Antioxidants and cardioprotective effects of ethyl acetate fraction of Canavalia rosea leaves in myocardial ischemia-reperfusion injury.

Different degrees in the biological activities of Canavalia rosea had been previously reported . In this study, our group assessed the cardioprotective effects of the ethyl acetate fraction (EAcF) of the Canavalia rosea leaves. Firstly, it was confirmed, by in vitro approach, that the EAcF has high antioxidant properties due to the presence of important secondary metabolites, as flavonoids. In order to explore their potential protector against cardiovascular disorders, hearts were previously perfused with EAcF (300 µg.mL-1) and submitted to the global ischemia followed by reperfusion in Langendorff system. The present findings have demonstrated that EAcF restored the left ventricular developed pressure and decreased the arrhythmias severity index. Furthermore, EAcF significantly increased the glutathiones peroxidase activity with decreased malondialdehyde and creatine kinase levels. EAcF was effective upon neither the superoxide dismutase, glutationes reductase nor the catalase activities. In addition, the Western blot analysis revealed that ischemia-reperfusion injury significantly upregulates caspase 3 protein expression, while EAcF abolishes this effect. These results provide evidence that the EAcF reestablishes the cardiac contractility and prevents arrhythmias; it is suggested that EAcF could be used to reduce injury caused by cardiac reperfusion. However more clinical studies should be performed, before applying it in the clinic.

Schinus terebinthifolius Raddi (Brazilian pepper) leaves extract: in vitro and in vivo evidence of anti-inflammatory and antioxidant properties.

The aim of this work was to evaluate the anti-inflammatory and antioxidant effects of ethyl acetate extract obtained from the leaves of Brazilian peppertree Schinus terebinthifolius Raddi (EAELSt). Total phenols and flavonoids, chemical constituents, in vitro antioxidant activity (DPPH and lipoperoxidation assays), and cytotoxicity in L929 fibroblasts were determined. In vivo anti-inflammatory and antioxidant properties were evaluated using TPA-induced ear inflammation model in mice. Phenol and flavonoid contents were 19.2 ± 0.4 and 93.8 ± 5.2 of gallic acid or quercetin equivalents/g, respectively. LC-MS analysis identified 43 compounds, of which myricetin-O-pentoside and quercetin-O-rhamnoside were major peaks of chromatogram. Incubation with EAELSt decreased the amount of DPPH radical (EC50 of 54.5 ± 2.4 µg/mL) and lipoperoxidation at 200-500 µg/mL. The incubation with EAELSt did not change fibroblast viability up to 100 µg/mL. Topical treatment with EAELSt significantly reduced edema and myeloperoxidase activity at 0.3, 1, and 3 mg/ear when compared to the vehicle-treated group. In addition, EAELSt decreased IL-6 and TNF-α levels and increased IL-10 levels. Besides, it modulated markers of oxidative stress (reduced total hydroperoxides and increased sulfhydryl contents and ferrium reduction potential) and increased the activity of catalase and superoxide dismutase, without altering GPx activity.

Omega-3 pleiad: The multipoint anti-inflammatory strategy.

Omega 3 (ω3) fatty acids have been described since the 1980s as promising anti-inflammatory substances. Prostaglandin and leukotriene modulation were exhaustively explored as the main reason for ω3 beneficial outcomes. However, during the early 2000s, after the human genome decoding advent, the nutrigenomic approaches exhibited an impressive plethora of ω3 targets, now under the molecular point of view. Different G protein-coupled receptors (GPCRs) recognizing ω3 and its derivatives appear to be responsible for blocking inflammation and insulin-sensitizing effects. A new class of ω3-derived substances, such as maresins, resolvins, and protectins, increases ω3 actions. Inflammasome disruption, the presence of GPR120 on immune cell surfaces, and intracellular crosstalk signaling mediated by PPARγ compose the last discoveries regarding the multipoint anti-inflammatory targets for this nutrient. This review shows a detailed mechanistic proposal to understand ω3 fatty acid action over the inflammatory environment in the background of several chronic diseases.

Reduced cutaneous inflammation associated with antioxidant action after topical application of the aqueous extract of Annona muricata leaves.

Annona muricata L. is used in folk medicine for treatment of diseases related to inflammatory and oxidative processes. This study investigated the effect of the aqueous extract of A. muricata leaves (AEAM) on TPA-induced ear inflammation and antioxidant capacity, both in vitro and in vivo. The in vitro antioxidant capacity of AEAM was measured by the 2,2-diphenyl-1-picrylhydrazyl (DPPH), ferric reducing/antioxidant power (FRAP) and lipoperoxidation assays. Cytotoxicity and reactive oxygen species (ROS) release were evaluated in the L929 fibroblasts. Swiss mice were submitted to TPA application and were topically treated with AEAM (0.3, 1 or 3 mg/ear). After 6 h, inflammatory and oxidative parameters were evaluated. Quercetin 3-glucoside, rutin, chlorogenic acid, catechin and gallic acid were identified in AEAM. It also presented antioxidant activity in all in vitro assays used. Incubation with AEAM did not cause cell cytotoxicity but reduced ROS release from fibroblasts. Compared with the control group, treatment with AEAM significantly reduced ear oedema and mieloperoxidase activity in inflamed ears, as well as histological parameters of inflammation. These results were associated with the reduction of total hydroperoxides and modulation of catalase, but not superoxide dismutase activity. These findings show the anti-inflammatory effect of AEAM is associated with antioxidant capacity.

Modulation of exercise-induced muscular damage and hyperalgesia by different 630 nm doses of light-emitting diode therapy (LEDT) in rats.

We compared the acute effects of different doses of 630 nm light-emitting diode therapy (LEDT) on skeletal muscle inflammation and hyperalgesia in rats submitted to exercise-induced muscle damage (EIMD). Wistar rats were divided into five experimental groups (n = 5-8/group): sedentary control (CON); exercise + passive recovery (PR); and exercise + LEDT (1.2 J/cm2, 1.8 J; 4.2 J/cm2, 6.3 J; 10.0 J/cm2, 15 J). After 100 min of swimming, the rats in the LEDT groups were exposed to phototherapy on the triceps surae muscle. For mechanical hyperalgesia evaluation, paw withdrawal threshold was assessed before and 24 h after swimming. Immediately after hyperalgesia tests, blood samples were collected to analyze creatine kinase (CK) activity and the soleus muscle was removed for histological and tumor necrosis factor (TNF)-α immunohistological analyses. In all LEDT groups, plasma CK activity was reduced to levels similar to those measured in the CON group. Paw withdrawal threshold decreased in the PR group (- 11.9 ± 1.9 g) when compared to the CON group (2.2 ± 1.5 g; p < 0.01) and it was attenuated in the group LEDT 4.2 J/cm2 (- 3.3 ± 2.4 g, p < 0.05). Less leukocyte infiltration and edema and fewer necrotic areas were found in histological sections of soleus muscle in LEDT (4.2 J/cm2) and LEDT (10.0 J/cm2) groups compared to the PR group. Also, LEDT (4.2 J/cm2) and LEDT (10.0 J/cm2) groups showed less immunostaining for TNF-α in macrophages or areas with necrosis of muscle fibers compared to the PR group. LEDT (4.2 J/cm2, 6.3 J)-reduced muscle inflammation and nociception in animals submitted to EIMD.

Medicinal Plants in the Treatment of Colitis: Evidence from Preclinical Studies.

Ulcerative colitis is a chronic inflammatory condition whose treatment includes aminosalicylates, corticosteroids, and immunomodulators. Medicinal plants seem to be an important alternative treatment for this condition. They have been the subject of a great number of studies in recent years. This study was conducted to systematically review the medicinal plants tested in experimental models of ulcerative colitis. We conducted a systematic literature search through specialized databases (PUBMED, SCOPUS, EMBASE, MEDLINE, LILACS, SCIELO, and SCISEARCH) and selected articles published between January 2000 and June 21, 2016 by using "medicinal plants" and "ulcerative colitis" as key words. Sixty-eight studies were included, and the families Asteraceae and Lamiaceae presented the largest number of studies, but plants from several other families were cited; many of them have shown good results in experimental animals. However, only a few species (such as Andrographis paniculata and Punica granatum) have undergone clinical tests against ulcerative colitis, and the observation that many preclinical studies reviewed are purely descriptive has certainly contributed to this fact. Chemical constituents (mainly flavonoids and terpenes) seem to play a role in the effects of the plants. Thus, the data herein reviewed reinforce the potential of medicinal plants as a source of alternative approaches to the treatment of ulcerative colitis.

Improvement of wound tissue repair by chitosan films containing (-)-borneol, a bicyclic monoterpene alcohol, in rats.

The aim of this study was to investigate the wound-healing activity of (-)-borneol (BOR) incorporated in chitosan film on healing protocol in rodents. To assess the BOR wound-healing potential, male Wistar rats were subjected to a full-thickness excisional wound. The animals were divided into three groups: dressed with chitosan-based film (QUIN); dressed with chitosan-based film containing 0·5% BOR (QUIBO05); or dressed with chitosan-based film containing 1% BOR (QUIBO1). Dressing the wound areas and histological analysis were performed on the 3rd, 7th, 14th, and 21st days. The myeloperoxidase (MPO) activity was assessed on the third and seventh days after surgical procedures. Wounds dressed with chitosan-based film containing BOR reduced significantly the MPO activity (P < 0·001), showed significantly larger wound retraction rates (7 days, P < 0·05), improved the granulation reaction, and also provided better collagenisation density and arrangement during wound healing. It is suggested that BOR modulates the wound-healing process and is a promising compound to be used in wound care. This product may be quite useful in improving wound healing and could be a new biotechnological product with healing properties and clinical application. Further ongoing studies will enable us to understand the precise mechanisms whereby BOR improves the wound-healing process.

Influence of Lemongrass Essential Oil (Cymbopogon flexuosus) Supplementation on Diabetes in Rat Model.

(1) Background: Species of the genus Cymbopogon and its essential oil are known for their antioxidant and hypoglycemic effects. This study aimed to investigate the impact of the essential oil of Cymbopogon flexuosus (EOCF), and its major component, citral, on glycemic, lipid, antioxidant parameters, and oxidative stress in a type 1 diabetes (DM1) rat model. (2) Methods: Initially, EOCF was analyzed by Gas chromatography-mass spectrometry (GC-MS) and the antioxidant activity of EOCF and citral was evaluated. Next, male Wistar rats (3 months old, 200-250 g) induced with DM1 using Streptozotocin (STZ) were divided into four groups: negative control supplemented with an 80% Tween solution, two groups of animals supplemented with EOCF (32 mg/kg and 64 mg/kg) and with citral (32 mg/kg), and treated for 14 days. Measurements of blood glucose levels and body weight were taken; after euthanasia, biochemical markers, including lipid profile, uric acid, alanine aminotransferase (ALT), and aspartate aminotransferase (AST), were evaluated. (3) Results: The predominant compounds in EOCF were α-citral (53.21%) and neral (19.42%), constituting 72.63% citral. EOCF showed good antioxidant activity, significantly greater than citral. EOCF supplementation demonstrated a mitigating effect on glycemic, lipid, and hepatic abnormalities induced by DM1. (4) Conclusions: EOCF emerges as a promising therapeutic option for the management of DM1.

The ethanol extract of the inner bark of Caesalpinia pyramidalis (Tul.) reduces urinary bladder damage during cyclophosphamide-induced cystitis in rats.

Hemorrhagic cystitis (HC) is a common side effect of cyclophosphamide therapy, which deserves new therapeutic strategies, such as those based on natural products. The ethanol extract of the inner bark of Caesalpinia pyramidalis (Tul.) (EECp) possesses anti-inflammatory, antinociceptive, and antioxidant activities as previously showed by our group. We have investigated the effect of EECp on the cyclophosphamide-induced HC. Cystitis was induced in male Wistar rats by the injection of cyclophosphamide. These animals were pretreated with EECp (100-400 mg/kg), vehicle, or mesna. Myeloperoxidase activity and malondialdehyde formation were measured in urinary bladder and other tissues. Bladder edema and histopathological alterations and serum nitric oxide metabolites concentration NOx- were also evaluated. Treatment with EECp (100-400 mg/kg) or mesna impaired the increase of myeloperoxidase activity in urinary bladder and the serum NOx- induced by cyclophosphamide but did not reduce edema in this tissue, as did mesna. Total histological score was reduced by EECp (100 mg/kg). Lung myeloperoxidase activity, which was increased by cyclophosphamide, was decreased significantly by EECp (400 mg/kg). EECp also diminished the malondialdehyde formation in bladder, lung, and spleen, although these parameters were not affected by cyclophosphamide. These results indicate that EECp reduced urinary bladder damage during cyclophosphamide-induced HC in rats.

Myrtenol Reduces Orofacial Nociception and Inflammation in Mice Through p38-MAPK and Cytokine Inhibition.

Orofacial pain is one of the commonest and most complex complaints in dentistry, greatly impairing life quality. Preclinical studies using monoterpenes have shown pharmacological potential to treat painful conditions, but the reports of the effects of myrtenol on orofacial pain and inflammation are scarce. The aim of this study was to evaluate the effect of myrtenol in experimental models of orofacial pain and inflammation. Orofacial nociceptive behavior and the immunoreactivity of the phosphorylated p38 (P-p38)-MAPK in trigeminal ganglia (TG) and spinal trigeminal subnucleus caudalis (STSC) were determined after the injection of formalin in the upper lip of male Swiss mice pretreated with myrtenol (12.5 and 25 mg/kg, i.p.) or vehicle. Orofacial inflammation was induced by the injection of carrageenan (CGN) in the masseter muscle of mice pretreated with myrtenol (25 and 50 mg/kg, i.p.) or its vehicle (0.02% Tween 80 in saline). Myeloperoxidase (MPO) activity and histopathological changes in the masseter muscle and interleukin (IL)-1ß levels in the TG and STSC were measured. The increase in face-rubbing behavior time induced by formalin and P-p38-MAPK immunostaining in trigeminal ganglia were significantly reduced by myrtenol treatment (12.5 and 25 mg/kg). Likewise, increased MPO activity and inflammatory histological scores in masseter muscle, as well as augmented levels of IL-1ß in the TG AND STSC, observed after CGN injection, were significantly decreased by myrtenol (25 and 50 mg/kg). Myrtenol has potential to treat orofacial inflammation and pain, which is partially related to IL-1ß levels in the trigeminal pathway and p38-MAPK modulation in trigeminal ganglia.

Gastroprotective action of the ethanol extract of Leonurus sibiricus L. (Lamiaceae) in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Leonurus sibiricus L. (Lamiaceae) is a medicinal plant known in Brazil as "rubim" or "erva de macaé". It is used for various purposes, including stomach disorders. AIM OF THE STUDY: To evaluate the effect of the ethanol extract of the aerial parts of L. sibiricus (EELs) in models of gastric damage in mice. MATERIAL AND METHODS: The effect of EELs (50, 100 and 300 mg/kg, p.o., 1 h before induction) was tested on acidified ethanol (ACEt)-induced gastric ulcers. Additionally, we tested the effect of EELs (by intraduodenal administration) in the pylorus ligation (PL) model. RESULTS: Pretreatment with EELs, at 300 mg/kg, but not 50 and 100 mg/kg, reduced the relative area of gastric ulcers induced by ACEt (p < 0.01) and lipoperoxidation (p < 0.001), and increased the sulfhydryl content (p < 0.01) in the stomach in comparison with the vehicle group. Pretreatment with N-ethylmaleimide (a blocker of non-protein sulfhydryl groups, 10 mg/kg, i.p.) or glibenclamide (a KATP channel blocker, 10 mg/kg, i.p.) inhibited the gastroprotective response caused by EELs (300 mg/kg; p < 0.001), but there were no alterations due to pretreatments with inhibitors of the synthesis of prostaglandins (indomethacin, 10 mg/kg), nitric oxide (L-NAME, 70 mg/kg) or hydrogen sulfide (DL-propargylglycine, 10 mg/kg). Treatment with EELs (300 mg/kg) reduced mucus production (p < 0.001) and the volume of gastric secretion (p < 0.001) after PL without affecting gastric acidity or pH. CONCLUSIONS: These results provide evidence that EELs exerts gastroprotective action in mice, with the participation of oxidative stress and mediation of NP-SH, KATP channels and mucus production.

Anti-inflammatory activity of hydroxydihydrocarvone.

Hydroxydihydrocarvone (HC) is a synthetic intermediate obtained by hydration of the natural compound (R)-(-)-carvone. The aim of the present study was to investigate the possible anti-inflammatory activity of orally administered HC. Toxicity, motor coordination, tail immersion test, as well as carrageenan-induced paw edema and myeloperoxidase (MPO) activity or peritonitis were all evaluated in rodents. HC was force-fed to the animals 1 h before the stimulus. The lethal dose 50% (LD50) of orally administered HC was 1259 mg/kg. No changes in motor coordination were recorded in HC-treated mice in the rotarod test. The time of response to the thermoceptive stimulus in the tail immersion test was longer in HC-treated animals (50, 100, and 200 mg/kg) than in the vehicle-treated group. HC also significantly decreased the area under curve of carrageenan-induced rat paw edema at 100 and 200 mg/kg and MPO activity at 200 mg/kg. Carrageenan-induced neutrophil recruitment to the peritoneal cavity was significantly reduced by HC at doses of 100 or 200 mg/kg, but not 50 mg/kg. These findings demonstrate that orally administered HC exerts antinociceptive and anti-inflammatory activities in rats and mice.

Natural products for orofacial nociception in pre-clinical studies: A systematic review.

OBJECTIVE: The aim of this systematic review was to summarize the use of natural products (NP) in the treatment of orofacial nociception in animal models. METHODS: Pre-clinical studies that have evaluated the efficacy of NPs in experimental orofacial nociception were sought in the Medline, Web of Science, Scopus, Embase, SciELO, LILACS and Scholar databases in January 2020, covering the period since the inception of each one. Two reviewers independently selected the studies, extracted the data and evaluated the risk of bias of the included studies. RESULTS: We included 41 records in qualitative synthesis. Fifty different NPs were investigated. All studies presented positive results for at least one orofacial nociception test. Regarding the risk of bias, most studies presented poor experimental design, mainly lack of randomization and blinding. The main class of isolated compounds tested was terpenes, of which monoterpenes were investigated in the majority of the studies. CONCLUSION: These results indicate that NPs are effective in treating experimental orofacial nociception and highlight some of these NPs, mainly terpenes, suggesting their potential for translational research.

Effects of the solid lipid nanoparticle of carvacrol on rodents with lung injury from smoke inhalation.

The aim of this present study was to evaluate the effect of solid lipid nanoparticles (SLN) containing carvacrol over the lung damage of airway smoke inhalation. The study was conducted with 30 rats subjected to smoke inhalation and divided into 5 groups such as, normal control, negative control, oxygen group, SLN alone, and SLN+CARV group. The animals were sacrificed 24 h after the induction of inhalation injury further, the tissues of larynx, trachea, and lungs were collected for the histological, hematological, myeloperoxidase, and malondialdehyde analysis. The obtained results showed that treatment with CARV+SLN minimized the inhalation injury, since it reduced malondialdehyde significantly, when compared to the negative control group and minimized the histological changes which proves the absence of pulmonary emphysema and exudate in laryngeal and tracheal lumen in the CARV+SLN-treated group. Meanwhile, the presence of lesion with chronic characteristics was observed in the negative control and oxygen groups. It is suggested that the SLN containing carvacrol minimized oxidative stress and histological damages generated from smoke inhalation in rodents.

Characterization of the mechanisms underlying the inflammatory response to Polistes lanio lanio (paper wasp) venom in mouse dorsal skin.

Stings by Polistes wasps can cause life-threatening allergic reactions, pain and inflammation. We examined the changes in microvascular permeability and neutrophil influx caused by the venom of Polistes lanio a paper wasp found in southeastern Brazil. The intradermal injection of wasp venom caused long-lasting paw oedema and dose-dependently increased microvascular permeability in mouse dorsal skin. SR140333, an NK(1) receptor antagonist, markedly inhibited the response, but the NK(2) receptor antagonist SR48968 was ineffective. The oedema was reduced in capsaicin-treated rats, indicating a direct activation of sensory fibres. Dialysis of the venom partially reduced the oedema and the remaining response was further inhibited by SR140333. Mass spectrometric analysis of the venom revealed two peptides (QPPTPPEHRFPGLM and ASEPTALGLPRIFPGLM) with sequence similarities to the C-terminal region of tachykinin-like peptides found in Phoneutria nigriventer spider venom and vertebrates. Wasp venom failed to release histamine from mast cells in vitro and spectrofluorometric assay of the venom revealed a negligible content of histamine in the usual dose of P. l. lanio venom (1nmol of histamine/7mug of venom) that was removed by dialysis. The histamine H(1) receptor antagonist pyrilamine, but not bradykinin B(1) or B(2) receptor antagonists, inhibited venom-induced oedema. In conclusion, P. l. lanio venom induces potent oedema and increases vascular permeability in mice, primarily through activation of tachykinin NK(1) receptors by substance P released from sensory C fibres, which in turn releases histamine from dermal mast cells. This is the first description of a neurovascular mechanism for P. l. lanio venom-mediated inflammation. The extent to which the two tachykinin-like peptides identified here contribute to this neurogenic inflammatory response remains to be elucidated.

Inflammatory oedema induced by Lachesis muta muta (Surucucu) venom and LmTX-I in the rat paw and dorsal skin.

The ability of crude venom and a basic phospholipase A(2) (LmTX-I) from Lachesis muta muta venom to increase the microvascular permeability in rat paw and skin was investigated. Crude venom or LmTX-I were injected subplantarly or intradermally and rat paw oedema and dorsal skin plasma extravasation were measured. Histamine release from rat peritoneal mast cell was also assessed. Crude venom or LmTX-I induced dose-dependent rat paw oedema and dorsal skin plasma extravasation. Venom-induced plasma extravasation was inhibited by the histamine H(1) antagonist mepyramine (6mg/kg), histamine/5-hydroxytriptamine antagonist cyproheptadine (2mg/kg), cyclooxygenase inhibitor indomethacin (5mg/kg), nitric oxide synthesis inhibitor l-NAME (100nmol/site), tachykinin NK(1) antagonist SR140333 (1nmol/site) and bradykinin B(2) receptor antagonist Icatibant (0.6mg/kg). Platelet-activating factor (PAF) antagonist PCA4248 (5mg/kg) had no effect. LmTX-I-induced skin extravasation was inhibited by cyproheptadine, mepyramine, indomethacin and PCA4248, while l-NAME and SR140333 had no effect. Additionally, both Lachesis muta muta venom and LmTX-I concentration-dependently induced histamine release from rat mast cells. In conclusion, Lachesis muta muta venom and LmTX-I increase microvascular permeability by mechanisms involving in vivo mast cell activation and arachidonic acid metabolites. Additionally, crude venom-induced responses also involve substance P, nitric oxide and bradykinin release, whether LmTX-I-induced responses involve PAF.

Modulation of the pharmacological effects of enzymatically-active PLA2 by BTL-2, an isolectin isolated from the Bryothamnion triquetrum red alga.

BACKGROUND: An interaction between lectins from marine algae and PLA2 from rattlesnake was suggested some years ago. We, herein, studied the effects elicited by a small isolectin (BTL-2), isolated from Bryothamnion triquetrum, on the pharmacological and biological activities of a PLA2 isolated from rattlesnake venom (Crotalus durissus cascavella), to better understand the enzymatic and pharmacological mechanisms of the PLA2 and its complex. RESULTS: This PLA2 consisted of 122 amino acids (approximate molecular mass of 14 kDa), its pI was estimated to be 8.3, and its amino acid sequence shared a high degree of similarity with that of other neurotoxic and enzymatically-active PLA2s. BTL-2 had a molecular mass estimated in approximately 9 kDa and was characterized as a basic protein. In addition, BTL-2 did not exhibit any enzymatic activity. The PLA2 and BTL-2 formed a stable heterodimer with a molecular mass of approximately 24-26 kDa, estimated by molecular exclusion HPLC. In the presence of BTL-2, we observed a significant increase in PLA2 activity, 23% higher than that of PLA2 alone. BTL-2 demonstrated an inhibition of 98% in the growth of the Gram-positive bacterial strain, Clavibacter michiganensis michiganensis (Cmm), but only 9.8% inhibition of the Gram-negative bacterial strain, Xanthomonas axonopodis pv passiflorae (Xap). PLA2 decreased bacterial growth by 27.3% and 98.5% for Xap and Cmm, respectively, while incubating these two proteins with PLA2-BTL-2 inhibited their growths by 36.2% for Xap and 98.5% for Cmm.PLA2 significantly induced platelet aggregation in washed platelets, whereas BTL-2 did not induce significant platelet aggregation in any assay. However, BTL-2 significantly inhibited platelet aggregation induced by PLA2. In addition, PLA2 exhibited strong oedematogenic activity, which was decreased in the presence of BTL-2. BTL-2 alone did not induce oedema and did not decrease or abolish the oedema induced by the 48/80 compound. CONCLUSION: The unexpected results observed for the PLA2-BTL-2 complex strongly suggest that the pharmacological activity of this PLA2 is not solely dependent on the presence of enzymatic activity, and that other pharmacological regions may also be involved. In addition, we describe for the first time an interaction between two different molecules, which form a stable complex with significant changes in their original biological action. This opens new possibilities for understanding the function and action of crude venom, an extremely complex mixture of different molecules.

Influence of acute pancreatitis on the in vitro responsiveness of rat mesenteric and pulmonary arteries.

BACKGROUND: Acute pancreatitis is an inflammatory disease characterized by local tissue injury and systemic inflammatory response leading to massive nitric oxide (NO) production and haemodynamic disturbances. Therefore, the aim of this work was to evaluate the vascular reactivity of pulmonary and mesenteric artery rings from rats submitted to experimental pancreatitis. Male Wistar rats were divided into three groups: saline (SAL); tauracholate (TAU) and phospholipase A2 (PLA2). Pancreatitis was induced by administration of TAU or PLA2 from Naja mocambique mocambique into the common bile duct of rats, and after 4 h of duct injection the animals were sacrificed. Concentration-response curves to acetylcholine (ACh), sodium nitroprusside (SNP) and phenylephrine (PHE) in isolated mesenteric and pulmonary arteries were obtained. Potency (pEC50) and maximal responses (EMAX) were determined. Blood samples were collected for biochemical analysis. RESULTS: In mesenteric rings, the potency for ACh was significantly decreased from animals treated with TAU (about 4.2-fold) or PLA2 (about 6.9-fold) compared to saline group without changes in the maximal responses. Neither pEC50 nor EMAX values for Ach were altered in pulmonary rings in any group. Similarly, the pEC50 and the EMAX values for SNP were not changed in both preparations in any group. The potency for PHE was significantly decreased in rat mesenteric and pulmonary rings from TAU group compared to SAL group (about 2.2- and 2.69-fold, for mesenteric and pulmonary rings, respectively). No changes were seen in the EMAX for PHE. The nitrite/nitrate (NOx-) levels were markedly increased in animals submitted to acute pancreatitis as compared to SAL group, approximately 76 and 68% in TAU and PLA2 protocol, respectively. CONCLUSION: Acute pancreatitis provoked deleterious effects in endothelium-dependent relaxing response for ACh in mesenteric rings that were strongly associated with high plasma NOx- levels as consequence of intense inflammatory responses. Furthermore, the subsensitivity of contractile response to PHE in both mesenteric and pulmonary rings might be due to the complications of this pathological condition in the early stage of pancreatitis.

The inclusion complex of carvacrol and ß-cyclodextrin reduces acute skeletal muscle inflammation and nociception in rats.

BACKGROUND: Skeletal muscle inflammation is strongly associated with pain and may impair regeneration and functional recovery after injury. Since anti-inflammatory and antinociceptive effects have been described for the inclusion complex of carvacrol and ß-cyclodextrin (ßCD-carvacrol), this study investigated the effects of ßCD-carvacrol in a model of acute skeletal muscle inflammation. METHODS: Muscle injury was induced in male Wistar rats by injection of 3% carrageenan in the gastrocnemius muscle. Rats were orally pretreated with saline (vehicle) or ßCD-carvacrol (20, 40, 80 and 180 mg/kg) one hour before administration of carrageenan. RESULTS: The injection of carrageenan in the gastrocnemius muscle increased tissue myeloperoxidase (MPO) activity (p < 0.001), edema (p < 0.001) and the levels of tumoral necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6, macrophage inflammatory protein (MIP-2), but not IL-10 levels. Also, it increased mechanical hyperalgesia and decreased the grip force of animals. Pretreatment with ßCD-carvacrol (80 or 160 mg/kg) significantly decreased muscle MPO activity and edema 24 h after injury in comparison to vehicle-pretreated group. Animals pretreated with ßCD-carvacrol (160 mg/kg) presented significantly lower levels of IL-1ß, IL-6 and MIP-2 and higher levels of IL-10 six hours after induction and lower levels of TNF-α and MIP-2 after 24 h when compared to the vehicle group. Pretreatment with ßCD-carvacrol also reduced mechanical hyperalgesia and limited the decrease of grip force (80 or 160 mg/kg; p < 0.001) 6 and 24 h after injury. CONCLUSION: These results show that ßCD-carvacrol reduces inflammation and nociception in a model of acute injury to skeletal muscles.

Vascular Kinin B1 and B2 Receptors Determine Endothelial Dysfunction through Neuronal Nitric Oxide Synthase.

B1- and B2-kinin receptors are G protein-coupled receptors that play an important role in the vascular function. Therefore, the present study was designed to evaluate the participation of kinin receptors in the acetylcholine (ACh)-induced vascular relaxation, focusing on the protein-protein interaction involving kinin receptors with endothelial and neuronal nitric oxide synthases (eNOS and nNOS). Vascular reactivity, nitric oxide (NO·) and reactive oxygen species (ROS) generation, co-immunoprecipitation were assessed in thoracic aorta from male wild-type (WT), B1- (B1R-/-), B2- (B2R-/-) knockout mice. Some vascular reactivity experiments were also performed in a double kinin receptors knockout mice (B1B2R-/-). For pharmacological studies, selective B1- and B2-kinin receptors antagonists, NOS inhibitors and superoxide dismutase (SOD) mimetic were used. First, we show that B1- and B2-kinin receptors form heteromers with nNOS and eNOS in thoracic aorta. To investigate the functionality of these protein-protein interactions, we took advantage of pharmacological tools and knockout mice. Importantly, our results show that kinin receptors regulate ACh-induced relaxation via nNOS signaling in thoracic aorta with no changes in NO· donor-induced relaxation. Interestingly, B1B2R-/- presented similar level of vascular dysfunction as found in B1R-/- or B2R-/- mice. In accordance, aortic rings from B1R-/- or B2R-/- mice exhibit decreased NO· bioavailability and increased superoxide generation compared to WT mice, suggesting the involvement of excessive ROS generation in the endothelial dysfunction of B1R-/- and B2R-/- mice. Alongside, we show that impaired endothelial vasorelaxation induced by ACh in B1R-/- or B2R-/- mice was rescued by the SOD mimetic compound. Taken together, our findings show that B1- and B2-kinin receptors regulate the endothelium-dependent vasodilation of ACh through nNOS activity and indicate that molecular disturbance of short-range interaction between B1- and B2-kinin receptors with nNOS might be involved in the oxidative pathogenesis of endothelial dysfunction.