RESUMO
Background: Copper metabolism dysfunction has been found to be associated with the progression of various malignant tumors. The aim of this study is to explore the prognostic value of copper metabolism-related genes (CMRGs) in hepatocellular carcinoma (HCC) and their impact on the immune microenvironment. Methods: We identified differentially expressed CMRGs in cancer and adjacent samples of HCC from The Cancer Genome Atlas (TCGA). Consensus clustering was performed to distinguish subgroups, and TIMER and CIBERSORT were applied to analyze the tumor immune microenvironment (TIME). We used the least absolute shrinkage and selection operator (LASSO) and multivariate Cox regression analysis to establish a prognostic risk model for CMRGs. Gene set enrichment analysis (GSEA) was performed to elucidate potential signaling mechanisms associated with the risk group, as well as to determine and compare the tumor mutation burden (TMB), immune cell infiltration levels, and immune checkpoint of the identified risk groups. Results: Two subgroups with significantly different survival rates were identified, with a better prognosis associated with high immune scores, high abundance of immune-infiltrating cells, and a relatively higher immune status. A prognostic risk model based on five CMRGs was constructed, which showed significant prognostic value. When combined with clinical feature column charts, this model can predict the prognosis of patients with HCC. Functional enrichment analysis showed that the low-risk group was enriched in a large number of metabolic pathways, while the high and low-risk groups exhibited different TMB and differential expression of immune checkpoint genes. The established model was validated in an independent International Cancer Genome Consortium (ICGC) dataset. Conclusions: The results indicate that the expression of CMRGs is associated with the prognosis of HCC and the tumor microenvironment, and can serve as a predictive indicator for evaluating the prognosis of HCC.
RESUMO
The ENCODE Consortium's efforts to annotate noncoding cis-regulatory elements (CREs) have advanced our understanding of gene regulatory landscapes. Pooled, noncoding CRISPR screens offer a systematic approach to investigate cis-regulatory mechanisms. The ENCODE4 Functional Characterization Centers conducted 108 screens in human cell lines, comprising >540,000 perturbations across 24.85 megabases of the genome. Using 332 functionally confirmed CRE-gene links in K562 cells, we established guidelines for screening endogenous noncoding elements with CRISPR interference (CRISPRi), including accurate detection of CREs that exhibit variable, often low, transcriptional effects. Benchmarking five screen analysis tools, we find that CASA produces the most conservative CRE calls and is robust to artifacts of low-specificity single guide RNAs. We uncover a subtle DNA strand bias for CRISPRi in transcribed regions with implications for screen design and analysis. Together, we provide an accessible data resource, predesigned single guide RNAs for targeting 3,275,697 ENCODE SCREEN candidate CREs with CRISPRi and screening guidelines to accelerate functional characterization of the noncoding genome.
Assuntos
Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Humanos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Sistemas CRISPR-Cas/genética , Genoma , Células K562 , RNA Guia de Sistemas CRISPR-CasRESUMO
BACKGROUND: Ischemia/reperfusion injury (IRI) is generally unavoidable following liver transplantation. Here, we investigated the role of protein phosphatase, Mg2+ /Mn2+ dependent 1G (PPM1G) in hepatic IRI. METHODS: Hepatic IRI was mimicked by employing a hypoxia/reperfusion (H/R) model in RAW 264.7 cells and a 70% warm ischemia model in C57BL/6 mice, respectively. In vitro, expression changes of tumor necrosis factor-α and interleukin were detected by quantitative real-time polymerase chain reaction (qRT-PCR), western blot analysis, and enzyme-linked immunosorbent assay. The protein expressions of PPM1G and the stimulator of interferon genes (STING) pathway components were analyzed by western blot. Interaction between PPM1G and STING was verified by coimmunoprecipitation (CO-IP). Immunofluorescence was applied for detection of p-IRF3. Flow cytometry, qRT-PCR and western blot were utilized to analyze markers of macrophage polarization. In vivo, histological analyses of mice liver were carried out by TUNEL and H&E staining. Changes in serum aminotransferases were also detected. RESULTS: Following H/R intervention, a steady decline in PPM1G along with an increase in inflammatory cytokines in vitro was observed. Addition of plasmid with PPM1G sequence limited the release of inflammatory cytokines and downregulated phosphorylation of STING. CO-IP validated the interaction between PPM1G and STING. Furthermore, inhibition of PPM1G with lentivirus enhanced phosphorylation of STING and its downstream components; meanwhile, p65, p38, and Jnk were also surged to phosphorylation. Expression of INOS and CD86 was surged, while CD206, Arg-1, and IL-10 were inhibited. In vivo, PPM1G inhibition further promoted liver damage, hepatocyte apoptosis, and transaminases release. Selective inhibition of STING with C-176 partially reversed the activation of STING pathway and inflammatory cytokines in vitro. M1 markers were also suppressed by C-176. In vivo, C-176 rescued liver damage and transaminase release caused by PPM1G inhibition. CONCLUSION: PPM1G suppresses hepatic IRI and macrophage M1 phenotype by repressing STING-mediated inflammatory pathways.
Assuntos
Hepatopatias , Proteína Fosfatase 2C , Traumatismo por Reperfusão , Animais , Camundongos , Citocinas/metabolismo , Isquemia/metabolismo , Hepatopatias/etiologia , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Proteína Fosfatase 2C/metabolismoRESUMO
BACKGROUND: Hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC) is one of the most common and deadly cancer and often accompanied by varying degrees of liver damage, leading to the dysfunction of fatty acid metabolism (FAM). This study aimed to investigate the relationship between FAM and HBV-associated HCC and identify FAM biomarkers for predicting the prognosis of HBV-associated HCC. METHODS: Gene Set Enrichment Analysis (GSEA) was used to analyze the difference of FAM pathway between paired tumor and adjacent normal tissue samples in 58 HBV-associated HCC patients from the Gene Expression Omnibus (GEO) database. Next, 117 HBV-associated HCC patients from The Cancer Genome Atlas (TCGA) database were analyzed to establish a prognostic signature based on 42 FAM genes. Then, the prognostic signature was validated in an external cohort consisting of 30 HBV-associated HCC patients. Finally, immune infiltration analysis was performed to evaluate the FAM-related immune cells in HBV-associated HCC. RESULTS: As a result, FAM pathway was clearly downregulated in tumor tissue of HBV-associated HCC, and survival analysis demonstrated that 12 FAM genes were associated with the prognosis of HBV-associated HCC. Lasso-penalized Cox regression analysis identified and established a five-gene signature (ACADVL, ACAT1, ACSL3, ADH4 and ECI1), which showed effective discrimination and prediction for the prognosis of HBV-associated HCC both in the TCGA cohort and the validation cohort. Immune infiltration analysis showed that the high-risk group, identified by FAM signature, of HBV-associated HCC had a higher ratio of Tregs, which was associated with the prognosis. CONCLUSIONS: Collectively, these findings suggest that there is a strong connection between FAM and HBV-associated HCC, indicating a potential therapeutic strategy targeting FAM to block the accumulation of Tregs into the tumor microenvironment of HBV-associated HCC.
Assuntos
Carcinoma Hepatocelular , Hepatite B , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/genética , Prognóstico , Neoplasias Hepáticas/genética , Hepatite B/complicações , Hepatite B/genética , Vírus da Hepatite B/genética , Ácidos Graxos , Microambiente TumoralRESUMO
The Encyclopedia of DNA elements (ENCODE) project is a collaborative effort to create a comprehensive catalog of functional elements in the human genome. The current database comprises more than 19000 functional genomics experiments across more than 1000 cell lines and tissues using a wide array of experimental techniques to study the chromatin structure, regulatory and transcriptional landscape of the Homo sapiens and Mus musculus genomes. All experimental data, metadata, and associated computational analyses created by the ENCODE consortium are submitted to the Data Coordination Center (DCC) for validation, tracking, storage, and distribution to community resources and the scientific community. The ENCODE project has engineered and distributed uniform processing pipelines in order to promote data provenance and reproducibility as well as allow interoperability between genomic resources and other consortia. All data files, reference genome versions, software versions, and parameters used by the pipelines are captured and available via the ENCODE Portal. The pipeline code, developed using Docker and Workflow Description Language (WDL; https://openwdl.org/) is publicly available in GitHub, with images available on Dockerhub (https://hub.docker.com), enabling access to a diverse range of biomedical researchers. ENCODE pipelines maintained and used by the DCC can be installed to run on personal computers, local HPC clusters, or in cloud computing environments via Cromwell. Access to the pipelines and data via the cloud allows small labs the ability to use the data or software without access to institutional compute clusters. Standardization of the computational methodologies for analysis and quality control leads to comparable results from different ENCODE collections - a prerequisite for successful integrative analyses.
RESUMO
BACKGROUND: Hepatic ischemia/reperfusion (I/R) injury is one of the major pathological processes associated with various liver surgeries. However, there is still a lack of strategies to protect against hepatic I/R injury because of the unknown underlying mechanism. The present study aimed to identify a potential strategy and provide a fundamental experimental basis for treating hepatic I/R injury. METHOD: A classic 70% ischemia/reperfusion injury was established. Immunoprecipitation was used to identify direct interactions between proteins. The expression of proteins from different subcellular localizations was detected by Western blotting. Cell translocation was directly observed by immunofluorescence. HE, TUNEL and ELISA were performed for function tests. RESULT: We report that tripartite motif containing 37 (TRIM37) aggravates hepatic I/R injury through the reinforcement of IKK-induced inflammation following dual patterns. Mechanistically, TRIM37 directly interacts with tumor necrosis factor receptor-associated factor 6 (TRAF6), inducing K63 ubiquitination and eventually leading to the phosphorylation of IKKß. TRIM37 enhances the translocation of IKKγ, a regulatory subunit of the IKK complex, from the nucleus to the cytoplasm, thereby stabilizing the cytoplasmic IKK complex and prolonging the duration of inflammation. Inhibition of IKK rescued the function of TRIM37 in vivo and in vitro. CONCLUSION: Collectively, the present study discloses some potential function of TRIM37 in hepatic I/R injury. Targeting TRIM37 might be potential for treatment against hepatic I/R injury.Targeting TRIM37 might be a potential treatment strategy against hepatic I/R injury.
Assuntos
Quinase I-kappa B , Proteínas Serina-Treonina Quinases , Humanos , Inflamação , Fígado , Isquemia , Proteínas com Motivo Tripartido , Ubiquitina-Proteína Ligases/genéticaRESUMO
The Encyclopedia of DNA elements (ENCODE) project is a collaborative effort to create a comprehensive catalog of functional elements in the human genome. The current database comprises more than 19000 functional genomics experiments across more than 1000 cell lines and tissues using a wide array of experimental techniques to study the chromatin structure, regulatory and transcriptional landscape of the Homo sapiens and Mus musculus genomes. All experimental data, metadata, and associated computational analyses created by the ENCODE consortium are submitted to the Data Coordination Center (DCC) for validation, tracking, storage, and distribution to community resources and the scientific community. The ENCODE project has engineered and distributed uniform processing pipelines in order to promote data provenance and reproducibility as well as allow interoperability between genomic resources and other consortia. All data files, reference genome versions, software versions, and parameters used by the pipelines are captured and available via the ENCODE Portal. The pipeline code, developed using Docker and Workflow Description Language (WDL; https://openwdl.org/) is publicly available in GitHub, with images available on Dockerhub (https://hub.docker.com), enabling access to a diverse range of biomedical researchers. ENCODE pipelines maintained and used by the DCC can be installed to run on personal computers, local HPC clusters, or in cloud computing environments via Cromwell. Access to the pipelines and data via the cloud allows small labs the ability to use the data or software without access to institutional compute clusters. Standardization of the computational methodologies for analysis and quality control leads to comparable results from different ENCODE collections - a prerequisite for successful integrative analyses.
RESUMO
Ischemia-reperfusion injury (IRI) is one of the most common complications in liver transplantation. METTL3 regulates inflammation and cellular stress response by modulating RNA m6A modification level. Here, the study aimed to investigate the role and mechanism of METTL3 in IRI after rat orthotopic liver transplantation. The total RNA m6A modification and METTL3 expression level was consistently down-regulated after 6â¯h or 24â¯h reperfusion in OLT, which is negatively associated with the hepatic cell apoptosis. Functionally, METTL3 pretreatment in donor significantly inhibited liver grafts apoptosis, improved liver function and depressed the proinflammatory cytokine/chemokine expression. Mechanistically, METTL3 inhibited apoptosis of grafts via upregulating HO-1. Moreover, m6A dot blot and MeRIP-qPCR assay revealed that METTL3 promoted HO-1 expression in an m6A-dependent manner. In vitro, METTL3 alleviated hepatocytes apoptosis by upregulating HO-1 under hypoxia/reoxygenation condition. Taken together, these findings demonstrate that METTL3 ameliorates rat OLT-stressed IRI by inducing HO-1 in an m6A-dependent manner, highlighting a potential target for IRI in liver transplantation.
Assuntos
Transplante de Fígado , Traumatismo por Reperfusão , Ratos , Animais , Transplante de Fígado/efeitos adversos , Fígado/metabolismo , Inflamação/metabolismo , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/metabolismo , RNA/metabolismoRESUMO
Objective: This is the first systematic review and meta-analysis to determine the factors that contribute to poor antibody response in organ transplant recipients after receiving the 2-dose severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine. Method: Data was obtained from Embase, PubMed, Web of Science, Cochrane Library, China National Knowledge Infrastructure (CNKI), and Chinese Biomedical Literature Database (CBM). Studies reporting factors associated with antibody responses to the 2-dose SARS-CoV-2 vaccine in solid organ transplant recipients were included in our study based on the inclusion and exclusion criteria. Two researchers completed the literature search, screening, and data extraction. Randomized models were used to obtain results. Egger's test was performed to determine publication bias. Sensitivity analysis was performed to determine the stability of the result. The heterogeneity was determined using the Galbraith plot and subgroup analysis. Results: A total of 29 studies were included in the present study. The factors included living donor, BNT162b2, tacrolimus, cyclosporine, antimetabolite, mycophenolic acid (MPA) or mycophenolate mofetil (MMF), azathioprine, corticosteroids, high-dose corticosteroids, belatacept, mammalian target of rapamycin (mTOR) inhibitor, tritherapy, age, estimated glomerular filtration rate (eGFR), hemoglobin, and tacrolimus level were significantly different. Multivariate analysis showed significant differences in age, diabetes mellitus, MPA or MMF, high-dose corticosteroids, tritherapy, and eGFR. Conclusion: The possible independent risk factors for negative antibody response in patients with organ transplants who received the 2-dose SARS-CoV-2 vaccine include age, diabetes mellitus, low eGFR, MPA or MMF, high-dose corticosteroids, and triple immunosuppression therapy. mTOR inhibitor can be a protective factor against weak antibody response. Systematic Review Registration: PROSPERO, identifier CRD42021257965.
Assuntos
COVID-19 , Diabetes Mellitus , Transplante de Rim , Adulto , Formação de Anticorpos , Vacina BNT162 , COVID-19/prevenção & controle , Vacinas contra COVID-19 , Diabetes Mellitus/tratamento farmacológico , Rejeição de Enxerto/prevenção & controle , Humanos , Transplante de Rim/métodos , Ácido Micofenólico , Fatores de Risco , SARS-CoV-2 , Serina-Treonina Quinases TOR , TacrolimoRESUMO
BACKGROUND: Hepatic ischemia reperfusion injury (IRI) is a major factor affecting the prognosis of liver transplantation through a series of severe cell death and inflammatory responses. However, the potential role of miR-141-3p in hepatic IRI is currently unknown. METHODS: We collected the serum of liver transplantation patients to study the relationship between miR-141-3p and liver injury. A mouse hepatic IRI model was established to measure hepatic dysfunction and cell apoptosis. MiR-141-3p mimic and inhibitor were transfected into hepatocytes to explore the characteristics of hypoxia/reoxygenation (H/R), a classical hepatic IRI in vitro model. RESULTS: We found that miR-141-3p levels were negatively correlated with alanine aminotransferase (ALT)/aspartate aminotransferase (AST) in liver transplantation patients. The results demonstrated that miR-141-3p was decreased in mouse liver tissue after hepatic IRI in mice and in hepatocytes after H/R. Overexpression of miR-141-3p directly decreased Kelch-like ECH-associated protein 1 (Keap1) levels and attenuated cell apoptosis in vivo and in vitro, while inhibition of miR-141-3p facilitated apoptosis. Further experiments revealed that overexpression of miR-141-3p also attenuated oxidative stress-induced damage in hepatocytes under H/R conditions. CONCLUSIONS: Our results indicate that miR-141-3p plays a major role in hepatic IRI through the Keap1 signaling pathway, and the present study suggests that miR-141-3p might have a protective effect on hepatic IRI to some extent.
Assuntos
Hepatopatias , MicroRNAs , Traumatismo por Reperfusão , Animais , Apoptose/genética , Modelos Animais de Doenças , Hipóxia/metabolismo , Isquemia , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Fígado/metabolismo , Hepatopatias/genética , Hepatopatias/metabolismo , Camundongos , MicroRNAs/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/metabolismoRESUMO
Neutrophil extracellular traps (NETs) play important roles in hepatic ischemic reperfusion injury (IRI) and acute rejection (AR)-induced immune responses to inflammation. After liver transplantation, HMGB1, an inflammatory mediator, contributes to the development of AR. Even though studies have found that HMGB1 can promote NET formation, the correlation between NETs and HMGB1 in the development of AR following liver transplantation has not been elucidated. In this study, levels of serum NETs were significantly elevated in patients after liver transplantation. Moreover, we found that circulating levels of NETs were negatively correlated with liver function. In addition, liver transplantation and elevated extracellular HMGB1 promoted NET formation. The HMGB1/TLR-4/MAPK signaling pathway, which is initiated by HMGB1, participates in NET processes. Moreover, in the liver, Kupffer cells were found to be the main cells secreting HMGB1. NETs induced Kupffer cell M1 polarization and decreased the intracellular translocation of HMGB1 by inhibiting DNase-1. Additionally, co-treatment with TAK-242 (a TLR-4 inhibitor) and rapamycin more effectively alleviated the damaging effects of AR following liver transplantation than either drug alone.
Assuntos
Armadilhas Extracelulares , Proteína HMGB1 , Transplante de Fígado , Armadilhas Extracelulares/metabolismo , Rejeição de Enxerto , Proteína HMGB1/metabolismo , Humanos , Células de Kupffer/metabolismo , Fígado/metabolismo , Neutrófilos , Receptor 4 Toll-Like/metabolismoRESUMO
Hepatic ischemia/reperfusion (I/R) injury plays a pivotal pathogenic role in trauma, hepatectomy, and liver transplantation. However, the whole mechanism remains undescribed. The objective of this study is to investigate the internal mechanism by which microRNA-22 (miR-22) targets family with sequence similarity 49 member B (FAM49B), thus aggravating hepatic I/R injury. Here, we found that miR-22 was upregulated while FAM49B was reduced in hepatic I/R injury. Inhibition of miR-22 in vitro was able to intensify expression of FAM49B, thus reducing phosphorylation of inhibitors of nuclear factor kappa-B kinase (IKK) and downstream pro-inflammatory proteins. A dual luciferase reporter assay indicated that miR-22 directly targeted FAM49B. Remission of hepatic pathologic alterations, apoptosis, and release of cytokines derived from constraints of miR-22 were abolished in vivo by repressing FAM49B. Further interference of Ras-related C3 botulinum toxin substrate 1 (Rac1) reversed the function of FAM49B inhibition, thus achieving anti-inflammatory consequences.
Assuntos
Quinase I-kappa B , Peptídeos e Proteínas de Sinalização Intracelular , Fígado , MicroRNAs , Traumatismo por Reperfusão , Fator 6 Associado a Receptor de TNF , Proteínas rac1 de Ligação ao GTP , Animais , Masculino , Camundongos , Regulação da Expressão Gênica , Quinase I-kappa B/antagonistas & inibidores , Quinase I-kappa B/metabolismo , Inflamação/genética , Inflamação/patologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fígado/irrigação sanguínea , Fígado/metabolismo , Fígado/patologia , Camundongos Endogâmicos C57BL , MicroRNAs/genética , MicroRNAs/metabolismo , Pirazóis/farmacologia , Proteínas rac1 de Ligação ao GTP/antagonistas & inibidores , Proteínas rac1 de Ligação ao GTP/metabolismo , Células RAW 264.7 , Traumatismo por Reperfusão/genética , Transdução de Sinais , Fator 6 Associado a Receptor de TNF/metabolismoRESUMO
Hepatic ischemia/reperfusion (I/R) injury occurs frequently in various liver operations and diseases, but its effective treatment remains inadequate because the key switch that leads to hepatic explosive inflammation has not been well disclosed. Dual specificity phosphatase 9 (DUSP9) is widely involved in the innate immune response of solid organs and is sometimes regulated by ubiquitin. In the present study, we find that DUSP9 is reduced in mouse hepatic I/R injury. DUSP9 enrichment attenuates hepatic inflammation both in vivo and in vitro as revealed by western blot analysis and qRT-PCR. In contrast, DUSP9 depletion leads to more severe I/R injury. Mechanistically, DUSP9 inhibits the phosphorylation of apoptosis signal-regulating kinase 1 (ASK1) by directly binding to ASK1, thereby decreasing tumor necrosis factor receptor-associated factor 6 (TRAF6), K63 ubiquitin and the phosphorylation of p38/JNK1 instead of ERK1. The present study documents a novel role of DUSP9 in hepatic I/R injury and implies the potential of targeting the DUSP9/ASK1 axis towards mitogen-activated protein kinase and TRAF6/inhibitor of κB kinase pathways.
Assuntos
Proteínas Quinases Ativadas por Mitógeno , Traumatismo por Reperfusão , Camundongos , Animais , Proteínas Quinases Ativadas por Mitógeno/metabolismo , MAP Quinase Quinase Quinase 5/genética , MAP Quinase Quinase Quinase 5/metabolismo , Fator 6 Associado a Receptor de TNF/metabolismo , Fígado/metabolismo , Inflamação , Ubiquitinas/metabolismo , Isquemia , Apoptose/fisiologia , Fosfatases de Especificidade Dupla/genética , Fosfatases de Especificidade Dupla/metabolismoRESUMO
Cytidine monophosphate kinase 2 (CMPK2) is a mitochondrial nucleotide monophosphate kinase which is important for the substrates of mitochondrial DNA synthesis and has been reported to participate in macrophage activation and the inflammatory response. The purpose of the present research was to determine the potential role of CMPK2 in hepatic ischemia/reperfusion (I/R) injury and to elucidate the underlying molecular mechanisms. The present study investigated the role of CMPK2 in regulating the NLRP3 pathway and liver dysfunction induced by hepatic I/R both in vivo and in vitro. It was revealed that hypoxia/reoxygenation (H/R) treatment enhanced the mRNA expression levels of CMPK2, NLRP3, IL-18, IL-1ß and TNF-α in RAW 264.7 cells. The protein expression levels of IL-18, IL-1ß and cleaved-caspase-1 were decreased following CMPK2 knockdown. Furthermore, the inhibition of AIM2 downregulated the expression level of IL-1ß, IL-18 and cleaved-caspase-1 in the CMPK2 knockdown group followed by H/R treatment, while the inhibition of NLRP3 did not. CMPK2 deficiency also decreased alanine aminotransferase and aspartate aminotransferase expression in mice serum, as well as the pathological changes in the liver. Similarly, the release of IL-18 and IL-1ß in mouse serum was also restrained with the decline of CMPK2. In conclusion, the results of the present study demonstrate that CMPK2 is indispensable for NLRP3 inflammasome activation, making CMPK2 an effective target to relieve the liver from I/R injury. In addition, the function of CMPK2 is closely associated with NLRP3 inflammasome activation, instead of AIM2.
RESUMO
Liver ischemia/reperfusion injury (IRI) is an inevitable pathological process exacerbating the occurrence of rejection in liver transplantation. At present, there is still a lack of sufficient cognition for the mechanism as well as effective clinical strategies. F-box/WD repeat-containing protein 5 (FBXW5), a key modulator of stress signalling, was recently reported to participate in hepatic immunity. However, the role of FBXW5 in liver IRI is still unclear. In the present study, we found expression of FBXW5 was increased in liver IRI both in vivo and in vitro. Inhibition of FBXW5 significantly alleviated both mitogen-activated protein kinase (MAPK) and inhibitor of nuclear factor kappa-B kinase (IKK) pathways, thus resulting in cytokine release, hepatic pathological injury and apoptosis. Over-expression of FBXW5 achieved an opposite effect. Investigations on the mechanism showed that FBXW5 intensified hepatic inflammation by promoting phosphorylation of ASK1, while blockade of TRAF6 could abolish this process. Moreover, reinforce of mTOR amplified the anti-inflammatory efficacy derived from inhibition of FBXW5, indicating the function of FBXW5/ASK1/TRAF6 axis in hepatic IRI might be relatively independent of mTOR-guided M2 polarization of Kupffer cell. Taken together, FBXW5 could be a key accelerator in liver IRI by enhancing activation of ASK1 in a TRAF6-dependent manner. The joint intervention towards both FBXW5 and mTOR might be a promising strategy to protect liver from IRI.
Assuntos
Proteínas F-Box/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , MAP Quinase Quinase Quinase 5/metabolismo , Traumatismo por Reperfusão/metabolismo , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Apoptose , Citocinas/metabolismo , Inibidores Enzimáticos/metabolismo , Proteínas F-Box/genética , Regulação da Expressão Gênica , Humanos , Células de Kupffer , Fígado , Sistema de Sinalização das MAP Quinases , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Modelos Animais , Fosforilação , Fosfotransferases/antagonistas & inibidoresRESUMO
BACKGROUND: Hepatic ischemia-reperfusion (I/R) injury (IRI) represents a crucial challenge in liver transplantation. Fisetin has anti-inflammatory, anti-aging and anti-oxidative properties. This study aimed to examine whether fisetin mitigates hepatic IRI and examine its underlying mechanisms. METHODS: Sham or warm hepatic I/R operated mice were pretreated with fisetin (5, 10 or 20 mg/kg). Hepatic histological assessments, TUNEL assays and serum aminotransferase measurements were performed. An in vitro hypoxia/reoxygenation (H/R) model using RAW264.7 macrophages pretreated with fisetin (2.5, 5 or 10 µmol/L) was also used. Serum and cell supernatant concentrations of interleukin-1ß (IL-1ß), IL-18 and tumor necrosis factor-α (TNF-α) were determined by enzyme-linked immunosorbent assay (ELISA). Protein levels of p-GSK3ß, p-AMPK and NLR family pyrin domain-containing 3 (NLRP3)-associated proteins were detected by Western blotting. RESULTS: Compared with the I/R group, fisetin pretreatment reduced pathological liver damage, serum aminotransferase levels, serum concentrations of IL-1ß, IL-18 and TNF-α in the murine IRI model. Fisetin also reduced the expression of NLRP3 inflammasome-associated proteins (NLRP3, cleaved caspase-1, IL-1ß and IL-18) in I/R-operated liver. The experiments in vitro showed that fisetin decreased the release of IL-1ß, IL-18 and TNF-α, and reduced the expression of NLRP3 inflammasome-associated proteins in H/R-treated RAW264.7 cells. Moreover, fisetin increased the expressions of p-GSK3ß and p-AMPK in both models, indicating that its anti-inflammatory effects were dependent on GSK3ß/AMPK signaling. The anti-inflammatory effects of fisetin were partially inhibited by the AMPK specific inhibitor compound C. CONCLUSIONS: Fisetin showed protective effects against hepatic IRI, countering inflammatory responses through mediating the GSK3ß/AMPK/NLRP3 inflammasome pathway.
Assuntos
Inflamassomos , Traumatismo por Reperfusão , Proteínas Quinases Ativadas por AMP , Animais , Anti-Inflamatórios , Flavonóis , Glicogênio Sintase Quinase 3 beta , Interleucina-18 , Interleucina-1beta , Fígado , Camundongos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Traumatismo por Reperfusão/prevenção & controle , Transaminases , Fator de Necrose Tumoral alfaRESUMO
Hepatic ischemia/reperfusion injury (IRI) is an inevitable pathological process in liver resection, shock and transplantation. However, the internal mechanism of hepatic IRI, including inflammatory transduction of multiple signaling pathways, is not fully understood. In the present study, we identified pleckstrin homology-like domain family member 1 (PHLDA1), suppressed by microRNA (miR)-194, as a critical intersection of dual inflammatory signals in hepatic IRI. PHLDA1 was upregulated in hepatic IRI with a concomitant downregulation of miR-194. Overexpression of miR-194 diminished PHLDA1 and inhibitors of the nuclear factor kappa-B kinase (IKK) pathway, thus leading to remission of hepatic pathological injury, apoptosis and release of cytokines. Further enrichment of PHLDA1 reversed the function of miR-194 both in vivo and in vitro. For an in-depth query, we verified PHLDA1 as a direct target of miR-194. Notably, inflammatory signal transduction of PHLDA1 was induced by activating TNF receptor-associated factor 6 (TRAF6), sequentially initiating IKK and mitogen-activated protein kinase (MAPK), both of which aggravate stress and inflammation in hepatic IRI. In conclusion, the miR-194/PHLDA1 axis was a key upstream regulator of IKK and MAPK in hepatic IRI. Targeting PHLDA1 might be a potential strategy for hepatic IRI therapy.