Rat Model Of Liver Fibrosis Research Articles

Role of phosphatase and tensin homolog deleted on chromosome ten in a rat model of carbon tetrachloride-induced liver fibrosis and the effect of qi-tonifying and blood-activating prescription

Role of phosphatase and tensin homolog deleted on chromosome ten in a rat model of carbon tetrachloride-induced liver fibrosis and the effect of qi-tonifying and blood-activating prescription

In vivo hepatic differentiation potential of human umbilical cord-derived mesenchymal stem cells: Therapeutic effect on liver fibrosis/cirrhosis.

AIMTo investigate the hepatic differentiation potential of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) and to evaluate their therapeutic effect on liver fibrosis/cirrhosis.METHODSA CCl4-induced liver fibrotic/cirrhotic rat model was used to assess the effect of hUC-MSCs. Histopathology was assessed by hematoxylin and eosin (H&E), Masson trichrome and Sirius red staining. The liver biochemical profile was measured using a Beckman Coulter analyzer. Expression analysis was performed using immunofluorescent staining, immunohistochemistry, Western blot, and real-time PCR.RESULTSWe demonstrated that the infused hUC-MSCs could differentiate into hepatocytes in vivo. Functionally, the transplantation of hUC-MSCs to CCl4-treated rats improved liver transaminases and synthetic function, reduced liver histopathology and reversed hepatobiliary fibrosis. The reversal of hepatobiliary fibrosis was likely due to the reduced activation state of hepatic stellate cells, decreased collagen deposition, and enhanced extracellular matrix remodeling via the up-regulation of MMP-13 and down-regulation of TIMP-1.CONCLUSIONTransplanted hUC-MSCs could differentiate into functional hepatocytes that improved both the biochemical and histopathologic changes in a CCl4-induced rat liver fibrosis model. hUC-MSCs may offer therapeutic opportunities for treating hepatobiliary diseases, including cirrhosis.

SENP1 attenuates the liver fibrosis through down-regulating the expression of SMAD2

To investigate whether SENP1 could play a regulating role in the liver fibrosis process, the Sprague-Dawley (SD) rats were used to establish the liver fibrosis rat models by intraperitoneally injecting with 1 ml/kg of 10% CCl4, while the control normal rats were injected with olive oil. Then confirmation experiments to verify the successful establishment of these models were conducted by detecting the cellular and lobular architecture, and liver function indexes using hematoxylin-eosin staining, Masson's trichrome staining and microplate method, respectively. In addition, the expression levels of fibrosis markers including collagen I, collagen III, α-SMA and TGF-β1 were inspected using quantitative real-time PCR (qRT-PCR), as well as SMAD2. Subsequently, the relative mRNA and protein level of SENP1 was also determined via qRT-PCR and western blot analysis. Next, the HSC-T6 cells of SENP1 knock-down were constructed and used to test the relative protein expression levels of α-SMA and SMAD2 in these cells. The results of hematoxylin-eosin staining, Masson's trichrome staining and microplate method turned out that the rat liver fibrosis models were constructed successfully, which was further confirmed by the increased expression of collagen I, collagen III, α-SMA and TGF-β1 in mRNA and protein level, as well as SMAD2. Then the expression of SENP1 was overexpressed in the rat liver fibrosis models induced by CCl4 and the TGF-β1 treatment could increase the protein expression level of collagen I, collagen III and α-SMA. Lastly, the SENP1 knockdown HSC-T6 cells were successfully constructed, while the silence of SENP1 down-regulated the protein expression of α-SMA and SMAD2. In conclusion, this study provided a new regulation mechanism about the liver fibrosis process.

Effect of human amniotic epithelial cells on pro-fibrogenic resident hepatic cells in a rat model of liver fibrosis.

Myofibroblasts are key fibrogenic cells responsible for excessive extracellular matrix synthesis characterizing the fibrotic lesion. In liver fibrosis, myofibroblasts derive either from activation of hepatic stellate cells (HSC) and portal fibroblasts (PF), or from the activation of fibroblasts that originate from ductular epithelial cells undergoing epithelial–mesenchymal transition. Ductular cells can also indirectly promote myofibroblast generation by activating TGF‐β, the main fibrogenic growth factor, through αvβ6 integrin. In addition, after liver injury, liver sinusoidal cells can lose their ability to maintain HSC quiescence, thus favouring HSC differentiation towards myofibroblasts. The amniotic membrane and epithelial cells (hAEC) derived thereof have been shown to decrease hepatic myofibroblast levels in rodents with liver fibrosis. In this study, in a rat model of liver fibrosis, we investigated the effects of hAEC on resident hepatic cells contributing to myofibroblast generation. Our data show that hAEC reduce myofibroblast numbers with a consequent reduction in fibronectin and collagen deposition. Interestingly, we show that hAEC strongly act on specific myofibroblast precursors. Specifically, hAEC reduce the activation of PF rather than HSC. In addition, hAEC target reactive ductular cells by inhibiting their proliferation and αvβ6 integrin expression, with a consequent decrease in TGF‐β activation. Moreover, hAEC counteract the transition of ductular cells towards fibroblasts, while it does not affect injury‐induced and fibrosis‐promoting sinusoidal alterations. In conclusion, among the emerging therapeutic applications of hAEC in liver diseases, their specific action on PF and ductular cells strongly suggests their application in liver injuries involving the expansion and activation of the portal compartment.

Hepatocyte-like Versus Mesenchymal Stem Cells in CCl4-induced Liver Fibrosis.

It is still a matter of debate as to whether in vitro mesenchymal stem cell (MSC)-derived hepatocytes may efficiently repopulate a host liver to provide adequate functional substitution. The aim of this study is to assess the efficacy and consistency of in vitro hepatic differentiation from Wharton jelly-derived MSCs, and to validate their therapeutic potential in experimentally induced liver fibrosis compared with nondifferentiated MSCs. Forty adult male albino rats were divided into 4 main groups: (I) normal control group; (II) carbon tetrachloride (CCl4)-treated group (injected CCl4 solution twice a week for 8 wk); (III) MSC-treated group (a single intravenous dose of MSCs from human umbilical cord at the fourth week of induction of fibrosis); and (IV) hepatocyte-like stem cells (HLCs)-treated group (a single intravenous dose of MSCs after in vitro conversion to hepatocyte at the fourth week of induction of fibrosis). Portal blood flow velocity and resistance, serum alanine transaminase, aspartate transaminase, albumin, and total bilirubin were measured. Liver homogenate was prepared for malondialdehyde, superoxide dismutase (SOD), nitric oxide (measured as nitrites), and TGFβ (transforming growth factor beta) assessment. Assessment of human cells homing into liver rat and their function was performed using immunohistochemistry for detection of human hepatocytes and α-fetoprotein antigens. Significant elevation of serum alanine transaminase, aspartate transaminase, and bilirubin, liver malondialdehyde, nitrites, TGFβ, fibrotic score, and significant reduction in serum albumin, liver SOD, and portal flow velocity in the CCl4-treated group were found when compared with normal rats. All these parameters significantly reversed in MSC-treated and hepatocyte-treated groups when compared with the CCl4-treated group. The MSC-treated group shows statistically better results in most liver function and oxidation parameters when compared with the HLC-treated group. Human MSCs can differentiate in vitro into functional HLCs. Transplantation of both MSCs and HLCs was feasible and effective in a liver fibrosis rat model. However, MSCs were better in regaining liver function. Future studies should address strategies to improve long-term implantation of MSCs and HLCs in the host liver.

High regenerative capacity of the liver and irreversible injury of male reproductive system in carbon tetrachloride-induced liver fibrosis rat model.

Liver fibrosis (LF) is a chronic disease, associated with many collateral diseases including reproductive dysfunction. Although the normal liver has a large regenerative capacity the complications of LF could be severe and irreversible. Hormone and sex-related issues of LF development and interactions with male reproductive have not been finally studied. The aim was to study the reproductive function of male rats in experimental CCl4-induced liver fibrosis rat model, and the capability for restoration of both the liver and male reproduction system. Studies were conducted on 20 3-month old Wistar male rats. The experimental animals were injected with freshly prepared 50% olive oil solution of carbohydrate tetrachloride (CCl4). On the 8th week after injection we noted the manifestations of liver fibrosis. The rats were left to self-healing of the liver for 8weeks. All male rats underwent ultrasound and biopsy of the liver and testes on the 8th and 16th weeks. The male rats were mated with healthy females before CCl4 injection, after modeling LF on the 8th week, and after self-healing of the liver. Pregnancy was monitored on ultrasound. On the 8th week of experiment we observed ultrasound manifestation of advanced liver fibrosis, including hepatosplenomegaly, portal hypertension. Ultrasound exam of the rat testes showed testicular degeneration, hydrocele, fibrosis, scarring, petrifications, size reduction, and restriction of testicular descent; testes size decreased from 1.24±0.62ml to 0.61±0.13, p<0.01. Liver histology showed granular dystrophy of hepatocytes, necrotic areas, lipid inclusions in parenchyma. Rats with liver fibrosis demonstrated severe injury of the reproductive system and altering of fertility: the offspring of male rats with advanced LF was 4.71±0.53 born alive vs 9.55±0.47 born from mating with healthy males, p<0.001. Eightweeks after last CCl4 injection, we revealed signs of liver regeneration, significant recovery of its structure. The ALT and AST levels significantly decreased and reached background measurements. As a result of the second interbreeding after liver self-healing no significant difference was found vs previous mating. Carbohydrate tetrachloride induces injury of liver parenchyma evoking fast and severe liver fibrosis, and is associated with irreversible structural and functional changes in testes, reducing fertility, decreasing potential pregnancy rate, and affecting its development. Liver showed high potential to regenerate, however the self-restoring after liver fibrosis was not accompanied with recovery of the reproductive system.

Mitigation of TGF-β/Smad signaling pathway-associated liver fibrosis by paeoniflorin

Purpose: To evaluate paeoniflorin (PF) as a possible protective agent against liver fibrosis and its likely mechanisms of action.Methods: A rat model of liver fibrosis was induced by carbon tetrachloride (CCl4) injection. Liver damage was determined by serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities. The hydroxyproline content of proteins was measured as an indirect way of assessing collagen deposition. TGF-β1 levels and TGF-β/Smad signaling pathway-related genes and proteins were analyzed by quantitative polymerase chain reaction (qPCR) assay and Western blot assay.Results: After PF administration, serum ALT and AST levels were significantly (p &lt; 0.01) reduced by 34.9 and 37.6 %, respectively. Collagen deposition was also significantly (p &lt; 0.01) reduced by 31.0 %. Hepatic stellate cell activation was significantly inhibited, as evidenced by suppressed α-SMA expression. PF inhibited phospo-Smad 2/3 by elevating Smad 7 level.Conclusion: PF alleviates CCl4-induced liver fibrosis in a dose-dependent manner probably by restoring the balance between activated Smad 2/3 and Smad7. Thus, PF might be a source of a novel anti-fibrotic agent.Keywords: Paeoniflorin, Fibrosis, Liver, TGF-β, Hepatic stellate cell, Smad, Hydroxyproline

Effects of aspirin and enoxaparin in a rat model of liver fibrosis

AIMTo examine the effects of aspirin and enoxaparin on liver function, coagulation index and histopathology in a rat model of liver fibrosis.METHODSForty-five male Sprague-Dawley rats were randomly divided into the control group (n = 5) and model group (n = 40). Thioacetamide (TAA) was used to induce liver fibrosis in the model group. TAA-induced fibrotic rats received TAA continuously (n = 9), TAA + low-dose aspirin (n = 9), TAA + high-dose aspirin (n = 9) or TAA + enoxaparin (n = 9) for 4 wk. All rats were euthanized after 4 wk, and both hematoxylin-eosin and Masson staining were performed to observe pathological changes in liver tissue.RESULTSLiver fibrosis was assessed according to the METAVIR score. Compared with untreated cirrhotic controls, a significant improvement in fibrosis grade was observed in the low-dose aspirin, high-dose aspirin and enoxaparin treated groups, especially in the high-dose aspirin treated group. Alanine aminotransferase and total bilirubin were higher, albumin was lower and both prothrombin time and international normalized ratio were prolonged in the four treatment groups compared to controls. No significant differences among the four groups were observed.CONCLUSIONAspirin and enoxaparin can alleviate liver fibrosis in this rat model.

Evodiamine ameliorates liver fibrosis in rats via TGF-β1/Smad signaling pathway.

Liver fibrosis is considered to be a result of chronic liver pathological changes, and hepatic stellate cells (HSCs) play an important role during this process. Evodiamine, an indole alkaloid derived from Evodia rutaecarpa, exhibits pharmacological activities. This study focused on the effects of evodiamine on carbon tetrachloride (CCl4)-induced liver fibrosis in rats and HSCs in vitro via the TGF-β1/Smad signaling pathway. A liver fibrosis rat model was established by the intraperitoneal injection of CCl4 (3ml/kg, 30% in olive oil). Evodiamine (15 and 25mg/kg) was administered orally for 8weeks. HSCs were treated with different evodiamine concentrations. The results indicated that evodiamine could improve the histopathological abnormalities in liver tissues and decrease the level of aspartate aminotransferase (AST), alanine aminotransferase (ALT), hydroxyproline, and total bilirubin (TBIL). Concentrations of IL-6, tumor necrosis factor-α (TNF-α), collagen-I (COL-I), and collagen-III (COL-III) were reduced by evodiamine. Western blotting and real-time PCR showed that protein expression of transforming growth factor-β (TGF-β1), p-Smad 2/3 (phosphorylation of Smad 2/3), and smooth muscle alpha-actin (α-SMA) as well as mRNA expression of TGF-β1 and α-SMA in liver tissues were downregulated by evodiamine. The cell proliferation, production of hydroxyproline, and the protein expression of TGF-β1, p-Smad 2/3, and α-SMA in HSCs were dose-dependently reduced by evodiamine. Collectively, evodiamine had an antifibrosis effect in CCl4-induced liver fibrosis, and reduced HSCs proliferation and collagen metabolism in vitro. The major mechanism was downregulation of relative expression of TGF-β1, p-Smad2/3, and α-SMA.

Identification of key metabolic changes during liver fibrosis progression in rats using a urine and serum metabolomics approach

Reversibility of hepatic fibrosis is an intrinsic response to chronic injury, and with on-going damage, fibrosis can progress to its end-stage consequence, cirrhosis. Non-invasive and reliable biomarkers for early detection of liver fibrosis are needed. Based on the CCl4-induced liver fibrosis rat model, urinary and serum metabolic profiling performed by LC-QTOF-MS associated with histological progression were utilized to identify liver fibrosis-specific potential biomarkers for early prediction and to reveal significant fibrotic pathways and their dynamic changes in different stages of liver fibrosis. Finally, nine differential metabolites in urine and ten in serum were selected and identified involving the most relevant metabolic pathways. Perturbations of tryptophan, valine, leucine, isoleucine, and citrate (TCA) cycle metabolites, along with sphingolipid and glycerophospholipid metabolites, occurred from the onset of liver fibrosis. Furthermore, dysregulation of valine and bile acid biosynthesis metabolites occurred in the intermediate and advanced stages. More importantly, among these metabolites, urinary kynurenic acid, 5-hydroxyindoleacetyl glycine, 4-(2-amino-3-hydroxyphenyl)-2,4-dioxobutanoic acid and serum sphinganine, sphingomyelin, L-leucine, L-tryptophan, and LysoPC(17:0) changed at all time points and may serve as potential early biomarkers for the diagnosis of hepatic fibrosis and as therapeutic targets. Overall, this work evaluates the potential of these metabolites for the early detection of liver fibrosis.

Measurement of Liver Stiffness Using Shear Wave Elastography in a Rat Model: Factors Impacting Stiffness Measurement with Multiple- and Single-Tracking-Location Techniques

The clinical use of elastography for monitoring fibrosis progression is challenged by the subtle changes in liver stiffness associated with early-stage fibrosis and the comparatively large variance in stiffness estimates provided by elastography. Single-tracking-location (STL) shear wave elasticity imaging (SWEI) is an ultrasound elastography technique previously found to provide improved estimate precision compared with multiple-tracking-location (MTL) SWEI. Because of the improved precision, it is reasonable to expect that STL-SWEI would provide improved ability to differentiate liver fibrosis stage compared with MTL-SWEI. However, this expectation has not been previously challenged rigorously. In this work, the performance of STL- and MTL-SWEI in the setting of a rat model of liver fibrosis is characterized, and the advantages of STL-SWEI in staging fibrosis are explored. The purpose of this study was to determine what advantages, if any, arise from using STL-SWEI instead of MTL-SWEI in the characterization of fibrotic liver. Thus, the ability of STL-SWEI to differentiate livers at various METAVIR fibrosis scores, for ex vivo postmortem measurements, is explored. In addition, we examined the effect of the common confounding factor of fluid versus solid boundary conditions in SWEI experiments. Sprague-Dawley rats were treated with carbon tetrachloride over several weeks to produce liver disease of varying severity. STL and MTL stiffness measurements were performed ex vivo and compared with the METAVIR scores from histological analysis and the duration of treatment. A strong association was observed between liver stiffness and weeks of treatment with the liver toxin carbon tetrachloride. Direct comparison of STL- and MTL-SWEI measurements revealed no significant difference in ability to differentiate fibrosis stages based on SWEI mean values. However, image interquartile range was greatly improved in the case of STL-SWEI, compared with MTL-SWEI, at small beam spacing.

Comprehensive Study of Multiple Stages Progressing to Nonalcoholic Steatohepatitis with Subsequent Fibrosis in SD Rats.

Because of the absence of the time course of histological nonalcoholic fatty hepatitis with subsequent fibrotic progression, the effective approaches available for controlling the onset and progression of non-alcoholic steatohepatitis (NASH) remain limited. Therefore, we detected the serum and liver tissue related lipid metabolism disorder, liver pathology and relative molecular makers alteration dynamically in a high fat-sucrose diet during different time points. High fat-sucrose diet significantly increased the serum lipid level on day 10. The excess lipid accumulation in liver was referred to as simple steatosis after the feeding of a high fat-sucrose diet for 20 days. The high fat-sucrose diet induced a hepatic inflammation response on day 30. Similarly, hepatic fibrosis was also initiated on day 30 and gradually formed from the 30th to the 50th day. Oxidative stress may be related with the process from NASH to liver fibrosis. Insulin resistance was involved in the progression from hepatic steatosis to NASH with hepatic fibrosis from the 20th to the 50th day. In conclusion, we established a high fat-sucrose diet induced nonalcoholic fatty hepatitis with liver fibrosis rat model, which presented the time course of histological nonalcoholic steatohepatitis and the initiation and progression change of characteristic molecular makers in the process from steatosis to hepatic fibrosis.

The different expressed serum proteins in rhCygb treated rat model of liver fibrosis by the optimized two-dimensional gel electrophoresis.

Liver fibrosis, a common pathological process of chronic liver diseases, is the final stage of liver dysfunction that has severely deleterious impact on human health. Cytoglobin was first discovered in 2001 by proteomic analysis in rat stellate cells and was reported to play an important role in controlling tissue fibrosis. However, the mechanism by which cytoglobin inhibits or reverses the progression of fibrosis remains unclear. The present study examines the effect of recombinant human cytoblobin (rhCygb) in a rat model of liver fibrosis. Proteomic approaches were employed to identify differentially expressed proteins in the fibrosis model. Optimized conditions for two-dimensional gel electrophoresis were developed to provide improved protein detection and separation. A total of 43 spots were obtained and, through the use of matrix-assisted laser desorption ionization time-of-flight mass spectrometry, 30 differentially expressed proteins were identified. Gene ontology term annotation and KEGG pathway analysis allowed us to explore the function of the represented proteins. Based on these results, we provide a theory of the molecular mechanism related to rhCygb reversion of fibrosis and which will assist in the identification of biomarkers in patient serum to improve early diagnosis of liver fibrosis.

Notch signaling mediated by TGF-β/Smad pathway in concanavalin A-induced liver fibrosis in rats.

AIMTo explore the exact interaction between Notch and transforming growth factor (TGF)-β signaling in liver fibrosis.METHODSWe established a rat model of liver fibrosis induced by concanavalin A. Peripheral blood mononuclear cells (PBMCs) were isolated from the modeled rats, and cultured with γ-secretase inhibitor DAPT and TGF-β inhibitor for 24 h. The mRNA levels of Notch and TGF-β signaling were detected by quantitative real-time polymerase chain reaction. Expression of Notch and TGF-β proteins was analyzed by western blotting.RESULTSCompared to control rats, Notch and TGF-β signaling was activated in PBMCs of model rats. Administration of DAPT and TGF-β inhibitor suppressed Notch and TGF-β signal transducer in PBMCs of model rats. DAPT reduced mRNA and protein expression of TGF-β signaling, such as TGF-β1 and Smad3. TGF-β inhibitor also downregulated Notch1, Hes1 and Hes5, and mRNA and protein expression of the Notch signaling pathway.CONCLUSIONNotch and TGF-β signaling play a role in liver fibrosis. TGF-β signaling upregulates Notch signaling, which promotes TGF-β signaling.

RNA Sequencing and Bioinformatics Analysis Implicate the Regulatory Role of a Long Noncoding RNA-mRNA Network in Hepatic Stellate Cell Activation

Background/Aims: To analyze the long noncoding (lncRNA)-mRNA expression network and potential roles in rat hepatic stellate cells (HSCs) during activation. Methods: LncRNA expression was analyzed in quiescent and culture-activated HSCs by RNA sequencing, and differentially expressed lncRNAs verified by quantitative reverse transcription polymerase chain reaction (qRT-PCR) were subjected to bioinformatics analysis. In vivo analyses of differential lncRNA-mRNA expression were performed on a rat model of liver fibrosis. Results: We identified upregulation of 12 lncRNAs and 155 mRNAs and downregulation of 12 lncRNAs and 374 mRNAs in activated HSCs. Additionally, we identified the differential expression of upregulated lncRNAs (NONRATT012636.2, NONRATT016788.2, and NONRATT021402.2) and downregulated lncRNAs (NONRATT007863.2, NONRATT019720.2, and NONRATT024061.2) in activated HSCs relative to levels observed in quiescent HSCs, and Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses showed that changes in lncRNAs associated with HSC activation revealed 11 significantly enriched pathways according to their predicted targets. Moreover, based on the predicted co-expression network, the relative dynamic levels of NONRATT013819.2 and lysyl oxidase (Lox) were compared during HSC activation both in vitro and in vivo. Our results confirmed the upregulation of lncRNA NONRATT013819.2 and Lox mRNA associated with the extracellular matrix (ECM)-related signaling pathway in HSCs and fibrotic livers. Conclusion: Our results detailing a dysregulated lncRNA-mRNA network might provide new treatment strategies for hepatic fibrosis based on findings indicating potentially critical roles for NONRATT013819.2 and Lox in ECM remodeling during HSC activation.

Protective effects of Ampelopsis brevipedunculata against in vitro hepatic stellate cells system and thioacetamide-induced liver fibrosis rat model

Context: Ampelopsis brevipedunculata Maxim (Vitaceae) is a traditional medicinal herb used for treating liver disorders. Objective: The hepatoprotective effects of A. brevipedunculata ethanol extract (ABE) was investigated in experimental models of fibrosis. Materials and methods: Hepatic stellate cells (HSCs) system in vitro and thioacetamide (TAA)-induced liver fibrosis rat model in vivo were used. Sprague–Dawley rats were divided into five groups of eight each (control, TAA, TAA with ABE 10 mg/kg, ABE 100 mg/kg and silymarin 50 mg/kg groups, respectively). Fibrosis was induced except to the control group by TAA (200 mg/kg, i.p.) twice per week for 13 weeks. ABE and silymarin was administered orally six times per week from the 7th week to the 13th week. Results: In HSC-T6 cells, ABE (0.1 mg/mL) and silymarin (0.05 mg/mL) significantly (p < 0.01) induced apoptosis (12.94 ± 5.72% and 14.9 ± 3.8%, respectively) compared with control group (7.51 ± 1.26%). The expression of fibrosis related genes (TGF-β, α-SMA and Col1A1) in HSC-T6 cells were significantly (p < 0.01) downregulated in ABE-treated groups compared with control group. In in vivo studies, ABE (10 and 100 mg/kg) treatment ameliorated the altered levels of serum biomarkers significantly (p < 0.01 and p < 0.001) in TAA-induced groups. Further, ABE (10 and 100 mg/kg) significantly (p < 0.01) attenuated the altered histopathological findings, glutathione content and the accumulation of hydroxyproline. Conclusion: These results collectively indicate that ABE can potentially be developed as a therapeutic agent in the treatment of hepatic fibrosis.

Metabolomics combined with pattern recognition and bioinformatics analysis methods for the development of pharmacodynamic biomarkers on liver fibrosis.

The major obstacle for the development of targeted therapies is the lack of pharmacodynamic (PD) biomarkers to provide an early readout of biological activities. As the modulation of metabolites may reflect the biological changes occurring in the targets, metabolomics is promising to be an efficient way to explore PD biomarkers. In the present study, a liver fibrosis rat model was established by intraperitoneal injection of CCl4 twice weekly for 6 weeks, the treatment of total aglycone extracts of Scutellaria baicalensis (TAES) was begun 4 weeks after the modeling, and gas chromatography-mass spectrometry (GC-MS) based metabolomics combined with pattern recognition and network analysis were carried out for the research on PD biomarkers of TAES on liver fibrosis. After 2 weeks of treatment, TAES shows positive effects on CCl4-induced liver fibrosis. In the metabolomics study, 63 urinary metabolites contributing to liver fibrosis were identified. Six metabolic pathways significantly enriched in metabolomics data were mapped onto a network to determine global patterns of metabolic alterations in liver fibrosis. By topological analysis, 6 metabolites with high centrality in the metabolic sub-network were selected as potential PD biomarkers. Within 24 h of the final administration, the 6 identified urine metabolic biomarkers with response to time variation of TAES were validated as PD biomarkers. This integrative study presents an attractive strategy to explore PD biomarkers, which may give insight into the actual pharmacological effect of target drugs, and the information from PD biomarkers can be combined with pharmacokinetics to select the optimal dose and a schedule of administration for the drugs.

Antifibrotic Effects of Human Amniotic Membrane Transplantation in Established Biliary Fibrosis Induced in Rats

Liver fibrosis is characterized by excessive accumulation of extracellular matrix components in the liver parenchyma that distorts the normal architecture and hepatic function. Progressive fibrosis could end in the advanced stage known as cirrhosis, resulting in the need to resort to liver transplantation. Amniotic membrane (AM) has emerged as an innovative therapeutic approach for chronic liver diseases due to its anti-inflammatory, antiscarring, and wound-healing effects. We have recently shown that AM can be used as a patch on the liver surface at the same time of fibrosis induction, resulting in significantly reduced progression and severity of biliary fibrosis. Here we investigated the effects of human AM on the established rat model of liver fibrosis, induced by the bile duct ligation (BDL). We also explored the effect of AM on the expression of transforming growth factor-1 (TGF-1), the main profibrogenic factor in hepatic fibrosis, and the proinflammatory cytokines, tumor necrosis factor- (TNF-), interleukin-6 (IL-6), and anti-inflammatory cytokine IL-10. Two weeks after BDL, the liver was covered with a fragment of AM or left untreated. Six weeks later, the fibrosis was first assessed by the semiquantitative Knodell and the METAVIR scoring systems and, thereafter, by CellProfiler digital image analysis to quantify the area occupied by collagen deposition, ductular reactions (DRs), activated myofibroblasts, and TGF-1. The hepatic cytokines were determined by ELISA. AM-treated rats showed a significantly lower score compared to the control BDL rats (2.50.9 vs. 3.50.3, respectively; p0.05). The collagen deposition, DRs, number of activated myofibroblasts, and TGF-1 were all reduced to about 50% of levels observed in untreated BDL rats. These findings suggest that AM, when applied as a patch onto the liver surface, is useful for treating well-established cholestatic fibrosis, and the mechanism was partly by means of downregulating the profibrotic factor TGF-1 and IL-6.

Genistein attenuates D-GalN induced liver fibrosis/chronic liver damage in rats by blocking the TGF-β/Smad signaling pathways

Background and aimGenistein is a major isoflavonoid abundantly found in soy. Earlier genistein has been reported to possess protective effect against a multitude of disorders including cancer. Previously we demonstrated the protective effects of Genistein in d-Galactosamine (D-GalN) induced fulminant hepatic failure (FHF) in rats. In present study, we evaluated the hepatoprotective activity of Genistein in rat model of chronic liver damage and liver fibrosis. MethodsLiver fibrosis was induced by intraperitoneal injection of D-GalN (250 mg/kg BW) twice a week for 12 weeks. Genistein (5 mg/kg BW) was given via intra-gastric route as co-treatment daily for 12 weeks. ResultsGenistein co-treatment significantly attenuated D-GalN-induced chronic liver damage and liver fibrosis as evident from a significant amelioration in functional impairment, including inhibition of the activation of Hepatic stellate cells (HSC), decreased expression in alpha smooth muscle actin (α-SMA) and accumulation of collagen matrix, and an elevation in serum alanine transaminase (ALT) and aspartate transaminase (AST) level. In addition Genistein co-treatment was associated with elevated expression of hepatic Smad7, which ultimately blunts the expression of TGF-β and the activation of TGF-β/Smad signaling. Furthermore Genistein significantly prevented the histopathological changes induced by D-GalN. ConclusionOur results suggest that Genistein could be a novel therapeutic/nutraceutical agent in treating chronic liver damage and liver fibrosis. In addition our study also suggests a possible mechanism of action in which Smad7-induced inhibition of TGF-β/Smad2/3 can be a central mechanism by which Genistein protects liver from chronic injury.

ROS-JNK1/2-dependent activation of autophagy is required for the induction of anti-inflammatory effect of dihydroartemisinin in liver fibrosis

Accumulating evidence identifies autophagy as an inflammation-related defensive mechanism against diseases including liver fibrosis. Therefore, autophagy may represent a new pharmacologic target for drug development to treat liver fibrosis. In this study, we sought to investigate the effect of dihydroartemisinin (DHA) on autophagy, and to further examine the molecular mechanisms of DHA-induced anti-inflammatory effects. We found that DHA appeared to play an essential role in controlling excessive inflammation. DHA suppressed inflammation in rat liver fibrosis model and inhibited the expression of proinflammatory cytokines in activated hepatic stellate cells (HSCs). Interestingly, DHA increased the autophagosome generation and autophagic flux in activated HSCs, which is underlying mechanism for the anti-inflammatory activity of DHA. Autophagy depletion impaired the induction of anti-inflammatory effect of DHA, while autophagy induction showed a synergistic effect with DHA. Importantly, our study also identified a crucial role for reactive oxygen species (ROS) in the facilitation of DHA-induced autophagy. Antioxidants, such as glutathione and N-acetyl cysteine, significantly abrogated ROS production, and in turn, prevented DHA-induced autophagosome generation and autophagic flux. Besides, we found that c-Jun N-terminal kinase1/2 (JNK1/2) was a downstream signaling molecule of ROS that mediated the induction of autophagy by DHA. Down-regulation of JNK1/2 activity, using selective JNK1/2 inhibitor (SP600125) or siJNK1/2, led to an inhibition of DHA-induced autophagy. Overall, these results provide novel implications to reveal the molecular mechanism of DHA-induced anti-inflammatory effects, by which points to the possibility of using DHA based proautophagic drugs for the treatment of inflammatory diseases.