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The stimulator of interferon genes (STING), an integral adaptor protein in the DNA-sensing pathway, plays a pivotal role in the innate immune response against infections. Additionally, it presents a valuable therapeutic target for infectious diseases and cancer. We observed that fangchinoline (Fan), a bis-benzylisoquinoline alkaloid (BBA), effectively impedes the replication of vesicular stomatitis virus (VSV), encephalomyocarditis virus (EMCV), influenza A virus (H1N1), and herpes simplex virus-1 (HSV-1) in vitro. Fan treatment significantly reduced the viral load, attenuated tissue inflammation, and improved survival in a viral sepsis mouse model. Mechanistically, Fan activates the antiviral response in a STING-dependent manner, leading to increased expression of interferon (IFN) and interferon-stimulated genes (ISGs) for potent antiviral effects in vivo and in vitro. Notably, Fan interacts with STING, preventing its degradation and thereby extending the activation of IFN-based antiviral responses. Collectively, our findings highlight the potential of Fan, which elicits antiviral immunity by suppressing STING degradation, as a promising candidate for antiviral therapy.
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Emerging evidence has linked the dysregulation of N6-methyladenosine (m6A) modification to inflammation and inflammatory diseases, but the underlying mechanism still needs investigation. Here, we found that high levels of m6A modification in a variety of hyperinflammatory states are p65-dependent because Wilms tumor 1-associated protein (WTAP), a key component of the "writer" complex, is transcriptionally regulated by p65, and its overexpression can lead to increased levels of m6A modification. Mechanistically, upregulated WTAP is more prone to phase separation to facilitate the aggregation of the writer complex to nuclear speckles and the deposition of m6A marks on transcriptionally active inflammatory transcripts, thereby accelerating the proinflammatory response. Further, a myeloid deficiency in WTAP attenuates the severity of LPS-induced sepsis and DSS-induced IBD. Thus, the proinflammatory effect of WTAP is a general risk-increasing mechanism, and interrupting the assembly of the m6A writer complex to reduce the global m6A levels by targeting the phase separation of WTAP may be a potential and promising therapeutic strategy for alleviating hyperinflammation.
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Adenosina , Proteínas de Ciclo Celular , Inflamação , Fatores de Processamento de RNA , Animais , Humanos , Camundongos , Adenosina/metabolismo , Adenosina/análogos & derivados , Modelos Animais de Doenças , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Lipopolissacarídeos , Camundongos Knockout , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo , Sepse/metabolismo , Sepse/genética , Sepse/patologia , Fator de Transcrição RelA/metabolismo , Fator de Transcrição RelA/genéticaRESUMO
Psoraleae Fructus (PF, Psoralea corylifolia L.), a traditional medicine with a long history of application, is widely used clinically for the treatment of various diseases. However, the reports of PF-related adverse reactions, such as hepatotoxicity, phototoxic dermatitis, and allergy, are increasing year by year, with liver injury being the mostly common. Our previous studies have demonstrated that PF and its preparations can cause liver injury in lipopolysaccharide (LPS)-mediated susceptibility mouse model, but the mechanism of PF-related liver injury is unclear. In this study, we showed that PF and bavachinin, a major component of PF, can directly induce the expression of caspase-1 and interleukin-1ß (IL-1ß), indicating that PF and bavachinin can directly triggered the activation of inflammasome. Furthermore, pretreatment with NLR family pyrin domain-containing 3 (NLRP3), NLR family CARD domain containing 4 (NLRC4) or absent in melanoma 2 (AIM2) inflammasome inhibitors, containing MCC950, ODN TTAGGG (ODN) and carnosol, all significantly reversed bavachinin-induced inflammasome activation. Mechanistically, bavachinin dose-dependently promote Gasdermin D (GSDMD) post-shear activation and then induce mitochondrial reactive oxygen species (mtROS) production and this effect is markedly inhibited by pretreatment with N-Acetylcysteine amide (NAC). In addition, combination treatment of LPS and bavachinin significantly induced liver injury in mice, but not LPS or bavachinin alone, and transcriptome analysis further validated these results. Thus, PF and bavachinin can induce the activation of inflammasome by promoting GSDMD cleavage and cause hepatotoxicity in mice. Therefore, PF, bavachinin, and PF-related preparations should be avoided in patients with inflammasome activation-associated diseases.
Assuntos
Inflamassomos , Proteínas de Ligação a Fosfato , Psoralea , Piroptose , Animais , Piroptose/efeitos dos fármacos , Camundongos , Proteínas de Ligação a Fosfato/metabolismo , Proteínas de Ligação a Fosfato/genética , Psoralea/química , Inflamassomos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Camundongos Endogâmicos C57BL , Lipopolissacarídeos/toxicidade , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Flavonoides/farmacologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Caspase 1/metabolismo , Interleucina-1beta/metabolismo , GasderminasRESUMO
BACKGROUND: Shaoyao Decoction (SYD) is a widely recognized herbal formula utilized in traditional Chinese medicine for the treatment of diarrhea. Although it has demonstrated significant effectiveness in clinical practice for treating ulcerative colitis, the precise mechanisms by which it operates remain largely elusive. METHODS: The active ingredients of SYD were obtained by ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS), which were used to explore the potential pharmacological mechanism based on TCMSP (Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform) and PANTHER (Protein Analysis Through Evolutionary Relationships) classification system. In a mouse model of dextran sulfate sodium (DSS)-induced colitis, mRNA sequencing, 16S rDNA sequencing and targeted metabolomics techniques were used to elucidate the mechanisms of SYD, and immunohistochemistry, immunofluorescence, enzyme linked immunosorbent assay, real time quantitative polymerase chain reaction and western blot were used to test the key targets. In addition, QGP-1 and H9 cells were performed to validate the discoveries from the animal experiments. RESULTS: In the mouse model of DSS-induced colitis, SYD effectively alleviated symptoms such as bloody stool, tissue damage, inflammation, intestinal flora dysbiosis and abnormal gene expression. Analyses of both differential expressed genes in colonic tissue and predicted 16S rDNA genes, as well as the analyses of targeted genes from TCMSP based on the active ingredients in UPLC-MS/MS of SYD, uncovered the enrichment of pathways involved in the biosynthesis and degredation of 5-hydroxytryptamine (5-HT). Interestingly, SYD suppressed the relative abundance of key genes in 5-HT synthesis, Tph1(Tryptophan hydroxylase 1) and Ddc (Dopa decarboxylase), in faeces from DSS-induced mice, leading to a reduction in the concentration of fecal 5-HT. Moreover, SYD augmented the production of butyric acid. Subsequently, increasing butyric acid influenced the metabolism of 5-HT in the organism through G protein-coupled receptor 43 by impeding its synthesis, facilitating its transport and degredation. These findings were additionally corroborated in a model utilizing enterochromaffin cell (QGP-1 cells). Furthermore, reduced levels of 5-HT hindered the activation of T lymphocytes (H9 cells) via the PKC (Protein kinase C) and NF-κB (Nuclear factor kappa-B) signaling pathways, by means of HTR1A (5-HT receptor 1A) and HTR3 (5-HT receptor 3). Additionally, diminished secretion of 5-HT resulted in reduced secretion of associated cytokines, thereby alleviating inflammation in the colon. CONCLUSION: Through modulation of T lymphocyte activation mediated by 5-HT metabolism in the local colon via the intestinal flora and its metabolite, SYD effectively mitigated colonic inflammation in DSS-induced mice.
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IL-1R-associated kinases (IRAKs) are signal transducers of the TLR/IL-1R-MyD88-TRAF6 pathways. Vertebrates possess two IRAK lineages, IRAK1/2/3 and IRAK4. In mammals, IRAK4/IRAK1 and IRAK4/IRAK2 are pathway enhancers, whereas IRAK3 is a repressor. However, in bony fish, IRAK2 is absent, and it remains elusive how fish IRAK1/3/4 functionally differ from their mammalian counterparts. In this study, we explored this using the zebrafish model. First, we showed that in human 293T cells, zebrafish IRAK1 and IRAK4 were components of the Myddosome (MyD88-IRAK4-IRAK1) complex, with IRAK1 serving as a potent pathway enhancer. Then, we discovered two zebrafish IRAK3 variants: one (IRAK3a) contains an N-terminal Death domain, a middle pseudokinase domain, and a C-terminal TRAF6-binding domain, whereas the other (IRAK3b) lost both the kinase and TRAF6-binding domains. This truncation of IRAK3 variants could be a conserved phenomenon in fish, because it is also observed in trout and grass carp. We proceeded to show that zebrafish IRAK3a acts as a pathway enhancer by binding with MyD88 and TRAF6, but its activity is milder than IRAK1, possibly because it has no kinase activity. Zebrafish IRAK3b, however, plays a sheer negative role, apparently because of its lack of kinase and TRAF6-binding domains. Moreover, zebrafish IRAK3a/3b inhibit the activity of IRAK1/4, not by interacting with IRAK1/4 but possibly by competing for MyD88 and TRAF6. Finally, we have verified the essential activities of zebrafish IRAK1/3a/3b/4 in zebrafish cells and embryos. In summary, to our knowledge, our findings provide new insights into the molecular functions of fish IRAKs and the evolution of the IRAK functional modes in vertebrates.
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Quinases Associadas a Receptores de Interleucina-1 , Fator 88 de Diferenciação Mieloide , Transdução de Sinais , Fator 6 Associado a Receptor de TNF , Proteínas de Peixe-Zebra , Peixe-Zebra , Animais , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Quinases Associadas a Receptores de Interleucina-1/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Fator 88 de Diferenciação Mieloide/genética , Fator 6 Associado a Receptor de TNF/metabolismo , Fator 6 Associado a Receptor de TNF/genética , Humanos , Transdução de Sinais/imunologia , Células HEK293 , Proteínas de Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genéticaRESUMO
Podocytes are specialized terminally differentiated cells in the glomerulus that are the primary target cells in many glomerular diseases. However, the current podocyte cell lines suffer from prolonged in vitro differentiation and limited survival time, which impede research progress. Therefore, it is necessary to establish a cell line that exhibits superior performance and characteristics. We propose a simple protocol to obtain an immortalized mouse podocyte cell (MPC) line from suckling mouse kidneys. Primary podocytes were cultured in vitro and infected with the SV40 tsA58 gene to obtain immortalized MPCs. The podocytes were characterized using Western blotting and quantitative real-time PCR. Podocyte injury was examined using the Cell Counting Kit-8 assay and flow cytometry. First, we successfully isolated an MPC line and identified 39 °C as the optimal differentiation temperature. Compared to undifferentiated MPCs, the expression of WT1 and synaptopodin was upregulated in differentiated MPCs. Second, the MPCs ceased proliferating at a nonpermissive temperature after day 4, and podocyte-specific proteins were expressed normally after at least 15 passages. Finally, podocyte injury models were induced to simulate podocyte injury in vitro. In summary, we provide a simple and popularized protocol to establish a conditionally immortalized MPC, which is a powerful tool for the study of podocytes.
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Diferenciação Celular , Podócitos , Animais , Podócitos/metabolismo , Podócitos/citologia , Camundongos , Proteínas WT1/metabolismo , Proteínas WT1/genética , Proteínas dos Microfilamentos/metabolismo , Proteínas dos Microfilamentos/genética , Linhagem Celular , Técnicas de Cultura de Células/métodos , Linhagem Celular Transformada , Proliferação de CélulasRESUMO
Generally shortened 3' UTR due to alternative polyadenylation (APA) is widely observed in cancer, but its regulation mechanisms for cancer are not well characterized. Here, with profiling of APA in colorectal cancer tissues and poly(A) signal editing, we firstly identified that the shortened 3' UTR of CTNNIBP1 in colorectal cancer promotes cell proliferation and migration. We found that liquid-liquid phase separation (LLPS) of PABPN1 is reduced albeit with higher expression in cancer, and the reduction of LLPS leads to the shortened 3' UTR of CTNNBIP1 and promotes cell proliferation and migration. Notably, the splicing factor SNRPD2 upregulated in colorectal cancer, can interact with glutamic-proline (EP) domain of PABPN1, and then disrupt LLPS of PABPN1, which attenuates the repression effect of PABPN1 on the proximal poly(A) sites. Our results firstly reveal a new regulation mechanism of APA by disruption of LLPS of PABPN1, suggesting that regulation of APA by interfering LLPS of 3' end processing factor may have the potential as a new way for the treatment of cancer.
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Regiões 3' não Traduzidas , Movimento Celular , Proliferação de Células , Neoplasias Colorretais , Proteína I de Ligação a Poli(A) , Poliadenilação , Humanos , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Proteína I de Ligação a Poli(A)/metabolismo , Proteína I de Ligação a Poli(A)/genética , Movimento Celular/genética , Regiões 3' não Traduzidas/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Separação de FasesRESUMO
The IL-17 pathway displays remarkably diverse functional modes between different subphyla, classes, and even orders, yet its driving factors remains elusive. Here, we demonstrate that the IL-17 pathway originated through domain shuffling between a Toll-like receptor (TLR)/IL-1R pathway and a neurotrophin-RTK (receptor-tyrosine-kinase) pathway (a Trunk-Torso pathway). Unlike other new pathways that evolve independently, the IL-17 pathway remains intertwined with its donor pathways throughout later evolution. This intertwining not only influenced the gains and losses of domains and components in the pathway but also drove the diversification of the pathway's functional modes among animal lineages. For instance, we reveal that the crustacean female sex hormone, a neurotrophin inducing sex differentiation, could interact with IL-17Rs and thus be classified as true IL-17s. Additionally, the insect prothoracicotropic hormone, a neurotrophin initiating ecdysis in Drosophila by binding to Torso, could bind to IL-17Rs in other insects. Furthermore, IL-17R and TLR/IL-1R pathways maintain crosstalk in amphioxus and zebrafish. Moreover, the loss of the Death domain in the pathway adaptor connection to IκB kinase and stress-activated protein kinase (CIKSs) dramatically reduced their abilities to activate nuclear factor-kappaB (NF-κB) and activator protein 1 (AP-1) in amphioxus and zebrafish. Reinstating this Death domain not only enhanced NF-κB/AP-1 activation but also strengthened anti-bacterial immunity in zebrafish larvae. This could explain why the mammalian IL-17 pathway, whose CIKS also lacks Death, is considered a weak signaling activator, relying on synergies with other pathways. Our findings provide insights into the functional diversity of the IL-17 pathway and unveil evolutionary principles that could govern the pathway and be used to redesign and manipulate it.
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Interleucina-17 , Transdução de Sinais , Receptores Toll-Like , Animais , Interleucina-17/metabolismo , Receptores Toll-Like/metabolismo , Fatores de Crescimento Neural/metabolismo , Fatores de Crescimento Neural/genética , Receptores de Interleucina-1/metabolismo , Receptores de Interleucina-1/genética , Evolução Molecular , Receptores de Interleucina-17/metabolismo , Receptores de Interleucina-17/genéticaRESUMO
There is an urgent need for novel therapies to treat end-stage liver disease due to the shortage of available organs. Although cell transplantation holds considerable promise, its availability is limited due to the low engrafted cell mass and lack of unifying cell transplantation strategies. Here, we optimally established human induced pluripotent stem cell-derived functional hepatobiliary organoids (HBOs) based on our previous research and transplanted them into a monkey model via liver subcapsular and submesenteric transplantation routes to assess their potential clinical application. Our study revealed that HBO transplantation could safely and effectively improve hepatoprotection effects by antiapoptotic and antifibrotic agents. In addition, we also discovered that while multiple HBO transplantation pathways may have a shared effector mechanism, their respective treatment approaches have distinct advantages. Transplantation of HBOs could promote the high expression of CTSV in hepatic sinusoid endothelial cells, which might halt the progression of hepatic sinusoidal capillarization and liver fibrosis. Liver subcapsular transplants had stronger pro-CTSV upregulation than HBO submesenteric transplants, which could be attributed to naturally high CTSV expression in HBOs. Interestingly, both transplantation routes of HBOs were targeted the injured liver and triggered a new pattern of ductular reaction to alleviate the degree of liver fibrosis by surrounding the area with CK19-positive labeled cells. These residing, homing and repairing effects might be related to the high expression of MMP family genes. By promoting a unique pattern of ductular reactions, submesenteric HBO transplantation has a more representative antifibrotic impact than liver subcapsular transplantation. Altogether, our data strongly imply that the treatment of severe liver diseases with liver subcapsular and submesenteric transplantation of HBOs may be clinically effective and safe. These findings provide new insight into HBOs for further experimental and clinical validation.
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Colestase , Células-Tronco Pluripotentes Induzidas , Animais , Humanos , Células Endoteliais , Cirrose Hepática/induzido quimicamente , Fígado/patologia , Colestase/patologia , Organoides , PrimatasRESUMO
Alternative polyadenylation (APA) is a widespread mechanism of gene regulation that generates mRNA isoforms with alternative 3' untranslated regions (3' UTRs). Our previous study has revealed the global 3' UTR shortening of host mRNAs through APA upon viral infection. However, how the dynamic changes in the APA landscape occur upon viral infection remains largely unknown. Here we further found that, the reduced protein abundance of CPSF6, one of the core 3' processing factors, promotes the usage of proximal poly(A) sites (pPASs) of many immune related genes in macrophages and fibroblasts upon viral infection. Shortening of the 3' UTR of these transcripts may improve their mRNA stability and translation efficiency, leading to the promotion of type I IFN (IFN-I) signalling-based antiviral immune responses. In addition, dysregulated expression of CPSF6 is also observed in many immune related physiological and pathological conditions, especially in various infections and cancers. Thus, the global APA dynamics of immune genes regulated by CPSF6, can fine-tune the antiviral response as well as the responses to other cellular stresses to maintain the tissue homeostasis, which may represent a novel regulatory mechanism for antiviral immunity.
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Poliadenilação , Viroses , Fatores de Poliadenilação e Clivagem de mRNA , Humanos , Regiões 3' não Traduzidas/genética , Regulação para Baixo , Imunidade/genética , Fatores de Poliadenilação e Clivagem de mRNA/genética , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Viroses/genética , Camundongos , AnimaisRESUMO
Cancer cells usually exhibit shortened 3' untranslated regions (UTRs) due to alternative polyadenylation (APA) to promote cell proliferation and migration. Upregulated CPSF6 leads to a systematic prolongation of 3' UTRs, but CPSF6 expression in tumors is typically higher than that in healthy tissues. This contradictory observation suggests that it is necessary to investigate the underlying mechanism by which CPSF6 regulates APA switching in cancer. Here, we find that CPSF6 can undergo liquid-liquid phase separation (LLPS), and elevated LLPS is associated with the preferential usage of the distal poly(A) sites. CLK2, a kinase upregulated in cancer cells, destructs CPSF6 LLPS by phosphorylating its arginine/serine-like domain. The reduction of CPSF6 LLPS can lead to a shortened 3' UTR of cell-cycle-related genes and accelerate cell proliferation. These results suggest that CPSF6 LLPS, rather than its expression level, may be responsible for APA regulation in cancer cells.
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Neoplasias , Poliadenilação , Regiões 3' não Traduzidas/genética , Proliferação de Células , Regulação da Expressão Gênica , Fatores de Poliadenilação e Clivagem de mRNA/genética , Neoplasias/genética , Humanos , Linhagem Celular TumoralRESUMO
A series of "molecular domestication" events are thought to have converted an invertebrate RAG-like (RAGL) transposase into the RAG1-RAG2 (RAG) recombinase, a critical enzyme for adaptive immunity in jawed vertebrates. The timing and order of these events are not well understood, in part because of a dearth of information regarding the invertebrate RAGL-A transposon family. In contrast to the abundant and divergent RAGL-B transposon family, RAGL-A most closely resembles RAG and is represented by a single orphan RAG1-like (RAG1L) gene in the genome of the hemichordate Ptychodera flava (PflRAG1L-A). Here, we provide evidence for the existence of complete RAGL-A transposons in the genomes of P. flava and several echinoderms. The predicted RAG1L-A and RAG2L-A proteins encoded by these transposons intermingle sequence features of jawed vertebrate RAG and RAGL-B transposases, leading to a prediction of DNA binding, catalytic, and transposition activities that are a hybrid of RAG and RAGL-B. Similarly, the terminal inverted repeats (TIRs) of the RAGL-A transposons combine features of both RAGL-B transposon TIRs and RAG recombination signal sequences. Unlike all previously described RAG2L proteins, RAG2L-A proteins contain an acidic hinge region, which we demonstrate is capable of efficiently inhibiting RAG-mediated transposition. Our findings provide evidence for a critical intermediate in RAG evolution and argue that certain adaptations thought to be specific to jawed vertebrates (e.g. the RAG2 acidic hinge) actually arose in invertebrates, thereby focusing attention on other adaptations as the pivotal steps in the completion of RAG domestication in jawed vertebrates.
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Elementos de DNA Transponíveis , Proteínas de Homeodomínio , Animais , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Vertebrados/genética , Vertebrados/metabolismo , Imunidade Adaptativa/genéticaRESUMO
BACKGROUND: Nonalcoholic steatohepatitis (NASH) is a progressive and inflammatory subtype of nonalcoholic fatty liver disease (NAFLD) characterized by hepatocellular injury, inflammation, and fibrosis in various stages. More than 20% of patients with NASH will progress to cirrhosis. Currently, there is a lack of clinically effective drugs for treating NASH, as improving liver histology in NASH is difficult to achieve and maintain through weight loss alone. Hence, the present study aimed to investigate potential therapeutic drugs for NASH. METHODS: BMDMs and THP1 cells were used to construct an inflammasome activation model, and then we evaluated the effect of epalrestat on the NLRP3 inflammasome activation. Western blot, real-time qPCR, flow cytometry, and ELISA were used to evaluate the mechanism of epalrestat on NLRP3 inflammasome activation. Next, MCD-induced NASH models were used to evaluate the therapeutic effects of epalrestat in vivo. In addition, to evaluate the safety of epalrestat in vivo, mice were gavaged with epalrestat daily for 14 days. RESULTS: Epalrestat, a clinically effective and safe drug, inhibits NLRP3 inflammasome activation by acting upstream of caspase-1 and inducing ASC oligomerization. Importantly, epalrestat exerts its inhibitory effect on NLRP3 inflammasome activation by inhibiting the activation of aldose reductase. Further investigation revealed that the administration of epalrestat inhibited NLRP3 inflammasome activation in vivo, alleviating liver inflammation and improving NASH pathology. CONCLUSIONS: Our study indicated that epalrestat, an aldose reductase inhibitor, effectively suppressed NLRP3 inflammasome activation in vivo and in vitro and might be a new therapeutic approach for NASH.
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Hepatopatia Gordurosa não Alcoólica , Humanos , Camundongos , Animais , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/patologia , Inflamassomos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Aldeído Redutase/uso terapêutico , Inflamação , Fibrose , Camundongos Endogâmicos C57BLRESUMO
Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) have the potential to be a therapeutic option for myocardium restoration. However, hiPSC-CMs of varying maturation and transplantation routes exhibit different reactivity and therapeutic effects. We previously demonstrated that the saponin+ compound induces more mature hiPSC-CMs. The safety and efficacy of multi-route transplantation of saponin+ compound-induced hiPSC-CMs in a nonhuman primate with myocardial infarction will be investigated for the first time in this study. Our findings indicate that optimized hiPSC-CMs transplanted via intramyocardial and intravenous routes may affect myocardial functions by homing or mitochondrial transfer to the damaged myocardium to play a direct therapeutic role as well as indirect beneficial roles via anti-apoptotic and pro-angiogenesis mechanisms mediated by different paracrine growth factors. Due to significant mural thrombosis, higher mortality, and unilateral renal shrinkage, intracoronary transplantation of hiPSC-CMs requires closer attention to anticoagulation and caution in clinical use. Collectively, our data strongly indicated that intramyocardial transplantation of hiPSC-CMs is the ideal technique for clinical application; multiple cell transfers are recommended to achieve steady and protracted efficacy because intravenous transplantation's potency fluctuates. Thus, our study offers a rationale for choosing a therapeutic cell therapy and the best transplantation strategy for optimally induced hiPSC-CMs.
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Although STAT3 has been reported as a negative regulator of type I interferon (IFN) signaling, the effects of pharmacologically inhibiting STAT3 on innate antiviral immunity are not well known. Capsaicin, approved for the treatment of postherpetic neuralgia and diabetic peripheral nerve pain, is an agonist of transient receptor potential vanilloid subtype 1 (TRPV1), with additional recognized potencies in anticancer, anti-inflammatory, and metabolic diseases. We investigated the effects of capsaicin on viral replication and innate antiviral immune response and discovered that capsaicin dose-dependently inhibited the replication of VSV, EMCV, and H1N1. In VSV-infected mice, pretreatment with capsaicin improved the survival rate and suppressed inflammatory responses accompanied by attenuated VSV replication in the liver, lung, and spleen. The inhibition of viral replication by capsaicin was independent of TRPV1 and occurred mainly at postviral entry steps. We further revealed that capsaicin directly bound to STAT3 protein and selectively promoted its lysosomal degradation. As a result, the negative regulation of STAT3 on the type I IFN response was attenuated, and host resistance to viral infection was enhanced. Our results suggest that capsaicin is a promising small-molecule drug candidate, and offer a feasible pharmacological strategy for strengthening host resistance to viral infection.
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Vírus da Influenza A Subtipo H1N1 , Interferon Tipo I , Infecções por Orthomyxoviridae , Camundongos , Animais , Capsaicina/farmacologia , Fator de Transcrição STAT3 , Transdução de Sinais , Proteínas de Transporte , Replicação ViralRESUMO
Inhibition of NLRP3 inflammasome activation produces potent therapeutic effects in a wide array of inflammatory diseases. Bergapten (BeG), a furocoumarin phytohormone present in many herbal medicines and fruits, exibits anti-inflammatory activity. In this study we characterized the therapeutic potential of BeG against bacterial infection and inflammation-related disorders, and elucidated the underlying mechanisms. We showed that pre-treatment with BeG (20 µM) effectively inhibited NLRP3 inflammasome activation in both lipopolysaccharides (LPS)-primed J774A.1 cells and bone marrow-derived macrophages (BMDMs), evidenced by attenuated cleaved caspase-1 and mature IL-1ß release, as well as reduced ASC speck formation and subsequent gasdermin D (GSDMD)-mediated pyroptosis. Transcriptome analysis revealed that BeG regulated the expression of genes involved in mitochondrial and reactive oxygen species (ROS) metabolism in BMDMs. Moreover, BeG treatment reversed the diminished mitochondrial activity and ROS production after NLRP3 activation, and elevated the expression of LC3-II and enhanced the co-localization of LC3 with mitochondria. Treatment with 3-methyladenine (3-MA, 5 mM) reversed the inhibitory effects of BeG on IL-1ß, cleaved caspase-1 and LDH release, GSDMD-N formation as well as ROS production. In mouse model of Escherichia coli-induced sepsis and mouse model of Citrobacter rodentium-induced intestinal inflammation, pre-treatment with BeG (50 mg/kg) significantly ameliorated tissue inflammation and injury. In conclusion, BeG inhibits NLRP3 inflammasome activation and pyroptosis by promoting mitophagy and maintaining mitochondrial homeostasis. These results suggest BeG as a promising drug candidate for the treatment of bacterial infection and inflammation-related disorders.
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Inflamassomos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Camundongos , Animais , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Piroptose , Espécies Reativas de Oxigênio/metabolismo , 5-Metoxipsoraleno/farmacologia , Mitofagia , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Caspase 1/metabolismo , Interleucina-1beta/metabolismoRESUMO
BACKGROUND: In recent decades, the prevalence of metabolic diseases, particularly diabetes, hyperlipidemia, obesity, and non-alcoholic fatty liver disease (NAFLD), has increased dramatically, causing great public health and economic burdens worldwide. Traditional Chinese medicine (TCM) serves as an effective therapeutic choice. Xiao-Ke-Yin (XKY) is a medicine and food homology TCM formula consisting of nine "medicine and food homology" herbs and is used to ameliorate metabolic diseases, such as insulin resistance, diabetes, hyperlipidemia and NAFLD. However, despite its therapeutic potential in metabolic disorders, the underlying mechanisms of this TCM remain unclear. This study aimed to evaluate the therapeutic effectiveness of XKY on glucolipid metabolism dysfunction and explore the potential mechanisms in db/db mice. METHODS: To verify the effects of XKY, db/db mice were treated with different concentrations of XKY (5.2, 2.6 and 1.3 g/kg/d) and metformin (0.2 g/kg/d, a hypoglycemic positive control) for 6 weeks, respectively. During this study, we detected the body weight (BW) and fasting blood glucose (FBG), oral glucose tolerance test (OGTT), insulin tolerance test (ITT), daily food intake and water intake. At the end of the animal experiment, blood samples, feces, liver and intestinal tissue of mice in all groups were collected. The potential mechanisms were investigated by using hepatic RNA sequencing, 16 S rRNA sequencing of the gut microbiota and metabolomics analysis. RESULTS: XKY efficiently mitigated hyperglycemia, IR, hyperlipidemia, inflammation and hepatic pathological injury in a dose dependent manner. Mechanistically, hepatic transcriptomic analysis showed that XKY treatment significantly reversed the upregulated cholesterol biosynthesis which was further confirmed by RT-qPCR. Additionally, XKY administration maintained intestinal epithelial homeostasis, modulated gut microbiota dysbiosis, and regulated its metabolites. In particular, XKY decreased secondary bile acid producing bacteria (Clostridia and Lachnospircaeae) and lowered fecal secondary bile acid (lithocholic acid (LCA) and deoxycholic acid (DCA)) levels to promote hepatic bile acid synthesis by inhibiting the LCA/DCA-FXR-FGF15 signalling pathway. Furthermore, XKY regulated amino acid metabolism including arginine biosynthesis, alanine, aspartate and glutamate metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, and tryptophan metabolism likely by increasing Bacilli, Lactobacillaceae and Lactobacillus, and decreasing Clostridia, Lachnospircaeae, Tannerellaceae and Parabacteroides abundances. CONCLUSION: Taken together, our findings demonstrate that XKY is a promising "medicine food homology" formula for ameliorating glucolipid metabolism and reveal that the therapeutic effects of XKY may due to its downregulation of hepatic cholesterol biosynthesis and modulation of the dysbiosis of the gut microbiota and metabolites.
RESUMO
Members of the gasdermin (GSDM) family are pore-forming effectors that cause membrane permeabilization and pyroptosis, a lytic proinflammatory type of cell death. To reveal the functional evolution of GSDM-mediated pyroptosis at the transition from invertebrates to vertebrates, we conducted functional characterization of amphioxus GSDME (BbGSDME) and found that it can be cleaved by distinct caspase homologs, yielding the N253 and N304 termini with distinct functions. The N253 fragment binds to cell membrane, triggers pyroptosis, and inhibits bacterial growth, while the N304 performs negative regulation of N253-mediated cell death. Moreover, BbGSDME is associated with bacteria-induced tissue necrosis and transcriptionally regulated by BbIRF1/8 in amphioxus. Interestingly, several amino acids that are evolutionarily conserved were found to be important for the function of both BbGSDME and HsGSDME, shedding new lights on the functional regulation of GSDM-mediated inflammation.
Assuntos
Anfioxos , Piroptose , Animais , Piroptose/fisiologia , Anfioxos/genética , Anfioxos/metabolismo , Morte Celular , Necrose , Caspase 3/metabolismoRESUMO
BACKGROUND: The transcription factor NRF2 is a master redox switch that regulates the cellular antioxidant response. However, recent advances have revealed new roles for NRF2, including the regulation of antiviral responses to various viruses, suggesting that pharmacological NRF2-activating agents may be a promising therapeutic drug for viral diseases. Isoliquiritigenin (ISL), a chalcone isolated from liquorice (Glycyrrhizae Radix) root, is reported to be a natural NRF2 agonist and has has antiviral activities against HCV (hepatitis C virus) and IAV (influenza A virus). However, the spectrum of antiviral activity and associated mechanism of ISL against other viruses are not well defined. PURPOSE: This study investigated the antiviral activity and underlying mechanism of ISL against vesicular stomatitis virus (VSV), influenza A virus (H1N1), encephalomyocarditis virus (EMCV), herpes simplex virus type 1 (HSV-1). METHODS: We evaluated the antiviral activity of ISL against VSV, H1N1, EMCV, and HSV-1 using flow cytometry and qRT-PCR analysis. RNA sequencing and bioinformatic analysis were performed to investigate the potential antiviral mechanism of ISL. NRF2 knockout cells were used to investigate whether NRF2 is required for the antiviral activity of ISL. The anti-apoptosis and anti-inflammatory activities of ISL were further measured by counting cell death ratio and assessing proinflammatory cytokines expression in virus-infected cells, respectively. In addition, we evaluated the antiviral effect of ISL in vivo by measuring the survival rate, body weights, histological analysis, viral load, and cytokine expression in VSV-infected mouse model. RESULTS: Our data demonstrated that ISL effectively suppressed VSV, H1N1, HSV-1, and EMCV replication in vitro. The antiviral activity of ISL could be partially impaired in NRF2-deficient cells. Virus-induced cell death and proinflammatory cytokines were repressed by ISL. Finally, we showed that ISL treatment protected mice against VSV infection by reducing viral titers and suppressing the expression of inflammatory cytokines in vivo. CONCLUSION: These findings suggest that ISL has antiviral and anti-inflammatory effects in virus infections, which are associated with its ability to activate NRF2 signaling, thus indicating that ISL has the potential to serve as an NRF2 agonist in the treatment of viral diseases.