Targeting cIAPs attenuates CCl4-induced liver fibrosis by increasing MMP9 expression derived from neutrophils.

AIMS: Liver fibrosis is a growing public health concern without effective medical treatment. Recent reports have indicated that inhibitors of apoptosis proteins (IAPs) were potential targets for idiopathic pulmonary fibrosis therapy. However, their roles have not been well identified in liver fibrosis. METHODS: The expression of IAPs were examined in human liver tissue and experimental mouse models. Liver fibrosis in CCl4-induced mouse models were investigated by Sirius red staining, RT-PCR, Western blotting after hepatocytes-specific cIAP2 knockout or IAPs inhibitor APG-1387 treatment. The underlying molecular mechanism of APG-1387 action was explored by apoptosis analysis, matrix metalloprotein 9 (MMP9) inhibition, neutrophils depletion, and CC Motif Chemokine Ligand 5 (CCL5) gene knockout in vitro and in vivo. FINDINGS: Our study showed that increased expression of cIAP2 was associated with liver fibrosis severity in liver tissues. Deletion of cIAP2 from hepatocytes or degrading cIAPs by APG-1387 ameliorated liver fibrosis induced by CCl4. APG-1387 treatment exhibited increased expression of MMP9 and resulted in higher ratio of MMP9 to tissue inhibitor of metalloproteinase-1. MMP9 was mainly derived from CCL5 chemotactic neutrophils. Further, MMP9 inhibition by CTT peptide, neutrophil depletion by Ly6G antibody or CCL5 deficiency blocked the anti-fibrotic effects of APG-1387 in vivo. SIGNIFICANCE: These results suggested that cIAPs, especially cIAP2, might play a novel role in the pathogenesis of liver fibrosis, and targeting cIAPs represented a promising therapeutic strategy for liver fibrosis by increasing MMP9 expression induced by CCL5 chemotactic neutrophils.

IGF-1 alleviates CCL4-induced hepatic cirrhosis and dysfunction of intestinal barrier through inhibition TLR4/NF-κB signaling mediated by down-regulation HMGB1.

INTRODUCTION AND OBJECTIVES: Cirrhosis has gradually become a serious public health issue, especially the national prevalence of cirrhosis was 29.2% in northwest China. Recent evidence has revealed that intestinal barrier (IB) dysfunction results from and contributes to cirrhosis. Our previous results have indicated that insulin-like growth factors (IGF-1) improved the impaired IB function and downregulated high mobility group protein box-1 (HMGB-1). Nevertheless, the role of the IGF-1/HMGB1 axis in cirrhosis remains largely unknown. MATERIALS AND METHODS: Western blotting and qRT-PCR were used to detect protein and mRNA levels of related genes. The levels of AST, ALT, IL-1ß, and TNF-α were examined using commercial kits. Immunofluorescence was used to evaluate the expression of HMGB1 in tissues. RESULTS: In carbon tetrachloride (CCl4)-treated rat, the levels of AST (380.12 vs. 183.97), ALT (148.12 vs. 53.56), IL-1ß (155.94 vs. 55.60), and TNF-α (155.00 vs. 48.90) were significantly increased compared with the control group, while IGF-1 treatment significantly alleviated CCL4-induced inflammatory response and IB dysfunction by downregulating HMGB1-mediated the TLR4/MyD88/NF-κB signaling pathway. In vitro experiments, HMGB1 treatment promoted inflammatory cytokines secretion and reduced cell viability and tight junctions by activating the TLR4/MyD88/NF-κB signaling pathway in Caco-2 cells, but IGF-1 alleviated these effects. CONCLUSION: Our findings suggest that IGF-1 might serve as a potential therapeutic target for cirrhosis and IB dysfunction via inactivation of the TLR4/MyD88/NF-κB pathway through down-regulation HMGB1.

FGF21 ameliorates hepatic fibrosis by multiple mechanisms.

BACKGROUND: Previous study reports that fibroblast growth factor 21 (FGF21) could ameliorate hepatic fibrosis, but its mechanisms have not been fully investigated. METHODS AND RESULTS: In this study, three models were used to investigate the mechanism by which FGF21 alleviates liver fibrosis. Hepatic fibrosis animal models were respectively induced by CCL4 and dimethylnitrosamine. Our results demonstrated that liver index and liver function were deteriorated in both models. Hematoxylin and eosin and Masson's staining showed that the damaged tissue architectonics were observed in the mice of both models. Treatment with FGF21 significantly ameliorated these changes. ELISA analysis showed that the serum levels of IL-1ß, IL-6 and TNF-α were significantly elevated in both models. However, administration of FGF21 significantly reduced these inflammatory cytokines. Real-time PCR and Western blot analysis showed that treatment with FGF21 significantly decreased mRNA and protein expressions of collagenI, α-SMA and TGF-ß. Platelet-derived growth factor-BB (PDGF-BB) stimulant was used to establish the experimental cell model in hepatic stellate cells (HSCs). Real-time PCR and Western blot analysis demonstrated that the expression of collagenI and α-SMA were significantly upregulated by this stimulant in model group. Interestingly, our results showed that mRNA and protein expressions of leptin were also significantly induced in PDGF-BB treated HSCs. Administration of FGF21 significantly reduced leptin expression in a dose dependent manner and these effects were reversed in siRNA (against ß-klotho) transfected HSCs. Furthermore, the leptin signaling pathways related protein p-ERK/t-ERK, p-STAT3/STAT3 and TGF-ß were significantly downregulated by FGF21 treatment in a dose dependent manner. The expressions of SOCS3 and Nrf-2 were enhanced by treatment with FGF21. The underlying mechanism may be that FGF21 regulates leptin-STAT3 axis via Nrf-2 and SOCS3 pathway in activated HSCs. CONCLUSIONS: FGF21 ameliorates hepatic fibrosis by multiple mechanisms.

RNA-Seq analysis of the protection by Dendrobium nobile alkaloids against carbon tetrachloride hepatotoxicity in mice.

OBJECTIVE: Dendrobium nobile is a genuine Chinese medicine. Dendrobium nobile Lindl. alkaloids (DNLA) protects against CCl4-induced acute liver injury. This study used RNA-Seq to explore the mechanisms. METHODS: Mice were pretreated with DNLA (10 and 20 mg/kg, po) for 7 days, and subsequently intoxicated with CCl4 (20 µL/kg, ip for 24 h). Liver RNA was extracted and subjected to RNA-Seq. The bioinformatics, including PCA, GO, KEGG, two-dimensional clustering, Ingenuity Pathways Analysis (IPA), and Illumina BaseSpace Correlation Engine (BSCE) were used to analyze the data. qPCR was performed on selected genes to verify RNA-Seq results. RESULTS: DNLA protection against CCl4 hepatotoxicity was confirmed by histopathology. PCA revealed the distinct gene expression patterns between the different treatment groups. GO showed that CCl4 induced the activation, adhesion and proliferation of immune cells. KEGG showed CCl4 induced oxidative stress, diseases and compromised adaptive responses. CCl4 induced differentially expressed genes (DEGs) were identified by DESeq2 with Padj < 0.05 and 2D-clustered with other groups. DNLA reverted CCl4-induced DEGs in a dose-dependent manner. qPCR analysis of S100 g, Sprr1, CCL3/7, Saa2/3, IL1rn, Cox7a2 and Rad15 confirmed RNA-Seq results. IPA showed that CCl4 treatment altered some signaling and metabolic pathways, which were ameliorated or returned to normal following DNLA treatment. The CCl4-activated mitochondrial oxidative phosphorylation was illustrated as an example. IPA Upstream Regulator Analysis further revealed the activated or inhibited molecules and chemicals that are responsible for CCl4-induced DEGs, and DNLA attenuated these changes. BSCE analysis verified that CCl4-induced DEGs were highly correlated with the GEO database of CCl4 hepatotoxicity in rodents, and DNLA dose-dependently attenuated such correlation. CONCLUSION: RNA-Seq revealed CCl4-induced DEGs, disruption of canonical pathways, activation or inhibition of upstream regulators, which are highly correlated with database for CCl4 hepatotoxicity. All these changes were attenuated or returned to normal by DNLA, demonstrating the mechanisms for DNLA to protect against CCl4 hepatotoxicity.

CAT1 silencing inhibits TGF-ß1-induced mouse hepatic stellate cell activation in vitro and hepatic fibrosis in vivo.

Hepatic fibrosis is characterized by abnormal accumulation of extracellular matrix (ECM). Hepatic stellate cells (HSCs) are the primary cells that produce ECM in response to hepatic injury, and transforming growth factor-beta (TGF-ß) has been regarded as the central stimulus responsible for HSC-mediated ECM production. In the present study, we attempted to identify a critical factor in HSC activation and the underlying mechanism. By analyzing online microarray expression profiles, we found that the expression of high-affinity cationic amino acid transporter 1 (CAT1) was upregulated in hepatic fibrosis models and activated HSCs. We isolated and identified mouse HSCs (MHSCs) and found that in these cells, CAT1 was most highly upregulated by TGF-ß1 stimulation in both time- and dose-dependent manners. In vitro, CAT1 overexpression further enhanced, while CAT1 silencing inhibited, the effect of TGF-ß1 in promoting MHSC activation. In vivo, CAT1 silencing significantly improved the hepatic fibrosis induced by both CCl4 and non-alcoholic fatty liver disease (NAFLD). In summary, CAT1 was significantly upregulated in TGF-ß1-activated MHSCs and mice with hepatic fibrosis. CAT1 silencing inhibited TGF-ß1-induced MHSC activation in vitro and fibrogenic changes in vivo. CAT1 is a promising target for hepatic fibrosis treatment that requites further investigation in human cells and clinical practice.

Chronic Carbon Tetrachloride Applications Induced Hepatocyte Apoptosis in Lipocalin 2 Null Mice Through Endoplasmic Reticulum Stress and Unfolded Protein Response.

The lack of Lipocalin (LCN2) provokes overwhelming endoplasmic reticulum (ER) stress responses in vitro and in acute toxic liver injury models, resulting in hepatocyte apoptosis. LCN2 is an acute phase protein produced in hepatocytes in response to acute liver injuries. In line with these findings we investigated ER stress responses of Lcn2-/- mice in chronic ER stress using a long-term repetitive carbon tetrachloride (CCl4) injection model. We found chronic CCl4 application to enhance ER stress and unfolded protein responses (UPR), including phosphorylation of eukaryotic initiation factor 2α (eIF2α), increased expression of binding immunoglobulin protein (BiP) and glucose-regulated protein 94 (GRP94). IRE1α/TRAF2/JNK signaling enhanced mitochondrial apoptotic pathways, and showed slightly higher in Lcn2-/- mice compared to the wild type counterparts, leading to increased hepatocyte apoptosis well evidenced by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Hepatocyte injuries were confirmed by significant high serum alanine transaminase (ALT) levels in CCl4-treated Lcn2-/- mice. Lcn2-/- mice furthermore developed mild hepatic steatosis, supporting our finding that ER stress promotes lipogenesis. In a previous report we demonstrated that the pharmacological agent tunicamycin (TM) induced ER stress through altered protein glycosylation and induced high amounts of C/EBP-homologous protein (CHOP), resulting in hepatocyte apoptosis. We compared TM-induced ER stress in wild type, Lcn2-/-, and Chop null (Chop-/-) primary hepatocytes and found Chop-/- hepatocytes to attenuate ER stress responses and resist ER stress-induced hepatocyte apoptosis through canonical eIF2α/GADD34 signaling, inhibiting protein synthesis. Unexpectedly, in later stages of TM incubation, Chop-/- hepatocytes resumed activation of IRE1α/JNK/c-Jun and p38/ATF2 signaling, leading to late hepatocyte apoptosis. This interesting observation indicates Chop-/- mice to be unable to absolutely prevent all types of liver injury, while LCN2 protects the hepatocytes by maintaining homeostasis under ER stress conditions.

Hepatoprotective effect of a fucoidan extract from Sargassum fluitans Borgesen against CCl4-induced toxicity in rats.

The in vivo antifibrotic effect of a fucoidan extract (FE) from Sargassum fluitans Borgesen was evaluated in a carbon tetrachloride-induced liver damage model in rats over twelve weeks. Chemical analysis showed the FE to contain carbohydrates, sulfates, uronic acids, protein, phenols, and to have a molecular weight of ~60 kDa. Physiological, biochemical, histological and genetic assays were done. Daily oral administration of FE (50 mg/kg) reduced liver enzymatic activity, liver infiltration of inflammatory cells, collagen fiber deposition and gene expression cytokines such as interleukin beta 1 (IL-ß1), tumor necrosis factor alpha (TNF-α), transforming growth factor beta 1 (TGF-ß1), Smad-3, Smad-2, collagen 1 alpha 1 (col1α1) and tissue inhibitor of metalloproteinase 1 (TIMP-1). It also increased RNA expression of Smad-7 and metalloproteinase 2 and 9 (MMP2 and MMP9). The fucoidan extract exhibited an antifibrotic effect mediated by the inhibiting TGF-ß1/Smad pathway, as well as anti-inflammatory effects.

Oct4 and Hnf4α-induced hepatic stem cells ameliorate chronic liver injury in liver fibrosis model.

Direct conversion from fibroblasts to generate hepatocyte like-cells (iHeps) bypassing the pluripotent state has been described in previous reports as an attractive method acquiring hepatocytes for cell-based therapy. The limited proliferation of iHeps, however, has hampered it uses in cell-based therapy. Since hepatic stem cells (HepSCs) possess self-renewal and bipotency with the capacity to differentiate into both hepatocytes and cholangiocytes, they have therapeutic potential for treating liver disease. Here, we investigated the therapeutic effects of induced HepSCs (iHepSCs) on a carbon tetrachloride (CCl4)-induced liver fibrosis model. We demonstrate that Oct4 and Hnf4a are sufficient to convert fibroblasts into expandable iHepSCs. Hepatocyte-like cells derived from iHepSCs (iHepSC-HEPs) exhibit the typical morphology of hepatocytes and hepatic functions, including glycogen storage, low-density lipoprotein (LDL) uptake, Indocyanine green (ICG) detoxification, drug metabolism, urea production, and albumin secretion. iHepSCs-derived cholangiocyte-like cells (iHepSC-CLCs) expressed cholangiocyte-specific markers and formed cysts and tubule-like structures with apical-basal polarity and secretory function in three-dimensional culture condition. Furthermore, iHepSCs showed anti-inflammatory and anti-fibrotic effects in CCl4-induced liver fibrosis. This study demonstrates that Oct4 and Hnf4α-induced HepSCs show typical hepatic and biliary functionality in vitro. It also presents the therapeutic effect of iHepSCs in liver fibrosis. Therefore, directly converting iHepSCs from somatic cells may facilitate the development of patient-specific cell-based therapy for chronic liver damage.

Therapeutic effects of a small molecule agonist of the relaxin receptor ML290 in liver fibrosis.

Fibrosis is an underlying cause of cirrhosis and hepatic failure resulting in end stage liver disease with limited pharmacological options. The beneficial effects of relaxin peptide treatment were demonstrated in clinically relevant animal models of liver fibrosis. However, the use of relaxin is problematic because of a short half-life. The aim of this study was to test the therapeutic effects of recently identified small molecule agonists of the human relaxin receptor, relaxin family peptide receptor 1 (RXFP1). The lead compound of this series, ML290, was selected based on its effects on the expression of fibrosis-related genes in primary human stellate cells. RNA sequencing analysis of TGF-ß1-activated LX-2 cells showed that ML290 treatment primarily affected extracellular matrix remodeling and cytokine signaling, with expression profiles indicating an antifibrotic effect of ML290. ML290 treatment in human liver organoids with LPS-induced fibrotic phenotype resulted in a significant reduction of type I collagen. The pharmacokinetics of ML290 in mice demonstrated its high stability in vivo, as evidenced by the sustained concentrations of compound in the liver. In mice expressing human RXFP1 gene treated with carbon tetrachloride, ML290 significantly reduced collagen content, α-smooth muscle actin expression, and cell proliferation around portal ducts. In conclusion, ML290 demonstrated antifibrotic effects in liver fibrosis.-Kaftanovskaya, E. M., Ng, H. H., Soula, M., Rivas, B., Myhr, C., Ho, B. A., Cervantes, B. A., Shupe, T. D., Devarasetty, M., Hu, X., Xu, X., Patnaik, S., Wilson, K. J., Barnaeva, E., Ferrer, M., Southall, N. T., Marugan, J. J., Bishop, C. E., Agoulnik, I. U., Agoulnik, A. I. Therapeutic effects of a small molecule agonist of the relaxin receptor ML290 in liver fibrosis.

Protective effect of RNA interference targeting Ikkß on hepatic fibrosis in rats.

Nuclear factor-κB (NF-κB) is an important regulatory factor in cells. NF-κB has a wide range of biological activities. After activation, it participates in the transcription and regulation of many genes and plays a role in infection, inflammatory response, oxidative stress, cell multiplication, and apoptosis. The activation of the NF-κB signal pathway is dependent on the degradation of the IκB kinase ß (IKKß) complex. IKK ß is the key kinase in the NF-κB activation pathway. After inhibition, it can block the activation of NF-κB. IKKß is a key regulator of NF-κB activation, also an early regulator of inflammation in all stages of the immune response. This study aimed to investigate the effect of IKKß-siRNA lentivirus vector treatment for hepatic fibrosis of rats. An IKKß-siRNA expression plasmid was constructed and injected in the tail vein of rats. Then, IKKß-siRNA distribution in the liver was observed by immunofluorescence, and the quantitative polymerase chain reaction was used to detect inflammation-related and fibrosis-related factors. IKKß-siRNA lentiviruses could be delivered to the liver and significantly decrease carbon tetrachloride-induced hepatic fibrosis. Furthermore, serum transaminase levels significantly decreased, and inflammation-related and fibrosis-related factors decreased. IKKß-siRNA can be an effective method of anti-fibrosis gene therapy for liver fibrosis.

Artificial MicroRNA-Mediated Tgfbr2 and Pdgfrb Co-Silencing Ameliorates Carbon Tetrachloride-Induced Hepatic Fibrosis in Mice.

Hepatic stellate cells (HSCs) are the primary cell type responsible for liver fibrogenesis. Transforming growth factor beta 1 (TGF-ß1) and platelet-derived growth factor (PDGF) are key profibrotic cytokines that regulate HSC activation and proliferation with functional convergence. Dual RNA interference against their receptors may achieve therapeutic effects. A novel RNAi strategy based on HSC-specific GFAP promoter-driven and lentiviral-expressed artificial microRNAs (amiRNAs) was devised that consists of an microRNA-30a backbone and effective shRNAs against mouse Pdgfrß and Tgfbr2. Then, its antifibrotic efficacy was tested in primary and cultured HSCs and in mice affected with carbon tetrachloride-induced hepatic fibrosis. The study shows that amiRNA-mediated Pdgfrß and Tgfbr2 co-silencing inhibits HSC activation and proliferation. After recombinant lentiviral particles were delivered into the liver via tail-vein injection, therapeutic amiRNAs were preferentially expressed in HSCs and efficiently co-knocked down in situ Tgfbr2 and Pdgfrß expression, which correlates with downregulated expression of target or effector genes of their signaling, which include Pai-1, P70S6K, and D-cyclins. amiRNA-based HSC-specific co-silencing of Tgfbr2 and Pdgfrß significantly suppressed hepatic expression of fibrotic markers α-Sma and Col1a1, extracellular matrix regulators Mmps and Timp1, and phenotypically ameliorated liver fibrosis, as indicated by reductions in serum alanine aminotransferase activity, collagen deposition, and α-Sma-positive staining. The findings provide proof of concept for the use of amiRNA-mediated co-silencing of two profibrogenic pathways in liver fibrosis treatment and highlight the therapeutic potential of concatenated amiRNAs for gene therapy.

Sirtuin3 deficiency exacerbates carbon tetrachloride-induced hepatic injury in mice.

Sirtuin3 (SIRT3) plays an important role in maintaining normal mitochondrial function and alleviating oxidative stress. After carbon tetrachloride (CCl4 ) administration, the expression of SIRT3 decreased in the liver of mice, which indicated that the SIRT3 might play a crucial role during chemical-induced acute hepatic injury. To verify the hypothesis, CCl 4 was given to induce acute hepatic injury in SIRT3 knockout (KO) mice and wild-type (WT) mice. CCl 4 -induced liver injury was more severe in SIRT3 KO mice compared with the WT mice. In addition, the oxidative stress induced by CCl 4 was enhanced in the SIRT3 KO mice. Furthermore, the increased expression of dynamin-related protein 1 was also aggravated in SIRT3 KO mice after CCl 4 administration. In conclusion, our study demonstrated that SIRT3 deficiency exacerbated CCl 4 -induced impairment of the liver in mice, and the mechanism might be related to enhanced oxidative stress.

Noncanonical farnesoid X receptor signaling inhibits apoptosis and impedes liver fibrosis.

BACKGROUND: Hepatocyte is particularly vulnerable to apoptosis, a hallmark of many liver diseases. Although pro-apoptotic mechanisms have been extensively explored, less is known about the hepatocyte-specific anti-apoptotic molecular events and it lacks effective approach to combat hepatocyte apoptosis. We investigated the anti-apoptotic effect and mechanism of farnesoid X receptor (FXR), and strategies of how to target FXR for inhibiting apoptosis implicated in liver fibrosis. METHODS: Sensitivity to apoptosis was compared between wild type and Fxr-/- mice and in cultured cells. Cell-based and cell-free assays were employed to identify the binding protein of FXR and to uncover the mechanism of its anti-apoptotic effect. Overexpression of FXR by adenovirus-FXR was employed to determine its anti-fibrotic effect in CCl4-treated mice. Specimens from fibrotic patients were collected to validate the relevance of FXR on apoptosis/fibrosis. FINDINGS: FXR deficiency sensitizes hepatocytes to death receptors (DRs)-engaged apoptosis. FXR overexpression, but not FXR ligands, inhibits apoptosis both in vitro and in vivo. Apoptotic stimuli lead to drastic reduction of FXR protein levels, a prerequisite for DRs-engaged apoptosis. Mechanistically, FXR interacts with caspase 8 (CASP8) in the cytoplasm, thus preventing the formation of death-inducing signaling complex (DISC) and activation of CASP8. Adenovirus-FXR transfection impedes liver fibrosis in CCl4-treated mice. Specimens from fibrotic patients are characterized with reduced FXR expression and compromised FXR/CASP8 colocalization. INTERPRETATION: FXR represents an intrinsic apoptosis inhibitor in hepatocytes and can be targeted via restoring its expression or strengthening FXR/CASP8 interaction for inhibiting hepatocytes apoptosis in liver fibrosis. FUND: National Natural Science Foundation of China.

Genome­wide identification of long noncoding RNAs in CCl4­induced liver fibrosis via RNA sequencing.

Liver fibrosis occurs as a result of chronic liver lesions, which may subsequently develop into liver cirrhosis and hepatocellular carcinoma. The involvement of long noncoding RNAs (lncRNAs) in liver fibrosis is being increasingly recognized. However, the exact mechanisms and functions of the majority of lncRNAs are poorly characterized. In the present study, the hepatotoxic substance carbon tetrachloride (CCl4) was employed to induce liver fibrosis in an animal model and agenome­wide identification of lncRNAs in fibrotic liver tissues compared with CCl4 untreated liver tissues was performed using RNA sequencing. Sprague­Dawley rats were treated with CCl4 for 8 weeks. Histopathogical alterations were observed in liver tissues, and serum levels of alanine aminotransferase, aspartate aminotransferase, transforming growth factor­ß1 and tumor necrosis factor­α were significantly higher, in the CCl4­treated group compared with the CCl4 untreated group. RNA sequencing of liver tissues demonstrated that 231 lncRNAs and 1,036 mRNAs were differentially expressed between the two groups. Furthermore, bioinformatics analysis demonstrated that the differentially expressed mRNAs were predominantly enriched in 'ECM­receptor interaction', 'PI3K­Akt signaling pathway' and 'focal adhesion' pathways, all of which are essential for liver fibrosis development. Validation of 12 significantly aberrant lncRNAs by reverse transcription­quantitative polymerase chain reaction indicated that the expression patterns of 11 lncRNAs were consistent with the sequencing data. Furthermore, overexpression of lncRNA NR_002155.1, which was markedly downregulated in CCl4­treated liver tissues, was demonstrated to inhibit HSC­T6 cell proliferation in vitro. In conclusion, the present study determined the expression patterns of mRNAs and lncRNAs in fibrotic liver tissue induced by CCl4. The identified differentially expressed lncRNAs may serve as novel diagnostic biomarkers and therapeutic targets for liver fibrosis.

Inhibition of lysyl oxidase-like 1 (LOXL1) expression arrests liver fibrosis progression in cirrhosis by reducing elastin crosslinking.

Mature crosslinked-poly-elastin deposition has been found to be associated with liver fibrosis. However, the regulation of crosslinked/insoluble elastin in liver fibrosis remains largely unknown. Here, we investigated the contribution of lysyl oxidases (LOXs) family, mediated elastin crosslinking, to liver fibrogenesis. We established carbon tetrachloride (CCl4)-induced liver fibrotic and cirrhotic models and found that crosslinked/insoluble elastin levels spiked only in cirrhosis stage during disease progression, in comparison to collagen Ι levels which increased continuously though all stages. Among the LOXs family members, only LOX-like 1 (LOXL1) levels were coincident with the appearance of crosslinked/insoluble elastin. These coincidences included that LOXL1 expression increased (34 fold) in cirrhosis, localized with α-smooth muscle actin (SMA) and was absent in normal and fibrotic livers. In LX-2 cells, LOXL1 silencing arrested expression of α-SMA, elastin and collagen Ι. Our previously characterized adeno-associated vector (AAV) 2/8 shRNA was shown to effectively downregulate LOXL1 expression in CCl4 induced fibrosis mice models. These resulted in delicate and thinner septa and less crosslinked elastin, with a 58% loss of elastin area and 51% decrease of collagen area. Our findings strongly suggested that elastin crosslinking and LOXL1 were co-associated with liver cirrhosis, while selective inhibition of LOXL1 arrested disease progression by reducing crosslinking of elastin.

Epigenetic Effects of an Adenosine Derivative in a Wistar Rat Model of Liver Cirrhosis.

The pathological characteristic of cirrhosis is scarring which results in a structurally distorted and dysfunctional liver. Previously, we demonstrated that Col1a1 and Pparg genes are deregulated in CCl4 -induced cirrhosis but their normal expression levels are recovered upon treatment with IFC-305, an adenosine derivative. We observed that adenosine was able to modulate S-adenosylmethionine-dependent trans-methylation reactions, and recently, we found that IFC-305 modulates HDAC3 expression. Here, we investigated whether epigenetic mechanisms, involving DNA methylation processes and histone acetylation, could explain the re-establishment of gene expression mediated by IFC-305 in cirrhosis. Therefore, Wistar rats were CCl4 treated and a sub-group received IFC-305 to reverse fibrosis. Global changes in DNA methylation, 5-hydroxymethylation, and histone H4 acetylation were observed after treatment with IFC-305. In particular, during cirrhosis, the Pparg gene promoter is depleted of histone H4 acetylation, whereas IFC-305 administration restores normal histone acetylation levels which correlates with an increase of Pparg transcript and protein levels. In contrast, the promoter of Col1a1 gene is hypomethylated during cirrhosis but gains DNA methylation upon treatment with IFC-305 which correlates with a reduction of Col1a1 transcript and protein levels. Our results suggest a model in which cirrhosis results in a general loss of permissive chromatin histone marks which triggers the repression of the Pparg gene and the upregulation of the Col1a1 gene. Treatment with IFC-305 restores epigenetic modifications globally and specifically at the promoters of Pparg and Col1a1 genes. These results reveal one of the mechanisms of action of IFC-305 and suggest a possible therapeutic function in cirrhosis. J. Cell. Biochem. 119: 401-413, 2018. © 2017 Wiley Periodicals, Inc.

Effects of Liver Fibrosis Progression on Tissue Relaxation Times in Different Mouse Models Assessed by Ultrahigh Field Magnetic Resonance Imaging.

Recently, clinical studies demonstrated that magnetic resonance relaxometry with determination of relaxation times T1 and T2⁎ may aid in staging and management of liver fibrosis in patients suffering from viral hepatitis and steatohepatitis. In the present study we investigated T1 and T2⁎ in different models of liver fibrosis to compare alternate pathophysiologies in their effects on relaxation times and to further develop noninvasive quantification methods of liver fibrosis. MRI was performed with a fast spin echo sequence for measurement of T1 and a multigradient echo sequence for determination of T2⁎. Toxic liver fibrosis was induced by injections of carbon tetrachloride (1.4 mL CCl4 per kg bodyweight and week, for 3 or 6 weeks) in BALB/cJ mice. Chronic sclerosing cholangitis was mimicked using the ATP-binding cassette transporter B4 knockout (Abcb4 -/-) mouse model. Untreated BALB/cJ mice served as controls. To assess hepatic fibrosis, we ascertained collagen contents and fibrosis scores after Sirius red staining. T1 and T2⁎ correlate differently to disease severity and etiology of liver fibrosis. T2⁎ shows significant decrease correlating with fibrosis in CCl4 treated animals, while demonstrating significant increase with disease severity in Abcb4 -/- mice. Measurements of T1 and T2⁎ may therefore facilitate discrimination between different stages and causes of liver fibrosis.

Thyroid hormones inhibit TGF-ß signaling and attenuate fibrotic responses.

TGF-ß, the most potent profibrogenic factor, acts by activating SMAD (mothers against decapentaplegic) transcription factors, which bind to SMAD-binding elements in target genes. Here, we show that the thyroid hormone triiodothyronine (T3), through binding to its nuclear receptors (TRs), is able to antagonize transcriptional activation by TGF-ß/SMAD. This antagonism involves reduced phosphorylation of SMADs and a direct interaction of the receptors with SMAD3 and SMAD4 that is independent of T3-mediated transcriptional activity but requires residues in the receptor DNA binding domain. T3 reduces occupancy of SMAD-binding elements in response to TGF-ß, reducing histone acetylation and inhibiting transcription. In agreement with this transcriptional cross-talk, T3 is able to antagonize fibrotic processes in vivo. Liver fibrosis induced by carbon tetrachloride is attenuated by thyroid hormone administration to mice, whereas aged TR knockout mice spontaneously accumulate collagen. Furthermore, skin fibrosis induced by bleomycin administration is also reduced by the thyroid hormones. These findings define an important function of the thyroid hormone receptors and suggest TR ligands could have beneficial effects to block the progression of fibrotic diseases.

Liver X receptor α is essential for the capillarization of liver sinusoidal endothelial cells in liver injury.

Liver X receptors (LXRs) play essential roles in lipogenesis, anti-inflammatory action and hepatic stellate cells (HSCs) activation in the liver. However, the effects of LXRs on the capillarization of liver sinusoidal endothelial cells (LSECs) in liver fibrosis remain undetermined. Here, we demonstrated that LXRα plays an important role in LSECs capillarization in a manner that involved Hedgehog (Hh) signaling. We found that LXRα expression in LSECs was increased in the carbon tetrachloride (CCl4)-induced fibrosis model. LXRα deletion markedly exacerbated CCl4-induced lesions assessed by histopathology, as well as inflammation and collagen deposition. Furthermore, capillarization of the sinusoids was aggravated in CCl4 -treated LXRα-deficient mice, as evidenced by increased CD34 expression, the formation of continuous basement membranes and aggravation of the loss of fenestrae. In vitro, LXR agonist could maintain freshly isolated LSECs differentiation on day 3. Furthermore, LXRα deletion led to increased expression of Hedgehog (Hh)-regulated gene in LSECs in the injured liver. Conversely, the LXR agonist could inhibit the Hh pathway in cultured LSECs. These responses indicated that LXRα suppressed the process of LSECs capillarization by repressing Hh signaling. Overall, our findings suggest that LXRα, by restoring the differentiation of LSECs, may be critical for the regression of liver fibrosis.

Role of NLRC5 in progression and reversal of hepatic fibrosis.

BACKGROUND: NLRC5, as the largest member of NLRs family, has recently been identified as a critical regulator of immune responses through negatively regulating NF-κB which is associated with the development of hepatic fibrosis. However, the expression and potential roles of NLRC5 in hepatic fibrosis and its reversal are still to be defined. METHODS: C57BL/6 mice were treatment with carbon tetrachloride (CCl4) induce hepatic fibrosis and its reversal. In vitro, models of hepatic fibrosis and its reversal are established by the treatment with TGF-ß and MDI. The expression of NLRC5 was determined by RT-PCR, Western blot and immunohistochemistry. Consequently, NLRC5 was overexpressed or knockdown by transfecting PEGFP-C2-NLRC5 or NLRC5-siRNA respectively in the reversal of hepatic fibrosis, and the expression of fibrogenic genes such as α-SMA and Col1α1 was quantified. The NF-κB activity was detected as well. RESULTS: Immunohistochemistry, RT-PCR and Western blot analysis with liver tissues and primary HSCs showed that NLRC5 was highly expressed in hepatic fibrosis and correspondingly decreased in the reversal stage. The differential expression of NLRC5 was confirmed in vitro. Enforced NLRC5 expression increased the expression of α-SMA and Col1α1, and blockade of NLRC5 reduced the fibrotic response. While the opposite expression of phosphorylated NF-кB p65 and phospho-IκBα was found. CONCLUSION: NLRC5 is differentially expressed in hepatic tissues and hepatic stellate cells during hepatic fibrosis and its reversal. All the data indicated that NLRC5 may play a crucial role in regulating the reversal of hepatic fibrosis through NF-κB signaling pathway.