RESUMO
Compared with young liver donors, aged liver donors are more susceptible to ischemia-reperfusion injury (IRI) following transplantation, which may be related to excessive inflammatory response and macrophage dysfunction, but the specific mechanism is unclear. Macrophage scavenger receptor 1 (MSR1) is a member of the scavenger receptor family, and plays an important regulatory role in inflammation response and macrophage function regulation. But its role in IRI following aged-donor liver transplantation is still unclear. This study demonstrates that MSR1 expression is decreased in macrophages from aged donor livers, inhibiting their efferocytosis and pro-resolving polarisation. Decreased MSR1 is responsible for the more severe IRI suffered by aged donor livers. Overexpression of MSR1 using F4/80-labeled AAV9 improved intrahepatic macrophage efferocytosis and promoted pro-resolving polarisation, ultimately ameliorating IRI following aged-donor liver transplantation. In vitro co-culture experiments further showed that overexpression of MSR1 promoted an increase in calcium concentration, which further activated the PI3K-AKT-GSK3ß pathway, and induced the upregulation of ß-catenin. Overall, MSR1-dependent efferocytosis promoted the pro-resolving polarisation of macrophages through the PI3K-AKT-GSK3ß pathway-induced up-regulating of ß-catenin leading to improved IRI following aged-donor liver transplantation.
RESUMO
BACKGROUND: The NOD-like receptor protein 3 (NLRP3) mediated pyroptosis of macrophages is closely associated with liver ischemia reperfusion injury (IRI). As a covalent inhibitor of NLRP3, Oridonin (Ori), has strong anti-inflammasome effect, but its effect and mechanisms for liver IRI are still unknown. METHODS: Mice and liver macrophages were treated with Ori, respectively. Co-IP and LC-MS/MS analysis of the interaction between PKM2 and NLRP3 in macrophages. Liver damage was detected using H&E staining. Pyroptosis was detected by WB, TEM, and ELISA. RESULTS: Ori ameliorated liver macrophage pyroptosis and liver IRI. Mechanistically, Ori inhibited the interaction between pyruvate kinase M2 isoform (PKM2) and NLRP3 in hypoxia/reoxygenationï¼H/Rï¼-induced macrophages, while the inhibition of PKM2/NLRP3 reduced liver macrophage pyroptosis and liver IRI. CONCLUSION: Ori exerted protective effects on liver IRI via suppressing PKM2/NLRP3-mediated liver macrophage pyroptosis, which might become a potential therapeutic target in the clinic.
Assuntos
Diterpenos do Tipo Caurano , Fígado , Macrófagos , Camundongos Endogâmicos C57BL , Proteína 3 que Contém Domínio de Pirina da Família NLR , Piroptose , Traumatismo por Reperfusão , Animais , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Piroptose/efeitos dos fármacos , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/tratamento farmacológico , Camundongos , Fígado/metabolismo , Fígado/efeitos dos fármacos , Fígado/patologia , Macrófagos/metabolismo , Macrófagos/efeitos dos fármacos , Diterpenos do Tipo Caurano/farmacologia , Masculino , Piruvato Quinase/metabolismo , Inflamassomos/metabolismo , Inflamassomos/efeitos dos fármacos , Hepatopatias/metabolismo , Hepatopatias/tratamento farmacológicoRESUMO
Eyes absent (Eya) is a highly conserved transcription cofactor and protein phosphatase that regulates multiple developmental processes throughout the metazoans. It is a dual function protein, working as a transcription factor in the nucleus and as a tyrosine phosphatase in the cytoplasm. In this study, we isolated EYA-1 of Caenorhabditis elegans, the only homolog of Eyes absent, and set up an effective feeding-based RNAi (RNA interference) against the gene. We found that knockdown of EYA-1 decreased heat and oxidative stress tolerance and accelerated the onset of paralysis mediated by Aß1-42 proteotoxicity and polyQ. Under heat stress (35°C), EYA-1 knockdown shortened the mean lifespan by 16.8%, which could be attributed to decrease in heat shock protein-16.2 (hsp-16.2) expression. Under oxidative stress, EYA-1 knockdown could shorten the mean lifespan by 18.7%, which could be attributed to intracellular ROS accumulation and the decrease of superoxide dismutase-3 (sod-3) protein expression. Moreover, EYA-1 knockdown animals also showed increased lipofuscin accumulation under oxidative stress. Further studies demonstrated that EYA-1 knockdown could not inhibit daf-16 nuclear accumulation in wild-type worms in response to stress. On the other hand, EYA-1 deficiency did not further reduce stress resistance of daf-16 mutants, which are stress sensitive. Quantitative real-time PCR results also showed that the expression of two daf-16 target genes, hsp-12.3 and sod-3, was downregulated in EYA-1 RNAi-treated worms under stress. All this evidence indicates EYA-1 is required for stress resistance of worms, and it might act downstream of daf-16 to regulate expression of stress resistance-associated genes.
Assuntos
Proteínas de Caenorhabditis elegans/fisiologia , Caenorhabditis elegans/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Proteínas Nucleares/fisiologia , Proteínas Tirosina Fosfatases/fisiologia , Adaptação Fisiológica , Animais , Proteínas de Caenorhabditis elegans/metabolismo , Fatores de Transcrição Forkhead , Genes Essenciais , Resposta ao Choque Térmico , Estresse Oxidativo , Fatores de Transcrição/metabolismoRESUMO
Objectives: Although a recent study reported that stimulator of interferon genes (STING) in macrophages has an important regulatory effect on liver ischemia-reperfusion injury (IRI), the underlying mechanism of STING-dependent innate immune activation in liver macrophages (Kupffer cells, KCs) remains unclear. Here, we investigated the effect of STING on liver macrophage pyroptosis and the associated regulatory mechanism of liver IRI. Methods: Clodronate liposomes were used to block liver macrophages. AAV-STING-RNAi-F4/80-EGFP, an adenoassociated virus (AAV), was transfected into the portal vein of mice in vivo, and the liver IRI model was established 14 days later. In vitro, liver macrophages were treated with STING-specific siRNA, and a hypoxia-reoxygenation (H/R) model was established. The level of STING was detected via Western blotting (WB), RT-PCR, and immunostaining. Liver tissue and blood samples were collected. Pathological changes in liver tissue were detected by hematoxylin and eosin (H&E) staining. Macrophage pyroptosis was detected by WB, confocal laser scanning microscopy (CLSM), transmission electron microscopy (TEM), and enzyme-linked immunosorbent assay (ELISA). The calcium concentration was measured by immunofluorescence and analyzed with a fluorescence microplate reader. Results: The expression of STING increased with liver IRI but decreased significantly after the clodronate liposome blockade of liver macrophages. After knockdown of STING, the activation of caspase 1-GSDMD in macrophages and liver IRI was alleviated. More interestingly, hypoxia/reoxygenation (H/R) increased the calcium concentration in liver macrophages, but the calcium concentration was decreased after STING knockdown. Furthermore, after the inhibition of calcium in H/R-induced liver macrophages by BAPTA-AM, pyroptosis was significantly reduced, but the expression of STING was not significantlydecreased. Conclusions: Knockdown of STING reduces calcium-dependent macrophage caspase 1-GSDMD-mediated liver IRI, representing a potential therapeutic approach in the clinic.
Assuntos
Caspase 1/metabolismo , Fígado/patologia , Macrófagos/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Traumatismo por Reperfusão/fisiopatologia , Animais , Humanos , Masculino , Camundongos , Transdução de Sinais , TransfecçãoRESUMO
OBJECTIVES: This study aimed to investigate the protective effect of SCARF1 on acute rejection (AR), phagocytic clearance of Kupffer cells (KCs), M2 polarization and the exact mechanism underlying these processes. METHODS: AAV was transfected into the portal vein of rats, and AR and immune tolerance (IT) models of liver transplantation were established. Liver tissue and blood samples were collected. The level of SCARF1 was detected via WB and immunohistochemical staining. Pathological changes in liver tissue were detected using HE staining. Apoptotic cells were detected using TUNEL staining. KC polarization was assessed via immunohistochemical staining. Primary KCs were isolated and co-cultured with apoptotic T lymphocytes. Phagocytosis of apoptotic cells and polarization of KCs were both detected using immunofluorescence. Calcium concentration was determined using immunofluorescence and a fluorescence microplate reader. The levels of PI3K, p-AKT and P-STAT3 were assessed via WB and immunofluorescence. RESULTS: Compared to the IT group, the level of SCARF1 was significantly decreased in the AR group. Overexpression of SCARF1 in KCs improved AR and liver function markers. Enhanced phagocytosis mediated by SCARF1 is beneficial for improving the apoptotic clearance of AR and promoting M2 polarization of KCs. SCARF1-mediated enhancement of phagocytosis promotes increased calcium concentration in KCs, thus further activating the PI3K-AKT-STAT3 signalling pathway. CONCLUSIONS: SCARF1 promotes the M2 polarization of KCs by promoting phagocytosis through the calcium-dependent PI3K-AKT-STAT3 signalling pathway.
Assuntos
Cálcio/metabolismo , Transplante de Fígado , Receptores Depuradores Classe F/metabolismo , Transdução de Sinais , Animais , Apoptose , Polaridade Celular , Células Cultivadas , Técnicas de Cocultura , Células de Kupffer/citologia , Células de Kupffer/metabolismo , Fígado/metabolismo , Fígado/patologia , Masculino , Fagocitose , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Endogâmicos Lew , Fator de Transcrição STAT3/metabolismo , Receptores Depuradores Classe F/genética , Linfócitos T/citologia , Linfócitos T/metabolismoRESUMO
PURPOSE: To investigate the roles of ER stress in Kupffer cells (KCs) and KC-derived TNF-α in the apoptosis of hepatic stellate cells (HSCs). METHODS: A rat model of liver fibrosis was established. Liver and blood serum samples were collected. Liver function assays, Masson staining, Sirius Red staining, ELISAs, and TUNEL and immunohistochemical staining were performed. Liver function, liver fibrosis, KC phenotype, inflammatory factors, and number of active HSCs were investigated. KCs were isolated, treated with tunicamycin, and then, cocultured with primary hepatic stellate cells. ELISAs, immunofluorescence staining, flow cytometry, and Western blotting were performed. KC phenotype, inflammatory factors, HSC apoptosis, and TNF-R1/caspase 8 pathway activity were examined. RESULT: s. ER stress in KCs reduced the levels of liver function markers, reduced the degree of liver fibrosis, and increased the number of KCs with the M1 phenotype and the expression of TNF-α. The increase in KC-derived TNF-α reduced the number of active HSCs and increased the activity of TNF-R1/caspase 8. Furthermore, ER stress in KCs promoted the polarization of KCs towards the M1 phenotype and increased the expression of TNF-α. The increase in KC-derived TNF-α triggered the apoptosis of HSCs and the activation of TNF-R1/caspase 8 in vitro, which was consistent with the in vivo results. CONCLUSION: ER stress in KCs promotes the polarization of these cells towards the M1 phenotype and increases the expression of TNF-α. Then, the increase in KC-derived TNF-α triggers the apoptosis of HSCs through TNF-R1/caspase 8.
Assuntos
Apoptose , Caspase 8/metabolismo , Estresse do Retículo Endoplasmático , Células Estreladas do Fígado/metabolismo , Células de Kupffer/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
The present study investigated the effects of matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) in transplantation-associated arteriosclerosis by observing their expression in transplanted aortas in rats. Allogenic and isogenic abdominal aortic transplantations were performed and grafts were removed from the recipients at the designated time points (day 7, 14, 28 and 56 post transplantation). Hematoxylin and eosin staining, immunohistochemistry, immunofluorescence and western blot analysis were used to evaluate the grafts. Significant proliferation of the intima was observed in the allogenic transplantation groups (P<0.05). The expressions of MMPs and TIMPs in the allografts were significantly increased compared with the isografts, and the suppression of MMP2 in allografts reduced injury after transplantation. The present study concluded that the imbalance of MMPs and TIMPs led to the disturbance of synthesis and the degradation of the extracellular matrix and it may represent a key cause of chronic rejection.
Assuntos
Aorta Abdominal/transplante , Arteriosclerose/etiologia , Metaloproteinase 2 da Matriz/análise , Inibidores Teciduais de Metaloproteinases/análise , Animais , Aorta Abdominal/patologia , Arteriosclerose/patologia , Masculino , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Transplante Homólogo , Túnica Íntima/patologiaRESUMO
OBJECTIVE: To investigate the effects of inhibiting TIM-4 function in Kupffer cells (KCs) on liver graft rejection in mice and explore the underlying mechanism. METHODS: Mouse models of orthotopic liver transplantation were treated with a control mAb group and TIM-4 mAb. The activated KCs were assayed with immunohistochemistry after operation. The expression of TIM-4 in KCs were assayed with Western blotting and RT-PCR and the levels of AST, ALT, TBIL, TNF-α, IFN-γ and CCL2 were assayed detected. The expression of TIM-4 in KCs was observed with laser confocal microscopy. HE staining was used to observe the microstructure of the liver tissues, and the number of CD25+Foxp3+T cells was determined using with flow cytometry; the proteins levels of p-P65and p-P38 were assayed with Western blotting. The donor mice were treated with clodronate liposomes to destroy the KCs in the liver before transplantation, and the liver grafts were examined for graft rejection. RESULTS: The number of activated KCs in the liver graft increased progressively over time. Compared with the sham-operated group, the liver graft showed significantly increased TIM-4 protein and mRNA levels at 1, 3, and 7 days after transplantation (P<0.05) and increased levels of AST, ALT, TBIL, TNF-α, IFN-γ and CCL2 at 7 days (P<0.05). The graft in TIM-4 mAb group showed mild pathological changes with a mean RAI score of 2.67∓0.75, which was significantly lower than that in control mAb group (P<0.05). The mean survival time of the recipient mice was 53.8∓6.4 days in TIM-4 mAb group, significantly longer than that in the control mAB group (14.5∓2.9 days, P<0.05). Donor treatment with clodronate liposomes resulted in comparable RAI scores in TIM-4 mAb and control mAb groups (8.01∓0.64 vs 7.93∓0.56, P>0.05). The protein levels of p-P65 and p-P38 in TIM-4 mAb group were significantly lower than those in control mAb group (P<0.05), and CD25+Foxp3+T cells in the liver graft increased significantly in TIM-4 mAb group. CONCLUSION: Inhibition of TIM-4 function in KCs reduces the production of inflammatory factors after liver transplantation possibly by inhibiting the NF-κB and MAPK signaling pathways and promoting the proliferation of Foxp3+Treg cells to induce allograft tolerance.
Assuntos
Anticorpos Monoclonais/farmacologia , Rejeição de Enxerto , Células de Kupffer/metabolismo , Transplante de Fígado , Proteínas de Membrana/antagonistas & inibidores , Animais , Imuno-Histoquímica , Células de Kupffer/efeitos dos fármacos , Fígado/cirurgia , Masculino , Camundongos , NF-kappa B/metabolismo , Linfócitos T Reguladores/imunologiaRESUMO
PURPOSE: To evaluate the influence of different root canal instrumentation size on disinfection of intracanal microbe of dental root canal. METHODS: 368 extracted human anterior teeth with single straight root were randomly divided into 8 groups of 46 roots in each. They were instrumented with K3 Ni-Ti files as follows: group A1 and group B1(#25/0.06), group A2 and group B2(#30/0.06), group A3 and group B3(#35/0.06), group A4 and group B4(#40/0.06). After being prepared and sterilized by autoclaving, group A was inoculated with Enterococcus faecalis, and group B was inoculated with Candida albicans. All groups were irrigated with Er:YAG laser combination of 3% NaOCl, 17% EDTA and 0.9% saline, and then the numbers of microbe on the surface of root canal walls were counted after the treatment, the absolute reduction of counting colony forming units(CFUs) and the relative residual rate of CFUs in the individual group was determined. The Date was analyzed with GraphPad Prism 5.02 software package by one-way analysis of variance. RESULTS: Levels of disinfection on E.faecalis and C.albicans increased when root canals were enlarged; #40/0.06 showed the best disinfection, #35/0.06 showed a significantly better disinfection than #30/0.06 and #25/0.06. Substantial reduction of microbe was obtained in #35/0.06 and #40/0.06 compared with #25/0.06 and #30/0.06(P<0.05). CONCLUSIONS: Within the root canal size of #25/0.06-#40/0.06, under the conditions of Er:YAG laser combination of 3% NaOCl, 17% EDTA and 0.9% saline, it was concluded that the reduction of E.faecalis and C.albicans of the anterior straight root canals could be predicted by increasing the root canal instrumentation size large than #30/0.06.
Assuntos
Cavidade Pulpar , Lasers de Estado Sólido , Irrigantes do Canal Radicular , Preparo de Canal Radicular/instrumentação , Candida albicans , Desinfecção/métodos , Enterococcus faecalis , Humanos , Tratamento do Canal Radicular , Hipoclorito de SódioRESUMO
The first total synthesis of (±)-pallambins C and D has been accomplished in a linear 38 step reaction from (±)-Wieland-Miescher ketone. The key conversions are featured as follows: a Grob fragmentation-intramolecular aldol cyclization and a thiourea/palladium-catalyzed carbonylative annulation.