Induction Of Hepatic Fibrosis Research Articles

Induction of hepatic fibrosis in mice with schistosomiasis by extracellular microRNA-30 derived from Schistosoma japonicum eggs.

Schistosomiasis is a zoonotic parasitic disorder induced by the infestation of schistosomes, a genus of trematodes. MicroRNAs (miRNAs) in egg-derived exosomes are crucial for modulating the host's immune responses and orchestrating the pathophysiological mechanisms. Although the exosomes secreted by S. japonicum contain abundant miRNAs, the specific roles of these miRNAs in the pathogenesis of schistosomiasis-induced hepatic fibrosis are yet to be comprehensively elucidated. The egg exosomes of S. japonicum secrete miRNA-30, a novel miRNA. In vitro, the effect of miRNA-30 was evaluated by transfecting HSCs with miRNA mimics. The target gene biosignature for miRNA-30 was predicted using the miRDB software. The effect of miRNA-30 in hepatic fibrosis was evaluated by either elevating its expression in healthy mice or by inhibiting its activity in infected mice by administration of recombinant adeno-associated virus serotype eight vectors expressing miRNA-30 or miRNA sponges. This novel miRNA can activate hepatic stellate cells (HSCs), the prinary effector cells of hepatic fibrosis, in vitro, i.e., it significantly increases the fibrogenic factors Col1(α1), Col3(α1), and α-SMA at both mRNA and protein levels. In addition, miRNA-30 may activate HSCs by targeting the host RORA gene. In addition, in vivo experiments were conducted by administering a recombinant adeno-associated viral vector to modulate the expression levels of miRNA-30. The overexpression of miRNA-30 in healthy mice significantly elevated the expression of Col1(α1), Col3(α1), and α-SMA at both the transcriptomic and proteomic scales. This overexpression was coupled with a pronounced augmentation in the hepatic hydroxyproline content. Conversely, the in vivo silencing of miRNA-30 in infected mice induced a considerable reduction in the size of hepatic granulomas and areas of collagen deposition. Hence, in vivo, modulation of miRNA-30 expression may play a pivotal role in ameliorating the severity of hepatic fibrosis in mice afflicted with S. japonica. The study results suggest that miRNA-30 may augment schistosomiasis-induced hepatic fibrosis through a probable interaction with the host RORA. Our study may improve the current theoretical framework regarding cross-species regulation by miRNAs of hepatic fibrosis in schistosomiasis.

A first experience of transduction for differentiated HepaRG cells using lentiviral technology

Currently, there is a lack of systems for studying the role of hepatitis B viral proteins, such as HBeAg and HBcAg, on liver injury. It is necessary to develop an original tool in order to clarify the role of these viral proteins in hepatic stellate cell activation, and to understand the molecular mechanisms of liver injury. HepaRG are the most reliable hepatocyte-like cells for studying liver functions or disorders. In this paper, we demonstrate that the transduction of differentiated HepaRG (dHepaRG) cells can be performed successfully using lentiviral particles. The production of a functional Green Fluorescent Protein (GFP) assessed by Fluorescence Activated Cell Sorting and fluorescence microscopy is up to 16% of GFP positive cells using a multiplicity of infection (MOI) of 2.4. We demonstrate that this technology can allow the stable expression of GFP during the long lifecycle of the cell (up to four weeks after the cell’s passage). With this innovative tool, we aim to express viral proteins such as HBeAg or HBcAg in dHepaRG cells. The preliminary results of this work shows that HBeAg can be efficiently produced in dHepaRG cells and that increased MOI allows a better production of this protein. Our future objective will be to study the role of HBc and HBe proteins on the induction of hepatic fibrosis.

Abstract 5248: Pioglitazone prevents hepatocellular carcinoma development in a rat model of cirrhosis

Abstract Introduction: Advanced hepatocellular carcinoma (HCC) is a leading cause of mortality worldwide with limited treatment options. There is a readily identifiable cohort of cirrhosis patients at risk and they are ideal candidates for chemoprevention. Anti-hyperglycemic agents have garnered interest for their potential anti-fibrotic as well as chemo-preventive effects. Pioglitazone, a selective PPAR-γ agonist, has been shown to reduce non-alcoholic steatohepatitis (NASH), but its role as an anti-fibrotic and chemopreventive agent has yet to be elucidated. The hypothesis of this study is that Pioglitazone reduces cirrhosis and subsequent HCC development in rats with diethylnitrosamine (DEN)-induced cirrhosis. Methods: Male Wistar received DEN 50mg/kg by intraperitoneal injection. DEN injury reliably recapitulates histological and molecular features of human HCC development with induction of hepatic fibrosis at 8 weeks, cirrhosis at 12 weeks, and HCC by 18 weeks. DEN-injured rats were randomized to receive oral gavage of pioglitazone at 3mg/kg/day (n=9) or vehicle control (n=9). Initiation of pioglitazone coincided with the development of liver fibrosis at 8 weeks. All animals were sacrificed at 18 weeks. Results: As expected, repeated injections of DEN in rats resulted in progressive fibrosis, cirrhosis, followed by HCC formation. Treatment with pioglitazone resulted in a 56% reduction of surface nodules relative to treatment with vehicle (7.4±4.9 vs. 17±7; p<0.005). Liver sections were stained by picrosirius red to assess fibrosis and pioglitazone significantly reduced collagen deposition in DEN-injured rats (collagen proportional area = 3.2±1.8% vs. 9.2±2%; p<0.035). This histologic observation was further confirmed by gene expression analysis with reductions in collagen-I, α-smooth muscle actin, and transforming growth factor beta in rats treated with pioglitazone. Finally, weekly injection of DEN also caused a significant decrease in overall body weight in comparison to untreated rats (398.1±60 vs. 598±46 grams; p<0.015), and pioglitazone treatment resulted in a trend for better protection of body weight relative to vehicle (398.1±60 vs. 427.5±56.3 grams). Conclusion: Overall our data supports the hypothesis that the anti-diabetic agent pioglitazone may be repurposed as a drug to reduce fibrosis and prevent HCC. This could be beneficiary in patient management given the low cost as well as minimal side effects. Citation Format: Shen Li, Sarani Ghoshal, Gunisha Arora, Derek J. Erstad, Michael Lanuti, Kenneth K. Tanabe, Bryan C. Fuchs. Pioglitazone prevents hepatocellular carcinoma development in a rat model of cirrhosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5248. doi:10.1158/1538-7445.AM2017-5248

Vascular Endothelial Growth Factor Expression in Hepatitis C Virus-Induced Liver Fibrosis: A Potential Biomarker.

The major complication of hepatitis C virus (HCV) infection is the induction of hepatic fibrosis. In this study, we investigated the correlation between the expression level of vascular endothelial growth factor (VEGFA) at mRNA and protein levels and the progression of HCV-related liver fibrosis. One hundred twenty subjects were selected for this study: 15 controls and 105 chronic HCV patients with different fibrosis grades (44 F0-F1 and 61 F2-F4). Quantitative real-time polymerase chain reaction (qRT-PCR) was used to measure VEGFA mRNA in peripheral blood mononuclear cells, while enzyme-linked immunosorbent assay (ELISA) was used to measure the secreted VEGFA protein in serum. Both qRT-PCR and ELISA results showed that HCV patients have significantly higher VEGFA expression than that of controls (P = 0.036 and 0.043, respectively). Moreover, patients with late fibrotic stages (F2-F4) exhibited the highest levels of VEGFA mRNA and protein (P = 0.008 and 0.041, respectively) when compared with controls. An area under the receiver operating characteristic curve (AUC of the ROC) for the circulatory VEGFA protein between HCV patients with fibrosis and healthy controls was 0.92 (P = 0.043). Our data suggest that VEGFA protein is a promising noninvasively diagnostic biomarker for HCV-induced liver fibrosis.

Attenuation of liver fibrosis by herbal compound 861 via upregulation of BMP-7/Smad signaling in the bile duct ligation model rat

Herbal compound861 (Cpd861) exerts an anti-fibrotic effect in patients with hepatic fibrosis; however, the anti-fibrotic mechanism has yet to be fully elucidated. The present study aimed to explore the mechanistic basis for the anti-fibrotic effect, with a focus on bone morphogenetic protein7 (BMP-7)/Smad signaling in a bile duct ligation (BDL)-induced liver fibrosis rat model. Following the induction of hepatic fibrosis, rats induced by BDL were treated with9g/kgCpd861 daily or an equal volume of saline for28days. Serum samples were prepared for monitoring the levels of alanine transaminase, aspartate transaminase and total bilirubin, and direct bilirubin analyses and liver samples were used to investigate gene expression, protein localization and protein expression analysis using real‑time quantitative polymerase chain reaction, immunohistochemistry and western blotting. The results revealed the attenuation of liver fibrosis by Cpd861 in the histological and biochemical experiments. BMP‑7 and phospho (p)‑Smad1/5/8 were localized predominantly in the cytoplasm of hepatocytes. In comparison with the saline‑treated BDL rats, Cpd861 markedly upregulated the gene expression of BMP‑7 and Smad5, as well as the protein expression of BMP‑7 and Smad1/5. In addition, p-Smad1/5/8 protein expression was markedly increased by Cpd861 in the BDL model. These results indicated that Cpd861 alleviates hepatic fibrosis possibly via the upregulation and activation of BMP-7/Smad signaling in hepatic fibrotic rats.

Identification of coinciding quantitative trait loci for the hepatic expression of SOCS3 and XBP-1 s during toxic and non-alcoholic fatty liver disease

Toxic and non-alcoholic fatty liver diseases are multifactorial (polygenic) disorders. A valuable tool to identify disease associated genetic loci is Quantitative Trait Locus (QTL) mapping, i.e. genome-wide linkage analysis of genetic and phenotypic variation in experimental crosses. Here we employ QTL mapping to analyze the regulation of two previously identified mediators of non-alcoholic fatty liver disease (NAFLD), the inflammatory mediator SOCS3 and the unfolded protein response regulator XBP-1 s. In the first study, F2 intercross progeny of the inbred mouse lines A/J and C57BL/6J were fed a high-fat/high caloric (HFHC) diet for eight weeks and then used to identify QTLs determining the hepatic expression of Socs3 and Xbp1 s (eQTLs). Overlapping eQTLs for Xbp1 s and Socs3 were identified on chromosomes (chr) 1 at (181.5 Mb) and 11 (56.2 and 58.4 MB), and a Socs3 QTL was localized on chr 12 (113.4 Mb). Microarray analysis identified differentially-expressed candidate genes within these QTLs. We then phenotyped 33 BXD recombinant inbred lines (Andreux et al. Cell 2012) after induction of hepatic fibrosis with the hepatotoxin CCl4 (1.4 mg/kg/week for 6 weeks). The transcriptome of each BXD line after CCl4 challenge was determined with Affymetrix Mouse Gene 1.0 ST microarrays. Using gene expression values as quantitative traits, we searched for coinciding regulatory loci within the Socs3 and Xbps1 QTLs on chr 1, 11, and 12. After CCl4 challenge, we mapped four eQTLs on chr 1 at 193.7 Mb, chr 4 at 63.4 Mb, chr 11 at 47.9 Mb, and chr 15 at 103 Mb. Among these, we found loci overlapping with the eQTLs that determine Socs3 and Xbps1 expression on the HFHC diet. Furthermore, we identified several cis-regulated genes from within these QTLs that were also identified to be differentially regulated after the HFHC challenge. In summary, the complementary genetic studies identify co-localizing regulatory loci for Socs3 and Xbps1 expression after liver injury induced by either dietary (high-fat) or toxic (CCl4) challenge. These results propose further investigation of the Socs3 and Xbps1 QTLs and their roles during chronic liver injury, as they might represent critical mediators in the pathobiology of steatohepatitis.

Observation of Esterase-Like-Albumin Activity during <i>N</i>'-Nitrosodimethyl amine Induced Hepatic Fibrosis in a Mammalian Model

Aim: Hepatic fibrosis (HF) is characterized by irregular growth and amassing of fibrous scar tissues in the liver causing weakened hepatocytes metabolism and protein level alterations, including albumin. Albumin with Mr ~68-70 kDa is unglycosylated soluble plasma protein with various biological roles. In this study, we demonstrate ‘esterase-like activity’ of albumin during NDMA-induced HF in rats. Material and Methods: In rats, HF was induced by weekly i.p. injections of NDMA in doses of 10 mg/kg b.wt. Sera of controls (untreated) and treated rats were processed for biochemical tests, electrophoretic profiling and in-gel esterase activity localization using a, b-naphthyl acetates. H&E staining of liver sections (~ 5 m m) was done to confirm induction of HF. Results: NDMA satisfactorily induces hepatic fibrosis within 21 days which is also evident by significant increase in SALP, SGOT, SGPT and bilirubin levels in rats. ‘Esterase-like activity’ of albumin detected in animal sera remains stable throughout the course of treatment irrespective of other biochemical changes. Conclusion: During pathogenesis of HF, formation of stable esterase-albumin complex may have some important role and hence, prior recommending the use of albumin as diagnostic marker we propose further investigations to elucidate the mechanism of its formation.

Arterialisation of the portal vein as a model for the induction of hepatic fibrosis: description of microsurgical models in the rat

Within the framework of liver transplantation, arterialisation of the portal vein in the case of non-recanalisable thrombosis has been reactivated. However, one of the consequences of this vascular reconstruction is the development of hepatic fibrosis. Clinical experience has shown that the development of fibrosis can be avoided by reducing portal inflow. We present, as a model for the induction of hepatic fibrosis, techniques of PVA, including transplantation. For PVA, several different techniques were used: the first with reduction of the portal inflow over a stent inserted in the right renal artery (PVA-B), the second with unrestricted flow using an aortic-portal segment (PVA-APS). The third technique was orthotopic liver transplantation with unrestricted portal arterialisation (OLTx-APS). Portal blood flow was measured with an ultrasonic flow probe. To determine the degree of hepatic fibrosis the amount of hydroxyproline was measured. Quantification of relative transcript levels of procollagen I was effected with real-time PCR using the TaqMan technology on a lightcycler instrument. The extracellular matrix was visualised with picro-sirius staining. Measurements with the ultrasonic probe showed a significant increase in flow rates, both with reduced (PVA-B) and unrestricted inflow (PVA-APS; OLTx-APS). The lowest survival rate (58%) was found in the group with unrestricted portal inflow. The reason for this was a high rate of thrombosis in the in the portal vascular tree (4 out of 12). In the OLTx-APS group four animals died within the first 3 postoperative days (69%), as a result of protracted postoperative shock. The overall survival rate was the highest (85%) in the group undergoing PVA with reduction of the portal inflow. PVA with unrestricted inflow was followed by a significant increase in extracellular collagen, which showed a clear correlation with the increase in the amount of hydroxyproline, the level of the mRNA for procollagen I and picro-sirius staining. With the operative PVA techniques presented herein, different arterial flow rates in the portal vein can be investigated. In our opinion these techniques represent an excellent animal model for studying the genesis of fibrosis and antifibrotic substances. By regulating the blood flow in the arterialised portal vein hepatic fibrosis can be reduced or even avoided. After a brief period of learning the microsurgical techniques, the surgeon can limit clamping times and achieve good results with these techniques.

Activation of Stellate Cells Before Induction of Hepatic Fibrosis – Precise Timing in Choline-deficient Diet-fed Rat Model

Introduction Quiescent hepatic stellate cells (HSCs) store vitamin A as lipid droplets in the cytoplasm. The activated HSCs by several stimuli have functions similar to that of myofibroblasts and play key roles in hepatic fibrosis [1-3]. However, precise timing between activation of the HSC and induction of hepatic fibrosis is still unknown. Cholinedeficient (CD) diet induces fatty liver and subsequently hepatic fibrosis in rats. We investigated the changes of HSCs in the progress of hepatic fibrosis induced by CD diet in rats, and analyzed the time course from the activation of the HSCs to the induction of hepatic fibrosis.

S-adenosylmethionine treatment prevents carbon tetrachloride-induced S-adenosylmethionine synthetase inactivation and attenuates liver injury.

Administration of carbon tetrachloride to rats resulted in induction of hepatic fibrosis and a 60% reduction of hepatic S-adenosylmethionine synthetase activity without producing any significant modification of hepatic levels of S-adenosylmethionine synthetase messenger RNA. The reduction of S-adenosylmethionine synthetase activity was corrected by treatment with S-adenosylmethionine (3 mg/kg/day, intramuscularly). Administration of carbon tetrachloride also produced a 45% depletion of liver glutathione (reduced form) that was corrected by S-adenosylmethionine treatment. After the rats received carbon tetrachloride, a 2.3-fold increase in liver collagen was observed; prolyl hydroxylase activity was 2.5 times greater than that seen in controls. These increases were attenuated in animals treated with carbon tetrachloride and S-adenosylmethionine. The attenuation by S-adenosylmethionine treatment of the fibrogenic effect of carbon tetrachloride was associated with a decrease in the number of rats in which cirrhosis developed.

Rapid induction of hepatic fibrosis in the gerbil after the parenteral administration of iron-dextran complex

Rapid induction of hepatic fibrosis in the gerbil after the parenteral administration of iron-dextran complex