Protective effect of Calendula officinalis extract against UVB-induced oxidative stress in skin: Evaluation of reduced glutathione levels and matrix metalloproteinase secretion

Background and purpose: Calendula officinalis flowers have long been employed time in folk therapy, and more than 35 properties have been attributed to decoctions and tinctures from the flowers. The main uses are as remedies for burns (including sunburns), bruises and cutaneous and internal inflammatory diseases of several origins. The recommended doses are a function both of the type and severity of the condition to be treated and the individual condition of each patient. Therefore, the present study investigated the potential use of Calendula officinalis extract to prevent UV irradiation-induced oxidative stress in skin. Methods: Firstly, the physico-chemical composition of marigold extract(ME) (hydroalcoholic extract)was assessed and the in vitro antioxidant efficacy was determined using different methodologies. Secondly, the cytotoxicity was evaluated in L929 and HepG2 cells with the MTT assay. Finally, the in vivo protective effect of ME against UVB-induced oxidative stress in the skin of hairless mice was evaluated by determining reduced glutathione (GSH) levels and monitoring the secretion/activity of metalloproteinases. Results and conclusions: The polyphenol, flavonoid, rutin and narcissin contents found in ME were 28.6 mg/g, 18.8 mg/g, 1.6 mg/g and 12.2 mg/g, respectively and evaluation of the in vitro antioxidant activity demonstrated a dose-dependent effect of ME against different radicals. Cytoxicity experiments demonstrated that ME was not cytotoxic for L929 and HepG2 cells at concentrations less than or equal to of 15 mg/mL However, concentrations greater than or equal to 30 mg/mL, toxic effects were observed. Finally, oral treatment of hairless mice with 150 and 300 mg/kg of ME maintained GSH levels close to non-irradiated control mice. In addition, this extract affects the activity/secretion of matrix metalloproteinases 2 and 9 (MMP-2 and -9) stimulated by exposure to UVB irradiation. However, additional studies are required to have a complete understanding of the protective effects of ME for skin. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

The ACB technique: a biomagentic tool for monitoring gastrointestinal contraction directly from smooth muscle in dogs

The aim of this paper was to verify whether AC biosusceptometry (ACB) is suitable for monitoring gastrointestinal (GI) contraction directly from smooth muscle in dogs, comparing with electrical recordings simultaneously. All experiments were performed in dogs with magnetic markers implanted under the serosa of the right colon and distal stomach, and their movements were recorded by ACB. Monopolar electrodes were implanted close to the magnetic markers and their electric potentials were recorded by electromyography (EMG). The effects of neostigmine, hyoscine butylbromide and meal on gastric and colonic parameters were studied. The ACB signal from the distal stomach was very similar to EMG; in the colonic recordings, however, within the same low-frequency band, ACB and EMG signals were characterized by simultaneity or a widely changeable frequency profile with time. ACB recordings were capable of demonstrating the changes in gastric and colonic motility determined by pharmacological interventions as well as by feeding. Our results reinforce the importance of evaluating the mechanical and electrical components of motility and show a temporal association between them. ACB and EMG arecomplementary for studying motility, with special emphasis on the colon. ACB offers an accurate method for monitoring in vivo GI motility.

Chronic Ethanol Consumption Induces Cavernosal Smooth Muscle Dysfunction in Rats

OBJECTIVES To investigate the effects of chronic ethanol consumption on nitric oxide (NO)-mediated relaxation in rat cavernosal smooth muscle (CSM). METHODS Male wistar rats were divided into 2 groups: control and ethanol. CSM obtained from both groups were mounted in organ chambers for measurement of isometric tension. Contraction of the strips was induced by electrical field stimulation (EFS, 1-32 Hertz) and phenylephrine. We also evaluated the effect of ethanol consumption on the relaxation induced by acetylcholine (0.01-1000 mu mol L(-1)), sodium nitroprusside (SNP, 0.01-1000 mu mol L(-1)), or EFS (1-32 Hz) in strips precontracted with phenylephrine (10 mu mol L(-1)). Blood ethanol, serum testosterone levels, and basal nitrate generation were determined. Immunoexpression of endothelial NO synthase (eNOS) and inducible NO synthase (iNOS) was also accessed. RESULTS Ethanol intake for 4 weeks significantly increased noradrenergic nerve-mediated contractions of CSM in response to EFS. The endothelium-dependent relaxation induced by acetylcholine decreased after the ethanol treatment. Ethanol consumption decreased serum testosterone levels but did not affect the nitrate levels on rat CSM. The mRNA and protein levels for eNOS and iNOS receptors were increased in CSM from ethanol-treated rats. CONCLUSIONS Ethanol consumption reduces endothelium-dependent relaxation induced by acetylcholine, but does not affect SNP or EFS-induced relaxation, suggesting that ethanol disrupts the endothelial function. Despite the overexpression of eNOS and iNOS in ethanol-treated rats, the impaired relaxation induced by acetylcholine may suggest that chronic ethanol consumption induces endothelial dysfunction. UROLOGY 74: 1250-1256, 2009. (C) 2009 Published by Elsevier Inc.

Blood Cardioplegia with N-Acetylcysteine May Reduce Coronary Endothelial Activation and Myocardial Oxidative Stress

Objectives: The aim of this prospective study was to compare the efficacy of intermittent antegrade blood cardioplegia with or without n-acetylcysteine (NAC) in reducing myocardial oxidative stress and coronary endothelial activation. Methods: Twenty patients undergoing elective isolated coronary artery bypass graft surgery were randomly assigned to receive intermittent antegrade blood cardioplegia (32 degrees C-34 degrees C) with (NAC group) or without (control group) 300 mg of NAC. For these 2 groups we compared clinical outcome, hemodynamic evolution, systemic plasmatic levels of troponin I, and plasma concentrations of malondialdehyde (MDA) and soluble vascular adhesion molecule 1 (sVCAM-1) from coronary sinus blood samples. Results: Patient demographic characteristics and operative and postoperative data findings in both groups were similar. There was no hospital mortality. Comparing the plasma levels of MDA 10 min after the aortic cross-clamping and of sVCAM-1 30 min after the aortic cross-clamping period with the levels obtained before the aortic clamping period, we observed increases of both markers, but the increase was significant only in the control group (P=.039 and P=.064 for MDA; P=.004 and P=.064 for sVCAM- 1). In both groups there was a significant increase of the systemic serum levels of troponin I compared with the levels observed before cardiopulmonary bypass (P<.001), but the differences between the groups were not significant (P=.570). Conclusions: Our investigation showed that NAC as an additive to blood cardioplegia in patients undergoing on-pump coronary artery bypass graft surgery may reduce oxidative stress and the resultant coronary endothelial activation.

Inhibition of COX 1 and 2 prior to Renal Ischemia/Reperfusion Injury Decreases the Development of Fibrosis

Ischemia and reperfusion injury (IRI) contributes to the development of chronic interstitial fibrosis/tubular atrophy in renal allograft patients, Cyclooxygenase (COX) 1 and 2 actively participate in acute ischemic injury by activating endothelial cells and inducing oxidative stress. Furthermore, blockade of COX I and 2 has been associated with organ improvement after ischemic damage. The aim of this study was to evaluate the role of COX I and 2 in the development of fibrosis by performing a COX I and 2 blockade immediately before IRI We subjected C57BI/6 male mice to 60 min of unilateral renal pedicle occlusion, Prior to surgery mice were either treated with indomethacin (IMT) at days -1 and 0 or were untreated. Blood and kidney samples were collected 6 wks after IRI. Kidney samples were analyzed by real-time reverse transcription-poly me rase chain reaction for expression of transforming growth factor beta (TGF-beta), monocyte chemoattractant protein 1 (MCP-1), osteopontin (OPN), tumor necrosis factor alpha (TNF-alpha), interleukin (IL)-1 beta, IL-10, heme oxygenose 1 (HO-1), vimentin, connective-tissue growth factor (CTGF), collagen 1, and bone morphogenic protein 7 (BMP-7), To assess tissue fibrosis we performed morphometric analyses and Sirius red staining. We also performed immunohistochemical analysis of anti-actin smooth muscle, Renal function did not significantly differ between groups. Animals pretreated with IMT showed significantly less interstitial fibrosis than nontreated animals. Gene transcript analyses showed decreased expression of TGF-beta, MCP-1,TNF-alpha, IL-1-beta, vimentin, collagen 1, CTGF and IL-10 mRNA (all P < 0.05), Moreover, HO-I mRNA was increased in animals pretreated with IMT (P < 0.05) Conversely, IMT treatment decreased osteopontin expression and enhanced BMP-7 expression, although these levels did rot reach statistical significance when compared with control expression levels, I he blockade of COX 1 and 2 resulted in less tissue fibrosis, which was associated with a decrease in proinflammatory cytokines and enhancement of the protective cellular response.

Genetic and environmental factors affecting ultrasound measures of longissimus muscle area and backfat thickness in Nelore cattle

The aim of the present study was to evaluate the genetic and environmental factors affecting records of longissimus muscle area (LMA) and back fat thickness (BF) obtained between the 12th and 13th ribs, and rump fat thickness (RF) between the hook and pin bones, measured by real-time ultrasound in Nelore cattle. Also, weight records of 22,778 animals born from 1998 to 2003, in ten farms across six Brazilian states were used. Carcass traits as measured by ultrasound of the live animal were recorded from 2002 to 2004 in 2590 males and females with ages varying from 450 to 599 days. Fixed models including farm, year and season of birth, sex and type of feed effects, and the covariates age of dam (AOD) and age of animal at measurement were used to study the effect of environmental factors on these traits. The genetic parameters for LMA, BF and RF were estimated with two and three-trait animal models with 120-day weights using a restricted maximum likelihood method. All environmental effects significantly affected carcass traits, with the exception of year of birth for BF and RF and AOD for LMA. The heritability estimates for LMA, BF and RF were 0.35, 0.51 and 0.39, respectively. Standard errors obtained in one-trait analyses were from 0.07 to 0.09. Genetic correlation estimates between LMA and the two traits of subcutaneous fat were low (close to zero) and 0.74 between BF and RF, indicating that the selection for LMA should not cause antagonism in the genetic improvement of subcutaneous fat measured by real-time ultrasound. (C) 2007 Elsevier B.V. All fights reserved.

Efficacy of Marigold Extract-Loaded Formulations Against UV-induced Oxidative Stress

The present study investigated the potential use of topical formulations containing marigold extract (ME) (Calendula officinalis extract) against ultraviolet (UV) B irradiation-induced skin damage. The physical and functional stabilities, as well as the skin penetration capacity, of the different topical formulations developed were evaluated. In addition, the in vivo capacity to prevent/treat the UVB irradiation-induced skin damage, in hairless mice, of the formulation with better skin penetration capacity was investigated. All of the formulations were physically and functionally stable. The gel formulation [Formulation 3 (F3)] was the most effective for the topical delivery of ME, which was detected as 0.21 mu g/cm(2) of narcissin and as 0.07 mu g/cm(2) of the rutin in the viable epidermis. This formulation was able to maintain glutathione reduced levels close to those of nonirradiated animals, but did not affect the gelatinase-9 and myeloperoxidase activities increased by exposure to UVB irradiation. In addition, F3 reduced the histological skin changes induced by UVB irradiation that appear as modifications of collagen fibrils. Therefore, the photoprotective effect in hairless mice achieved with the topical application of ME in gel formulation is most likely associated with a possible improvement in the collagen synthesis in the subepidermal connective tissue. (C) 2010 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:2182-2193, 2011

Effects of Recession versus Tenotomy Surgery without Recession in Adult Rabbit Extraocular Muscle

PURPOSE. Surgical recession of an extraocular muscle (EOM) posterior to its original insertion is a common form of strabismus surgery, weakening the rotational force exerted by the muscle on the globe and improving eye alignment. The purpose of this study was to assess myosin heavy chain (MyHC) isoform expression and satellite cell activity as defined by Pax7 expression in recessed EOMs of adult rabbits compared with that in muscles tenotomized but not recessed and with that in normal control muscles. METHODS. The scleral insertion of the superior rectus muscle was detached and sutured either 7 mm posterior to its original insertion site (recession surgery) or at the same site (tenotomy). One day before euthanatization, the rabbits received bromodeoxyuridine (BrdU) injections. After 7 and 14 days, selected EOMs from both orbits were examined for changes in fast, slow, neonatal, and developmental MyHC isoform expression, Pax7 expression, and BrdU incorporation. RESULTS. Recession and tenotomy surgery resulted in similar changes in the surgical EOMs. These included a decreased proportion of fast MyHC myofibers, an increased proportion of slow MyHC myofibers, and increased BrdU-positive satellite cells. Similar changes were seen in the non-operated contralateral superior rectus muscles. The ipsilateral inferior rectus showed reciprocal changes to the surgical superior rectus muscles. CONCLUSIONS. The EOMs are extremely adaptive to changes induced by recession and tenotomy surgery, responding with modulations in fiber remodeling and myosin expression. These adaptive responses could be manipulated to improve surgical success rates. (Invest Ophthalmol Vis Sci. 2010;51:5646-5656) DOI:10.1167/iovs.10-5523

Antioxidant status and biomarkers of oxidative stress in bovine leukemia virus-infected dairy cows

Bovine leukemia virus (BLV) is among the most widespread livestock pathogens in many countries. Despite advances in understanding the pathogenesis of this disease, little is known about the involvement of oxidative stress. Therefore, this study examined the antioxidant status and the markers of oxidative stress in BLV-infected dairy cows. BLV infection was associated with an increase in triacylglycerol levels, a decrease in glutathione peroxidase (GSH-Px) activity and a tendency toward lower superoxide dismutase activity in the infected animals. No significant difference was observed in other markers of oxidative stress (i.e., conjugated dienes, hydroperoxides and malondialdehyde) in the infected animals compared to controls. A novel method for the analysis of oxidative stress, Z-scan based on the measurement of the mean-value of 9 in low density lipoprotein indicated that the infected animals had low-density lipoprotein particles that were slightly less modified than those from the healthy group. Thus, we conclude that BLV infection is associated with a selective decrease in GSH-Px activity without any alteration in the common plasma markers of oxidative stress. (C) 2011 Elsevier B.V. All rights reserved.

Evaluation of the lateral pterygoid muscle using magnetic resonance imaging

Objectives: The aims of this study were to evaluate the visibility of the lateral pterygoid muscle (LPM) in temporomandibular joint (TMJ) images obtained by MRI, using different projections and to compare image findings with clinical symptoms of patients with and without temporomandibular disorders (TMD). Methods: In this study, LPM images of 50 participants with and without TMDs were investigated by MRI. The images of the LPM in different projections of 100 TMJs from 35 participants (70 TMJs) with and 15 participants (30 TMJs) without clinical signs and symptoms of TMD were visible and analysed. Results: The oblique sagittal and axial images of the TMJ clearly showed the LPM. Hypertrophy (1.45%), atrophy (2.85%) and contracture (2.85%) were the abnormalities found in the LPM. TMD signs, such as hypermobility (11.4%), hypomobility (12.9%) and disc displacement (20.0%), could be seen in TMJ images. Related clinical symptoms, such as pain (71.4%), articular sounds (30.4%), bruxism (25.7%) and headache (22.9%), were observed. Conclusions: Patients with TMD can present with alterations in the LPM thickness. Patients without TMD also showed alterations, such as atrophy and contracture, in TMJ images. Recognition of alterations in the LPM will improve our understanding of clinical symptoms and pathophysiology of TMD, and may lead to a more specific diagnosis of these disorders. Dentomaxillofacial Radiology (2010) 39, 494-500. doi: 10.1259/dmfr/80928433

Morphological Study of Myoneural Interaction between the Levator Labii Superioris Muscle and its Motor Nerve in Rats

The progress of science in search of new techniques of the nerve regeneration and the functional repair in reinnervated muscle has been the target of many researchers around the world. Consequently, nerves and muscles in different body segments asked for more enlightenment of their morphology, their interrelation with other anatomic structures and their peculiarities. One of the most significant areas that need deeper studies is the region of the head and neck, since they are often affected by important pathologies. In order to offer the researcher`s community a morphological myoneural interaction model, this study elected the levator labii superioris muscle and its motor nerve, the buccal branch of the facial nerve (VII pair) not only for its special characteristics, but also its value on the facial expression. The rat was chosen for this investigation for being easy to obtain, to keep, to manipulate and to compare this experiment with many others studies previously published. The techniques used were Mesoscopic (dissection), histoenzymologic and morphometric ones. In the results the muscle proved to have a predominance of fast twich fibers (FG and FOG) and superficial location, with a proximal bone and a distal cutaneous insertion. Its motor nerve, the buccal branch of the facial nerve (VII pair), breaks through the muscle belly into its deep face, and comprised a heterogeneous group of myelinic nerve fibers disposed in a regular form in all fascicle. Near the motor point, the nerve showed to be composed of two fascicles with different sizes. Due to the small nerve dimensions, the nerve fibers have a smaller diameter if compared to the motor nerve of pectineus muscle of the cat. Further studies with neural tracers have already had a start in order to provide more information about the distribution and the architecture of these fibers.

gamma-Radiation induces apoptosis via sarcoplasmatic reticulum in guinea pig ileum smooth muscle cells

We investigated the effects of gamma-radiation on cells isolated from the longitudinal smooth muscle layer of the guinea pig ileum, a relatively radioresistant tissue. Single doses (up to 50 Gy) reduced the amount of sarcoplasmatic reticulum and condensed the myofibrils, as shown by electron microscopy 3 days post-irradiation. After that, contractility of smooth muscle strips was reduced. Ca(2+) handling was altered after irradiation, as shown in fura-2 loaded cells, with elevated basal intracellular Ca(2+), reduced amount of intrareticular Ca(2+), and reduced capacitive Ca(2+) entry. Radiation also induced apoptosis, judged from flow cytometry of cells loaded with proprium iodide. Electron microscopy showed that radiation caused condensation of chromatin in dense masses around the nuclear envelope, the presence of apoptotic bodies, fragmentation of the nucleus, detachment of cells from their neighbors, and reductions in cell volume. Radiation also caused activation of caspase 12. Apoptosis was reduced by the administration of the caspase inhibitor Z-Val-Ala-Asp-fluoromethyl-ketone methyl ester (Z-VAD-FIVIK) during the 3 day period after irradiation, and by the chelator of intracellular Ca(2+), 1,2-bis(o-aminophenoxy)ethane-N,N,N`,N`-tetraacetic acid (BAPTA), from 1 h before until 2 h after irradiation. BAPTA also reduced the effects of radiation on contractility, basal intracellular Ca(2+), amount of intrareticular Ca(2+), capacitative Ca(2+) entry, and apoptosis. In conclusion, the effects of gamma radiation on contractility, Ca(2+) handling, and apoptosis appear due to a toxic action of intracellular Ca(2+). Ca(2+)-induced damage to the sarcoplasmatic reticulum seems a key event in impaired Ca(2+) handling and apoptosis induced by gamma-radiation. (c) 2008 Elsevier B.V. All rights reserved.

Bcl-X-L translocation in renal tubular epithelial cells in vitro protects distal cells from oxidative stress

Background. The molecular pathogenesis of different sensitivities of the renal proximal and distal tubular cell populations to ischemic injury, including ischemia-reperfusion (IR)-induced oxidative stress, is not well-defined. An in vitro model of oxidative stress was used to compare the survival of distal [Madin-Darby canine kidney (MDCK)] and proximal [human kidney-2 (HK-2)] renal tubular epithelial cells, and to analyze for links between induced cell death and expression and localization of selected members of the Bcl-2 gene family (anti-apoptotic Bcl-2 and Bcl-X-L, pro-apoptotic Bax and Bad), Methods. Cells were treated with 1 mmol/L hydrogen peroxide (H2O2) Or were grown in control medium for 24 hours. Cell death (apoptosis) was quantitated using defined morphological criteria. DNA gel electrophoresis was used for biochemical identification. Protein expression levels and cellular localization of the selected Bcl-2 family proteins were analyzed (West ern immunoblots, densitometry, immunoelectron microscopy). Results. Apoptosis was minimal in control cultures and was greatest in treated proximal cell cultures (16.93 +/- 4.18% apoptosis) compared with treated distal cell cultures (2.28 +/- 0.85% apoptosis, P < 0.001). Endogenous expression of Bcl-X-L and Bax, but not Bcl-2 or Bad, was identified in control distal cells, Bcl-X-L and Bax had nonsignificant increases (P > 0.05) in these cells. Bcl-2, Bax, and Bcl-X-L, but not Bad, were endogenously expressed in control proximal cells. Bcl-X-L was significantly decreased in treated proximal cultures (P < 0.05), with Bas and Bcl-2 having nonsignificant increases (P > 0.05). Immunoelectron microscopy localization indicated that control and treated hut surviving proximal cells had similar cytosolic and membrane localization of the Bcl-2 proteins. In comparison, surviving cells in the treated distal cultures showed translocation of Bcl-X-L from cytosol to the mitochondria after treatment with H2O2, a result that was confirmed using cell fractionation and analysis of Bcl-XL expression levels of the membrane and cytosol proteins. Bax remained distributed evenly throughout the surviving distal cells, without particular attachment to any cellular organelle. Conclusion. The results indicate that in this in vitro model, the increased survival of distal compared with proximal tubular cells after oxidative stress is best explained by the decreased expression of anti-apoptotic Bcl-X-L in proximal cells, as well as translocation of Bcl-X-L protein to mitochondria within the surviving distal cells.

Pyrrolidine dithiocarbamate down-regulates vascular matrix metalloproteinases and ameliorates vascular dysfunction and remodelling in renovascular hypertension

BACKGROUND AND PURPOSE Mounting evidence implicates matrix metalloproteinase (MMP) in the vascular dysfunction and remodelling associated with hypertension. We tested the hypothesis that treatment with pyrrolidine dithiocarbamate (PDTC), which interferes with NF-kappa B-induced MMPs gene transcription, could exert antihypertensive effects, prevent MMP-2 and MMP-9 up-regulation, and protect against the functional alterations and vascular remodelling of two-kidney, one clip (2K1C) hypertension. EXPERIMENTAL APPROACH Sham-operated or hypertensive rats were treated with vehicle or PDTC (100 mg.Kg(-1).day(-1)) by gavage for 8 weeks. Systolic blood pressure (SBP) was monitored weekly. Aortic rings were isolated to assess endothelium-dependent relaxations. Quantitative morphometry of structural alterations of the aortic wall was carried out in haematoxylin/eosin sections. Formation of vascular reactive oxygen species (ROS), and inducible (i) NOS and phosphorylated-p65 NF-kappa B subunit expression were measured in the aortas. MMP-2 and MMP-9 aortic levels and gelatinolytic activity were determined by gelatin and in situ zymography and by immunofluorescence. KEY RESULTS Treatment with PDTC attenuated the increases in SBP and prevented the endothelial dysfunction associated with 2K1C hypertension. Moreover, PDTC reversed the vascular aortic remodelling, the increases in aortic ROS levels and in iNOS and phosphorylated-p65 NF-kappa B expression found in 2K1C rats. These effects were associated with attenuation of 2K1C up-regulation of aortic MMP-2 and MMP-9 levels and gelatinolytic activity. CONCLUSION AND IMPLICATIONS These findings suggest that PDTC down-regulates vascular MMPs and ameliorates vascular dysfunction and remodelling in renovascular hypertension, thus providing evidence supporting the suggestion that PDTC is probably a good candidate to be used to treat hypertension.