XIAP-mediated degradation of IFT88 disrupts HSC cilia to stimulate HSC activation and liver fibrosis.

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-ß (TGF-ß), a profibrotic factor, enhances XIAP-mediated ubiquitination of IFT88, promoting its proteasomal degradation. Blocking XIAP-mediated IFT88 degradation ablates TGF-ß-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.

Agalactosyl IgG induces liver fibrogenesis via Fc gamma receptor 3a on human hepatic stellate cells.

The relevance of aberrant serum IgG N-glycosylation in liver fibrosis has been identified; however, its causal effect remains unclear. Because hepatic stellate cells (HSCs) contribute substantially to liver fibrosis, we investigated whether and through which mechanisms IgG N-glycosylation affects the fibrogenic properties of HSCs. Analysis of serum IgG1 N-glycome from 151 patients with chronic hepatitis B or liver cirrhosis revealed a positive correlation between Ishak fibrosis grading and IgG1 with agalactosyl N-glycoforms on the crystallizable fragment (Fc). Fc gamma receptor (FcγR) IIIa was observed in cultured human HSCs and HSCs in human liver tissues, and levels of FcγRIIIa in HSCs correlated with the severity of liver fibrosis. Additionally, agalactosyl IgG treatment caused HSCs to have a fibroblast-like morphology, enhanced migration and invasion capabilities, and enhanced expression of the FcγRIIIa downstream tyrosine-protein kinase SYK. Furthermore, agalactosyl IgG treatment increased fibrogenic factors in HSCs, including transforming growth factor (TGF)-ß1, total collagen, platelet-derived growth factor subunit B and its receptors, pro-collagen I-α1, α-smooth muscle actin, and matrix metalloproteinase 9. These effects were more pronounced in HSCs that stably expressed FCGR3A and were reduced in FCGR3A knockout cells. Agalactosyl IgG and TGF-ß1 each increased FCGR3A in HSCs. Furthermore, serum TGF-ß1 concentrations in patients were positively correlated with agalactosyl IgG1 levels and liver fibrosis severity, indicating a positive feedback loop involving agalactosyl IgG, HSC-FcγRIIIa, and TGF-ß1. In conclusion, agalactosyl IgG promotes fibrogenic characteristics in HSCs through FcγRIIIa. © 2024 The Pathological Society of Great Britain and Ireland.

Neutrophil extracellular traps activate hepatic stellate cells and monocytes via NLRP3 sensing in alcohol-induced acceleration of MASH fibrosis.

OBJECTIVE: Alcohol use in metabolic dysfunction-associated steatohepatitis (MASH) is associated with an increased risk of fibrosis and liver-related death. Here, we aimed to identify a mechanism through which repeated alcohol binges exacerbate liver injury in a high fat-cholesterol-sugar diet (MASH diet)-induced model of MASH. DESIGN: C57BL/6 mice received either chow or the MASH diet for 3 months with or without weekly alcohol binges. Neutrophil infiltration, neutrophil extracellular traps (NETs) and fibrosis were evaluated. RESULTS: We found that alcohol binges in MASH increase liver injury and fibrosis. Liver transcriptomic profiling revealed differential expression of genes involved in extracellular matrix reorganisation, neutrophil activation and inflammation compared with alcohol or the MASH diet alone. Alcohol binges specifically increased NET formation in MASH livers in mice, and NETs were also increased in human livers with MASH plus alcohol use. We discovered that cell-free NETs are sensed via Nod-like receptor protein 3 (NLRP3). Furthermore, we show that cell-free NETs in vitro induce a profibrotic phenotype in hepatic stellate cells (HSCs) and proinflammatory monocytes. In vivo, neutrophil depletion using anti-Ly6G antibody or NET disruption with deoxyribonuclease treatment abrogated monocyte and HSC activation and ameliorated liver damage and fibrosis. In vivo, inhibition of NLRP3 using MCC950 or NLRP3 deficiency attenuated NET formation, liver injury and fibrosis in MASH plus alcohol diet-fed mice (graphical abstract). CONCLUSION: Alcohol binges promote liver fibrosis via NET-induced activation of HSCs and monocytes in MASH. Our study highlights the potential of inhibition of NETs and/or NLRP3, as novel therapeutic strategies to combat the profibrotic effects of alcohol in MASH.

ATF3 is involved in rSjP40-mediated inhibition of HSCs activation in Schistosoma japonicum-infected mice.

Schistosomiasis is a parasitic disease characterized by liver fibrosis, a process driven by the activation of hepatic stellate cells (HSCs) and subsequent collagen production. Previous studies from our laboratory have demonstrated the ability of Schistosoma japonicum protein P40 (SjP40) to inhibit HSCs activation and exert an antifibrotic effect. In this study, we aimed to elucidate the molecular mechanism underlying the inhibitory effect of recombinant SjP40 (rSjP40) on HSCs activation. Using a cell model in which rSjP40 inhibited LX-2 cell activation, we performed RNA-seq analyses and identified ATF3 as the most significantly altered gene. Further investigation revealed that rSjP40 inhibited HSCs activation partly by suppressing ATF3 activation. Knockdown of ATF3 in mouse liver significantly alleviated S. japonicum-induced liver fibrosis. Moreover, our results indicate that ATF3 is a direct target of microRNA-494-3p, a microRNA associated with anti-liver fibrosis effects. rSjP40 was found to downregulate ATF3 expression by upregulating microRNA-494-3p in LX-2 cells. This downregulation led to the inhibition of the expression of liver fibrosis proteins α-SMA and COL1A1, ultimately alleviating liver fibrosis caused by S. japonicum.

The well-developed actin cytoskeleton and Cthrc1 expression by actin-binding protein drebrin in myofibroblasts promote cardiac and hepatic fibrosis.

Fibrosis is mainly triggered by inflammation in various tissues, such as heart and liver tissues, and eventually leads to their subsequent dysfunction. Fibrosis is characterized by the excessive accumulation of extracellular matrix proteins (e.g., collagens) produced by myofibroblasts. The well-developed actin cytoskeleton of myofibroblasts, one of the main features differentiating them from resident fibroblasts in tissues under inflammatory conditions, contributes to maintaining their ability to produce excessive extracellular matrix proteins. However, the molecular mechanisms via which the actin cytoskeleton promotes the production of fibrosis-related genes in myofibroblasts remain unclear. In this study, we found, via single-cell analysis, that developmentally regulated brain protein (drebrin), an actin-binding protein, was specifically expressed in cardiac myofibroblasts with a well-developed actin cytoskeleton in fibrotic hearts. Moreover, our immunocytochemistry analysis revealed that drebrin promoted actin cytoskeleton formation and myocardin-related transcription factor-serum response factor signaling. Comprehensive single-cell analysis and RNA-Seq revealed that the expression of collagen triple helix repeat containing 1 (Cthrc1), a fibrosis-promoting secreted protein, was regulated by drebrin in cardiac myofibroblasts via myocardin-related transcription factor-serum response factor signaling. Furthermore, we observed the profibrotic effects of drebrin exerted via actin cytoskeleton formation and the Cthrc1 expression regulation by drebrin in liver myofibroblasts (hepatic stellate cells). Importantly, RNA-Seq demonstrated that drebrin expression levels increased in human fibrotic heart and liver tissues. In summary, our results indicated that the well-developed actin cytoskeleton and Cthrc1 expression due to drebrin in myofibroblasts promoted cardiac and hepatic fibrosis, suggesting that drebrin is a therapeutic target molecule for fibrosis.

Lipidomic profiling of rat hepatic stellate cells during activation reveals a two-stage process accompanied by increased levels of lysosomal lipids.

Hepatic stellate cells (HSCs) are liver-resident cells best known for their role in vitamin A storage under physiological conditions. Upon liver injury, HSCs activate into myofibroblast-like cells, a key process in the onset of liver fibrosis. Lipids play an important role during HSC activation. Here, we provide a comprehensive characterization of the lipidomes of primary rat HSCs during 17 days of activation in vitro. For lipidomic data interpretation, we expanded our previously described Lipid Ontology (LION) and associated web application (LION/Web) with the LION-PCA heatmap module, which generates heatmaps of the most typical LION-signatures in lipidomic datasets. Furthermore, we used LION to perform pathway analysis to determine the significant metabolic conversions in lipid pathways. Together, we identify two distinct stages of HSC activation. In the first stage, we observe a decrease of saturated phosphatidylcholine, sphingomyelin, and phosphatidic acid and an increase in phosphatidylserine and polyunsaturated bis(monoacylglycero)phosphate (BMP), a lipid class typically localized at endosomes and lysosomes. In the second activation stage, BMPs, hexosylceramides, and ether-linked phosphatidylcholines are elevated, resembling a lysosomal lipid storage disease profile. The presence of isomeric structures of BMP in HSCs was confirmed ex vivo in MS-imaging datasets of steatosed liver sections. Finally, treatment with pharmaceuticals targeting the lysosomal integrity led to cell death in primary HSCs but not in HeLa cells. In summary, our combined data suggest that lysosomes play a critical role during a two-stage activation process of HSCs.

Cross-talk between gastric cancer and hepatic stellate cells promotes invadopodia formation during liver metastasis.

In gastric cancer (GC), the liver is a common organ for distant metastasis, and patients with gastric cancer with liver metastasis (GCLM) generally have poor prognosis. The mechanism of GCLM is unclear. Invadopodia are special membrane protrusions formed by tumor cells that can degrade the basement membrane and ECM. Herein, we investigated the role of invadopodia in GCLM. We found that the levels of invadopodia-associated proteins were significantly higher in liver metastasis than in the primary tumors of patients with GCLM. Furthermore, GC cells could activate hepatic stellate cells (HSCs) within the tumor microenvironment of liver metastases through the secretion of platelet-derived growth factor subunit B (PDGFB). Activated HSCs secreted hepatocyte growth factor (HGF), which activated the MET proto-oncogene, MET receptor of GC cells, thereby promoting invadopodia formation through the PI3K/AKT pathway and subsequently enhancing the invasion and metastasis of GC cells. Therefore, cross-talk between GC cells and HSCs by PDGFB/platelet derived growth factor receptor beta (PDGFRß) and the HGF/MET axis might represent potential therapeutic targets to treat GCLM.

Inflammasomes in chronic liver disease: hepatic injury, fibrosis progression and systemic inflammation.

Chronic liver disease (CLD) leads to hepatocellular injury that triggers a pro-inflammatory state in several parenchymal and non-parenchymal hepatic cell types ultimately resulting in liver fibrosis, cirrhosis, portal hypertension (PH) and liver failure. Thus, an improved understanding of the inflammasomes - as key molecular drivers of liver injury - supports the development of novel diagnostic or prognostic biomarkers and effective therapeutics. In liver disease, innate immune cells respond to hepatic noxes by activating cell-intrinsic inflammasomes via toll-like receptors (TLRs) and nuclear factor kappa-B (NF-κB) and release of pro-inflammatory cytokines (such as IL-1ß, IL-18, TNF-α and IL-6). Subsequently, cells of the adaptive immune system are recruited to fuel hepatic inflammation, and liver parenchymal cells may undergo programmed cell-death mediated by gasdermin D, termed pyroptosis. With liver disease progression, there is a shift towards a type 2 inflammatory response, which promotes tissue repair but also fibrogenesis. Inflammasome activation may also occur at extrahepatic sites, such as the white adipose tissue in metabolic dysfunction-associated steatohepatitis (MASH). In end-stage liver disease, flares of inflammation (e.g., in severe alcohol-related hepatitis) that spark on a dysfunctional immune system, contribute to inflammasome-mediated liver injury and potentially result in organ dysfunctions/failures, as seen in acute-on-chronic liver failure (ACLF). This review provides an overview on current concepts regarding inflammasome activation in liver disease progression and related biomarkers and therapeutic approaches that are being developed for patients with liver disease.

Blueberry anthocyanins improve liver fibrosis by regulating NCOA4 ubiquitination through TRIM7 to affect ferroptosis of hepatic stellate cells.

This study aims to investigate the role and molecular mechanism of anthocyanin in improving liver fibrosis through ferroptosis, providing a basis for drug development and targeted therapy. In this study, a mouse model of liver fibrosis was established using CCl4, and the anthocyanin treatment groups were administered 100 mg/kg anthocyanin daily via gavage. Furthermore, real-time fluorescent quantitative PCR (qRT-PCR), Western blotting (WB), and enzyme-linked immunosorbent assay were used to assess liver fibrosis indicators and liver injury markers. Histopathological methods were used to confirm the morphology of liver injury in different treatment groups. The effects of anthocyanins on ferroptosis markers, NCOA4 and FTH1 expression, were examined through qRT-PCR, WB, and Co-IP. Confocal microscopy was used to validate the colocalization of ferritin and lysosomes. A differential expression model of TRIM7 was constructed to verify its impact on the progression of liver fibrosis. The present study demonstrates the hepatoprotective effects of anthocyanins in liver fibrosis, highlighting their ability to enhance hepatic stellate cell (HSC) ferroptosis and regulate ferritin autophagy. Moreover, TRIM7 is identified as a key mediator of anthocyanin-induced regulation of hepatic stellate cells activation for liver fibrosis treatment through modulation of ferroautophagy. Mechanistic investigations further reveal that TRIM7 exerts its influence on the process of ferroautophagy by controlling NCOA4 ubiquitination. Our study discovered that anthocyanins could improve liver fibrosis by regulating NCOA4 ubiquitination through TRIM7, thereby affecting hepatic stellate cells' ferroptosis levels.NEW & NOTEWORTHY This was the first study to demonstrate that anthocyanins can improve the progression of liver fibrosis by promoting hepatic stellate cell (HSC) ferroptosis. Anthocyanins could affect the content of Fe2+ by promoting ferroautophagy in HSCs, thereby promoting the level of ferroptosis. This study demonstrates for the first time that anthocyanins can inhibit the expression of TRIM7 and then affect the ubiquitination of NCOA4 to regulate the level of ferritin autophagy and ferroptosis.

Apigenin intervenes in liver fibrosis by regulating PKM2-HIF-1α mediated oxidative stress.

Apigenin (API) is a natural flavonoid compound with antioxidant, anti fibrotic, anti-inflammatory and other effects, but there is limited research on the effect of API on liver fibrosis. This study aims to explore the effect and potential mechanism of API on liver fibrosis induced by CCl4 in mice. The results indicate that API reduces oxidative stress levels, inhibits hepatic stellate cell (HSC) activation, and exerts anti liver fibrosis effects by regulating the PKM2-HIF-1α pathway. We observed that API alleviated liver tissue pathological damage and collagen deposition in CCl4 induced mouse liver fibrosis model, promoting the recovery of liver function in mice with liver fibrosis. In addition, the API inhibits the transition of Pyruvate kinase isozyme type M2 (PKM2) from dimer to tetramer formation by regulating the EGFR-MEK1/2-ERK1/2 pathway, thereby preventing dimer from entering the nucleus and blocking PKM2-HIF-1α access. This change leads to a decrease in malondialdehyde (MDA) and Catalase (CAT) levels and an increase in glutathione (GSH), superoxide dismutase (SOD), glutathione peroxidase (GSH-PX) levels, as well as total antioxidant capacity (T-AOC) in the liver of liver fibrosis mice. At the same time, API downregulated the expression of α-smooth muscle actin (α-SMA), Vimentin and Desmin in the liver tissue of mice with liver fibrosis, inhibited the activation of HSC, and reduced collagen deposition. These results indicate that API can inhibit HSC activation and alleviate CCl4 induced liver fibrosis by inhibiting the PKM2-HIF-1α pathway and reducing oxidative stress, laying an important foundation for the development and clinical application of API as a novel drug for treating liver fibrosis.

Knockdown of circ_0044226 promotes endoplasmic reticulum stress-mediated autophagy and apoptosis in hepatic stellate cells via miR-4677-3p/SEC61G axis.

Downregulation of circ_0044226 has been demonstrated to reduce pulmonary fibrosis, but the role of circ_0044226 in liver fibrosis remains to be explored. In this work, we found that circ_0044226 expression was upregulated during liver fibrosis. Knockdown of circ_0044226 inhibited proliferation, promoted autophagy and apoptosis of hepatic stellate cell LX-2. Bioinformatic analysis and dual luciferase reporter assays confirmed the interaction between circ_0044226, miR-4677-3p and SEC61G. Mechanistically, knockdown of circ_0044226 suppressed SEC61G expression by releasing miR-4677-3p, thereby enhancing endoplasmic reticulum stress. Overexpression of SEC61G or endoplasmic reticulum stress inhibitor 4-phenylbutiric acid partially reversed the effect of knockdown circ_0044226 on LX-2 cell function. In vivo experiments showed that inhibition of circ_0044226 attenuated CCL4-induced liver fibrosis in mice. These imply that circ_0044226 may be a potential target for the treatment of liver fibrosis.

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

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

Single-Cell Transcriptomic Analysis Revealed the Cell Population Changes and Cell-Cell Communication in the Liver of a Carnivorous Fish in Response to High-Carbohydrate Diet.

BACKGROUND: Carnivorous fish have a low carbohydrate utilization ability, and the physiologic and molecular basis of glucose intolerance has not been fully illustrated. OBJECTIVES: This study aimed to use largemouth bass as a model to investigate the possible mechanism of glucose intolerance in carnivorous fish with the help of single-nuclei RNA sequencing (snRNA-seq). METHODS: Two diets were formulated, a low-carbohydrate (LC) diet and a high-carbohydrate (HC) diet. The feeding trial lasted for 6 wk, and then, growth performance, biochemical parameters, liver histology, and snRNA-seq were performed. RESULTS: Growth performance of fish was not affected by the HC diet, while liver glucolipid metabolism disorder and liver injury were observed. A total of 13,247 and 12,848 cells from the liver derived from 2 groups were isolated and sequenced, and 7 major liver cell types were annotated by the marker genes. Hepatocytes and cholangiocytes were lower and hepatic stellate cells (HSCs) and immune cells were higher in the HC group than those in the LC group. Reclustering analysis identified 7 subtypes of hepatocytes and immune cells, respectively. The HSCs showed more cell communication with other cell types, and periportal hepatocytes showed more cell communication with other hepatocyte subtypes. Cell-cell communication mainly focused on cell junction-related signaling pathways. Uncovered by the pseudotime analysis, midzonal hepatocytes were differentiated into 2 major branches-biliary epithelial hepatocytes and hepatobiliary hybrid progenitor. Cell junction and liver fibrosis-related genes were highly expressed in the HC group. HC diet induced the activation of HSCs and, therefore, led to the liver fibrosis of largemouth bass. CONCLUSIONS: HC diet induces liver glucolipid metabolism disorder and liver injury of largemouth bass. The increase and activation of HSCs might be the main reason for the liver injury. In adaption to HC diet, midzonal hepatocytes differentiates into 2 major branches-biliary epithelial hepatocytes and hepatobiliary hybrid progenitors.

Hepatocyte GPCR signaling regulates IRF3 to control hepatic stellate cell transdifferentiation.

BACKGROUND: Interferon Regulatory Factor 3 (IRF3) is a transcription factor that plays a crucial role in the innate immune response by recognizing and responding to foreign antigens. Recently, its roles in sterile conditions are being studied, as in metabolic and fibrotic diseases. However, the search on the upstream regulator for efficient pharmacological targeting is yet to be fully explored. Here, we show that G protein-coupled receptors (GPCRs) can regulate IRF3 phosphorylation through of GPCR-Gα protein interaction. RESULTS: IRF3 and target genes were strongly associated with fibrosis markers in liver fibrosis patients and models. Conditioned media from MIHA hepatocytes overexpressing IRF3 induced fibrogenic activation of LX-2 hepatic stellate cells (HSCs). In an overexpression library screening using active mutant Gα subunits and Phos-tag immunoblotting, Gαs was found out to strongly phosphorylate IRF3. Stimulation of Gαs by glucagon or epinephrine or by Gαs-specific designed GPCR phosphorylated IRF3. Protein kinase A (PKA) signaling was primarily responsible for IRF3 phosphorylation and Interleukin 33 (IL-33) expression downstream of Gαs. PKA phosphorylated IRF3 on a previously unrecognized residue and did not require reported upstream kinases such as TANK-binding kinase 1 (TBK1). Activation of Gαs signaling by glucagon induced IL-33 production in hepatocytes. Conditioned media from the hepatocytes activated HSCs, as indicated by α-SMA and COL1A1 expression, and this was reversed by pre-treatment of the media with IL-33 neutralizing antibody. CONCLUSIONS: Gαs-coupled GPCR signaling increases IRF3 phosphorylation through cAMP-mediated activation of PKA. This leads to an increase of IL-33 expression, which further contributes to HSC activation. Our findings that hepatocyte GPCR signaling regulates IRF3 to control hepatic stellate cell transdifferentiation provides an insight for understanding the complex intercellular communication during liver fibrosis progression and suggests therapeutic opportunities for the disease. Video Abstract.

Targeted inhibition of autophagy in hepatic stellate cells by hydroxychloroquine: An effective therapeutic approach for the treatment of liver fibrosis.

BACKGROUND AND PURPOSE: Liver fibrosis is a wound-healing reaction which is the main cause of chronic liver diseases worldwide. The activated hepatic stellate cell (aHSC) is the main driving factor in the development of liver fibrosis. Inhibiting autophagy of aHSC can prevent the progression of liver fibrosis, but inhibiting autophagy of other liver cells has opposite effects. Hence, targeted inhibition of autophagy in aHSC is quite necessary for the treatment of liver fibrosis, which prompts us to explore the targeted delivery system of small molecule autophagy inhibitor hydroxychloroquine (HCQ) that can target aHSC and alleviate the liver fibrosis. METHODS: The delivery system of HCQ@retinol-liposome nanoparticles (HCQ@ROL-LNPs) targeting aHSC was constructed by the film dispersion and pH-gradient method. TGF-ß-induced HSC activation and thioacetamide (TAA)-induced liver fibrosis mice model were established, and the targeting ability and therapeutic effect of HCQ@ROL-LNPs in liver fibrosis were studied subsequently in vitro and in vivo. RESULTS: HCQ@ROL-LNPs have good homogeneity and stability. They inhibited the autophagy of aHSC selectively by HCQ and reduced the deposition of extracellular matrix (ECM) and the damage to other liver cells. Compared with the free HCQ and HCQ@LNPs, HCQ@ROL-LNPs had good targeting ability, showing enhanced therapeutic effect and low toxicity to other organs. CONCLUSION: Construction of HCQ@ROL-LNPs delivery system lays a theoretical and experimental foundation for the treatment of liver fibrosis and promotes the development of clinical therapeutic drugs for liver diseases.

Human umbilical cord-derived mesenchymal stem cells attenuate hepatic stellate cells activation and liver fibrosis.

BACKGROUND: Liver cirrhosis, a prevalent chronic liver disease, is characterized by liver fibrosis as its central pathological process. Recent advancements highlight the clinical efficacy of umbilical cord mesenchymal stem cell (UC-MSC) therapy in the treatment of liver cirrhosis. METHODS AND RESULTS: We investigated the pharmacodynamic effects of UC-MSCs and MSC conditional medium (MSC-CM) in vivo, utilizing a carbon tetrachloride (CCl4)-induced fibrotic rat model. Concurrently, we assessed the in vitro impact of MSCs and MSC-CM on various cellular process of hepatic stellate cells (HSCs), including proliferation, apoptosis, activation, immunomodulatory capabilities, and inflammatory factor secretion. Our results indicate that both MSCs and MSC-CM significantly ameliorate the pathological extent of fibrosis in animal tissues, reducing the collagen content, serum biochemical indices and fibrosis biomarkers. In vitro, MSC-CM significantly inhibited the activation of the HSC line LX-2. Notably, MSC-CM modulated the expression of type I procollagen and TGFß-1 while increasing MMP1 expression. This modulation restored the MMP1/TIMP1 ratio imbalance and extracellular matrix deposition in TGFß-1 induced fibrosis. Both MSCs and MSC-CM not only induced apoptosis in HSCs but also suppressed proliferation and inflammatory cytokine release from activated HSCs. Furthermore, MSCs and MSC-CM exerted a suppressive effect on total lymphocyte activation. CONCLUSIONS: UC-MSCs and MSC-CM primarily modulate liver fibrosis severity by regulating HSC activation. This study provides both in vivo and in vitro pharmacodynamic evidence supporting the use of MSCs in liver fibrosis treatment.

Exogenous S1P via S1P receptor 2 induces CTGF expression through Src-RhoA-ROCK-YAP pathway in hepatic stellate cells.

BACKGROUND: Hepatic fibrosis, a prevalent chronic liver condition, involves excessive extracellular matrix production associated with aberrant wound healing. Hepatic stellate cells (HSCs) play a pivotal role in liver fibrosis, activated by inflammatory factors such as sphingosine 1-phosphate (S1P). Despite S1P's involvement in fibrosis, its specific role and downstream pathway in HSCs remain controversial. METHODS: In this study, we investigated the regulatory role of S1P/S1P receptor (S1PR) in Hippo-YAP activation in both LX-2 cell lines and primary HSCs. Real-time PCR, western blot, pharmacological inhibitors, siRNAs, and Rho activity assays were adopted to address the molecular mechanisms of S1P mediated YAP activation. RESULTS: Serum and exogenous S1P significantly increased the expression of YAP target genes in HSCs. Pharmacologic inhibitors and siRNA-mediated knockdowns of S1P receptors showed S1P receptor 2 (S1PR2) as the primary mediator for S1P-induced CTGF expression in HSCs. Results using siRNA-mediated knockdown, Verteporfin, and Phospho-Tag immunoblots showed that S1P-S1PR2 signaling effectively suppressed the Hippo kinases cascade, thereby activating YAP. Furthermore, S1P increased RhoA activities in cells and ROCK inhibitors effectively blocked CTGF induction. Cytoskeletal-perturbing reagents were shown to greatly modulate CTGF induction, suggesting the important role of actin cytoskeleton in S1P-induced YAP activation. Exogeneous S1P treatment was enough to increase the expression of COL1A1 and α-SMA, that were blocked by YAP specific inhibitor. CONCLUSIONS: Our data demonstrate that S1P/S1PR2-Src-RhoA-ROCK axis leads to Hippo-YAP activation, resulting in the up-regulation of CTGF, COL1A1 and α-SMA expression in HSCs. Therefore, S1PR2 may represent a potential therapeutic target for hepatic fibrosis.

TET1 inhibits liver fibrosis by blocking hepatic stellate cell activation.

Hepatic stellate cells (HSCs) are critical regulator contributing to the onset and progression of liver fibrosis. Chronic liver injury triggers HSCs to undergo vast changes and trans-differentiation into a myofibroblast HSCs, the mechanism remains to be elucidated. This study investigated that the involvement of hydroxymethylase TET1 (ten-eleven translocation 1) in HSC activation and liver fibrosis. It is revealed that TET1 levels were downregulated in the livers in mouse models of liver fibrosis and patients with cirrhosis, as well as activated HSCs in comparison to quiescent HSCs. In vitro data showed that the inhibition of TET1 promoted the activation HSC, whereas TET1 overexpression inhibited HSC activation. Moreover, TET1 could regulate KLF2 (Kruppel-like transcription factors) transcription by promoting hydroxymethylation of its promoter, which in turn suppressed the activation of HSCs. In vivo, it is confirmed that liver fibrosis was aggravated in Tet1 knockout mice after CCl4 injection, accompanied by excessive activation of primary stellate cells, in contrast to wild-type mice. In conclusion, we suggested that TET1 plays a significant role in HSC activation and liver fibrosis, which provides a promising target for anti-fibrotic therapies.

Hydronidone induces apoptosis in activated hepatic stellate cells through endoplasmic reticulum stress-associated mitochondrial apoptotic pathway.

BACKGROUND AND AIM: Hydronidone (HDD) is a novel pirfenidone derivative developed initially to reduce hepatotoxicity. Our previous studies in animals and humans have demonstrated that HDD treatment effectively attenuates liver fibrosis, yet the underlying mechanism remains unclear. This study aimed to investigate whether HDD exerts its anti-fibrotic effect by inducing apoptosis in activated hepatic stellate cells (aHSCs) through the endoplasmic reticulum stress (ERS)-associated mitochondrial apoptotic pathway. METHODS: The carbon tetrachloride (CCl4)- and 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-induced liver fibrosis models were used for in vivo studies. In vitro studies were conducted using the human hepatic stellate cell line LX-2. The apoptotic effect of HDD on aHSCs was examined using TUNEL and flow cytometry assays. The small interfering RNA (siRNA) technique was employed to downregulate the expression of interest genes. RESULTS: HDD treatment significantly promoted apoptosis in aHSCs in both the CCl4- and DDC-induced liver fibrosis in mice and LX-2 cells. Mechanistic studies revealed that HDD triggered ERS and subsequently activated the IRE1α-ASK1-JNK pathway. Furthermore, the influx of cytochrome c from the mitochondria into the cytoplasm was increased, leading to mitochondrial dysfunction and ultimately triggering apoptosis in aHSCs. Notably, inhibition of IRE1α or ASK1 by siRNA partially abrogated the pro-apoptotic effect of HDD in aHSCs. CONCLUSIONS: The findings of both in vivo and in vitro studies suggest that HDD induces apoptosis in aHSCs via the ERS-associated mitochondrial apoptotic pathway, potentially contributing to the amelioration of liver fibrosis.

Novel protein C6ORF120 promotes liver fibrosis by activating hepatic stellate cells through the PI3K/Akt/mTOR pathway.

BACKGROUND AND AIM: The role of C6ORF120 in promoting CCL4-induced hepatic fibrosis and its possible mechanisms were explored in C6orf120 knockout rats (C6orf120-/-) and LX-2 cells (a type of human hepatic stellate cell line). METHODS: In vivo experiments, wild-type and C6orf120-/- rats were used to investigate the function of C6ORF120. In the in vitro experiments, C6ORF120 recombinant protein (rC6ORF120) at a concentration of 200 ng/mL was used to stimulate LX-2 cells. Sirius Red staining, Masson staining, western blotting, polymerase chain reaction, immunohistochemistry, and immunofluorescence were used to explore fibrosis-associated factors. RESULTS: C6orf120-/- rats showed mild fibrosis and liver injury in the CCL4-induced liver fibrosis model. Furthermore, RNA-seq revealed that C6orf120-/- rats had less extracellular matrix deposition and activated stellate cells. Consistent with the in vivo, the rC6ORF120 induced LX-2 cell activation. Moreover, mechanistic studies revealed that the p-PI3K/PI3K, p-Akt/Akt, and p-mTOR/mTOR levels were significantly elevated and LY294002 (a PI3K/Akt/mTOR typical pathway inhibitor) reversed the function of C6ORF120 in activating LX-2 cells. CONCLUSION: C6ORF120 could activate hepatic stellate cells and promote hepatic fibrosis via the PI3K/Akt/mTOR signaling pathway.