Carmen De Caro

Gut-brain Axis: Role of Lipids in the Regulation of Inflammation, Pain and CNS Diseases

The human gut is a composite anaerobic environment with a large, diverse and dynamic enteric microbiota, represented by more than 100 trillion microorganisms, including at least 1000 distinct species. The discovery that a different microbial composition can influence behavior and cognition, and in turn the nervous system can indirectly influence enteric microbiota composition, has significantly contributed to establish the well-accepted concept of gut-brain axis. This hypothesis is supported by several evidence showing mutual mechanisms, which involve the vague nerve, the immune system, the hypothalamic-pituitaryadrenal (HPA) axis modulation and the bacteria-derived metabolites. Many studies have focused on delineating a role for this axis in health and disease, ranging from stress-related disorders such as depression, anxiety and irritable bowel syndrome (IBS) to neurodevelopmental disorders, such as autism, and to neurodegenerative diseases, such as Parkinson Disease, Alzheimers Disease etc. Based on this background, and considering the relevance of alteration of the symbiotic state between host and microbiota, this review focuses on the role and the involvement of bioactive lipids, such as the N-acylethanolamine (NAE) family whose main members are N-arachidonoylethanolamine (AEA), palmitoylethanolamide (PEA) and oleoilethanolamide (OEA), and short chain fatty acids (SCFAs), such as butyrate, belonging to a large group of bioactive lipids able to modulate peripheral and central pathologic processes. Their effective role has been studied in inflammation, acute and chronic pain, obesity and central nervous system diseases. A possible correlation has been shown between these lipids and gut microbiota through different mechanisms. Indeed, systemic administration of specific bacteria can reduce abdominal pain through the involvement of cannabinoid receptor 1 in the rat; on the other hand, PEA reduces inflammation markers in a murine model of inflammatory bowel disease (IBD), and butyrate, producted by gut microbiota, is effective in reducing inflammation and pain in irritable bowel syndrome and IBD animal models. In this review, we underline the relationship among inflammation, pain, microbiota and the different lipids, focusing on a possible involvement of NAEs and SCFAs in the gut-brain axis and their role in the central nervous system diseases.

Palmitoylethanolamide protects mice against 6-OHDA-induced neurotoxicity and endoplasmic reticulum stress: In vivo and in vitro evidence.

Several pathogenetic factors have been involved in the onset and progression of Parkinsons disease (PD), including inflammation, oxidative stress, unfolded protein accumulation, and apoptosis. Palmitoylethanolamide (PEA), an endogenous N-acylethanolamine, has been shown to be a neuroprotective and anti-inflammatory molecule, acting as a peroxisome proliferator activated receptor (PPAR)-α agonist. In this study we investigated the effects of PEA on behavioral alterations and the underlying pathogenic mechanisms in the 6-hydroxydopamine (6-OHDA)-induced model of PD in male mice. Additionally, we showed the involvement of PPAR-α in PEA protective effect on SH-SY5Y neuroblastoma against 6-OHDA damage. Here, we report that PEA (3-30mg/kg/days.c.) improved behavioral impairments induced by unilateral intrastriatal injection of 6-OHDA. This effect was accompanied by a significant increase in tyrosine hydroxylase expression at striatal level, indicating PEA preserving effect on dopaminergic neurons. Moreover, we found a reduction in the expression of pro-inflammatory enzymes, i.e. inducible nitric oxide synthase and cyclooxygenase-2, a modulation between pro- and anti-apoptotic markers, suggestive of PEA capability in controlling neuroinflammation and cell death. Interestingly, PEA also showed protective scavenging effect, through superoxide dismutase induction, and dampened unfolding protein response, interfering on glucose-regulated protein 78 expression and PERK-eIF2α pathway. Similar data were found in in vitro studies, where PEA treatment was found to rescue SH-SY5Y neuroblastoma cells from 6-OHDA-induced damage and death, partly by inhibiting endoplasmic reticulum stress detrimental response. Therefore, PEA, counteracting the pathogenetic aspects involved in the development of PD, showed its therapeutic potential, possibly integrating current treatments correcting dopaminergic deficits and motor dysfunction.

Nanoparticles prolong N-palmitoylethanolamide anti-inflammatory and analgesic effects in vivo

N-Palmitoylethanolamide showed great therapeutic potential in the treatment of inflammation and pain but its unfavourable pharmacokinetics properties will hinder its use in the clinical practice. A nanotechnology-based formulation was developed to enhance the probability of N-palmitoylethanolamide therapeutic success, especially in skin disease management. Lipid nanoparticles were produced and characterized to evaluate their mean size, ζ-potential, thermal behaviour, and morphology. The ability of N-palmitoylethanolamide to diffuse across the epidermis as well as anti-inflammatory and analgesic effects were investigated. Particles had a mean size of about 150 nm and a ζ-potential of -40 mV. DSC data confirmed the solid state of the matrix and the embedding of N-palmitoylethanolamide while electron microscopy have evidenced a peculiar internal structure (i.e., low-electrondense spherical objects within the matrix) that can be reliably ascribed to the presence of oil nanocompartments. Lipid nanoparticles increased N-palmitoylethanolamide percutaneous diffusion and prolonged the anti-inflammatory and analgesic effects in vivo. Lipid nanoparticles seem a good nanotechnology-based strategy to bring N-palmitoylethanolamide to clinics.

Sodium butyrate and its synthetic amide derivative modulate nociceptive behaviors in mice

In the present study we investigated the role of sodium butyrate (butyrate), and its more palatable derivative, the N-(1-carbamoyl-2-phenyl-ethyl) butyramide (FBA), in animal models of acute and chronic pain. We found that oral administrations of butyrate (10-200mg/Kg) or equimolecular FBA (21.2-424mg/Kg) reduced visceral pain in a dose- and time-dependent manner. Both drugs were also effective in the formalin test, showing an antinociceptive effect. This analgesic effect was blocked by glibenclamide, suggesting the involvement of ATP-dependent K(+) channels. Moreover, following repeated administration butyrate (100-200mg/Kg) and FBA (212-424mg/Kg) retained their analgesic properties in a model of neuropathic pain, reducing mechanical and thermal hyperalgesia in the chronic constriction injury (CCI) model. The involvement of peroxisome proliferator-activated receptor (PPAR) -α and -γ for the analgesic effect of butyrate was also investigated by using PPAR-α null mice or the PPAR-γ antagonist GW9662. Western blot analysis, confirmed the role of peroxisome receptors in butyrate effects, evidencing the increase of PPAR-α and -γ expression, associated to the reduction of inflammatory markers (COX-2, iNOS, TNF-α and cFOS). In conclusion, we describe the role of butyrate-based drugs in pain, identifying different and converging non-genomic and genomic mechanisms of action, which cooperate in nociception maintenance.

Down regulation of pro-inflammatory pathways by tanshinone IIA and cryptotanshinone in a non-genetic mouse model of Alzheimer's disease

Graphical abstract Figure. No caption available. &NA; Alzheimers disease (AD) is a common form of dementia mainly characterized by the deposition of neurofibrillary tangles and &bgr;‐amyloid (A&bgr;) peptides in the brain. Additionally, increasing evidence demonstrates that a neuro‐inflammatory state plays a key role in the development of this disease. Beside synthetic drugs, the use of natural compounds represents an alternative for the development of new potential drugs for the treatment of AD. Among these, the root of Salvia miltiorhiza Bunge (also known as Danshen) used for the treatment of cardiovascular, cerebrovascular disease and CNS functional decline in Chinese traditional medicine is one of the most representative examples. We therefore evaluated the effects of tanshinone IIA (TIIA) and cryptotanshinone (CRY) (the two major lipophilic compounds of Danshen) in a non‐genetic mouse model of &bgr;‐amyloid (A&bgr;)‐induced AD, which is mainly characterized by reactive gliosis and neuro‐inflammation in the brain. To this aim, mice were injected intracerebroventricularly (i.c.v.) with A&bgr;1–42 peptide (3 &mgr;g/3 &mgr;l) and after with TIIA and CRY (1, 3, or 10 mg/kg) intraperitoneally (i.p.) 3 times weekly for 21 days following the induction of experimental AD. Spatial working memory was assessed as a measure of short‐term memory in mice, whereas the level of GFAP, S100&bgr;, COX‐2, iNOS and NF‐kBp65 monitored by western blot and ELISA assay, were selected as markers of reactive gliosis and neuro‐inflammation. Finally, by docking studies, the modulation of key pro‐inflammatory enzymes and pathways involved in the AD‐related neuro‐inflammation were also investigated. Results indicate that TIIA and CRY alleviate memory decline in A&bgr;1‐42‐injected mice, in a dose dependent manner. Moreover, the analysis of gliosis‐related and neuro‐inflammatory markers in the hippocampal tissues reveal a remarkable reduction in the expression of GFAP, S100&bgr;, COX‐2, iNOS and NF‐kBp65 after CRY (10 mg/kg) treatment. These effects were less evident, but still significant, after TIIA (10 mg/kg). Finally, in silico analysis also revealed that both compounds were able to interact with the binding sites of NF‐kBp65 endorsing the data from biochemical analysis. We conclude that TIIA and CRY display anti‐inflammatory and neuroprotective effect in a non‐genetic mouse model of AD, thus playing a role in slowing down the course and onset of AD.

Specific signatures of the gut microbiota and increased levels of butyrate in children treated with fermented cow's milk containing heat-killed Lactobacillus paracasei CBA L74

ABSTRACT We recently demonstrated that cows milk fermented with the probiotic Lactobacillus paracasei CBA L74 (FM-CBAL74) reduces the incidence of respiratory and gastrointestinal tract infections in young children attending school. This effect apparently derives from a complex regulation of non-immune and immune protective mechanisms. We investigated whether FM-CBAL74 could regulate gut microbiota composition and butyrate production. We randomly selected 20 healthy children (12 to 48 months) from the previous randomized controlled trial, before (t0) and after 3 months (t3) of dietary treatment with FM-CBAL74 (FM) or placebo (PL). Fecal microbiota was profiled using 16S rRNA gene amplicon sequencing, and the fecal butyrate concentration was also measured. Microbial alpha and beta diversities were not significantly different between groups prior to treatment. FM-CBAL74 but not PL treatment increased the relative abundance of Lactobacillus. Individual Blautia, Roseburia, and Faecalibacterium oligotypes were associated with FM-CBAL74 treatment and demonstrated correlative associations with immune biomarkers. Accordingly, PICRUSt analysis predicted an increase in the proportion of genes involved in butyrate production pathways, consistent with an increase in fecal butyrate observed only in the FM group. Dietary supplementation with FM-CBAL74 induces specific signatures in gut microbiota composition and stimulates butyrate production. These effects are associated with changes in innate and acquired immunity. IMPORTANCE The use of a fermented milk product containing the heat-killed probiotic strain Lactobacillus paracasei CBAL74 induces changes in the gut microbiota, promoting the development of butyrate producers. These changes in the gut microbiota composition correlate with increased levels of innate and acquired immunity biomarkers.

Extensively hydrolyzed casein formula alone or with L. rhamnosus GG reduces β‐lactoglobulin sensitization in mice

Extensively hydrolyzed casein formula (EHCF) has been proposed for the prevention and is commonly used for the treatment of cows milk allergy (CMA). The addition of the probiotic Lactobacillus rhamnosus GG (LGG) to EHCF may induce faster acquisition of tolerance to cows milk. The mechanisms underlying this effect are largely unexplored. We investigated the effects of EHCF alone or in combination with LGG on β‐lactoglobulin (BLG) sensitization in mice.

Palmitoylethanolamide Treatment Reduces Blood Pressure in Spontaneously Hypertensive Rats: Involvement of Cytochrome P450-Derived Eicosanoids and Renin Angiotensin System

Palmitoylethanolamide (PEA), a peroxisome proliferator-activated receptor-α agonist, has been demonstrated to reduce blood pressure and kidney damage secondary to hypertension in spontaneously hypertensive rat (SHR). Currently, no information is available concerning the putative effect of PEA on modulating vascular tone. Here, we investigate the mechanisms underpinning PEA blood pressure lowering effect, exploring the contribution of epoxyeicosatrienoic acids, CYP-dependent arachidonic acid metabolites, as endothelium-derived hyperpolarizing factors (EDHF), and renin angiotensin system (RAS) modulation. To achieve this aim SHR and Wistar-Kyoto rats were treated with PEA (30 mg/kg/day) for five weeks. Functional evaluations on mesenteric bed were performed to analyze EDHF-mediated vasodilation. Moreover, mesenteric bed and carotid were harvested to measure CYP2C23 and CYP2J2, the key isoenzymes in the formation of epoxyeicosatrienoic acids, and the soluble epoxide hydrolase, which is responsible for their degradation in the corresponding diols. Effect of PEA on RAS modulation was investigated by analyzing angiotensin converting enzyme and angiotensin receptor 1 expression. Here, we showed that EDHF-mediated dilation in response to acetylcholine was increased in mesenteric beds of PEA-treated SHR. Western blot analysis revealed that the increase in CYP2C23 and CYP2J2 observed in SHR was significantly attenuated in mesenteric beds of PEA-treated SHR, but unchanged in the carotids. Interestingly, in both vascular tissues, PEA significantly decreased the soluble epoxide hydrolase protein level, accompanied by a reduced serum concentration of its metabolite 14-15 dihydroxyeicosatrienoic acid, implying a reduction in epoxyeicosatrienoic acid hydrolisis. Moreover, PEA treatment down-regulated angiotensin receptor 1 and angiotensin converting enzyme expression, indicating a reduction in angiotensin II-mediated effects. Consistently, a damping of the activation of angiotensin receptor 1 underlying pathways in mesenteric beds was shown in basal conditions in PEA-treated SHR. In conclusion, our data demonstrate the involvement of epoxyeicosatrienoic acids and renin angiotensin system in the blood pressure lowering effect of PEA.

Antinociceptive effect of two novel transient receptor potential melastatin 8 antagonists in acute and chronic pain models in rat

Transient receptor potential (TRP) channels are a superfamily of non‐selective cation permeable channels involved in peripheral sensory signalling. Animal studies have shown that several TRPs are important players in pain modulation. Among them, the TRP melastatin 8 (TRPM8) has elicited more interest for its controversial role in nociception. This channel, expressed by a subpopulation of sensory neurons in dorsal root ganglia (DRG) and trigeminal ganglia (TG), is activated by cold temperatures and cooling agents. In experimental neuropathic pain models, an up‐regulation of this receptor in DRG and TG has been observed, suggesting a key role for TRPM8 in the development and maintenance of pain. Consistent with this hypothesis, TRPM8 knockout mice are less responsive to pain stimuli.

The potential role of cannabinoids in epilepsy treatment

ABSTRACT Introduction: Epilepsy is one of the world’s oldest recognized and prevalent neurological diseases. It has a great negative impact on patients’ quality of life (QOL) as a consequence of treatment resistant seizures in about 30% of patients together with drugs’ side effects and comorbidities. Therefore, new drugs are needed and cannabinoids, above all cannabidiol, have recently gathered attention. Areas covered: This review summarizes the scientific data from human and animal studies on the major cannabinoids which have been of interest in the treatment of epilepsy, including drugs acting on the endocannabinoid system. Expert commentary: Despite the fact that cannabis has been used for many purposes over 4 millennia, the development of drugs based on cannabinoids has been very slow. Only recently, research has focused on their potential effects and CBD is the first treatment of this group with clinical evidence of efficacy in children with Dravet syndrome; moreover, other studies are currently ongoing to confirm its effectiveness in patients with epilepsy. On the other hand, it will be of interest to understand whether drugs acting on the endocannabinoid system will be able to reach the market and prove their known preclinical efficacy also in patients with epilepsy.