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Background: Absent in melanoma 2 (AIM2) inflammasome-dependent pyroptosis and neutrophil extracellular traps (NETs) have been implicated in chronic liver disease (CLD). However, the specific intrahepatic cell type that undergoes AIM2 inflammasome-dependent pyroptosis and how their interaction augments hepatic inflammation/fibrosis remains unclear., Methods: The expression and correlation of AIM2 inflammasome-dependent pyroptosis-related indicators and NETs were analyzed in biopsy tissue and blood specimens from chronic hepatitis patients (CHs). Cell-based experiments were conducted to investigate their interaction. In vitro and in vivo experiments were used to analyze their effects on the progression of hepatic inflammation/fibrosis as well as their clinical importance., Results: Elevated levels of AIM2 inflammasome-dependent pyroptosis indicators and NETs were detected in biopsy tissue and blood specimens. Circulating NETs were positively correlated with pyroptosis-related indicators, and both were related with disease severity. Confocal imaging revealed that AIM2 was mainly localized to hepatic macrophages, indicating that hepatic macrophages were the major cell type that underwent pyroptosis. NETs were directly engulfed by macrophages and then stimulated AIM2 inflammasome-dependent macrophage pyroptosis in vitro, which amplified the activation of hepatic stellate cells (HSCs) and increased collagen deposition. Administration of the NETs degradation agent DNase I or the AIM2 inflammasome activation inhibitor ODN A151 effectively alleviated chronic liver inflammation/fibrosis progression in vivo., Conclusions: NETs-induced AIM2 inflammasome-dependent pyroptosis in macrophages facilitates liver inflammation/fibrosis progression. The identified NET-AIM2 inflammasome cascade could serve as a novel therapeutic target for hepatic inflammation/fibrosis progression., Competing Interests: Declarations. Ethics approval and consent to participate: This study was approved by the Ethical Committee of the Second Hospital Affiliated with Chongqing Medical University (No. 2023–019). Consent for publication: All listed authors consent to the submission, and all data are used with the consent of the person generating the data. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Background: SIRT7 is a class III HDACs deacetylase which plays critical roles in various biological processes. Aberrant SIRT7 expression is associated with tumorigenesis and disease progression while role of SIRT7 in hepatic fibrosis remain elusive., Methods: SIRT7 expression was examined in fibrotic liver sample via WB and IHC. Myeloid cell-specific knockout (SIRT7 MKO ) mice were generated by crossing SIRT7 flox/flox mice with LysM-Cre mice. Primary hepatic stellate cells (HSCs) was isolated to examine stellate cells activation. SIRT7 and SMAD2/3 interaction were analyzed by immunoprecipitation. SB525334 was used to prevent SMAD2/3 phosphorylation., Results: SIRT7 expression was decreased during chronic liver disease progression but was increased in liver cancer. IHC staining indicated that SIRT7 was primarily expressed in non-parenchymal cells in both fibrotic and cirrhotic liver. Knockout SIRT7 in myeloid cells resulted in significant elevation of serum ALT and liver fibrosis, but mildly affected hepatic inflammation after CCl4 treatment. We further observed significant elevation of elevation of stellate cell activation and SMAD2/3 activation in SIRT7 MKO mice. By using primary HSCs and stellate cell line, we confirmed that SIRT7 interacted with SMAD2/3, induced its deacetylation and was critical in regulation of SMAD2/3 activation and stellate cell activation upon TGF-β stimulation. Pharmacological inhibition of SMAD2/3 reversed the hyperactivation of SIRT7 MKO HSCs after TGF-β stimulation, and abolished stellate cell activation and liver fibrosis in SIRT7 MKO mice., Conclusion: Our findings revealed previously unidentified role of SIRT7 in regulating HSCs activation via modulating TGF-β/SMAD2/3 signaling pathway. Targeting SIRT7 might offer novel therapeutic option against liver fibrosis., Competing Interests: Declaration of Competing Interest The authors declare that they have no affiliations with or involvement in any organization or entity with any financial interest in the subject matter or materials discussed in this manuscript., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Primary biliary cholangitis (PBC) is a chronic autoimmune liver disease characterized by multilineage immune dysregulation, which subsequently causes inflammation, fibrosis, and even cirrhosis of liver. Due to the limitation of traditional assays, the local hepatic immunopathogenesis of PBC has not been fully characterized. Here, we utilize single-cell RNA sequencing technology to depict the immune cell landscape and decipher the molecular mechanisms of PBC patients. We reveal that cholangiocytes and hepatic stellate cells are involved in liver inflammation and fibrosis. Moreover, Kupffer cells show increased levels of inflammatory factors and decreased scavenger function related genes, while T cells exhibit enhanced levels of inflammatory factors and reduced cytotoxicity related genes. Interestingly, we identify a liver-resident Th1-like population with JAK-STAT activation in the livers of both PBC patients and murine PBC model. Finally, blocking the JAK-STAT pathway alleviates the liver inflammation and eliminates the liver-resident Th1-like cells in the murine PBC model. In conclusion, our comprehensive single-cell transcriptome profiling expands the understanding of pathological mechanisms of PBC and provides potential targets for the treatment of PBC in patients., (© 2024. The Author(s).)

The peptide hormone kisspeptin attenuates liver steatosis, metabolic dysfunction-associated steatohepatitis (MASH), and fibrosis in mouse models by signaling via the kisspeptin 1 receptor (KISS1R). However, whether kisspeptin impacts fibrogenesis in the human liver is not known. We investigated the impact of a potent kisspeptin analog (KPA) on fibrogenesis using human precision-cut liver slices (hPCLS) from fibrotic livers from male patients, in human hepatic stellate cells (HSCs), LX-2, and in primary mouse HSCs. In hPCLS, 48 h and 72 h of KPA (3 nM, 100 nM) treatment decreased collagen secretion and lowered the expression of fibrogenic and inflammatory markers. Immunohistochemical studies revealed that KISS1R is expressed and localized to HSCs in MASH/fibrotic livers. In HSCs, KPA treatment reduced transforming growth factor b (TGFβ)-the induced expression of fibrogenic and inflammatory markers, in addition to decreasing TGFβ-induced collagen secretion, cell migration, proliferation, and colony formation. Mechanistically, KISS1R signaling downregulated TGFβ signaling by decreasing SMAD2/3 phosphorylation via the activation of protein phosphatases, PP2A, which dephosphorylates SMAD 2/3. This study revealed for the first time that kisspeptin reverses human hepatic fibrogenesis, thus identifying it as a new therapeutic target to treat hepatic fibrosis.

Schistosomiasis affects over 250 million people worldwide, with the highest prevalence at the age of 10-14 years. The influence of the host's age on the severity of liver damage is unclear. We infected male 8, 14, and 20-week-old mice with S. mansoni . Hepatic damage, inflammation, fibrosis, and metabolism were analyzed by RT-qPCR, Western blotting, ELISA, immunohistochemistry, and mechanistic transwell chamber experiments using S. mansoni eggs and human hepatic stellate cells (HSCs) or primary mouse hepatocytes. Major results were validated in human biopsies. We found that hepatosplenomegaly, granuloma size, egg load, inflammation, fibrosis, and glycogen stores all improved with the increasing age of the host. However, serum alanine transaminase (ALT) levels were lowest in young mice infected with S. mansoni . Hepatic carbohydrate exploitation was characterized by a shift towards Warburg-like glycolysis in S. mansoni -infected animals. Notably, S. mansoni eggs stimulated hepatic stellate cells to an alternatively activated phenotype (GFAP + /desmin + /αSMA - ) that secretes IL-6 and MCP-1. The reduction of fibrosis in older age likely depends on the fine-tuning of regulatory and inflammatory cytokines, alternative HSC activation, and the age-dependent preservation of hepatic energy stores. The current results emphasize the significance of investigations on the clinical relevance of host age-dependent liver damage in patients with schistosomiasis.

The role of peroxisome proliferator-activated receptor (PPAR)β/δ in hepatic fibrosis remains a subject of debate. Here, we examined the effects of a PPARβ/δ agonist on the pathogenesis of liver fibrosis and the activation of hepatic stellate cells (HSCs), the main effector cells in liver fibrosis, in response to the pro-fibrotic stimulus transforming growth factor-β (TGF-β). The PPARβ/δ agonist GW501516 completely prevented glucose intolerance and peripheral insulin resistance, blocked the accumulation of collagen in the liver, and attenuated the expression of inflammatory and fibrogenic genes in mice fed a choline-deficient high-fat diet (CD-HFD). The antifibrogenic effect of GW501516 observed in the livers CD-HFD-fed mice could occur through an action on HSCs since primary HSCs isolated from Ppard -/- mice showed increased mRNA levels of the profibrotic gene Col1a1. Moreover, PPARβ/δ activation abrogated TGF-β1-mediated cell migration (an indicator of cell activation) in LX-2 cells (immortalized activated human HSCs). Likewise, GW501516 attenuated the phosphorylation of the main downstream intracellular protein target of TGF-β1, suppressor of mothers against decapentaplegic (SMAD)3, as well as the levels of the SMAD3 co-activator p300 via the activation of AMP-activated protein kinase (AMPK) and the subsequent inhibition of extracellular signal-regulated kinase-1/2 (ERK1/2) in LX-2 cells. Overall, these findings uncover a new mechanism by which the activation of AMPK by a PPARβ/δ agonist reduces TGF-β1-mediated activation of HSCs and fibrosis via the reduction of both SMAD3 phosphorylation and p300 levels., Competing Interests: Declaration of Competing Interest None., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Background & Aims: Toll-like receptor 9 (Tlr9) is a pathogen recognition receptor detecting unmethylated DNA derivatives of pathogens and damaged host cells. It is therefore an important modulator of innate immunity. Here we investigated the role of Tlr9 in fibrogenesis and progression of hepatocellular carcinoma in chronic liver disease., Materials and Methods: We treated mice with a constitutive deletion of Tlr9 (Tlr9 -/- ) with DEN/CCl 4 for 24 weeks. As a second model, we used hepatocyte-specific Nemo knockout (Nemo Δhepa ) mice and generated double knockout (Nemo Δhepa Tlr9 -/- ) animals., Results: We show that Tlr9 is in the liver primarily expressed in Kupffer cells, suggesting a key role of Tlr9 in intercellular communication during hepatic injury. Tlr9 deletion resulted in reduced liver fibrosis as well as tumor burden. We observed down-regulation of hepatic stellate cell activation and consequently decreased collagen production in both models. Tlr9 deletion was associated with decreased apoptosis and compensatory proliferation of hepatocytes, modulating the initiation and progression of hepatocarcinogenesis. These findings were accompanied by a decrease in interferon-β and an increase in chemokines having an anti-tumoral effect., Conclusions: Our data define Tlr9 as an important receptor involved in fibrogenesis, but also in the initiation and progression of hepatocellular carcinoma during chronic liver diseases., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

The mechanisms underlying the remarkable capacity of the liver to regenerate are still not completely understood. Particularly, the cross-talk between cytokines and cellular components of the process is of utmost importance because they represent potential avenues for diagnostics and therapeutics. WNT1-inducible-signaling pathway protein 1 (WISP1) is a cytokine member of the CCN family, a family of proteins that play many different roles in liver pathophysiology. WISP1 also belongs to the earliest and strongest upregulated genes in mouse livers after CCl 4 intoxication and has recently been shown to be secreted by tumor cells and to bind to type 1 collagen to cause its linearization in vitro and in tumor tissue in vivo. We show that WISP1 expression is strongly induced by TGFβ, a critical cytokine in wound healing processes. Additionally, secretion of WISP1 protein by hepatic stellate is increased in cells upon TGFβ stimulation (~seven-fold increase). Furthermore, WISP1 facilitates the migration of mouse hepatic stellate cells through collagen in vitro. However, in WISP1 knockout mice, no difference in stellate cell accumulation in damaged liver tissue and no influence on fibrosis was obtained, probably because the knockout of WISP1 was compensated by other factors in vivo.

Background: The tumor microenvironment (TME) exerts profound effects on tumor progression and therapeutic efficacy. In hepatocellular carcinoma (HCC), the TME is enriched with cancer-associated fibroblasts (CAFs), which secrete a plethora of cytokines, chemokines, and growth factors that facilitate tumor cell proliferation and invasion. However, the intricate architecture of the TME in HCC, as well as the mechanisms driving interactions between tumor cells and CAFs, remains largely enigmatic., Methods: We analyzed 10 spatial transcriptomics and 12 single-cell transcriptomics samples sourced from public databases, complemented by 20 tumor tissue samples from liver cancer patients obtained in a clinical setting., Results: Our findings reveal that tumor cells exhibiting high levels of SPP1 are preferentially localized adjacent to hepatic stellate cells (HSCs). The SPP1 secreted by these tumor cells interacts with the CD44 receptor on HSCs, thereby activating the PI3K/AKT signaling pathway, which promotes the differentiation of HSCs into CAFs. Notably, blockade of the CD44 receptor effectively abrogates this interaction. Furthermore, in vivo studies demonstrate that silencing SPP1 expression in tumor cells significantly impairs HSC differentiation into CAFs, leading to a reduction in tumor volume and collagen deposition within the tumor stroma., Conclusions: This study delineates the SPP1-CD44 signaling axis as a pivotal mechanism underpinning the interaction between tumor cells and CAFs. Targeting this pathway holds potential to mitigate liver fibrosis and offers novel therapeutic perspectives for liver cancer management., (© 2024. The Author(s).)

Background: Dysregulated lipid oxidation occurs in several pathological processes characterized by cell proliferation and migration. Nonetheless, the molecular mechanism of lipid oxidation is not well appreciated in liver fibrosis, which is accompanied by enhanced fibroblast proliferation and migration., Methods: We investigated the causes and consequences of lipid oxidation in liver fibrosis using cultured cells, animal models, and clinical samples., Results: Increased ecto-nucleotide pyrophosphatase/phosphodiesterase (ENPP1) expression caused increased lipid oxidation, resulting in the proliferation and migration of hepatic stellate cells (HSCs) that lead to liver fibrosis, whereas fibroblast-specific ENPP1 knockout reversing these results. Elevated ENPP1 and N 6 -methyladenosine (m 6 A) levels were associated with high expression of Wilms tumor 1 associated protein (WTAP). Mechanistically, WTAP-mediated m 6 A methylation of the 3'UTR of ENPP1 mRNA and induces its translation dependent of YTH domain family proteins 1 (YTHDF1). Additionally, ENPP1 could interact with hypoxia inducible lipid droplet associated (HILPDA) directly; overexpression of ENPP1 further recruits HILPDA-mediated lipid oxidation, thereby promotes HSCs proliferation and migration, while inhibition of ENPP1 expression produced the opposite effect. Clinically, increased expression of WTAP, YTHDF1, ENPP1, and HILPDA, and increased m 6 A mRNA content, enhanced lipid oxidation, and increased collagen deposition in human liver fibrosis tissues., Conclusions: We describe a novel mechanism in which WTAP catalyzes m 6 A methylation of ENPP1 in a YTHDF1-dependent manner to enhance lipid oxidation, promoting HSCs proliferation and migration and liver fibrosis., (© 2024. The Author(s).)

Infection with Schistosoma japonicum (S. japonicum) is an important zoonotic parasitic disease that causes liver fibrosis in both human and domestic animals. The activation of hepatic stellate cells (HSCs) is a crucial phase in the development of liver fibrosis, and inhibiting their activation can alleviate this progression. Total flavonoids of litchi seed (TFL) is a naturally extracted drug, and modern pharmacological studies have shown its anti-fibrotic and liver-protective effects. However, the role of TFL in schistosomiasis liver fibrosis is still unclear. This study investigated the therapeutic effects of TFL on liver fibrosis in S. japonicum infected mice and explored its potential mechanisms. Animal study results showed that TFL significantly reduced the levels of Interleukin-1β (IL-1β), Tumor Necrosis Factor-α (TNF-α), Interleukin-4 (IL-4), and Interleukin-6 (IL-6) in the serum of S. japonicum infected mice. TFL reduced the spleen index of mice and markedly improved the pathological changes in liver tissues induced by S. japonicum infection, decreasing the expression of alpha-smooth muscle actin (α-SMA), Collagen I and Collagen III protein in liver tissues. In vitro studies indicated that TFL also inhibited the activation of HCSs induced by Transforming Growth Factor-β1 (TGF-β1) and reduced the levels of α-SMA. Gut microbes metagenomics study revealed that the composition, abundance, and functions of the mice gut microbiomes changed significantly after S. japonicum infection, and TLF treatment reversed these changes. Therefore, our study indicated that TFL alleviated granulomatous lesions and improved S. japonicum induced liver fibrosis in mice by inhibiting the activation of HSCs and by improving the gut microbiomes., Competing Interests: Declaration of Competing Interest We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work. There is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Background: A two-way relationship exists between type 2 diabetes (T2DM) and human nonalcoholic steatohepatitis (NASH). Several diabetic NASH models have the disadvantages of long cycles or inconsistent with the actual incidence of human disease, which would be costly and time-consuming to investigate disease pathogenesis and develop drugs. Therefore, there is an urgent need to establish a diabetic NASH mouse model., Methods: The combination between Fructose-palmitate-cholesterol diet (FPC) and Streptozotocin (STZ) (FPC+STZ) was used to construct diabetic NASH mouse model. The in vivo effects of silencing acid-sensitive Ion Channel 1a (ASIC1a) were examined with an adeno-associated virus 9 (AAV9) carrying ASIC1a short hairpin RNA (shRNA) in FPC+STZ model., Results: The mice fed with FPC for 12 weeks had insulin resistance, hyperinsulinemia, lipid accumulation, and increased hepatic levels of inflammatory factors. However, it still did not develop remarkable liver fibrosis. Most interestingly, noticeable fibrotic scars were observed in the liver of mice from FPC+STZ group. Furthermore, insulin therapy significantly ameliorated FPC+STZ-induced NASH-related liver fibrosis, indicating that hyperglycemia is of great significance in NASH development and progression. Importantly, ASIC1a was found to be involved in the pathogenesis of diabetic NASH as demonstrated that silencing ASIC1a in HSCs significantly ameliorated FPC+STZ-induced NASH fibrosis. Mechanistically, ASIC1a interacted with Poly Adp-adenosine ribose polymerase (PARP1) to promote HSC activation by inducing autophagy., Conclusion: A FPC diet combined with an injection of STZ induces a diabetic NASH mouse model in a shorter period. Targeting ASIC1a may provide a novel therapeutic target for the treatment of diabetic NASH., Competing Interests: Declaration of Competing Interest The author declares that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Masson SAS.)

Hepatic stellate cells (HSCs) secrete extracellular matrix for collagen deposition, contributing to liver fibrosis. Ferroptosis is a novel type of programmed cell death induced by iron overload-dependent lipid peroxidation. Regulation of ferroptosis in hepatic stellate cells (HSCs) may have therapeutic potential for liver fibrosis. Here, we found that Maf bZIP transcription factor G (MafG) was upregulated in human and murine liver fibrosis. Interestingly, MafG knockdown increased HSCs ferroptosis, while MafG overexpression conferred resistance of HSCs to ferroptosis. Mechanistically, MafG physically interacted with non-muscle myosin heavy chain IIa (MYH9) to transcriptionally activate lipocalin 2 (LCN2) expression, a known suppressor for ferroptosis. Site-directed mutations of MARE motif blocked the binding of MafG to LCN2 promoter. Re-expression of LCN2 in MafG knockdown HSCs restored resistance to ferroptosis. In bile duct ligation (BDL)-induced mice model, we found that treatment with erastin alleviated murine liver fibrosis by inducing HSC ferroptosis. HSC-specific knowdown MafG based on adeno-associated virus 6 (AAV-6) improved erastin-induced HSC ferroptosis and alleviation of liver fibrosis. Taken together, MafG inhibited HSCs ferroptosis to promote liver fibrosis through transcriptionally activating LCN2 expression. These results suggest that MafG/MYH9-LCN2 signaling pathway could be a novel targets for the treatment of liver fibrosis., (© 2024. The Author(s), under exclusive licence to ADMC Associazione Differenziamento e Morte Cellulare.)

Hepatic stellate cells (HSCs) are a major source of fibrogenic cells and play a central role in liver fibrogenesis. HSC activation depends on metabolic activation, for which it is well established that fatty acid oxidation (FAO) sustains their rapid proliferative rate. Studies have indicated that tanshinones inhibit HSC activation, however, the anti-fibrosis mechanisms of tanshinones are remain unclear. Herein, we reported that cryptotanshinone (CTS), a lipid-soluble ingredient of Salvia miltiorrhiza Bunge, exhibited the strongest inhibitory effects on HSC-LX2 proliferation and activation. CTS could induce lipocyte phenotype in mouse primary HSC and HSC-LX2. Transcriptomic sequencing and qPCR revealed that CTS regulated fatty acid metabolism and inhibited CPT1A and CPT1B expression. Target prediction suggested CTS regulates lipid metabolism by targeting STAT3. Mechanistically, the level of ATP and acetyl-CoA were reduced by the treatment of CTS, indicating that CTS could inhibit the level of FAO. Furthermore, CTS could inhibit the phosphorylation and nuclear translocation of STAT3. Additionally, CPT1A overexpression reversed the efficacy of CTS. Finally, CTS (40 mg/kg/day) attenuated CCl 4 -induced liver fibrosis and inhibited collagen production and HSC activation. Moreover, the results of immunofluorescence showed that α-SMA and p-STAT3 were co-located, and CTS could reduce the levels of p-STAT3 and α-SMA. In summary, CTS alleviated liver fibrosis by inhibiting the p-STAT3/CPT1A-dependent FAO both in vitro and in vivo, making it a potential candidate drug for the treatment of liver fibrosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The activation of hepatic stellate cells (HSCs) is central to the occurrence and development of liver fibrosis. Our previous studies showed that autophagy promotes HSC activation and ultimately accelerates liver fibrosis. Unc-51-like autophagy activating kinase 1 (ULK1) is an autophagic initiator in mammals, and N 6 -methyladenosine (m 6 A) modification is closely related to autophagy. In this study, we find that the m 6 A demethylase fat mass and obesity-associated protein (FTO), which is the m 6 A methylase with the most significant difference in expression, is upregulated during HSC activation and bile duct ligation (BDL)-induced hepatic fibrosis. Importantly, we identify that FTO overexpression aggravates HSC activation and hepatic fibrosis via autophagy. Mechanistically, compared with other autophagy-related genes, ULK1 is a target of FTO because FTO mainly mediates the m 6 A demethylation of ULK1 and upregulates its expression, thereby enhancing autophagy and the activation of HSCs. Notably, the m 6 A reader YTH domain-containing protein 2 (YTHDC2) decreases ULK1 mRNA level by recognizing the m 6 A binding site and ultimately inhibiting autophagy and HSC activation. Taken together, our findings highlight m 6 A-dependent ULK1 as an essential regulator of HSC autophagy and reveal that ULK1 is a novel potential therapeutic target for hepatic fibrosis treatment.

Sulfenylation is a reversible oxidative posttranslational modification (PTM) of proteins on cysteine residues. Despite the dissection of various biological functions of cysteine sulfenylation, its roles in hepatic fibrosis remain elusive. Here, we report that EphB2, a receptor tyrosine kinase previously implicated in liver fibrosis, is regulated by cysteine sulfenylation during the fibrotic progression of liver. Specifically, EphB2 is sulfenylated at the residues of Cys636 and Cys862 in activated hepatic stellate cells (HSCs), leading to the elevation of tyrosine kinase activity and protein stability of EphB2 and stronger interactions with focal adhesion kinase for the activation of downstream mitogen-activated protein kinase signaling. The inhibitions of both EphB2 kinase activity and cysteine sulfenylation by idebenone (IDE), a marketed drug with potent antioxidant activity, can markedly suppress the activation of HSCs and ameliorate hepatic injury in two well-recognized mouse models of liver fibrosis. Collectively, this study reveals cysteine sulfenylation as a new type of PTM for EphB2 and sheds a light on the therapeutic potential of IDE for the treatment of liver fibrosis., (© 2024. The Author(s).)

The combination of lineage tracing and immunohistochemistry has helped to identify subpopulations and fate of hepatic stellate cells (HSC) in murine liver. HSC are sinusoidal pericytes that act as myofibroblast precursors after liver injury. Single cell RNA sequencing approaches have recently helped to differentiate central and portal HSC. A specific Cre line to lineage trace portal HSC has not yet been described. We used three Cre lines (Lrat-Cre, PDGFRβ-CreER T2 and SMMHC-CreER T2 ) known to label mesenchymal cells including HSC in combination with a tdTomato-expressing reporter. All three Cre lines labeled populations of HSC as well as smooth muscle cells (SMC). Using the SMMHC-CreER T2 , we identified a subtype of HSC in the periportal area of the hepatic lobule (termed zone 1-HSC). We lineage traced tdTomato-expressing zone 1-HSC over 1 year, described fibrotic behavior in two fibrosis models and investigated their possible role during fibrosis. This HSC subtype resides in zone 1 under healthy conditions; however, zonation is disrupted in preclinical models of liver fibrosis (CCl 4 and MASH). Zone 1-HSC do not transform into αSMA-expressing myofibroblasts. Rather, they participate in sinusoidal capillarization. We describe a novel subtype of HSC restricted to zone 1 under physiological conditions and its possible function after liver injury. In contrast to the accepted notion, this HSC subtype does not transform into αSMA-positive myofibroblasts; rather, zone 1-HSC adopt properties of capillary pericytes, thereby participating in sinusoidal capillarization., (© 2024. The Author(s).)

The liver is a complex organ that performs vital functions in the body. Despite its extraordinary regenerative capacity compared to other organs, exposure to chemical, infectious, metabolic and immunologic insults and toxins renders the liver vulnerable to inflammation, degeneration and fibrosis. Abnormal wound healing response mediated by aberrant signaling pathways causes chronic activation of hepatic stellate cells (HSCs) and excessive accumulation of extracellular matrix (ECM), leading to hepatic fibrosis and cirrhosis. Fibrosis plays a key role in liver carcinogenesis. Once thought to be irreversible, recent clinical studies show that hepatic fibrosis can be reversed, even in the advanced stage. Experimental evidence shows that removal of the insult or injury can inactivate HSCs and reduce the inflammatory response, eventually leading to activation of fibrolysis and degradation of ECM. Thus, it is critical to understand the role of gene-environment interactions in the context of liver fibrosis progression and regression in order to identify specific therapeutic targets for optimized treatment to induce fibrosis regression, prevent HCC development and, ultimately, improve the clinical outcome.

Non-alcoholic fatty liver disease (NAFLD) is a clinicopathologic syndrome characterized by excessive fat deposition in hepatocytes and a major cause of end-stage liver disease. Autophagy is a metabolic pathway responsible for degrading cytoplasmic products and damaged organelles, playing a pivotal role in maintaining the homeostasis and functionality of hepatocytes. Recent studies have shown that pharmacological intervention to activate or restore autophagy provides benefits for liver function recovery by promoting the clearance of lipid droplets (LDs) in hepatocytes, decreasing the production of pro-inflammatory factors, and inhibiting activated hepatic stellate cells (HSCs), thus improving liver fibrosis and slowing down the progression of NAFLD. This article summarizes the physiological process of autophagy, elucidates the close relationship between NAFLD and autophagy, and discusses the effects of drugs on autophagy and signaling pathways from the perspectives of hepatocytes, kupffer cells (KCs), and HSCs to provide assistance in the clinical management of NAFLD., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Shen, Yang, Wang, Chen, Chen, Zhang, Xiong and Leng.)

Objective: This study aimed to evaluate the effect of exosomes derived from gastric cancer cells on the phenotypic transformation of hepatic stellate cells (HSCs) and the effect of HSC activation on the malignant behavior of gastric cancer cells, including its molecular mechanism., Methods: Exosomes derived from the human gastric adenocarcinoma cell line AGS were extracted and purified by polymer precipitation and ultrafiltration, respectively. The exosomes' morphologic characteristics were observed using transmission electron microscopy, particle size was determined through nanoparticle-tracking analysis, and marker proteins were detected using western blotting. Exosome uptake by LX-2 HSCs was observed through fluorescence-based tracing. Reverse transcription quantitative PCR (RT-qPCR) was used to detect the messenger RNA (mRNA) expression of alpha-smooth muscle actin (α-SMA) and fibroblast activation protein (FAP). Using functional assays, the effects of LX-2 HSC activation on the biological behavior of malignant gastric cancer cells were evaluated. The effects of LX-2 HSC activation on the protein expression of epithelial-mesenchymal transition (EMT)-related genes and β-catenin were evaluated via western blotting., Results: The extracted particles conformed to the definitions of exosomes and were thus considered gastric cancer cell-derived exosomes. Fluorescence-based tracing successfully demonstrated that exosomes were enriched in LX-2 HSCs. RT-qPCR revealed that the mRNA expression of the cancer-associated fibroblast markers α-SMA and FAP was significantly increased. LX-2 HSC activation considerably enhanced gastric cancer cell proliferation, invasion, and migration. Western blotting showed that the expression of the EMT-related epithelial marker E-cadherin was significantly downregulated, whereas the expression of interstitial markers (N-cadherin and vimentin) and β-catenin was remarkably upregulated in gastric cancer cells., Conclusion: Exosomes derived from gastric cancer cells promoted phenotypic transformation of HSCs and activated HSCs to become tumor-associated fibroblasts. Gastric cancer cell-derived cells significantly enhanced gastric cancer cell proliferation, invasion, and migration after HSC activation, which may promote EMT of gastric cancer cells through the Wnt/β-catenin pathway., (Copyright © 2024 Copyright: © 2024 Journal of Cancer Research and Therapeutics.)

Background: Non-alcoholic steatohepatitis (NASH) and liver fibrosis are progressive conditions associated with non-alcoholic fatty liver disease (NAFLD), characterized by hepatocyte pyroptosis and hepatic stellate cell (HSC) activation. Gentiopicroside (GPS) has emerged as a potential treatment for NASH, yet its underlying mechanism remains unclear., Aim: To confirm that GPS can improve NASH and liver fibrosis by blocking the NLRP3 signaling pathway STUDY DESIGN: Initially, different animal models were used to study the effects and mechanisms of GPS on NASH and fibrosis. Subsequent in vitro experiments utilized co-cultures and other techniques to delve deeper into its mechanism, followed by validation of the findings in mouse liver tissues., Methods: C57BL/6 mice were fed high-fat, high-cholesterol (HFHC), or methionine-choline-deficient (MCD) diets to induce NASH and fibrosis. RAW264.7 cells and born marrow bone marrow-derived macrophages (BMDMs) were stimulated with LPS and ATP to induce inflammation, then co-cultured with primary hepatocytes and HSCs, treated with GPS, and its efficacy and mechanism were analyzed., Results: In vivo, GPS alleviated NASH and liver fibrosis by inhibiting the NLRP3 pathway. In vitro, GPS attenuated inflammation induced by BMDMs by inhibiting TLR4 and NLRP3 signaling pathways, and Co-culture studies suggested that GPS reduced hepatocyte pyroptosis and HSC activation, which was also confirmed in liver tissues CONCLUSION: GPS improves NASH and liver fibrosis by inhibiting the TLR4 and NLRP3 signaling pathways. The specific mechanism may be related to the suppression of macrophage-mediated inflammatory responses, thereby reducing hepatocyte pyroptosis and HSC activation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

The burden of chronic liver disease is globally increasing at an alarming rate. Chronic liver injury leads to liver inflammation and fibrosis (LF) as critical determinants of long-term outcomes such as cirrhosis, liver cancer, and mortality. LF is a wound-healing process characterized by excessive deposition of extracellular matrix (ECM) proteins due to the activation of hepatic stellate cells (HSCs). In the healthy liver, quiescent HSCs metabolize and store retinoids. Upon fibrogenic activation, quiescent HSCs transdifferentiate into myofibroblasts; lose their vitamin A; upregulate α-smooth muscle actin; and produce proinflammatory soluble mediators, collagens, and inhibitors of ECM degradation. Activated HSCs are the main effector cells during hepatic fibrogenesis. In addition, the accumulation and activation of profibrogenic macrophages in response to hepatocyte death play a critical role in the initiation of HSC activation and survival. The main source of myofibroblasts is resident HSCs. Activated HSCs migrate to the site of active fibrogenesis to initiate the formation of a fibrous scar. Single-cell technologies revealed that quiescent HSCs are highly homogenous, while activated HSCs/myofibroblasts are much more heterogeneous. The complex process of inflammation results from the response of various hepatic cells to hepatocellular death and inflammatory signals related to intrahepatic injury pathways or extrahepatic mediators. Inflammatory processes modulate fibrogenesis by activating HSCs and, in turn, drive immune mechanisms via cytokines and chemokines. Increasing evidence also suggests that cellular stress responses contribute to fibrogenesis. Recent data demonstrated that LF can revert even at advanced stages of cirrhosis if the underlying cause is eliminated, which inhibits the inflammatory and profibrogenic cells. However, despite numerous clinical studies on plausible drug candidates, an approved antifibrotic therapy still remains elusive. This state-of-the-art review presents cellular and molecular mechanisms involved in hepatic fibrogenesis and its resolution, as well as comprehensively discusses the drivers linking liver injury to chronic liver inflammation and LF.

More than 90% of hepatocellular carcinoma (HCC) cases develop in the presence of fibrosis or cirrhosis, making the tumor microenvironment (TME) of HCC distinctive due to the intricate interplay between cancer-associated fibroblasts (CAFs) and cancer stem cells (CSCs), which collectively regulate HCC progression. However, the mechanisms through which CSCs orchestrate the dynamics of the tumor stroma during HCC development remain elusive. Our study unveils a significant upregulation of Sema3C in fibrotic liver, HCC tissues, peripheral blood of HCC patients, as well as sorafenib-resistant tissues and cells, with its overexpression correlating with the acquisition of stemness properties in HCC. We further identify NRP1 and ITGB1 as pivotal functional receptors of Sema3C, activating downstream AKT/Gli1/c-Myc signaling pathways to bolster HCC self-renewal and tumor initiation. Additionally, HCC cells-derived Sema3C facilitated extracellular matrix (ECM) contraction and collagen deposition in vivo, while also promoting the proliferation and activation of hepatic stellate cells (HSCs). Mechanistically, Sema3C interacted with NRP1 and ITGB1 in HSCs, activating downstream NF-kB signaling, thereby stimulating the release of IL-6 and upregulating HMGCR expression, consequently enhancing cholesterol synthesis in HSCs. Furthermore, CAF-secreted TGF-β1 activates AP1 signaling to augment Sema3C expression in HCC cells, establishing a positive feedback loop that accelerates HCC progression. Notably, blockade of Sema3C effectively inhibits tumor growth and sensitizes HCC cells to sorafenib in vivo. In sum, our findings spotlight Sema3C as a novel biomarker facilitating the crosstalk between CSCs and stroma during hepatocarcinogenesis, thereby offering a promising avenue for enhancing treatment efficacy and overcoming drug resistance in HCC., (© 2024. The Author(s).)

Hepatic fibrosis (HF) is a pathological process of structural and functional impairment of the liver and is a key component in the progression of chronic liver disease. There are no specific anti-hepatic fibrosis (anti-HF) drugs, and HF can only be improved or prevented by alleviating the cause. Autophagy of hepatic stellate cells (HSCs) is closely related to the development of HF. In recent years, traditional Chinese medicine (TCM) has achieved good therapeutic effects in the prevention and treatment of HF. Several active ingredients from TCM (AITCM) can regulate autophagy in HSCs to exert anti-HF effects through different pathways, but relevant reviews are lacking. This paper reviewed the research progress of AITCM regulating HSCs autophagy against HF, and also discussed the relationship between HSCs autophagy and HF, pointing out the problems and limitations of the current study, in order to provide references for the development of anti-HF drugs targeting HSCs autophagy in TCM. By reviewing the literature in PubMed, Web of Science, Embase, CNKI and other databases, we found that the relationship between autophagy of HSCs and HF is currently controversial. HSCs autophagy may promote HF by consuming lipid droplets (LDs) to provide energy for their activation. However, in contrast, inducing autophagy in HSCs can exert the anti-HF effect by stimulating their apoptosis or senescence, reducing type I collagen accumulation, inhibiting the extracellular vesicles release, degrading pro-fibrotic factors and other mechanisms. Some AITCM inhibit HSCs autophagy to resist HF, with the most promising direction being to target LDs. While, others induce HSCs autophagy to resist HF, with the most promising direction being to target HSCs apoptosis. Future research needs to focus on cell targeting research, autophagy targeting research and in vivo verification research, and to explore the reasons for the contradictory effects of HSCs autophagy on HF., Competing Interests: The authors report no conflicts of interest in this work., (© 2024 Liu et al.)

Albumin has a variety of biological functions, such as immunomodulatory and antioxidant activity, which depends largely on its thiol activity. However, in clinical trials, the treatment of albumin by injection of commercial human serum albumin (HSA) did not achieve the desired results. Here, we constructed reduced modified albumin (SH-Alb) for in vivo and in vitro experiments to investigate the reasons why HSA did not achieve the expected effects. SH-Alb was found to delay the progression of liver fibrosis in mice by alleviating liver inflammation and oxidative stress. Although R-Alb also has some of the above roles, the effect of SH-Alb is more remarkable. Mechanism studies have shown that SH-Alb reduces the release of pro-inflammatory and pro-fibrotic cytokine through the mitogen-activated protein kinase (MAPK) signaling pathway. In addition, SH-Alb deacetylates SOD2, a key enzyme of mitochondrial reactive oxygen species (ROS) production, by promoting the expression of SIRT3, thereby reducing the accumulation of ROS. Finally, macrophages altered by R-Alb or SH-Alb can inhibit the activation of hepatic stellate cells and endothelial cells, further delaying the progression of liver fibrosis. These results indicate that SH-Alb can remodel the phenotype of macrophages, thereby affecting the intrahepatic microenvironment and delaying the process of liver fibrosis. It provides a good foundation for the application of albumin in clinical treatment., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper, (Copyright © 2024. Published by Elsevier Masson SAS.)

Liver sinusoidal endothelial cells (LSECs) are liver-specific endothelial cells with the highest permeability than other mammalian endothelial cells, characterized by the presence of fenestrae on their surface, the absence of diaphragms and the lack of basement membrane. Located at the interface between blood and other liver cell types, LSECs mediate the exchange of substances between the blood and the Disse space, playing a crucial role in maintaining substance circulation and homeostasis of multicellular communication. As the initial responders to chronic liver injury, the abnormal LSEC activation not only changes their own physicochemical properties but also interrupts their communication with hepatic stellate cells and hepatocytes, which collectively aggravates the process of liver fibrosis. In this review, we have comprehensively updated the various pathways by which LSECs were involved in the initiation and aggravation of liver fibrosis, including but not limited to cellular phenotypic change, the induction of capillarization, decreased permeability and regulation of intercellular communications. Additionally, the intervention effects and latest regulatory mechanisms of anti-fibrotic drugs involved in each aspect have been summarized and discussed systematically. As we studied deeper into unraveling the intricate role of LSECs in the pathophysiology of liver fibrosis, we unveil a promising horizon that pave the way for enhanced patient outcomes.

Liver fibrosis is characterized by the activation of perivascular hepatic stellate cells (HSCs), the release of fibrogenic nanosized extracellular vesicles (EVs), and increased HSC glycolysis. Nevertheless, how glycolysis in HSCs coordinates fibrosis amplification through tissue zone-specific pathways remains elusive. Here, we demonstrate that HSC-specific genetic inhibition of glycolysis reduced liver fibrosis. Moreover, spatial transcriptomics revealed a fibrosis-mediated up-regulation of EV-related pathways in the liver pericentral zone, which was abrogated by glycolysis genetic inhibition. Mechanistically, glycolysis in HSCs up-regulated the expression of EV-related genes such as Ras-related protein Rab-31 ( RAB31 ) by enhancing histone 3 lysine 9 acetylation on the promoter region, which increased EV release. Functionally, these glycolysis-dependent EVs increased fibrotic gene expression in recipient HSC. Furthermore, EVs derived from glycolysis-deficient mice abrogated liver fibrosis amplification in contrast to glycolysis-competent mouse EVs. In summary, glycolysis in HSCs amplifies liver fibrosis by promoting fibrogenic EV release in the hepatic pericentral zone, which represents a potential therapeutic target.

Liver fibrosis is the excessive accumulation of extracellular matrix proteins that occurs in most types of chronic liver disease. At the cellular level, liver fibrosis is associated with the activation of hepatic stellate cells (HSCs) which transdifferentiate into a myofibroblast-like phenotype that is contractile, proliferative and profibrogenic. HSC transdifferentiation induces genome-wide changes in gene expression that enable the cell to adopt its profibrogenic functions. We have previously identified that the deubiquitinase ubiquitin C-terminal hydrolase 1 (UCHL1) is highly induced following HSC activation; however, the cellular targets of its deubiquitinating activity are poorly defined. Here, we describe a role for UCHL1 in regulating the levels and activity of hypoxia-inducible factor 1 (HIF1), an oxygen-sensitive transcription factor, during HSC activation and liver fibrosis. HIF1 is elevated during HSC activation and promotes the expression of profibrotic mediator HIF target genes. Increased HIF1α expression correlated with induction of UCHL1 mRNA and protein with HSC activation. Genetic deletion or chemical inhibition of UCHL1 impaired HIF activity through reduction of HIF1α levels. Furthermore, our mechanistic studies have shown that UCHL1 elevates HIF activity through specific cleavage of degradative ubiquitin chains, elevates levels of pro-fibrotic gene expression and increases proliferation rates. As we also show that UCHL1 inhibition blunts fibrogenesis in a pre-clinical 3D human liver slice model of fibrosis, these results demonstrate how small molecule inhibitors of DUBs can exert therapeutic effects through modulation of HIF transcription factors in liver disease. Furthermore, inhibition of HIF activity using UCHL1 inhibitors may represent a therapeutic opportunity with other HIF-related pathologies., (© 2024 The Author(s).)

Background: Biliary atresia (BA) is a devastating congenital disease characterized by inflammation and progressive liver fibrosis. Activation of hepatic stellate cells (HSCs) plays a central role in the pathogenesis of hepatic fibrosis. Our study aimed to investigate the pharmacological effect and potential mechanism of pirfenidone (PFD) and andrographolide (AGP) separately and together on liver fibrosis of BA., Materials and Methods: The bile ducts of male C57BL/6J mice were ligated or had the sham operation. The in vivo effects of PFD and/or AGP on liver fibrosis of BA were evaluated. Human hepatic stellate cells (LX-2) were also treated with PFD and/or AGP in vitro ., Results: PFD and/or AGP ameliorates liver fibrosis and inflammation in the mice model of BA, as evidenced by significant downregulated in the accumulation of collagen fibers, hepatic fibrosis markers ( α -SMA, collagen I, and collagen IV), and inflammatory markers (IL-1 β , IL-6, and TNF- α ). Moreover, compared with monotherapy, these changes are more obvious in the combined treatment of PFD and AGP. Consistent with animal experiments, hepatic fibrosis markers ( α -SMA, collagen I, and CTGF) and inflammatory markers (IL-1 β , IL-6, and TNF- α ) were significantly decreased in activated LX-2 cells after PFD and/or AGP treatment. In addition, PFD and/or AGP inhibited the activation of HSCs by blocking the TGF- β /Smad signaling pathway, and the combined treatment of PFD and AGP synergistically inhibited the phosphorylation of Smad2 and Smad3., Conclusion: The combined application of PFD and AGP exerted superior inhibitive effects on HSC activation and liver fibrosis by mediating the TGF- β /Smad signaling pathway as compared to monotherapy. Therefore, the combination of PFD and AGP may be a promising treatment strategy for liver fibrosis in BA., Competing Interests: The authors declare that there are no conflicts of interest in this study., (Copyright © 2024 Guang Xu et al.)

Aging enhances numerous processes that compromise homeostasis and pathophysiological processes. Among these, activated HSCs play a pivotal role in advancing liver fibrosis. This research delved into how aging impacts liver fibrosis mechanisms. The study involved 32 albino rats categorized into four groups: Group I (young controls), Group II (young with liver fibrosis), Group III (old controls), and Group IV (old with liver fibrosis). Various parameters including serum ALT, adiponectin, leptin, and cholesterol levels were evaluated. Histopathological analysis was performed, alongside assessments of TGF-β, FOXP3, and CD133 gene expressions. Markers of fibrosis and apoptosis were the highest in group IV. Adiponectin levels significantly decreased in Group IV compared to all other groups except Group II, while cholesterol levels were significantly higher in liver fibrosis groups than their respective control groups. Group III displayed high hepatic expression of desmin, α-SMA, GFAP and TGF- β and in contrast to Group I. Increased TGF-β and FOXP3 gene expressions were observed in Group IV relative to Group II, while CD133 gene expression decreased in Group IV compared to Group II. In conclusion, aging modulates immune responses, impairs regenerative capacities via HSC activation, and influences adipokine and cholesterol levels, elevating the susceptibility to liver fibrosis., (© 2024. The Author(s).)

Liver fibrosis/cirrhosis is a pathological state caused by excessive extracellular matrix deposition. Sustained activation of hepatic stellate cells (HSC) is the predominant cause of liver fibrosis, but the detailed mechanism is far from clear. In this study, we found that long noncoding RNA Fendrr is exclusively increased in hepatocytes in the murine model of CCl 4 - and bile duct ligation-induced liver fibrosis, as well as in the biopsies of liver cirrhosis patients. In vivo, ectopic expression of Fendrr aggravated the severity of CCl 4 -induced liver fibrosis in mice. In contrast, inhibiting Fendrr blockaded the activation of HSC and ameliorated CCl 4 -induced liver fibrosis. Our mechanistic study showed that Fendrr binds to STAT2 and enhances its enrichment in the nucleus, which then promote the expression of interleukin 6 (IL-6), and, ultimately, activates HSC in a paracrine manner. Accordingly, disrupting the interaction between Fendrr and STAT2 by ectopic expression of a STAT2 mutant attenuated the profibrotic response inspired by Fendrr in the CCl 4 -induced liver fibrosis. Notably, the increase of Fendrr in patient fibrotic liver is positively correlated with the severity of fibrosis and the expression of IL-6. Meanwhile, hepatic IL-6 positively correlates with the extent of liver fibrosis and HSC activation as well, thus suggesting a causative role of Fendrr in HSC activation and liver fibrosis. In conclusion, these observations identify an important regulatory cross talk between hepatocyte Fendrr and HSC activation in the progression of liver fibrosis, which might represent a potential strategy for therapeutic intervention., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

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-1β 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-1β 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., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: The role of exosomes derived from HepG2.2.15 cells, which express hepatitis B virus (HBV)-related proteins, in triggering the activation of LX2 liver stellate cells and promoting liver fibrosis and cell proliferation remains elusive. The focus was on comprehending the relationship and influence of differentially expressed microRNAs (DE-miRNAs) within these exosomes., Aim: To elucidate the effect of exosomes derived from HepG2.2.15 cells on the activation of hepatic stellate cell (HSC) LX2 and the progression of liver fibrosis., Methods: Exosomes from HepG2.2.15 cells, which express HBV-related proteins, were isolated from parental HepG2 and WRL68 cells. Western blotting was used to confirm the presence of the exosomal marker protein CD9. The activation of HSCs was assessed using oil red staining, whereas DiI staining facilitated the observation of exosomal uptake by LX2 cells. Additionally, we evaluated LX2 cell proliferation and fibrosis marker expression using 5-ethynyl-2'-deoxyuracil staining and western blotting, respectively. DE-miRNAs were analyzed using DESeq2. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were used to annotate the target genes of DE-miRNAs., Results: Exosomes from HepG2.2.15 cells were found to induced activation and enhanced proliferation and fibrosis in LX2 cells. A total of 27 miRNAs were differentially expressed in exosomes from HepG2.2.15 cells. GO analysis indicated that these DE-miRNA target genes were associated with cell differentiation, intracellular signal transduction, negative regulation of apoptosis, extracellular exosomes, and RNA binding. KEGG pathway analysis highlighted ubiquitin-mediated proteolysis, the MAPK signaling pathway, viral carcinogenesis, and the toll-like receptor signaling pathway, among others, as enriched in these targets., Conclusion: These findings suggest that exosomes from HepG2.2.15 cells play a substantial role in the activation, proliferation, and fibrosis of LX2 cells and that DE-miRNAs within these exosomes contribute to the underlying mechanisms., Competing Interests: Conflict-of-interest statement: All authors declare that they have no conflict of interest., (©The Author(s) 2024. Published by Baishideng Publishing Group Inc. All rights reserved.)

Non-alcoholic steatohepatitis (NASH), caused by fat buildup, can lead to liver inflammation and damage. Elucidation of the spatial distribution of fibrotic tissue in the fatty liver in NASH can be immensely useful to understand its pathogenesis. Thus, we developed a novel serial section-3D (SS3D) technique that combines high-resolution image acquisition with 3D construction software, which enabled highly detailed analysis of the mouse liver and extraction and quantification of stained tissues. Moreover, we studied the underexplored mechanism of fibrosis progression in the fatty liver in NASH by subjecting the mice to a high-fat diet (HFD), followed by lipopolysaccharide (LPS) administration. The HFD/LPS (+) group showed extensive fibrosis compared with control; additionally, the area of these fibrotic regions in the HFD/LPS (+) group was almost double that of control using our SS3D technique. LPS administration led to an increase in Tnfα and Il1β mRNA expression and the number of macrophages in the liver. On the other hand, transforming growth factor-β1 (Tgfβ1) mRNA increased in HFD group compared to that of control group without LPS-administration. In addition, COL1A1 levels increased in hepatic stellate cell (HSC)-like XL-2 cells when treated with recombinant TGF-β1, which attenuated with recombinant latency-associated protein (rLAP). This attenuation was rescued with LPS-activated macrophages. Therefore, we demonstrated that fatty liver produced "latent-form" of TGF-β1, which activated by macrophages via inflammatory cytokines such as TNFα and IL1β, resulting in activation of HSCs leading to the production of COL1A1. Moreover, we established the effectiveness of our SS3D technique in creating 3D images of fibrotic tissue, which can be used to study other diseases as well., (© 2024. The Author(s).)

Aim: Metabolic dysfunction-associated steatohepatitis (MASH) is one of the most prevalent liver diseases and is characterized by steatosis and the accumulation of bioactive lipids. This study aims to understand the specific lipid species responsible for the progression of liver fibrosis in MASH., Methods: Changes in bioactive lipid levels were examined in the livers of MASH mice fed a choline-deficient diet (CDD). Additionally, sphingosine kinase (SphK)1 mRNA, which generates sphingosine 1 phosphate (S1P), was examined in the livers of patients with MASH., Results: CDD induced MASH and liver fibrosis were accompanied by elevated levels of S1P and increased expression of SphK1 in capillarized liver sinusoidal endothelial cells (LSECs) in mice. SphK1 mRNA also increased in the livers of patients with MASH. Treatment of primary cultured mouse hepatic stellate cells (HSCs) with S1P stimulated their activation, which was mitigated by the S1P receptor (S1PR)2 inhibitor, JTE013. The inhibition of S1PR2 or its knockout in mice suppressed liver fibrosis without reducing steatosis or hepatocellular damage., Conclusion: S1P level is increased in MASH livers and contributes to liver fibrosis via S1PR2., Competing Interests: Tatsuya Kanto has personal financial interests from Gilead Sciences for Lecture fees. Hideo Shindou was collaborated with ONO Pharmaceutical company, but the collaboration is not related to this manuscript. This does not alter our adherence to PLOS ONE policies on sharing data and materials., (Copyright: © 2024 Osawa et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Hepatic stellate cell (HSC) activation and hepatic fibrosis mediated biliary atresia (BA) development, but the underlying molecular mechanisms are poorly understood. This study aimed to investigate the roles of circRNA hsa_circ_0009096 in the regulation of HSC proliferation and hepatic fibrosis., Methods: A cellular hepatic fibrosis model was established by treating LX-2 cells with transforming growth factor β (TGF-β1). RNaseR and actinomycin D assays were performed to detect hsa_circ_0009096 stability. Expression of hsa_circ_0009096, miR-370-3p, and target genes was detected using reverse transcription-qPCR. Direct binding of hsa_circ_0009096 to miR-370-3p was validated using dual luciferase reporter assay. Cell cycle progression and apoptosis of LX-2 cells were assessed using flow cytometry. The alpha-smooth muscle actin (α-SMA), collagen 1A1 (COL1A1), and TGF beta receptor 2 (TGFBR2) protein levels in LX-2 cells were analyzed using immunocytochemistry and western blotting., Results: Hsa_circ_0009096 exhibited more resistance to RNase R and actinomycinD digestion than UTRN mRNA. Hsa_circ_0009096 expression increased significantly in LX-2 cells treated with TGF-β1, accompanied by elevated α-SMA and COL1A1 expression. Hsa_circ_0009096 siRNAs effectively promoted miR-370-3p and suppressed TGFBR2 expression in LX-2 cells, mediated by direct association of hsa_circ_0009096 with miR-370-3p. Hsa_circ_0009096 siRNA interfered with the cell cycle progression, promoted apoptosis, and reduced α-SMA and COL1A1 expression in LX-2 cells treated with TGF-β1. MiR-370-3p inhibitors mitigated the alterations in cell cycle progression, apoptosis, and α-SMA, COL1A1, and TGFBR2 expression in LX-2 cells caused by hsa_circ_0009096 siRNA. In conclusion, hsa_circ_0009096 promoted HSC proliferation and hepatic fibrosis during BA pathogenesis by accelerating TGFBR2 expression by sponging miR-370-3p., Competing Interests: The authors declare that they have no competing interests., (© 2024 Wu et al.)

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 (TGFβ) 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., Competing Interests: The authors declare no competing financial interests., (Copyright: © 2024 Pan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Liver fibrosis is a chronic liver disease caused by prolonged liver injuries. Excessive accumulation of extracellular matrix replaces the damaged hepatocytes, leading to fibrous scar formation and fibrosis induction. Lactoferrin (LF) is a glycoprotein with a conserved, monomeric signal polypeptide chain, exhibiting diverse physiological functions, including antioxidant, anti-inflammatory, antibacterial, antifungal, antiviral, and antitumoral activities. Previous study has shown LF's protective role against chemically-induced liver fibrosis in rats. However, the mechanisms of LF in liver fibrosis are still unclear. In this study, we investigated LF's mechanisms in thioacetamide (TAA)-induced liver fibrosis in rats and TGF-β1-treated HSC-T6 cells. Using ultrasonic imaging, H&E, Masson's, and Sirius Red staining, we demonstrated LF's ability to improve liver tissue damage and fibrosis induced by TAA. LF reduced the levels of ALT, AST, and hydroxyproline in TAA-treated liver tissues, while increasing catalase levels. Additionally, LF treatment decreased mRNA expression of inflammatory factors such as Il-1β and Icam-1, as well as fibrogenic factors including α-Sma, Collagen I, and Ctgf in TAA-treated liver tissues. Furthermore, LF reduced TAA-induced ROS production and cell death in FL83B cells, and decreased α-SMA, Collagen I, and p-Smad2/3 productions in TGF-β1-treated HSC-T6 cells. Our study highlights LF's ability to ameliorate TAA-induced hepatocyte damage, oxidative stress, and liver fibrosis in rats, potentially through its inhibitory effect on HSC activation. These findings suggest LF's potential as a therapeutic agent for protecting against liver injuries and fibrosis., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

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.

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., Competing Interests: Conflict-of-interest statement: All the authors report no relevant conflicts of interest for this article., (©The Author(s) 2024. Published by Baishideng Publishing Group Inc. All rights reserved.)

Purpose: Human umbilical cord mesenchymal stem cell (hucMSC)-derived small extracellular vesicles (sEVs) are natural nanocarriers with promising potential in treating liver fibrosis and have widespread applications in the fields of nanomedicine and regenerative medicine. However, the therapeutic efficacy of natural hucMSC-sEVs is currently limited owing to their non-specific distribution in vivo and partial removal by mononuclear macrophages following systemic delivery. Thus, the therapeutic efficacy can be improved through the development of engineered hucMSC-sEVs capable to overcome these limitations., Patients and Methods: To improve the anti-liver fibrosis efficacy of hucMSC-sEVs, we genetically engineered hucMSC-sEVs to overexpress the anti-fibrotic gene bone morphogenic protein 7 ( BMP7 ) in parental cells. This was achieved using lentiviral transfection, following which BMP7-loaded hucMSC-sEVs were isolated through ultracentrifugation. First, the liver fibrosis was induced in C57BL/6J mice by intraperitoneal injection of 50% carbon tetrachloride (CCL4) twice a week for 8 weeks. These mice were subsequently treated with BMP7+sEVs via tail vein injection, and the anti-liver fibrosis effect of BMP7+sEVs was validated using small animal in vivo imaging, immunohistochemistry (IHC), tissue immunofluorescence, and enzyme-linked immunosorbent assay (ELISA). Finally, cell function studies were performed to confirm the in vivo results., Results: Liver imaging and liver histopathology confirmed that the engineered hucMSC-sEVs could reach the liver of mice and aggregate around activated hepatic stellate cells (aHSCs) with a significantly stronger anti-liver fibrosis effect of BMP7-loaded hucMSC-sEVs compared to those of blank or negative control-transfected hucMSC-sEVs. In vitro, BMP7-loaded hucMSC-sEVs promoted the phenotypic reversal of aHSCs and inhibited their proliferation to enhance the anti-fibrotic effects., Conclusion: These engineered BMP7-loaded hucMSC-sEVs offer a novel and promising strategy for the clinical treatment of liver fibrosis., Competing Interests: The authors report no conflicts of interest in this work., (© 2024 Zhu et al.)

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-β/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-β/Smad axis as a potential target for treatment of schistosomiasis-related liver fibrosis., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Zhong et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Sterol regulatory element-binding protein (SREBP)-1c is involved in cellular lipid homeostasis and cholesterol biosynthesis and is highly increased in nonalcoholic steatohepatitis (NASH). However, the molecular mechanism by which SREBP-1c regulates hepatic stellate cells (HSCs) activation in NASH animal models and patients have not been fully elucidated. In this study, we examined the role of SREBP-1c in NASH and the regulation of LCN2 gene expression. Wild-type and SREBP-1c knockout (1cKO) mice were fed a high-fat/high-sucrose diet, treated with carbon tetrachloride (CCl 4 ), and subjected to lipocalin-2 (LCN2) overexpression. The role of LCN2 in NASH progression was assessed using mouse primary hepatocytes, Kupffer cells, and HSCs. LCN2 expression was examined in samples from normal patients and those with NASH. LCN2 gene expression and secretion increased in CCl 4 -induced liver fibrosis mice model, and SREBP-1c regulated LCN2 gene transcription. Moreover, treatment with holo-LCN2 stimulated intracellular iron accumulation and fibrosis-related gene expression in mouse primary HSCs, but these effects were not observed in 1cKO HSCs, indicating that SREBP-1c-induced LCN2 expression and secretion could stimulate HSCs activation through iron accumulation. Furthermore, LCN2 expression was strongly correlated with inflammation and fibrosis in patients with NASH. Our findings indicate that SREBP-1c regulates Lcn2 gene expression, contributing to diet-induced NASH. Reduced Lcn2 expression in 1cKO mice protects against NASH development. Therefore, the activation of Lcn2 by SREBP-1c establishes a new connection between iron and lipid metabolism, affecting inflammation and HSCs activation. These findings may lead to new therapeutic strategies for NASH., (© 2024. The Author(s).)

Alcohol-associated liver disease (ALD) is a substantial cause of morbidity and mortality worldwide and represents a spectrum of liver injury beginning with hepatic steatosis (fatty liver) progressing to inflammation and culminating in cirrhosis. Multiple factors contribute to ALD progression and disease severity. Here, we overview several crucial mechanisms related to ALD end-stage outcome development, such as epigenetic changes, cell death, hemolysis, hepatic stellate cells activation, and hepatic fatty acid binding protein 4. Additionally, in this review, we also present two clinically relevant models using human precision-cut liver slices and hepatic organoids to examine ALD pathogenesis and progression.

Activation of hepatic stellate cells (HSCs) plays a critical role in liver fibrosis. However, the molecular basis for HSC activation remains poorly understood. Herein, we demonstrate that primary cilia are present on quiescent HSCs but exhibit a significant loss upon HSC activation which correlates with decreased levels of the ciliary protein intraflagellar transport 88 (IFT88). Ift88-knockout mice are more susceptible to chronic carbon tetrachloride-induced liver fibrosis. Mechanistic studies show that the X-linked inhibitor of apoptosis (XIAP) functions as an E3 ubiquitin ligase for IFT88. Transforming growth factor-β (TGF-β), a profibrotic factor, enhances XIAP-mediated ubiquitination of IFT88, promoting its proteasomal degradation. Blocking XIAP-mediated IFT88 degradation ablates TGF-β-induced HSC activation and liver fibrosis. These findings reveal a previously unrecognized role for ciliary homeostasis in regulating HSC activation and identify the XIAP-IFT88 axis as a potential therapeutic target for liver fibrosis., (© 2024. The Author(s).)

The p63 protein has pleiotropic functions and, in the liver, participates in the progression of nonalcoholic fatty liver disease (NAFLD). However, its functions in hepatic stellate cells (HSCs) have not yet been explored. TAp63 is induced in HSCs from animal models and patients with liver fibrosis and its levels positively correlate with NAFLD activity score and fibrosis stage. In mice, genetic depletion of TAp63 in HSCs reduces the diet-induced liver fibrosis. In vitro silencing of p63 blunts TGF-β1-induced HSCs activation by reducing mitochondrial respiration and glycolysis, as well as decreasing acetyl CoA carboxylase 1 (ACC1). Ectopic expression of TAp63 induces the activation of HSCs and increases the expression and activity of ACC1 by promoting the transcriptional activity of HER2. Genetic inhibition of both HER2 and ACC1 blunt TAp63-induced activation of HSCs. Thus, TAp63 induces HSC activation by stimulating the HER2-ACC1 axis and participates in the development of liver fibrosis., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Three-dimensional (3D) geometrical models are potent tools for quantifying complex tissue features and exploring structure-function relationships. However, these models are generally incomplete due to experimental limitations in acquiring multiple (> 4) fluorescent channels in thick tissue sections simultaneously. Indeed, predictive geometrical and functional models of the liver have been restricted to few tissue and cellular components, excluding important cellular populations such as hepatic stellate cells (HSCs) and Kupffer cells (KCs). Here, we combined deep-tissue immunostaining, multiphoton microscopy, deep-learning techniques, and 3D image processing to computationally expand the number of simultaneously reconstructed tissue structures. We then generated a spatial single-cell atlas of hepatic architecture (Hep3D), including all main tissue and cellular components at different stages of post-natal development in mice. We used Hep3D to quantitatively study 1) hepatic morphodynamics from early post-natal development to adulthood, and 2) the effect on the liver's overall structure when changing the hepatic environment after removing KCs. In addition to a complete description of bile canaliculi and sinusoidal network remodeling, our analysis uncovered unexpected spatiotemporal patterns of non-parenchymal cells and hepatocytes differing in size, number of nuclei, and DNA content. Surprisingly, we found that the specific depletion of KCs results in morphological changes in hepatocytes and HSCs. These findings reveal novel characteristics of liver heterogeneity and have important implications for both the structural organization of liver tissue and its function. Our next-gen 3D single-cell atlas is a powerful tool to understand liver tissue architecture, opening up avenues for in-depth investigations into tissue structure across both normal and pathological conditions., (© 2024. The Author(s).)

Background & Aims: Sphingosine kinase 1 (SphK1) has distinct roles in the activation of Kupffer cells and hepatic stellate cells in liver fibrosis. Here, we aim to investigate the roles of SphK1 on hepatic macrophage recruitment and polarization in liver fibrosis., Methods: Liver fibrosis was induced by carbon tetrachloride in wild-type and SphK1 -/- mice to study the recruitment and polarization of macrophages. The effects of SphK1 originated from macrophages or other liver cell types on liver fibrosis were further strengthened by bone marrow transplantation. The direct effects of SphK1 on macrophage polarization were also investigated in vitro. Expression analysis of SphK1 and macrophage polarization index was conducted with human liver samples., Results: SphK1 deletion attenuated the recruitment of hepatic macrophages along with reduced M1 and M2 polarization in mice induced by carbon tetrachloride. SphK1 deficiency in endogenous liver cells attenuated macrophage recruitment via C-C motif chemokine ligand 2. Macrophage SphK1 activated the ASK1-JNK1/2-p38 signaling pathway to promote M1 polarization. Furthermore, macrophage SphK1 downregulated small ubiquitin-like modifier-specific peptidase1 to decrease de-SUMOylation of Kruppel-like factor 4 to promote M2 polarization. Finally, we confirmed that SphK1 expression was elevated and positively correlated with macrophage M1 and M2 polarization in human fibrosis livers., Conclusions: Our findings demonstrated that SphK1 aggravated liver fibrosis by promoting macrophage recruitment and M1/M2 polarization. SphK1 in macrophages is a potential therapeutic target for the treatment of liver fibrosis., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background & Aims: Cirrhotic portal hypertension (CPH) is the leading cause of mortality in patients with cirrhosis. Over 50% of patients with CPH treated with current clinical pharmacotherapy still present variceal bleeding or sometimes death owing to insufficient reduction in portal pressure. Elevated intrahepatic vascular resistance (IHVR) plays a fundamental role in increasing portal pressure. Because of its potent effect in reducing portal pressure and maintaining normal portal inflow to preserve liver function, lowering the IHVR is acknowledged as an optimal anti-CPH strategy but without clinical drugs. We aimed to investigate the protective effect of microbial-derived Urolithin A (UroA) in IHVR and CPH., Methods: Carbon tetrachloride or bile duct ligation surgery was administered to mice to induce liver fibrosis and CPH. 16S rRNA gene sequencing was used for microbial analysis. Transcriptomics and metabolomics analyses were employed to study the host and cell responses., Results: UroA was remarkably deficient in patients with CPH and was negatively correlated with disease severity. UroA deficiency was also confirmed in CPH mice and was associated with a reduced abundance of UroA-producing bacterial strain (Lactobacillus murinus, L. murinus). Glutaminolysis of hepatic stellate cells (HSCs) was identified as a previously unrecognized target of UroA. UroA inhibited the activity of glutaminase1 to suppress glutaminolysis, which counteracted fibrogenesis and contraction of HSCs and ameliorated CPH by relieving IHVR. Supplementation with UroA or L. murinus effectively ameliorated CPH in mice., Conclusions: We for the first time identify the deficiency of gut microbial metabolite UroA as an important cause of CPH. We demonstrate that UroA exerts an excellent anti-CPH effect by suppressing HSC glutaminolysis to lower the IHVR, which highlighted its great potential as a novel therapeutic agent for CPH., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background & Aims: Transforming growth factor-β1 (TGF-β1) plays important roles in chronic liver diseases, including metabolic dysfunction-associated steatotic liver disease (MASLD). MASLD involves various biological processes including dysfunctional cholesterol metabolism and contributes to progression to metabolic dysfunction-associated steatohepatitis and hepatocellular carcinoma. However, the reciprocal regulation of TGF-β1 signaling and cholesterol metabolism in MASLD is yet unknown., Methods: Changes in transcription of genes associated with cholesterol metabolism were assessed by RNA sequencing of murine hepatocyte cell line (alpha mouse liver 12/AML12) and mouse primary hepatocytes treated with TGF-β1. Functional assays were performed on AML12 cells (untreated, TGF-β1 treated, or subjected to cholesterol enrichment [CE] or cholesterol depletion [CD]), and on mice injected with adenovirus-associated virus 8-control/TGF-β1., Results: TGF-β1 inhibited messenger RNA expression of several cholesterol metabolism regulatory genes, including rate-limiting enzymes of cholesterol biosynthesis in AML12 cells, mouse primary hepatocytes, and adenovirus-associated virus-TGF-β1-treated mice. Total cholesterol levels and lipid droplet accumulation in AML12 cells and liver tissue also were reduced upon TGF-β1 treatment. Smad2/3 phosphorylation after 2 hours of TGF-β1 treatment persisted after CE or CD and was mildly increased after CD, whereas TGF-β1-mediated AKT phosphorylation (30 min) was inhibited by CE. Furthermore, CE protected AML12 cells from several effects mediated by 72 hours of incubation with TGF-β1, including epithelial-mesenchymal transition, actin polymerization, and apoptosis. CD mimicked the outcome of long-term TGF-β1 administration, an effect that was blocked by an inhibitor of the type I TGF-β receptor. In addition, the supernatant of CE- or CD-treated AML12 cells inhibited or promoted, respectively, the activation of LX-2 hepatic stellate cells., Conclusions: TGF-β1 inhibits cholesterol metabolism whereas cholesterol attenuates TGF-β1 downstream effects in hepatocytes., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)