Anti-hyperalgesic effects of photobiomodulation therapy (904 nm) on streptozotocin-induced diabetic neuropathy imply MAPK pathway and calcium dynamics modulation.

Several recent studies have established the efficacy of photobiomodulation therapy (PBMT) in painful clinical conditions. Diabetic neuropathy (DN) can be related to activating mitogen-activated protein kinases (MAPK), such as p38, in the peripheral nerve. MAPK pathway is activated in response to extracellular stimuli, including interleukins TNF-α and IL-1ß. We verified the pain relief potential of PBMT in streptozotocin (STZ)-induced diabetic neuropathic rats and its influence on the MAPK pathway regulation and calcium (Ca2+) dynamics. We then observed that PBMT applied to the L4-L5 dorsal root ganglion (DRG) region reduced the intensity of hyperalgesia, decreased TNF-α and IL-1ß levels, and p38-MAPK mRNA expression in DRG of diabetic neuropathic rats. DN induced the activation of phosphorylated p38 (p-38) MAPK co-localized with TRPV1+ neurons; PBMT partially prevented p-38 activation. DN was related to an increase of p38-MAPK expression due to proinflammatory interleukins, and the PBMT (904 nm) treatment counteracted this condition. Also, the sensitization of DRG neurons by the hyperglycemic condition demonstrated during the Ca2+ dynamics was reduced by PBMT, contributing to its anti-hyperalgesic effects.

The edematogenic effect of Micrurus lemniscatus venom is dependent on venom phospholipase A2 activity and modulated by non-neurogenic factors.

Coral snakes mainly cause neurotoxic symptoms in human envenomation, but experimental studies have already demonstrated several pharmacological activities in addition to these effects. This investigation was carried out with the aim of evaluating (1) non-neurogenic mechanisms involved in the inflammatory response induced by Micrurus lemniscatus venom (MLV) in rat hind paws, (2) participation of PLA2 in this response, and (3) neutralizing efficiency of commercial anti-elapid antivenom on edema. MLV promoted a rapid, significant increase in vascular permeability, influx of leukocytes, and disorganization of collagen bundles, as demonstrated by histological analysis. Several pretreatments were applied to establish the involvement of inflammatory mediators in MLV-induced edema (5 µg/paw). Treatment of animals with chlorpromazine reduced MLV-induced edema, indicating participation of TNF-α. However, the inefficiency of other pharmacological treatments suggests that eicosanoids, leukotrienes, and nitric oxide have no role in this type of edema formation. In contrast, PAF negatively modulates this venom-induced effect. MLV was recognized by anti-elapid serum, but this antivenom did not neutralize edema formation. Chemical modification of MLV with p-bromophenacyl bromide abrogated the phospholipase activity and markedly reduced edema, demonstrating PLA2 participation in MLV-induced edema. In conclusion, the non-neurogenic inflammatory profile of MLV is characterized by TNF-α-mediated edema, participation of PLA2 activity, and down-regulation by PAF. MLV induces an influx of leukocytes and destruction of collagen fibers at the site of its injection.

Evaluation of Protection by Caffeic Acid, Chlorogenic Acid, Quercetin and Tannic Acid against the In Vitro Neurotoxicity and In Vivo Lethality of Crotalus durissus terrificus (South American Rattlesnake) Venom.

Systemic envenomation by Crotalus durissus terrificus (South American rattlesnake) can cause coagulopathy, rabdomyolysis, acute kidney injury, and peripheral neuromuscular blockade, the latter resulting in flaccid paralysis. Previous studies have shown that plant products such as tannic acid and theaflavin can protect against the neuromuscular blockade caused by C. d. terrificus venom in vitro. In this work, we used mouse-isolated phrenic nerve-diaphragm preparations to examine the ability of caffeic acid, chlorogenic acid, and quercetin to protect against C. d. terrificus venom-induced neuromuscular blockade in vitro. In addition, the ability of tannic acid to protect against the systemic effects of severe envenomation was assessed in rats. Preincubation of venom with caffeic acid (0.5 mg/mL), chlorogenic acid (1 mg/mL), or quercetin (0.5 mg/mL) failed to protect against venom (10 µg/mL)-induced neuromuscular blockade. In rats, venom (6 mg kg-1, i.p.) caused death in ~8 h, which was prevented by preincubation of venom with tannic acid or the administration of antivenom 2 h post-venom, whereas tannic acid given 2 h post-venom prolonged survival (~18.5 h) but did not prevent death. Tannic acid (in preincubation protocols or given 2 h post-venom) had a variable effect on blood creatinine and urea and blood/urine protein levels and prevented venom-induced leukocytosis. Tannic acid attenuated the histological lesions associated with renal damage in a manner similar to antivenom. The protective effect of tannic acid appeared to be mediated by interaction with venom proteins, as assessed by SDS-PAGE. These findings suggest that tannic acid could be a potentially useful ancillary treatment for envenomation by C. d. terrificus.

Benefits of Sebastiania hispida (Euphorbiaceae) extract and photobiomodulation therapy as potentially adjunctive strategies to be explored against snake envenoming.

The purpose of this study was to assess the topic use of Sebastiania hispida extract and low-level gallium-arsenide laser irradiation (GaAs, 904 nm) to reduce the local myonecrosis and edema of Bothrops moojeni snake venom-injected gastrocnemius. Wistar rats receiving intramuscular venom injection (VBm) were compared with saline control (S) and envenomed rats receiving local exposure to plant extract (VExt) or laser irradiation (VL). The phytochemistry and thin-layer chromatography of S. hispida extract indicated the presence of phenolic compounds like gallic acid and flavonoids including quercetin. Gastrocnemius of VExt and VL groups had a significant reduction of edema and creatine kinase (CK) activities and a greater Myogenin (MyoG) expression compared to VBm group, with the plant extract efficacy better than laser exposure. Reduction of edema and serum CK activities reflects a lessening of muscle damage, whereas the increase of MyoG indicates myoblast differentiation and acceleration of muscle repair. The S. hispida richness in phenolic compounds and flavonoids, such as the light modulatory ability to triggering a multitude of cell signalings likely underlie the positive outcomes. Our findings suggest both treatments as potential auxiliary tools to be explored in clinical trials in combination with anti-venom therapy after Bothropic snakebites.

Exploring the ability of low-level laser irradiation to reduce myonecrosis and increase Myogenin transcription after Bothrops jararacussu envenomation.

Envenoming caused by snakebites is a very important neglected tropical disease worldwide. The myotoxic phospholipases present in the bothropic venom disrupt the sarcolemma and compromise the mechanisms of energy production, leading to myonecrosis. Photobiomodulation therapy (PBMT) has been used as an effective tool to treat diverse cases of injuries, such as snake venom-induced myonecrosis. Based on that, the aim of this study was to analyze the effects of PBMT through low-level laser irradiation (904 nm) on the muscle regeneration after the myonecrosis induced by Bothrops jararacussu snake venom (Bjssu) injection, focusing on myogenic regulatory factors expression, such as Pax7, MyoD, and Myogenin (MyoG). Male Swiss mice (Mus musculus), 6-8-week-old, weighing 22 ± 3 g were used. Single sub-lethal Bjssu dose or saline was injected into the right mice gastrocnemius muscle. At 3, 24, 48, and 72 h after injections, mice were submitted to PBMT treatment. When finished the periods of 48 and 72 h, mice were euthanized and the right gastrocnemius were collected for analyses. We observed extensive inflammatory infiltrate in all the groups submitted to Bjssu injections. PBMT was able to reduce the myonecrotic area at 48 and 72 h after envenomation. There was a significant increase of MyoG mRNA expression at 72 h after venom injection. The data suggest that beyond the protective effect promoted by PBMT against Bjssu-induced myonecrosis, the low-level laser irradiation was able to stimulate the satellite cells, thus enhancing the muscle repair by improving myogenic differentiation.

VEGF/VEGFR-2 system exerts neuroprotection against Phoneutria nigriventer spider envenomation through PI3K-AKT-dependent pathway.

This study was undertaken to elucidate why VEGF/VEGFR-2 is elevated in the hippocampus of rats injected with Phoneutria nigriventer spider venom (PNV). PNV delays Na+ channels inactivation; blocks Ca2+ and K+ channels, increases glutamate release, causes blood-brain breakdown (BBBb), brain edema and severe excitotoxicity. Analytical FT-IR spectroscopy showed profound alteration in molecular biochemical state, with evidences for VEGFR-2 (KDR/Flk-1) signaling mediation. By blocking VEGF/VEGFR-2 binding via pre-treatment with itraconazole we demonstrated that animals' condition was deteriorated soon at 1-2 h post-PNV exposure concurrently with decreased expression of VEGF, BBB-associated proteins, ZO-1, ß-catenin, laminin, P-gp (P-glycoprotein), Neu-N (neuron's viability marker) and MAPKphosphorylated-p38, while phosphorylated-ERK and Src pathways were increased. At 5 h and coinciding with incipient signs of animals' recuperation, the proteins associated with protection (HIF-1α, VEGF, VEGFR-1, VEGFR-2, Neu-N, occludin, ß-catenin, laminin, P-gp efflux protein, phosphorylated-p38) increased thus indicating p38 pathway activation together with paracellular route strengthening. However, the BBB transcellular trafficking and caspase-3 increased (pro-apoptotic pathway activation). At 24 h, the transcellular route reestablished physiological state but the pro-survival pathway PI3K/(p-Akt) dropped in animals underwent VEGF/VEGFR-2 binding inhibition, whereas it was significantly activated at matched interval in PNV group without prior itraconazole; these results demonstrate impaired VEGF' survival effects at 24 h. The inhibition of VEGF/VEGFR-2 binding identified 5 h as turning point at which multi-level dynamic interplay was elicited to reverse hippocampal damage. Collectively, the data confirmed VEGFR-2 signaling via serine-threonine kinase Akt as neuroprotective pathway against PNV-induced damage. Further studies are needed to elucidate mechanisms underlying PNV effects.

Muscle proteolysis via ubiquitin-proteasome system (UPS) is activated by BthTx-I Lys49 PLA2 but not by BthTx-II Asp49 PLA2 and Bothrops jararacussu venom.

Bites by viperid snakes belonging to Bothrops genus produce fast and intense local edema, inflammation, bleeding and myonecrosis. In this study, we investigated the role of Myogenic Regulatory Factors (MRFs: MyoD; Myog), negatively regulated by GDF-8 (Myostatin), and ubiquitin-proteasome system pathway (UPS: MuRF-1; Fbx-32) in gastrocnemius muscle regeneration after Bothrops jararacussu snake venom (Bjussu) or its isolated phospholipase A2 myotoxins, BthTx-I (Lys-49 PLA2) and BthTx-II (Asp-49 PLA2) injection. Male Swiss mice received a single intra-gastrocnemius injection of crude Bjussu, at a dose/volume of 0.83 mg/kg/20 µl, and BthTx-I or BthTx-II, at a dose/volume of 2.5 mg/kg/20 µl. Control mice (Sham) received an injection of sterile saline solution (NaCl 0.9%; 20 µl). At 24, 48, 72 and 96 h post injection, right gastrocnemius was collected for protein expression analyses. Based on the temporal expressional dynamics of MyoD, Myog and GDF-8/Myostatin, it was possible to propose that the myogenesis pathway was impacted most badly by BthTx-II followed by BthTx-I and lastly by B. jararacussu venom, thus suggesting that catalytic activity has likely inhibitory role on the satellite cells-mediated reparative myogenesis pathway. Inversely, the catalytic activity seems to be not a determinant for the activation of proteins ubiquitination by MuRF-1 and Fbx-32/Atrogin-1 E3 proteasome ligases, given proteolysis pathway through UPS was activated neither after Bjussu, nor after BthTx-II, but just after the catalytically-inactive BthTx-I Lys-49 PLA2-homologue exposure. The findings of this study disclose interesting perspective for further mechanistic studies about pathways that take part in the atrophy and repair after permanent damage induced by bothropic snakebites.

Gait analysis correlates mechanical hyperalgesia in a model of streptozotocin-induced diabetic neuropathy: A CatWalk dynamic motor function study.

Peripheral neuropathy is a complication of diabetes commonly associated with pain and decline in motor compound action potential, leading to alterations in plantar pressure during gait. We identified motor impairments in streptozotocin (STZ)-induced diabetic neuropathic rats and correlated with mechanical withdrawal thresholds, establishing this correlation as a complementary method to investigate the development of chronic hyperalgesia in diabetic neuropathy. METHODS: UNICAMP's Ethics Committee (protocol number 3902-1) approved all experiments. Male Lewis rats (200-250 g) received a STZ-low-dose (25 mg/kg/day) (STZ group) or 0.1 M sodium citrate buffer (SCB, control group) once a day, during five consecutive days. Diabetic rats (250 mg/dL blood glucose) were submitted to electronic von Frey and CatWalk tests at 0, 7, 14, 21, and 28 days after treatment. RESULTS: STZ, but not SCB, induced diabetes. After the 14th day (STZ)-induced diabetic rats showed mechanical hyperalgesia and a reduction in the hind limbs footprint intensities. At the 28th day, rats presented alterations in spatial parameters (Maximum Contact Area; Stride Length; Print Area), which showed a strong correlation with mechanical withdrawal thresholds (r2 = 0.97; 0.99, and 0.93, respectively). CONCLUSIONS: Correlation between gait parameters and mechanical withdrawal thresholds enables a better experimental approach to evaluate the development of chronic hyperalgesia in the STZ-induced diabetes model. It allows a concise crosstalk of motor and sensorial functions, which are usually analyzed individually. CatWalk gait parameters can be used as a complementary tool to investigate the development of hyperalgesia in STZ-induced diabetic neuropathic rats.

The in vivo toxicological profile of cationic solid lipid nanoparticles.

Cationic solid lipid nanoparticles (cSLNs) are considered as one of the most effective lipid nanocarriers for delivery of low water-solubility compounds and genetic materials. As the excipients used in the cSLN production are generally regarded as safe (GRAS), the formulations are granted as non-toxic. However, the toxicological profile of new SLN-based formulations should always be performed to confirm that the delivery systems themselves may not impose risks to the human health. Therefore, in this study, we delineate the toxicological profile of the cSLN formulation at 24 and 72 h after single intravenous injection to male Wistar rats. Hematological, biochemical, and histopathological evaluations of the spleen, lungs, liver, and kidneys indicated short-lived alterations including neutrophilia. We found increases in the population of macrophages in the lungs, liver, and spleen and also migration of circulating neutrophils into inflamed tissue and a decrease in blood urea nitrogen. We also observed the presence of cSLNs within the brain parenchyma without any sign of damage to the blood-brain barrier. These side effects appeared to be mild and transitory (< 72 h). These findings reinforce the importance of investigating the toxicity of SLN-based formulations before the incorporation of drugs/genetic material to the formulation and its translation to the clinic.

Aqueous Extract of Brazilian Berry (Myrciaria jaboticaba) Peel Improves Inflammatory Parameters and Modulates Lactobacillus and Bifidobacterium in Rats with Induced-Colitis.

Natural compounds could be a complementary alternative to inflammatory bowel disease (IBD) management. This study determined the effects of an aqueous extract of Myrciaria jaboticaba peel (EJP) (50 g L-1) on 2,4,6-trinitrobenzenesulfonic acid-induced colitis. Wistar rats were randomized into five groups: HC-healthy control, CC-colitis control, DC-drug control, SJ-short-term treatment with EJP, and LJ-long-term treatment with EJP. The EJP treatments reduced body weight loss, stool consistency score, and spleen enlargement. Gut microbiota was modulated through increased Lactobacillus and Bifidobacterium counts after EJP treatment. Short-chain fatty acids were also higher in the EJP treatment groups. The antioxidant enzyme activities were greater than CC or DC controls. Myeloperoxidase activity (LJ), inducible nitric oxide synthase (LJ/SJ), and intercellular adhesion molecule (SJ) levels were lower than in the CC group. EJP decreased histological scoring, mucosal thickness, and preserved the crypts and histological structure. Therefore, EJP showed beneficial effects and could be potentially used as an adjuvant in IBD treatment.

Venom of the Phoneutria nigriventer spider alters the cell cycle, viability, and migration of cancer cells.

The mechanisms of cancer involve changes in multiple biological pathways. Multitarget molecules, which are components of animal venoms, are therefore a potential strategy for treating tumors. The objective of this study was to screen the effects of Phoneutria nigriventer spider venom (PnV) on tumor cell lines. Cultured human glioma (NG97), glioblastoma (U-251) and cervix adenocarcinoma (HeLa) cells, and nontumor mouse fibroblasts (L929) were treated with low (14 µg/ml) and high (280 µg/ml) concentrations of PnV, and analyzed through assays for cell viability (thiazolyl blue tetrazolium blue), proliferation (carboxyfluorescein succinimidyl ester), death (annexin V/propidium iodide [Pi]), the cell cycle (Pi), and migration (wound healing and transwell assay). The venom decreased the viability of U-251 cells, primarily by inducing cell death, and reduced the viability of NG97 cells, primarily by inhibiting the cell cycle. The migration of all the tumor cell lines was delayed when treated with venom. The venom significantly affected all the tumor cell lines studied, with no cytotoxic effect on normal cells (L929), although the nonglial tumor cell (HeLa) was less sensitive to PnV. The results of the current study suggest that PnV may be composed of peptides that are highly specific for the multiple targets involved in the hallmarks of cancer. Experiments are underway to identify these molecules.

Inhibition of VEGF-Flk-1 binding induced profound biochemical alteration in the hippocampus of a rat model of BBB breakdown by spider venom. A preliminary assessment using FT-IR spectroscopy.

Phoneutria nigriventer spider venom (PNV) contains ion channels-acting neuropeptides that in rat induces transitory blood-brain barrier breakdown (BBBb) in hippocampus in parallel with VEGF upregulation. We investigated whether VEGF has a neuroprotective role by inhibiting its binding to receptor Flk-1 by itraconazole (ITZ). FT-IR spectroscopy examined the biochemical status of hippocampus and evaluated BBBb in rats administered PNV or ITZ/PNV at periods with greatest toxicity (1-2h), recovery (5h) and visual absence of symptoms (24h), and compared to saline and ITZ controls. The antifungal treatment before venom intoxication aggravated the venom effects and increased BBB damage. FT-IR spectra of venom, hippocampi of controls, PNV and ITZ-PNV showed a 1400 cm-1 band linked to symmetric stretch of carboxylate and 1467 cm-1 band (CH2 bending: mainly lipids) that were considered biomarker and reference bands, respectively. Inhibition of VEGF/Flk-1 binding produced marked changes in lipid/protein stability at 1-2h. The largest differences were observed in spectra regions assigned to lipids, both symmetric (2852 cm-1) and asymmetric (2924 and 2968 cm-1). Quantitative analyses showed greatest increases in the 1400 cm-1/1467 cm-1 ratio also at 1h. Such changes at period of rats' severe intoxication referred to wavenumber region from 3106 cm-1 to 687 cm-1 assigning for C-H and N-H stretching of protein, Amide I, C=N cytosine, N-H adenine, Amide II, CH2 bending: mainly lipids, C-O stretch: glycogen, polysaccharides, glycolipids, z-type DNA, C-C, C-O and CH out-of-plane bending vibrations. We conclude that VEGF has a neuroprotective role and can be a therapeutic target in PNV envenomation. FT-IR spectroscopy showed to be instrumental for monitoring biochemical changes in this model of P. nigriventer venom-induced BBB disruption.

PnTx2-6 (or δ-CNTX-Pn2a), a toxin from Phoneutria nigriventer spider venom, releases l-glutamate from rat brain synaptosomes involving Na+ and Ca2+ channels and changes protein expression at the blood-brain barrier.

PhTx2 is the most toxic fraction from the venom of the spider Phoneutria nigriventer, being responsible to sodium entry into cortical synaptosomes, increasing the release of neurotransmitters, such as l-glutamate (L-Glu) and; acetylcholine. In this study, we investigated the action of a toxin purified from; PhTx2 fraction, called PnTx2-6 or δ-CNTX-Pn2a, on L-Glu release from rat; brain cortex synaptosomes, as well as its ability to induce blood-brain barrier permeability. PnTx2-6 increased L-Glu release from rat cortical brain synaptosomes in a time- and dose-dependent manner (EC50 = ∼20 nM; Tm = 16min), as measured by a fluorimetric method. The increase of L-Glu by PnTx2-6 was inhibited by tetrodotoxin. And partially inhibited by EGTA. Calcium channel blockers ω-conotoxin MVIIC (P/Q-types) and ω-conotoxin GVIA (N-type), were able to reduce the PnTx2-6-induced release of L-Glu, while nifedipine (L-type) did not show any inhibition. These findings suggest that thew release of L-Glu by PnTx2-6 is due its primary action on sodium channels, well-known to be target of this toxin. PnTx2-6 is able to potentiate penile erection and this effect may be related with the release of l-glutamate from the CNS, besides a local effect on corpus carvenosum, as previously shown by our group. If L-Glu release and penile erection potentiation are indeed correlated, then this toxin should be able to cross the blood brain barrier (BBB). Results by immunoblotting assays indicated a change in the expression of proteins associated with the paracellular and transcellular transport at the blood-brain barrier, suggesting a BBB dysfunction mediated by PnTx2-6. Therefore, PnTx2-6 may induce the release l-glutamate in the central nervous system, when injected peripherally.

Vibrational spectroscopy of muscular tissue intoxicated by snake venom and exposed to photobiomodulation therapy.

The pathogenesis of myonecrosis caused by myotoxins from bothropic venom is associated with local extracellular matrix (ECM) disintegration, hemorrhage, and inflammation. Search for alternative methods associated with serum therapy is mandatory to neutralize the fast development of local damage following snakebites. The experimental use of photobiomodulation therapy (PBMT) in murine models has shown promising results relative to structural and functional recovery from bothropic snakebite-induced myonecrosis. This study pioneered in using Raman and Fourier transform infrared (FTIR) spectroscopies to characterize biochemical alterations in the gastrocnemius that had been injected with Bothrops jararacussu venom and exposed to local PBMT. Results show that vibrational spectra from lyophilized and diluted venom (1307 cm -1) was also found in the envenomed gastrocnemius indicating venom presence in the unirradiated muscle 48 h post-injection; but any longer visible after PBMT at this time exposure or 72 h post-injection regardless irradiated or not. Raman and FTIR analyses indicated that the bands with higher area and intensity were 1657 and 1547 cm-1 and 1667 and 1452 cm-1, respectively; all are assignments for proteins, especially collagen, and are higher in the PBMT-exposed gastrocnemius. The infrared spectra suggest that laser treatment was able to change protein in tissue and that such change indicates collagen as the main target. We hypothesize that the findings reflect remodeling of ECM with key participation of collagen and faster tissue recovery for an anabolic condition.

Effects of photobiomodulation therapy on Bothrops moojeni snake-envenomed gastrocnemius of mice using enzymatic biomarkers.

Bothropic venom contains a range of biologically active substances capable of causing severe local and systemic envenoming symptomatology within its victims. The snake anti-venom is effective against systemic effects but has no neutralizing effect against the fast developing local effects. Herein, mice gastrocnemius injected with Bothrops moojeni venom (40 µg/kg) or saline solution were irradiated with HeNe (632.8 nm) and GaAs (904 nm) lasers (daily energy density of 4 J/cm2; 0.03/0.21 power density; 0.07/0.16 spot size; 1.2/0.04 total energy, 1 cm off contact, for HeNe and GaAs lasers, respectively) and euthanized in periods ranging from 3 h to 21 days. Blood biochemistry for creatine kinase (CK), alkaline phosphatase (ALP), acid phosphatase (AP), lactate dehydrogenase (LDH), aspartate transaminase (AST), and myoglobin and histopathological analysis, for assessing the degree of myonecrosis and regeneration of gastrocnemius, were done at every time interval. GaAs laser promoted faster photobiomodulation therapy (PBMT) effects, and the GaAs group exhibited a better clinical outcome than the HeNe group. Within the GaAs group, the serum levels of CK, LDH, AP, AST, and myoglobin, which were increased by the physiological effects of the venom, were reduced to initial baseline before snake envenomation in less time than those irradiated by the HeNe laser. However, the group receiving irradiation from the HeNe laser returned the levels of ALP activity to baseline faster than those of the GaAs group. Histopathological analysis revealed enhanced muscle regeneration in mice groups treated with both lasers. PBM promoted by GaAs and HeNe showed well-developed centrally nucleate regenerating cells and an increased number of newly formed blood vessels when compared to unirradiated muscle. We therefore suggest that GaAs had the best outcomes likely derived from a deeper penetrating longer wavelength. We conclude that PMBT is a promising, non-invasive approach to be further tested in pre-clinical studies with a goal to further its clinical use in skeletal muscle recovery in snakebite victims.

Jaboticaba berry peel intake prevents insulin-resistance-induced tau phosphorylation in mice.

The hyperphosphorylation of microtubule-associated protein tau (tau) in the hippocampus can be caused by central and peripheral insulin resistance and these alterations are related to the development of tauopathies, such as Alzheimer's disease. In this study, we used a high-fat diet to induce obesity and insulin resistance in adult Swiss mice and checked whether supplementation with Myrciaria jaboticaba berry peel for 10 weeks could improve insulin sensitivity, learning/memory performance, and prevent tau phosphorylation in the hippocampus. Furthermore, adipocytokines, inflammatory markers, and oxidative stress were assessed. Myrciaria jaboticaba peel has phenolic compounds (e.g., cyanidin, ellagic acid), dietary fiber and carotenoids, which contribute to great antioxidant capacity. Supplementation of the high-fat diet with 4% M. jaboticaba peel prevented fat weight gain and reduced peripheral insulin resistance. The treated group also showed lower tau phosphorylation in the hippocampus corroborating better learning/memory performance in the Morris water maze test. Maintenance of neuronal viability, lower levels of hippocampal inflammatory markers, and improved brain antioxidant defenses were also related to the consumption of M. jaboticaba peel. These findings contribute to a better understanding of how a high-fat diet supplemented with jaboticaba berry peel counteracts the impairment of cognitive functions caused by high-fat diet intake and diet-induced insulin resistance.

A survey on some biochemical and pharmacological activities of venom from two Colombian colubrid snakes, Erythrolamprus bizona (Double-banded coral snake mimic) and Pseudoboa neuwiedii (Neuwied's false boa).

Colombian colubrid snake venoms have been poorly studied. They represent a great resource of biological, ecological, toxinological and pharmacological research. We assessed some enzymatic properties and neuromuscular effects of Erythrolamprus bizona and Pseudoboa neuwiedii venoms from Colombia. Proteolytic, amidolytic and phospholipase A2 (PLA2) activities were analyzed using colorimetric assays and the neuromuscular activity was analyzed in chick biventer cervicis (BC) preparations. The venom of both species showed very low PLA2 and amidolytic activities; however, both exhibited high proteolytic activity, which in E. bizona venom surpassed that of P. neuwiedii venom. E. bizona and P. neuwiedii venoms provoked partial neuromuscular blockade, which was more prominent in P. neuwiedii venom. E. bizona venom (30 µg/ml) induced a significant potentiation of the contracture response to exogenous ACh (110 µM), which was not accompanied by twitch height alteration, whereas the highest venom concentration (100 µg/ml) inhibited contracture responses to both ACh and KCl (40 mM). In contrast, P. neuwiedii venom (30 and 100 µg/ml) caused significant reduction in the contracture responses to exogenous ACh and KCl. The morphological analyses showed high myotoxic effects in the muscle fibers of BC incubated with either venoms; however, they are more prominent in the P. neuwiedii venom. Our results suggest that the myotoxicity of the venom of the two Colombian species can be ascribed to their high proteolytic activity. An interesting data was the potentiation of the ACh-induced contracture, but not the twitch height, caused by E. bizona venom, at a concentration that is harmless to muscle fibers integrity. This phenomenon remains to be further elucidated, and suggest that a possible involvement of post-synaptic receptors cannot be discarded. This work is a contribution to expand the knowledge on colubrid venoms; it allows envisaging that the two venoms offer the potential to go further in the identification of their components and biological targets.

PEGylation of Reduced Graphene Oxide Induces Toxicity in Cells of the Blood-Brain Barrier: An in Vitro and in Vivo Study.

Polyethylene glycol (PEG) coating has been frequently used to improve the pharmacokinetic behavior of nanoparticles. Studies that contribute to better unravel the effects of PEGylation on the toxicity of nanoparticle formulation are therefore highly relevant. In the present study, reduced graphene oxide (rGO) was functionalized with PEG, and its effects on key components of the blood-brain barrier, such as astrocytes and endothelial cells, were analyzed in culture and in an in vivo rat model. The in vitro studies demonstrated concentration-dependent toxicity. The highest concentration (100 µg/mL) of non-PEGylated rGO had a lower toxic influence on cell viability in primary cultures of astrocytes and rat brain endothelial cells, while PEGylated rGO induced deleterious effects and cell death. We assessed hippocampal BBB integrity in vivo by evaluating astrocyte activation and the expression of the endothelial tight and adherens junctions proteins. From 1 h to 7 days post-rGO-PEG systemic injection, a notable and progressive down-regulation of protein markers of astrocytes (GFAP, connexin-43), the endothelial tight (occludin), and adherens (ß-catenin) junctions and basal lamina (laminin) were observed. The formation of intracellular reactive oxygen species demonstrated by increases in the enzymatic antioxidant system in the PEGylated rGO samples was indicative of oxidative stress-mediated damage. Under the experimental conditions and design of the present study the PEGylation of rGO did not improve interaction with components of the blood-brain barrier. In contrast, the attachment of PEG to rGO induced deleterious effects in comparison with the effects caused by non-PEGylated rGO.

Presynaptic Proteins as Markers of the Neurotoxic Activity of BmjeTX-I and BmjeTX-II Toxins from Bothrops marajoensis (Marajó Lancehead) Snake Venom.

Neuromuscular preparations exposed to B. marajoensis venom show increases in the frequency of miniature end-plate potentials and twitch tension facilitation followed by presynaptic neuromuscular paralysis, without evidences of muscle damage. Considering that presynaptic toxins interfere into the machinery involved in neurotransmitter release (synaptophysin, synaptobrevin, and SNAP25 proteins), the main objective of this communication is to analyze, by immunofluorescence and western blotting, the expression of the synaptic proteins, synaptophysin, synaptobrevin, and SNAP25 and by myography, light, and transmission electron microscopy the pathology of motor nerve terminals and skeletal muscle fibres of chick biventer cervicis preparations (CBC) exposed in vitro to BmjeTX-I and BmjeTX-II toxins from B. marajoensis venom. CBC incubated with toxins showed irreversible twitch tension blockade and unaffected KCl- and ACh-evoked contractures, and the positive colabelling of acetylcholine receptors confirmed that their action was primarily at the motor nerve terminal. Hypercontraction and loose myofilaments and synaptic vesicle depletion and motor nerve damage indicated that the toxins displayed both myotoxic and neurotoxic effect. The blockade resulted from interference on synaptophysin, synaptobrevin, and SNAP25 proteins leading to the conclusion that BmjeTX-I and BmjeTX-II affected neurotransmitter release machinery by preventing the docking of synaptic vesicles to the axolemma of the nerve terminal.

Sildenafil (Viagra(®)) prevents and restores LPS-induced inflammation in astrocytes.

Astrocytes are effectively involved in the pathophysiological processes in the central nervous system (CNS) and may contribute to or protect against development of inflammatory and degenerative diseases. Sildenafil is a potent and selective phosphodiesterase-5 (PDE-5) inhibitor, which induces cyclic GMP accumulation. However, the mechanisms of actions on glial cells are not clear. The aim of the present work is to evaluate the role of sildenafil in lipopolysaccharide (LPS)-stimulated astrocytes. The cytoskeleton integrity and Ca(2+) waves were assessed as indicators of inflammatory state. Two main groups were done: (A) one prevention and (B) one restoration. Each of these groups: A1: control; A2: LPS for 24h; A3: sildenafil 1 or 10µM for 4h and then sildenafil 1 or 10µM+LPS for 24h. B1: control; B2: LPS for 24h; B3: LPS for 24h and then LPS+sildenafil 1 or 10µM for 24h. Cytoskeleton integrity was analyzed through GFAP immunolabeling and actin labeling with an Alexa 488-conjugated phalloidin probe. Calcium responses were assessed through a Ca(2+)-sensitive fluorophore Fura-2/AM. The results show that both preventive and restorative treatments with sildenafil (in both concentrations) reduced the Ca(2+) responses in intensity and induced a more organized actin fiber pattern, compared to LPS treated cells. This work demonstrated for the first time that astrocytes are a key part of the sildenafil protective effects in the CNS.