Protective Actions of Cannabidiol on Aging-Related Inflammation, Oxidative Stress and Apoptosis Alterations in Liver and Lung of Long Evans Rats.

BACKGROUND: Aging is characterised by the progressive accumulation of oxidative damage which leads to inflammation and apoptosis in cells. This affects all tissues in the body causing the deterioration of several organs. Previous studies observed that cannabidiol (CBD) could extend lifespan and health span by its antioxidant, anti-inflammatory and autophagy properties. However, research on the anti-aging effect of CBD is still in the beginning stages. This study aimed to investigate the role of cannabidiol (CBD) in the prevention of age-related alterations in liver and lung using a murine model. METHODS: 15-month-old Long Evans rats were treated with 10 mg/kg b.w./day of CBD for 10 weeks and compared to animals of the same age as old control and 2-month-old animals as young control. Gene and/or protein expressions, by RT-qPCR and Western blotting, respectively, were assessed in terms of molecules related to oxidative stress (GST, GPx, GR and HO-1d), inflammation (NFκB, IL-1ß and TNF-α) and apoptosis (BAX, Bcl-2, AIF, and CASP-1). In addition, MDA and MPO levels were measured by colorimetric assay. Results were analysed by ANOVA followed by Tukey-Kramer test, considering statistically significant a p < 0.05. RESULTS: GST, GPx and GR expressions were significantly reduced (p < 0.01) in liver samples from old animals compared to young ones and CBD treatment was able to revert it. A significant increase was observed in old animals compared to young ones in relation to oxidative stress markers (MDA and HO-1d), proinflammatory molecules (NFκB, IL-1ß and TNF-α), MPO levels and proapoptotic molecules (BAX, AIF and CASP-1), while no significant alterations were observed in the antiapoptotic molecules (Bcl-2). All these changes were more noticeable in the liver, while the lung seemed to be less affected. In almost all the measured parameters, CBD treatment was able to revert the alterations caused by age restoring the levels to those observed in the group of young animals. CONCLUSIONS: Chronic treatment with CBD in 15-month-old rats showed beneficial effects in lung and more significantly in liver by reducing the levels of inflammatory, oxidative and apoptotic mediators, and hence the cell damage associated with these three processes inherent to aging.

Activation of NLRP3 Inflammasome in Liver of Long Evans Lactating Rats and Its Perinatal Effects in the Offspring after Bisphenol F Exposure.

The liver is the organ responsible for the metabolism and detoxification of BPF, the BPA analogue that is replacing it in plastic-based products. It is not known whether BPF can trigger inflammatory responses via the NLRP3 inflammasome, which plays a major role in the development of liver disease. The aim of this study was to evaluate nitrosative stress species (RNS) and NLRP3 inflammasome activation in the liver of lactating dams after BPF exposure. Moreover, it was studied whether this effect could also be observed in the liver of female and male offspring at postnatal day 6 (PND6). 36 Long Evans rats were randomly distributed according to oral treatment into three groups: Control, BPF-low dose (LBPF; 0.0365 mg/kg b.w./day) group and BPF-high dose (HBPF; 3.65 mg/kg b.w./day) group. The levels of nitrosative stress-inducing proteins (eNOS, iNOS, HO-1d), NLRP3 inflammasome components (NLRP3, PyCARD, CASP1) and proinflammatory cytokines (IL-1ß, IL-18, IFN-γ and TNF-α) were measured by gene and protein expression in the liver of lactating dams and in female and male PND6 offspring. Lactating dams treated with LBPF showed a significant increase in iNOS and HO-1d, activation of NLRP3 components (NLRP3, PyCARD, CASP1) and promoted the release of proinflammatory cytokines such as IL-1ß, IL-18, IFN-γ and TNF-α. Similar effects were found in female and male PND6 offspring after perinatal exposure. LBPF oral administration and perinatal exposure caused an increase of nitrosative stress markers and proinflammatory cytokines. Also, NLRP3 inflammasome activation was significantly increased in in the liver of lactating dams and PND6 offspring.

S-Adenosylmethionine Decreases Bacterial Translocation, Proinflammatory Cytokines, Oxidative Stress and Apoptosis Markers in Hepatic Ischemia-Reperfusion Injury in Wistar Rats.

Hepatic ischemia/reperfusion injury (IRI) can seriously impair liver function. It is initiated by oxidative stress, resulting in inflammation and apoptosis-induced cellular damage. Glutathione (GSH) prevents oxidative stress. S-Adenosylmethionine (SAMet) is a GSH synthesis precursor that avoids the deficit in SAMet-synthetase activity and contributes to intracellular ATP repletion. It also acts as a methyl group donor, stabilizing hepatocyte membranes, among other functions. This study investigated the effect of SAMet on bacterial translocation and levels of proinflammatory cytokines, oxidative stress and apoptosis markers in male Wistar rats subjected to hepatic IRI. Animals were randomly divided into six groups: (1) sham operation, (3) animals undergoing 60 min of ischemia of the right lateral lobe for temporary occlusion of the portal vein and hepatic artery plus 10 min of reperfusion, and (5) the same as (3) but with a reperfusion period of 120 min. Groups 2, 4 and 6, respectively, are the same as (1), (3) and (5), except that animals received SAMet (20 mg/kg) 15 min before ischemia. GSH, ATP, lipid peroxidation (LPO), TNF-α, IL-1ß, IL-6, total caspase-1 and caspase-9, total and cleaved caspase-3, and phosphatidylcholine were determined in the liver. Endotoxin, TNF-α, IL-1ß, IL-6, IL-10 and LPO in vena cava and portal vein blood samples were also measured. Endotoxin and LPO levels as well as proinflammatory cytokines and apoptotic markers increased significantly in animals undergoing IRI, both after 10 and 120 min of reperfusion. IRI produced a significant decrease in GSH, ATP, portal IL-10 and phosphatidylcholine. SAMet treatment prevented these effects significantly and increased survival rate. The study suggests that SAMet exerts protective effects in hepatic IRI.

Oxidative stress increases in liver of lactating rats after BPF-low-dose exposure: perinatal effects in the offspring.

Bisphenol F (BPF) is replacing Bisphenol A (BPA) in the manufacture of products due to endocrine-disrupting effects. BPF monomers can also be released into the environment and enter the food chain, resulting in human exposure to low doses. Since bisphenols are primarily metabolized by the liver, this organ is more vulnerable to lower doses of bisphenols than others. Exposure during prenatal development may increase the risk of diseases in adulthood. The aim was to evaluate whether BPF administration could generate oxidative stress in liver of lactating rats, and whether these effects may be also observed in female and male postnatal day 6 (PND6) offspring. Long Evans rats received oral treatment: Control, BPF-low-dose (LBPF) 0.0365 mg/kg b.w./day, and BPF-high-dose (HBPF) 3.65 mg/kg b.w./day. The levels of antioxidant enzymes (CAT, SOD, GR, GPx and GST), glutathione system (GSH, GSSG) and lipid damage markers (MDA, LPO) were measured using colorimetric methods in liver of both lactating dams and in PND6 offspring. Mean values were analyzed using Prism-7. LBPF affected liver defense mechanisms (antioxidant enzymes and glutathione system), increasing ROS levels and producing lipid peroxidation in lactating dams. Similar effects were found in female and male PND6 offspring as a consequence of perinatal exposure.

Low Dose of BPA Induces Liver Injury through Oxidative Stress, Inflammation and Apoptosis in Long-Evans Lactating Rats and Its Perinatal Effect on Female PND6 Offspring.

Bisphenol A (BPA) is a phenolic compound used in plastics elaboration for food protection or packaging. BPA-monomers can be released into the food chain, resulting in continuous and ubiquitous low-dose human exposure. This exposure during prenatal development is especially critical and could lead to alterations in ontogeny of tissues increasing the risk of developing diseases in adulthood. The aim was to evaluate whether BPA administration (0.036 mg/kg b.w./day and 3.42 mg/kg b.w./day) to pregnant rats could induce liver injury by generating oxidative stress, inflammation and apoptosis, and whether these effects may be observed in female postnatal day-6 (PND6) offspring. Antioxidant enzymes (CAT, SOD, GR, GPx and GST), glutathione system (GSH/GSSG) and lipid-DNA damage markers (MDA, LPO, NO, 8-OHdG) were measured using colorimetric methods. Inducers of oxidative stress (HO-1d, iNOS, eNOS), inflammation (IL-1ß) and apoptosis (AIF, BAX, Bcl-2 and BCL-XL) were measured by qRT-PCR and Western blotting in liver of lactating dams and offspring. Hepatic serum markers and histology were performed. Low dose of BPA caused liver injury in lactating dams and had a perinatal effect in female PND6 offspring by increasing oxidative stress levels, triggering an inflammatory response and apoptosis pathways in the organ responsible for detoxification of this endocrine disruptor.

Effects of GH on the Aging Process in Several Organs: Mechanisms of Action.

In order to investigate the possible beneficial effects of GH administration on the aging process, 24-month-old rats of both sexes and 10-month-old SAMP8 mice were used. Male rats showed increased fat content and decreased lean body mass together with enhanced vasoconstriction and reduced vasodilation of their aortic rings compared to young adult animals. Chronic GH treatment for 10 weeks increased lean body mass and reduced fat weight together with inducing an enhancement of the vasodilatory response by increasing eNOS and a reduction of the constrictory responses. Old SAMP8 male mice also showed insulin resistance together with a decrease in insulin production by the endocrine pancreas and a reduced expression of differentiation parameters. GH treatment decreased plasma levels and increased pancreatic production of insulin and restored differentiation parameters in these animals. Ovariectomy plus low calcium diet in rabbits induced osteoporosis Titanium implants inserted into these rabbit tibiae showed after one month lesser bone to implant (BIC) surface and bone mineral density (BMD). Local application of GH in the surgical opening was able to increase BIC in the osteoporotic group. The hippocampus of old rats showed a reduction in the number of neurons and also in neurogenesis compared to young ones, together with an increase of caspases and a reduction of Bcl-2. GH treatment was able to enhance significantly only the total number of neurons. In conclusion, GH treatment was able to show beneficial effects in old animals on all the different organs and metabolic functions studied.

A Novel Circulating MicroRNA for the Detection of Acute Myocarditis.

BACKGROUND: The diagnosis of acute myocarditis typically requires either endomyocardial biopsy (which is invasive) or cardiovascular magnetic resonance imaging (which is not universally available). Additional approaches to diagnosis are desirable. We sought to identify a novel microRNA for the diagnosis of acute myocarditis. METHODS: To identify a microRNA specific for myocarditis, we performed microRNA microarray analyses and quantitative polymerase-chain-reaction (qPCR) assays in sorted CD4+ T cells and type 17 helper T (Th17) cells after inducing experimental autoimmune myocarditis or myocardial infarction in mice. We also performed qPCR in samples from coxsackievirus-induced myocarditis in mice. We then identified the human homologue for this microRNA and compared its expression in plasma obtained from patients with acute myocarditis with the expression in various controls. RESULTS: We confirmed that Th17 cells, which are characterized by the production of interleukin-17, are a characteristic feature of myocardial injury in the acute phase of myocarditis. The microRNA mmu-miR-721 was synthesized by Th17 cells and was present in the plasma of mice with acute autoimmune or viral myocarditis but not in those with acute myocardial infarction. The human homologue, designated hsa-miR-Chr8:96, was identified in four independent cohorts of patients with myocarditis. The area under the receiver-operating-characteristic curve for this novel microRNA for distinguishing patients with acute myocarditis from those with myocardial infarction was 0.927 (95% confidence interval, 0.879 to 0.975). The microRNA retained its diagnostic value in models after adjustment for age, sex, ejection fraction, and serum troponin level. CONCLUSIONS: After identifying a novel microRNA in mice and humans with myocarditis, we found that the human homologue (hsa-miR-Chr8:96) could be used to distinguish patients with myocarditis from those with myocardial infarction. (Funded by the Spanish Ministry of Science and Innovation and others.).

Determination of SARS-CoV-2 RNA in different particulate matter size fractions of outdoor air samples in Madrid during the lockdown.

BACKGROUND: Previous studies described the presence of SARS-CoV-2 in outdoor air particulate matter (PM) in urban areas of northern Italy and USA. The city of Madrid was heavily affected by COVID-19 during March-June 2020. Also, this city usually displays high concentrations of PM under several atmospheric situations. This is mandatory to assess the presence of viral RNA in PM, as an indicator of epidemic recurrence. Our study was aimed at investigating the presence of SARS-CoV-2 RNA in outdoor air samples (on PM10, PM2.5 and PM1). METHODS: Six samples of PM10, PM2.5 and PM1 were collected between the May 4th and 22nd 2020 in Madrid, on quartz fiber filters by using MCV high volume samplers (30 m3 h-1 flow) with three inlets (Digitel DHA-80) for sampling PM10, PM2.5 and PM1. RNA extraction and amplification was performed according to the protocol recently set by Setti et al.2020c in Italy. Up to three highly specific molecular marker genes (N1, N2, and RP) were used to test the presence of SARS-CoV-2 RNA. RESULTS: After RNA extraction and expression measurements of N1, N2 and RP genes from all the collected filters, no presence of SARS-CoV-2 RNA was observed. Control tests to exclude false positive results were successfully accomplished. CONCLUSIONS: No presence of SARS-CoV-2 in quartz fiber filters samplers for PM10, PM2.5 and PM1 fractions was observed in our study carried out in Madrid during the month of May 2020. Nevertheless, the absence of viral genomes could be due to different factors including: limited social interactions and economic activities resulting in reduced circulation of the coronavirus, lower daily PM concentration in outdoor air, as well as to meteorological stability and higher temperature that characterize spring season. Further research should be carried out during winter, in presence of higher viral circulation and daily PM exceedances.

Thymus-Derived Regulatory T Cell Development Is Regulated by C-Type Lectin-Mediated BIC/MicroRNA 155 Expression.

Thymus-derived regulatory T (tTreg) cells are key to preventing autoimmune diseases, but the mechanisms involved in their development remain unsolved. Here, we show that the C-type lectin receptor CD69 controls tTreg cell development and peripheral Treg cell homeostasis through the regulation of BIC/microRNA 155 (miR-155) and its target, suppressor of cytokine signaling 1 (SOCS-1). Using Foxp3-mRFP/cd69+/- or Foxp3-mRFP/cd69-/- reporter mice and short hairpin RNA (shRNA)-mediated silencing and miR-155 transfection approaches, we found that CD69 deficiency impaired the signal transducer and activator of transcription 5 (STAT5) pathway in Foxp3+ cells. This results in BIC/miR-155 inhibition, increased SOCS-1 expression, and severely impaired tTreg cell development in embryos, adults, and Rag2-/- γc-/- hematopoietic chimeras reconstituted with cd69-/- stem cells. Accordingly, mirn155-/- mice have an impaired development of CD69+ tTreg cells and overexpression of the miR-155-induced CD69 pathway, suggesting that both molecules might be concomitantly activated in a positive-feedback loop. Moreover, in vitro-inducible CD25+ Treg (iTreg) cell development is inhibited in Il2rγ-/-/cd69-/- mice. Our data highlight the contribution of CD69 as a nonredundant key regulator of BIC/miR-155-dependent Treg cell development and homeostasis.