RESUMO
OBJECTIVES: The aim of this study was to investigate the regulatory role of G protein subunit alpha i3 (GNAI3) in periodontitis. DESIGN: Following the induction of human periodontal ligament stem cells (hPDLSCs) with lipopolysaccharide (LPS), the mRNA and protein expressions of GNAI3 and Lin28A were detected by real-time quantitative polymerase chain reaction (RT-qPCR) and western blot. The transfection efficiency of Oe-GNAI3 and sh-Lin28A was examined by virtue of RT-qPCR and western blot. With the application of ELISA and flow cytometry, the releases of inflammatory cytokines and cell apoptosis were appraised. Alkaline phosphatase (ALP) staining and alizarin red S (ARS) staining were conducted to evaluate osteogenic differentiation. Next, the binding ability of Lin28A with GNAI3 mRNA was estimated by radioimmunoprecipitation (RIP) assay while the stability of GNAI3 mRNA was assessed utilizing RT-qPCR. Western blot was employed for the measurement of inflammation-, apoptosis- and nuclear factor-kappaB (NF-κB)/NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome pathway-related proteins and osteogenic markers. RESULTS: The expression of GNAI3 was down-regulated in LPS-induced hPDLSCs. After the transfection with Oe-GNAI3, the inflammation and apoptosis in LPS-induced hPDLSCs were inhibited while osteogenic differentiation was promoted. Moreover, Lin28A could stabilize GNAI3 mRNA and Lin28A knockdown significantly reduced GNAI3 expression. Further experiments verified that the inhibitory effects of GNAI3 overexpression on LPS-induced cellular inflammation and cell apoptosis as well as the promotive effects on osteogenic differentiation in hPDLSCs were all partially counteracted by Lin28A depletion, which may possibly be mediated via the regulation of the NF-κB/NLRP3 inflammasome pathway. CONCLUSION: GNAI3 that mediated by Lin28A regulates the inflammation and osteogenic differentiation in LPS-induced hPDLSCs by mediating the NF-κB/NLRP3 inflammasome pathway.
Assuntos
Diferenciação Celular , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP , Inflamassomos , NF-kappa B , Proteína 3 que Contém Domínio de Pirina da Família NLR , Osteogênese , Ligamento Periodontal , Proteínas de Ligação a RNA , Células-Tronco , Humanos , Apoptose/efeitos dos fármacos , Western Blotting , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Inflamassomos/metabolismo , Inflamação/metabolismo , Lipopolissacarídeos/farmacologia , NF-kappa B/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Osteogênese/efeitos dos fármacos , Ligamento Periodontal/citologia , Ligamento Periodontal/metabolismo , Periodontite/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais , Células-Tronco/metabolismo , Células-Tronco/efeitos dos fármacos , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismoRESUMO
BACKGROUND: NLRP3 (NOD-, LRR- and pyrin domain-containing protein 3)-induced pyroptosis is involved in the development of a variety of autoimmune diseases, but its role in IgG4-related sialadenitis (IgG4-RS) is unclear. METHODS: Salivary gland tissues from 19 patients with IgG4-RS were designated the experimental group, and peritumoral tissues from 20 patients with benign salivary gland tumours were designated the control group. The cell morphology and fibrosis in the IgG4-RS samples were observed by haematoxylin-eosin (H&E) and Masson trichrome (MT) staining. Immunohistochemical (IHC) staining was used to determine pyroptosis-related proteins (NLRP3, ASC (apoptosis-associated speck-like protein containing a CARD), Caspase-1, GSDMD (gasdermin family members, including digestive dermatin D), interleukin 1ß (IL-1ß), and interleukin 18 (IL-18)) expression levels. RESULTS: Increased lymphoid follicle proliferation, germinal centre plasma cell infiltration, and irregular fibrosis were observed in the experimental group compared with the control group. The NLRP3, ASC, Caspase-1, GSDMD, IL-1ß, and IL-18 levels were significantly higher in the experimental group than in the control group (p < 0.0001). CONCLUSION: This study suggested that pyroptosis-related proteins might be involved in IgG4-RS pathogenesis. However, the specific cellular pathway involved and whether multiple cell death pathways contribute to the occurrence of IgG4-RS still need to be further studied.
Assuntos
Interleucina-18 , Sialadenite , Caspase 1/metabolismo , Fibrose , Humanos , Imunoglobulina G , Interleucina-1beta/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR , PiroptoseRESUMO
PURPOSE: A characteristic of prion diseases which affect both animals and humans is the aggregation of PrP amyloid fibrils in the brain, associated with a chronic inflammatory response dominated by microglial activation. In this study, we hypothesised that specific ligands of the 18-kDa translocator protein (TSPO) would be effective in the evaluation of microglial activation related to PrP(sc) deposits in prion disease. PROCEDURES: Chronological studies using in vitro autoradiography were carried out with [(3)H]-PK11195 and [(125)I]-IMPY on frozen cerebral sections from scrapie-infected mice and controls. Accumulation of prion deposits was confirmed by histoblot staining with prion protein-specific monoclonal antibody. Ex vivo autoradiographic studies were carried out with [(125)I]-CLINDE and [(125)I]-IMPY at the terminal stage of infection. RESULTS: Chronological studies using in vitro autoradiography showed that PrP(sc) deposits were co-localised with activated microglia as early as 60 days post-inoculation. Progressive levels of PrP(sc) and TSPO staining were successively observed in the hippocampus, cortex and left thalamus of infected mouse brain sections in the course of the disease and were correlated with the signals obtained by histoblot staining. Significant TSPO labelling was also observed ex vivo in the cortex, hippocampus and thalamus of scrapie-infected mice. In parallel, [(125)I]-IMPY showed labelling in the same cerebral regions but with high background staining. CONCLUSIONS: These findings indicate the ability of [(125)I]-IMPY and [(125)I]-CLINDE to evaluate prion deposits and microglial activation in vitro and ex vivo in scrapie-infected mice at different stages of the disease.
Assuntos
Microglia/metabolismo , Imagem Molecular/métodos , Príons/metabolismo , Scrapie/diagnóstico por imagem , Animais , Autorradiografia , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes/farmacocinética , Radioisótopos do Iodo/farmacocinética , Camundongos , Camundongos Endogâmicos C57BL , Microglia/patologia , Sondas Moleculares/farmacocinética , Placa Amiloide/diagnóstico por imagem , Placa Amiloide/metabolismo , Príons/análise , Piridinas , Cintilografia , Scrapie/metabolismo , Scrapie/patologiaRESUMO
INTRODUCTION: A potential single-photon emission computed tomography imaging agent for labeling of A beta plaques of Alzheimer's disease, IMPY (2-(4'-dimethylaminophenyl)-6-iodo-imidazo[1,2-a]pyridine), would be effective in detection of prion amyloid deposits in transmissible spongiform encephalopathies (TSEs). METHODS: In vitro autoradiographic studies were carried out with [125 I]IMPY on brain sections from scrapie-infected mice and age-matched controls. Competition study was performed to evaluate the prion deposit binding specificity with nonradioactive IMPY. RESULTS: Binding of [125 I]IMPY was observed in infected brain sections, while on age-matched control brain sections, there was no or very low labeling. Prion deposit binding was confirmed by histoblots with prion protein-specific monoclonal antibody 2D6. In the presence of nonradioactive IMPY, the binding of [125 I]IMPY was significantly inhibited in all regions studied. CONCLUSIONS: These findings indicate that IMPY can detect the prion deposits in vitro in scrapie-infected mice. Labeled with 123 I, this ligand may be useful to quantitate prion deposit burdens in TSEs by in vivo imaging.