RESUMO
Endogenous cannabinoids and peripheral cannabinoid CB2 receptors (CB2Rs) are involved in the antinociceptive effect of electroacupuncture (EA) on inflammatory pain. However, it is not clear how CB2R activation contributes to the antinociceptive effect of EA. The major proinflammatory cytokines, such as tumour necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß) and IL-6, are involved in inflammatory pain. Here we determined the effects of CB2R activation and EA on the expression level of IL-1ß, IL-6 and TNF-α in inflamed skin tissues. Inflammatory pain was induced by injection of complete Freund's adjuvant into the left hindpaw of rats. Thermal hyperalgesia was tested with a radiant heat stimulus, and mechanical allodynia was quantified using von Frey filaments. The mRNA and protein levels of IL-1ß, IL-6 and TNF-α in inflamed skin tissues were measured using real-time polymerase chain reaction and Western blot, respectively. Local injection of the selective CB2R agonist AM1241 or EA applied to GB30 and GB34 significantly reduced thermal hyperalgesia and mechanical allodynia induced by tissue inflammation. The specific CB2R antagonist AM630 significantly attenuated the antinociceptive effect of EA. Furthermore, EA or AM1241 treatment significantly decreased the mRNA and protein levels of IL-1ß, IL-6 and TNF-α in inflamed skin tissues. In addition, pretreatment with AM630 significantly reversed the inhibitory effect of EA on these cytokine levels in inflamed skin tissues. Our results suggest that EA reduces inflammatory pain and proinflammatory cytokines in inflamed skin tissues through activation of CB2Rs.
Assuntos
Eletroacupuntura , Hiperalgesia/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Manejo da Dor/métodos , Receptor CB2 de Canabinoide/metabolismo , Pele/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Canabinoides/farmacologia , Hiperalgesia/induzido quimicamente , Indóis/farmacologia , Inflamação/metabolismo , Interleucina-1beta/genética , Interleucina-6/genética , Masculino , Medição da Dor , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor CB2 de Canabinoide/agonistas , Receptor CB2 de Canabinoide/antagonistas & inibidores , Pele/efeitos dos fármacos , Fator de Necrose Tumoral alfa/genéticaRESUMO
Galectins can recognize and specifically bind to ß-galactoside residues, playing crucial roles in innate immune responses of vertebrates and invertebrates. We cloned the cDNA of a tandem-repeat galectin from the pearl oyster Pinctada fucata (designated as PoGal2). PoGal2 cDNA is 1347 bp long and consists of a 5'-untranslated region (UTR) of 3 bp, a 3'-UTR of 297 bp with one cytokine RNA instability motif (ATTTA), and an open reading frame of 1047 bp, encoding a polypeptide of 349 amino acids, with an estimated molecular mass of 38.1 kDa and a theoretical isoelectric point of 8.5. PoGal2 contains two carbohydrate recognition domains (CRDs); both have the conserved carbohydrate-binding motifs H-NPR and WG-EE. PoGal2 shares 50.6 and 50.9% identity with those of abalone (Haliotis discus) and the Manila clam (Venerupis philippinarum), respectively. Phylogenetic analysis revealed that the tandem-repeat galectins formed two clades for the different species. Molluscan tandem-repeat galectins were clustered into a single clade, and nematode tandem-repeat galectins were clustered into another single clade. In both clades, CRD-N and CRD-C were divided into different groups. PoGal2 mRNA was constitutively expressed in all tissues analyzed, and the expression level of PoGal2 mRNA was found to be significantly up-regulated in digestive glands, gills and hemocytes after Vibrio alginolyticus stimulation/infection. Expression profile analysis showed that the expression level of PoGal2 mRNA was significantly up-regulated at 8, 12 and 24 h after V. alginolyticus infection. These results suggest that PoGal2 is a constitutive and inducible acute-phase protein involved in the innate immune response of pearl oysters.
Assuntos
Galectinas/genética , Pinctada/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar , Etiquetas de Sequências Expressas , Galactosídeos/metabolismo , Galectinas/química , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Biblioteca Gênica , Imunidade Inata , Dados de Sequência Molecular , Filogenia , Pinctada/classificação , RNA Mensageiro/biossíntese , Alinhamento de Sequência , Análise de Sequência de DNA , Sequências de Repetição em TandemRESUMO
Two new plasmids pNQ216 (4.1kp) and pNQ402 (2.8 kb) were constructed by combining the replication origin of the plasmid pNK289, a cryptic plasmid resides in B. pumilus 289, and the cat-86 gene from plasmid pPL601. These two plasmids can be maintained steadily in both B. subtilis and B. pumilus. The penetrance of these plasmids on LB medium with Cm (20 micrograms/ml) is 30% higher than that of pPL600. Thus both of these plasmids can be used as new cloning vectors in Bacillus.
