Oxidative stress promotes liver fibrosis by modulating the microRNA-144 and SIN3A-p38 pathways in hepatic stellate cells.

Background & Aims: Reactive oxygen species (ROS) act as modulators triggering cellular dysfunctions and organ damage including liver fibrosis in which hepatic stellate cell (HSC) activation plays a key role. Previous studies suggest that microRNA-144 (miR-144) acts as a pro-oxidant molecule; however, whether and how miR-144 affects HSC activation and liver fibrosis remain unknown. Methods: Carbon tetrachloride (CCl4) and bile duct ligation (BDL)-induced experimental liver fibrosis models were used. Hepatic miR-144 expression was analyzed by miRNA in situ hybridization with RNAscope probe. The in vivo effects of silencing or overexpressing miR-144 were examined with an adeno-associated virus 6 (AAV6) carrying miR-144 inhibitor or mimics in fibrotic mouse experimental models. Results: In this study, we demonstrated that ROS treatment significantly upregulated miR-144 in HSCs, which further promoted HSC activation in vitro. Interestingly, miR-144 was preferentially elevated in HSCs of experimental liver fibrosis in mice and in human liver fibrotic tissues. Furthermore, in vivo loss or gain-of-function experiments via AAV6 carrying miR-144 antagomir or agomir revealed that blockade of miR-144 in HSCs mitigated, while overexpression of miR-144 in HSCs accelerated the development of experimental liver fibrosis. Mechanistically, SIN3 transcription regulator family member A (SIN3A), a transcriptional repressor, was identified to be the target of miR-144 in HSCs. MiR-144 downregulated Sin3A, and in line with this result, specific knockdown of Sin3a in HSCs remarkedly activated p38 MAPK signaling pathway to promote HSC activation, eventually exacerbating liver fibrosis. Conclusions: Oxidative stress-driven miR-144 fuels HSC activation and liver fibrogenesis by limiting the SIN3A-p38 axis. Thus, a specific inhibition of miR-144 in HSCs could be a novel therapeutic strategy for the treatment of liver fibrosis.

Uncovering a Novel Functional Interaction Between Adult Hepatic Progenitor Cells, Inflammation and EGFR Signaling During Bile Acids-Induced Injury.

Chronic cholestatic damage is associated to both accumulation of cytotoxic levels of bile acids and expansion of adult hepatic progenitor cells (HPC) as part of the ductular reaction contributing to the regenerative response. Here, we report a bile acid-specific cytotoxic response in mouse HPC, which is partially impaired by EGF signaling. Additionally, we show that EGF synergizes with bile acids to trigger inflammatory signaling and NLRP3 inflammasome activation in HPC. Aiming at understanding the impact of this HPC specific response on the liver microenvironment we run a proteomic analysis of HPC secretome. Data show an enrichment in immune and TGF-ß regulators, ECM components and remodeling proteins in HPC secretome. Consistently, HPC-derived conditioned medium promotes hepatic stellate cell (HSC) activation and macrophage M1-like polarization. Strikingly, EGF and bile acids co-treatment leads to profound changes in the secretome composition, illustrated by an abolishment of HSC activating effect and by promoting macrophage M2-like polarization. Collectively, we provide new specific mechanisms behind HPC regulatory action during cholestatic liver injury, with an active role in cellular interactome and inflammatory response regulation. Moreover, findings prove a key contribution for EGFR signaling jointly with bile acids in HPC-mediated actions.

Exportin 4 DNA promoter methylation in liver fibrosis.

A role for exportin 4 (XPO4) in the pathogenesis of liver fibrosis was recently identified. We sought to determine changes in hepatic XPO4 promoter methylation levels during liver fibrosis. The quantitative real-time RT-PCR technique was used to quantify the mRNA level of XPO4. Additionally, pyrosequencing was utilized to assess the promoter methylation status of XPO4. The methylation rate of the XPO4 promoter was significantly increased with fibrosis in human and mouse models, while XPO4 mRNA expression negatively correlated with methylation of its promoter. DNA methyltransferases (DNMTs) levels (enzymes that drive DNA methylation) were upregulated in patients with liver fibrosis compared to healthy controls and in hepatic stellate cells upon transforming growth factor beta (TGFß) stimulation. The DNA methylation inhibitor 5-Aza or specific siRNAs for these DNMTs led to restoration of XPO4 expression. The process of DNA methylation plays a crucial role in the repression of XPO4 transcription in the context of liver fibrosis development.

Novel Inositol 1,4,5-Trisphosphate Receptor Inhibitor Antagonizes Hepatic Stellate Cell Activation: A Potential Drug to Treat Liver Fibrosis.

Liver fibrosis, characterized by excessive extracellular matrix (ECM) deposition, can progress to cirrhosis and increases the risk of liver cancer. Hepatic stellate cells (HSCs) play a pivotal role in fibrosis progression, transitioning from a quiescent to activated state upon liver injury, wherein they proliferate, migrate, and produce ECM. Calcium signaling, involving the inositol 1,4,5-trisphosphate receptor (IP3R), regulates HSC activation. This study investigated the efficacy of a novel IP3R inhibitor, desmethylxestospongin B (dmXeB), in preventing HSC activation. Freshly isolated rat HSCs were activated in vitro in the presence of varying dmXeB concentrations. The dmXeB effectively inhibited HSC proliferation, migration, and expression of fibrosis markers without toxicity to the primary rat hepatocytes or human liver organoids. Furthermore, dmXeB preserved the quiescent phenotype of HSCs marked by retained vitamin A storage. Mechanistically, dmXeB suppressed mitochondrial respiration in activated HSCs while enhancing glycolytic activity. Notably, methyl pyruvate, dimethyl α-ketoglutarate, and nucleoside supplementation all individually restored HSC proliferation despite dmXeB treatment. Overall, dmXeB demonstrates promising anti-fibrotic effects by inhibiting HSC activation via IP3R antagonism without adverse effects on other liver cells. These findings highlight dmXeB as a potential therapeutic agent for liver fibrosis treatment, offering a targeted approach to mitigate liver fibrosis progression and its associated complications.

Fluorescence imaging sheds light on the immune evasion mechanisms of hepatic stellate cells mediated by superoxide anion.

Whether and how the reactive oxygen species generated by hepatic stellate cells (HSCs) promote immune evasion of hepatocellular carcinoma (HCC) remains mysterious. Therefore, investigating the function of superoxide anion (O2•-), the firstly generated reactive oxygen species, during the immune evasion become necessary. In this work, we establish a novel in situ imaging method for visualization of O2•- changes in HSCs based on a new two-photon fluorescence probe TPH. TPH comprises recognition group for O2•- and HSCs targeting peptides. We observe that O2•- in HSCs gradually rose, impairing the infiltration of CD8+ T cells in HCC mice. Further studies reveal that the cyclin-dependent kinase 4 is deactivated by O2•-, and then cause the up-regulation of PD-L1. Our work provides molecular insights into HSC-mediated immune evasion of HCC, which may represent potential targets for HCC immunotherapy.

Hepatic recruitment of myeloid-derived suppressor cells upon liver injury promotes both liver regeneration and fibrosis.

BACKGROUND: The liver regeneration is a highly complicated process depending on the close cooperations between the hepatocytes and non-parenchymal cells involving various inflammatory cells. Here, we explored the role of myeloid-derived suppressor cells (MDSCs) in the processes of liver regeneration and liver fibrosis after liver injury. METHODS: We established four liver injury models of mice including CCl4-induced liver injury model, bile duct ligation (BDL) model, concanavalin A (Con A)-induced hepatitis model, and lipopolysaccharide (LPS)-induced hepatitis model. The intrahepatic levels of MDSCs (CD11b+Gr-1+) after the liver injury were detected by flow cytometry. The effects of MDSCs on liver tissues were analyzed in the transwell co-culture system, in which the MDSCs cytokines including IL-10, VEGF, and TGF-ß were measured by ELISA assay and followed by being blocked with specific antibodies. RESULTS: The intrahepatic infiltrations of MDSCs with surface marker of CD11b+Gr-1+ remarkably increased after the establishment of four liver injury models. The blood served as the primary reservoir for hepatic recruitment of MDSCs during the liver injury, while the bone marrow appeared play a compensated role in increasing the number of MDSCs at the late stage of the inflammation. The recruited MDSCs in injured liver were mainly the M-MDSCs (CD11b+Ly6G-Ly6Chigh) featured by high expression levels of cytokines including IL-10, VEGF, and TGF-ß. Co-culture of the liver tissues with MDSCs significantly promoted the proliferation of both hepatocytes and hepatic stellate cells (HSCs). CONCLUSIONS: The dramatically and quickly infiltrated CD11b+Gr-1+ MDSCs in injured liver not only exerted pro-proliferative effects on hepatocytes, but also accounted for the activation of profibrotic HSCs.

Bystander Effects and Profibrotic Interactions in Hepatic Stellate Cells during HIV and HCV Coinfection.

This study aims to explore the influence of coinfection with HCV and HIV on hepatic fibrosis. A coculture system was set up to actively replicate both viruses, incorporating CD4 T lymphocytes (Jurkat), hepatic stellate cells (LX-2), and hepatocytes (Huh7.5). LX-2 cells' susceptibility to HIV infection was assessed through measurements of HIV receptor expression, exposure to cell-free virus, and cell-to-cell contact with HIV-infected Jurkat cells. The study evaluated profibrotic parameters, including programed cell death, ROS imbalance, cytokines (IL-6, TGF-ß, and TNF-α), and extracellular matrix components (collagen, α-SMA, and MMP-9). The impact of HCV infection on LX-2/HIV-Jurkat was examined using soluble factors released from HCV-infected hepatocytes. Despite LX-2 cells being nonsusceptible to direct HIV infection, bystander effects were observed, leading to increased oxidative stress and dysregulated profibrotic cytokine release. Coculture with HIV-infected Jurkat cells intensified hepatic fibrosis, redox imbalance, expression of profibrotic cytokines, and extracellular matrix production. Conversely, HCV-infected Huh7.5 cells exhibited elevated profibrotic gene transcriptions but without measurable effects on the LX-2/HIV-Jurkat coculture. This study highlights how HIV-infected lymphocytes worsen hepatic fibrosis during HCV/HIV coinfection. They increase oxidative stress, profibrotic cytokine levels, and extracellular matrix production in hepatic stellate cells through direct contact and soluble factors. These insights offer valuable potential therapies for coinfected individuals.

[Arbutin ameliorates liver fibrosis in mice by inhibiting macrophage recruitment and regulating the Akt/NF-κB and Smad signaling pathways].

OBJECTIVE: To investigate the protective effect of arbutin against CCl4-induced hepatic fibrosis in mice and explore the underlying mechanisms. METHODS: Twenty-four C57BL/6 mice were randomly divided into control group, model group, and low- and high-dose arbutin treatment (25 and 50 mg/kg, respectively) groups. Mouse models of liver fibrosis were established by intraperitoneal injection of CCl4, and arbutin was administered daily via gavage for 6 weeks. After the treatments, serum biochemical parameters of the mice were tested, and liver tissues were taken for HE staining, Sirius Red staining and immunohistochemical staining. RT-qPCR was used to detect the mRNA levels of α-SMA, Pdgfb, Col1α1, Timp-1, Ccl2 and Tnf-a, and Western blotting was performed to detect α-SMA protein expression in the liver tissues. In the cell experiment, the effect of arbutin treatment for 24 h on THP-1 and RAW264.7 cell migration and recruitment was examined using Transwell migration assay and DAPI staining; The changes in protein levels of Akt, p65, Smad3, p-Akt, p-p65, p-Smad3 and α-SMA in arbutintreated LX-2 cells were detected with Western blotting. RESULTS: Arbutin treatment significantly lowered serum alanine aminotransferase and aspartate aminotransferase levels, alleviated liver tissue damage and collagen deposition, and reduced macrophage infiltration and α-SMA protein expression in the liver of the mouse models (P < 0.05 or 0.001). Arbutin treatment also significantly reduced CCl4-induced elevation of a-SMA, Pdgfb, Col1α1, Timp-1, Ccl2 and Tnf-a mRNA levels in mice (P < 0.05). In the cell experiment, arbutin treatment obviously inhibited migration and recruitment of THP-1 and RAW264.7 cells and lowered the phosphorylation levels of Akt, p65 and Smad3 and the protein expression level of α-SMA in LX-2 cells. CONCLUSION: Arbutin ameliorates liver inflammation and fibrosis in mice by inhibiting hepatic stellate cell activation via reducing macrophage recruitment and infiltration and suppressing activation of the Akt/NF-κB and Smad signaling pathways.

Effects of Mesenchymal Stem Cells-Derived Extracellular Vesicles on Inhibition of Hepatic Fibrosis by Delivering miR-200a.

BACKGROUND: Hepatic fibrosis (HF) is a common pathological feature of chronic hepatic diseases. We aimed to illuminate the significance of amniotic mesenchymal stem cells (AMSCs)-derived extracellular vesicles (AMSCs-EVs) in HF. METHODS: Human AMSCs-EVs were isolated and identified. HF mice were constructed and treated with EVs. The fibrosis was observed by staining experiments and Western blot (WB) assay. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), and hepatic hydroxyproline (Hyp) were detected to confirm liver function. For the in vitro experiments, human hepatic stellate cells were induced with transforming growth factor-ß and treated with EVs. To measure the degree of HF, the expression of alpha-smooth muscle actin (α-SMA) and Collagen I was detected by WB assay, and cell proliferation was detected by cell counting kit 8 assay. The levels of miR-200a, Zinc finger E-box binding homeobox 1 (ZEB1), and phosphoinositide-3-kinase regulatory subunit 3 (PIK3R3) were detected by WB and real-time quantitative polymerase chain reaction. The binding of ZEB1 to PIK3R3 and miR-200a to ZEB1 was analyzed by chromatin immunoprecipitation and dual luciferase assays to validate their relationships. RESULTS: Human AMSCs and AMSCs-EVs were obtained. Serum ALT, AST, TBIL, and hepatic Hyp were increased, implying the fibrosis degree was aggravated in HF mice, which was decreased again after EV treatment. EVs inhibited HF degree by reducing α-SMA and Collagen I and promoting cell proliferation. AMSCs-EVs delivered miR-200a into hepatocytes, which up-regulated miR-200a expression, inhibited ZEB1 expression, and reduced its enrichment on the PIK3R3 promoter, therefore inhibiting PIK3R3 expression and alleviating HF. Overexpression of ZEB1 or PIK3R3 attenuated the anti-fibrotic effect of AMSCs-EVs. CONCLUSION: Human AMSCs-derived EVs mediated miR-200a delivery and inhibition of intracellular ZEB1/PIK3R3 axis to exert anti-fibrosis effects.

Sja-let-7 suppresses the development of liver fibrosis via Schistosoma japonicum extracellular vesicles.

Schistosomiasis is a fatal zoonotic parasitic disease that also threatens human health. The main pathological features of schistosomiasis are granulomatous inflammation and subsequent liver fibrosis, which is a complex, chronic, and progressive disease. Extracellular vesicles (EVs) derived from schistosome eggs are broadly involved in host-parasite communication and act as important contributors to schistosome-induced liver fibrosis. However, it remains unclear whether substances secreted by the EVs of Schistosoma japonicum, a long-term parasitic "partner" in the hepatic portal vein of the host, also participate in liver fibrosis. Here, we report that EVs derived from S. japonicum worms attenuated liver fibrosis by delivering sja-let-7 into hepatic stellate cells (HSCs). Mechanistically, activation of HSCs was reduced by targeting collagen type I alpha 2 chain (Col1α2) and downregulation of the TGF-ß/Smad signaling pathway both in vivo and in vitro. Overall, these results contribute to further understanding of the molecular mechanisms underlying host-parasite interactions and identified the sja-let-7/Col1α2/TGF-ß/Smad axis as a potential target for treatment of schistosomiasis-related liver fibrosis.

LncRNA FRMD6-AS1/miR-491-5p/USP13 pathway attenuated ferroptosis and contributed to liver fibrosis.

Liver fibrosis is an invertible pathophysiologic process featured by excessive accumulation of extracellular matrix (ECM) which injures liver cells and activates hepatic stellate cells (HSCs). Besides, inducing ferroptosis in activated HSCs can alleviate liver fibrosis. LncRNAs modulate ferroptosis in activated HSCs and ECM deposition in liver fibrosis. However, the role of lncRNA FRMD6-AS1 in liver fibrosis is not discovered. In this study, lncRNA FRMD6-AS1 was dramatically up-regulated in activated HSCs. Knockdown of FRMD6-AS1 markedly increased iron ion, ROS and MDA levels, decreased GSH level, SLC7A11 and GPX4 protein expressions in activated HSCs. In addition, HSCs activation markers α-SMA and COL1α1 expressions were up-regulated in activated HSCs; knockdown of FRMD6-AS1 markedly down-regulated α-SMA and COL1α1 expressions in HSCs. Besides, lncRNA FRMD6-AS1 could interact with miR-491-5p, and negatively modulate miR-491-5p expression. USP13 was a target of miR-491-5p, and could be negatively modulated by miR-491-5p. Moreover, FRMD6-AS1 knockdown increased iron ion and ROS levels, decreased SLC7A11 and GPX4 protein expressions, facilitated HSCs viability, and up-regulated α-SMA and COL1α1 expressions via miR-491-5p/USP13 pathway. Finally, FRMD6-AS1 knockdown restored liver tissue structure and abrogated fibrosis in livers in a CCL4 liver fibrosis mouse model. Hence, lncRNA FRMD6-AS1/miR-491-5p/USP13 pathway repressed ferroptosis, promoted ECM deposition and facilitated liver fibrosis in vitro and in vivo models.

Single-cell multiomics guided mechanistic understanding of Fontan-associated liver disease.

The Fontan operation is the current standard of care for single-ventricle congenital heart disease. Individuals with a Fontan circulation (FC) exhibit central venous hypertension and face life-threatening complications of hepatic fibrosis, known as Fontan-associated liver disease (FALD). The fundamental biology and mechanisms of FALD are little understood. Here, we generated a transcriptomic and epigenomic atlas of human FALD at single-cell resolution using multiomic snRNA-ATAC-seq. We found profound cell type-specific transcriptomic and epigenomic changes in FC livers. Central hepatocytes (cHep) exhibited the most substantial changes, featuring profound metabolic reprogramming. These cHep changes preceded substantial activation of hepatic stellate cells and liver fibrosis, suggesting cHep as a potential first "responder" in the pathogenesis of FALD. We also identified a network of ligand-receptor pairs that transmit signals from cHep to hepatic stellate cells, which may promote their activation and liver fibrosis. We further experimentally demonstrated that activins A and B promote fibrotic activation in vitro and identified mechanisms of activin A's transcriptional activation in FALD. Together, our single-cell transcriptomic and epigenomic atlas revealed mechanistic insights into the pathogenesis of FALD and may aid identification of potential therapeutic targets.

Role of Epiregulin on Lipopolysaccharide-Induced Hepatocarcinogenesis as a Mediator via EGFR Signaling in the Cancer Microenvironment.

Lipopolysaccharides (LPSs) have been reported to be important factors in promoting the progression of hepatocellular carcinoma (HCC), but the corresponding molecular mechanisms remain to be elucidated. We hypothesize that epiregulin (EREG), an epidermal growth factor (EGF) family member derived from hepatic stellate cells (HSCs) and activated by LPS stimulation, is a crucial mediator of HCC progression with epidermal growth factor receptor (EGFR) expression in the tumor microenvironment. We used a mouse xenograft model of Huh7 cells mixed with half the number of LX-2 cells, with/without intraperitoneal LPS injection, to elucidate the role of EREG in LPS-induced HCC. In the mouse model, LPS administration significantly enlarged the size of xenografted tumors and elevated the expression of EREG in tumor tissues compared with those in negative controls. Moreover, CD34 immunostaining and the gene expressions of angiogenic markers by a reverse transcription polymerase chain reaction revealed higher vascularization, with increased interleukin-8 (IL-8) expression in the tumors of the mice group treated with LPS compared to those without LPS. Our data collectively suggested that EREG plays an important role in the cancer microenvironment under the influence of LPS to increase not only the tumor cell growth and migration/invasion of EGFR-positive HCC cells but also tumor neovascularization via IL-8 signaling.

DCDC2 inhibits hepatic stellate cell activation and ameliorates CCl4-induced liver fibrosis by suppressing Wnt/ß-catenin signaling.

Liver fibrosis, as a consequence of chronic liver disease, involves the activation of hepatic stellate cell (HSC) caused by various chronic liver injuries. Emerging evidence suggests that activation of HSC during an inflammatory state can lead to abnormal accumulation of extracellular matrix (ECM). Investigating novel strategies to inhibit HSC activation and proliferation holds significant importance for the treatment of liver fibrosis. As a member of the doublecortin domain-containing family, doublecortin domain containing 2 (DCDC2) mutations can lead to neonatal sclerosing cholangitis, but its involvement in liver fibrosis remains unclear. Therefore, this study aims to elucidate the role of DCDC2 in liver fibrosis. Our findings revealed a reduction in DCDC2 expression in both human fibrotic liver tissues and carbon tetrachloride (CCl4)-induced mouse liver fibrotic tissues. Furthermore, exposure to transforming growth factor beta-1(TGF-ß1) stimulation resulted in a dose- and time-dependent decrease in DCDC2 expression. The overexpression of DCDC2 inhibited the expression of α-smooth muscle actin (α-SMA) and type I collagen alpha 1 (Col1α1), and reduced the activation of HSC stimulated with TGF-ß1. Additionally, we provided evidence that the Wnt/ß-catenin signaling pathway was involved in this process, wherein DCDC2 was observed to inhibit ß-catenin activation, thereby preventing its nuclear translocation. Furthermore, our findings demonstrated that DCDC2 could attenuate the proliferation and epithelial-mesenchymal transition (EMT)-like processes of HSC. In vivo, exogenous DCDC2 could ameliorate CCl4-induced liver fibrosis. In summary, DCDC2 was remarkably downregulated in liver fibrotic tissues of both humans and mice, as well as in TGF-ß1-activated HSC. DCDC2 inhibited the activation of HSC induced by TGF-ß1 in vitro and fibrogenic changes in vivo, suggesting that it is a promising therapeutic target for liver fibrosis and warrants further investigation in clinical practice.

The heavy subunit of ferritin stimulates NLRP3 inflammasomes in hepatic stellate cells through ICAM-1 to drive hepatic inflammation.

Serum ferritin concentrations increase during hepatic inflammation and correlate with the severity of chronic liver disease. Here, we report a molecular mechanism whereby the heavy subunit of ferritin (FTH) contributes to hepatic inflammation. We found that FTH induced activation of the NLRP3 inflammasome and secretion of the proinflammatory cytokine interleukin-1ß (IL-1ß) in primary rat hepatic stellate cells (HSCs) through intercellular adhesion molecule-1 (ICAM-1). FTH-ICAM-1 stimulated the expression of Il1b, NLRP3 inflammasome activation, and the processing and secretion of IL-1ß in a manner that depended on plasma membrane remodeling, clathrin-mediated endocytosis, and lysosomal destabilization. FTH-ICAM-1 signaling at early endosomes stimulated Il1b expression, implying that this endosomal signaling primed inflammasome activation in HSCs. In contrast, lysosomal destabilization was required for FTH-induced IL-1ß secretion, suggesting that lysosomal damage activated inflammasomes. FTH induced IL-1ß production in liver slices from wild-type mice but not in those from Icam1-/- or Nlrp3-/- mice. Thus, FTH signals through its receptor ICAM-1 on HSCs to activate the NLRP3 inflammasome. We speculate that this pathway contributes to hepatic inflammation, a key process that stimulates hepatic fibrogenesis associated with chronic liver disease.

Taurine attenuates activation of hepatic stellate cells by inhibiting autophagy and inducing ferroptosis.

BACKGROUND: Liver fibrosis is a compensatory response during the tissue repair process in chronic liver injury, and finally leads to liver cirrhosis or even hepatocellular carcinoma. The pathogenesis of hepatic fibrosis is associated with the progressive accumulation of activated hepatic stellate cells (HSCs), which can transdifferentiate into myofibroblasts to produce an excess of the extracellular matrix (ECM). Myofibroblasts are the main source of the excessive ECM responsible for hepatic fibrosis. Therefore, activated hepatic stellate cells (aHSCs), the principal ECM producing cells in the injured liver, are a promising therapeutic target for the treatment of hepatic fibrosis. AIM: To explore the effect of taurine on aHSC proliferation and the mechanisms involved. METHODS: Human HSCs (LX-2) were randomly divided into five groups: Normal control group, platelet-derived growth factor-BB (PDGF-BB) (20 ng/mL) treated group, and low, medium, and high dosage of taurine (10 mmol/L, 50 mmol/L, and 100 mmol/L, respectively) with PDGF-BB (20 ng/mL) treated group. Cell Counting Kit-8 method was performed to evaluate the effect of taurine on the viability of aHSCs. Enzyme-linked immunosorbent assay was used to estimate the effect of taurine on the levels of reactive oxygen species (ROS), malondialdehyde, glutathione, and iron concentration. Transmission electron microscopy was applied to observe the effect of taurine on the autophagosomes and ferroptosis features in aHSCs. Quantitative real-time polymerase chain reaction and Western blot analysis were performed to detect the effect of taurine on the expression of α-SMA, Collagen I, Fibronectin 1, LC3B, ATG5, Beclin 1, PTGS2, SLC7A11, and p62. RESULTS: Taurine promoted the death of aHSCs and reduced the deposition of the ECM. Treatment with taurine could alleviate autophagy in HSCs to inhibit their activation, by decreasing autophagosome formation, downregulating LC3B and Beclin 1 protein expression, and upregulating p62 protein expression. Meanwhile, treatment with taurine triggered ferroptosis and ferritinophagy to eliminate aHSCs characterized by iron overload, lipid ROS accumulation, glutathione depletion, and lipid peroxidation. Furthermore, bioinformatics analysis demonstrated that taurine had a direct targeting effect on nuclear receptor coactivator 4, exhibiting the best average binding affinity of -20.99 kcal/mol. CONCLUSION: Taurine exerts therapeutic effects on liver fibrosis via mechanisms that involve inhibition of autophagy and trigger of ferroptosis and ferritinophagy in HSCs to eliminate aHSCs.

TM7SF3 controls TEAD1 splicing to prevent MASH-induced liver fibrosis.

The mechanisms of hepatic stellate cell (HSC) activation and the development of liver fibrosis are not fully understood. Here, we show that deletion of a nuclear seven transmembrane protein, TM7SF3, accelerates HSC activation in liver organoids, primary human HSCs, and in vivo in metabolic-dysfunction-associated steatohepatitis (MASH) mice, leading to activation of the fibrogenic program and HSC proliferation. Thus, TM7SF3 knockdown promotes alternative splicing of the Hippo pathway transcription factor, TEAD1, by inhibiting the splicing factor heterogeneous nuclear ribonucleoprotein U (hnRNPU). This results in the exclusion of the inhibitory exon 5, generating a more active form of TEAD1 and triggering HSC activation. Furthermore, inhibiting TEAD1 alternative splicing with a specific antisense oligomer (ASO) deactivates HSCs in vitro and reduces MASH diet-induced liver fibrosis. In conclusion, by inhibiting TEAD1 alternative splicing, TM7SF3 plays a pivotal role in mitigating HSC activation and the progression of MASH-related fibrosis.

Aflatoxin B1-exposed hepatocyte-derived extracellular vesicles: Initiating hepatic stellate cell-mediated liver fibrosis through a p53-Parkin-dependent mitophagy pathway.

Environmental aflatoxin B1 (AFB1) exposure has been proposed to contribute to hepatocellular carcinoma by promoting liver fibrosis, but the potential mechanisms remain to be further elucidated. Extracellular vesicles (EVs) were recognized as crucial traffickers for hepatic intercellular communication and play a vital role in the pathological process of liver fibrosis. The AFB1-exposed hepatocyte-derived EVs (AFB1-EVs) were extracted, and the functional effects of AFB1-EVs on the activation of hepatic stellate cells (HSCs) were explored to investigate the molecular mechanism of AFB1 exposure-induced liver fibrogenesis. Our results revealed that an environment-level AFB1 exposure induced liver fibrosis via HSCs activation in mice, while the AFB1-EVs mediated hepatotoxicity and liver fibrogenesis in vitro and in vivo. AFB1 exposure in vitro increased PINK1/Parkin-dependent mitophagy in hepatocytes, where upregulated transcription of the PARK2 gene via p53 nuclear translocation and mitochondrial recruitment of Parkin, and promoted AFB1-EVs-mediated mitochondria-trafficking communication between hepatocytes and HSCs. The knockdown of Parkin in HepaRG cells reversed HSCs activation by blocking the mitophagy-related AFB1-EVs trafficking. This study further revealed that the hepatic fibrogenesis of AFB1 exposure was rescued by genetic intervention with siPARK2 or p53's Pifithrin-α (PFTα) inhibitors. Furthermore, AFB1-EVs-induced HSCs activation was relieved by GW4869 pharmaceutic inhibition of EVs secretion. These results revealed a novel mechanism that AFB1 exposure-induced p53-Parkin signal axis regulated mitophagy-dependent hepatocyte-derived EVs to mediate the mitochondria-trafficking intercellular communication between hepatocytes and HSCs in the local hepatotoxic microenvironment to promote the activated HSCs-associated liver fibrogenesis. Our study provided insight into p53-Parkin-dependent pathway regulation and promised an advanced strategy targeting intervention to EVs-mediated mitochondria trafficking for preventing xenobiotics-induced liver fibrosis.

SLC7A11-ROS/αKG-AMPK axis regulates liver inflammation through mitophagy and impairs liver fibrosis and NASH progression.

The changes of inflammation and metabolism are two features in nonalcoholic steatohepatitis (NASH). However, how they interact to regulate NASH progression remains largely unknown. Our works have demonstrated the importance of solute carrier family 7 member 11 (SLC7A11) in inflammation and metabolism. Nevertheless, whether SLC7A11 regulates NASH progression through mediating inflammation and metabolism is unclear. In this study, we found that SLC7A11 expression was increased in liver samples from patients with NASH. Upregulated SLC7A11 level was also detected in two murine NASH models. Functional studies showed that SLC7A11 knockdown or knockout had augmented steatohepatitis with suppression of inflammatory markers in mice. However, overexpression of SLC7A11 dramatically alleviated diet-induced NASH pathogenesis. Mechanically, SLC7A11 decreased reactive oxygen species (ROS) level and promoted α-ketoglutarate (αKG)/prolyl hydroxylase (PHD) activity, which activated AMPK pathway. Furthermore, SLC7A11 impaired expression of NLRP3 inflammasome components through AMPK-mitophagy axis. IL-1ß release through NLRP3 inflammasome recruited myeloid cells and promoted hepatic stellate cells (HSCs) activation, which contributed to the progression of liver injury and fibrosis. Anti-IL-1ß and anakinra might attenuate the hepatic inflammatory response evoked by SLC7A11 knockdown. Moreover, the upregulation of SLC7A11 in NASH was contributed by lipid overload-induced JNK-c-Jun pathway. In conclusions, SLC7A11 acts as a protective factor in controlling the development of NASH. Upregulation of SLC7A11 is protective by regulating oxidation, αKG and energy metabolism, decreasing inflammation and fibrosis.

PFKFB3-mediated glycolysis in hepatic stellate cells promotes liver regeneration.

Hepatic stellate cells (HSCs) perform a significant function in liver regeneration (LR) by becoming active. We propose to investigate if activated HSCs enhance glycolysis via PFKFB3, an essential glycolytic regulator, and whether targeting this pathway could be beneficial for LR. The liver and isolated HSCs of mice subjected to 2/3 partial hepatectomy (PHx) exhibited a significant rise in PFKFB3 expression, as indicated by quantitative RT-PCR analyses and Western blotting. Also, the primary HSCs of mice subjected to PHx have a significant elevation of the glycolysis level. Knocking down PFKFB3 significantly diminished the enhancement of glycolysis by PDGF in human LX2 cells. The hepatocyte proliferation in mice treated with PHx was almost completely prevented when the PFKFB3 inhibitor 3PO was administered, emerging that PFKFB3 is essential in LR. Furthermore, there was a decline in mRNA expression of immediate early genes and proinflammatory cytokines. In terms of mechanism, both the p38 MAP kinase and ERK1/2 phosphorylation in LO2 cells and LO2 proliferation were significantly reduced by the conditioned medium (CM) obtained from LX2 cells with either PFKFB3 knockdown or inhibition. Compared to the control group, isolated hepatocytes from 3PO-treated mice showed decreased p38 MAP kinase and ERK1/2 phosphorylation and proliferation. Thus, LR after PHx involves the activation of PFKFB3 in HSCs, which enhances glycolysis and promotes lactate production, thereby facilitating hepatocyte proliferation via the p38/ERK MAPK signaling pathway.