Pharmacological effects of palmitoylethanolamide on hypertension, insulin-resistance and obesity in murine models

N-Palmitoylethanolamide (PEA) is an endogenous N-acylethanolamine, first identified in lipid extracts from brain, liver, and muscle of rat and guinea pig. PEA is formed “on demand” from membrane phospholipids and it is gaining ever-increasing interest not only for its anti-inflammatory and analgesic effects mediated by peroxisome-proliferator activated receptor (PPAR)-α, but also for its novel metabolic effects. Overweight and obesity are defined as abnormal or excessive fat accumulation that may impair health. Main consequence of obesity is cardiovascular disease (CVD). The sum of the risk factors that predisposes to CVD goes by the name of “metabolic syndrome” (MetS). Hypertension is an important hallmark of MetS and a common cause of kidney disease. In the first part of this thesis, we investigated the mechanisms underpinning PEA blood pressure lowering effect, exploring the contribution of epoxyeicosatrienoic acids (EETs), CYP-dependent arachidonic acid (AA) metabolites, as endothelium derived hyperpolarizing factors (EDHF), and renin-angiotensin system (RAS) modulation. To achieve this aim, SHR and Wistar-Kyoto normotensive (WKY) rats were treated with PEA (30 mg/kg/day, s.c.) for five weeks. Functional evaluations on mesenteric bed were performed to analyze EDHF mediated vasodilation. Moreover, mesenteric bed and carotid were harvested to measure the soluble epoxide hydrolase (sEH), which is the enzyme responsible for EETs degradation in their corresponding inactive diols. Effect of PEA on RAS modulation was investigated by analyzing angiotensin converting enzyme (ACE) and angiotensin receptor (AT)1 expression. We showed that EDHF-mediated dilation in response to acetylcholine (Ach) was increased in mesenteric beds of PEA-treated SHR. Interestingly, in both vascular tissues, PEA significantly decreased the sEH protein level, accompanied by a reduced serum concentration of its metabolite 14-15 dihydroxyeicosatrienoic acid (DHET), implying a reduction in EET hydrolysis. Moreover, PEA treatment down-regulated AT1 and ACE expression, indicating a reduction in Ang II-mediated effects. Our data clearly demonstrate the involvement of EETs and RAS in the blood pressure lowering effect of PEA. The relationship between obesity, insulin-resistance (IR) type 2 diabetes mellitus (T2DM) and MetS is well known. IR is defined as an inefficient glucose uptake and utilization in peripheral tissues in response to insulin stimulation. IR in the prediabetes stage is a feature of glucose intolerance, which includes impaired fasting glucose and/or impaired glucose tolerance. When insulin binds to its transmembrane receptor (InsR), promotes its autophosphorylation (pInsR). Activated pInsR recruits insulin receptor substrate (IRS), leading to insulin signaling cascade. A potential link between inflammation and IR has been shown. Indeed, obesity is characterized by chronic low grade inflammation, where the release of adipose tissue-derived cytokines can block insulin action and cause systemic IR. In fact, serum tumor necrosis factor (TNF)-α and interleukin (IL)-6 are significantly increased in serum from obese patients. All cytokines induce IRS1 protein degradation, which suppresses insulin signaling pathway and subsequently suppresses glucose transporter (GLUT) translocation and glycogen synthesis, contributing to IR and hyperglycemia. Our study was focused on the pharmacological effect of PEA in an animal model of diet-induced obesity (DIO), feeding mice with a high-fat diet (HFD), and on the mechanisms by which this lipid mediator could modulate the storage and availability of energy sources, restoring lipid/glucose homeostasis. To achieve this aim, mice were fed a standard chow diet (STD group) or HFD (DIO group). After twelve weeks, both STD or HFD mice were treated with PEA (30 mg/kg/day, o.s.) for ten weeks. At the end of the experimental period, body parameters were determined, and serum and tissues collected for following determinations. Interestingly, PEA caused a reduction in body weight and fat mass, improved glucose tolerance and prevented IR, induced by HFD feeding. Moreover, PEA restored the alterations of serum biochemical and inflammatory parameters, inducing a marked reduction of ALT, AST, cholesterol, and pro-inflammatory cytokines, such as TNF-α, IL-1 and monocyte chemoattractant protein (MCP)-1. PEA also normalized metabolic hormone levels and restored insulin sensitivity. At hepatic level, PEA treatment significantly induced an increase in the activation AMPK/ACC pathway, stimulating fatty acid oxidation, compromised in obese mice. To evaluate tissue insulin-sensitivity, we determined the hepatic expression of the InsR, whose expression decreased in liver of DIO mice compared to that of STD animals, and increased in PEA-treated mice. Then, we evaluated the effectiveness of hepatic insulin signaling through the evaluation of InsR and Akt phosphorylated state and the expression of GLUT-2. PEA treatment restored insulin signaling. The protective effect of PEA was strengthened by the evaluation of hepatic IL-6 and TNF-α, whose transcription, upregulated by HFD feeding, was reduced. To address the direct effect of PEA on hepatic insulin-sensitivity, we evaluated the restoration of insulin signaling, altered by the induction of IR, in HepG2 cells, a human hepatocarcinoma cell line. Therefore, we demonstrated in vitro that PEA increased the phosphorylation of Akt in insulin resistant cells, following insulin stimulation. PEA was also able to modulate glucose homeostasis at hypothalamic level. Therefore, we examined neuronal activation at the arcuate (ARC) and ventromedial (VMH) nuclei, evaluating c-fos immunostaining. In the ARC nucleus of DIO mice, a decrease in c-fos labeling was found. Interestingly, in the PEA-treated DIO group, a trend of c-fos labeling increase was evidenced. Consistently, in the VHM of DIO mice a significant decrease in the neuronal activation was shown compared to STD mice, although, no differences were found between DIO and PEA-treated DIO mice. The involvement of the hypothalamic control of glucose homeostasis by PEA was confirmed in in vitro experiments, using human SH-SY5Y neuroblastoma cell line. When insulin-resistant cells were treated with PEA, the re-stimulation with insulin showed a restoration of Akt phosphorylation, and therefore of insulin-sensitivity. These findings show that this acylethanolamide also displays a central effect on glucose homeostasis, reducing neuronal IR. Our data strengthened evidence on the metabolic activity of PEA, through the involvement of central and peripheral mechanisms. PEA clearly ameliorates glucose-tolerance and insulin-sensitivity, indicating its therapeutic potential for the treatment of metabolic dysfunctions associated to obesity, such as IR and T2DM

Occurrence of Legionella spp. in thermal environments: Virulence factors and biofilm formation in isolates from a spa☆ , ☆☆

Abstract The aim of the study was to evaluate the occurrence of Legionella spp. in the water system of a spa in the city of Naples by analyzing water, air and surface samples. On the whole, 312 samples were collected and analyzed in the course of 10 months. Legionella CYE Agar Base and Legionella Latex Test (Oxoid©) were used to identify and serotype presumptive Legionella pneumophila strains. A further identification was carried out by rDNA16S and ITS region amplification followed by a sequence analysis by DNA Sequencing Analysis software (Applied Biosystems). Similarity search was performed using BLAST algorithm against the GenBank database (NCBI GenBank). Specific in-vitro tests aimed to evaluate the production of esoenzymes (hemolysins, collagenases, mucinases, lipases, proteinases, DNAses, elastases) on GC-FC Agar were also carried out. Finally, a crystal violet staining method (absorbance at 570 nm) was used to evaluate the ability of the strains to produce biofilm in a 96-multiwell polyethylene plate. All samples were negative for L. pneumophila. Six different Legionella strains were isolated from water samples and identified as Legionella londiniensis and Legionella spp. A significant (from 1000 to 10,000 CFU/L) and a low to moderate (from 100 to 1000 CFU/L) contamination were detected respectively in the 5% and 4% of samples; 91% of water samples showed a Legionella spp. amount less than 100 CFU/L. Two Legionella londiniensis isolates showed collagenases, caseinases, proteinases and gelatinases activities, being classified as potentially pathogenic bacteria. None of the isolates were classified as strong biofilm producer but they showed a moderate to weak ability to form biofilm on polyethylene. This result is significant because large part of the spa pipelines is plastic-coated. The highest frequency of isolation of Legionella spp. was detected in the unit for Thermal Mud Therapy, which showed a relative risk value equal to 1.69 (CI 95% 0.60–4.70). Although our results proved a moderate contamination in different water samples, the presence of potentially pathogenic environmental strains of Legionella spp. should not be underestimated because most part of costumers attending the spa are old and sick people, and Legionella strains can represent a real risk

Butyrate Modulates Inflammation in Chondrocytes via GPR43 Receptor

Background/Aims: Osteoarthritis (OA) is a joint degenerative biomechanical disorder involving immunity, metabolic alterations, inflammation, and cartilage degradation, where chondrocytes play a pivotal role. OA has not effective pharmacological treatments and new therapeutic targets are needed. Adipokines contribute to the low-grade systemic inflammation in OA. Here, we explored novel molecular mechanisms of sodium butyrate (BuNa) in modulating inflammation and chemotaxis in chondrocytes, demonstrating the direct involvement of its G protein-coupled receptor (GPR)-43. Methods: ATDC5 murine chondrocytes were stimulated with interleukin (IL)-1β, in the presence or not of BuNa, for 24 h. RT-PCR and Western blot analysis was performed to evaluate the expression of inflammatory mediators and structural proteins. Results: Butyrate reduced the expression of canonic pro-inflammatory mediators (Nos2, COX-2, IL-6), pro-inflammatory adipokines (lipocalin-2 and nesfatin-1) and adhesion molecule (VCAM-1 and ICAM-1) in IL-1β-stimulated chondrocytes, inhibiting several inflammatory signalling pathways (NFκB, MAPKinase, AMPK-α, PI3K/Akt). Butyrate also reduced metalloproteinase production and limited the loss of type II collagen in IL-1β-inflamed chondrocytes. The chemoattractant effect of butyrate, after different inflammatory challenges, was revealed by increased annexin (AnxA)1 levels and chemokines expression. The chemoattractant and anti-inflammatory activities of butyrate were completely blunted by GPR43 silencing using RNA interference. Conclusion: Taken together, our data suggest the potential application of sodium butyrate as a novel candidate in a multi-target approach for the treatment of chondrocyte inflammation and cartilage degenerative process

Altered gut microbiota and endocannabinoid system tone in vitamin D deficiency-mediated chronic pain

Abstract Recent evidence points to the gut microbiota as a regulator of brain and behavior, although it remains to be determined if gut bacteria play a role in chronic pain. The endocannabinoid system is implicated in inflammation and chronic pain processing at both the gut and central nervous system (CNS) levels. In the present study, we used low Vitamin D dietary intake in mice and evaluated possible changes in gut microbiota, pain processing and endocannabinoid system signaling. Vitamin D deficiency induced a lower microbial diversity characterized by an increase in Firmicutes and a decrease in Verrucomicrobia and Bacteroidetes. Concurrently, vitamin D deficient mice showed tactile allodynia associated with neuronal hyperexcitability and alterations of endocannabinoid system members (endogenous mediators and their receptors) at the spinal cord level. Changes in endocannabinoid (anandamide and 2-arachidonoylglycerol) levels were also observed in the duodenum and colon. Remarkably, the anti-inflammatory anandamide congener, palmitoylethanolamide, counteracted both the pain behaviour and spinal biochemical changes in vitamin D deficient mice, whilst increasing the levels of Akkermansia, Eubacterium and Enterobacteriaceae, as compared with vehicle-treated mice. Finally, induction of spared nerve injury in normal or vitamin D deficient mice was not accompanied by changes in gut microbiota composition. Our data suggest the existence of a link between Vitamin D deficiency – with related changes in gut bacterial composition – and altered nociception, possibly via molecular mechanisms involving the endocannabinoid and related mediator signaling systems

Epigenetic fingerprint in endometrial carcinogenesis: the hypothesis of a uterine field cancerization.

"Abstract. Transcriptional silencing by CpG island hypermethylation plays a critical role in endometrial carcinogenesis. In a collection of benign, premalignant and malignant endometrial lesions, a methylation profile of a complete gene panel, such steroid receptors (ERα, PR), DNA mismatch repair (hMLH1), tumor-suppressor genes (CDKN2A\/P16 and CDH1\/E-CADHERIN) and WNT pathway inhibitors (SFRP1, SFRP2, SFRP4, SFRP5) was investigated in order to demonstrate their pathogenetic role in endometrial lesions. Our results indicate that gene hypermethylation may be an early event in endometrial endometrioid tumorigenesis. Particularly, ERα, PR, hMLH1, CDKN2A\/P16, SFRP1, SFRP2 and SFRP5 revealed a promoter methylation status in endometrioid carcinoma, whereas SFRP4 showed demethylation in cancer. P53 immunostaining showed weak-focal protein expression level both in hyperplasic lesions and in endometrioid cancer. Non-endometrioid cancers showed very low levels of epigenetic methylations, but strong P53 protein positivity. Fisher exact test revealed a statistically significant association between hMLH1, CDKN2A\/P16 and SFRP1 genes methylation and endometrioid carcinomas and between hMLH1 gene methylation and peritumoral endometrium (p < 0.05). Our data confirm that the methylation profile of the peritumoral endometrium is different from the altered molecular background of benign endometrial polyps and hyperplasias. Therefore, our findings suggest that the methylation of hMLH1, CDKN2A\/P16 and SFRP1 may clearly distinguish between benign and malignant lesions. Finally, this study assessed that the use of an epigenetic fingerprint may improve the current diagnostic tools for a better clinical management of endometrial lesions.

2-Pentadecyl-2-oxazoline ameliorates memory impairment and depression-like behaviour in neuropathic mice: possible role of adrenergic alpha2- and H3 histamine autoreceptors

Neuropathic pain (NP) remains an untreatable disease due to the complex pathophysiology that involves the whole pain neuraxis including the forebrain. Sensory dysfunctions such as allodynia and hyperalgesia are only part of the symptoms associated with neuropathic pain that extend to memory and affectivity deficits. The development of multi-target molecules might be a promising therapeutic strategy against the symptoms associated with NP. 2-pentadecyl-2-oxazoline (PEA-OXA) is a plant-derived agent, which has shown effectiveness against chronic pain and associated neuropsychiatric disorders. The molecular mechanisms by which PEA-OXA exerts its effects are, however, only partially known. In the current study, we show that PEA-OXA, besides being an alpha2 adrenergic receptor antagonist, also acts as a modulator at histamine H3 receptors, and report data on its effects on sensory, affective and cognitive symptoms associated with the spared nerve injury (SNI) model of neuropathic pain in mice. Treatment for 14&nbsp;days with PEA-OXA after the onset of the symptoms associated with neuropathic pain resulted in the following effects: (i) allodynia was decreased; (ii) affective/cognitive impairment associated with SNI (depression, spatial, and working memories) was counteracted; (iii) long-term potentiation in vivo in the lateral entorhinal cortex-dentate gyrus (perforant pathway, LPP) was ameliorated, (iv) hippocampal glutamate, GABA, histamine, norepinephrine and dopamine level alterations after peripheral nerve injury were reversed, (v) expression level of the TH positive neurons in the Locus Coeruleus were normalized. Thus, a 16-day treatment with PEA-OXA alleviates the sensory, emotional, cognitive, electrophysiological and neurochemical alterations associated with SNI-induced neuropathic pain

Cervical cancer benefits from trabectedin combination with the β-blocker propranolol: in vitro and ex vivo evaluations in patient-derived organoids

Background: Cervical cancer (CC) is characterized by genomic alterations in DNA repair genes, which could favor treatment with agents causing DNA double-strand breaks (DSBs), such as trabectedin. Hence, we evaluated the capability of trabectedin to inhibit CC viability and used ovarian cancer (OC) models as a reference. Since chronic stress may promote gynecological cancer and may hinder the efficacy of therapy, we investigated the potential of targeting β-adrenergic receptors with propranolol to enhance trabectedin efficacy and change tumor immunogenicity.Methods: OC cell lines, Caov-3 and SK-OV-3, CC cell lines, HeLa and OV2008, and patient-derived organoids were used as study models. MTT and 3D cell viability assays were used for drug(s) IC50 determination. The analysis of apoptosis, JC-1 mitochondrial membrane depolarization, cell cycle, and protein expression was performed by flow cytometry. Cell target modulation analyses were carried out by gene expression, Western blotting, immunofluorescence, and immunocytochemistry.Results: Trabectedin reduced the proliferation of both CC and OC cell lines and notably of CC patient-derived organoids. Mechanistically, trabectedin caused DNA DSBs and S-phase cell cycle arrest. Despite DNA DSBs, cells failed the formation of nuclear RAD51 foci and underwent apoptosis. Under norepinephrine stimulation, propranolol enhanced trabectedin efficacy, further inducing apoptosis through the involvement of mitochondria, Erk1/2 activation, and the increase of inducible COX-2. Notably, trabectedin and propranolol affected the expression of PD1 in both CC and OC cell lines.Conclusion: Overall, our results show that CC is responsive to trabectedin and provide translational evidence that could benefit CC treatment options. Our study pointed out that combined treatment offset trabectedin resistance caused by β-adrenergic receptor activation in both ovarian and cervical cancer models

Clinical and molecular epidemiology of erythropoietic protoporphyria in Italy

Background: Erythropoietic protoporphyria (EPP) is a rare inherited disease associated with heme metabolism, characterized by severe life-long photosensitivity and liver involvement. Objectives: To provide epidemiological data of EPP in Italy. Materials and Methods: Prospective/retrospective data of EPP patients were collected by an Italian network of porphyria specialist centres (Gruppo Italiano Porfiria, GrIP) over a 20-year period (1996-2017). Results: In total, 179 patients (79 females) with a clinical and biochemical diagnosis of EPP were assessed, revealing a prevalence of 3.15 cases per million persons and an incidence of 0.13 cases per million persons/year. Incidence significantly increased after 2009 (due to the availability of alfa-melanotide, which effectively limits skin photosensitivity). Mean age at diagnosis was 28 years, with only 22 patients (12.2%) diagnosed 6410 years old. Gene mutations were assessed in 173 (96.6%) patients; most (164; 91.3%) were FECH mutations on one allele in association with the hypomorphic variant, c.315-48C, on the other (classic EPP), and nine (5.2%) were ALAS2 mutations (X-linked EPP). Only one case of autosomal recessive EPP was observed. Of the 42 different FECH mutations, 15 are novel, three mutations collectively accounted for 45.9% (75/164) of the mutations (c.215dupT [27.2%], c.901_902delTG [11.5%] and c.67\u2009+\u20095G\u2009&gt;\u2009A [7.2%]), and frameshift mutations were prevalent (33.3%). A form of light protection was used by 109/179 (60.8%) patients, and 100 (56%) had at least one \u3b1-melanotide implant. Three cases of severe acute liver involvement, requiring OLT, were observed. Conclusions: These data define, for the first time, the clinical and molecular epidemiology of EPP in Italy

Butyrate Regulates Liver Mitochondrial Function, Efficiency, and Dynamics in Insulin-Resistant Obese Mice

Fatty liver, oxidative stress, and mitochondrial dysfunction are key pathophysiological features of insulin resistance and obesity. Butyrate, produced by fermentation in the large intestine by gut microbiota, and its synthetic derivative, the N-(1-carbamoyl-2-phenyl-ethyl) butyramide, FBA, have been demonstrated to be protective against insulin resistance and fatty liver. Here, hepatic mitochondria were identified as the main target of the beneficial effect of both butyrate-based compounds in reverting insulin resistance and fat accumulation in diet-induced obese mice. In particular, butyrate and FBA improved respiratory capacity and fatty acid oxidation, activated the AMPK-acetyl-CoA carboxylase pathway, and promoted inefficient metabolism, as shown by the increase in proton leak. Both treatments consistently increased utilization of substrates, especially fatty acids, leading to the reduction of intracellular lipid accumulation and oxidative stress. Finally, the shift of the mitochondrial dynamic toward fusion by butyrate and FBA resulted in the improvement not only of mitochondrial cell energy metabolism but also of glucose homeostasis. In conclusion, butyrate and its more palatable synthetic derivative, FBA, modulating mitochondrial function, efficiency, and dynamics, can be considered a new therapeutic strategy to counteract obesity and insulin resistance

Hydroxytyrosol prevents metabolic impairment reducing hepatic inflammation and restoring duodenal integrity in a rat model of NAFLD.

The potential mechanisms of action of polyphenols in nonalcoholic fatty liver disease (NAFLD) are overlooked. Here, we evaluate the beneficial therapeutic effects of hydroxytyrosol (HT), the major metabolite of the oleuropein, in a nutritional model of insulin resistance (IR) and NAFLD by high-fat diet. Young male rats were divided into three groups receiving (1) standard diet (STD; 10.5% fat), (2) high-fat diet (HFD; 58.0% fat) and (3) HFD+HT (10 mg/kg/day by gavage). After 5 weeks, the oral glucose tolerance test was performed, and at 6th week, blood sample and tissues (liver and duodenum) were collected for following determinations. The HT-treated rats showed a marked reduction in serum AST, ALT and cholesterol and improved glucose tolerance and insulin sensitivity, reducing homeostasis model assessment index. HT significantly corrected the metabolic impairment induced by HFD, increasing hepatic peroxisome proliferator activated receptor PPAR-α and its downstream-regulated gene fibroblast growth factor 21, the phosphorylation of acetyl-CoA carboxylase and the mRNA carnitine palmitoyltransferase 1a. HT also reduced liver inflammation and nitrosative/oxidative stress decreasing the nitrosylation of proteins, reactive oxygen species production and lipid peroxidation. Moreover, HT restored intestinal barrier integrity and functions (fluorescein isothiocyanate-dextran permeability and mRNA zona occludens ZO-1). Our data demonstrate the beneficial effect of HT in the prevention of early inflammatory events responsible for the onset of IR and steatosis, reducing hepatic inflammation and nitrosative/oxidative stress and restoring glucose homeostasis and intestinal barrier integrity