Murine Model Of Liver Fibrosis Research Articles

Dual blockade of interleukin-17A and interleukin-17F as a therapeutic strategy for liver fibrosis: Investigating the potential effect and mechanism of brodalumab

Liver fibrosis is a terminal manifestation of various chronic liver diseases. There are no drugs that can reverse the condition. Recently, the importance of interleukin-17 (IL17) in the pathophysiology has been revealed and has attracted attention as a therapeutic target. We aimed to reveal the roles of IL17A and IL17F in liver fibrosis, and to validate the potential of their dual blockade as therapeutic strategy. First, we retrospectively reviewed the longitudinal change of FIB-4 index, a clinical indicator of liver fibrosis, among psoriasis patients treated by brodalumab, which blocks IL17 receptor A (IL17RA). Next, we examined anti-fibrotic efficacy of anti-IL17RA antibody (Ab) in two murine liver fibrosis models by histopathological investigation and real-time reverse transcription polymerase chain reaction (RT-PCR). Finally, we analyzed the effect of IL17A and IL17F upon human hepatic stellate cells with RNA sequencing, real-time RT-PCR, western blotting, chromatin immunoprecipitation, and flow cytometry. Clinical data showed that FIB-4 index significantly decreased among psoriasis patients treated by brodalumab. In vivo studies additionally demonstrated that anti-IL17RA Ab ameliorates liver fibrosis induced by tetrachloride and methionine-choline deficient diet. Furthermore, in vitro experiments revealed that both IL17A and IL17F enhance cell-surface expression of transforming growth factor-β receptor II and promote pro-fibrotic gene expression via the JUN pathway in human hepatic stellate cells. Our insights suggest that IL17A and IL17F share their pro-fibrotic function in the context of liver fibrosis, and moreover, dual blockade of IL17A and IL17F by anti-IL17RA Ab would be a promising strategy for the management of liver fibrosis.

Age-dependent loss of hepatic SIRT1 enhances NLRP3 inflammasome signaling and impairs capacity for liver fibrosis resolution.

Our studies indicate that the longevity factor SIRT1 is implicated in metabolic disease; however, whether and how hepatocyte-specific SIRT1 signaling is involved in liver fibrosis remains undefined. We characterized a functional link of age-mediated defects in SIRT1 to the NLRP3 inflammasome during age-related liver fibrosis. In multiple experimental murine models of liver fibrosis, we compared the development of liver fibrosis in young and old mice, as well as in liver-specific SIRT1 knockout (SIRT1 LKO) mice and wild-type (WT) mice. Liver injury, fibrosis, and inflammation were assessed histologically and quantified by real-time PCR analysis. In a model of hepatotoxin-induced liver fibrosis, old mice displayed more severe and persistent liver fibrosis than young mice during liver injury and after injury cessation, as characterized by inhibition of SIRT1, induction of NLRP3, infiltration of macrophages and neutrophils, activation of hepatic stellate cells (HSCs), and excessive deposition and remodeling of the extracellular matrix. Mechanistically, deletion of SIRT1 in hepatocytes resulted in NLRP3 and IL-1β induction, pro-inflammatory response, and severe liver fibrosis in young mice, mimicking the ability of aging to impair the resolution of established fibrosis. In an aging mouse model, chronic-plus-binge alcohol feeding-induced liver fibrosis was attenuated by treatment with MCC950, a selective NLRP3 inhibitor. NLRP3 inhibition ameliorated alcoholic liver fibrosis in old mice by repressing inflammation and reducing hepatocyte-derived danger signaling-ASK1 and HMGB1.In conclusion, age-dependent SIRT1 defects lead to NLRP3 activation and inflammation, which in turn impairs the capacity to resolve fibrosis during aging.

Highlighting sex dimorphism in the molecular mechanisms responsible for sarcopenia in a murine model of liver fibrosis

Highlighting sex dimorphism in the molecular mechanisms responsible for sarcopenia in a murine model of liver fibrosis

GDF15 Ameliorates Liver Fibrosis by Metabolic Reprogramming of Macrophages to Acquire Anti-Inflammatory Properties

Liver fibrosis/cirrhosis is significant health burden worldwide, resulting in liver failure or cancer and accounting for many deaths each year. The pathogenesis of liver fibrosis is very complex, which makes treatment challenging. Growth differentiation factor 15 (GDF15), a cysteine knot protein belonging to the transforming growth factor β (TGF-β) superfamily, has been shown to play a protective role after tissue injury and to promote a negative energy balance during obesity and diabetes. However, paucity of literature is available about GDF15 function in liver fibrosis. This study aimed to investigate the immunomodulatory role and therapeutic potential of GDF15 in progression of hepatic fibrosis. GDF15 expression was studied in patients with fibrosis/cirrhosis and in 2 murine models of liver fibrosis, including mice treated with CCl4 or DDC diet. GDF15 involvement in the pathogenesis of liver fibrosis was assessed in Gdf15 knockout mouse using both CCl4 and DDC diet experimental models. We used the CCl4 and/or DDC diet-induced liver fibrosis model to examine the antifibrotic and anti-inflammatory effects of AAV8-mediated GDF15 overexpression in hepatocytes or recombinant mouse GDF15. GDF15 expression is decreased in the liver of animal models and patients with liver fibrosis/cirrhosis compared with those without liver disease. Invivo studies showed that GDF15 deficiency aggravated CCl4 and DDC diet-induced liver fibrosis, while GDF15 overexpression mediated by AAV8 or its recombinant protein alleviated CCl4 and/or DDC diet-induced liver fibrosis. In Gdf15 knockout mice, the intrahepatic microenvironment that developed during fibrosis showed relatively more inflammation, as demonstrated by enhanced infiltration of monocytes and neutrophils and increased expression of proinflammatory factors, which could be diminished by AAV8-mediated GDF15 overexpression in hepatocytes. Intriguingly, GDF15 exerts its effects by metabolic reprogramming macrophages to acquire an oxidative phosphorylation-dependent anti-inflammatory functional fate. Furthermore, adoptive transfer of GDF15-preprogrammed macrophages to mouse models of liver fibrosis induced by CCl4 attenuated inflammation and alleviated the progression of liver fibrosis. GDF15 ameliorates liver fibrosis via modulation of liver macrophages. Our data implicate the importance of the liver microenvironment in macrophage programming during liver fibrosis and suggest GDF15 that is a potentially attractive therapeutic target for the treatment of patients with liver fibrosis.

P20-10 A new murine liver fibrosis model induced by polyhexamethylene guanidine-phosphate

P20-10 A new murine liver fibrosis model induced by polyhexamethylene guanidine-phosphate

MiR-488-5p mitigates hepatic stellate cell activation and hepatic fibrosis via suppressing TET3 expression

Background and aimsNumerous studies have demonstrated that hepatic fibrosis, a progressive condition as an endpoint of multiple chronic hepatic diseases, is largely characterized with the extensive activation of hepatic stellate cells (HSCs). The precise effect of miR-488-5p in HSCs during hepatic fibrosis has not been elucidated.MethodsIn our study, qRT‐PCR was applied to assess the level of miR-488-5p in activated HSCs stimulated by TGF-β1. We built murine liver fibrosis models with carbon tetrachloride (CCl4), high-fat diet (HFD) and bile duct ligation (BDL). In vitro, the effects of miR-488-5p in HSCs were examined through cell proliferation assay and apoptosis. Luciferase reporter assay was applied to identify the underlying target of miR-488-5p. In vivo, the effects of miR-488-5p were explored through mouse liver fibrosis models.ResultsThe reduction of miR-488-5p in the activated HSCs induced by TGF-β1 and three mouse hepatic fibrosis models were identified. The in vitro functional experimentations verified that miR-488-5p restrained expression of fibrosis-related markers and proliferative capacity in HSCs. Mechanically, we identified that miR-488-5p inhibited tet methylcytosine dioxygenase 3 (TET3) expression via straightly binding onto the 3′ UTR of its mRNA, which sequentially restrained the TGF-β/Smad2/3 pathway. TET3 inhibition induced by the overexpression of miR-488-5p reduced extracellular matrix deposition, which contributed to mitigating mouse liver fibrosis.ConclusionWe highlight that miR-488-5p restrains the activation of HSCs and hepatic fibrosis via targeting TET3 which is involved in the TGF-β/Smad2/3 signaling pathway. Collectively, miR-488-5p is identified as a potential therapeutic target for hepatic fibrosis.

TRPM8 deficiency attenuates liver fibrosis through S100A9-HNF4α signaling

BackgroundLiver fibrosis represent a major global health care burden. Data emerging from recent advances suggest TRPM8, a member of the transient receptor potential (TRP) family of ion channels, plays an essential role in various chronic inflammatory diseases. However, its role in liver fibrosis remains unknown. Herein, we assessed the potential effect of TRPM8 in liver fibrosis.MethodsThe effect of TRPM8 was evaluated using specimens obtained from classic murine models of liver fibrosis, namely wild-type (WT) and TRPM8−/− (KO) fibrotic mice after carbon tetrachloride (CCl4) or bile duct ligation (BDL) treatment. The role of TRPM8 was systematically evaluated using specimens obtained from the aforementioned animal models after various in vivo and in vitro experiments.ResultsClinicopathological analysis showed that TRPM8 expression was upregulated in tissue samples from cirrhosis patients and fibrotic mice. TRPM8 deficiency not only attenuated inflammation and fibrosis progression in mice but also helped to alleviate symptoms of cholangiopathies. Moreover, reduction in S100A9 and increase in HNF4α expressions were observed in liver of CCl4- and BDL- treated TRPM8−/− mice. A strong regulatory linkage between S100A9 and HNF4α was also noticed in L02 cells that underwent siRNA-mediated S100A9 knockdown and S100A9 overexpressing plasmid transfection. Lastly, the alleviative effect of a selective TRPM8 antagonist was confirmed in vivo.ConclusionsThese findings suggest TRPM8 deficiency may exert protective effects against inflammation, cholangiopathies, and fibrosis through S100A9-HNF4α signaling. M8-B might be a promising therapeutic candidate for liver fibrosis.Graphical

EP3 enhances adhesion and cytotoxicity of NK cells toward hepatic stellate cells in a murine liver fibrosis model

Natural killer (NK) cells exhibit antifibrotic properties in liver fibrosis (LF) by suppressing activated hepatic stellate cell (HSC) populations. Prostaglandin E2 (PGE2) plays a dual role in innate and adaptive immunity. Here, we found that E-prostanoid 3 receptor (EP3) was markedly downregulated in NK cells from liver fibrosis mice and patients with liver cirrhosis. NK cell-specific deletion of EP3 aggravated hepatic fibrogenesis in mouse models of LF. Loss of EP3 selectively reduced the cytotoxicity of the CD27+CD11b+ double positive (DP) NK subset against activated HSCs. Mechanistically, deletion of EP3 impaired the adhesion and cytotoxicity of DP NK cells toward HSCs through modulation of Itga4-VCAM1 binding. EP3 upregulated Itga4 expression in NK cells through promoting Spic nuclear translocation via PKC-mediated phosphorylation of Spic at T191. Activation of EP3 by sulprostone alleviated CCL4-induced liver fibrosis in mice. Thus, EP3 is required for adhesion and cytotoxicity of NK cells toward HSCs and may serve as a therapeutic target for the management of LF.

FSTL1 promotes liver fibrosis by reprogramming macrophage function through modulating the intracellular function of PKM2

ObjectiveFollistatin-like protein 1 (FSTL1) is widely recognised as a secreted glycoprotein, but its role in modulating macrophage-related inflammation during liver fibrosis has not been documented. Herein, we aimed to characterise...

Honokiol Acts as a Potent Anti-Fibrotic Agent in the Liver through Inhibition of TGF-β1/SMAD Signaling and Autophagy in Hepatic Stellate Cells.

Chronic liver injury may result in hepatic fibrosis, which can progress to cirrhosis and eventually liver failure. There are no drugs that are specifically approved for treating hepatic fibrosis. The natural product honokiol (HNK), a bioactive compound extracted from Magnolia grandiflora, represents a potential tool in the management of hepatic fibrosis. Though HNK has been reported to exhibit suppressive effects in a rat fibrosis model, the mechanisms accounting for this suppression remain unclear. In the present study, the anti-fibrotic effects of HNK on the liver were evaluated in vivo and in vitro. In vivo studies utilized a murine liver fibrosis model, in which fibrosis is induced by treatment with carbon tetrachloride (CCl4). For in vitro studies, LX-2 human hepatic stellate cells (HSCs) were treated with HNK, and expression of markers of fibrosis, cell viability, the transforming growth factor-β (TGF-β1)/SMAD signaling pathway, and autophagy were analyzed. HNK was well tolerated and significantly attenuated CCl4-induced liver fibrosis in vivo. Moreover, HNK decreased HSC activation and collagen expression by downregulating the TGF-β1/SMAD signaling pathway and autophagy. These results suggest that HNK is a new potential candidate for the treatment of hepatic fibrosis through suppressing both TGF-β1/SMAD signaling and autophagy in HSCs.

A New Murine Liver Fibrosis Model Induced by Polyhexamethylene Guanidine-Phosphate.

Liver fibrosis is part of the wound healing process to help the liver recover from the injuries caused by various liver-damaging insults. However, liver fibrosis often progresses to life-threatening cirrhosis and hepatocellular carcinoma. To overcome the limitations of current in vivo liver fibrosis models for studying the pathophysiology of liver fibrosis and establishing effective treatment strategies, we developed a new mouse model of liver fibrosis using polyhexamethylene guanidine phosphate (PHMG-p), a humidifier sterilizer known to induce lung fibrosis in humans. Male C57/BL6 mice were intraperitoneally injected with PHMG-p (0.03% and 0.1%) twice a week for 5 weeks. Subsequently, liver tissues were examined histologically and RNA-sequencing was performed to evaluate the expression of key genes and pathways affected by PHMG-p. PHMG-p injection resulted in body weight loss of ~15% and worsening of physical condition. Necropsy revealed diffuse fibrotic lesions in the liver with no effect on the lungs. Histology, collagen staining, immunohistochemistry for smooth muscle actin and collagen, and polymerase chain reaction analysis of fibrotic genes revealed that PHMG-p induced liver fibrosis in the peri-central, peri-portal, and capsule regions. RNA-sequencing revealed that PHMG-p affected several pathways associated with human liver fibrosis, especially with upregulation of lumican and IRAK3, and downregulation of GSTp1 and GSTp2, which are closely involved in liver fibrosis pathogenesis. Collectively we demonstrated that the PHMG-p-induced liver fibrosis model can be employed to study human liver fibrosis.

Histone H3K27 methyltransferase EZH2 and demethylase JMJD3 regulate hepatic stellate cells activation and liver fibrosis.

Rationale: As the central hallmark of liver fibrosis, transdifferentiation of hepatic stellate cells (HSCs), the predominant contributor to fibrogenic hepatic myofibroblast responsible for extracellular matrix (ECM) deposition, is characterized with transcriptional and epigenetic remodeling. We aimed to characterize the roles of H3K27 methyltransferase EZH2 and demethylase JMJD3 and identify their effective pathways and novel target genes in HSCs activation and liver fibrosis.Methods: In primary HSCs, we analyzed effects of pharmacological inhibitions and genetic manipulations of EZH2 and JMJD3 on HSCs activation. In HSCs cell lines, we evaluated effects of EZH2 inhibition by DZNep on proliferation, cell cycling, senescence and apoptosis. In CCl4 and BDL murine models of liver fibrosis, we assessed in vivo effects of DZNep administration and Ezh2 silencing. We profiled rat primary HSCs transcriptomes with RNA-seq, screened the pathways and genes associated with DZNep treatment, analyzed EZH2 and JMJD3 regulation towards target genes by ChIP-qPCR.Results: EZH2 inhibition by DZNep resulted in retarded growth, lowered cell viability, cell cycle arrest in S and G2 phases, strengthened senescence, and enhanced apoptosis of HSCs, decreased hepatic collagen deposition and rescued the elevated serum ALT and AST activities of diseased mice, and downregulated cellular and hepatic expressions of H3K27me3, EZH2, α-SMA and COL1A. Ezh2 silencing by RNA interference in vitro and in vivo showed similar effects. JMJD3 inhibition by GSK-J4 and overexpression of wild-type but not mutant Jmjd3 enhanced or repressed HSCs activation respectively. EZH2 inhibition by DZNep transcriptionally inactivated TGF-β1 pathway, cell cycle pathways and vast ECM components in primary HSCs. EZH2 inhibition decreased H3K27me3 recruitment at target genes encoding TGF-β1 pseudoreceptor BAMBI, anti-inflammatory cytokine IL10 and cell cycle regulators CDKN1A, GADD45A and GADD45B, and increased their expressions, while Jmjd3 overexpression manifested alike effects.Conclusions: EZH2 and JMJD3 antagonistically modulate HSCs activation. The therapeutic effects of DZNep as epigenetic drug in liver fibrosis are associated with the regulation of EZH2 towards direct target genes encoding TGF-β1 pseudoreceptor BAMBI, anti-inflammatory cytokine IL10 and cell cycle regulators CDKN1A, GADD45A and GADD45B, which are also regulated by JMJD3. Our present study provides new mechanistic insight into the epigenetic modulation of EZH2 and JMJD3 in HSCs biology and hepatic fibrogenesis.

Effects of Combined Admistration of Imatinib and Sorafenib in a Murine Model of Liver Fibrosis.

Liver fibrosis is defined as excessive extracellular matrix deposition in the hepatic parenchyma as a consequence of complex interactions among matrix-producing hepatic stellate cells (HSCs) and liver-resident and infiltrating cells. In addition to the liver, the process of fibrosis may represent end-stage disease of several diseases including kidneys, lungs, spleens, heart, muscles and at certain extent, the central nervous system and the peripheral nerves. To date, antifibrotic treatment of fibrosis represents an unconquered area for drug development. The aim of the present study was to test the efficacy of a new drug combination for the treatment of hepatic fibrosis in order to provide a proof-of-concept for the use of therapeutic agents in clinical practice. For this purpose, we have studied the effects of the PDGF inhibitor imatinib and the angiogenesis inhibitor sorafenib, administered alone or in combination, in reducing the progression of the fibrogenetic process in a pre-clinical model of liver damage induced in mice by repeated administration of Concanavalin A (ConA), resembling long-tern autoimmune hepatitis. Our results suggest that treatments with imatinib and sorafenib can modulate potently and, in a superimposable fashion, the fibrinogenic process when administered alone. However, and in agreement with the computational data presently generated, they only exert partial overlapping antifibrotic effects in modulating the main pathways involved in the process of liver fibrosis, without significant additive or synergist effects, when administered in combination.

PSMP/MSMP promotes hepatic fibrosis through CCR2 and represents a novel therapeutic target

C-C motif chemokine receptor 2 (CCR2) has been recognized as a promising target for the treatment of liver fibrosis. PC3-secreted microprotein (PSMP)/microseminoprotein (MSMP) is a novel chemotactic cytokine and its receptor is CCR2. In the present study we investigated the expression and role of PSMP in liver fibrosis/cirrhosis. PSMP expression was studied in patients with fibrosis/cirrhosis and in 3 murine models of liver fibrosis, including mice treated with carbon tetrachloride (CCl4), bile-duct ligation, or a 5-diethoxycarbonyl-1,4-dihydrocollidine diet. The role of PSMP was evaluated in Psmp-/- mice and after treatment with a PSMP antibody in wild-type mice. The direct effects of PSMP on macrophages and hepatic stellate cells were studied invitro. In this study, we found that PSMP was highly expressed in fibrotic/cirrhotic tissues from patients with different etiologies of liver disease and in the 3 experimental mouse models of fibrosis. Damage-associated molecular pattern molecules HMGB-1 and IL-33 induced hepatocytes to produce PSMP. PSMP deficiency resulted in a marked amelioration of hepatic injury and fibrosis. In CCl4-induced hepatic injury, the infiltration of macrophages and CCR2+ monocytes into the liver was significantly decreased in Psmp-/- mice. Consistent with the decreased levels of intrahepatic macrophages, proinflammatory cytokines were significantly reduced. Moreover, adeno-associated virus-8 vectors successfully overexpressing human PSMP in Psmp-/- mouse livers could reverse the attenuation of liver injury and fibrosis induced by CCl4 in a CCR2-dependent manner. Treatment with a specific PSMP-neutralizing antibody, 3D5, prevented liver injury and fibrosis induced by CCl4 in mice. At the cellular level, PSMP directly promoted M1 polarization of macrophages and activation of LX-2 cells. PSMP enhances liver fibrosis through its receptor, CCR2. PSMP is a potentially attractive therapeutic target for the treatment of patients with liver fibrosis. Our present study identifies the essential role of the protein PSMP for the development and progression of liver fibrosis in humans and mice. PSMP promotes liver fibrosis through inflammatory macrophage infiltration, polarization and production of proinflammatory cytokines, as well as direct activation of hepatic stellate cells via its receptor CCR2. A PSMP antibody can significantly reduce liver fibrosis development invivo. These findings indicate that PSMP is a potential therapeutic target and its antibody is a potential therapeutic agent for the treatment of liver fibrosis.

Growth differentiation factor 11 attenuates liver fibrosis via expansion of liver progenitor cells

ObjectiveLiver fibrosis and cirrhosis resulting from chronic liver injury represent a major healthcare burden worldwide. Growth differentiation factor (GDF) 11 has been recently investigated for its role in rejuvenation of...

THU-079-Study of anti-fibrotic activity of human umbilical-cord tissue-derived mesenchymal stromal cells during fibrogenesis or resolution in murine models of liver fibrosis

THU-079-Study of anti-fibrotic activity of human umbilical-cord tissue-derived mesenchymal stromal cells during fibrogenesis or resolution in murine models of liver fibrosis

γδT Cells Suppress Liver Fibrosis via Strong Cytolysis and Enhanced NK Cell-Mediated Cytotoxicity Against Hepatic Stellate Cells.

Liver fibrosis is the excessive accumulation of extracellular matrix proteins, resulting from maladaptive wound healing responses to chronic liver injury. γδT cells are important in chronic liver injury pathogenesis and subsequent liver fibrosis; however, their role and underlying mechanisms are not fully understood. The present study aims to assess whether γδT cells contribute to liver fibrosis regression. Using a carbon tetrachloride (CCl4)-induced murine model of liver fibrosis in wild-type (WT) and γδT cell deficient (TCRδ−/−) mice, we demonstrated that γδT cells protected against liver fibrosis and exhibited strong cytotoxicity against activated hepatic stellate cells (HSCs). Further study show that chronic liver inflammation promoted hepatic γδT cells to express NKp46, which contribute to the direct killing of activated HSCs by γδT cells. Moreover, we identified that an IFNγ-producing γδT cell subset (γδT1) cells exhibited stronger cytotoxicity against activated HSCs than the IL-17-producing subset (γδT17) cells upon chronic liver injury. In addition, γδT cells promoted the anti-fibrotic ability of conventional natural killer (cNK) cells and liver-resident NK (lrNK) cells by enhancing their cytotoxicity against activated HSCs. The cell crosstalk between γδT and NK cells was shown to depend partly on co-stimulatory receptor 4-1BB (CD137) engagement. In conclusion, our data confirmed the protective effects of γδT cells, especially the γδT1 subset, by directly killing activated HSCs and increasing NK cell-mediated cytotoxicity against activated HSCs in CCl4-induced liver fibrosis, which suggest valuable therapeutic targets to treat liver fibrosis.

Antifibrotic Effect of Marine Ovothiol in an In Vivo Model of Liver Fibrosis.

Liver fibrosis is a complex process caused by chronic hepatic injury, which leads to an excessive increase in extracellular matrix protein accumulation and fibrogenesis. Several natural products, including sulfur-containing compounds, have been investigated for their antifibrotic effects; however, the molecular mechanisms underpinning their action are partially still obscure. In this study, we have investigated for the first time the effect of ovothiol A, π-methyl-5-thiohistidine, isolated from sea urchin eggs on an in vivo murine model of liver fibrosis. Mice were intraperitoneally injected with carbon tetrachloride (CCl4) to induce liver fibrosis and treated with ovothiol A at the dose of 50 mg/kg 3 times a week for 2 months. Treatment with ovothiol A caused a significant reduction of collagen fibers as observed by histopathological changes and serum parameters compared to mice treated with control solution. This antifibrotic effect was associated to the decrease of fibrogenic markers involved in liver fibrosis progression, such as the transforming growth factor (TGF-β), the α-smooth muscle actin (α-SMA), and the tissue metalloproteinases inhibitor (TIMP-1). Finally, we provided evidence that the attenuation of liver fibrosis by ovothiol A treatment can be regulated by the expression and activity of the membrane-bound γ-glutamyl-transpeptidase (GGT), which is a key player in maintaining intracellular redox homoeostasis. Overall, these findings indicate that ovothiol A has significant antifibrotic properties and can be considered as a new marine drug or dietary supplement in potential therapeutic strategies for the treatment of liver fibrosis.

The therapeutic effects of interleukin 10 gene-modified bone marrow-derived dendritic cells in a murine model of liver fibrosis

Objective: To investigate the therapeutic effects and mechanisms of interleukin-10 (IL-10) gene-modified dendritic cells (DC-IL-10) in mice with liver fibrosis. Methods: DC-IL-10 was constructed in vitro, the phenotype and function of which were evaluated by flow cytometry. BALB/c mice were treated with intraperitoneal injection of carbon tetrachloride (CCl4) to establish liver fibrotic model. DC-IL-10 was administrated via tail vein. Animals were divided into 4 groups including normal dendritic cell(DC) control, liver fibrosis only, negative lentiviral transfection DC (DC-mock) and DC-IL-10. Liver function, cytokine secretion, T lymphocyte differentiation and liver histomorphology were tested. Real-time PCR and western blot were used to analyze the effect of DC-IL-10 on Wnt/β-catenin signaling pathway and its role in liver fibrosis. Results: When compared with DC control and DC-mock, the expression of DC-IL-10 surface stimulating molecules (major histocompatibity complex-Ⅱ, CD(80), CD(86)) were significantly decreased (F=14.708, 22.503, 12.595, respectively, all P<0.05), and DC-IL-10 significantly inhibited T lymphocyte proliferation (F=50.295, P<0.05). When compared with liver fibrosis group, serum alanine aminotransferase and aspartate transaminase were decreased in DC-IL-10 treated group (all P<0.05), other parameters including inflammatory factors (tumor necrosis factor α, IL-6, IL-1β) reduced (all P <0.05), the proportion of regulatory T cells (Treg) increased (F=6.742, P<0.05), pathological damage improved, the expression of Wnt3a, α-SMA and β-catenin mRNA and protein significantly reduced in DC-IL-10 treatment group (all P<0.001) . Conclusions: DC-IL-10 induces elevation of Treg for immune tolerance, as well as inhibition of inflammatory response, block of Wnt/β-catenin signaling pathway, which translates into improvement of liver fibrosis.

Activation of autophagy is required for Oroxylin A to alleviate carbon tetrachloride-induced liver fibrosis and hepatic stellate cell activation

Liver fibrosis is a reversible pathophysiological process correlated with intense repair and cicatrization mechanisms, and its end-stage cirrhosis is responsible for high morbidity and mortality worldwide. Interestingly, the use of natural products as a realistic option for the treatment of liver fibrosis has broadly been accepted. Oroxylin A, a safe and natural product, shows a wide range of pharmacological activities such as anti-inflammatory, anti-oxidant, and anti-tumor properties. However, the effects of Oroxylin A on liver fibrosis remain poorly understood. In the present study, we sought to determine the effect of Oroxylin A on carbon tetrachloride (CCl4)-induced liver fibrosis, and to further examine the molecular mechanisms. We found that treatment with Oroxylin A markedly decreased the level of liver injury markers, alkaline phosphatase (ALP), aspartate aminotransferase (AST), and alanine aminotransferase (ALT), in a dose dependent manner. Moreover, Oroxylin A treatment remarkably inhibited extracellular matrix (ECM) deposition, and significantly down-regulated the mRNA and protein expression of liver fibrosis markers including α1(I)collagen, fibronectin, alpha-smooth muscle actin (α-SMA), PDGF-βR, and TGF-βR1 in CCl4-induced murine model of liver fibrosis. Furthermore, experimental results in vitro showed that Oroxylin A treatment reduced the mRNA and protein expression of HSC activation markers, α-SMA, desmin, α1 (I) collagen, fibronectin, TGF-β, and TNF-α, in a dose dependent manner. Attractively, Oroxylin A treatment also markedly up-regulated the expression of autophagy makers, LC3-B, Atg3, Atg4, Atg5, Beclin1/Atg6, Atg7, Atg9, ATG12, and Atg14, and apparently reduced the expression of autophagy substrate p62 in both CCl4-induced murine model of liver fibrosis and PDGF-BB-treated HSCs. Importantly, inhibition of autophagy by specific inhibitor 3-methyladenine (3-MA) completely abolished Oroxylin A-induced anti-fibrosis effect, indicating that activation of autophagy was required for Oroxylin A to alleviate liver fibrosis. Overall, these results provide novel implications to reveal the molecular mechanism of Oroxylin A-induced anti-fibrosis properties, by which points to the possibility of using Oroxylin A for the treatment of liver fibrosis.