Bone protective effect of sinomenine against monosodium iodoacetate induced knee and hip injury in rat model: an inflammatory pathway.

PURPOSE: Osteoarthritis (OA) is a degenerative joint disease which is categorized via destruction of joint cartilage and it also affects the various joints, especially knees and hips. Sinomenine active phytoconstituents isolated from the stem of Sinomenium acutum and already proof anti-inflammatory effect against the arthritis model of rodent. In this experimental protocol, we scrutinized the anti-osteoarthritis effect of sinomenine against monosodium iodoacetate (MIA) induced OA in rats. METHODS: MIA (3 mg/50 µL) was used for inducing the OA in the rats, and rats received the oral administration of sinomenine (2.5, 5 and 7.5 mg/kg body weight) up to the end of the experimental study (four weeks). The body and organs weight were estimated. Aggrecan, C-terminal cross-linked telopeptide of type II collagen (CTX-II), glycosaminoglycans (GCGs), monocyte chemoattractant protein-1 (MCP-1), Interferon gamma (IFN-γ), antioxidant, inflammatory cytokines, inflammatory mediators and matrix metalloproteinases (MMP) were analyzed. RESULTS: Sinomenine significantly (P < 0.001) boosted the body weight and reduced the heart weight, but the weight of spleen and kidney remain unchanged. Sinomenine significantly (P < 0.001) reduced the level of nitric oxide, MCP-1 and improved the level of aggrecan, IFN-γ and GCGs. Sinomenine remarkably upregulated the level of glutathione, superoxide dismutase and suppressed the level of malonaldehyde. It effectually modulated the level of inflammatory cytokines and inflammatory mediators and significantly (P < 0.001) reduced the level of MMPs, like MMP-1, 2, 3, 9 and 13. CONCLUSIONS: Sinomenine is a beneficial active agent for the treatment of OA disease.

The blockade of kappa opioid receptors exacerbates alveolar bone resorption in rats.

OBJECTIVES: Bone resorption associated to chronic diseases, such as arthritis and periodontitis, results from exacerbated immuno-inflammatory host response that leads to tissue breakdown. The significance of opioid pathways as endogenous modulators of inflammatory events has already been described. Thus, the aim of this work is to determine whether some of the main three opioid receptors are endogenously activated to prevent bone loss during experimentally-induced alveolar bone resorption. DESIGN: This study used an experimental model of alveolar bone resorption induced by ligature in rats. A silk thread was placed around the 2nd maxillary molar of male Wistar rats. In the 3rd, 4th and 5th day after ligation the rats received a local injection of different concentrations of opioid antagonists Cyprodime, Naltrindole, or Nor-binaltorphimine, which specifically block mü, delta and kappa opioid receptors, respectively. In the 7th experimental day, rats were euthanized and their maxillae collected for evaluation of alveolar bone and fiber attachment loss, morphometric counting of osteoclasts and osteoblasts, as well as the levels of cytokines IL-1ß, IFN-γ, and IL-6 by ELISA. RESULTS: Selective antagonism of kappa opioid receptors, but not mü and delta, exacerbated alveolar bone resorption induced by ligature in rats. The increased bone loss associated with higher number of osteoclasts surrounding alveolar bone, although osteoblasts' counting remained unchanged. The concentrations of IL-1ß and IL-6 in periodontal tissues were also significantly higher in the rats treated with the kappa antagonist. CONCLUSION: Inhibiting kappa opioid receptors exacerbates alveolar bone resorption.

Milonine, an Alkaloid of Cissampelos sympodialis Eichl. (Menispermaceae) Inhibits Histamine Release of Activated Mast Cells.

Milonine is an alkaloid of Cissampelos sympodialis Eichl. (Menispermaceae), a plant used in the northeast of Brazil to treat allergies such as asthma, rhinitis, and other conditions. Previously, several alkaloids were isolated from its roots and leaves with pharmacological properties in asthma and acute inflammation models. Therefore, the aim of this study was to evaluate the milonine effect on mast cells degranulation in vivo and in vitro. Swiss mice (n = 8) were used in models of paw edema induced by carrageenan, compound 48/80, or histamine. One hour before challenge, the animals were treated with milonine (at different doses) or standard drugs and, at different time points, the edema formation was measured. In addition, other different methods, such as anaphylactic shock reaction and scratching behavior models both induced by compound 48/80, a mast cell degranulator, were used to assess milonine effect histamine release in vivo. Moreover, milonine effect on mast cell degranulation in vitro was also carried out. Firstly, it was observed that milonine significantly decreased the carrageenan edema formation only at the beginning of the reaction (i.e., up to 2 h after challenge). Furthermore, this alkaloid decreased the edema induced by compound 48/80, maintained the paw tissue integrity, without modulating histamine-induced paw edema. In anaphylactic shock reaction, milonine increased the time of animal survival when compared with compound 48/80 group. Milonine also significantly decreased the scratching behavior induced by compound 48/80 with decreasing of mast cell degranulation in vitro. Therefore, these data indicated that milonine presents anti-allergic properties by decreasing mast cell degranulation rather than acting on histamine effect.

Milonine, a Morphinandienone Alkaloid, Has Anti-Inflammatory and Analgesic Effects by Inhibiting TNF-α and IL-1ß Production.

Milonine is a morphinandienone alkaloid from Cissampelos sympodialis Eichl (Menispermaceae), a plant used in Brazil to treat inflammatory disorders. In this study, we evaluated the anti-inflammatory and analgesic activity of milonine (MIL) by using classical experimental models of inflammation and nociception. The results showed that MIL reduced the paw edema formation induced by lipopolysaccharide, prostaglandin E2, and bradykinin, without interfering with the serotonin-induced edema. With respect to the nociception experiments, MIL decreased the exudate into the peritoneum induced by acetic acid, maintaining the tissue morphology. The alkaloid was able to inhibit the peritonitis induced by carrageenan, decreasing mainly the migration of polymorphonuclear cells, without altering the mononuclear cell number, and reduced the levels of TNF-α and IL-1ß in the peritoneum. In addition, MIL was able to decrease the frequency of abdominal writhing induced by acetic acid but did not increase the latency time of the animals in the hot plate test. MIL significantly reduced the nociceptive behavior of paw licking induced by formalin only at the second phase of the test. In conclusion, we demonstrate that milonine has anti-inflammatory and anti-nociceptive activities by inhibiting mediators essential for the inflammatory process.

Effect of sinomenine on the expression of rheumatoid arthritis fibroblast-like synoviocytes MyD88 and TRAF6.

The effect of sinomenine (SIN) on the toll-like receptor (TLR) signal transduction pathway as well as the expression of myeloid differentiation factor 88 (MyD88) and tumor necrosis factor (TNF) receptor-associated factor-6 (TRAF6) was investigated. SIN inhibition of rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) proliferation and RA cartilage and subchondral bone destruction was also investigated. RA-FLS were cultured in vitro and the intracellular alkaline phosphatase (ALP) activity was determined in order to obtain the optimal drug concentration. The rate of cell proliferation was determined. Fluorescence quantitative polymerase chain reaction (PCR) was applied to determine the MyD88 and TRAF-6 gene expression and western blot was used to detect the MyD88 and TRAF-6 protein expression. The ALP activity in the SIN groups was lower than that in the control group, among which the 0.5 mM SIN group had the lowest ALP activity (P < 0.01). The rate of RA-FLS proliferation detected by CCK-8 assay in the 0.5-mM SIN group was lower than that in the control group (P < 0.01) and was the highest 4 days after SIN induction. Gene and protein expression of MyD88 and TRAF-6 were downregulated significantly in the 0.5-mM SIN group compared to that in the control group (P < 0.01). SIN effectively inhibited MyD88 and TRAF-6 expression in RA-FLS, which may be one of the important molecular mechanisms involved in RA treatment and prevention of cartilage and subchondral bone destruction.

Opioid-like antinociceptive effects of oral administration of a lectin purified from the seeds of Canavalia brasiliensis.

The objective of this study was to evaluate the antinociceptive effects of a lectin from Canavalia brasiliensis (ConBr) when administered orally to murine models of chemical and thermal nociception. ConBr up to 100 mg/kg produced significant and dose-dependent antinociceptive effects: 81% reduction in abdominal writhing induced by 0.6% acetic acid; 26 and 52% reduction in early- and late-stage paw licking, respectively, induced by 2.5% formalin; and 155% increase in reaction latency (heightened thermal pain threshold). In all models, the antinociceptive effect was reversed by the lectin-binding carbohydrate α-d-methyl-mannoside and by the nonselective opioid antagonist naloxone. The antinociceptive effect observed in the formalin test was inhibited by the δ-selective antagonist naltrindole and the κ-selective antagonist nor-binaltorphimine but not by the µ-selective antagonist cyprodime. In conclusion, when administered orally to Swiss mice, the ConBr lectin displayed antinociceptive activity, both peripheral and central, mediated by the opioid system and involving δ-and κ-receptors and the lectin domain.

Early role of the κ opioid receptor in ethanol-induced reinforcement.

Effects of early ethanol exposure on later ethanol intake emphasize the importance of understanding the neurobiology of ethanol-induced reinforcement early in life. Infant rats exhibit ethanol-induced appetitive conditioning and ethanol-induced locomotor activation, which have been linked in theory and may have mechanisms in common. The appetitive effects of ethanol are significantly modulated by µ and δ opioid receptors, whereas µ but not δ receptors are involved in the motor stimulant effects of ethanol during early development. The involvement of the κ opioid receptor (KOR) system in the motivational effects of ethanol has been much less explored. The present study assessed, in preweanling (infant) rats, the modulatory role of the KOR system in several paradigms sensitive to ethanol-induced reinforcement. Kappa opioid activation and blockade were examined in second-order conditioned place preference with varied timing before conditioning and with varied ethanol doses. The role of KOR on ethanol-induced locomotion and ethanol-induced taste conditioning was also explored. The experiments were based on the assumption that ethanol concurrently induces appetitive and aversive effects and that the latter may be mediated by activation of kappa receptors. The main result was that blockade of kappa function facilitated the expression of appetitive ethanol reinforcement in terms of tactile and taste conditioning. The effects of kappa activation on ethanol conditioning seemed to be independent from ethanol's stimulant effects. Kappa opioid activation potentiated the motor depressing effects of ethanol but enhanced motor activity in control subjects. Overall, the results support the hypothesis that a reduced function of the KOR system in nondependent subjects should attenuate the aversive consequences of ethanol.

A validated method for the simultaneous quantitation of bioactive alkaloid markers in the leaf ethanolic extract of Cissampelos sympodialis Eichl.: a phenological variation study.

INTRODUCTION: The leaf hydroalcoholic extract of Cissampelos sympodialis Eichl. (Menispermaceae) has shown promising activity in different animal models of asthma. Several alkaloids have been identified in the extract, including warifteine and methylwarifteine (bisbenzylisoquinoline), as well as milonine (morphinandienone). OBJECTIVE: To develop and validate an analytical method for the simultaneous quantitation of the bioactive markers of C. sympodialis hydroalcoholic leaf extract and to apply the method to a seasonal (phenological) study of the concentration of the alkaloid markers. METHODOLOGY: The method used reversed phase high performance liquid chromatography with UV detection and calibration by standard addition. Separation was achieved using a C18-column (250 × 4.6 mm, 5 µm) and a mobile phase consisting of a mixture of 0.05% aqueous (Et)3NH2 (A):MeOH(B) in gradient mode at a flow rate of 1.0 mL/min. RESULTS: The method proved to be linear in the concentration range tested (2-100 µg/mL, r² > 0,99), precise (RSD ≤ 15%), accurate (85-115%), selective and robust. Detection limits for warifteine, methyl-warifteine and milonine were 0.39, 1.10 and 1.77 µg/mL respectively. The highest concentration of total alkaloids (determined as the sum of the three alkaloids) in the hydroalcoholic extract of the leaves was 2.9 ± 0.2 mg/g extract (n = 3), prior to fruit development. Both warifteine and methylwarifteine were detected in the total alkaloid fraction of the ripened fruits. CONCLUSION: The results demonstrated that significant variations in the concentration of the biomarkers occurred throughout the vegetative cycle. The lowest concentration of the alkaloids in the leaves coincided with their appearance in the ripened fruits.

Cardiovascular effects elicited by milonine, a new 8,14-dihydromorphinandienone alkaloid.

The mechanisms underlying the cardiovascular responses evoked by milonine (i.v.), an alkaloid, were investigated in rats. In normotensive rats, milonine injections produced hypotension and tachycardia, which were attenuated after N(w) -nitro-L-arginine methyl esther (L-NAME; 20 mg/kg, i.v.). In phenylephrine (10 µM), pre-contracted mesenteric artery rings, milonine (10⁻¹° M to 3 × 10⁻4 M) caused a concentration-dependent relaxation (EC50 = 1.1 × 10⁻6 M, E(max) = 100 ± 0.0%) and this effect was rightward shifted after either removal of the vascular endothelium (EC50 = 1.6 × 10⁻5, p < 0.001), or after L-NAME 100 µM (EC50 = 6.2 × 10⁻5, p < 0.001), hydroxocobalamin 30 µM (EC50 = 1.1 × 10⁻4, p < 0.001) or ODQ 10 µM (EC50 = 1.9 × 10⁻4 p < 0.001). In addition, in rabbit aortic endothelial cells, milonine increased NO3⁻ levels. The relaxant effect induced by milonine was attenuated in the presence of KCl (20 mM), a modulator efflux K(+) (EC50 = 1.2 × 10⁻5, p < 0.001), or different potassium channel blockers such as glibenclamide (10 µM) (EC50 = 6.3 × 10⁻5, p < 0.001), TEA (1 mM) (EC50 = 2.3 × 10⁻5 M, n = 6) or Charybdotoxin (0.2 µM) plus apamin (0.2 µM) (EC50 = 3.9 × 10⁻4 M, n = 7). In addition, pre-contraction with high extracellular potassium concentration prevented milonine-induced vasorelaxation (EC50 = 1.0 × 10⁻4, p < 0.001). Milonine also reduced CaCl2 -induced contraction in Ca²(+) -free solution containing KCl (60 mM). In conclusion, using combined functional and biochemical approaches, we demonstrated that the hypotensive and vasorelaxant effects produced by milonine are, at least in part, mediated by the endothelium, likely via nitric oxide release, activation of nitric oxide-cGMP pathway and opening of K(+) channels.

QSAR study on the antinociceptive activity of some morphinans.

Quantitative structure-activity relationship studies were performed to describe and predict the antinociceptive activity of 31 morphinan derivatives reported by the US Drug Evaluation Committee in 2005 and 2006. From these, three data sets were constructed and several models were calculated following the multiple linear regression and Leave-One-Out Cross-Validation (LOO-CV) tests. In general, these models achieved good descriptive power (approximately 92%) as well as predictive power (approximately 76%), but were unable to predict an external validation set of morphinan derivatives. When artificial neural networks were applied to these models, an improvement of the predictive and external validation values was obtained. It was observed that the results of the NN models are significantly better that those obtained by multiple linear regression. In spite that the problem under investigation can be handled adequately by a linear model, a neural network does bring slight improvements in the predictive power.

Bloqueo combinado de los nervios ciático y femoral para la analgesia postoperatoria en el reemplazo total de rodilla / Combined sciatic and femoral nerves block for postoperative analgesia in toal knee replacement

Objetivos: Comparar la calidad y duración de la analgesia postoperatoria en pacientes programados para cirugía de reemplazo total de rodilla unilateral (RTR) luego de anestesia subaracnoidea (AS), anestesia subaracnoidea y bloqueo femoral (BF), y anestesia subaracnoidea y bloqueo femoral combinado con un bloqueo del nervio ciático por la vía parasacra (BFPS). Métodos: Estudiamos en forma prospectiva, randomizada y ciego simple a 44 pacientes programados para cirugía de RTR. Todos recibieron una anestesia subaracnoidea. A los pacientes de los grupos BF y BFPS se les realizó un bloqueo del nervio femoral previo al bloqueo subaracnoideo. Concluído el procedimiento quirúrgico, a los pacientes del grupo BFPS se les practicó un bloqueo del nervio ciático por vía parasacra. Se midió el tiempo transcurrido hasta el primer pedido de analgésicos, la cantidad total de pedidos y el consumo acumulado de meperidina durante las primeras 48 horas postoperatorias. En los grupos BF y BFPS se midió el dolor con Escala Visual Análoga (EVA) al momento de la resolución total del bloqueo central y a las 4, 8 Y 24 horas. Un tercer grupo (AS) fue conformado en forma retrospectiva para control y comparación de los resultados. Resultados: El tiempo transcurrido hasta el primer pedido de opioides se prolongó en forma significativa (p < 0.001) en el grupo BFPS, siendo mayor que en los otros dos grupos. El consumo de opioides durante las primeras 18 h fue significativa mente menor (p < 0.001) en el grupo BFPS que en los grupos AS y BF. El consumo de opioides fue significativamente superior en el grupo AS durante las primeras 24 horas pero similar al BF y BFPS luego. Los valores de EVA al disiparse el bloqueo central y a las 4, 8 Y 24 horas fueron significativamente menores en el grupo BNFPS que en el BF. Conclusión: El agregado de un bloqueo del nervio ciático por vía parasacra al bloqueo femoral luego del reemplazo total de rodilla unilateral resultó en una analgesia postoperatoria mejor y más prolongada, y un menor consumo de opioides durante las primeras 24 horas en comparación con los otros grupos estudiados. (AU)

Bloqueo combinado de los nervios ciático y femoral para la analgesia postoperatoria en el reemplazo total de rodilla / Combined sciatic and femoral nerves block for postoperative analgesia in toal knee replacement

Objetivos: Comparar la calidad y duración de la analgesia postoperatoria en pacientes programados para cirugía de reemplazo total de rodilla unilateral (RTR) luego de anestesia subaracnoidea (AS), anestesia subaracnoidea y bloqueo femoral (BF), y anestesia subaracnoidea y bloqueo femoral combinado con un bloqueo del nervio ciático por la vía parasacra (BFPS). Métodos: Estudiamos en forma prospectiva, randomizada y ciego simple a 44 pacientes programados para cirugía de RTR. Todos recibieron una anestesia subaracnoidea. A los pacientes de los grupos BF y BFPS se les realizó un bloqueo del nervio femoral previo al bloqueo subaracnoideo. Concluído el procedimiento quirúrgico, a los pacientes del grupo BFPS se les practicó un bloqueo del nervio ciático por vía parasacra. Se midió el tiempo transcurrido hasta el primer pedido de analgésicos, la cantidad total de pedidos y el consumo acumulado de meperidina durante las primeras 48 horas postoperatorias. En los grupos BF y BFPS se midió el dolor con Escala Visual Análoga (EVA) al momento de la resolución total del bloqueo central y a las 4, 8 Y 24 horas. Un tercer grupo (AS) fue conformado en forma retrospectiva para control y comparación de los resultados. Resultados: El tiempo transcurrido hasta el primer pedido de opioides se prolongó en forma significativa (p < 0.001) en el grupo BFPS, siendo mayor que en los otros dos grupos. El consumo de opioides durante las primeras 18 h fue significativa mente menor (p < 0.001) en el grupo BFPS que en los grupos AS y BF. El consumo de opioides fue significativamente superior en el grupo AS durante las primeras 24 horas pero similar al BF y BFPS luego. Los valores de EVA al disiparse el bloqueo central y a las 4, 8 Y 24 horas fueron significativamente menores en el grupo BNFPS que en el BF. Conclusión: El agregado de un bloqueo del nervio ciático por vía parasacra al bloqueo femoral luego del reemplazo total de rodilla unilateral resultó en una analgesia postoperatoria mejor y más prolongada, y un menor consumo de opioides durante las primeras 24 horas en comparación con los otros grupos estudiados.