RESUMO
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.
Assuntos
Cisteína , Células Estreladas do Fígado , Cirrose Hepática , Receptor EphB2 , Transdução de Sinais , Animais , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Cisteína/metabolismo , Transdução de Sinais/efeitos dos fármacos , Receptor EphB2/metabolismo , Receptor EphB2/genética , Humanos , Camundongos , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/patologia , Camundongos Endogâmicos C57BL , Masculino , Processamento de Proteína Pós-Traducional , Modelos Animais de DoençasRESUMO
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.
Assuntos
Células Estreladas do Fígado , Cirrose Hepática , Humanos , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Animais , Miofibroblastos/metabolismo , Miofibroblastos/patologiaRESUMO
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-ß1 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.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Semaforinas , Microambiente Tumoral , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/metabolismo , Humanos , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/metabolismo , Microambiente Tumoral/genética , Semaforinas/genética , Semaforinas/metabolismo , Integrina beta1/genética , Integrina beta1/metabolismo , Camundongos , Transdução de Sinais/genética , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Neuropilina-1/genética , Neuropilina-1/metabolismo , Linhagem Celular Tumoral , Células-Tronco Neoplásicas/patologia , Células-Tronco Neoplásicas/metabolismo , Animais , Regulação Neoplásica da Expressão Gênica/genética , Sorafenibe/farmacologia , Fibroblastos Associados a Câncer/metabolismo , Fibroblastos Associados a Câncer/patologia , Progressão da DoençaRESUMO
Metabolic dysfunction-associated steatohepatitis (MASH) is characterized by infiltration of monocyte-derived macrophages (MdMs) into the liver; however, the function of these macrophages is largely unknown. We previously demonstrated that a population of MdMs, referred to as hepatic lipid-associated macrophages (LAMs), assemble into aggregates termed hepatic crown-like structures in areas of liver fibrosis. Intriguingly, decreasing MdM recruitment resulted in increased liver fibrosis, suggesting that LAMs contribute to antifibrotic pathways in MASH. In this study, we determined that hepatic crown-like structures are characterized by intimate interactions between activated hepatic stellate cells (HSCs) and macrophages in a collagen matrix in a mouse model of MASH. MASH macrophages displayed collagen-degrading capacities, and HSCs derived from MASH livers promoted expression of LAM marker genes and acquisition of a collagen-degrading phenotype in naive macrophages. These data suggest that crosstalk between HSCs and macrophages may contribute to collagen degradation MASH.
Assuntos
Células Estreladas do Fígado , Cirrose Hepática , Macrófagos , Fenótipo , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/imunologia , Células Estreladas do Fígado/patologia , Animais , Camundongos , Macrófagos/imunologia , Macrófagos/metabolismo , Cirrose Hepática/patologia , Cirrose Hepática/metabolismo , Cirrose Hepática/imunologia , Camundongos Endogâmicos C57BL , Colágeno/metabolismo , Modelos Animais de Doenças , Humanos , Fígado/patologia , Fígado/metabolismo , Fígado/imunologia , Masculino , Fígado Gorduroso/patologia , Fígado Gorduroso/metabolismo , Fígado Gorduroso/imunologiaRESUMO
Excessive transforming growth factor ß1 (TGF-ß1) secreted by activated hepatic stellate cells (aHSCs) aggravates liver fibrosis via over-activation of TGF-ß1-mediated signaling pathways in a TGF-ß type I receptor (TßRI) dependent manner. TßRI with the C-terminal valine truncated (RIPΔ), as a novel TßRI-mimicking peptide, is an appealing anti-fibrotic candidate by competitive binding of TGF-ß1 to block TGF-ß1 signal transduction. Platelet-derived growth factor receptor ß (PDGFßR) is highly expressed on the surface of aHSCs in liver fibrosis. Herein, we designed a novel RIPΔ variant Z-RIPΔ (PDGFßR-specific affibody ZPDGFßR fused to the N-terminus of RIPΔ) for liver fibrosis therapy, and expect to improve the anti-liver fibrosis efficacy by specifically inhibiting the TGF-ß1 activity in aHSCs. Target peptide Z-RIPΔ was prepared in Escherichia coli by SUMO fusion system. Moreover, Z-RIPΔ specifically bound to TGF-ß1-activated aHSCs, inhibited cell proliferation and migration, and reduced the expression of fibrosis markers (α-SMA and FN) and TGF-ß1 pathway-related effectors (p-Smad2/3 and p-p38) in vitro. Furthermore, Z-RIPΔ specifically targeted the fibrotic liver, alleviated the liver histopathology, mitigated the fibrosis responses, and blocked TGF-ß1-mediated Smad and p38 MAPK cascades. More importantly, Z-RIPΔ exhibited a higher fibrotic liver-targeting capacity and stronger anti-fibrotic effects than its parent RIPΔ. Besides, Z-RIPΔ showed no obvious toxicity effects in treating both an in vitro cell model and an in vivo mouse model of liver fibrosis. In conclusion, Z-RIPΔ represents a promising targeted candidate for liver fibrosis therapy.
Assuntos
Células Estreladas do Fígado , Cirrose Hepática , Receptor do Fator de Crescimento Transformador beta Tipo I , Transdução de Sinais , Proteínas Smad , Fator de Crescimento Transformador beta1 , Proteínas Quinases p38 Ativadas por Mitógeno , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Animais , Fator de Crescimento Transformador beta1/metabolismo , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/patologia , Cirrose Hepática/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Camundongos , Proteínas Smad/metabolismo , Masculino , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Peptídeos/farmacologia , Peptídeos/química , Humanos , Camundongos Endogâmicos C57BLRESUMO
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.
Assuntos
Cirrose Hepática , Macrófagos , Sirtuína 3 , Superóxido Dismutase , Animais , Humanos , Masculino , Camundongos , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Fígado/patologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Cirrose Hepática/patologia , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/metabolismo , Macrófagos/metabolismo , Macrófagos/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Estresse Oxidativo/efeitos dos fármacos , Fenótipo , Células RAW 264.7 , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Sirtuína 3/metabolismo , Superóxido Dismutase/metabolismoRESUMO
Chronic liver diseases, primarily metabolic dysfunction-associated steatotic liver disease (MASLD), harmful use of alcohol, or viral hepatitis, may result in liver fibrosis, cirrhosis, and cancer. Hepatic fibrogenesis is a complex process with interactions between different resident and non-resident heterogeneous liver cell populations, ultimately leading to deposition of extracellular matrix and organ failure. Shifts in cell phenotypes and functions involve pronounced transcriptional and protein synthesis changes that require metabolic adaptations in cellular substrate metabolism, including glucose and lipid metabolism, resembling changes associated with the Warburg effect in cancer cells. Cell activation and metabolic changes are regulated by metabolic stress responses, including the unfolded protein response, endoplasmic reticulum stress, autophagy, ferroptosis, and nuclear receptor signaling. These metabolic adaptations are crucial for inflammatory and fibrogenic activation of macrophages, lymphoid cells, and hepatic stellate cells. Modulation of these pathways, therefore, offers opportunities for novel therapeutic approaches to halt or even reverse liver fibrosis progression.
Assuntos
Cirrose Hepática , Humanos , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Animais , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Transdução de Sinais , Reprogramação MetabólicaRESUMO
BACKGROUND: Hepatic stellate cells (HSCs) serve as the crucial accelerating factor in the progression of liver fibrosis (LF). In contrast to HSCs, adult-derived human liver stem/progenitor cells (ADHLSCs) exhibit greater potency in terms of differentiation and proliferation, rendering them highly applicable in LF treatment. The objective of this study is to identify new therapeutic targets for LF by comparing differentially expressed genes (DEGs) between ADHLSCs and HSCs. METHODS: We investigated DEGs between ADHLSCs and HSCs using the GSE49995 dataset obtained from the Gene Expression Omnibus (GEO) database, aiming to identify new therapeutic targets for LF. Subsequently, we activated HSCs to delve deeper into the mesenchyme homeobox 2 (MEOX2), PH domain Leucine-rich repeat protein phosphatase (PHLPP), and Phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling pathways in LF progression, employing platelet-derived growth factor (PDGF), and conducted infection with Overexpression (OE)-MEOX2 and shRNA-MEOX2 (sh-MEOX2) lentiviruses. Cell viability was assessed using the Cell Counting Kit-8 (CCK-8) assay, while cell proliferation was evaluated through 5-ethynyl-2'-deoxyuridine (EdU) staining and flow cytometry. Relative mRNA expression levels were determined via qPCR. Western blot analysis was performed to measure protein expression levels, and the regulatory role of MEOX2 was investigated using dual luciferase reporter assays. RESULTS: We identified 332 DEGs that were down-regulated and 201 DEGs that were up-regulated between ADHLSCs and HSCs. Notably, MEOX2 expression in ADHLSCs was significantly reduced. These DEGs primarily participated in the collagen-containing extracellular matrix and the PI3K/AKT signaling pathway. MEOX2 could inhibit cancer cell proliferation via the PI3K/AKT signaling pathway. Additionally, the JASRPAR2022 database predicted the target gene PHLPP of MEOX2. Our results indicated that OE-MEOX2 significantly inhibited HSCs' cell vitality and proliferation. Further analysis revealed that MEOX2 binds to PHLPP promoters, thereby up-regulating its transcription. This action led to the inhibition of p-AKT expression, consequently reducing HSC proliferation and slowing the progression of LF. CONCLUSIONS: MEOX2 up-regulates PHLPP expression and inhibits AKT phosphorylation, thereby reducing the cell activity and proliferation ability of HSCs and inhibiting the progression of LF.
Assuntos
Células Estreladas do Fígado , Proteínas de Homeodomínio , Cirrose Hepática , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Transdução de Sinais , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Humanos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/genética , Cirrose Hepática/patologia , Cirrose Hepática/genética , Cirrose Hepática/metabolismo , Proteínas de Homeodomínio/metabolismo , Proteínas de Homeodomínio/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proliferação de Células/genéticaRESUMO
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/CCl4 for 24 weeks. As a second model, we used hepatocyte-specific Nemo knockout (NemoΔhepa) mice and generated double knockout (NemoΔhepaTlr9-/-) 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-ß 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.
Assuntos
Carcinoma Hepatocelular , Modelos Animais de Doenças , Neoplasias Hepáticas , Camundongos Knockout , Receptor Toll-Like 9 , Animais , Receptor Toll-Like 9/metabolismo , Receptor Toll-Like 9/genética , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/genética , Camundongos , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/genética , Cirrose Hepática/patologia , Cirrose Hepática/metabolismo , Cirrose Hepática/genética , Apoptose , Camundongos Endogâmicos C57BL , Masculino , Células de Kupffer/metabolismo , Células de Kupffer/patologia , Doença Crônica , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Hepatócitos/metabolismo , Hepatócitos/patologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proliferação de Células , Hepatopatias/patologia , Hepatopatias/metabolismo , Hepatopatias/genética , Fígado/patologia , Fígado/metabolismoRESUMO
Liver fibrosis is a significant health concern globally due to its association with severe liver conditions like cirrhosis and liver cancer. Histone lactylation has been implicated in the progression of hepatic fibrosis, but its specific role in liver fibrosis, particularly regarding H3K18 lactylation, remained unclear. To investigate this, we established in vivo and in vitro models of liver fibrosis using carbon tetrachloride (CCl4) injection in rats and stimulation of hepatic stellate cells (HSCs) with TGF-ß1, respectively. We found that histone lactylation, particularly H3K18 lactylation, was upregulated in both CCl4-induced rats and TGF-ß1-activated HSCs, indicating its potential involvement in liver fibrosis. Further experiments revealed that lactate dehydrogenase A (LDHA) knockdown inhibited H3K18 lactylation and had a beneficial effect on liver fibrosis by suppressing HSC proliferation, migration, and extracellular matrix (ECM) deposition. This suggests that H3K18 lactylation promotes liver fibrosis progression. Chromatin immunoprecipitation (ChIP) and luciferase reporter assays demonstrated that H3K18 lactylation facilitated the transcription of SOX9, a transcription factor associated with fibrosis. Importantly, overexpression of SOX9 counteracted the effects of LDHA silencing on activated HSCs, indicating that SOX9 is downstream of H3K18 lactylation in promoting liver fibrosis. In summary, this study uncovers a novel mechanism by which H3K18 lactylation contributes to liver fibrosis by activating SOX9 transcription. This finding opens avenues for exploring new therapeutic strategies for hepatic fibrosis targeting histone lactylation pathways.
Assuntos
Progressão da Doença , Células Estreladas do Fígado , Histonas , Cirrose Hepática , Ratos Sprague-Dawley , Fatores de Transcrição SOX9 , Animais , Humanos , Masculino , Ratos , Tetracloreto de Carbono , Movimento Celular/genética , Proliferação de Células , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Histonas/metabolismo , Histonas/genética , L-Lactato Desidrogenase/metabolismo , L-Lactato Desidrogenase/genética , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Cirrose Hepática/genética , Cirrose Hepática/induzido quimicamente , Fatores de Transcrição SOX9/metabolismo , Fatores de Transcrição SOX9/genética , Transcrição Gênica , Fator de Crescimento Transformador beta1/metabolismoRESUMO
The prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD) is increasing rapidly due to the obesity epidemic. In the inflammatory stages of MASLD (MASH), activation of hepatic stellate cells (HSCs) leads to initiation and progression of liver fibrosis. Extracellular vesicles (EVs) are released from all cell types and play an important role in intercellular communication. However, the role of EVs released from hepatocytes in the context of MASLD is largely unknown. Therefore, the present study aimed to investigate the role of EVs derived from both normal and steatotic (free fatty acid-treated) hepatocytes on the phenotype of HSCs via the senescence pathway. Primary rat hepatocytes were treated with free fatty acids (FFAs: oleic acid and palmitic acid). EVs were collected by ultracentrifugation. EVs markers and HSCs activation and senescence markers were assessed by Western blot analysis, qPCR and cytochemistry. Reactive oxygen species (ROS) production was assessed by fluorescence assay. RNA profiles of EVs were evaluated by sequencing. We found that EVs from hepatocytes treated with FFAs (FFA-EVs) inhibit collagen type 1 and α-smooth muscle actin expression, increase the production of ROS and the expression of senescence markers (IL-6, IL-1ß, p21 and senescence-associated ß-galactosidase activity) in early activating HSCs via the AKT-mTOR pathway. Sequencing showed differentially enriched RNA species between the EVs groups. In conclusion, EVs from FFA-treated hepatocytes inhibit HSC activation by inducing senescence via the AKT-mTOR signaling pathway. Determining the components in EVs from steatotic hepatocytes that induce HSC senescence may lead to the identification of novel targets for intervention in the treatment of MASLD in the future.
Assuntos
Senescência Celular , Vesículas Extracelulares , Células Estreladas do Fígado , Hepatócitos , Proteínas Proto-Oncogênicas c-akt , Transdução de Sinais , Serina-Treonina Quinases TOR , Animais , Vesículas Extracelulares/metabolismo , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Células Estreladas do Fígado/efeitos dos fármacos , Ratos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Senescência Celular/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Hepatócitos/metabolismo , Hepatócitos/patologia , Hepatócitos/efeitos dos fármacos , Masculino , Fígado Gorduroso/metabolismo , Fígado Gorduroso/patologia , Espécies Reativas de Oxigênio/metabolismo , Células Cultivadas , Ratos Sprague-DawleyRESUMO
Hepatic stellate cells (HSCs) activation is a key event in the development of liver fibrosis, and blockage of the activation of HSCs has been shown to alleviate liver fibrosis. Sophoridine, a bioactive alkaloid found in many Chinese herbs, exhibits a broad spectrum of pharmacological effects, but its activities are not strong. In this study, a series of structurally modified derivatives of sophoridine were designed and synthesized. Among them, sophoridine α-aryl propionamide derivative ZM600 displayed a significant inhibitory effect on the activation of HSCs. The in vivo experiment demonstrated that ZM600 markedly ameliorated carbon tetrachloride (CCl4) and bile duct ligation (BDL)-induced liver fibrosis with a significant improvement of extracellular matrix deposition. Mechanism investigations revealed that ZM600 specifically inhibited the activation of NF-κB, PI-3K/AKT, and TGF-ß/Smads signaling pathways. These results suggest that ZM600 has a protective effect on liver fibrosis, which provides a new candidate for the treatment of liver fibrosis.
Assuntos
Alcaloides , Células Estreladas do Fígado , Cirrose Hepática , Matrinas , Quinolizinas , Animais , Quinolizinas/farmacologia , Quinolizinas/síntese química , Quinolizinas/química , Quinolizinas/uso terapêutico , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/patologia , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Alcaloides/farmacologia , Alcaloides/química , Alcaloides/síntese química , Alcaloides/uso terapêutico , Masculino , NF-kappa B/metabolismo , NF-kappa B/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Tetracloreto de Carbono , Camundongos , Relação Estrutura-Atividade , Ratos , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Descoberta de Drogas , Antifibróticos/farmacologia , Antifibróticos/uso terapêutico , Antifibróticos/química , Antifibróticos/síntese química , Ratos Sprague-DawleyRESUMO
Liver fibrosis, a progressive process of fibrous scarring, results from the accumulation of extracellular matrix proteins (ECM). If left untreated, it often progresses to diseases such as cirrhosis and hepatocellular carcinoma. Lycorine, a natural alkaloid derived from medicinal plants, has shown diverse bioactivities by targeting JAK2/STAT3 signaling, but its pharmacological effects and potential molecular mechanisms in liver fibrosis remains largely unexplored. The purpose of this study is to elucidate the pharmacological activity and molecular mechanism of lycorine in anti-hepatic fibrosis. Findings indicate that lycorine significantly inhibited hepatic stellate cells (HSCs) activation by reducing the expression of α-SMA and collagen-1. In vivo, lycorine treatment alleviated carbon tetrachloride (CCl4) -induced mice liver fibrosis, improving liver function, decreasing ECM deposition, and inhibiting fibrosis-related markers' expression. Mechanistically, it was found that lycorine exerts protective activity through the JAK2/STAT3 and PI3K/AKT signaling pathways, as evidenced by transcriptome sequencing technology and small molecule inhibitors. These results underscore lycorine's potential as a therapeutic drug for liver fibrosis.
Assuntos
Alcaloides de Amaryllidaceae , Tetracloreto de Carbono , Células Estreladas do Fígado , Janus Quinase 2 , Cirrose Hepática , Fenantridinas , Proteínas Proto-Oncogênicas c-akt , Fator de Transcrição STAT3 , Transdução de Sinais , Animais , Janus Quinase 2/metabolismo , Fator de Transcrição STAT3/metabolismo , Alcaloides de Amaryllidaceae/farmacologia , Tetracloreto de Carbono/toxicidade , Transdução de Sinais/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Camundongos , Masculino , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/patologia , Cirrose Hepática/metabolismo , Fenantridinas/farmacologia , Fenantridinas/uso terapêutico , Fosfatidilinositol 3-Quinases/metabolismo , Camundongos Endogâmicos C57BL , Linhagem CelularRESUMO
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.
Assuntos
Proliferação de Células , Células Estreladas do Fígado , Receptores de Inositol 1,4,5-Trifosfato , Cirrose Hepática , Animais , Humanos , Ratos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/antagonistas & inibidores , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/patologia , Cirrose Hepática/metabolismoRESUMO
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 Il1ß mRNA expression and the number of macrophages in the liver. On the other hand, transforming growth factor-ß1 (Tgfß1) 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-ß1, 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-ß1, which activated by macrophages via inflammatory cytokines such as TNFα and IL1ß, 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.
Assuntos
Dieta Hiperlipídica , Lipopolissacarídeos , Cirrose Hepática , Macrófagos , Hepatopatia Gordurosa não Alcoólica , Fator de Crescimento Transformador beta1 , Animais , Fator de Crescimento Transformador beta1/metabolismo , Camundongos , Macrófagos/metabolismo , Macrófagos/efeitos dos fármacos , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Cirrose Hepática/patologia , Cirrose Hepática/metabolismo , Dieta Hiperlipídica/efeitos adversos , Masculino , Fígado/metabolismo , Fígado/patologia , Camundongos Endogâmicos C57BL , Ativação de Macrófagos , Imageamento Tridimensional/métodos , Modelos Animais de Doenças , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Interleucina-1beta/metabolismoRESUMO
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.
Assuntos
Apigenina , Subunidade alfa do Fator 1 Induzível por Hipóxia , Cirrose Hepática , Estresse Oxidativo , Animais , Estresse Oxidativo/efeitos dos fármacos , Apigenina/farmacologia , Apigenina/uso terapêutico , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Cirrose Hepática/metabolismo , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/patologia , Camundongos , Masculino , Piruvato Quinase/metabolismo , Camundongos Endogâmicos C57BL , Tetracloreto de Carbono/toxicidade , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/patologia , Proteínas de Ligação a Hormônio da Tireoide , Fígado/metabolismo , Fígado/efeitos dos fármacos , Fígado/patologia , Hormônios Tireóideos/metabolismo , Antioxidantes/farmacologia , Antioxidantes/metabolismo , Receptores ErbBRESUMO
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.
Assuntos
Proliferação de Células , Exossomos , Células Estreladas do Fígado , Cirrose Hepática , MicroRNAs , Humanos , Exossomos/metabolismo , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Células Hep G2 , Cirrose Hepática/patologia , Cirrose Hepática/metabolismo , Cirrose Hepática/genética , MicroRNAs/metabolismo , MicroRNAs/genética , Vírus da Hepatite B/genética , Transdução de Sinais , Fígado/patologia , Fígado/metabolismoRESUMO
Drug-induced liver injury (DILI) is one of the major concerns during drug development. Wide acceptance of the 3â¯R principles and the innovation of in-vitro techniques have introduced various novel model options, among which the three-dimensional (3D) cell spheroid cultures have shown a promising prospect in DILI prediction. The present study developed a 3D quadruple cell co-culture liver spheroid model for DILI prediction via self-assembly. Induction by phorbol 12-myristate 13-acetate at the concentration of 15.42â¯ng/mL for 48â¯hours with a following 24-hour rest period was used for THP-1 cell differentiation, resulting in credible macrophagic phenotypes. HepG2 cells, PUMC-HUVEC-T1 cells, THP-1-originated macrophages, and human hepatic stellate cells were selected as the components, which exhibited adaptability in the designated spheroid culture conditions. Following establishment, the characterization demonstrated the competence of the model in long-term stability reflected by the maintenance of morphology, viability, cellular integration, and cell-cell junctions for at least six days, as well as the reliable liver-specific functions including superior albumin and urea secretion, improved drug metabolic enzyme expression and CYP3A4 activity, and the expression of MRP2, BSEP, and P-GP accompanied by the bile acid efflux transport function. In the comparative testing using 22 DILI-positive and 5 DILI-negative compounds among the novel 3D co-culture model, 3D HepG2 spheroids, and 2D HepG2 monolayers, the 3D culture method significantly enhanced the model sensitivity to compound cytotoxicity compared to the 2D form. The novel co-culture liver spheroid model exhibited higher overall predictive power with margin of safety as the classifying tool. In addition, the non-parenchymal cell components could amplify the toxicity of isoniazid in the 3D model, suggesting their potential mediating role in immune-mediated toxicity. The proof-of-concept experiments demonstrated the capability of the model in replicating drug-induced lipid dysregulation, bile acid efflux inhibition, and α-SMA upregulation, which are the key features of liver steatosis and phospholipidosis, cholestasis, and fibrosis, respectively. Overall, the novel 3D quadruple cell co-culture spheroid model is a reliable and readily available option for DILI prediction.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas , Técnicas de Cocultura , Esferoides Celulares , Humanos , Esferoides Celulares/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas/patologia , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Células Hep G2 , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Células THP-1 , Fígado/efeitos dos fármacos , Fígado/patologia , Fígado/metabolismo , Sobrevivência Celular/efeitos dos fármacosRESUMO
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 CCl4- 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 CCl4-induced liver fibrosis in mice. In contrast, inhibiting Fendrr blockaded the activation of HSC and ameliorated CCl4-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 CCl4-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.
Assuntos
Hepatócitos , Interleucina-6 , Cirrose Hepática , RNA Longo não Codificante , Animais , Humanos , Masculino , Camundongos , Tetracloreto de Carbono/toxicidade , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Hepatócitos/metabolismo , Hepatócitos/patologia , Interleucina-6/metabolismo , Interleucina-6/genética , Cirrose Hepática/metabolismo , Cirrose Hepática/genética , Cirrose Hepática/patologia , Camundongos Endogâmicos C57BL , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Fator de Transcrição STAT2/metabolismo , Fator de Transcrição STAT2/genéticaRESUMO
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.