Pharmacological evaluation of a traditional Brazilian medicinal plant, Monteverdia ilicifolia. Part I - Preclinical safety study.

ETHNOPHARMACOLOGICAL RELEVANCE: Monteverdia ilicifolia (Maytenus ilicifolia, Celastraceae), known as "espinheira-santa", has been widely used in Brazil to manage mainly gastrointestinal diseases. This species has been listed in the Brazilian Pharmacopeia and in the National List of Essential Medicines (RENAME). Considering that clinical studies about M. ilicifolia are rare, our group has been performing a broader project designed to evaluate the efficacy of M. ilicifolia capsules in a clinical trial, for this reason, approaches to provide safety to those patients are relevant. AIM OF THE STUDY: We aimed to investigate the potential pharmacokinetic interaction and hepatotoxicity and intestinal toxicity of an aqueous extract of M. ilicifolia and its main phytocompounds, catequin, epicatequin, and quercetin. METHODS AND MATERIALS: Slices of liver and intestine of Wistar rats were incubated with different concentrations of M. ilicifolia extract or isolated compounds (catechin, epicatechin and quercetin). Commercial kits were used to evaluate enzyme activities of CYP2D6 and CYP3A4 isoforms, as well as cell viability (MTT) assay and intracellular enzymes leakage, specifically lactate dehydrogenase (LDH), alkaline phosphatase (AP), aspartate aminotransferase (AST), alanine aminotransferase (ALT) were studied. RESULTS: Incubation with M. ilicifolia extract, catechin, epicatechin and quercetin did not affect significantly any evaluated parameter in intestines. The intracellular enzymes leakages, CYP2D6, LDH and AST, were increased with M. ilicifolia extract and quercetin in liver slices. CONCLUSIONS: Our in vitro findings highlighted, for the first time, the potential hepatotoxicity induced by an aqueous extract of M. ilicifolia, consequently this species and its products should be avoided in liver diseases, supporting that studies of safety must be performed including in the context of traditional medicinal plants.

Circulating Total Extracellular Vesicles Cargo Are Associated with Age-Related Oxidative Stress and Susceptibility to Cardiovascular Diseases: Exploratory Results from Microarray Data.

Aging is a risk factor for many non-communicable diseases such as cardiovascular and neurodegenerative diseases. Extracellular vesicles and particles (EVP) carry microRNAs that may play a role in age-related diseases and may induce oxidative stress. We hypothesized that aging could impact EVP miRNA and impair redox homeostasis, contributing to chronic age-related diseases. Our aims were to investigate the microRNA profiles of circulating total EVPs from aged and young adult animals and to evaluate the pro- and antioxidant machinery in circulating total EVPs. Plasma from 3- and 21-month-old male Wistar rats were collected, and total EVPs were isolated. MicroRNA isolation and microarray expression analysis were performed on EVPs to determine the predicted regulation of targeted mRNAs. Thirty-one mature microRNAs in circulating EVPs were impacted by age and were predicted to target molecules in canonical pathways directly related to cardiovascular diseases and oxidative status. Circulating total EVPs from aged rats had significantly higher NADPH oxidase levels and myeloperoxidase activity, whereas catalase activity was significantly reduced in EVPs from aged animals. Our data shows that circulating total EVP cargo-specifically microRNAs and oxidative enzymes-are involved in redox imbalance in the aging process and can potentially drive cardiovascular aging and, consequently, cardiac disease.

Achyrocline satureioides as an adjuvant therapy for the management of mild viral respiratory infections in the context of COVID-19: Preliminary results of a randomized, placebo-controlled, and open-label clinical trial.

We evaluated the impact of an Achyrocline satureioides inflorescence infusion on the clinical outcomes of viral respiratory infections, including those caused by SARS-CoV-2, in a monocentric, randomized, open-label, placebo-controlled clinical trial. Patients with symptoms of viral respiratory infection, including suspected cases of COVID-19, were included and assigned to receive either A. satureioides (n = 57) or Malus domestica (n = 67) infusions twice a day for 14 days. All participants were included before the RT-PCR results, performed using a nasopharyngeal swab. The patients were further divided into subgroups according to real-time polymerase chain reaction results: SARS-CoV-2-positive and SARS-CoV-2-negative subgroups for statistical analyses. We assessed clinical outcomes, such as the latency to resolution of cough, dyspnea, fever, sore throat, chest pain, smell and taste dysfunctions, diarrhea, nausea, abdominal pain, and loss of appetite; hospitalization; and mortality with questionnaires and medical records. The subjects that received early A. satureioides infusion showed a significant reduction in the average number of days with respiratory and neurological symptoms compared with the control group (M. domestica infusion). We conclude that A. satureioides is a safe agent and, in combination with standard care, improves viral respiratory infection symptoms, especially those related to COVID-19.

MiRNAs that target amyloid precursor protein processing machinery in extracellular vesicles and particles derived from oral squamous cells carcinoma.

BACKGROUND: Considering that microRNAs (miRNAs), extracellular vesicles and particles (EVPs) and the amyloid precursor protein (APP) processing have been shown to be altered in oral squamous cells carcinoma (OSCC), it is possible that miRNAs that target APP processing pathways in EVPs are impacted in tumor cells. Our aim was to evaluate miRNAs that target APP itself or disintegrin and metalloproteinase domain 10 (ADAM10), which generate a trophic compound, sAPPα, in EVPs derived from OSCC cell lines, an aggressive and non-invasive, compared to normal keratinocytes. METHODS: We used two OSCC cell lines, an aggressive human oral squamous cell carcinoma cell line (SCC09) and a less aggressive cell line (CAL27) compared with a keratinocyte lineage (HaCaT). Cells were maintained in cell media, from which we isolated EVPs. EVPs were evaluated regarding their size and concentration using Nanotracking Analysis. We measured the levels of miRNAs which had as potential downstream target APP or ADAM10, specifically miR-20a-5p, miR-103a-3p, miR-424-5p, miR-92b-3p, miR-31-5p, and miR-93-5. RESULTS: There were no differences on size distributions and concentration of isolated EVPs. OSCC cell lines-derived EVPs miR-20a-5p, miR-92b-3p, and miR-93-5p were upregulated in comparison to HaCaT-derived EVPs; while miR-31-5p was reduced in EVPs obtained from CAL27 cells. CONCLUSION: Our results indicate changes in miRNAs that target APP machinery processing in EVPs derived from OSCC cell lines of different aggressiveness, which may be involved with abnormal miRNA expression in OSCC tissue and/or releasing tumor suppressor miRNA.

Potential involvement of circulating extracellular vesicles and particles on exercise effects in malignancies.

Physical activity and exercise have been widely related to prevention, treatment, and control for several non-communicable diseases. In this context, there are innumerous pre-clinical and clinical evidence indicating the potential role of exercise, beyond cancer prevention and survival, improved quality of life, including on psychological components, bone health and cachexia, from cancer survivors is described as well. This mini-review raises the potential role of circulating extracellular and particles vesicles (EVPs) cargo, as exerkines, conducting several positive effects on adjacent and/or distant tissues such as tumor, immune, bone and muscle cells. We highlighted new perspectives about microRNAs into EVPs changes induced by exercise and its benefits on malignancies, since microRNAs can be implicated with intricated physiopathological processes. Potential microRNAs into EVPs were pointed out here as players spreading beneficial effects of exercise, such as miR-150-5p, miR-124, miR-486, and miRNA-320a, which have previous findings on involvement with clinical outcomes and as well as tumor microenvironment, regulating intercellular communication and tumor growth. For example, high-intensity interval aerobic exercise program seems to increase miR-150 contents in circulating EVPs obtained from women with normal weight or overweight. In accordance circulating EVPs miR-150-5p content is correlated with prognosis colorectal cancer, and ectopic expression of miR-150 may reduce cell proliferation, invasion and metastasis. Beyond the involvement of bioactive miRNAs into circulating EVPs and their pathways related to clinical and preclinical findings, this mini review intends to support further studies on EVPs cargo and exercise effects in oncology.

Aerobic exercise, alone or combined with an anti-inflammatory drug, reduces the severity of epileptic seizures and levels of central pro-inflammatory cytokines in an animal model of epileptic seizures.

Epilepsy is a chronic neurological disorder and there is increasing evidence about the role of inflammation in epileptogenesis. These findings have spurred the search for new immunomodulatory approaches that can improve prognosis. Using an animal model of chemically-induced epileptic seizures, we tested exercise alone as non-pharmacological therapy, and exercise combined with an anti-inflammatory drug. Five groups were used: sedentary, diazepam, aerobic exercise alone, aerobic exercise combined with an anti-inflammatory drug, and naive control. Our goal was to compare the severity of the epileptic seizures between groups as well as seizure latency in a pentylenetetrazole-induced paradigm. Cytokine levels (IL-1ß, TNF-α, and IL-10) were measured. Both exercise groups showed a reduction in seizure severity and lower levels of pro-inflammatory cytokines in the cortex, while the levels of cytokines in the hippocampus remained unaffected.

Circulating Extracellular Vesicles and Particles Derived From Adipocytes: The Potential Role in Spreading MicroRNAs Associated With Cellular Senescence.

Aging is associated with adipose tissue dysfunction and is recognized as a risk factor for shortened life span. Considering that in vitro findings have shown the involvement of microRNA in extracellular vesicles and particles (EVPs) on senescence, we hypothesized that circulating EVPs derived from adipocytes can be involved in the aging process via their microRNA cargo. We aimed to determine the microRNA profiles of circulating EVPs derived from adipocytes (FABP4+) from aged and young adult animals and to perform in silico prediction of their downstream signaling effects. Plasma was obtained from Wistar rats (3 and 21 months old), and adipocyte-derived EVPs were isolated using the commercially available kit. Fatty acid-binding protein 4 (FABP4) was used for adipocyte-derived EVPs isolation; microRNA isolation and microarray expression analysis were performed. The analysis revealed 728 miRNAs, 32 were differentially between groups (p < 0.05; fold change ≥ |1.1|), of which 15 miRNAs were upregulated and 17 were downregulated in circulating EVPs from aged animals compared to young adults. A conservative filter was applied, and 18 microRNAs had experimentally validated and highly conserved predicted mRNA targets, with a total of 2,228 mRNAs. Canonical pathways, disease and functions, and upstream regulator analyses were performed using IPA-QIAGEN, allowing a global and interconnected evaluation. IPA categories impacted negatively were cell cycle, cellular development, cellular growth and proliferation, and tissue development, while those impacted positively were "digestive system cancer" and "endocrine gland tumor." Interestingly, the upregulated miR-15-5p targets several cyclins, such as CCND1 and CCND2, and miR-24-3p seems to target CDK4 (cyclin-dependent kinase 4); then potentially inhibiting their expression, both miRNAs can induce a negative regulation of cell cycle progression. In contrast, silencing of negative cell cycle checkpoint regulators, such as p21 and p16, can be predicted, which can induce impairment in response to genotoxic stressors. In addition, predicted targets, such as SMAD family members, seem to be involved in the positive control of digestive and endocrine tumors. Taken together, this exploratory study indicates that miRNA signature in circulating adipocyte-derived EVPs may be involved with the double-edged sword of cellular senescence, including irreversible proliferation arrest and tissue-dependent cancer, and seems to be suitable for further validation and confirmatory studies.

Correlation Between Inflammatory and Epigenetic Marks With Aerobic Performance in 10-km Runners.

Purpose: Our goals were to evaluate the effect of a 10-km running trial on inflammatory and epigenetic markers of 10-km runners and correlate the biochemical findings with anthropometric variables and performance. Methods: Twenty trained 10-km runners and seven sedentary male volunteers were recruited. Venous blood samples were collected at different times: under resting conditions, before the 10 Km race, and immediately after the finish. Inflammatory markers (IL-6, IL-10, and IL-ß) and cortisol levels were evaluated in plasma, while DNA methyltransferases (DNMT1 and DNMT3b) contents were measured in peripheral blood mononuclear cells (PBMCs). Results: Higher levels of plasma IL-6 levels were observed in 10-km runners compared to the sedentary group. After the trial, the runners had a significant increase on IL-6, IL-10, and cortisol plasma levels compared to baseline. Conclusion: Our findings suggest that inflammatory profile, but not DNMT content, influences aerobic performance in 10-km runners.

Circulating extracellular vesicles delivering beneficial cargo as key players in exercise effects.

Exercise has been recognized as an effective preventive and therapeutic approach for numerous diseases. This review addresses the potential role of circulating extracellular vesicles (EV) cargo that is modulated by physical activity. EV transport and deliver beneficial molecules to adjacent and distant tissues as a whole-body phenomenon, resulting in a healthier global status. Several candidate EV molecules, especially miRNAs, are summarized here as mediators of the beneficial effects of exercise, using different modalities, frequencies, volumes, and intensities. The following are among the candidate miRNAs: miR-21, miR-146, miR-486, miR-148a-3p, miR-223-3p, miR-142-3p, and miR-191a-5p. We highlight the relationship between EV cargo modifications, their targets and pathway interactions, in clinical outcomes, for example, on cardiovascular or immune diseases. This review brings an innovative perspective providing evidence for an intricate biological basis of the relationship between EV cargo and exercise-induced benefits on several diseases. Moreover, specific changes on circulating EV content might potentially be used as biomarkers of exercise efficacy.

Acrobatic exercise recovers object recognition memory impairment in hypoxic-ischemic rats.

Neonatal hypoxia-ischemia (HI) can lead to cognitive impairments and motor dysfunction. Acrobatic exercises (AE) were proposing as therapeutic option to manage HI motor deficits, however, the cognitive effects after this treatment are still poorly understood. Therefore, we evaluated the effects of AE protocol on memory impairments and brain plasticity markers after Rice-Vannucci HI rodent model. Wistar rats on the 7th postnatal day (PND) were submitted to HI model and after weaning (PND22) were trained for 5 weeks with AE protocol, then subsequently submitted to cognitive tests. Our results showed recovery in novel object recognition (NOR) memory, but not, spatial Morris Water Maze (WM) memory after AE treatment in HI rats. BDNF and synaptophysin neuroplasticity markers indicate plastic alterations in the hippocampus and striatum, with maintenance of synaptophysin despite the reduction of total volume tissue, besides, hippocampal HI-induced ipsilateral BDNF increased, and striatum contralateral BDNF decreased were noted. Nevertheless, the exercise promoted functional recovery and seems to be a promising strategy for HI treatment, however, future studies identifying neuroplastic pathway for this improvement are needed.

Effects of exercise modalities on BDNF and IL-1ß content in circulating total extracellular vesicles and particles obtained from aged rats.

There are evidences about the involvement of systemic factors, such as brain-derived neurotrophic factor (BDNF), on functional exercise effects. Although aerobic exercise can impact circulating extracellular vesicles and particles (EVPs) cargo, other exercise modalities were not studied. Taken that BDNF and anti-inflammatory effects have been related to functional outcomes, and BDNF and IL-1ß have been detected in circulating EVPs, our aim was to evaluate circulating total EVPs profile from adult and aged Wistar rats submitted to exercise modalities, namely aerobic, acrobatic, resistance or combined for 20 min, 3 times a week, during 12 weeks. A modality- and age-dependent effect on total EVPs cargo was observed; aerobic exercise induced an augment in BDNF and IL-1ß in EVPs from aged rats, while acrobatic and combined exercise modalities reduced IL-1ß content in EVPs from adult ones. Besides, all exercise modalities attenuated aging-induced CD63 changes in circulating total EVPs; this finding can be involved with reduced mortality rate and improved memory performance previously observed. Changes on EVPs profile, such as increased CD63 levels can be related, at least in part, to an exercise-induced healthier global status. Additionally, aerobic exercise-induced effects on BDNF and IL-1ß levels might indicate additional benefits in aged individuals.

Early life exposure to hypercaloric diet impairs eating behavior during weaning: The role of BDNF signaling and astrocyte marks.

Literature shows that gestational and/or lactational exposure to hypercaloric diets induces long term effects on eating behavior and the involvement of neurochemical mechanisms. We hypothesized that the effects of hypercaloric diets in early development phases can precede an overweight or an obesity status. The aim of the present study was to evaluate the impact of gestational and lactational exposure to cafeteria diet on eating behavior and neurochemical parameters, BDNF signaling, epigenetic and astrocyte marks in the hippocampus and olfactory bulb during the weaning phase. Pregnant female rats were randomized between standard and cafeteria diet, the respective diet was maintained through the lactational period. The framework of feeding pattern, meal, and its microstructure, was observed in postnatal day 20. Exposure to cafeteria diet increased the number of meals, associated with a lower first inter-meal interval and higher consumption in both genders, without any changes in body weight. Diet exposure also reduced the number of grooming, a behavior typically found at the end of meals. Hypercaloric diet exposure reduced BDNF levels in the olfactory bulb and hippocampus from rats of both sexes and increased the content of the TrkB receptor in hippocampi. It was observed an increase in HDAC5 levels, an epigenetic mark. Still, early exposure to the hypercaloric diet reduced hippocampal GFAP and PPARγ levels, without any effect on NeuN content, indicating that alterations in astrocytes can precede those neuronal outcomes. Our results showed that changes in interrelated neurochemical signaling, BDNF, and astrocyte marks, induced by hypercaloric diet in early stages of development may be related to impairment in the temporal distribution of eating pattern and consequent amounts of consumed food during the weaning phase.

Methylphenidate treatment increases hippocampal BDNF levels but does not improve memory deficits in hypoxic-ischemic rats.

BACKGROUND: Methylphenidate (MPH) is a stimulant drug mainly prescribed to treat cognitive impairments in attention-deficit/hyperactivity disorder (ADHD). We demonstrated that neonatal hypoxia-ischemia (HI) induced attentional deficits in rats and MPH administration reversed these deficits. However, MPH effects on memory deficits after the HI procedure have not been evaluated yet. AIMS: We aimed to analyze learning and memory performance of young hypoxic-ischemic rats after MPH administration and associate their performance with brain-derived neurotrophic factor (BDNF) levels in the prefrontal cortex and hippocampus. METHODS: Male Wistar rats were divided into four groups (n=11-13/group): control saline (CTS), control MPH (CTMPH), HI saline (HIS) and HIMPH. The HI procedure was conducted at post-natal day (PND) 7 and memory tasks between PND 30 and 45. MPH administration (2.5 mg/kg, i.p.) occurred 30 min prior to each behavioral session and daily, for 15 days, for the BDNF assay (n=5-7/group). RESULTS: As expected, hypoxic-ischemic animals demonstrated learning and memory deficits in the novel-object recognition (NOR) and Morris water maze (MWM) tasks. However, MPH treatment did not improve learning and memory deficits of these animals in the MWM-and even disrupted the animals' performance in the NOR task. Increased BDNF levels were found in the hippocampus of HIMPH animals, which seem to have been insufficient to improve memory deficits observed in this group. CONCLUSIONS: The MPH treatment was not able to improve memory deficits resulting from the HI procedure considering a dose of 2.5 mg/kg. Further studies investigating different MPH doses would be necessary to determine a dose-response relationship in this model.

Exercise Modalities Improve Aversive Memory and Survival Rate in Aged Rats: Role of Hippocampal Epigenetic Modifications.

We aimed to investigate the effects of aging and different exercise modalities on aversive memory and epigenetic landscapes at brain-derived neurotrophic factor, cFos, and DNA methyltransferase 3 alpha (Bdnf, cFos, and Dnmt3a, respectively) gene promoters in hippocampus of rats. Specifically, active epigenetic histone markers (H3K9ac, H3K4me3, and H4K8ac) and a repressive mark (H3K9me2) were evaluated. Adult and aged male Wistar rats (2 and 22 months old) were subjected to aerobic, acrobatic, resistance, or combined exercise modalities for 20 min, 3 times a week, during 12 weeks. Aging per se altered histone modifications at the promoters of Bdnf, cFos, and Dnmt3a. All exercise modalities improved both survival rate and aversive memory performance in aged animals (n = 7-10). Exercise altered hippocampal epigenetic marks in an age- and modality-dependent manner (n = 4-5). Aerobic and resistance modalities attenuated age-induced effects on hippocampal Bdnf promoter H3K4me3. Besides, exercise modalities which improved memory performance in aged rats were able to modify H3K9ac or H3K4me3 at the cFos promoter, which could increase gene transcription. Our results highlight biological mechanisms which support the efficacy of all tested exercise modalities attenuating memory deficits induced by aging.

The amyloid precursor protein (APP) processing as a biological link between Alzheimer's disease and cancer.

Aging is a risk factor for several illnesses, such as Alzheimer's Disease and various cancers. However, an inverse correlation between malignancies and Alzheimer's Disease has been suggested. This review addressed the potential role of non-amyloidogenic and amyloidogenic pathways of amyloid precursor protein processing as a relevant biochemical mechanism to clarify this association. Amyloidogenic and non-amyloidogenic pathways have been related to Alzheimer's Disease and certain malignancies, respectively. Several known molecules involved in APP processing, including its regulation and final products, were summarized. Among them some candidate mechanisms emerged, such as extracellular-regulated kinase (Erk) and protein kinase C (PKC). Therefore, the imbalance of APP processing may be involved with the negative correlation between cancer and Alzheimer Disease.

Epigenetic marks are modulated by gender and time of the day in the hippocampi of adolescent rats: A preliminary study.

Although the involvement of gender in epigenetic machinery in peripheral tissues during the neonatal period has been suggested, the gender-related epigenetic profile of brain areas during the adolescent period is rarely exploited. Furthermore, the influence of time of day on hippocampal acetylation marks has been demonstrated in young adult and aged rats; however, there are no studies reporting epigenetic changes in the adolescent period. Therefore, this study aimed to investigate the effects of gender on hippocampal DNA methyltransferase 1 content and histone deacetylase (HDAC) activity of adolescent rats at different time points, specifically early morning and afternoon. Both epigenetic markers increased significantly in the hippocampi of female rats compared to the male group, an indicator of reduced transcriptional activity. In addition, HDAC activity during the early morning was higher compared to afternoon groups in both male and female rats, while DNA methyltransferase 1 content was not altered by the time of day. Our findings demonstrate that hippocampal DNA methylation and histone acetylation status can be influenced by gender during the adolescent period, while the time of the day impacts HDAC activity.

Folic acid supplementation during pregnancy prevents cognitive impairments and BDNF imbalance in the hippocampus of the offspring after neonatal hypoxia-ischemia.

Folic acid (FA) supplementation (400 µg/day) has been recommended during pregnancy to prevent neural tube defects. However, in some countries, flours are required to be fortified with FA, possibly increasing the levels of this vitamin in pregnant women. Our previous studies have evidenced a dual effect of the FA treatment in a rat model of neonatal hypoxia-ischemia (HI). Aiming to better correlate with humans, this paper evaluated the effects of two different levels of FA supplementation during pregnancy on memory parameters and neuronal survival and plasticity in the hippocampus of rats submitted to the neonatal HI. During pregnancy, female Wistar rats received one of these diets: standard (SD), supplemented with 2 mg/kg of FA or with 20 mg/kg of FA. At the 7th PND, rats suffered the HI procedure. At the 60th PND rats were evaluated in the open field, Morris water maze, novel-object recognition and inhibitory avoidance tasks. Furthermore, neuronal density, synaptophysin densitometry and BDNF concentration were assessed in the hippocampus. Both doses of FA prevented the HI-induced memory impairments. The supplementation reversed the BDNF late increase in the hippocampus of the HI rats, but did not inhibit the neuronal death. In conclusion, FA supplementation during pregnancy prevented memory deficits and BDNF imbalance after neonatal HI. These findings are particularly relevant because neuroprotection was achieved even in the high level of FA supplementation during pregnancy, indicating that this intervention would be considered secure for the offspring development.