RESUMO
Acetaminophen (APAP) overdose is the primary cause of druginduced acute liver failure in numerous Western countries. NLR family pyrin domain containing 3 (NLRP3) inflammasome activation serves a pivotal role in the pathogenesis of various forms of acute liver injury. However, the cellular source for NLRP3 induction and its involvement during APAPinduced hepatotoxicity have not been thoroughly investigated. In the present study, hematoxylin and eosin staining was performed to assess histopathological changes of liver tissue. Immunohistochemistry staining(NLRP3, Caspase1, IL1ß, GSDMD and Caspase3), western blotting (NLRP3, Caspase1, IL1ß, GSDMD and Caspase3) and RTqPCR (NLRP3, Caspase1 and IL1ß) were performed to assess the expression of NLRP3/GSDMD signaling pathway. TUNEL staining was performed to assess apoptosis of liver tissue. The serum expression levels of inflammatory factors (IL6, IL18, IL1ß and TNFα) were assessed using ELISA and inflammation of liver tissue was assessed using immunohistochemistry (Ly6G and CD68) and RTqPCR (TNFα, Il6, Mcp1, Cxcl1, Cxcl2). A Cell Counting Kit8 was performed to assess cell viability and apoptosis. Protein and gene expression were analyzed by western blotting (PCNA, CCND1) and RTqPCR (CyclinA2, CyclinD1 and CyclinE1). Through investigation of an APAPinduced acute liver injury model (AILI), the present study demonstrated that APAP overdose induced activation of NLRP3 and cleavage of gasdermin D (GSDMD) in hepatocytes, both in vivo and in vitro. Additionally, mice with hepatocytespecific knockout of Nlrp3 exhibited reduced liver injury and lower mortality following APAP intervention, accompanied by decreased infiltration of inflammatory cells and attenuated inflammatory response. Furthermore, pharmacological blockade of NLRP3/GSDMD signaling using MCC950 or disulfiram significantly ameliorated liver injury and reduced hepatocyte death. Notably, hepatocyte Nlrp3 deficiency promoted liver recovery by enhancing hepatocyte proliferation. Collectively, the present study demonstrated that inhibition of the NLRP3 inflammasome protects against APAPinduced acute liver injury by reducing hepatocyte pyroptosis and suggests that targeting NLRP3 may hold therapeutic potential for treating AILI.
Assuntos
Doença Hepática Crônica Induzida por Substâncias e Drogas , Proteína 3 que Contém Domínio de Pirina da Família NLR , Camundongos , Animais , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Inflamassomos/metabolismo , Acetaminofen/efeitos adversos , Piroptose , Caspase 3 , Fator de Necrose Tumoral alfa , Interleucina-6 , Hepatócitos/metabolismoRESUMO
BACKGROUND: Adipose tissue-derived stem cell (ADSC) transplantation has been shown to be effective for the management of severe liver disorders. Preactivation of ADSCs enhanced their therapeutic efficacy. However, these effects have not yet been examined in relation to cholestatic liver injury. METHODS: In the present study, a cholestatic liver injury model was established by bile duct ligation (BDL) in male C57BL/6 mice. Human ADSCs (hADSCs) with or without tumor necrosis factor-alpha (TNF-α) and interleukin-1beta (IL-1ß) pretreatment were administrated into the mice via tail vein injections. The efficacy of hADSCs on BDL-induced liver injury was assessed by histological staining, real-time quantitative PCR (RT-qPCR), Western blot, and enzyme-linked immune sorbent assay (ELISA). In vitro, the effects of hADSC conditioned medium on the activation of hepatic stellate cells (HSCs) were investigated. Small interfering RNA (siRNA) was used to knock down cyclooxygenase-2 (COX-2) in hADSCs. RESULTS: TNF-α/IL-1ß preconditioning could downregulate immunogenic gene expression and enhance the engraftment efficiency of hADSCs. Compared to control hADSCs (C-hADSCs), TNF-α/IL-1ß-pretreated hADSCs (P-hADSCs) significantly alleviated BDL-induced liver injury, as demonstrated by reduced hepatic cell death, attenuated infiltration of Ly6G + neutrophils, and decreased expression of pro-inflammatory cytokines TNF-α, IL-1ß, C-X-C motif chemokine ligand 1 (CXCL1), and C-X-C motif chemokine ligand 2 (CXCL2). Moreover, P-hADSCs significantly delayed the development of BDL-induced liver fibrosis. In vitro, conditioned medium from P-hADSCs significantly inhibited HSC activation compared to that from C-hADSCs. Mechanistically, TNF-α/IL-1ß upregulated COX-2 expression and increased prostaglandin E2 (PGE2) secretion. The blockage of COX-2 by siRNA transfection reversed the benefits of P-hADSCs for PGE2 production, HSC activation, and liver fibrosis progression. CONCLUSION: In conclusion, our results suggest that TNF-α/IL-1ß pretreatment enhances the efficacy of hADSCs in mice with cholestatic liver injury, partially through the COX-2/PGE2 pathway.
Assuntos
Doença Hepática Crônica Induzida por Substâncias e Drogas , Colestase , Camundongos , Masculino , Humanos , Animais , Fator de Necrose Tumoral alfa/metabolismo , Dinoprostona/metabolismo , Ciclo-Oxigenase 2/metabolismo , Doença Hepática Crônica Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Crônica Induzida por Substâncias e Drogas/patologia , Meios de Cultivo Condicionados/farmacologia , Ligantes , Camundongos Endogâmicos C57BL , Fígado/metabolismo , Colestase/patologia , Cirrose Hepática/patologia , Fibrose , Quimiocinas/metabolismoRESUMO
In this work, N-benzylarylamide-dithiocarbamate based derivatives were designed, synthesized, and their biological activities as anticancer agents were explored. Some of the 33 target compounds displayed significant antiproliferative activities with IC50 values at the double-digit nanomolar level. The representative compound I-25 (also named MY-943) not only showed the most effective inhibitory effects on three selected cancer cells MGC-803 (IC50 = 0.017 µM), HCT-116 (IC50 = 0.044 µM) and KYSE450 (IC50 = 0.030 µM), but also exhibited low nanomolar IC50 values from 0.019 to 0.253 µM against the other 11 cancer cells. Compound I-25 (MY-943) effectively inhibited tubulin polymerization and suppressed LSD1 at the enzymatic levels. Compound I-25 (MY-943) could act on the colchicine binding site of ß-tubulin, thus disrupting the construction of cell microtubule network and affecting the mitosis. In addition, compound I-25 (MY-943) could dose-dependently induce the accumulation of H3K4me1/2 (MGC-803 and SGC-7091 cells) and H3K9me2 (SGC-7091 cells). Compound I-25 (MY-943) could induce G2/M phase arrest and cell apoptosis, and suppress migration in MGC-803 and SGC-7901 cells. In addition, compound I-25 (MY-943) significantly modulated the expression of apoptosis- and cycle-related proteins. Furthermore, the binding modes of compound I-25 (MY-943) with tubulin and LSD1 were explored by molecular docking. The results of in vivo anti-gastric cancer assays using in situ tumor models showed that compound I-25 (MY-943) effectively reduced the weight and volume of gastric cancer in vivo without obvious toxicity. All these findings suggested that the N-benzylarylamide-dithiocarbamate based derivative I-25 (MY-943) was an effective dual inhibitor of tubulin polymerization and LSD1 that inhibited gastric cancers.
Assuntos
Antineoplásicos , Neoplasias Gástricas , Humanos , Tubulina (Proteína)/metabolismo , Linhagem Celular Tumoral , Simulação de Acoplamento Molecular , Polimerização , Proliferação de Células , Moduladores de Tubulina/farmacologia , Moduladores de Tubulina/química , Antineoplásicos/farmacologia , Antineoplásicos/química , Neoplasias Gástricas/tratamento farmacológico , Histona Desmetilases/metabolismo , Relação Estrutura-Atividade , Ensaios de Seleção de Medicamentos AntitumoraisRESUMO
Liver regeneration is a complex process that needs orchestration of multiple nonparenchymal cells including sinusoid endothelial cells. Vascular endothelial growth factor (VEGF) serves a crucial role in angiogenesis and liver regeneration. However, the lack of an highefficiency delivery system target to the injured site reduces the local therapeutic efficacy of VEGF. In our previous study, collagen binding VEGF (CBDVEGF) was established by fusing collagen binding domain (CBD) into the Nterminal of native VEGF and improved cardiac function after myocardial infraction. The present study investigated the therapeutic effect of CBDVEGF on liver regeneration by a mouse model of partial hepatectomy. After injection through portal vein following 2/3 hepatectomy, CBDVEGF was largely retained in the hepatic extracellular matrix for 48 h. Furthermore, CBDVEGF application significantly promoted sinusoidal regeneration and remodeling in remanent liver tissue 48 h after hepatectomy. In addition, CBDVEGF treatment significantly enhanced the proliferation of hepatocytes at 2 and 3 days postsurgery compared with native VEGF, concomitant with attenuated liver injury. In conclusion, these results demonstrated that CBDVEGF could be a promising therapeutic strategy for liver regeneration.
Assuntos
Hepatectomia , Regeneração Hepática , Animais , Colágeno/metabolismo , Células Endoteliais/metabolismo , Hepatócitos/metabolismo , Hiperplasia/patologia , Fígado/metabolismo , Camundongos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Purpose: To explore the effect and mechanism of NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasomes on corneal fibrosis. Methods: The wild-type, NLRP3 knockout (KO), and myeloid cell-specific NLRP3 KO (NLRP3 Lyz-KO) C57 mice were used to establish a corneal scarring model. NLRP3 inhibitor, IL-1ß neutralizing antibody, and an IL-1R antagonist were used to investigate the role of NLRP3 and IL-1ß in corneal fibrosis. The expression of the NLRP3 signaling pathway related proteins, alpha-smooth muscle actin, TGF-ß was determined by quantitative real-time polymerase chain reaction, Western blotting, and immunofluorescence staining. Flow cytometry was used to detect the infiltration of macrophages during corneal fibrosis. Results: The components of the NLRP3 inflammasomes were elevated and activated during corneal scarring. Additionally, genetic or chemical-mediated blocking of NLRP3 as well as IL-1ß significantly alleviated corneal fibrosis. Moreover, neutrophil (CD45+Ly6G+) and macrophage (CD45+ F4/80+) accumulation increased in the cornea during the progression of corneal fibrosis. Intriguingly, the increased concentrations of NLRP3 and IL-1ß were prominently colocalized with the infiltrating F4/80+ macrophages. Expectedly, NLRP3 Lyz-KO mice exhibited a marked decrease in their corneal fibrosis symptoms. Mechanistically, the activation of IL-1ß or macrophage NLRP3 stimulated the expression of TGF-ß1 in the corneal epithelial cells, whereas an NLRP3 deficiency decreased its expression in the corneal epithelium. Conclusions: These observations revealed that the NLRP3 inflammasome activation in infiltrating macrophages contributes to corneal fibrosis by regulating corneal epithelial TGF-ß1 expression. Targeting the NLRP3 inflammasome might be a promising strategy for the treatment of corneal scarring.
Assuntos
Epitélio Corneano , Inflamassomos , Animais , Cicatriz/metabolismo , Epitélio Corneano/metabolismo , Inflamassomos/metabolismo , Interleucina-1beta/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Fator de Crescimento Transformador beta1/metabolismoRESUMO
The chalcone and quinoline scaffolds are frequently utilized to design novel anticancer agents. As the continuation of our work on effective anticancer agents, we assumed that linking chalcone fragment to the quinoline scaffold through the principle of molecular hybridization strategy could produce novel compounds with potential anticancer activity. Therefore, quinoline-chalcone derivatives were designed and synthesized, and we explored their antiproliferative activity against MGC-803, HCT-116, and MCF-7 cells. Among these compounds, compound 12e exhibited a most excellent inhibitory potency against MGC-803, HCT-116, and MCF-7 cells with IC50 values of 1.38, 5.34, and 5.21 µM, respectively. The structure-activity relationship of quinoline-chalcone derivatives was preliminarily explored in this report. Further mechanism studies suggested that compound 12e inhibited MGC-803 cells in a dose-dependent manner and the cell colony formation activity of MGC-803 cells, arrested MGC-803 cells at the G2/M phase and significantly upregulated the levels of apoptosis-related proteins (Caspase3/9 and cleaved-PARP) in MGC-803 cells. In addition, compound 12e could significantly induce ROS generation, and was dependent on ROS production to exert inhibitory effects on gastric cancer cells. Taken together, all the results suggested that directly linking chalcone fragment to the quinoline scaffold could produce novel anticancer molecules, and compound 12e might be a valuable lead compound for the development of anticancer agents.
Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Chalconas/síntese química , Chalconas/farmacologia , Desenho de Fármacos , Quinolinas/síntese química , Quinolinas/farmacologia , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Chalconas/química , Humanos , Quinolinas/química , Espécies Reativas de Oxigênio/metabolismo , Relação Estrutura-AtividadeRESUMO
NEDDylation process regulates multiple physiological functions and signaling pathways, which are still in an equilibrium that favors the survival and proliferation of tumor cells. Unlike inhibitors, NEDDylation agonists are rarely studied. In this work, novel 1,2,4-triazine-dithiocarbamate derivatives were synthesized and evaluated for antiproliferative activity against MGC-803, PC-3 and EC-109 cells. Among them, compound K3 displayed the most potent activity MGC-803, PC-3 and EC-109 cells with IC50 values of 2.35, 5.71 and 10.1 µM, respectively, which were more potent than 5-FU. Further cellular mechanisms suggested that compound K3 inhibited the cell viability, induced proliferation inhibition, arrested cell cycle at G2/M phase and induced cell apoptosis in MGC-803 and HGC-27 cells. Importantly, compound K3 could interact with NAE1 to promote the NEDDylation of MGC-803 and HGC-27 cells. The promotion of NEDDylation resulted in the degradation of c-IAP and YAP/TAZ, which leads to the induction of cell apoptosis and inhibition of proliferation in MGC-803 and HGC-27 cells. Therefore, as a NEDDylation agonist, compound K3 could effectively inhibit gastric cancer cells. Here, we reported NEDDylation promotion induced by compound K3, which could inhibit the cancer cell lines MGC-803 and HGC-27 and induce the cancer cell apoptosis via prompting the degradation of c-IAP and YAP/TAZ.