Joana Liberal

Propolis is a bee product with numerous biological and pharmacological properties, such as immunomodulatory and anti-inflammatory activities. It has been used in folk medicine as a healthy drink and in food to improve health and prevent inflammatory diseases. However, little is known about its mechanism of action. Thus, the goal of this study was to verify the antioxidant activity and to explore the anti-inflammatory properties of propolis by addressing its intracellular mechanism of action. Caffeic acid was investigated as a possible compound responsible for propolis action.The antioxidant properties of propolis and caffeic acid were evaluated by using the 2,2-Diphenyl-1-picrylhydrazyl free radical (DPPH) scavenging method. To analyze the anti-inflammatory activity, Raw 264.7 macrophages were treated with different concentrations of propolis or caffeic acid, and nitric oxide (NO) production, a strong pro-inflammatory mediator, was evaluated by the Griess reaction. The concentrations of propolis and caffeic acid that inhibited NO production were evaluated on intracellular signaling pathways triggered during inflammation, namely p38 mitogen-activated protein kinase (MAPK), c-jun NH2-terminal kinase (JNK1/2), the transcription nuclear factor (NF)-κB and extracellular signal-regulated kinase (ERK1/2), through Western blot using specific antibodies. A possible effect of propolis on the cytotoxicity of hepatocytes was also evaluated, since this product can be used in human diets.Caffeic acid showed a higher antioxidant activity than propolis extract. Propolis and caffeic acid inhibited NO production in macrophages, at concentrations without cytotoxicity. Furthermore, both propolis and caffeic acid suppressed LPS-induced signaling pathways, namely p38 MAPK, JNK1/2 and NF-κB. ERK1/2 was not affected by propolis extract and caffeic acid. In addition, propolis and caffeic acid did not induce hepatotoxicity at concentrations with strong anti-inflammatory potential.Propolis exerted an antioxidant and anti-inflammatory action and caffeic acid may be involved in its inhibitory effects on NO production and intracellular signaling cascades, suggesting its use as a natural source of safe anti-inflammatory drugs.

OBJECTIVE Calcium dobesilate (CaD) has been used in the treatment of diabetic retinopathy in the last decades, but its mechanisms of action are not elucidated. CaD is able to correct the excessive vascular permeability in the retina of diabetic patients and in experimental diabetes. We investigated the molecular and cellular mechanisms underlying the protective effects of CaD against the increase in blood–retinal barrier (BRB) permeability induced by diabetes. RESEARCH DESIGN AND METHODS Wistar rats were divided into three groups: controls, streptozotocin-induced diabetic rats, and diabetic rats treated with CaD. The BRB breakdown was evaluated using Evans blue. The content or distribution of tight junction proteins (occludin, claudin-5, and zonula occluden-1 [ZO-1]), intercellular adhesion molecule-1 (ICAM-1), and p38 mitogen-activated protein kinase (p38 MAPK) was evaluated by Western blotting and immunohistochemistry. Leukocyte adhesion was evaluated in retinal vessels and in vitro. Oxidative stress was evaluated by the detection of oxidized carbonyls and tyrosine nitration. NF-κB activation was measured by enzyme-linked immunosorbent assay. RESULTS Diabetes increased the BRB permeability and retinal thickness. Diabetes also decreased occludin and claudin-5 levels and altered the distribution of ZO-1 and occludin in retinal vessels. These changes were inhibited by CaD treatment. CaD also inhibited the increase in leukocyte adhesion to retinal vessels or endothelial cells and in ICAM-1 levels, induced by diabetes or elevated glucose. Moreover, CaD decreased oxidative stress and p38 MAPK and NF-κB activation caused by diabetes. CONCLUSIONS CaD prevents the BRB breakdown induced by diabetes, by restoring tight junction protein levels and organization and decreasing leukocyte adhesion to retinal vessels. The protective effects of CaD are likely to involve the inhibition of p38 MAPK and NF-κB activation, possibly through the inhibition of oxidative/nitrosative stress.

Breast cancer is the most prevalent malignant tumor and main oncologic cause of mortality in women. Although most diagnosis of breast pathology is accomplished using hematoxylin and eosin stained sections, some cases require immunohistochemistry for proper evaluation. We investigated the latter cases including distinctions between ductal and lobular carcinoma, in situ and invasive carcinoma, typical ductal hyperplasia and atypical ductal hyperplasia/ductal carcinoma in situ, papillary and spindle cell lesion assessment, metastasis evaluation, and assessment of prognostic and therapy markers. E-cadherin is used to differentiate ductal and lobular carcinoma; 34βE12, CK8, p120 catenin and β-catenin also produce consistent results. Myoepithelial cell (MEC) stains are used to evaluate in situ and invasive carcinoma; calponin, smooth muscle myosin heavy chain and p63 are sensitive/specific markers. 34βE12 and CK5/6 are positive in ductal hyperplasia, which enables its differentiation from atypical ductal hyperplasia and ductal carcinoma in situ. CK 5/6, ER and MEC markers are consistent options for evaluating papillary lesions. Spindle cell lesions can be assessed using β-catenin, SMA, CD34, p63, CKs and hormone receptors. It is important to differentiate primary carcinomas from metastases; the most commonly used markers to identify breast origin include mammaglobin, GCDFP-15, GATA3 and ER, although none of these is completely sensitive or specific. Immunohistochemistry can be used to evaluate central prognostic and predictive factors including molecular subtypes, HER2, hormone receptors, proliferation markers (Ki-67) and lymph-vascular invasion markers including ERG, CD31, CD34, factor VIII and podoplanin. Owing to the complexity of mammary lesions, diagnosis also depends on each particular situation, evaluation of cytological characteristics revealed by immunochemistry and correlation with histological findings.

Flavonoids from lemongrass – Cymbopogon citratus (DC.) Stapf – leaves infusion, a commonly consumed beverage for the treatment of inflammatory-related conditions, were investigated in this work. Luteolin O-, C- and O,C-glycosides were isolated and identified by nuclear magnetic resonance, being the cassiaoccidentalin B structure fully characterized for the first time in lemongrass. The anti-inflammatory activity of luteolin and its glycosides was evaluated in lipopolysaccharide-stimulated macrophages. Luteolin glycosides demonstrated less cytotoxicity than luteolin itself. Although glycosylation decreases luteolin anti-inflammatory properties, being higher to C-glycosylation, an inhibitory effect on inflammatory mediator production (nitric oxide and IL-1β) was verified for the luteolin 7-O-β-glucopyranoside, without cytotoxic effects. Therefore, luteolin glycosides from lemongrass infusion are evidenced as a less toxic alternative to current anti-inflammatory drugs with promising use in pharmaceutical and food supplement industries. Additionally, this work establishes structure–activity relationships, which constitutes valuable information in the design of anti-inflammatory luteolin glycosides devoid of toxicity.

Urtica dioica and other less studied Urtica species (Urticaceae) are often used as a food ingredient. Fifteen hydroxycinnamic acid derivatives and sixteen flavonoids, flavone and flavonol-type glycosides were identified in hydroalcoholic extracts from aerial parts of Urtica dioica L., Urtica urens L. and Urtica membranacea using HPLC-PDA-ESI/MSn. Among them, the 4-caffeoyl-5-p-coumaroylquinic acid and three statin-like 3-hydroxy-3-methylglutaroyl flavone derivatives were identified for the first time in Urtica urens and U. membranacea respectively. Urtica membranacea showed the higher content of flavonoids, mainly luteolin and apigenin C-glycosides, which are almost absent in the other species studied. In vitro, Urtica dioica exhibited greater antioxidant activity but Urtica urens exhibited stronger anti-inflammatory potential. Interestingly, statin-like compounds detected in Urtica membranacea have been associated with hypocholesterolemic activity making this plant interesting for future investigations. None of the extracts were cytotoxic to macrophages and hepatocytes in bioactive concentrations (200 and 350 μg/mL), suggesting their safety use in food applications.

Despite being one of the most commonly prescribed antiepileptic drugs, levetiracetam is marketed in oral and intravenous dosage forms, which are associated to drug-drug interactions and drug-resistant epilepsy (DRE). The purpose of the present study was to assess the potential of the intranasal route to deliver levetiracetam into the brain, due to the particular anatomical features of the nasal cavity. After development and characterization of the drug formulation, a thermoreversible gel loaded with levetiracetam was administered to CD-1 male mice by intranasal route and its pharmacokinetics compared to those observed after intravenous administration. Similar plasma pharmacokinetic profiles were obtained and the intranasal absolute bioavailability was 107.44%, underscoring that a high drug fraction was systemically absorbed. In brain tissue, maximum drug concentrations were 4.48 and 4.02 μg/g (intranasal vs intravenous) and the mean cerebral concentrations were significantly higher after intranasal administration. The percentage of drug targeting efficiency was 182.35% while direct transport percentage was 46.38%, suggesting that almost 50% of levetiracetam undergoes direct nose-to-brain delivery. Complementarily, an in vivo intranasal repeated dose toxicity study was performed and no relevant histopathological alterations were observed. The herein proposed non-invasive and safe intranasal administration route allowed a direct nose-to-brain delivery of levetiracetam and is a promising strategy for the treatment of DRE.

Inflammation is a defensive response of the organism to manage harmful stimuli sensed by innate immune cells. The signal alarm is triggered by the recognition of pathogen-associated molecular patterns, such as microbial components, or host-derived damage-associated molecular patterns (DAMPs), namely high-mobility group box 1 protein (HMGB1) and purine metabolites, through a set of highly conserved receptors in immune cells termed pattern recognition receptors. Among these receptors, membrane-associated toll-like receptors (TLRs) and cytosolic nucleotide binding and oligomerization domain (nod)-like receptors (NLRs) assume particular relevance in the inflammatory process. Once activated, NLRs induce the assembly of multiprotein complexes called inflammasomes, leading to production of proinflammatory cytokines (e.g., interleukin-1) and induction of inflammatory cell death (pyroptosis) through the activation of caspase-1. Although these processes intend to protect the body from insults, prolonged or exacerbated inflammatory responses associated with inflammasome activation are related to a growing number of diseases. Recently, inflammasome activation and autophagy were shown to be linked and to mutually influence each other. Therefore, we aim, in this review, to discuss the recent evidences concerning the cross talk between autophagy and inflammasome activation and its potential roles in disease progression.

Lavandulaviridis L´Hér. is an endemic Iberian species with a high essential oil yield and a pleasant lemon scent. Despite these interesting features, this species remains unrecognized and poorly explored by the food and pharmaceutical industries. Nevertheless, it has been valued in traditional medicine being used against flu, circulatory problems and to relieve headaches. Since these disorders trigger inflammatory responses, it is relevant to determine the anti-inflammatory potential of L. viridis L´Hér. essential oil in an attempt to validate its traditional use and concomitantly to increment its industrial exploitation. Therefore, in the present study the chemical composition of this volatile extract as well as the effect on ROS production, inflammatory response and proteasome activity on LPS-stimulated macrophages were disclosed. Also, its safety profile on keratinocytes, hepatocytes and alveolar epithelial cells was depicted, envisioning a future human administration. The essential oil was characterized by high quantities of 1,8-cineole, camphor and α-pinene. From a pharmacological point of view, the essential oil showed a potent antioxidant effect and inhibited nitric oxide production through down-modulation of nuclear factor kappa B-dependent Nos2 transcription and consequently iNOS protein expression as well as a decrease in proteasomal activity. The anti-inflammatory activity was also evidenced by a strong inhibition of LPS-induced Il1b and Il6 transcriptions and downregulation of COX-2 levels. Overall, bioactive safe concentrations of L. viridis L´Hér. essential oil were disclosed, thus corroborating the traditional usage of this species and paving the way for the development of plant-based therapies.

Luteolin is a dietary flavonoid with medicinal properties including antioxidant, antimicrobial, anticancer, antiallergic, and anti-inflammatory. However, the effect of luteolin on liver X receptors (LXRs), oxysterol sensors that regulate cholesterol homeostasis, lipogenesis, and inflammation, has yet to be studied. To unveil the potential of luteolin as an LXRα/β modulator, we investigated by real-time RT-PCR the expression of LXR-target genes, namely, sterol regulatory element binding protein 1c (SREBP-1c) in hepatocytes and ATP-binding cassette transporter (ABC)A1 in macrophages. The lipid content of hepatocytes was evaluated by Oil Red staining. The results demonstrated, for the first time, that luteolin abrogated the LXRα/β agonist-induced LXRα/β transcriptional activity and, consequently, inhibited SREBP-1c expression, lipid accumulation, and ABCA1 expression. Therefore, luteolin could abrogate hypertriglyceridemia associated with LXR activation, thus presenting putative therapeutic effects in diseases associated with deregulated lipid metabolism, such as hepatic steatosis, cardiovascular diseases, and diabetes.

Agrimony ( Agrimonia eupatoria L.) (Ae) is used in traditional medicine to treat inflammatory and oxidative related diseases. Therefore, this study focuses on the anti-inflammatory and analgesic potential of Ae infusion (AeI). Phenolic compounds characterization was achieved by HPLC-PDA-ESI/<mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M1"><mml:mrow><mml:msup><mml:mrow><mml:mi mathvariant="normal">M</mml:mi><mml:mi mathvariant="normal">S</mml:mi></mml:mrow><mml:mrow><mml:mi>n</mml:mi></mml:mrow></mml:msup></mml:mrow></mml:math>. To evaluate antioxidant potential, 2,2-diphenyl-1-picrylhydrazyl (DPPH), superoxide anion, hydroxyl radical, and SNAP assays were used. In vitro anti-inflammatory activity of AeI was investigated in LPS-stimulated macrophages by measuring the NO production. In vivo anti-inflammatory activity was validated using the mouse carrageenan-induced paw edema model. Peripheral and central analgesic potential was evaluated using the acetic acid-induced writhing and hot-plate tests, respectively, as well as the formalin assay to assess both activities. The safety profile was disclosed in vitro and in vivo, using MTT and hematoxylin assays, respectively. Vitexin, quercetin O -galloyl-hexoside, and kaempferol O -acetyl-hexosyl-rhamnoside were referred to in this species for the first time. AeI and mainly AePF (Ae polyphenolic fraction) showed a significant antiradical activity against all tested radicals. Both AeI and AePF decreased NO levels in vitro, AePF being more active than AeI. In vivo anti-inflammatory and analgesic activities were verified for both samples at concentrations devoid of toxicity. Agrimony infusion and, mainly, AePF are potential sources of antiradical and anti-inflammatory polyphenols.

Ellagitannins have been gaining attention as potential anticancer molecules. However, the low bioavailability of ellagitannins and their extensive metabolization in the gastrointestinal tract into ellagic acid and urolithins suggest that the health benefits of consuming ellagitannins rely on the direct effects of their metabolites. Recently, chemopreventive and chemotherapeutic activities were ascribed to urolithins. Nonetheless, there is still a need to screen and evaluate the selectivity of these molecules and to elucidate their cellular mechanisms of action. Therefore, this work focused on the antiproliferative effects of urolithins A, B and C and ellagic acid on different human tumor cell lines. The evaluation of cell viability and the determination of the half-maximal inhibitory concentrations indicated that the sensitivity to the studied urolithins varied markedly between the different cell lines, with the bladder cancer cells (UMUC3) being the most susceptible. In UMUC3 cells, urolithin A was the most active molecule, promoting cell cycle arrest at the G2/M checkpoint, increasing apoptotic cell death and inhibiting PI3K/Akt and MAPK signaling. Overall, the present study emphasizes the chemopreventive/chemotherapeutic potential of urolithins, highlighting the stronger effects of urolithin A and its potential to target transitional bladder cancer cells.

Fragaria vesca leaves have been used in folk medicine for the treatment of several diseases, namely gastrointestinal, cardiovascular and urinary disorders, which could be related with the potential anti-inflammatory properties of the extract. This work aims to disclose the bioactivity and the underlying action mechanism of an extract from Fragaria vesca leaves in order to support its traditional uses.A hydroalcoholic extract was prepared from Fragaria vesca leaves and its anti-inflammatory potential was evaluated through inhibition of nitric oxide production and expression of several pro-inflammatory proteins in lipopolysaccharide-triggered macrophages. Nitric oxide scavenger activity was also assessed using a standard nitric oxide donor. Since numerous inflammatory proteins are tightly regulated by ubiquitination and proteasomal degradation, the putative effect of the extract on these cellular proteolytic pathways was also disclosed. The phytochemical characterization was performed by HPLC-PDA-ESI/MSn and compared with an infusion prepared according to the traditional method.For non-cytotoxic concentrations (80 and 160µg/mL) the extract inhibited nitrite production, probably due to a direct nitric oxide scavenging. Furthermore, inhibition of proteasome activity was verified, leading to accumulation of ubiquitinated proteins. The extract also increased the conversion of the microtubule-associated protein light chain LC3-I to LC3-II, a marker of autophagy. Polyphenols, namely ellagitannins, proanthocyanidins, and quercetin and kaempferol glucuronide derivatives were identified in Fragaria vesca leaves extract. Most of the identified phenolic compounds matched with those found in traditional preparation, the infusion.The extract has a direct nitric oxide scavenging activity giving support to the traditional use of this plant for the treatment of inflammatory disorders. Furthermore, the extract affects the proteolytic systems but its role in cancer treatment requires further studies.

A few studies have reported the existence of depletion of synaptic vesicles, and changes in neurotransmitter release and in the content of exocytotic proteins in the hippocampus of diabetic rats. Recently, we found that diabetes alters the levels of synaptic proteins in hippocampal nerve terminals. Hyperglycemia is considered the main trigger of diabetic complications, although other factors, such as low insulin levels, also contribute to diabetes-induced changes. Thus, the aim of this work was to evaluate whether long-term elevated glucose per se, which mimics prolonged hyperglycemia, induces significant changes in the content and localization of synaptic proteins involved in exocytosis in hippocampal neurons. Hippocampal cell cultures were cultured for 14 days and were exposed to high glucose (50 mM) or mannitol (osmotic control; 25 mM plus 25 mM glucose), for 7 days. Cell viability and nuclear morphology were evaluated by MTT and Hoechst assays, respectively. The protein levels of vesicle-associated membrane protein-2 (VAMP-2), synaptosomal-associated protein-25 (SNAP-25), syntaxin-1, synapsin-1, synaptophysin, synaptotagmin-1, rabphilin 3a, and also of vesicular glutamate and GABA transporters (VGluT-1 and VGAT), were evaluated by immunoblotting, and its localization was analyzed by immunocytochemistry. The majority of the proteins were not affected. However, elevated glucose decreased the content of SNAP-25 and increased the content of synaptotagmin-1 and VGluT-1. Moreover, there was an accumulation of syntaxin-1, synaptotagmin-1 and VGluT-1 in the cell body of some hippocampal neurons exposed to high glucose. No changes were detected in mannitol-treated cells. In conclusion, elevated glucose per se did not induce significant changes in the content of the majority of the synaptic proteins studied in hippocampal cultures, with the exception of SNAP-25, synaptotagmin-1 and VGluT-1. However, there was an accumulation of some proteins in cell bodies of hippocampal neurons exposed to elevated glucose, suggesting that the trafficking of these proteins to the synapse may be compromised. Moreover, these results also suggest that other factors, in addition to hyperglycemia, certainly contribute to alterations detected in synaptic proteins in diabetic animals.

Helicobacter pylori (H. pylori) plays a causal role in chronic gastritis, peptic ulcer and gastric cancer. Antibiotic resistance and side effects of therapy have led to research for new candidates. Susceptibility and virulence factors were studied in H. pylori 12 clinical isolates. Agrimonia eupatoria L. (Ag), Fragaria vesca (Fv) extracts and a Fv ellagitannin-enriched fraction (Fve) activity were tested against these isolates. Ag, Fv extracts and Fve presented activity against H. pylori multi-resistant and virulent. Fve demonstrated the best activity (5 mg/mL inhibited 67% of isolates), follow by Fv (12.5 mg/mL inhibited 58%) and by Ag (25 mg/mL inhibited 40%). This is the first report of Fv extract and Fve fraction activity on H. pylori isolates. These activity and that of Ag were independently of the different patterns of virulence and susceptibility, suggesting potential of these plants as new natural drugs or as bioactive agents in association with antibiotic therapy.

The hepatocellular carcinoma, a primary malignancy of the liver, has a very poor prognosis and a lower survival rate. Moreover, the inefficacy of conventional therapies emphasizes the importance of discovering new bioactive compounds. Several studies clearly state that plant-derived polyphenols, namely ellagitannins, have several health benefits. Fragaria vesca leaves contain high amounts of polyphenols, being especially rich in ellagitannins. Therefore, this study aimed to characterize an ellagitannin-enriched fraction (EEF) from F. vesca leaves and to unveil the anticancer potential of this fraction on human hepatocellular carcinoma cells. The analysis of EEF by HPLC-PDA-ESI/MSn allowed the detection of 12 ellagitannins. The cell viability of both EEF and crude extract was determined after 24 h of cells treatment and the half-maximal inhibitory concentration (IC50) was evaluated. The IC50 of the EEF (113 μg/mL) was about 6 times lower than the IC50 of the crude extract (690 μg/mL). Furthermore, EEF induced cell cycle arrest at G2/M checkpoint and decreased cell proliferation in a dose-dependent way. This fraction also induced an accumulation of LC3-II protein through blockage of autophagic flux, and inhibited chymotrypsin-like activity of 26S proteasome. These results showed, for the first time, that EEF from F. vesca leaves inhibits both, autophagic and ubiquitin-proteasome system pathways, two main intracellular protein degradation systems that are targets for anticancer therapies. Additionally, a proteomic analysis allowed the identification of 914 proteins, among which 133 were modulated after cells treatment with EEF, most of them related to metabolic pathways. Overall, this study shows that the EEF from F. vesca leaves decreased cell proliferation, inhibited the proteolytic mechanisms and modulated the metabolic pathways of the cell. Additionally this study points out F. vesca as a source of valuable molecules with anticancer potential, suggesting that ellagitannins, the polyphenols identified in this fraction, could be useful in the development of new fine-tuned therapeutic strategies against carcinogenesis.

Cymbopogon citratus (Cc), commonly known as lemongrass, is a very important crop worldwide, being grown in tropical countries. It is widely used in the food, pharmaceutical, cosmetic and perfumery industries for its essential oil. Cc aqueous extracts are also used in traditional medicine. They contain high levels of polyphenols, which are known for their antioxidant and anti-inflammatory properties. Hydrodistillation of lemongrass essential oil produces an aqueous waste (CcHD) which is discarded. Therefore a comparative study between CcHD and Cc infusion (CcI) was performed to characterize its phytochemical profile and to research its antioxidant and anti-inflammatory potential.HPLC-PDA/ESI-MS(n) analysis showed that CcI and CcHD have similar phenolic profiles, with CcHD presenting a higher amount of polyphenols. Additionally, both CcI and CcHD showed antioxidant activity against DPPH (EC50 of 41.72 ± 0.05 and 42.29 ± 0.05 µg mL(-1) respectively) and strong anti-inflammatory properties, by reducing NO production and iNOS expression in macrophages and through their NO-scavenging activity, in a dose-dependent manner. Furthermore, no cytotoxicity was observed.The data of this study encourage considering the aqueous solution from Cc leaf hydrodistillation as a source of bioactive compounds, which may add great industrial value to this crop.

Diabetic retinopathy is a leading cause of vision loss and blindness. Increasing evidence has shown that the neuronal components of the retina are affected even before the detection of vascular lesions. Hyperglycemia is considered the main pathogenic factor for the development of diabetic complications. Nevertheless, other factors like neuroinflammation, might also contribute for neural changes. To clarify whether hyperglycemia can be the main trigger of synaptic changes, we evaluated whether prolonged elevated glucose per se, mimicking chronic hyperglycemia, is able to change the content and distribution of several exocytotic proteins and vesicular glutamate and GABA transporters in retinal neurons. Moreover, we also tested the hypothesis that an inflammatory stimulus (interleukin-1β) could exacerbate the effects induced by exposure to elevated glucose, contributing for changes in synaptic proteins in retinal neurons. Rat retinal neural cells were cultured for 9 days. Cells were exposed to elevated d-glucose (30 mM) or d-mannitol (osmotic control), for 7 days, or were exposed to interleukin-1β (10 ng/ml) or LPS (1 μg/ml) for 24 h. The protein content and distribution of SNARE proteins (SNAP-25, syntaxin-1, VAMP-2), synapsin-1, synaptotagmin-1, rabphilin 3a, VGluT-1 and VGAT, were evaluated by western blotting and immunocytochemistry. The protein content and immunoreactivity of syntaxin-1, synapsin-1, rabphilin 3a and VGAT increased in retinal neural cells exposed to high glucose. No changes were detected when cells were exposed to interleukin-1β, LPS or mannitol per se. Particularly, exposure to interleukin-1β for 24 h did not exacerbate the effect of high glucose on the content and immunoreactivity of exocytotic proteins, suggesting the primordial role of hyperglycemia for neuronal changes. In summary, prolonged exposure to elevated glucose alters the total content of several proteins involved in exocytosis, suggesting that hyperglycemia per se is a fundamental factor for neuronal changes caused by diabetes.

Diabetic retinopathy and diabetic encephalopathy are two common complications of diabetes mellitus. The impairment of glutamatergic neurotransmission in the retina and hippocampus has been suggested to be involved in the pathogenesis of these diabetic complications. In this study, we investigated the effect of elevated glucose concentration and diabetes on the protein content and surface expression of AMPA receptor subunits in the rat retina and hippocampus. We have used two models, cultured retinal and hippocampal cells exposed to elevated glucose concentration and an animal model of streptozotocin-induced type 1 diabetes. The immunoreactivity of GluA1, GluA2 and GluA4 was evaluated by Western blot and immunocytochemistry. The levels of these subunits at the plasma membrane were evaluated by biotinylation and purification of plasma membrane-associated proteins. Elevated glucose concentration increased the total levels of GluA2 subunit of AMPA receptors in retinal neural cells, but not of the subunits GluA1 or GluA4. However, at the plasma membrane, elevated glucose concentration induced an increase of all AMPA receptor subunits. In cultured hippocampal neurons, elevated glucose concentration did not induce significant alterations in the levels of AMPA receptor subunits. In the retinas of diabetic rats there were no persistent changes in the levels of AMPA receptor subunits comparing to aged-matched control retinas. Also, no consistent changes were detected in the levels of GluA1, GluA2 or GluA4 in the hippocampus of diabetic rats. We demonstrate that elevated glucose concentration induces early changes in AMPA receptor subunits, mainly in GluA2 subunit, in retinal neural cells. Conversely, hippocampal neurons seem to remain unaffected by elevated glucose concentration, concerning the expression of AMPA receptors, suggesting that AMPA receptors are more susceptible to the stress caused by elevated glucose concentration in retinal cells than in hippocampal neurons.

Calcium dobesilate (CaD) has been prescribed to some patients in the early stages of diabetic retinopathy to delay its progression. We previously reported that the treatment of diabetic animals (4 weeks of diabetes) with CaD, during the last 10 days of diabetes, prevents blood-retinal barrier breakdown. Here, we aimed to investigate whether later treatment of diabetic rats with CaD would reverse inflammatory processes in the retina. Diabetes was induced with streptozotocin, and 6 weeks after diabetes onset, CaD (100 mg/kg/day) was administered for 2 weeks. The treatment with CaD significantly increased glial fibrillary acidic protein (GFAP) levels in the retina of nondiabetic animals (138.6 ± 12.8% of control) and enhanced the diabetes-induced increase in GFAP levels (174.8 ± 5.6% of control). In addition, CaD prevented the increase in mRNA and protein expression of tumor necrosis factor and interleukin-1β, as well as the formation of oxidized carbonyl residues and the increase in nitrotyrosine immunoreactivity, particularly in the ganglion cell layer of diabetic animals. We demonstrate that the treatment of diabetic animals with CaD can reverse the established proinflammatory processes in the retina. These beneficial effects appear to be attributed, at least partially, to the antioxidant properties of CaD.

Background: An intricate interplay between innate and adaptive immune cells is crucial for an effective immune response during disease, infection and vaccination. This interplay is mainly performed by dendritic cells (DCs), which are professional antigen presenting cells with unparalleled capacity to translate innate to adaptive immunity. They effectively recognize and uptake antigens, migrate to lymphoid tissues, and activate naïve T-cells. Indeed, DCs have numerous germline encoded pattern recognition receptors (PRR) that recognize conserved pathogen associated molecular patterns (PAMPs) or danger associated molecular patterns (DAMPs). While some PRRs like Toll-like receptors (TLRs) recognize PAMPs and DAMPs at the cell surface and in endosomal/lysosomal compartments, others, such as NOD-like receptors (NLRs), act as cytosolic sensors. NLRs activation through recognition of PAMPs and DAMPs leads to the assembly of signaling multimeric protein complexes named inflammasomes. Inflammasomes are important regulators of caspase 1, the enzyme responsible for the proteolytically cleavage of precursors’ pro-IL-1β and pro-IL-18 into their active form. Keywords: Adjuvanticity, dendritic cells, inflammasome, NLR, TLR, NLRP3, DAMPs.

As part of our ongoing study on the valorization of aromatic plants, the present study was designed to elucidate the composition, scavenging potential, anti-inflammatory activity and cytotoxicity of Margotia gummifera essential oils. Umbels were submitted to hydrodistillation in a Clevenger-type apparatus and the oils were analyzed by GC and GC–MS. For the anti-inflammatory activity, an in vitro model of lipopolysaccharide-stimulated macrophages was used and the inhibition of nitric oxide (NO) production was quantified through the Griess reagent, in the presence of the essential oil or its main compounds. NO scavenging potential was assessed using an NO donor and the cytotoxicity was evaluated on macrophages, keratinocytes and alveolar epithelial cells. The oils were characterized by high contents of monoterpene hydrocarbons, being the major compounds myrcene (20.4–23.0%) and sabinene (21.0–23.5%). The oil, myrcene and sabinene significantly inhibited NO production without affecting cell viability and showed a very effective NO scavenging potential, sabinene being the most active compound. These results suggest that M. gummifera essential oil, sabinene and myrcene should be explored as a natural source of new antioxidant and anti-inflammatory drugs for the development of food supplements, nutraceuticals or plant-based medicines.

A new C15-acetogenin, sagonenyne (20), exhibiting an unusual single tetrahydropyran ring was isolated from an ethyl acetate extract of Laurencia obtusa collected on the Corsican coastline. Its structure was established by detailed NMR spectroscopic analysis, mass spectrometry, and comparison with literature data. Twenty-three known compounds were identified in the same extract by means of column chromatography steps, using a 13C-NMR computer aided method developed in our laboratory. In addition to sesquiterpenes, which represent the main chemical class of this extract, diterpenes, sterols, and C15-acetogenins were identified. The crude extract was submitted to a cytotoxicity assay and was particularly active against THP-1 cells, a human leukemia monocytic cell line.

The impairment of glutamatergic neurotransmission has been associated with diabetic complications in the central nervous system, such as diabetic retinopathy. Here, we investigated the effect of elevated glucose exposure and diabetes on α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor composition, subunit phosphorylation, and the association of the GluA2 subunit with accessory proteins in the retina.The subunit composition of AMPA receptors and the association of the GluA2 subunit with modulatory proteins were evaluated with coimmunoprecipitation in retinal neural cell cultures and in the retina of experimentally induced-diabetic rats. The phosphorylation status of AMPA receptor subunits was evaluated with western blotting.In retinal neural cell cultures, elevated glucose did not significantly alter the composition of AMPA receptors, namely, the interactions between the GluA1, GluA2, and GluA4 subunits, but reduced GluA2 association with GRIP1. Moreover, elevated glucose did not cause changes on the level of GluA1 phosphorylated at serine residues 831 and 845. Diabetes induced early transitory changes in the interaction between AMPA receptor subunits GluA1, GluA2, and GluA4. At 8 weeks of diabetes, the content of GluA1 phosphorylated at serine 831 or serine 845 in the retina increased, compared to age-matched controls.Taken together, these results suggest that diabetes induces dynamic changes in AMPA receptor subunit composition, which could affect glutamatergic transmission in the rat retina.

Uncaria tomentosa (Ut), commonly known as Cat's claw or “uña de gato”, is a medicinal plant with immunostimulant, cytotoxic and antioxidant properties. The aim of this work is to investigate the immunostimulant activity of Ut bark decoction and its tannin-rich fraction (TF) on human macrophages. Ut decoction and TF were obtained as previously [1]. The production of inflammatory-related cytokines, namely interleukin (IL)-1β, IL-6, IL-8, IL-10, IL-12 (p70), granulocyte-macrophage colony-stimulating factor (GM-CSF), chemokine (C-C motif) ligand (CCL) 2, CCL3, CCL4, and tumor necrosis factor (TNF)-α, in the culture medium of human macrophages treated with Ut decoction and TF, were determined by Bio-Plex suspension array system (Bio-Rad, Hercules, CA). We found that Ut decoction increases the expression of all the mentioned cytokines, revealing an immunostimulant action. Tannins partially contributed to this activity. In conclusion, our data supports the traditional use of Uncaria tomentosa as immunostimulant plant and points its tannins as immunomodulating compounds that could play a significant role in human disease prevention and treatment.

As part of our ongoing work on the valorization of Portuguese lavenders we explored both the anti-inflammatory potential of Lavandula viridis essential oil (EO) and its mechanism of action.

Body mass index has been studied as one of the factors that negatively influences COVID-19. In this work we intend to analyze this influence. A representative sample of the population of Beira Interior was used (around 2%), to which immunity research and a socio-demographic survey were carried out. It was found that obesity influences the vaccination rate, and all other variables analyzed were not influenced by the body mass index.

Introduction: SARS-CoV-2 affects the epithelial cells of the respiratory tract, causing severe infections. Since it is a pathology with a high level of transmissibility, it becomes central to mass testing. In addition, there was also a need to monitor the epidemic through serological tests. Objective: Evaluate the presence of antibodies against SARS-CoV-2 in the community residing in Beira Baixa through immunological screening tests. Materials and methods: Analytical, cross-sectional, and observational study, whose sample consists of 206 individuals. Data collection took place between February and April 2021, in the laboratories of the Dr. Lopes Dias Higher School of Health. Verbal informed consent, a questionnaire to collect sociodemographic data and the serological test were applied. Results: Of the total number of participants, 15.5% admitted to having had COVID-19, of which 0.5% suspected they had been infected and 84% said they had never been infected. Regarding the presence of antibodies, 2.9% of the tests performed were positive for the presence of IgM's while 30.1% were positive for the presence of IgG's. Regarding vaccination, at the time of the investigation only 10.2% of the participants were vaccinated, of which 9.7% had IgG antibodies. Conclusion: Rapid serological tests can provide information about the presence of antibodies to SARS-CoV-2, thus being a very advantageous tool for immunity studies.

The thermal waters have numerous medicinal advantages, highlighting their anti-inflammatory and immuno-modulatory properties, useful in the treatment of various pathologies of the respiratory, gastrointestinal, muscoloskeletal, skin, among others (Prandelli et al, 2013; Zajac, 2021; Franz et al, 2021). Several studies demonstrate the importance that this type of water presents in cardiovascular pathologies, namely in its use as protectors of the most important risk factors for the triggering of these pathologies (Laukkanen et al, 2015; Laukkanen et al, 2018). Some authors report that balneotherapy is associated with normalization of lipid profile parameters (Heinonen &amp;; Laukkanen, 2018; Esperland et al, 2022) as well as reducing susceptibility to viral infections (Kunutsor et al., 2017; Kunutsor et al., 2021). There is also the component associated with skin and the delay of aging, as demonstrated in studies in the area (Vaz et al, 2022). Of course, there are studies that demonstrate the possibility of the existence of some intercurrences, with special incidence in the cardiac area and in the psychiatric area - note, however, that the studies referred to point to the pre-existence of pathology (Zaccardi et al, 2017; Laukkanen et al 2018).

Body mass index has been studied as one of the factors that negatively influences COVID-19. In this work, we intend to analyze this influence. A representative sample of the population of Beira Interior was used (around 2%), on which immunity research and a socio-demographic survey were carried out. It was found that obesity influences the vaccination rate, and that all other variables analyzed were not influenced by body mass index.

Alzheimer´s disease is a neurodegenerative disease and the most common cause of dementia with no cure or treatment. Therefore, the investigation to find a disease-modifying therapeutic is crucial. From a pathophysiological point of view, AD is characterized by the loss of homeostatic functions that control redox and energy metabolism, neuroinflammation, and proteostasis. The transcription factor nuclear factor erythroid 2–related factor 2 (Nrf2) is a master controller of these functions and, in the past decades, recent reports have shown that its overall activity is compromised in AD. Thus, Nrf2 has been considered an attractive molecular target for AD therapeutic research. Most pharmacological Nrf2 activators are small electrophilic molecules that react with cysteine residues present in Kelch-like ECH-Associating protein 1 (Keap1), thereby inducing Nrf2 release and further nuclear translocation and activation. Accordingly, low molecular weight skin allergens, such as dimethyl fumarate, currently used to treat the relapsing forms of multiple sclerosis and with positive results in preventing spatial memory impairments and hippocampal neurodegeneration in rats, are able to activate Nrf2. This activation is well explained by their direct reactivity towards key cysteine residues of Keap1, leading to its dissociation from Nrf2, which in turn is translocated to the nucleus inducing the transcription of over 250 protective genes. Hence, we conducted a ground-breaking study where the potential of Isoeugenol, a skin allergen with electrophilic properties, to activate Nrf2 and revert some AD hallmarks, was investigated. This work was conducted in vitro (in mice microglia cells exposed to LPS and neuronal cells overexpressing the human APP with Swedish mutation, N2a-APPswe) and in vivo (in the AD double transgenic mice, APP/PS1, intranasally administered with Isoeugenol), at an early (6-month-old animals) and late (11-month-old animals) AD stage. Overall, the results showed that Isoeugenol exhibit a good pharmacokinetic and pharmacodynamic profile. Isoeugenol activates Nrf2 and displays antioxidant and anti-inflammatory effects and reduced the levels of Aβ peptides in in vitro and in vivo models of AD. In addition, its positive effect on metabolism was also demonstrated in vivo, as it reduced the triglyceride and LDL cholesterol levels in treated AD mice. Importantly, Isoeugenol improved the memory deficits observed in APP/PS1 mice, which was more evident in older animals (11-month-old), reinforcing its potential in ameliorating AD hallmarks, even at a late stage.

Historically, extracts and preparations of plants are the basis of traditional medicine and the starting point for the discovery of new therapeutic agents [1]. Cytisus multiflorus and Eriocephalus africanus are small shrubs native from Iberian Peninsula and South Africa, respectively, and distributed in Mediterranean region. Despite their common application in folk medicine and claimed health benefits, including antioxidant and anti-inflammatory properties [2, 3, 4], there is still a lack of scientific data supporting this [5, 6]. In this work, phenolic-enriched extracts of Cytisus multiflorus and Eriocephalus africanus were obtained and evaluated in chemical models for their phenolic content, reducing power capacity, scavenging ability for DPPH radical, nitric oxide and hypochlorous acid, as well as for their ability to inhibit 5-Lipoxygenase activity i.e., a central enzyme in inflammatory process. Overall, the results from the chemical tests indicated that Cytisus multiflorus extract was more promising regarding antioxidant and anti-inflammatory properties reason why this extract was then further tested for these activities in biological models. The present communication will focus on the experimental data obtained so far by our group for the phenolic-enriched extracts of Cytisus multiflorus and Eriocephalus africanus, as a valid contribution to clarify the mechanisms of action of the claimed antioxidant and anti-inflammatory activities of the two plants.

Quercus suber L. cork has various industrial applications and from these activities many by-products are formed. Different phytoconstituents, namely polyphenols were identified1, and significant anti-inflammatory properties have been attributed to some polyphenols. In order to increase value to the cork industrial wastes, the aim of the current study was to evaluate the effect of a polyphenol-rich extract from a cork by-product on the production and/or scavenging of the pro-inflammatory mediator nitric oxide (NO) and on inducible NO synthase (iNOS) expression. A hydroalcoholic extract – EXTRACTCORK AM – was used in the macrophage cell line RAW 264.7 for evaluation of NO production by Griess reaction, cytotoxicity by MTT and iNOS expression by Western blot. NO scavenging activity was also assessed using S-nitroso-N-acetylpenicillamine (SNAP), a NO donor. Phytochemical characterization was performed by HPLC-PDA-ESI/MSn. Ellagitannins were the main polyphenols in the analyzed extract. For no cytotoxic concentration this extract inhibited the production of NO, as well as iNOS expression triggered by the potent immunostimulator lipopolysaccharide (LPS). NO scavenging activity was not observed, suggesting that the anti-inflammatory properties verified for this extract are due to inhibition of iNOS expression instead of scavenging of NO. In conclusion, our results show that the extract obtained from an industrial cork by-product has significant anti-inflammatory activity and can be a valuable source of molecules for the treatment of inflammatory diseases.

Agrimony (Agrimonia eupatoria L.) is used in traditional medicine as infusion, decoction and tincture for oxidative stress-related diseases. In our previous works, we found that agrimony infusion has significant scavenging capacity against the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical, eventually related to their phenolic compounds, namely tannins and flavonoids, such as flavan-3-ols, flavonols and flavones derivatives1. The aim of this work was to evaluate the contribution of the phenolic compounds for the antioxidant activity, by using two different radical scavenging assays: DPPH and superoxide anion. A polyphenol-enriched fraction (AePEF) and two sub-fractions (FI, containing phenolic acids and tannins, and FII, containing flavonoids) were obtained from the infusion fractioning. Both AePEF and FII exhibited a marked activity against the DPPH and superoxide radicals. In conclusion, our results point that the agrimony flavonoids significantly contribute for antioxidant activity, which could be good candidates for the treatment of inflammation and cancer.

Plant polyphenols are well-known antioxidants, and recent studies have reported that they play an important role in prevention and treatment of oxidative-related disorders, such as inflammatory diseases and cancer.

Purpose Calcium dobesilate (CaD) is used in the treatment of diabetic retinopathy, but its mechanism of action is not completely elucidated. We investigated the molecular and cellular mechanisms underlying the protective effects of CaD against the blood-retinal barrier (BRB) breakdown induced by diabetes. Methods Wistar rats were divided into: controls, diabetic (1 month diabetes duration) and diabetic treated with CaD (100 mg/kg/day; orally given) during the last 10 days. The BRB breakdown was evaluated using Evans blue. The content or distribution of occludin, claudin-5, ZO-1, ICAM-1 and p38 MAPK was evaluated by western blotting and immunohistochemistry. Leukocyte adhesion was evaluated in retinal vessels and in vitro. Oxidative stress was evaluated by the detection of oxidized carbonyls and tyrosine nitration. NF-κB activation was measured by ELISA. Results Diabetes increased the BRB permeability and retinal thickness, decreased occludin and claudin-5 levels, and altered the distribution of ZO-1 and occludin in retinal vessels. CaD inhibited these changes, as well as the increase in ICAM-1 levels and leukocyte adhesion to retinal vessels or endothelial cells induced by diabetes or high glucose. Moreover, CaD decreased oxidative stress, and p38 MAPK and NF-κB activation caused by diabetes. Conclusion CaD prevents the BRB breakdown induced by diabetes by restoring tight junction protein levels and organization and decreasing leukocyte adhesion to retinal vessels. The protective effects of CaD are likely to involve the inhibition of p38 MAPK and NF-κB activation, possibly through the inhibition of oxidative/nitrosative stress. Support: OM Pharma SA, Switzerland

se "reinventar" para atender ao novo cenário.

se "reinventar" para atender ao novo cenário.

Abstract Introduction The high emergency in the control of the pandemic has determined that the scientific community is dedicated to the production of effective vaccines for immunization against SARS-CoV-2. With the emergence of new variants of SARS-CoV-2, there was a need to increase immunity in the vaccinated population through a booster dose. The booster dose is allowed to increase the immunity of individuals and, consequently, reduce the probability of reinfection, as well as severe symptoms associated with COVID-19. Objective To evaluate the acquired immunity to COVID-19 through vaccination, and to verify the influence of the booster dose on the antibody’s concentration. Materials and Methods Data were collected from 965 individuals of both genders and vaccinated for COVID-19. Results 91.8% of the sample had neutralizing antibodies against SARS-CoV-2, and the group of fully vaccinated individuals had a higher percentage of neutralized antibodies compared to individuals who did not have a booster dose (53.2% vs. 38 .7%). Discussion Neutralizing antibodies were found in 91.8% of the sample, which demonstrates the effectiveness of vaccination in the production of antibodies. The results demonstrated the effectiveness of the booster dose in the production of neutralizing antibodies against SARS-CoV-2. Conclusion Vaccination was quite effective in producing neutralizing antibodies. In addition, booster vaccination increased humoral immunity.