Human Fibrotic Liver Research Articles

Oxidative stress-mediated mitochondrial fission promotes hepatic stellate cell activation via stimulating oxidative phosphorylation

Previous studies have demonstrated dysregulated mitochondrial dynamics in fibrotic livers and hepatocytes. Little is currently known about how mitochondrial dynamics are involved, nor is it clear how mitochondrial dynamics participate in hepatic stellate cell (HSC) activation. In the present study, we investigated the role of mitochondrial dynamics in HSC activation and the underlying mechanisms. We verified that mitochondrial fission was enhanced in human and mouse fibrotic livers and active HSCs. Moreover, increased mitochondrial fission driven by fis1 overexpression could promote HSC activation. Inhibiting mitochondrial fission using mitochondrial fission inhibitor-1 (Mdivi-1) could inhibit activation and induce apoptosis of active HSCs, indicating that increased mitochondrial fission is essential for HSC activation. Mdivi-1 treatment also induced apoptosis in active HSCs in vivo and thus ameliorated CCl4-induced liver fibrosis. We also found that oxidative phosphorylation (OxPhos) was increased in active HSCs, and OxPhos inhibitors inhibited activation and induced apoptosis in active HSCs. Moreover, increasing mitochondrial fission upregulated OxPhos, while inhibiting mitochondrial fission downregulated OxPhos, suggesting that mitochondrial fission stimulates OxPhos during HSC activation. Next, we found that inhibition of oxidative stress using mitoquinone mesylate (mitoQ) and Tempol inhibited mitochondrial fission and OxPhos and induced apoptosis in active HSCs, suggesting that oxidative stress contributes to excessive mitochondrial fission during HSC activation. In conclusion, our study revealed that oxidative stress contributes to enhanced mitochondrial fission, which triggers OxPhos during HSC activation. Importantly, inhibiting mitochondrial fission has huge prospects for alleviating liver fibrosis by eliminating active HSCs.

Mesenchymal stem cells improve liver fibrosis and protect hepatocytes by promoting microRNA-148a-5p-mediated inhibition of Notch signaling pathway

BackgroundMesenchymal stem cells (MSCs) are considered to be a potential therapeutic tool for liver fibrosis. Inhibiting the activation of hepatic stellate cells (HSCs) and protecting hepatocytes are important mechanisms for the anti-fibrotic effect of MSCs. However, how MSCs inhibit liver fibrosis by regulating the expression of microRNAs (miRNAs) has not been fully clarified.MethodsTransforming growth factor-β1 (TGF-β1)-activated HSCs LX-2 were single cultured or co-cultured with human umbilical cord mesenchymal stem cells (HUC-MSCs). High-throughput sequencing was used to evaluate the differentially expressed microRNAs (DEMs) between the two groups. Quantitative real-time PCR (qRT-PCR), Western blot, and transfection experiments were used to investigate and screen the most significantly up-regulated DEM. Bioinformatics analysis was used to predict the target mRNAs and the potential functions of the DEM. The possible mechanism of HUC-MSCs against liver fibrosis was analyzed by co-culture experiment of HUC-MSCs with LX-2 cells, and HUC-MSCs treatment of Bile duct ligation (BDL)-induced liver fibrosis in mice. Finally, the mechanism of the DEM regulating liver fibrosis was confirmed in human liver fibrosis specimens.ResultsMicroRNA-148a-5p (miR-148a-5p) was the most significantly up-regulated DEM in activated LX-2 cells co-cultured with HUC-MSCs compared with LX-2 cells single cultured. Up-regulation of the expression of miR-148a-5p in activated LX-2 cells could significantly inhibit the expression of hepatic fibrosis markers α-SMA and Col1α1. Notch2 was one target gene of miR-148a-5p. Co-cultured with HUC-MSCs could inhibit the activation of LX-2 cells by inhibiting the expression of the Notch2 and the Notch signaling pathway. In addition, HUC-MSCs treatment could up-regulate the expression of miR-148a-5p in liver tissue and hepatocytes, promote the proliferation and avoid the apoptosis of hepatocytes, and reduce the degree of fibrosis by inhibiting expression of the Notch2 and the Notch signaling pathway in BDL-induced liver fibrosis mice. Moreover, miR-148a-5p was down-regulated and Notch2 was up-regulated in fibrotic human liver tissues compared with the normal livers.ConclusionsHUC-MSCs treatment could inhibit HSCs activation, protect hepatocytes, and alleviate BDL-induced liver fibrosis in mice by up-regulating the expression of miR-148-5p and inhibiting the Notch signaling pathway. The down-regulation of miR-148-5p and up-regulation of Notch2 could be used as biomarkers to monitor the progression of liver fibrosis.

LncRNA XIST promotes mitochondrial dysfunction of hepatocytes to aggravate hepatic fibrogenesis via miR-539-3p/ADAMTS5 axis.

A previous study indicated that long non-coding RNA X-inactive-specific transcript (XIST) promoted ethanol-induced HSCs autophagy and activation. Considering the critical role of HSC activation in hepatic fibrosis, the aim of the present study was to reveal the exact role of XIST in liver fibrosis and its underlying mechanism. The expression of XIST in the liver from CCL4-induced mice and control mice as well as human fibrotic liver tissue and healthy liver tissue was examined. The mitochondrial reactive oxygen species (mtROS), mitochondrial membrane potential (MMP), and mitochondrial morphology were measured to assess the mitochondrial damage. The relationship between XIST and miR-539-3p as well as between miR-539-3p and ADAMTS5 was verified by a dual-luciferase reporter assay. The expression levels of HSCs activation markers were examined by Western blot. The results showed that the XIST was upregulated in fibrotic liver tissue, and overexpression of XIST induced mitochondrial dysfunction in hepatocytes. miR-539-3p directly targeted XIST, and ADAMTS5 mRNA was a downstream target of miR-539-3p. Knockdown of miR-539-3p led to an increased mitochondrial damage in hepatocytes in terms of reduced mitochondrial length, decreased MMP, and increased ROS production. However, the depletion of ADAMTS5 reversed the regulatory effect of XIST on mitochondrial damage in hepatocytes and the activation of HSCs. Our study revealed the critical role of the XIST/miR-539-3p/ADAMTS5 axis in regulating mitochondrial damage in hepatocytes and the activation of HSCs. This study may provide a potential therapeutic strategy for the treatment of liver fibrosis.

Hyaluronan synthase 2, a target of miR-200c, promotes carbon tetrachloride-induced acute and chronic liver inflammation via regulation of CCL3 and CCL4

Liver fibrosis occurs during wound healing after repeated liver injury and is characterized by extensive extracellular matrix deposition. We previously identified hyaluronan synthase 2 (HAS2) as a driver of liver fibrosis and hepatic stellate cell (HSC) activation. Developing strategies to suppress HSC activation is key to alleviating liver fibrosis, and HAS2 is an attractive candidate for intervention. To gain insight into the molecular function of HAS2, we investigated its posttranscriptional regulation. We found that miR-200c directly targets the 3’ untranslated regions of HAS2. Moreover, miR-200c and HAS2 were inversely expressed in fibrotic human and mouse livers. After establishing the direct interaction between miR-200c and HAS2, we investigated the functional outcome of regulating HAS2 expression in three murine models: CCl4-induced acute liver injury, CCl4-induced chronic liver fibrosis, and bile duct ligation-induced liver fibrosis. Hepatic Has2 expression was induced by acute and chronic CCl4 treatment. In contrast, miR-200c expression was decreased after CCl4 treatment. HSC-specific Has2 deletion reduced the expression of inflammatory markers and infiltration of macrophages in the models. Importantly, hyaluronidase-2 (HYAL2) but not HYAL1 was overexpressed in fibrotic human and murine livers. HYAL2 is an enzyme that can cleave the extracellular matrix component hyaluronan. We found that low-molecular-weight hyaluronan stimulated the expression of inflammatory genes. Treatment with the HA synthesis inhibitor 4-methylumbelliferone alleviated bile duct ligation-induced expression of these inflammatory markers. Collectively, our results suggest that HAS2 is negatively regulated by miR-200c and contributes to the development of acute liver injury and chronic liver inflammation via hyaluronan-mediated immune signaling.

The Unfolded Protein Response Regulates ER‐phagy in Fibrogenic Hepatic Stellate Cells

IntroductionLiver injury activates Hepatic Stellate Cells (HSCs) which secrete fibrogenic proteins such as collagen I to promote scarring. Increased translation of the collagen I precursor procollagen I by HSCs causes ER stress due to 30% of nascent procollagen I failing to fold correctly, placing a burden on the ER; however, it is unclear how HSCs adapt to this stress. ER stress activates the Unfolded Protein Response (UPR) which signals through pathways mediated by Activating Transcription Factor 6α (ATF6α), Inositol Requiring Enzyme 1 (IRE1α), or Protein Kinase R‐like ER kinase (PERK). ATF6α or IRE1α inhibition limits HSC activation and promotes apoptosis in vitro, while deletion of ATF6α or IRE1α limits fibrogenesis and reduces HSC number in vivo. While it is clear that the UPR plays a crucial role in HSC activation and survival, the mechanisms that facilitate this role are unknown. Recent work shows that misfolded procollagen I can undergo ER‐phagy, where receptors on the ER recruit autophagic membranes to engulf portions of the ER containing misfolded proteins and target them for degradation. ER‐phagy can be activated by ER stress, but the fibrogenic role and regulation of ER‐phagy in HSCs is unknown. We hypothesized that UPR induction of ER‐phagy targets misfolded procollagen I for degradation, thus promoting HSC survival and fibrogenesis.MethodsExpression of ER‐phagy receptors (Cell‐cycle progression gene 1 (CCPG1), Family with sequence similarity 134B (FAM134B), and Atlastin 3 (ATL3)) was assessed in 1) livers from patients with advanced fibrosis or controls (GSE25097), 2) murine livers harvested from age‐ and sex‐matched mice following bile‐duct ligation (3 weeks) or sham controls; and 3) primary hHSCs or mHSCs, or immortalized hHSCs (LX‐2) following TGFβ treatment (2ng/mL, 24h). ER‐phagic flux was measured in LX‐2 cells expressing a fluorescent ER‐phagy reporter (RAMP4‐GFP‐mCherry), with RAMP4 as a known ER‐phagic cargo. UPR signaling was disrupted using inhibitors targeting ATF6α (6µM Ceapin‐A7) or IRE1α (0.5µM 4µ8C), or RNAi targeting PERK. CCPG1 or FAM134B were knocked out from LX‐2 cells using CRISPR‐Cas9. HSC activation and UPR signaling were measured by qPCR and Western blot.ResultsExpression of CCPG1 and ATL3 increased in fibrotic human livers compared to controls, while CCPG1 mRNA, and FAM134B protein and mRNA levels increased in fibrotic mouse livers compared to controls. In vitro activation of primary hHSCs or mHSCs also increased CCPG1, FAM134B, and ATL3 protein and mRNA levels, as well as increased ER‐phagic flux in LX‐2 cells. Regulation of ER‐phagic flux and expression of ER‐phagy receptors was UPR‐dependent, with inhibition of ATF6α or IRE1α blocking TGFβ‐induced ER‐phagic flux, while PERK knockdown increased ER‐phagic flux. Interestingly, CCPG1 or FAM134B loss did not impact HSC activation, or induce the pro‐apoptotic UPR.ConclusionsER‐phagy receptors increased in fibrotic human and murine livers, and TGFβ upregulated ER‐phagy receptors and increased ER‐phagic flux in HSCs through UPR‐dependent mechanisms. Deletion of CCPG1 or FAM134B did not impact HSC activation, suggesting redundancy between ER‐phagic receptors. Future studies will focus on understanding the fibrogenic role of ER‐phagy in HSCs.

Inhibition of ASCT2 induces hepatic stellate cell senescence with modified proinflammatory secretome through an IL-1α/NF-κB feedback pathway to inhibit liver fibrosis

Senescence of activated hepatic stellate cells (aHSCs) is a stable growth arrest that is implicated in liver fibrosis regression. Senescent cells often accompanied by a multi-faceted senescence-associated secretory phenotype (SASP). But little is known about how alanine-serine-cysteine transporter type-2 (ASCT2), a high affinity glutamine transporter, affects HSC senescence and SASP during liver fibrosis. Here, we identified ASCT2 is mainly elevated in aHSCs and positively correlated with liver fibrosis in human and mouse fibrotic livers. We first discovered ASCT2 inhibition induced HSCs to senescence in vitro and in vivo. The proinflammatory SASP were restricted by ASCT2 inhibition at senescence initiation to prevent paracrine migration. Mechanically, ASCT2 was a direct target of glutaminolysis-dependent proinflammatory SASP, interfering IL-1α/NF-κB feedback loop via interacting with precursor IL-1α at Lys82. From a translational perspective, atractylenolide III is identified as ASCT2 inhibitor through directly bound to Asn230 of ASCT2. The presence of –OH group in atractylenolide III is suggested to be favorable for the inhibition of ASCT2. Importantly, atractylenolide III could be utilized to treat liver fibrosis mice. Taken together, ASCT2 controlled HSC senescence while modifying the proinflammatory SASP. Targeting ASCT2 by atractylenolide III could be a therapeutic candidate for liver fibrosis.

Rab31 promotes activation of hepatic stellate cells by accelerating TGF-β receptor II complex endocytosis.

Hepatic stellate cells activation is the key process of liver fibrosis, revealing the molecular mechanism of which is helpful to provide an effective target for inhibiting liver fibrosis. Rab31, a small GTPase, regulates the specificity of intracellular vesicular transport system, and is crucial for signal transduction. However, whether Rab31 is involved in hepatic stellate cells activation is unknown. Analysis of the differences in gene expression between human healthy and fibrotic liver tissues by sequencing revealed that Rab31 was significantly upregulated in fibrotic tissues. Immunohistochemistry and immunofluorescence analysis confirmed that Rab31 positively correlated with hepatic fibrosis. Next, mouse primary hepatic stellate cells were prepared, and their continuous activation was accompanied by Rab31 expression increased. Interestingly, knockdown of Rab31 by lentivirus can significantly restrict those cell activation. Subsequently, the vary of signal transduction after Rab31 knockdown was detected, its presented that the TGF-β/Smads signaling was obviously affected. Following experiments identified that Rab31 knockdown significantly inhibited the TGF-β activation and led to the failure of hepatic stellate cells activation. Importantly, we revealed that Rab31 knockdown could inhibit TGF-β receptor II complex endocytosis, a prerequisite for the activation of TGF-β signaling. Finally, in a mouse CCl4 fibrosis model, we proved that Rab31 knockdown markedly inhibited hepatic fibrosis. Our study demonstrated that Rab31 could promote hepatic stellate cells activation by accelerating TGF-β Receptor II complex endocytosis, suggesting that interfering with Rab31 could be an effectively strategy to inhibit hepatic fibrosis progression.

Focal adhesion kinase-related non-kinase ameliorates liver fibrosis by inhibiting aerobic glycolysis via the FAK/Ras/c-myc/ENO1 pathway.

BACKGROUNDHepatic stellate cell (HSC) hyperactivation is a central link in liver fibrosis development. HSCs perform aerobic glycolysis to provide energy for their activation. Focal adhesion kinase (FAK) promotes aerobic glycolysis in cancer cells or fibroblasts, while FAK-related non-kinase (FRNK) inhibits FAK phosphorylation and biological functions.AIMTo elucidate the effect of FRNK on liver fibrosis at the level of aerobic glycolytic metabolism in HSCs.METHODSMouse liver fibrosis models were established by administering CCl4, and the effect of FRNK on the degree of liver fibrosis in the model was evaluated. Transforming growth factor-β1 was used to activate LX-2 cells. Tyrosine phosphorylation at position 397 (pY397-FAK) was detected to identify activated FAK, and the expression of the glycolysis-related proteins monocarboxylate transporter 1 (MCT-1) and enolase1 (ENO1) was assessed. Bioinformatics analysis was performed to predict putative binding sites for c-myc in the ENO1 promoter region, which were validated with chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays.RESULTSThe pY397-FAK level was increased in human fibrotic liver tissue. FRNK knockout promoted liver fibrosis in mouse models. It also increased the activation, migration, proliferation and aerobic glycolysis of primary hepatic stellate cells (pHSCs) but inhibited pHSC apoptosis. Nevertheless, opposite trends for these phenomena were observed after exogenous FRNK treatment in LX-2 cells. Mechanistically, the FAK/Ras/c-myc/ENO1 pathway promoted aerobic glycolysis, which was inhibited by exogenous FRNK.CONCLUSIONFRNK inhibits aerobic glycolysis in HSCs by inhibiting the FAK/Ras/c-myc/ENO1 pathway, thereby improving liver fibrosis. FRNK might be a potential target for liver fibrosis treatment.

Targeting ER protein TXNDC5 in hepatic stellate cell mitigates liver fibrosis by repressing non-canonical TGFβ signalling

Background and objectivesLiver fibrosis (LF) occurs following chronic liver injuries. Currently, there is no effective therapy for LF. Recently, we identified thioredoxin domain containing 5 (TXNDC5), an ER protein disulfide...

Silencing IQGAP1 alleviates hepatic fibrogenesis via blocking bone marrow mesenchymal stromal cell recruitment to fibrotic liver

IQ motif-containing guanosine triphosphatase (GTPase)-activating protein 1 (IQGAP1) is a cytosolic scaffolding protein involved in cell migration. Our previous studies suggest sphingosine 1-phosphate (S1P) triggers bone marrow (BM) mesenchymal stromal cells (BMSCs) to damaged liver, thereby promoting liver fibrosis. However, the role of IQGAP1 in S1P-induced BMSC migration and liver fibrogenesis remains unclear. Chimeric mice of BM cell labeled by EGFP were used to build methionine-choline-deficient and high-fat (MCDHF)-diet-induced mouse liver fibrosis. IQGAP1 small interfering RNA (siRNA) was utilized to silence IQGAP1 in vivo. IQGAP1 expression is significantly elevated in MCDHF-diet-induced mouse fibrotic livers. Positive correlations are presented between IQGAP1 and fibrosis hallmarks expressions in human and mouse fibrotic livers. In vitro, depressing IQGAP1 expression blocks S1P-induced motility and cytoskeleton remodeling of BMSCs. S1P facilitates IQGAP1 aggregating to plasma membrane via S1P receptor 3 (S1PR3) and Cdc42/Rac1. In addition, IQGAP1 binds to Cdc42/Rac1, regulating S1P-induced activation of Cdc42/Rac1 and mediating BMSC migration in concert. In vivo, silencing IQGAP1 reduces the recruitment of BMSCs to impaired liver and effectively alleviates liver fibrosis induced by MCDHF diet. Together, silencing IQGAP1 relieves liver fibrosis by blocking BMSC migration, providing an effective therapeutic strategy for liver fibrosis.

Effect of miR-183-5p on Cholestatic Liver Fibrosis by Regulating Fork Head Box Protein O1 Expression.

Liver fibrosis is a common pathological feature of end-stage liver disease and has no effective treatment. MicroRNAs (miRNAs) have been found to modulate gene expression in liver disease. But the potential role of miRNA in hepatic fibrosis is still unclear. The objective of this research is to study the potential mechanism and biological function of miR-183-5p in liver fibrosis. In this study, we used high-throughput sequencing to find that miR-183-5p is upregulated in human fibrotic liver tissues. In addition, miR-183-5p was upregulated both in rat liver fibrosis tissue induced by bile-duct ligation (BDL) and activated LX-2 cells (human hepatic stellate cell line) according to the result of quantitative real-time PCR (RT-qPCR). Moreover, the inhibition of miR-183-5p alleviated liver fibrosis, decreased the fibrotic biomarker levels in vitro and in vivo, and led toLX-2 cell proliferation inhibition and, apoptosis induction. The result of dual-luciferase assay revealed that miR-183-5p suppressed fork head box protein O1 (FOXO1) expression by binding to its 3′UTR directly. Next, we used lentivirus to overexpress FOXO1 in LX-2 cells, and we found that overexpression of FOXO1 reversed the promotion of miR-183-5p on liver fibrosis, reducing the fibrotic biomarker levels inLX-2 cells, inhibitingLX-2 cell proliferation, and promoting apoptosis. Furthermore, overexpression of FOXO1 prevented the activation of the transforming growth factor (TGF)-β signaling pathway in TGF-β1-induced LX-2 cells according to the result of western blotting. In conclusion, the findings showed thatmiR-183-5p might act as a key regulator of liver fibrosis, and miR-183-5p could promote cholestatic liver fibrosis by inhibiting FOXO1 expression through the TGF-β signaling pathway. Thus, inhibition of miR-183-5pmay be a new way to prevent and improve liver fibrosis.

Transforming growth factor beta-1 upregulates glucose transporter 1 and glycolysis through canonical and noncanonical pathways in hepatic stellate cells.

BACKGROUNDHepatic stellate cells (HSCs) are the key effector cells mediating the occurrence and development of liver fibrosis, while aerobic glycolysis is an important metabolic characteristic of HSC activation. Transforming growth factor-β1 (TGF-β1) induces aerobic glycolysis and is a driving factor for metabolic reprogramming. The occurrence of glycolysis depends on a high glucose uptake level. Glucose transporter 1 (GLUT1) is the most widely distributed glucose transporter in the body and mainly participates in the regulation of carbohydrate metabolism, thus affecting cell proliferation and growth. However, little is known about the relationship between TGF-β1 and GLUT1 in the process of liver fibrosis and the molecular mechanism underlying the promotion of aerobic glycolysis in HSCs.AIMTo investigate the mechanisms of action of GLUT1, TGF-β1 and aerobic glycolysis in the process of HSC activation during liver fibrosis.METHODSImmunohistochemical staining and immunofluorescence assays were used to examine GLUT1 expression in fibrotic liver tissue. A Seahorse extracellular flux (XF) analyzer was used to examine changes in aerobic glycolytic flux, lactate production levels and glucose consumption levels in HSCs upon TGF-β1 stimulation. The mechanism by which TGF-β1 induces GLUT1 protein expression in HSCs was further explored by inhibiting/promoting the TGF-β1/mothers-against-decapentaplegic-homolog 2/3 (Smad2/3) signaling pathway and inhibiting the p38 and phosphoinositide 3-kinase (PI3K)/AKT signaling pathways. In addition, GLUT1 expression was silenced to observe changes in the growth and proliferation of HSCs. Finally, a GLUT1 inhibitor was used to verify the in vivo effects of GLUT1 on a mouse model of liver fibrosis.RESULTSGLUT1 protein expression was increased in both mouse and human fibrotic liver tissues. In addition, immunofluorescence staining revealed colocalization of GLUT1 and alpha-smooth muscle actin proteins, indicating that GLUT1 expression was related to the development of liver fibrosis. TGF-β1 caused an increase in aerobic glycolysis in HSCs and induced GLUT1 expression in HSCs by activating the Smad, p38 MAPK and P13K/AKT signaling pathways. The p38 MAPK and Smad pathways synergistically affected the induction of GLUT1 expression. GLUT1 inhibition eliminated the effect of TGF-β1 on HSC proliferation and migration. A GLUT1 inhibitor was administered in a mouse model of liver fibrosis, and GLUT1 inhibition reduced the degree of liver inflammation and liver fibrosis.CONCLUSIONTGF-β1 induces GLUT1 expression in HSCs, a process related to liver fibrosis progression. In vitro experiments revealed that TGF-β1-induced GLUT1 expression might be one of the mechanisms mediating the metabolic reprogramming of HSCs. In addition, in vivo experiments also indicated that the GLUT1 protein promotes the occurrence and development of liver fibrosis.

SEAM is a spatial single nuclear metabolomics method for dissecting tissue microenvironment.

Spatial metabolomics can reveal intercellular heterogeneity and tissue organization. Here we report on the spatial single nuclear metabolomics (SEAM) method, a flexible platform combining high-spatial-resolution imaging mass spectrometry and a set of computational algorithms that can display multiscale and multicolor tissue tomography together with identification and clustering of single nuclei by their in situ metabolic fingerprints. We first applied SEAM to a range of wild-type mouse tissues, then delineated a consistent pattern of metabolic zonation in mouse liver. We further studied the spatial metabolic profile in the human fibrotic liver. We discovered subpopulations of hepatocytes with special metabolic features associated with their proximity to the fibrotic niche, and validated this finding by spatial transcriptomics with Geo-seq. These demonstrations highlighted SEAM's ability to explore the spatial metabolic profile and tissue histology at the single-cell level, leading to a deeper understanding of tissue metabolic organization.

18F]MAGL-4-11 positron emission tomography molecular imaging of monoacylglycerol lipase changes in preclinical liver fibrosis models

Monoacylglycerol lipase (MAGL) is a pivotal enzyme in the endocannabinoid system, which metabolizes 2-arachidonoylglycerol (2-AG) into the proinflammatory eicosanoid precursor arachidonic acid (AA). MAGL and other endogenous cannabinoid (EC) degrading enzymes are involved in the fibrogenic signaling pathways that induce hepatic stellate cell (HSC) activation and ECM accumulation during chronic liver disease. Our group recently developed an 18F-labeled MAGL inhibitor ([18F]MAGL-4-11) for PET imaging and demonstrated highly specific binding in vitro and in vivo. In this study, we determined [18F]MAGL-4-11 PET enabled imaging MAGL levels in the bile duct ligation (BDL) and carbon tetrachloride (CCl4) models of liver cirrhosis; we also assessed the hepatic gene expression of the enzymes involved with EC system including MAGL, NAPE-PLD, FAAH and DAGL that as a function of disease severity in these models; [18F]MAGL-4-11 autoradiography was performed to assess tracer binding in frozen liver sections both in animal and human. [18F]MAGL-4-11 demonstrated reduced PET signals in early stages of fibrosis and further significantly decreased with disease progression compared with control mice. We confirmed MAGL and FAAH expression decreases with fibrosis severity, while its levels in normal liver tissue are high; in contrast, the EC synthetic enzymes NAPE-PLD and DAGL are enhanced in these different fibrosis models. In vitro autoradiography further supported that [18F]MAGL-4-11 bound specifically to MAGL in both animal and human fibrotic liver tissues. Our PET ligand [18F]MAGL-4-11 shows excellent sensitivity and specificity for MAGL visualization in vivo and accurately reflects the histological stages of liver fibrosis in preclinical models and human liver tissues.

Death-Associated Protein 6 (Daxx) Alleviates Liver Fibrosis by Modulating Smad2 Acetylation.

Transforming growth factor-β (TGF-β) has been identified as an inducer of hepatocyte epithelial–mesenchymal transition (EMT), which triggers liver fibrosis. Death-associated protein 6 (Daxx) is known to be associated with the TGF-β-induced apoptotic pathway, but the function of Daxx in liver fibrosis remains unknown. This study aimed to elucidate the role of Daxx in liver fibrosis. We used liver fibrosis tissues from humans and mice to assess Daxx expression. EMT properties and TGF-β signaling pathway activation were investigated in the Daxx-overexpressing FL83B cell line. The therapeutic effect of Daxx was investigated in a mouse model of liver fibrosis by the hydrodynamic injection of plasmids. The expression of Daxx was markedly decreased in hepatocytes from fibrotic human and mouse livers, as well as in hepatocytes treated with TGF-β in vitro. The overexpression of Daxx inhibited the EMT process by interfering with the TGF-β-induced phosphorylation of Smad2. Coimmunoprecipitation analysis confirmed that Daxx reduced the transcriptional activity of Smad2 by binding to its MH1 domain and interfering with Smad2 acetylation. In addition, the therapeutic delivery of Daxx alleviated liver fibrosis in a thioacetamide-induced fibrosis mouse model. Overall, our results indicate that Daxx could be a potential therapeutic target to modulate fibrogenesis, as well as a useful biomarker for liver fibrosis.

KRT17 Promotes the Activation of HSCs via EMT in Liver Fibrosis

Background and AimsAlthough activation of hepatic stellate cells (HSCs) plays a central role in the development of liver fibrosis, the mechanism underlying the activation of HSCs remains unclear. Keratin 17 (KRT17), a member of the intermediate filament family, can regulate tumor cell proliferation and migration. The current study aimed to elucidate the role of KRT17 in the activation of HSCs and the mechanisms underlying liver fibrosis.MethodsThe expression of KRT17 was determined using immunohistochemistry in tissue microarray. Western blotting and qRT-PCR assays were used to determine the KRT17 expression in fibrotic liver tissues obtained from human subjects and mice. LX-2 cells were treated with TGF-β1 recombinant protein and adipocyte differentiation mixture (MDI) mix to induce and reverse LX-2 cell activation, respectively, in order to explore the correlation between KRT17 and HSC activation. Additionally, cell proliferation and migration abilities of LX-2 cells transfected with KRT17-overexpressing plasmid or small interfering RNA were determined using CCK-8, flow cytometry, Transwell, and wound healing assays. Finally, rescue assay was used to explore the role of KRT17 in HSC activation and epithelial-mesenchymal transition (EMT).ResultsThe expression of KRT17 was higher in the human and mouse fibrotic liver tissues than in healthy liver tissues, and it was positively correlated with HSC activation. Upregulated KRT17 enhanced proliferation, migration, HSC activation and EMT in LX-2 cells, while knockdown of KRT17 reversed these effects. TGF-β1 recombinant protein accelerated KRT17-mediated EMT, HSC activation and proliferation, while TGF-β1 inhibitor counteracted the effect of KRT17 in vitro.ConclusionsKRT17 promoted HSC activation, proliferation and EMT in hepatic fibrosis probably via TGF-β1 signaling, and KRT17 might serve as a therapeutic target for the treatment of liver fibrosis.

EZH2-mediated inhibition of KLF14 expression promotes HSCs activation and liver fibrosis by downregulating PPARγ.

ObjectivesInduction of deactivation and apoptosis of hepatic stellate cells (HSCs) are principal therapeutic strategies for liver fibrosis. Krüppel‐like factor 14 (KLF14) regulates various biological processes, however, roles, mechanisms and implications of KLF14 in liver fibrosis are unknown.Materials and MethodsKLF14 expression was detected in human, rat and mouse fibrotic models, and its effects on HSCs were assessed. Chromatin immunoprecipitation assays were utilized to investigate the binding of KLF14 to peroxisome proliferator‐activated receptor γ (PPARγ) promoter, and the binding of enhancer of zeste homolog 2 (EZH2) to KLF14 promoter. In vivo, KLF14‐overexpressing adenovirus was injected via tail vein to thioacetamide (TAA)‐treated rats to investigate the role of KLF14 in liver fibrosis progression. EZH2 inhibitor EPZ‐6438 was utilized to treat TAA‐induced rat liver fibrosis.ResultsKLF14 expression was remarkably decreased in human, rat and mouse fibrotic liver tissues. Overexpression of KLF14 increased LD accumulation, inhibited HSCs activation, proliferation, migration and induced G2/M arrest and apoptosis. Mechanistically, KLF14 transactivated PPARγ promoter activity. Inhibition of PPARγ blocked the suppressive role of KLF14 overexpression in HSCs. Downregulation of KLF14 in activated HSCs was mediated by EZH2‐regulated histone H3 lysine 27 trimethylation. Adenovirus‐mediated KLF14 overexpression ameliorated TAA‐induced rat liver fibrosis in PPARγ‐dependent manner. Furthermore, EPZ‐6438 dramatically alleviated TAA‐induced rat liver fibrosis. Importantly, KLF14 expression was decreased in human with liver fibrosis, which was significantly correlated with EZH2 upregulation and PPARγ downregulation.ConclusionsKLF14 exerts a critical anti‐fibrotic role in liver fibrosis, and targeting the EZH2/KLF14/PPARγ axis might be a novel therapeutic strategy for liver fibrosis.

Liver fibrosis promotes immune escape in hepatocellular carcinoma via GOLM1-mediated PD-L1 upregulation

Immune checkpoint blockade is considered a breakthrough in cancer treatment. However, with the low response rates and therapeutic resistance of patients with hepatocellular carcinoma (HCC), the challenges facing the application of this treatment are tremendous. Liver fibrosis is a key driver of tumor immune escape, the underlying mechanism has never been clarified. This study sought to explore the role of liver fibrosis in regulating tumor-infiltrating lymphocytes (TILs) and inducing tumor immunosuppression. Ninety-nine fixed HCC tissue samples were used to analyze the association between liver fibrosis and immune escape using immunohistochemistry. In HCC patients, low FIB-4 values and high CD8+ T cell infiltration were correlated with prolonged survival. Elevated expression of immune checkpoints and attenuated antitumor immunity were observed in CCl4-induced mice liver fibrosis models and human fibrotic livers compared to control group. GOLM1 levels were increased in livers of patients with fibrosis and mice in response to CCl4-induced liver fibrosis. CD8+ T cell infiltrations were significantly decreased and PD-L1 expression was significantly increased in tumor tissues from hepatocyte-specific GOLM1 transgenic mice (Alb/GOLM1 mice) inducing chemical carcinogenesis compared to their corresponding control WT mice. GOLM1 induced PD-L1 expression via EGFR pathway activation. EGFR inhibitors, especially together with anti-PD-L1 therapy, improved the efficacy of immunotherapy in HCC. These findings illustrate the importance of liver fibrosis-induced immunosuppression as a tumor-promoting mechanism. GOLM1, which is highly upregulated in the fibrotic liver, regulates tumor microenvironmental immune escape via the EGFR/PD-L1 signaling pathway. EGFR blockade may bolster the efficacy of immune checkpoint inhibitors for HCC treatment.

Inhibition of Cytochrome P450 1B1 Prevents Hepatic Stellate Cell Activation and Liver Fibrosis

Background : Liver fibrosis often results from chronic liver injury and it is characterized by the accumulation of extracellular matrix. Activation of the hepatic stellate cell (HSC) is a key fibrogenic event. Cytochrome P450 1b1 (Cyp1b1) is a heme-thiolate monooxygenase involved in the metabolism of endogenous compounds and xenobiotics. The goal of this study is to determine whether and how Cyp1b1 plays a role in hepatic stellate cell (HSC) activation and liver fibrosis. Methods : The effects of Cyp1b1 on HSC activation and fibrogenesis were assessed by real-time PCR, immunofluorescence, and RNA-seq analysis. Wild-type (WT) and Cyp1b1 knockout (Cyp1b1-/-) mice were subjected to thioacetamide (TAA), carbon tetrachloride (CCl4), or bile duct ligation (BDL) models of liver fibrosis, and the fibrosis phenotypes were analyzed at the histology and gene expression levels. Hepatic retinoid levels were measured by liquid chromatography-mass spectrometry (LC-MS/MS). Results : Cyp1b1 is highly expressed in HSCs. The expression of Cyp1b1 was elevated in fibrotic mouse and human livers, as well as in activated mouse and human HSCs. In contrast, down-regulation or pharmacological inhibition of Cyp1b1 by 2,4,3′,5′-tetramethoxystilbene (TMS) attenuated HSC activation in vitro and in vivo. Cyp1b1-/- mice or WT mice treated with TMS were protected from liver fibrosis. Mechanistically, the fibrogenic induction of Cyp1b1 is likely mediated by the transcriptional factor wilms' tumor 1 (WT1). The pro-fibrogenic activity of Cyp1b1 may have been accounted for by its metabolism of retinoids in HSCs. Conclusion : Our study has uncovered an important endobiotic function of Cyp1b1 in promoting liver fibrosis. Cyp1b1 promotes HSC activation and liver fibrogenesis by facilitating retinoids depletion. Pharmacological inhibition of Cyp1b1 might be a potential strategy for the treatment of liver fibrosis.

RNF2 Mediates Hepatic Stellate Cells Activation by Regulating ERK/p38 Signaling Pathway in LX-2 Cells.

The therapeutic approach of liver fibrosis is still an unsolved clinical problem worldwide. Notably, the accumulation of extracellular matrix (ECM) in the liver is mediated by the production of cytokines and growth factors, such as transforming growth factor-β1 (TGF-β1) in hepatic stellate cells (HSCs). Ring finger protein 2 (RNF2) was identified as the catalytic subunit of polycomb repressive complex 1 (PRC1), mediating the monoubiquitination of histone H2A. In recent years, a growing amount of evidence suggests that RNF2 may play an important role in multiple pathological processes involved in cancer. Here, we explored the role of RNF2 in liver fibrogenesis and its potential mechanisms. The results showed that RNF2 was up-regulated in human fibrotic liver tissue. Knockdown of RNF2 led to a decreasing expression of collagen1 and α-smooth muscle actin (α-SMA) in LX-2 cells, which was upregulated by RNF2 overexpression. Moreover, RNF2 overexpression significantly promoted TGF-β1-induced LX-2 cell proliferation but decreased apoptosis. Furthermore, knockdown of RNF2 inhibited the activation of ERK/p38 signaling pathways induced by TGF-β1. These data suggested that RNF2 is an effective pro-fibrogenic factor for HSC activation via ERK/p38 signaling pathway. RNF2 inhibition might be a promising therapeutic target for liver fibrosis.