RESUMO
OBJECTIVES: The aim of this study was to investigate the role of P2X7R (purinergic 2X7 receptor) and NLRP3 (NACHT, LRR, and PYD domains-containing protein 3) inflammasome activation in the process of pancreatic fibrosis in a mouse model of chronic pancreatitis (CP). METHODS: Chronic pancreatitis was induced by repeated intraperitoneal injections of 50 µg/kg cerulein for 6 weeks in mice. P2X7R antagonist oxidized ATP (OxATP) or brilliant blue G (BBG) was administered after the last cerulein injection for 2 weeks. Pancreatic chronic inflammation and fibrosis were evaluated by histological score, Sirius red staining, and alpha-smooth muscle actin immunohistochemical staining. We further determined pancreatic P2X7R, NLRP3, and caspase-1 expressions in gene and protein levels and the pancreatic concentrations of caspase-1, interleukin 1ß (IL-1ß), and IL-18. RESULTS: The pancreatic P2X7R, NLRP3, and caspase-1 expressions in gene and protein levels and the pancreatic concentrations of caspase-1, IL-1ß, and IL-18 were all reduced significantly in both the OxATP and BBG groups (P < 0.05). The pancreatic chronic inflammation and the fibrosis indices were all remarkably attenuated (P < 0.05). CONCLUSIONS: P2X7R antagonist OxATP and BBG significantly decreased pancreatic chronic inflammation and fibrosis in a mouse CP model and suggested that blockade of P2X7R-NLRP3 inflammasome signaling pathway may represent a novel therapeutic strategy for CP and its fibrotic process.
Assuntos
Modelos Animais de Doenças , Inflamassomos/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Pâncreas/metabolismo , Pancreatite Crônica/genética , Receptores Purinérgicos P2/genética , Animais , Caspase 1/genética , Caspase 1/metabolismo , Ceruletídeo , Citocinas/metabolismo , Fibrose , Expressão Gênica/efeitos dos fármacos , Humanos , Inflamassomos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Pâncreas/efeitos dos fármacos , Pâncreas/patologia , Pancreatite Crônica/induzido quimicamente , Pancreatite Crônica/metabolismo , Receptores Purinérgicos P2/metabolismo , Corantes de Rosanilina/farmacologiaRESUMO
In the study of the regulation of incubation, broodiness and laying performance in pigeons (Columba liva), a cDNA library, which was enriched with full-length brooding-related genes, was constructed by SMART LD-PCR techniques using the pituitary glands of incubating White King pigeons. The titers of optimal primary libraries were 1.54x10(6) pfu/mL and 1.80x10(6) pfu/mL and the titers of amplified libraries were 1.89x10(8) pfu/mL and 2.32x10(9 )pfu/mL. The percentages of recombinant clones of primary libraries and amplified libraries were all over 90%. A positive clone was sequenced and named ubiquitin based on the highly similar from other species. The fragment has the four initial codons of ATG, a termination codon of TAA and a signal sequence of AATAAA for adding the poly-A tail. The open reading frame of 918bp encodes 305 amino acids (NCBI accession number is EU981283). Recombinant pigeon ubiquitin protein was efficiently expressed with the form of insoluble inclusion bodies in E. coli BL21 transformed with a pET28a(+) expression vector containing the DNA sequence encoding mature pigeon ubiquitin. The molecular weight of expressed protein is the same as predicted size of approximately 35kD. To improve the efficiency of cloning full-length cDNA, strategies of RACE combined with cDNA library were used. The length of pigeons ubiquitin-conjugating enzyme gene obtained was 1263 bp containing a complete open reading frame of 435 bp that encodes 144 aa (NCBI accession number is EU914824). The results of this study not only provide a starting point for further study of ubiquitin function in pigeon species, but also provide a starting point for investigating the brooding mechanisms of pigeons.