group (n=43), Ad-EGFP group (n=43) and normal group (n=12).Rats were injected once with Ad-IGFBPrP1(4×109pfu per rat), Ad-EGFP (4×109pfu per rat), or were injected with the same doses of saline via the tail vein. Serumand liver tissue was obtained for analysis at week2/7,1,2,4,6and9. Three animalswere allocated to each group at week2/7. Eight animals were allocated to each groupat other time point. Enhanced green fluorescent protein (EGFP) in rat liver tissue wasobserved by fluorescence microscope. The percentage of EGFP positive cells wasdetected by flow cytometry (FCM). The expressions of IGFBPrP1mRNA and proteinwere detected by real-time quantitative PCR (qRT-PCR) and Western blot,respectively.Results:1. In the preliminary experiment, EGFP was detected in rat liver tissue afterinfection of4×109pfu or2×109pfu of Ad-EGFP via the tail vein and at week2,after a second dose of2×109pfu of Ad-EGFP was injected. A basal expression of EGFP was observed in this two ways. The magnitude of transfected cells was higherwith a single dose of4×109pfu Ad-EGFP in the liver than that with repetitiveinjection of2×109pfu Ad-EGFP (60.4%vs46.3%) detected by FCM. Therefore, ratswere transfected with4×109pfu Ad-EGFP in subsequent experiments.2. The bright green fluorescence of EGFP was observed by fluorescence microscopeand expression levels of EGFP in liver peaked at1week after adenoviral infection.The green fluorescence of EGFP gradually weakened at2weeks and dispeared by4weeks after the infection.3. The percentage of EGFP positive cells was the most (60.4%) and efficiency ofadenoviral-mediated gene expression peaked at1week after adenoviral infection.EGFP positive cells gradually decreased at2weeks after adenoviral infection.4. Adenoviral-mediated gene transfer of IGFBPrP1augmented IGFBPrP1mRNAexpression, which was much greater than in Ad-EGFP group and in normal group.IGFBPrP1mRNA expression in liver tissue peaked (5.57±1.52) at1week afteradenoviral infection.5. Western blot analysis showed that expression of IGFBPrP1protein peaked (1.07±0.11) at2weeks after injection and IGFBPrP1level later gradually downregulated.Conclusion: An adenovirus successfully deliver IGFBPrP1gene to the rat liverthrough tail veins.Part2Gene transfer of IGFBPrP1contributes tohepatic fibrosis in ratsObjective: To determine whether IGFBPrP1induced hepatic fibrosis.Methods: The experimental group was the same as part1. Liver sections from ratswere stained with Haematoxylin and eosin staining (H&E staining) and Sirius redstaining to determine histopathology changes and fibrillar collagen. Western blot wasused to detect the expressions of α-SMA. Collagen content of the liver was evaluated by determining the amount of hydroxyproline. The liver function was detected toobserve the liver tissue damage by automatic biochemical analyzer.Results:1. Light microscopy of HE and Sirius red staining showed that there washydropic degeneration, fatty degeneration, necrosis, infiltration of inflammatory cellsand bile duct proliferation after infection with Ad-IGFBPrP1. Rats infected4weeksafter injection of Ad-IGFBPrP1showed collagen fibrils in the portal and central veinarea. During the process of liver fibrogenesis, collagen deposition gradually expandedwith time extended. Rats administered with Ad-IGFBPrP1displayed a periportalfibrosis characterized by portal-portal and central-portal fibrous linkages (lesiondegree of fibrosis stage3).2. Western blot analysis showed that the protein level of α-SMA increased in livertissue with the increase of the degree of hepatic fibrosis. At9weeks afterAd-IGFBPrP1infection α-SMA expression peaked, rising by20-fold compared withthe Ad-EGFP group.3. Quantitative analysis indicated that the hydroxyproline content in Ad-IGFBPrP1group was gradually increased accompanied by the extent of fibrosis.4. Compared with the normal and Ad-EGFP group, serum ALT and TBIL levels weresignificantly increased in Ad-IGFBPrP1group (P＜0.05). Serum TP and ALB levelswere not significantly different between the Ad-IGFBPrP1group, the Ad-EGFP groupand normal group(P＞0.05).Conclusion: Gene transfer of IGFBPrP1induced hepatic fibrosis in rats.Part3Screening differential expression genes ofhepatic fibrosis-related signaling pathwayinduced by IGFBPrP1using PCR ArrayObjective: To Screen differential expression genes of hepatic fibrosis-related signaling pathway induced by IGFBPrP1using PCR Array.Methods: SD rats were randomly divided into Ad-IGFBPrP1group, Ad-EGFP groupand normal group. Three animals were allocated to each group. Liver tissue wasobtained at2weeks. Total RNA from liver tissue was extracted and cDNA wassynthesized. Differential expression genes of signal transduction-related werescreened by qRT-PCR.Results: Eight-four signal-related genes were analyzed by Signal TransductionPathwayFinder PCR Array. There were33differential expression genes (39.29%),17of them were high expressing and16were low express. Differential expression geneswere included in16signaling pathways, including: Egr1, Hhip of Hedgehog pathwayand PTEN of PI3K/AKT pathway.Conclusion: IGFBPrP1may contribute to hepatic fibrosis by means of thecooperative effects of different signaling pathways. Egr1, Hhip and PTEN may be theCandidate Genes of hepatic fibrosis-related signal transduction pathway induced byIGFBPrP1.Part4Differential expression genes of MAPK pathwayin fibrotic livers induced by IGFBPrP1Objective: To Screen differential expression genes of MAPK pathway in fibroticlivers induced by IGFBPrP1.Methods: The experimental group was the same as part3. Total RNA from livertissue was extracted and cDNA was synthesized. Differential expression genes ofMAPK pathway in fibrotic livers induced by IGFBPrP1were screened by qRT-PCR.Results: Twenty-four differential expression genes were screened out (28.57%), ofwhich19were up-regulated and5were down-regulated. These genes werecategorized according to their functions as transcription factors, Raf regulating proteins and cell cycle proteins regulated by the ERK1/2pathway. Map2k2(MEK2)and Mapk3(ERK1) high expressed in liver tissue.Conclusion: IGFBPrP1may induce hepatic fibrosis by activating different stages ofMAPK pathway. Map2k2(MEK2) and Mapk3(ERK1) may be the Candidate Genes ofhepatic fibrosis-related signal transduction pathway induced by IGFBPrP1.Part5The validation of some differential expression genesin rat fibrotic livers induced by IGFBPrP1Objective: To validate the expression changes of some differential genes in ratfibrotic livers induced by IGFBPrP1.Methods: The experimental group was the same as part1. The expression ofdifferential expression genes was measured by qRT-PCR and Western blot,respectively.Results:1. As compared with that in normal and Ad-EGFP group, Map2k2(MEK2)and Mapk3(ERK1) mRNA expression was up-regulated (P＜0.05). As comparedwith that in normal and Ad-EGFP group, Hhip and PTEN mRNA level wasdown-regulated (P＜0.05), while Egr1mRNA level was up-regulated (P＜0.05).2. Western blot analysis showed that the protein level of PTEN decreased in livertissue with the increase of the degree of hepatic fibrosis. The expression of PTENprotein at different time points was remarkably reduced compared with that in normaland Ad-EGFP group(P＜0.05). The expression of Egr1protein increased and peakedat2weeks after the infection(P＜0.05), then gradually deceased.Conclusion: IGFBPrP1may contribute to hepatic fibrosis by means of thecooperative effects of differential expression genes including Map2k2(MEK2),Mapk3(ERK1), Hhip, PTEN and Egr1. This may at least in part clarify thepathogenesis of hepatic fibrosis induced by IGFBPrP1. Part6Preliminary exploration of the relation betweenIGFBPrP1and TGFβ1in hepatic fibrosisObjective: To explore the relation between IGFBPrP1and TGFβ1in hepatic fibrosis.Methods: The experimental group was the same as part1. The protein expression ofIGFBPrP1, α-SMA and TGFβ1in rat fibrotic livers induced by IGFBPrP1wasdetected by Western blot. The change of protein expression was observed in theadvanced stage of hepatic fibrosis.Results: Western blot analysis showed that the expression of IGFBPrP1proteinincreased and peaked at2weeks after the infection, then gradually deceased. Theprotein level of α-SMA and TGFβ1increased with the increase of the degree ofhepatic fibrosis. At9weeks after the infection α-SMA and TGFβ1expression peaked.The hydroxyproline content was gradually increased accompanied by the extent offibrosis and peaked at9weeks after the infection.Conclusion: IGFBPrP1stimulated TGFβ1production in fibrotic livers, which maybe the main pathway of hepatic fibrosis induced by IGFBPrP1.