RESUMO
AIM OF THE STUDY: Toona sinensis Roem. (Meliaceae; Toona sinensis; Chinese toon) is a type of arbor that is widely distributed in Asia. The fruits of Toona sinensis Roem has been traditionally recognized for treatment of cerebrovascular diseases. To evaluate the potential clinical use of the fruits of Toona sinensis Roem, we determined the dose dependence of the neuroprotective efficacy in a focal cerebral ischemic reperfusion model of rats and explored the underlying mechanisms. MATERIALS AND METHODS: Rats were subjected to occlusion of the middle cerebral artery (MCAO) by a nylon filament and treated with different doses (20mg/kg and 30 mg/kg) of n-butanol soluble fraction of the water extract of Chinese toon fruit or the vehicle for 1 week before induction of ischemia, s.i.d.. RESULTS: n-Butanol soluble fraction of the water extract of Chinese toon fruit reduced in a dose-dependent manner the ischemia-induced cerebral infarct and edema volume and attenuated neurological deficits observed at 6h point after ischemia. n-Butanol soluble fraction of the water extract of Chinese toon fruit reduced the levels of nitrate, nitrite, lipid peroxidation, cyclooxygenase-1, thromboxane in post-ischemic brain. n-Butanol soluble fraction of the water extract of Chinese toon fruit adjusted the elevation of the activity of glutathione peroxidase and superoxide dismutase in ischemic brain. CONCLUSIONS: The present study was the first evidence of effectiveness of n-butanol soluble fraction of the water extract of Chinese toon fruit in the rat stroke models, as it reduced infarct volume, inhibited the oxidative stress and inflammation.
Assuntos
Isquemia Encefálica/tratamento farmacológico , Frutas/química , Inflamação/tratamento farmacológico , Meliaceae/química , Estresse Oxidativo/efeitos dos fármacos , Extratos Vegetais/farmacologia , 1-Butanol/química , Animais , Epoprostenol/genética , Epoprostenol/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Glutationa Peroxidase/metabolismo , Inflamação/metabolismo , Masculino , Malondialdeído/metabolismo , Extratos Vegetais/química , Prostaglandina-Endoperóxido Sintases/genética , Prostaglandina-Endoperóxido Sintases/metabolismo , Ratos , Ratos Sprague-Dawley , Superóxido Dismutase/metabolismo , Tromboxano A2/genética , Tromboxano A2/metabolismo , Água/químicaRESUMO
Prostacyclin (PGI2) is a potent vasodilator and important mediator of vascular homeostasis; however, its clinical use is limited because of its short (<2-min) half-life. Thus, we hypothesize that the use of engineered endothelial progenitor cells (EPCs) that constitutively secrete high levels of PGI2 may overcome this limitation of PGI2 therapy. A cDNA encoding COX-1-10aa-PGIS, which links human cyclooxygenase-1 (COX-1) to prostacyclin synthase (PGIS), was delivered via nucleofection into outgrowth EPCs derived from rat bone marrow mononuclear cells. PGI2-secreting strains (PGI2-EPCs) were established by continuous subculturing of transfected cells under G418 selection. Genomic PCR, RT-PCR, and Western blot analyses confirmed the overexpression of COX-1-10aa-PGIS in PGI2-EPCs. PGI2-EPCs secreted significantly higher levels of PGI2 in vitro than native EPCs (P < 0.05) and showed higher intrinsic angiogenic capability; conditioned medium (CM) from PGI2-EPCs promoted better tube formation than CM from native EPCs (P < 0.05). Cell- and paracrine-mediated in vitro angiogenesis was attenuated when COX-1-10aa-PGIS protein expression was knocked down. Whole-cell patch-clamp studies showed that 4-aminopyridine-sensitive K(+) current density was increased significantly in rat smooth muscle cells (rSMCs) cocultured under hypoxia with PGI2-EPCs (7.50 ± 1.59 pA/pF; P < 0.05) compared with rSMCs cocultured with native EPCs (3.99 ± 1.26 pA/pF). In conclusion, we successfully created EPC strains that overexpress an active novel enzyme resulting in consistent secretion of PGI2. PGI2-EPCs showed enhanced intrinsic proangiogenic properties and provided favorable paracrine-mediated cellular protections, including promoting in vitro angiogenesis of native EPCs and hyperpolarization of SMCs under hypoxia.
Assuntos
Engenharia Celular/métodos , Endotélio Vascular/metabolismo , Epoprostenol/biossíntese , Epoprostenol/genética , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Células-Tronco/metabolismo , 4-Aminopiridina/metabolismo , Animais , Apoptose/genética , Processos de Crescimento Celular/genética , Meios de Cultivo Condicionados/metabolismo , Ciclo-Oxigenase 1/genética , Sistema Enzimático do Citocromo P-450/genética , DNA Complementar/genética , Endotélio Vascular/citologia , Epoprostenol/metabolismo , Meia-Vida , Hipóxia/genética , Hipóxia/metabolismo , Oxirredutases Intramoleculares/genética , Proteínas de Membrana/genética , Músculo Liso Vascular/citologia , Neovascularização Fisiológica , Fenótipo , Canais de Potássio/metabolismo , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transfecção/métodosRESUMO
Curcumin regulates prostaglandin (PG) synthesis in a variety of cells. PGE2 and PGI2 are generated from arachidonic acid (AA) by cyclooxygenases 1 and 2 (COX-1 and COX-2) and the synthase (PGES and PGI2S) pathways. This study evaluates the in vitro effect of curcumin on the expression of COX-1, COX-2, PGI2S and microsomal PGES-1 (mPGES-1), and the production of PGE2 and PGI2 in human coronary artery endothelial cells (HCAEC). HCAEC monolayers were incubated with curcumin and the expression of mRNA, protein and the production of PGI2 and PGE2 were quantified. Incubation of HCAEC with curcumin led to a time and concentration-dependent increases in COX-2 mRNA with a small but significant decrease in COX-1 mRNA expression. Curcumin also stimulated the expression of PGI2S and mPGES-1 mRNA. Although curcumin stimulated COX-2, PGI2S and mPGES-1 gene expression, it failed to increase PGI2 or PGE2 production. Interestingly, supplementation of the culture medium with AA increased prostanoid production by both quiescent and curcumin-treated cells. However, in comparison to the quiescent cells, the prostanoid production by curcumin-treated cells was markedly enhanced as AA concentrations in the medium were increased, and the enhanced prostanoid production was blocked by the presence of COX-2 specific inhibitor. Taken together, these results suggest that curcumin regulates prostanoid homeostasis in HCAEC by modulating multiple steps including the expression of COX-1, COX-2, PGI2S and mPGES-1.
Assuntos
Vasos Coronários/metabolismo , Curcumina/farmacologia , Ciclo-Oxigenase 1/biossíntese , Ciclo-Oxigenase 2/biossíntese , Dinoprostona/biossíntese , Células Endoteliais/metabolismo , Epoprostenol/biossíntese , Ácido Araquidônico/farmacologia , Células Cultivadas , Vasos Coronários/citologia , Vasos Coronários/efeitos dos fármacos , Ciclo-Oxigenase 1/genética , Ciclo-Oxigenase 1/metabolismo , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Inibidores de Ciclo-Oxigenase 2/farmacologia , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Dinoprostona/antagonistas & inibidores , Dinoprostona/genética , Dinoprostona/metabolismo , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Epoprostenol/antagonistas & inibidores , Epoprostenol/genética , Epoprostenol/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Oxirredutases Intramoleculares/genética , Oxirredutases Intramoleculares/metabolismo , Prostaglandina-E SintasesRESUMO
To better define the role of the various prostanoid synthases in the adjuvant-induced arthritis (AIA) model, we have determined the temporal expression of the inducible PGE synthase (mPGES-1), mPGES-2, the cytosolic PGES (cPGES/p23), and prostacyclin synthase, and compared with that of cyclooxygenase-1 (COX-1) and COX-2. The profile of induction of mPGES-1 (50- to 80-fold) in the primary paw was similar to that of COX-2 by both RNA and protein analysis. Quantitative PCR analysis indicated that induction of mPGES-1 at day 15 was within 2-fold that of COX-2. Increased PGES activity was measurable in membrane preparations of inflamed paws, and the activity was inhibitable by MK-886 to >or=90% with a potency similar to that of recombinant rat mPGES-1 (IC(50) = 2.4 microM). The RNA of the newly described mPGES-2 decreased by 2- to 3-fold in primary paws between days 1 and 15 postadjuvant. The cPGES/p23 and COX-1 were induced during AIA, but at much lower levels (2- to 6-fold) than mPGES-1, with the peak of cPGES/p23 expression occurring later than that of COX-2 and PGE(2) production. Prostacyclin (measured as 6-keto-PGF(1alpha)) was transiently elevated on day 1, and prostacyclin synthase was down-regulated at the RNA level after day 3, suggesting a diminished role of prostacyclin during the maintenance of chronic inflammation in the rat AIA. These results show that mPGES-1 is up-regulated throughout the development of AIA and suggest that it plays a major role in the elevated production of PGE(2) in this model.
Assuntos
Artrite Experimental/enzimologia , Dinoprostona/biossíntese , Oxirredutases Intramoleculares/biossíntese , Isoenzimas/fisiologia , Microssomos/enzimologia , Prostaglandina-Endoperóxido Sintases/fisiologia , Regulação para Cima , Adjuvantes Imunológicos/administração & dosagem , Animais , Antígenos de Bactérias/administração & dosagem , Artrite Experimental/imunologia , Artrite Experimental/patologia , Ciclo-Oxigenase 2 , Citosol/efeitos dos fármacos , Citosol/enzimologia , Dinoprostona/genética , Modelos Animais de Doenças , Edema/enzimologia , Edema/patologia , Epoprostenol/biossíntese , Epoprostenol/genética , Membro Posterior , Indóis/farmacologia , Injeções Intradérmicas , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/enzimologia , Oxirredutases Intramoleculares/genética , Isoenzimas/genética , Microssomos/efeitos dos fármacos , Mycobacterium/imunologia , Antagonistas de Prostaglandina/biossíntese , Antagonistas de Prostaglandina/farmacologia , Prostaglandina-E Sintases , Prostaglandina-Endoperóxido Sintases/genética , RNA Mensageiro/biossíntese , Ratos , Ratos Sprague-Dawley , Regulação para Cima/genética , Regulação para Cima/imunologiaRESUMO
Antithrombin (AT) supplementation in patients with severe sepsis has been shown to improve organ failures in which activated leukocytes are critically involved. However, the precise mechanism(s) for the therapeutic effects of AT is not well understood. We examined in rats whether AT reduces ischemia/reperfusion (I/R)-induced renal injury by inhibiting leukocyte activation. AT markedly reduced the I/R-induced renal dysfunction and histologic changes, whereas neither dansyl glutamylglycylarginyl chloromethyl ketone-treated factor Xa (DEGR-F.Xa), a selective inhibitor of thrombin generation, nor Trp49-modified AT, which lacks affinity for heparin, had any effect. Renal tissue levels of 6-keto-PGF(1 alpha), a stable metabolite of prostacyclin (PGI(2)), increased after renal I/R. AT enhanced the I/R-induced increases in renal tissue levels of 6-keto-PGF(1 alpha), whereas neither DEGR-F.Xa nor Trp49-modified AT had any effect. AT significantly inhibited I/R-induced decrease in renal tissue blood flow and the increase in the vascular permeability. Ischemia/reperfusion-induced increases in renal tissue levels of tumor necrosis factor-alpha, cytokine-induced neutrophil chemoattractant, and myeloperoxidase were significantly inhibited in animals given AT. Pretreatment of animals with indomethacin reversed the effects induced by AT. Iloprost, an analog of PGI(2), produced effects similar to those induced by AT. These observations strongly suggest that AT reduces the I/R-induced renal injury by inhibiting leukocyte activation. The therapeutic effects of AT might be mainly mediated by PGI(2) released from endothelial cells through interaction of AT with cell surface glycosaminoglycans.