Fibrotic Mouse Livers Research Articles

The de-sulfinylation enzyme sulfiredoxin-1 attenuates hepatic stellate cell activation and liver fibrosis by modulating the PTPN12-NLRP3 axis.

Liver fibrosis is characterized by the progressive scarring of liver tissue. Oxidative stress is a critical causal factor of hepatic stellate cell (HSC) activation and the subsequent liver fibrogenesis, but the mechanism is not fully understood. Cysteine sulfinic acid (Cys-SO2H), a modification of reactive cysteine residues, is a unique form of oxidative response that alters the structure and function of proteins. Sulfiredoxin 1 (SRXN1) is responsible for ATP-dependent reduction of the Cys-SO2H to sulfenic acid (Cys-SOH). We found that the expression of SRXN1 was increased in activated HSCs and in human and mouse fibrotic livers. HSC-specific ablation of Srxn1 or pharmacological inhibition of Srxn1 exacerbated HSC activation and sensitized mice to liver fibrosis. Mechanistically, SRXN1 inhibited HSC activation by de-sulfinylating the phosphatase protein tyrosine phosphatase non-receptor type 12 (PTPN12), which enhanced its phosphatase activity and protein stability, leading to decreased tyrosine phosphorylation and reduced activation of the pro-fibrotic inflammasome protein NLRP3. The anti-fibrotic effect of SRXN1 was abolished when NLRP3 was inhibited. In contrast, overexpression of PTPN12 attenuated NLRP3 activation, and this effect was further amplified by the C164A S-sulfinylation resistant mutant of PTPN12. Our findings have uncovered an important role of SRXN1 and protein S-sulfinylation in HSC activation and liver fibrosis. The SRXN1-PTPN12-NLRP3 axis represents potential therapeutic targets for liver fibrosis.

Inhibition of heme-thiolate monooxygenase CYP1B1 prevents hepatic stellate cell activation and liver fibrosis by accumulating trehalose.

Activation of extracellular matrix-producing hepatic stellate cells (HSCs) is a key event in liver fibrogenesis. We showed that the expression of the heme-thiolate monooxygenase cytochrome P450 1B1 (CYP1B1) was elevated in human and mouse fibrotic livers and activated HSCs. Systemic or HSC-specific ablation and pharmacological inhibition of CYP1B1 attenuated HSC activation and protected male but not female mice from thioacetamide (TAA)-, carbon tetrachloride (CCl4)-, or bile duct ligation (BDL)-induced liver fibrosis. Metabolomic analysis revealed an increase in the disaccharide trehalose in CYP1B1-deficient HSCs resulting from intestinal suppression of the trehalose-metabolizing enzyme trehalase, whose gene we found to be a target of RARα. Trehalose or its hydrolysis-resistant derivative lactotrehalose exhibited potent antifibrotic activity in vitro and in vivo by functioning as an HSC-specific autophagy inhibitor, which may account for the antifibrotic effect of CYP1B1 inhibition. Our study thus reveals an endobiotic function of CYP1B1 in liver fibrosis in males, mediated by liver-intestine cross-talk and trehalose. At the translational level, pharmacological inhibition of CYP1B1 or the use of trehalose/lactotrehalose may represent therapeutic strategies for liver fibrosis.

System analysis based on weighted gene co-expression analysis identifies SOX7 as a novel regulator of hepatic stellate cell activation and liver fibrosis.

Hepatic stellate cell (HSC) activation is the essential pathological process of liver fibrosis (LF). The molecular mechanisms regulating HSC activation and LF are incompletely understood. Here, we explored the effect of transcription factor SRY-related high mobility group box7 (SOX7) on HSC activation and LF, and the underlying molecular mechanism. We found the expression levels of SOX7 were decreased in human and mouse fibrotic livers, particularly at the fibrotic foci. SOX7 was also downregulated in primary activated HSCs and TGF-β1 stimulated LX-2 cells. SOX7 knockdown promoted activation and proliferation of LX-2 cells while inhibiting their apoptosis. On the other hand, overexpression of SOX7 suppressed the activation and proliferation of HSCs. Mechanistically, SOX7 attenuates HSC activation and LF by decreasing the expression of β-catenin and phosphorylation of Smad2 and Smad3 induced by TGF-β1. Furthermore, overexpression of SOX7 using AAV8-SOX7 mouse models ameliorated the extent of LF in response to CCl4 treatment invivo. Collectively, SOX7 suppressed HSC activation and LF. Targeting SOX7, therefore, could be a potential novel strategy to protect against LF.

Preclinical targeting of liver fibrosis with a 89Zr-labeled Fibrobody® directed against platelet derived growth factor receptor-β.

Hepatic fibrosis develops as a response to chronic liver injury, resulting in the formation of fibrous scars. This process is initiated and driven by collagen-producing activated myofibroblasts which reportedly express high levels of platelet derived growth factor receptor-β (PDGFRβ). We therefore regard PDGFRβ as an anchor for diagnosis and therapy. The Fibrobody® SP02SP26-ABD is a biparatopic VHH-construct targeting PDGFRβ. Here, we explore its potential as a theranostic vector for liver fibrosis. Specificity, cross-species binding, and cellular uptake of SP02SP26-ABD was assessed using human, mouse and rat PDGFRβ ectodomains and PDGFRβ-expressing cells. Cellular uptake by PDGFRβ-expressing cells was also evaluated by equipping the Fibrobody® with auristatinF and reading out in vitro cytotoxicity. The validity of PDGFRβ as a marker for active fibrosis was confirmed in human liver samples and 3 mouse models of liver fibrosis (DDC, CCl4, CDA-HFD) through immunohistochemistry and RT-PCR. After radiolabeling of DFO*-SP02SP26-ABD with 89Zr, its in vivo targeting ability was assessed in healthy mice and mice with liver fibrosis by PET-CT imaging, ex vivo biodistribution and autoradiography. SP02SP26-ABD shows similar nanomolar affinity for human, mouse and rat PDGFRβ. Cellular uptake and hence subnanomolar cytotoxic potency of auristatinF-conjugated SP02SP26-ABD was observed in PDGFRβ-expressing cell lines. Immunohistochemistry of mouse and human fibrotic livers confirmed co-localization of PDGFRβ with markers of active fibrosis. In all three liver fibrosis models, PET-CT imaging and biodistribution analysis of [89Zr]Zr-SP02SP26-ABD revealed increased PDGFRβ-specific uptake in fibrotic livers. In the DDC model, liver uptake was 12.15 ± 0.45, 15.07 ± 0.90, 20.23 ± 1.34, and 20.93 ± 4.35%ID/g after 1,2,3 and 4 weeks of fibrogenesis, respectively, compared to 7.56 ± 0.85%ID/g in healthy mice. Autoradiography revealed preferential uptake in the fibrotic (PDGFRβ-expressing) periportal areas. The anti-PDGFRβ Fibrobody® SP02SP26-ABD shows selective and high-degree targeting of activated myofibroblasts in liver fibrosis, and qualifies as a vector for diagnostic and therapeutic purposes.

Liver sinusoidal endothelial cells contribute to portal hypertension through collagen type IV-driven sinusoidal remodeling.

Portal hypertension (PHTN) is a severe complication of liver cirrhosis and is associated with intrahepatic sinusoidal remodeling induced by sinusoidal resistance and angiogenesis. Collagen type IV (COL4), a major component of basement membrane, forms in liver sinusoids upon chronic liver injury. However, the role, cellular source, and expression regulation of COL4 in liver diseases are unknown. Here, we examined how COL4 is produced and how it regulates sinusoidal remodeling in fibrosis and PHTN. Human cirrhotic liver sample RNA sequencing showed increased COL4 expression, which was further verified via immunofluorescence staining. Single-cell RNA sequencing identified liver sinusoidal endothelial cells (LSECs) as the predominant source of COL4 upregulation in mouse fibrotic liver. In addition, COL4 was upregulated in a TNF-α/NF-κB-dependent manner through an epigenetic mechanism in LSECs in vitro. Indeed, by utilizing a CRISPRi-dCas9-KRAB epigenome-editing approach, epigenetic repression of the enhancer-promoter interaction showed silencing of COL4 gene expression. LSEC-specific COL4 gene mutation or repression in vivo abrogated sinusoidal resistance and angiogenesis, which thereby alleviated sinusoidal remodeling and PHTN. Our findings reveal that LSECs promote sinusoidal remodeling and PHTN during liver fibrosis through COL4 deposition.

Ba-Qi-Rougan formula alleviates hepatic fibrosis by suppressing hepatic stellate cell activation via the MSMP/CCR2/PI3K pathway

Ethnopharmacological relevanceThe Ba-Qi-Rougan formula (BQRGF) is a traditional and effective compound prescription from Traditional Chinese Medicine (TCM) utilized in treating hepatic fibrosis (HF). Aim of the studyWe aimed to evaluate the therapeutic efficacy of BQRGF on HF and explore the underlying mechanisms of action. Materials and methodsUPLC-Q-TOF-MS technology was employed to identify the material basis of BQRGF. Mice with carbon tetrachloride (CCl4)-induced HF received BQRGF at three doses (3.87, 7.74, and 15.48 g/kg per day). We examined serum and liver biochemical indicators and liver histology to assess the therapeutic impact. Primary mouse cells were isolated and utilized for experimental analysis. MSMP expression levels were examined in vitro and in vivo experimental models, including human and mouse tissue. Furthermore, lentivirus and small interfering RNA (siRNA) transfections were employed to manipulate microseminoprotein (MSMP) expression in LO2 cells (human normal liver cells). These manipulated LO2 cells were then co-cultured with LX2 human hepatic stellate cells (HSCs). Through the modulation of MSMP expression in co-cultured cells, administering recombinant MSMP (rMSMP) with or without BQRGF-medicated serum, and using specific pathway inhibitors or agonists in LX2 cells, we elucidated the underlying mechanisms. ResultsA total of 48 compounds were identified from BQRGF, with 12 compounds being absorbed into the bloodstream and 9 compounds being absorbed into the liver. Four weeks of BQRGF treatment in the HF mouse model led to significant improvements in biochemical and molecular assays and histopathology, particularly in the medium and high-dose groups. These improvements included a reduction in the level of liver injury and fibrosis-related factors. MSMP levels were elevated in human and mouse fibrotic liver tissues, and this increase was mitigated in HF mice treated with BQRGF. Moreover, primary cells and co-culture studies revealed that BQRGF reduced MSMP expression, decreased the expression of the hepatic stellate cell (HSC) activation markers, and suppressed critical phosphorylated protein levels in the CCR2/PI3K/AKT pathway. These findings were further validated using CCR2/PI3K/AKT signaling inhibitors and agonists in MSMP-activated LX2 cells. ConclusionsCollectively, our results suggest that BQRGF combats HF by diminishing MSMP levels and inhibiting MSMP-induced HSC activation through the CCR2/PI3K/AKT pathway.

Lysine specific demethylase 1 inhibits sodium arsenite activation of HSCs by regulating SESN2/AMPK/ULK1 signaling pathway activity.

Lysine specific demethylase 1 (LSD1) is a histone demethylase that specifically catalyzes the demethylation of histone H3K4 (H3K4me1/2) and regulates gene expression. In addition, it can mediate the process of autophagy through its demethylase activity. Sestrin2 (SESN2) is a stress-induced protein and a positive regulator of autophagy. In NaAsO2-induced mouse fibrotic livers and activated hepatic stellate cells (HSCs), LSD1 expression is decreased, SESN2 expression is increased, and autophagy levels are also increased. Overexpression of LSD1 and silencing of SESN2 decreased the level of autophagy and attenuated the activation of HSCs induced by NaAsO2. LSD1 promoted SESN2 gene transcription by increasing H3K4me1/2 in the SESN2 promoter region. 3-methyladenine (3-MA) and chloroquine were used to inhibit autophagy of HSCs, and the degree of activation was also alleviated. Taken together, LSD1 positively regulates SESN2 by increasing H3K4me1/2 enrichment in the SESN2 promoter region, which in turn increases the level of autophagy and promotes the activation of HSCs. Our results may provide new evidence for the importance of LSD1 in the process of autophagy and activation of HSCs induced by arsenic poisoning. Increasing the expression and activity of LSD1 is expected to be an effective way to reverse the autophagy and activation of HSCs induced by arsenic poisoning.

Activated hepatic stellate cell-derived Bmp-1 induces liver fibrosis via mediating hepatocyte epithelial-mesenchymal transition

Liver fibrosis is a reparative response to injury that arises from various etiologies, characterized by activation of hepatic stellate cells (HSCs). Periostin, a secreted matricellular protein, has been reported to participate in tissue development and regeneration. However, its involvement in liver fibrosis remains unknown. This study investigated the roles and mechanisms of Periostin in phenotypic transition of HSCs and relevant abnormal cellular crosstalk during liver fibrosis. The fate of hepatic stellate cells (HSCs) during liver fibrogenesis was investigated using single-cell and bulk RNA sequencing profiles, which revealed a significant proliferation of activated HSCs (aHSCs) in fibrotic livers of both humans and mice. αSMA-TK mice were used to demonstrate that depletion of proliferative aHSCs attenuates liver fibrosis induced by carbon tetrachloride and 3,5-diethoxycarbonyl-1,4-dihydrocollidine. Through integrating data from single-cell and bulk sequencing, Periostin was identified as a distinctive hallmark of proliferative aHSC subpopulation. Elevated levels of Periostin were detected in fibrotic livers of both humans and mice, primarily within aHSCs. However, hepatic Periostin levels were decreased along with depletion of proliferative aHSCs. Deficiency of Periostin led to reduced liver fibrosis and suppressed hepatocyte epithelial-mesenchymal transition (EMT). Periostin-overexpressing HSCs, exhibiting a proliferative aHSC phenotype, release bone morphogenetic protein-1 (Bmp-1), which activates EGFR signaling, inducing hepatocyte EMT and contributing to liver fibrosis. In conclusion, Periostin in aHSCs drives their acquisition of a proliferative phenotype and the release of Bmp-1. Proliferative aHSC subpopulation-derived Bmp-1 induces hepatocyte EMT via EGFR signaling, promoting liver fibrogenesis. Bmp-1 and Periostin should be potential therapeutic targets for liver fibrosis.

Platelet-derived growth factor receptor β-targeted positron emission tomography imaging for the noninvasive monitoring of liver fibrosis.

Noninvasive quantifying activated hepatic stellate cells (aHSCs) by molecular imaging is helpful for assessing disease progression and therapeutic responses of liver fibrosis. Our purpose is to develop platelet-derived growth factor receptor β (PDGFRβ)-targeted radioactive tracer for assessing liver fibrosis by positron emission tomography (PET) imaging of aHSCs. Comparative transcriptomics, immunofluorescence staining and flow cytometry were used to evaluate PDGFRβ as biomarker for human aHSCs and determine the correlation of PDGFRβ with the severity of liver fibrosis. The high affinity affibody for PDGFRβ (ZPDGFRβ) was labeled with gallium-68 (68Ga) for PET imaging of mice with carbon tetrachloride (CCl4)-induced liver fibrosis. Binding of the [68Ga]Ga-labeled ZPDGFRβ ([68Ga]Ga-DOTA-ZPDGFRβ) for aHSCs in human liver tissues was measured by autoradiography. PDGFRβ overexpressed in aHSCs was highly correlated with the severity of liver fibrosis in patients and CCl4-treated mice. The 68Ga-labeled ZPDGFRβ affibody ([68Ga]Ga-DOTA-ZPDGFRβ) showed PDGFRβ-dependent binding to aHSCs. According to the PET imaging, hepatic uptake of [68Ga]Ga-DOTA-ZPDGFRβ increased with the accumulation of aHSCs and collagens in the fibrotic livers of mice. In contrast, hepatic uptake of [68Ga]Ga-DOTA-ZPDGFRβ decreased with spontaneous recovery or treatment of liver fibrosis, indicating that the progression and therapeutic responses of liver fibrosis in mice could be visualized by PDGFRβ-targeted PET imaging. [68Ga]Ga-DOTA-ZPDGFRβ also bound human aHSCs and visualized fibrosis in patient-derived liver tissues. PDGFRβ is a reliable biomarker for both human and mouse aHSCs. PDGFRβ-targeted PET imaging could be used for noninvasive monitoring of liver fibrosis in mice and has great potential for clinical translation.

Portulaca oleracea L. extract relieve mice liver fibrosis by inhibiting TLR-4/NF-κB, Bcl-2/Bax and TGF-β1/Smad2 signalling transduction

Portulaca oleracea L. are annual herb, which has various pharmacological effects including hepatoprotective property. However, the effect of Portulaca oleracea L. (POL-1) in mice with carbon tetrachloride (CCl4)-induced liver fibrosis and its mechanism of action have not been clarified. POL-1 ameliorated the CCl4-induced liver fibrosis in mice, as shown by decreased collagen deposition and the decreased expression of liver fibrosis marker collagen I and α-smooth muscle actin (α-SMA) mRNA. In addition, treatment with POL-1 suppressed the proliferation of activated human hepatic stellate cell line (LX-2). POL-1 inhibited the oxidative stress and inflammation in fibrotic livers of mice. Mechanistically, POL-1 inhibited the CCl4-induced expression of toll-like receptor-4 (TLR4), myeloid differentiation factor 88 (MyD88), nuclear factor kappa-B (NF-κBp65) p65, Bcl2-associated X (Bax), transforming growth factor-β1 (TGF-β1) and drosophila mothers against decapentaplegic 2 (Smad2) proteins, upregulated B-cell lymphoma −2 (Bcl-2) proteins in livers of mice. These findings suggested that POL-1 attenuated liver fibrosis.

The C-type lectin COLEC10 is predominantly produced by hepatic stellate cells and involved in the pathogenesis of liver fibrosis

Hepatic stellate cell is one of the major nonparenchymal cell types in liver. It has been proved the hepatic stellate cells are activated upon liver injury and produce excessive extracellular matrix to induce liver fibrosis. Single-cell RNA sequencing has been introduced to identify the subpopulations and function of hepatic stellate cells for its remarkable resolution of representation of single-cell transcriptome. According to the re-analysis of single-cell RNA sequencing data and pseudotime trajectory inference, we have found the C-type lectins including Colec10 and Colec11 are not produced by hepatocytes but predominantly produced by hepatic stellate cells, especially quiescent ones in the mice livers. In addition, the expression of Colec10 is decreased in the fibrotic livers of CCl4-challenged mice. COLEC10 is also mainly expressed in the hepatic stellate cells of human livers and the expression of COLEC10 is decreased with the progression of liver fibrosis. The bulk RNA sequencing data of the lentivirus transfected LX-2 cells indicates the function of COLEC10 is associated with inflammation, angiogenesis and extracellular matrix alteration. Surprisingly, the in vitro overexpression of COLEC10 in LX-2 cells promotes the mRNA expression of extracellular matrix components including COL1A1, COL1A2 and COL3A1 and the extracellular matrix degradation enzyme MMP2. To further investigate the role of COLEC10 in the pathogenesis of liver fibrosis, the serum concentration of COLEC10 in patients with chronic liver disease and healthy donors is measured. The serum concentration of COLEC10 is elevated in the patients with chronic liver disease compared to the healthy donors and positively correlated with serum concentration of the D-dimer but not the most of liver function markers. Altogether, we conclude that the C-type lectin COLEC10 is predominantly produced by the hepatic stellate cells and involved in the pathogenesis of liver fibrosis.

Targeting GPR65 alleviates hepatic inflammation and fibrosis by suppressing the JNK and NF-κB pathways

BackgroundG-protein coupled receptors (GPCRs) are recognized as attractive targets for drug therapy. However, it remains poorly understood how GPCRs, except for a few chemokine receptors, regulate the progression of liver fibrosis. Here, we aimed to reveal the role of GPR65, a proton-sensing receptor, in liver fibrosis and to elucidate the underlying mechanism.MethodsThe expression level of GPR65 was evaluated in both human and mouse fibrotic livers. Furthermore, Gpr65-deficient mice were treated with either bile duct ligation (BDL) for 21 d or carbon tetrachloride (CCl4) for 8 weeks to investigate the role of GPR65 in liver fibrosis. A combination of experimental approaches, including Western blotting, quantitative real-time reverse transcription‑polymerase chain reaction (qRT-PCR), and enzyme-linked immunosorbent assay (ELISA), confocal microscopy and rescue studies, were used to explore the underlying mechanisms of GPR65’s action in liver fibrosis. Additionally, the therapeutic potential of GPR65 inhibitor in the development of liver fibrosis was investigated.ResultsWe found that hepatic macrophages (HMs)-enriched GPR65 was upregulated in both human and mouse fibrotic livers. Moreover, knockout of Gpr65 significantly alleviated BDL- and CCl4-induced liver inflammation, injury and fibrosis in vivo, and mouse bone marrow transplantation (BMT) experiments further demonstrated that the protective effect of Gpr65 knockout is primarily mediated by bone marrow-derived macrophages (BMMs). Additionally, in vitro data demonstrated that Gpr65 silencing and GPR65 antagonist inhibited, while GPR65 overexpression and application of GPR65 endogenous and exogenous agonists enhanced the expression and release of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and transforming growth factor-β (TGF-β), all of which subsequently promoted the activation of hepatic stellate cells (HSCs) and the damage of hepatocytes (HCs). Mechanistically, GPR65 overexpression, the acidic pH and GPR65 exogenous agonist induced up-regulation of TNF-α and IL-6 via the Gαq-Ca2+-JNK/NF-κB pathways, while promoted the expression of TGF-β through the Gαq-Ca2+-MLK3-MKK7-JNK pathway. Notably, pharmacological GPR65 inhibition retarded the development of inflammation, HCs injury and fibrosis in vivo.ConclusionsGPR65 is a major regulator that modulates the progression of liver fibrosis. Thus, targeting GPR65 could be an effective therapeutic strategy for the prevention of liver fibrosis.

LncRNA-Gm9866 promotes liver fibrosis by activating TGFβ/Smad signaling via targeting Fam98b

ObjectiveThe exact mechanism and target molecules of liver fibrosis have remained largely elusive. Here, we investigated the role of long noncoding RNA Gm9866(lncRNA-Gm9866) on liver fibrosis.MethodsThe transcription of lncRNA-Gm9866 in activated cells and mouse fibrotic livers was determined by quantitative polymerase chain reaction (qRT-PCR). The effects of lentivirus-mediated knockdown or overexpression of lncRNA-Gm9866 in liver fibrosis were examined in vitro and in vivo. Furthermore, bioinformatics analysis, cell samples validation, fluorescence in situ hybridization (FISH) co-localization, RNA binding protein immunoprecipitation (RIP), actinomycin D test and Western blot (WB) were carried out to explore the potential mechanism of lncRNA-Gm9866.ResultsThe expression of α-smooth muscle actin (α-SMA), Collagen I (COL-1) and lncRNA-Gm9866 were significantly increased in tissues and cells. Overexpressing lncRNA-Gm9866 promoted the activation of hepatic stellate cells (HSCs). Silencing lncRNA-Gm9866 inhibited the activation of HSCs and transforming growth factor-β1 (TGFβ1) induced fibrosis. Overexpressing lncRNA-Gm9866 promoted hepatocytes (HCs) apoptosis and the expression of pro-fibrogenic genes, inhibited the proliferation and migration of HCs. Knockdown of lncRNA-Gm9866 inhibited the apoptosis of HCs, the expression of pro-fibrogenic genes, TGFβ1 induced fibrosis and the occurrence of carbon tetrachloride (CCl4)-induced liver fibrosis, and promoted the proliferation and migration of HCs. Mechanistically, lncRNA-Gm9866 may directly bine with Fam98b. Silencing Fam98b in stably overexpressing lncRNA-Gm9866 cell lines reversed the increase of pro-fibrogenic genes and pro-apoptotic genes, fibrosis related pathway protein TGFβ1, Smad2/3, p-Smad2/3 and Notch3 induced by overexpressing lncRNA-Gm9866.ConclusionsLncRNA-Gm9866 may regulate TGFβ/Smad and Notch pathways by targeting Fam98b to regulate liver fibrosis. LncRNA-Gm9866 may be a new target for diagnosis and treatment of liver fibrosis.

LncRNA Helf promotes hepatic inflammation and fibrosis by interacting with PTBP1 to facilitate PIK3R5 mRNA stabilization

BackgroundHepatic fibrosis is a common consequence of chronic liver diseases without approved antifibrotic therapies. Long noncoding RNAs (lncRNAs) play an important role in various pathophysiological processes. However, the functions of certain lncRNAs involved in mediating the antifibrotic role remain largely unclear.MethodsThe RNA level of lnc-High Expressed in Liver Fibrosis (Helf) was detected in both mouse and human fibrotic livers. Furthermore, lnc-Helf-silenced mice were treated with carbon tetrachloride (CCl4) or bile duct ligation (BDL) to investigate the function of lnc-Helf in liver fibrosis.ResultsWe found that lnc-Helf has significantly higher expression in human and mouse fibrotic livers as well as M1 polarized hepatic macrophages (HMs) and activated hepatic stellate cells (HSCs). In vivo studies showed that silencing lnc-Helf by AAV8 vector alleviates CCl4- and BDL-induced hepatic inflammation and fibrosis. Furthermore, in vitro experiments revealed that lnc-Helf promotes HSCs activation and proliferation, as well as HMs M1 polarization and proliferation in the absence or presence of cytokine stimulation. Mechanistically, our data illustrated that lnc-Helf interacts with RNA binding protein PTBP1 to promote its interaction with PIK3R5 mRNA, resulting in increased stability and activating the AKT pathway, thus promoting HSCs and HMs activation and proliferation, which augments hepatic inflammation and fibrosis.ConclusionOur results unveil a lnc-Helf/PTBP1/PIK3R5/AKT feedforward, amplifying signaling that exacerbates the process of hepatic inflammation and fibrosis, thus providing a possible therapeutic strategy for hepatic fibrosis.

Hepatic stellate cell-intrinsic role of SOCS1 in controlling hepatic fibrogenic response and the pro-inflammatory macrophage compartment during liver fibrosis.

Hepatic stellate cells (HSC) become activated, differentiate to myofibroblasts and produce extracellular fibrillar matrix during liver fibrosis. The hepatic fibrogenic response is orchestrated by reciprocal interactions between HSCs and macrophages and their secreted products. SOCS1 can regulate several cytokines and growth factors implicated in liver fibrosis. Here we investigated the role of SOCS1 in regulating HSC activation. Mice lacking SOCS1 in HSCs (Socs1ΔHSC) were generated by crossing Socs1fl/fl and LratCre mice. Liver fibrosis was induced by carbon tetrachloride and evaluated by Sirius red staining, hydroxyproline content and immunostaining of myofibroblasts. Gene expression of pro-fibrogenic factors, cytokines, growth factors and chemokines were quantified by RT-qPCR. The phenotype and the numbers of intrahepatic leukocyte subsets were studied by flow cytometry. The impact of fibrosis on the development of diethyl nitrosamine-induced hepatocellular carcinoma was evaluated. Socs1ΔHSC mice developed more severe liver fibrosis than control Socs1fl/fl mice that was characterized by increased collagen deposition and myofibroblast differentiation. Socs1ΔHSC mice showed a significant increase in the expression of smooth muscle actin, collagens, matrix metalloproteases, cytokines, growth factors and chemokines in the liver following fibrosis induction. The fibrotic livers of Socs1ΔHSC mice displayed heightened inflammatory cell infiltration with increased proportion and numbers of Ly6ChiCCR2+ pro-inflammatory macrophages. This macrophage population contained elevated numbers of CCR2+CX3CR1+ cells, suggesting impaired transition towards restorative macrophages. Fibrosis induction following exposure to diethyl nitrosamine resulted in more numerous and larger liver tumor nodules in Socs1ΔHSC mice than in Socs1fl/fl mice. Our findings indicate that (i) SOCS1 expression in HSCs is a critical to control liver fibrosis and development of hepatocaellular carcinoma, and (ii) attenuation of HSC activation by SOCS1 regulates pro-inflammatory macrophage recruitment and differentiation during liver fibrosis.

Parkin-Mediated Mitophagy by TGF-β Is Connected with Hepatic Stellate Cell Activation.

Hepatic stellate cells (HSCs) are the main contributors to the development and progression of liver fibrosis. Parkin is an E3 ligase involved in mitophagy mediated by lysosomes that maintains mitochondrial homeostasis. Unfortunately, there is little information regarding the regulation of parkin by transforming growth factor-β (TGF-β) and its association with HSC trans-differentiation. This study showed that parkin is upregulated in fibrotic conditions and elucidated the underlying mechanism. Parkin was observed in the cirrhotic region of the patient liver tissues and visualized using immunostaining and immunoblotting of mouse fibrotic liver samples and primary HSCs. The role of parkin-mediated mitophagy in hepatic fibrogenesis was examined using TGF-β-treated LX-2 cells with mitophagy inhibitor, mitochondrial division inhibitor 1. Parkin overexpression and its colocalization with desmin in human tissues were found. Increased parkin in fibrotic liver homogenates of mice was observed. Parkin was expressed more abundantly in HSCs than in hepatocytes and was upregulated under TGF-β. TGF-β-induced parkin was due to Smad3. TGF-β facilitated mitochondrial translocation, leading to mitophagy activation, reversed by mitophagy inhibitor. However, TGF-β did not change mitochondrial function. Mitophagy inhibitor suppressed profibrotic genes and HSC migration mediated by TGF-β. Collectively, parkin-involved mitophagy by TGF-β facilitates HSC activation, suggesting mitophagy may utilize targets for liver fibrosis.

Single-cell RNA sequencing reveals the heterogeneity and intercellular communication of hepatic stellate cells and macrophages during liver fibrosis.

Uncontrolled and excessive progression of liver fibrosis is thought to be the prevalent pathophysiological cause of liver cirrhosis and hepatocellular cancer, and there are currently no effective antifibrotic therapeutic options available. Intercellular communication and cellular heterogeneity in the liver are involved in the progression of liver fibrosis, but the exact nature of the cellular phenotypic changes and patterns of interregulatory remain unclear. Here, we performed single-cell RNA sequencing on nonparenchymal cells (NPCs) isolated from normal and fibrotic mouse livers. We identified eight main types of cells, including endothelial cells, hepatocytes, dendritic cells, B cells, natural killer/T (NK/T) cells, hepatic stellate cells (HSCs), cholangiocytes and macrophages, and revealed that macrophages and HSCs exhibit the most variance in transcriptional profile. Further analyses of HSCs and macrophage subpopulations and ligand-receptor interaction revealed a high heterogeneity characterization and tightly interregulated network of these two groups of cells in liver fibrosis. Finally, we uncovered a profibrotic Thbs1+ macrophage subcluster, which expands in mouse and human fibrotic livers, activating HSCs via PI3K/AKT/mTOR signaling pathway. Our findings decode unanticipated insights into the heterogeneity of HSCs and macrophages and their intercellular crosstalk at a single-cell level, and may provide potential therapeutic strategies in liver fibrosis.

Farnesoid X receptor activation is required for the anti-inflammatory and anti-oxidative stress effects of Alisol B 23-acetate in carbon tetrachloride-induced liver fibrosis in mice

Previous studies have shown that Alisol B 23-acetate (23ABA) had potent liver-protection effects, however, its roles and potential mechanisms in carbon tetrachloride (CCl4)-induced liver fibrosis remain to be determined. The present study aimed to investigate the effects of 23ABA on CCl4-induced liver fibrosis and tried to elucidate the underlying mechanisms by focusing on regulating of farnesoid X receptor (FXR). In vivo study found that 23ABA alleviated the CCl4-induced liver injury, and showed no obvious systemic toxicity on mice. 23ABA inhibited the collagen production, decreased sera levels of hyaluronic acid (HA) and procollagen type III (PC-III), lowered mRNA expression of α-smooth muscle actin (α-SMA), fibronectin, collagen I and collagen III in livers of mice. 23ABA inhibited the mRNA expressions and the sera levels of interleukin-6 (IL-6), IL-1β, and tumor necrosis factor-α (TNF-α), as well as decreased the expression of cyclooxygenase 2 (COX-2) in fibrotic livers of mice. Besides, 23ABA decreased levels of reactive oxygen species (ROS) and malondialdehyde (MDA), increased glutathione (GSH) level, enhanced activities of superoxide dismutase (SOD) and glutathione reductase (GR) as well as increased mRNA expression of nuclear factor-E2-related factor 2 (Nrf2), glutamate-cysteine ligase, catalytic subunit (GCLC) and glutamate-cysteine ligase, modifier subunit (GCLM). Further study showed that the anti-liver injury and anti-fibrotic effects of 23ABA were abrogated by FXR antagonist guggulsterone (GS) in vivo. In addition, the inhibition effects of 23ABA on liver inflammation and oxidative stress were also weakened by treatment with GS in CCl4-induced fibrotic mice livers. In conclusion, the protective effects of 23ABA against CCl4-induced liver injury and fibrosis, due to FXR-mediated regulation of liver inflammation and oxidative stress.

Caveolin-1 depletion attenuates hepatic fibrosis via promoting SQSTM1-mediated PFKL degradation in HSCs.

The key glycolytic enzyme phosphofructokinase (PFK) is responsible for maintaining glycolytic stability and an important energy source for activating hepatic stellate cells (HSCs). However, its regulation in activated HSCs remains unclear. Caveolin-1 (Cav1), a major constituent of caveolae, has emerged as a key target for triggering glycolysis. However, the relationship between Cav1 and glycolysis during HSC activation is not well established. In this study, Cav1 was upregulated in mouse and human fibrotic liver tissues. We concluded that HSC-specific Cav1 knockdown markedly alleviates liver injury and fibrosis. Mechanistically, Cav1 was elevated during primary mouse HSC activation, competing with SQSTM1 for the regulatory subunit of PFK liver type and inhibiting the SQSTM1-mediated autophagy-independent lysosomal degradation pathway to sustain HSC activation. We also identified the heptapeptide alamandine as a promising therapeutic agent that downregulates Cav1 protein levels via proteasomal degradation and may impair glycolysis. Our study provides evidence of the crucial role and mechanism of Cav1 in the glucose metabolic network in HSCs and highlights Cav1 as a critical therapeutic target for the treatment of liver fibrosis.

RNF41 orchestrates macrophage-driven fibrosis resolution and hepatic regeneration.

Hepatic inflammation is a common trigger of chronic liver disease. Macrophage activation is a predictive parameter for survival in patients with cirrhosis. Ring finger protein 41 (RNF41) negatively regulates proinflammatory cytokines and receptors; however, the precise involvement of macrophage RNF41 in liver cirrhosis remains unknown. Here, we sought to understand how RNF41 dictates macrophage fate in hepatic fibrosis and repair within the inflammatory milieu. We found that RNF41 expression is down-regulated in CD11b+ macrophages recruited to mouse fibrotic liver and to patient cirrhotic liver regardless of cirrhosis etiology. Prolonged inflammation with TNF-α progressively reduced macrophage RNF41 expression. We designed a macrophage-selective gene therapy with dendrimer-graphite nanoparticles (DGNPs) to explore the influence of macrophage RNF41 restoration and depletion in liver fibrosis and regeneration. RNF41 expression induced in CD11b+ macrophages by DGNP-conjugated plasmids ameliorated liver fibrosis, reduced liver injury, and stimulated hepatic regeneration in fibrotic mice with or without hepatectomy. This therapeutic effect was mainly mediated by the induction of insulin-like growth factor 1. Conversely, depletion of macrophage RNF41 worsened inflammation, fibrosis, hepatic damage, and survival. Our data reveal implications of macrophage RNF41 in the control of hepatic inflammation, fibrosis, and regeneration and provide a rationale for therapeutic strategies in chronic liver disease and potentially other diseases characterized by inflammation and fibrosis.