CCl4-induced Liver Fibrosis In Mice Research Articles

SB431542-Loaded Liposomes Alleviate Liver Fibrosis by Suppressing TGF-β Signaling.

Liver fibrosis is a common outcome of most chronic liver diseases, but there is no clinically approved drug for its treatment. Previous studies have reported the potential of SB431542 as an inhibitor of TGF-β signaling in the treatment of liver fibrosis, but it shows poor water solubility and low bioavailability. Here, we improve these characteristics of SB431542 by loading it into liposomes (SB-Lips) with two FDA-approved excipients: soya phosphatidyl S100 and Solutol HS15. In vitro, SB-Lips had stronger inhibitory effects on the proliferation and activation of hepatic stellate cells LX-2 than free SB. After an intravenous injection in a CCl4-induced liver fibrosis mouse model, SB-Lips accumulated preferentially in the liver, its area under the concentration-time curve was significantly higher than that of free SB431542, and it alleviated hepatic fibrosis significantly more than free drug, which was associated with greater inhibition of TGF-β signaling. Furthermore, SB-Lips did not cause significant injury to other organs. These results suggest that our liposomal system is safe and effective for delivering SB431542 to fibrotic liver.

Protective Effect of Spore Powder of Antrodia camphorata ATCC 200183 on CCl4-Induced Liver Fibrosis in Mice.

Liver fibrosis is a pathological process with intrahepatic diffused deposition of the excess extracellular matrix, which leads to various chronic liver diseases. Drugs with high efficacy and low toxicity for liver fibrosis are still unavailable. Antrodia camphorata has antioxidant, antivirus, antitumor and anti-inflammation roles, and has been used to treat liver diseases in the population. However, the hepatoprotective effects of A. camphorata spores and the mechanisms behind it have not been investigated. In this study, we evaluate the hepatoprotective effect of spore powder of A. camphorata (SP, 100 mg/kg/day or 200 mg/kg/day) on carbon tetrachloride (CCl4)-induced liver fibrosis in mice. SP groups reduced serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities compared with the CCl4 group. SP also showed a decrease in hydroxyproline (Hyp) content in liver tissues. SP improved cell damage and reduced collagen deposition by H&E, Sirius red and Masson staining. Furthermore, SP down-regulated the mRNA levels of α-SMA and Col 1, and the protein expression of α-smooth muscle actin (α-SMA), collagen I (Col 1), tumor necrosis factor alpha (TNF-α), toll like receptor 4 (TLR4) and nuclear factor-Κb (NF-κB) p65. In summary, SP has an ameliorative effect on hepatic fibrosis, probably by inhibiting the activation of hepatic stellate cells, reducing the synthesis of extracellular matrix.

Baihe Wuyao decoction ameliorates CCl4-induced chronic liver injury and liver fibrosis in mice through blocking TGF-β1/Smad2/3 signaling, anti-inflammation and anti-oxidation effects

Ethnopharmacological relevanceBaihe Wuyao decoction (BWD), a prescription of Traditional Chinese Medicines, composed of Lilium brownii var. viridulum Baker.(Lilii Bulbus) and Lindera aggregata (Sims) Kosterm. (Linderae Radix), has been used to treat epigastric pain and superficial gastritis for hundreds of years in China. Recently, some compounds obtained from Lilii Bulbus and Linderae Radix had active effects of hepatic protection or liver fibrosis alleviation. Thus, we aim to evaluate the effects of BWD on treatment of chronic liver injury and liver fibrosis induced by carbon tetrachloride (CCl4) and to elucidate the possible molecular mechanism. Materials and methodsMice were treated with BWD (low, medium and high dose), diammonium glycyrrhizinate or vehicle by oral gavage once daily, simultaneously intraperitoneal injected with a single dose of CCl4 (1 μl/g body weight) twice a week for consecutive 6 weeks. Next, all mice were sacrificed after fasted 12 h, and serums and liver tissues were harvested for analysis. The hepatic injury was detected by serum biomarker assay, including aspartate aminotransferase (AST) and alanine aminotransferase (ALT). The hepatic histology and collagen were illustrated by hematoxylin-eosin staining and Sirius red staining respectively. The antioxidant capacity of liver tissues was evaluated by the contents of superoxide dismutase (SOD) and malondialdehyde (MDA) in liver homogenization. The mRNA gene or protein expressions related to fibrosis, oxidative stress and inflammation molecules were performed by real-time quantitative PCR (RT-PCR) or Western-blot. ResultsBWD exhibited a good hepatic protection with ameliorating liver histological changes, decreasing serum AST and ALT contents, and reducing hepatic fibrosis with stimulation ECMs (such as Collagen1 and Collagen3) degradation. BWD inhibited hepatic stellate cells (HSCs) activation, promoted matrix metalloproteinase-2 (MMP2), MMP9, and MMP12 while suppressing tissue inhibitors of matrix metalloproteinase-1 (TIMP1) expression, and blocked traditional fibrosis TGF-β1/Smad2/3 signal pathway. Moreover, BWD exhibited anti-inflammation effect proved by the reduction of liver Interleukin-1β (IL-1β), TNF-α, IL-11 mRNA levels and promoted anti-oxidation effects determined by inhibition of liver MDA and iNOS levels while promoting liver SOD and Mn-SOD. ConclusionBWD ameliorates CCl4-induced CLI and liver fibrosis which is correlated to its blocking TGF-β1/Smad2/3 signaling, anti-inflammation, and anti-oxidation effects. BWD, as a small traditional prescription, is a promising treatment for CLI and liver fibrosis through multiple pharmacological targets.

Ph-responsive polymersomes for matrix metalloproteinase-1 delivery as a promising therapeutic strategy for the treatment of liver fibrosis

Background & Aims: Liver fibrosis is a growing health problem affecting millions of people worldwide. Chronic liver injury leads to the formation of scar tissue due to the excessive accumulation of extracellular matrix, mainly collagen-I and -III produced by activated hepatic stellate cells (HSCs). Currently, there are no therapies available for liver fibrosis. Matrix metalloproteinase-1 (MMP-1) is an enzyme that degrades the scar tissue by degrading collagen-I and -III favoring fibrolysis. We hypothesized liver-specific delivery of MMP-1 to degrade collagen as a promising approach for the treatment of liver fibrosis. Using state-of-the-art technologies, we synthesized innovative pH-responsive smart MMPsomes with the MMP1 decorated on the polymersome surface. Finally, we investigated the therapeutic efficacy of MMP1 and MMPsomes on human HSCs in vitro and on liver fibrosis mouse models in vivo, and ex vivo on fibrotic mouse livers. Methods: Polymersomes (Psomes) were fabricated using the pH switch method and MMP-1 post-loading at pH 5-6 was performed to increase the interaction between MMP-1 and the Psome membrane. Physiochemical analysis and enzymatic assays were performed to characterize the attachment and functionalization of enzyme on the Psomes. In vitro studies were performed on TGF-β activated human HSCs to evaluate the effects of MMP1 and MMPsomes on the cell viability, functionality and the gene and protein expression of collagen and HSCs activation marker α-SMA. Finally, the efficacy of MMP-1 and MMPsomes was tested in vivo and ex vivo on CCl4-induced liver fibrosis mouse models Results: Decoration of MMP-1 on the surface of the polymersome was successfully established with an affinity of 30%, without inhibition of function. Synthesized MMPsomes showed favorable size (~180nm at pH 6 and ~145 nm at pH 8) and charge (positive at pH 6 and negative at pH 8). MMP-1 and more significantly MMPsomes showed dose-dependent inhibition of collagen-I, -III and α-SMA gene expression, and collagen-I protein expression in TGF-β activated human HSCs with no significant effects on cell viability. The studies on CCl4-induced liver fibrosis mouse models have been performed, showed promising preliminary results with no in vivo toxicity however still under investigation. Conclusions: In conclusion, we present an innovative approach of MMP-1 delivery for the treatment of liver fibrosis.

Hsa_circ_0004018 suppresses the progression of liver fibrosis through regulating the hsa-miR-660-3p/TEP1 axis.

Efforts have been made in the prevention and treatment of liver fibrosis. The inhibition or depletion of the hepatic stellate cells (HSCs) has been considered as a potential approach. Recently, there are numbers of studies about the role of the circular RNA in the disease progression. However, the role of circular RNA in the regulation of HSCs and the progression of liver fibrosis remained elusive. In this study, we constructed a CCl4-induced liver fibrosis mouse model and overexpressed hsa_circ_0004018 in HSCs. Then, salvianolic acid B was used to treat HSCs in vitro. We found that hsa_circ_0004018 is downregulated in liver fibrogenesis. Luciferase reporter assay was performed to verify the interaction of hsa_circ_0004018, hsa-miR-660-3p and TEP1. It showed that hsa_circ_0004018 may act as a sponge of hsa-miR-660-3p, which can target and downregulate the expression of TEP1. hsa_circ_0004018 expressing lentivirus was used to investigate the in-vivo function of hsa_circ_0004018 in CCl4-induced liver fibrosis mice. We also reveal that the hsa_circ_0004018/hsa-miR-660-3p/TEP1 axis contributes to the proliferation and activation of HSCs. In addition, the overexpression of hsa_circ_0004018 alleviated the progression of liver fibrosis. In conclusion, our study highlights hsa_circ_0004018 as a potential biomarker and therapeutic target for liver fibrosis.

Exosomes derived from natural killer cells inhibit hepatic stellate cell activation and liver fibrosis.

Activation of hepatic stellate cells (HSCs) is a prominent driver of liver fibrosis. This study was designed to investigate the effect of exosomes derived from natural killer (NK) cells on HSC activation and liver fibrosis. The exosomes were isolated from NK-92MI cells (NK-Exo) and identified by transmission electron microscopy, nanoparticle tracking analysis, and western blot. Then NK-Exo was administered into TGF-β1-treated LX-2 cells (human HSC line) and mice with CCl4-induced liver fibrosis. LX-2 cell proliferation was determined by CCK-8 assay. The levels of α-SMA and CoL1A1 were measured by qRT-PCR and western blot to evaluate HSC activation. Serum levels of AST and ALT were measured. Hematoxylin-eosin, Masson staining, and Sirius Red staining were performed to assess the pathological changes and collagen deposition. Cell supernatant derived from NK-92MI cells inhibited TGF-β1-induced HSC proliferation and activation in LX-2 cells, and this effect was counteracted by the exosome inhibitor GW4869. Further assays confirmed that NK-Exo treatment significantly inhibited TGF-β1-induced HSC proliferation and activation. Moreover, NK-Exo administration alleviated CCl4-induced liver fibrosis in mice. NK-Exo inhibited TGF-β1-induced HSC activation and CCl4-induced liver fibrosis.

The in vitro effects of phospholipase D1-mTOR axis in liver fibrogenesis

AimsThe activation of hepatic stellate cells (HSCs) plays a central role in liver fibrosis progression. Phospholipase D (PLD) enzymes participate in multiple cellular activities. However, whether and how PLD regulates HSCs activation remain elusive. Main methodsThe expression of intrahepatic PLD1 and PLD2 was determined in CCl4-induced mouse liver fibrosis models by western blot and immunohistochemistry. Cell model of liver fibrogenesis was constructed using rat HSCs line (HSC-T6) treated with recombinant transforming growth factor β1 (TGFβ1). Fibrogenesis was evaluated on the aspects of proliferation, expression of pro-fibrogenic markers and migration. The effects mediated by PLD1-mTOR axis on TGFβ1-induced fibrogenesis were evaluated using HSC-T6 treated with small-molecular PLD1 inhibitors, PLD1-SiRNA, rapamycin (mTOR inhibitor) and MHY1485 (mTOR activator). Key findingsSignificant increase of PLD1, not PLD2 was documented in CCl4-induced cirrhotic compared to normal liver tissues. Suppression of PLD1 activities by PLD inhibitors or down-regulation of PLD1 expression in HSC-T6 could significantly restrain TGFβ1-induced fibrogenesis, as reflected by decreased cell proliferation and reduced expression of pro-fibrogenic markers. Besides, either PLD1 inhibitor or PLD1-SiRNA significantly inhibited mTOR activity of HSC-T6. Moreover, PLD1 inhibitors not only exhibited similar effects with rapamycin in TGFβ1-induced fibrogenesis, but also blunted MHY1485 enhanced cell proliferation of HSC-T6. SignificanceThe PLD1-mTOR axis of HSCs could be therapeutically targeted in advanced liver fibrosis.

A model study for the manufacture and validation of clinical-grade deciduous dental pulp stem cells for chronic liver fibrosis treatment

BackgroundHuman deciduous pulp stem cells (hDPSCs) have remarkable stem cell potency associated with cell proliferation, mesenchymal multipotency, and immunosuppressive function and have shown beneficial effects in a variety of animal disease models. Recent studies demonstrated that hDPSCs exhibited in vivo anti-fibrotic and anti-inflammatory action and in vivo hepatogenic-associated liver regeneration, suggesting that hDPSCs may offer a promising source with great clinical demand for treating liver diseases. However, how to manufacture ex vivo large-scale clinical-grade hDPSCs with the appropriate quality, safety, and preclinical efficacy assurances remains unclear.MethodsWe isolated hDPSCs from human deciduous dental pulp tissues formed by the colony-forming unit-fibroblast (CFU-F) method and expanded them under a xenogeneic-free and serum-free (XF/SF) condition; hDPSC products were subsequently stored by two-step banking including a master cell bank (MCB) and a working cell bank (WCB). The final products were directly thawed hDPSCs from the WCB. We tested the safety and quality check, stem cell properties, and preclinical potentials of final hDPSC products and hDPSC products in the MCB and WCB.ResultsWe optimized manufacturing procedures to isolate and expand hDPSC products under a XF/SF culture condition and established the MCB and the WCB. The final hDPSC products and hDPSC products in the MCB and WCB were validated the safety and quality including population doubling ability, chromosome stability, microorganism safety, and stem cell properties including morphology, cell surface marker expression, and multipotency. We also evaluated the in vivo immunogenicity and tumorigenicity and validated in vivo therapeutic efficacy for liver regeneration in a CCl4-induced chronic liver fibrosis mouse model in the final hDPSC products and hDPSC products in the WCB.ConclusionThe manufacture and quality control results indicated that the present procedure could produce sufficient numbers of clinical-grade hDPSC products from a tiny deciduous dental pulp tissue to enhance clinical application of hDPSC products in chronic liver fibrosis.

Total phenolic extract of Euscaphis konishii hayata Pericarp attenuates carbon tetrachloride (CCl4)-induced liver fibrosis in mice

Liver fibrosis is a crucial pathological process involved in the hepatogenic morbidity and mortality. The pericarp of Euscaphis konishii Hayata is usually used in the cooking soup to improve liver function in Southern China, and high level of phenolic compounds has been found in the E. konishii pericarp. The total phenolic compounds extracted from E. konishii pericarp (TPEP) was obtained by polyamide column chromatograph, and 9 phenolic compounds of TPEP were identified through LC/MS and NMR. TPEP exhibited strong free radicals scavenging activity in vitro, and the chronic CCl4-induced liver fibrosis mice were established to elucidate the hepatoprotective mechanism of TPEP in vivo. TPEP treatment (50, 100 and 200 mg/kg) ameliorated the oxidative stress, immune dysfunction, inflammatory response and hepatic fibrosis induced by CCl4 introduction, alleviated the histopathological alteration and hepatocyte apoptosis in the liver tissue. Pretreatment with TPEP suppressed the activation of NADPH oxidase (NOX) signaling to attenuate oxidative stress in the liver tissue. TPEP administration inhibited the translocation of NF-κB into the nucleus to prevent the expression of downstream proinflammatory cytokines. TPEP treatment downregulated the activation of TGF-β/Smad pathway, and facilitated the degradation of extracellular matrix through enhancing matrix metalloproteinases (MMPs) activity and decreasing the expression of matrix metalloproteinase inhibitors (TIMPs). In conclusion, TPEP inhibited CCl4-induced hepatic fibrosis through its antioxidant, anti-inflammatory and anti-fibrotic activities.

Identification of Lineage-Specific Transcription Factors That Prevent Activation of Hepatic Stellate Cells and Promote Fibrosis Resolution

Development of liver fibrosis is associated with activation of quiescent hepatic stellate cells (HSCs) into collagen type I-producing myofibroblasts (activated HSCs). Cessation of liver injury often results in fibrosis resolution and inactivation of activated HSCs/myofibroblasts into a quiescent-like state (inactivated HSCs). We aimed to identify molecular features of phenotypes of HSCs from mice and humans. We performed studies with LratCre, Ets1-floxed, Nf1-floxed, Pparγ-floxed, Gata6-floxed, Rag2-/-γc-/-, and C57/Bl6 (control) mice. Some mice were given carbon tetrachloride (CCl4) to induce liver fibrosis, with or without a peroxisome proliferator-activated receptor-γ (PPARγ) agonist. Livers from mice were analyzed by immunohistochemistry. Quiescent, activated, and inactivated HSCs were isolated from livers of Col1α1YFP mice and analyzed by chromatin immunoprecipitation and sequencing. Human HSCs were isolated from livers denied for transplantation. We compared changes in gene expression patterns and epigenetic modifications (histone H3 lysine 4 dimethylation and histone H3 lysine 27 acetylation) in primary mouse and human HSCs. Transcription factors were knocked down with small hairpin RNAs in mouse HSCs. Motif enrichment identified E26 transcription-specific transcription factors (ETS) 1, ETS2, GATA4, GATA6, interferon regulatory factor (IRF) 1, and IRF2 transcription factors as regulators of the mouse and human HSC lineage. Small hairpin RNA-knockdown of these transcription factors resulted in increased expression of genes that promote fibrogenesis and inflammation, and loss of HSC phenotype. Disruption of Gata6 or Ets1, or Nf1 or Pparγ (which are regulated by ETS1), increased the severity of CCl4-induced liver fibrosis in mice compared to control mice. Only mice with disruption of Gata6 or Pparγ had defects in fibrosis resolution after CCl4 administration was stopped, associated with persistent activation of HSCs. Administration of a PPARγ agonist accelerated regression of liver fibrosis after CCl4 administration in control mice but not in mice with disruption of Pparγ. Phenotypes of HSCs from humans and mice are regulated by transcription factors, including ETS1, ETS2, GATA4, GATA6, IRF1, and IRF2. Activated mouse and human HSCs can revert to a quiescent-like, inactivated phenotype. We found GATA6 and PPARγ to be required for inactivation of human HSCs and regression of liver fibrosis in mice.

Preventive effect of ethanol extract from Chinese sumac fruits against tetrachloromethane-induced liver fibrosis in mice

Chinese sumac (Rhus chinensis Mill.) fruits are traditionally used as a condiment and herb. The aim of this study is to evaluate the preventive effect of the ethanolic extract from Chinese sumac fruits against CCl4-induced liver fibrosis in mice and to delineate the underlying mechanisms. The results showed that the ethanolic extract substantially decreased AST, ALT, MDA, and hydroxyprolin (HYP), restored GSH, SOD, and CAT, and prevented liver fibrosis induced by CCl4. Further, investigation revealed that the prevention against liver fibrosis may be related to heightened inflammation due to suppression of the release of IL-1β, IL-6, and TNF-α, and the downregulation of COX-2, iNOS, p-NF-κB, and p-P38; the prevention of hepatocyte apoptosis was due to the upregulation of Bcl-2 and downregulation of Bax; the reduction of HSCs activation was due to the downregulation of TGF-β1 and upregulation of PPAR-γ; and the decrease in the extracellular matrix (ECM) deposition was due to the regulation of MMP9 and TIMP2. These findings proved that Chinese sumac fruits could effectively prevent CCl4-induced liver fibrosis in mice and may be used to develop functional foods and/or nutraceuticals to prevent or alleviate liver fibrosis.

Fuzheng Huayu Recipe Prevented and Treated CCl4-Induced Mice Liver Fibrosis through Regulating Polarization and Chemotaxis of Intrahepatic Macrophages via CCL2 and CX3CL1.

Background Fuzheng Huayu recipe (FZHY) is an original Chinese patent medicine which was developed and marketed by our institute. It could markedly improve liver tissue inflammation and ameliorate hepatic fibrosis in the clinical study. The intrahepatic macrophages recruitment and polarization play an important role in the progress of liver inflammation and fibrosis. Whether FZHY exerted its antiliver fibrosis effects through regulating intrahepatic macrophages phenotypic ratios is still unknown. This study aims to explore the antifibrosis mechanism of FZHY on regulating the recruitment and polarization of intrahepatic macrophages. Methods C57/B6 mice were used for the establishment of the CCl4-induced mice liver fibrosis model. Liver inflammation and fibrosis were evaluated by HE and Sirius red staining, hydroxyproline assays, and biochemical tests. The levels of chemokines and inflammatory cytokines in liver tissue were measured by RNA-Seq transcriptome analysis, western blot assay, RT-qPCR, and immunofluorescence assay. The macrophages recruitment and phenotypic polarization were observed by flow cytometry. Results FZHY significantly improved liver inflammation and reduced liver fibrosis degree. TNF signaling pathway, involved in macrophages recruitment and phenotypic polarization, was discovered by RNA-Seq transcriptome analysis. In TNF signaling pathway, CCL2 expression was significantly decreased and CX3CL1 expression was significantly upregulated by FZHY in liver tissue and primary intrahepatic macrophages. The ratio of proinflammatory hepatic resident macrophage-Kupffer cells (F4/80+CD11b−CD86+) was downregulated by FZHY, while the proportion of anti-inflammatory Kupffer cells (F4/80+CD11b−CD206+) was upregulated. Meanwhile, the ratio of proinflammatory Ly6Chigh macrophages (F4/80+CD11b+Ly6Chigh) which were recruited from blood circulation by CCL2 was reduced by FZHY, while the ratio of restorative Ly6Clow macrophages (F4/80+CD11b+Ly6Clow) which were recruited from blood circulation or induced from Ly6Chigh macrophages polarization by CX3CL1 was significantly increased. Conclusions FZHY could regulate the recruitment and polarization of intrahepatic macrophages via CCL2 and CX3CL1, so as to play its anti-inflammation and antifibrosis pharmacological effects in the liver.

4-methylumbelliferone Prevents Liver Fibrosis by Affecting Hyaluronan Deposition, FSTL1 Expression and Cell Localization.

4-methylumbelliferone (4MU) is an inhibitor of hyaluronan deposition and an active substance of hymecromone, a choleretic and antispasmodic drug. 4MU reported to be anti-fibrotic in mouse models; however, precise mechanism of action still requires further investigation. Here we describe the cellular and molecular mechanisms of 4MU action on CCl4-induced liver fibrosis in mice using NGS transcriptome, Q-PCR and immunohistochemical analysis. Collagen and hyaluronan deposition were prevented by 4MU. The CCl4 stimulated expression of Col1a and αSMA were reduced, while the expression of the ECM catabolic gene Hyal1 was increased in the presence of 4MU. Bioinformatic analysis identified an activation of TGF-beta and Wnt/beta-catenin signaling pathways, and inhibition of the genes associated with lipid metabolism by CCL4 treatment, while 4MU restored key markers of these pathways to the control level. Immunohistochemical analysis reveals the suppression of hepatic stellate cells (HSCs) transdifferentiation to myofibroblasts by 4MU treatment. The drug affected the localization of HSCs and macrophages in the sites of fibrogenesis. CCl4 treatment induced the expression of FSTL1, which was downregulated by 4MU. Our results support the hypothesis that 4MU alleviates CCl4-induced liver fibrosis by reducing hyaluronan deposition and downregulating FSTL1 expression, accompanied by the suppression of HSC trans-differentiation and altered macrophage localization.

PRC1 promotes GLI1-dependent osteopontin expression in association with the Wnt/\u03b2-catenin signaling pathway and aggravates liver fibrosis

BackgroundPRC1 (Protein regulator of cytokinesis 1) regulates microtubules organization and functions as a novel regulator in Wnt/β-catenin signaling pathway. Wnt/β-catenin is involved in development of liver fibrosis (LF). We aim to investigate effect and mechanism of PRC1 on liver fibrosis.MethodsCarbon tetrachloride (CCl4)-induced mice LF model was established and in vitro cell model for LF was induced by mice primary hepatic stellate cell (HSC) under glucose treatment. The expression of PRC1 in mice and cell LF models was examined by qRT-PCR (quantitative real-time polymerase chain reaction), western blot and immunohistochemistry. MTT assay was used to detect cell viability, and western blot to determine the underlying mechanism. The effect of PRC1 on liver pathology was examined via measurement of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and hydroxyproline, as well as histopathological analysis.ResultsPRC1 was up-regulated in CCl4-induced mice LF model and activated HSC. Knockdown of PRC1 inhibited cell viability and promoted cell apoptosis of activated HSC. PRC1 expression was regulated by Wnt3a signaling, and PRC1 could regulate downstream β-catenin activation. Moreover, PRC1 could activate glioma-associated oncogene homolog 1 (GLI1)-dependent osteopontin expression to participate in LF. Adenovirus-mediated knockdown of PRC1 in liver attenuated LF and reduced collagen deposition.ConclusionsPRC1 aggravated LF through regulating Wnt/β-catenin mediated GLI1-dependent osteopontin expression, providing a new potential therapeutic target for LF treatment.

TAF and TDF attenuate liver fibrosis through NS5ATP9, TGFβ1/Smad3, and NF-κB/NLRP3 inflammasome signaling pathways.

This study aimed to investigate the roles and mechanisms of tenofovir alafenamide fumarate (TAF)/tenofovir disoproxil fumarate (TDF) in treating liver fibrosis. The effects of TAF/TDF on carbon tetrachloride (CCl4)-induced liver fibrosis in C57BL/6 wild-type or nonstructural protein 5A transactivated protein 9 (NS5ATP9) knockout mice were studied. The differentiation, activation, and proliferation of LX-2 cells after TAF/TDF treatment were tested in vitro. The expression of NS5ATP9 and activities of transforming growth factor-β1 (TGFβ1)/Sekelsky mothers against decapentaplegic homolog 3 (Smad3) and NF-κB/NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasome signaling pathways were detected in TAF/TDF-treated mice and LX-2 cells. The genes related to extracellular matrix accumulation were detected in vivo and in vitro after NS5ATP9 silencing or knockout. TAF/TDF significantly inhibited CCl4-induced liver fibrosis in mice, and regulated the differentiation, activation, and proliferation of hepatic stellate cells (HSCs). Furthermore, TAF/TDF suppressed the activities of TGFβ1/Smad3 and NF-κB/NLRP3 inflammasome signaling pathways in vivo and in vitro. NS5ATP9 inhibited liver fibrosis through TGFβ1/Smad3 and NF-κB signaling pathways. TAF/TDF upregulated the expression of NS5ATP9 in vivo and in vitro. Finally, TAF/TDF could only show marginal therapeutic effects when NS5ATP9 was silenced and knocked out in vivo and in vitro. TAF/TDF prevented progression and promoted reversion of liver fibrosis through assembling TGFβ1/Smad3 and NF-κB/NLRP3 inflammasome signaling pathways via upregulating the expression of NS5ATP9. TAF/TDF also regulated the differentiation, activation, and proliferation of HSCs. The findings provided strong evidence for the role of TAF/TDF as a new promising therapeutic strategy in liver fibrosis.

Serine Protease HtrA2/Omi Deficiency Impairs Mitochondrial Homeostasis and Promotes Hepatic Fibrogenesis via Activation of Hepatic Stellate Cells.

The loss of mitochondrial function impairs intracellular energy production and potentially results in chronic liver disease. Increasing evidence suggests that mitochondrial dysfunction in hepatocytes contributes to the activation of hepatic stellate cells (HSCs), thereby resulting in hepatic fibrogenesis. High-temperature requirement protein A2 (HtrA2/Omi), a mitochondrial serine protease with various functions, is responsible for quality control in mitochondrial homeostasis. However, little information is available regarding its role in mitochondrial damage during the development of liver fibrosis. This study examined whether HtrA2/Omi regulates mitochondrial homeostasis in hepatocyte during the development of hepatic fibrogenesis. In this study, we demonstrated that HtrA2/Omi expression considerably decreased in liver tissues from the CCl4-induced liver fibrotic mice model and from patients with liver cirrhosis. Knockdown of HtrA2/Omi in hepatocytes induced the accumulation of damaged mitochondria and provoked mitochondrial reactive oxygen species (mtROS) stress. We further show that the damaged mtDNA isolated from HtrA2/Omi-deficient hepatocytes as a form of damage-associated molecular patterns can induce HSCs activation. Moreover, we found that motor neuron degeneration 2-mutant mice harboring the missense mutation Ser276Cys in the protease domain of HtrA2/Omi displayed altered mitochondrial morphology and function, which increased oxidative stress and promoted liver fibrosis. Conversely, the overexpression of HtrA2/Omi via hydrodynamics-based gene transfer led to the antifibrotic effects in CCl4-induced liver fibrosis mice model through decreasing collagen accumulation and enhancing anti-oxidative activity by modulating mitochondrial homeostasis in the liver. These results suggest that suppressing HtrA2/Omi expression promotes hepatic fibrogenesis via modulating mtROS generation, and these novel mechanistic insights involving the regulation of mitochondrial homeostasis by HtrA2/Omi may be of importance for developing new therapeutic strategies for hepatic fibrosis.

Ablation of serum response factor in hepatic stellate cells attenuates liver fibrosis.

Trans-differentiation, or activation, of hepatic stellate cells (HSCs) is a hallmark event in liver fibrosis although the underlying mechanism is not fully appreciated. Serum response factor (SRF) is a pleiotropic sequence-specific transcription factor with a ubiquitous expression pattern. In the present study, we investigated the effect of HSC-specific ablation of SRF on liver fibrosis in vivo and the underlying mechanism. We report that SRF bound to the promoter regions of pro-fibrogenic genes, including collagen type I (Col1a1/Col1a2) and alpha smooth muscle actin (Acta2), with greater affinity in activated HSCs compared to quiescent HSCs. Ablation of SRF in HSCs in vitro downregulated the expression of fibrogenic genes by dampening the accumulation of active histone marks. SRF also interacted with MRTF-A, a well-documented co-factor involved in liver fibrosis, on the pro-fibrogenic gene promoters during HSC activation. In addition, SRF directly regulated MRTF-A transcription in activated HSCs. More importantly, HSC conditional SRF knockout (CKO) mice developed a less robust pro-fibrogenic response in the liver in response to CCl4 injection and BDL compared to wild-type littermates. In conclusion, our data demonstrate that SRF may play an essential role in HSC activation and liver fibrosis. KEY MESSAGES: • SRF deficiency decelerates activation of hepatic stellate cells (HSCs) in vitro. • SRF epigenetically activates pro-fibrogenic transcription to promote HSC maturation. • SRF interacts with MRTF-A and contributes to MRTF-A transcription. • Conditional SRF deletion in HSCs attenuates BDL-induced liver fibrosis in mice. • Conditional SRF ablation in HSCs attenuates CCl4-induced liver fibrosis in mice.

Procyanidin B2 inhibits the activation of hepatic stellate cells and angiogenesis via the Hedgehog pathway during liver fibrosis.

BackgroundLiver fibrosis is a wound‐healing process of liver featured by the over‐deposition of extracellular matrix (ECM) and angiogenesis. However, the effective treatment is lacking. Procyanidin B2 (PB2) is a flavonoid extract abundant in grape seeds with anti‐oxidant, anti‐inflammatory and anti‐cancer properties. The present study aimed to determine effects of PB2 on liver fibrosis.MethodThe CCl4‐induced mouse liver fibrosis model and a human hepatic stellate cell (HSC) line (LX2 cells) were used to study the activation, ECM production and angiogenesis of HSCs through Western blotting analysis, immunohistochemistry, immunofluorescence staining, flow cytometry and tubulogenesis assay. A Hedgehog (Hh) pathway inhibitor (cyclopamine) and Smoothened agonist (SAG) were used to investigate the role of PB2 on Hh pathway.ResultsThe results showed that PB2 could inhibit the proliferation and induce apoptosis of HSCs. PB2 could also down‐regulate the expressions of VEGF‐A, HIF‐1α, α‐SMA, Col‐1 and TGF‐β1 of HSCs in vivo and in vitro. The application of SAG and cyclopamine proved that PB2 targets on Hh pathway.ConclusionsPB2 inhibited the Hh pathway to suppress the activation, ECM production and angiogenesis of HSCs, therefore reverses the progression of liver fibrosis in vivo and in vitro.

RIP3 deficiency alleviates liver fibrosis by inhibiting ROCK1-TLR4-NF-κB pathway in macrophages.

Liver fibrosis is an important pathologic process in injured liver tissues. A protein kinase, receptor-interacting protein (RIP)3, plays a crucial role in mediating different diseases. However, the role of RIP3 in macrophages in liver fibrosis has not yet been studied. In our study, we found that RIP3 expression was up-regulated in liver tissues and macrophages of humans and mice with liver fibrosis. Absence of RIP3 in macrophages could alleviate inflammation and macrophage or neutrophil accumulation in mice after carbon tetrachloride (CCl4) or bile duct ligation (BDL) treatment. Importantly, RIP3 deficiency in macrophages could decrease CCl4-induced and BDL-induced liver fibrosis in mice. Moreover, RIP3 deficiency could inhibit the TLR4-NF-κB pathway through suppressing Rho-associated coiled-coil containing protein kinase (ROCK)1 in macrophages. To explore the connection of ROCK1 and RIP3 in macrophages of mice with liver fibrosis in vivo, ROCK1-overexpressed macrophages were infused to RIP3-deficient mice, which resulted in increased inflammation and liver fibrosis. In conclusion, our findings suggest that RIP3 plays a crucial proinflammatory role in liver fibrosis by regulating the ROCK1-TLR4-NF-κB signaling pathway in macrophages and therefore may be a potential therapeutic target for immune-mediated liver fibrosis.-Wei, S., Zhou, H., Wang, Q., Zhou, S., Li, C., Liu, R., Qiu, J., Shi, C., Lu, L. RIP3 deficiency alleviates liver fibrosis by inhibiting ROCK1-TLR4-NF-κB pathway in macrophages.

Geniposide, a sonic hedgehog signaling inhibitor, inhibits the activation of hepatic stellate cell

Liver fibrosis is a continuous wound-healing process, which is due to excessive deposition of extracellular matrix (ECM) caused by activated Hepatic Stellate Cells (HSCs). Geniposide (GP) is a naturally occurring iridoid glucoside and is extracted from Gardenia jasminoides Ellis. GP has long been speculated to play a vital role in the treatment of hyperlipidemia and fatty liver. Emerging evidence has demonstrated that GP may be importantly associated with the pathophysiology of liver fibrosis. Nevertheless, the fundamental mechanism is yet uncertain. This studywas designed to explore the possible mechanism for the inhibitory effect of GP on CCl4-induced mice liver fibrosis. In line with several clinical reports, GP significantly reduced the levels of hyaluronic acid (HA), laminin (LN), hydroxyproline (HYP), aspartate aminotransferase (AST) and alanine aminotransferase (ALT). Meanwhile, liver fibrosis was significantly alleviated by GP as indicated by decreased α-smooth muscle actin (α-SMA) expression and type I collagen alpha-1 (Col I α1) deposition. In addition, GP could reduce the cell viability (IC50 = 77.11 and 42.88 μM at 24 and 48 h respectively) and cause G2/M cell arrest of the activated HSC-T6 cells. Noteworthy, GP prominently suppressed the Sonic hedgehog (Shh) signaling pathway and it might inhibit the activation and proliferation of HSC-T6 cells through Shh signaling pathway. Taken together, the current research indicate that GP has a promising antifibrotic effect which could be partially attributed to its suppression of Shh signaling pathway. Hence GP could be employed as a promising therapeutic strategy for the treatment of clinical hepatic injury.