Chemogenetic inhibition of central amygdala CRF-expressing neurons decreases alcohol intake but not trauma-related behaviors in a rat model of post-traumatic stress and alcohol use disorder.

Post-traumatic stress disorder (PTSD) and alcohol use disorder (AUD) are often comorbid. Few treatments exist to reduce comorbid PTSD/AUD. Elucidating the mechanisms underlying their comorbidity could reveal new avenues for therapy. Here, we employed a model of comorbid PTSD/AUD, in which rats were subjected to a stressful shock in a familiar context followed by alcohol drinking. We then examined fear overgeneralization and irritability in these rats. Familiar context stress elevated drinking, increased fear overgeneralization, increased alcohol-related aggressive signs, and elevated peripheral stress hormones. We then examined transcripts of stress- and fear-relevant genes in the central amygdala (CeA), a locus that regulates stress-mediated alcohol drinking. Compared with unstressed rats, stressed rats exhibited increases in CeA transcripts for Crh and Fkbp5 and decreases in transcripts for Bdnf and Il18. Levels of Nr3c1 mRNA, which encodes the glucocorticoid receptor, increased in stressed males but decreased in stressed females. Transcripts of Il18 binding protein (Il18bp), Glp-1r, and genes associated with calcitonin gene-related peptide signaling (Calca, Ramp1, Crlr-1, and Iapp) were unaltered. Crh, but not Crhr1, mRNA was increased by stress; thus, we tested whether inhibiting CeA neurons that express corticotropin-releasing factor (CRF) suppress PTSD/AUD-like behaviors. We used Crh-Cre rats that had received a Cre-dependent vector encoding hM4D(Gi), an inhibitory Designer Receptors Exclusively Activated by Designer Drugs. Chemogenetic inhibition of CeA CRF neurons reduced alcohol intake but not fear overgeneralization or irritability-like behaviors. Our findings suggest that CeA CRF modulates PTSD/AUD comorbidity, and inhibiting CRF neural activity is primarily associated with reducing alcohol drinking but not trauma-related behaviors that are associated with PTSD/AUD.

Ethanol withdrawal-induced adaptations in prefrontal corticotropin releasing factor receptor 1-expressing neurons regulate anxiety and conditioned rewarding effects of ethanol.

Prefrontal circuits are thought to underlie aberrant emotion contributing to relapse in abstinence; however, the discrete cell-types and mechanisms remain largely unknown. Corticotropin-releasing factor and its cognate type-1 receptor, a prominent brain stress system, is implicated in anxiety and alcohol use disorder (AUD). Here, we tested the hypothesis that medial prefrontal cortex CRF1-expressing (mPFCCRF1+) neurons comprise a distinct population that exhibits neuroadaptations following withdrawal from chronic ethanol underlying AUD-related behavior. We found that mPFCCRF1+ neurons comprise a glutamatergic population with distinct electrophysiological properties and regulate anxiety and conditioned rewarding effects of ethanol. Notably, mPFCCRF1+ neurons undergo unique neuroadaptations compared to neighboring neurons including a remarkable decrease in excitability and glutamatergic signaling selectively in withdrawal, which is driven in part by the basolateral amygdala. To gain mechanistic insight into these electrophysiological adaptations, we sequenced the transcriptome of mPFCCRF1+ neurons and found that withdrawal leads to an increase in colony-stimulating factor 1 (CSF1) in this population. We found that selective overexpression of CSF1 in mPFCCRF1+ neurons is sufficient to decrease glutamate transmission, heighten anxiety, and abolish ethanol reinforcement, providing mechanistic insight into the observed mPFCCRF1+ synaptic adaptations in withdrawal that drive these behavioral phenotypes. Together, these findings highlight mPFCCRF1+ neurons as a critical site of enduring adaptations that may contribute to the persistent vulnerability to ethanol misuse in abstinence, and CSF1 as a novel target for therapeutic intervention for withdrawal-related negative affect.

Cannabidiol-rich non-psychotropic Cannabis sativa L. oils attenuate peripheral neuropathy symptoms by regulation of CB2-mediated microglial neuroinflammation.

Neuropathic pain (NP) is a chronic disease that affects the normal quality of life of patients. To date, the therapies available are only symptomatic and they are unable to reduce the progression of the disease. Many studies reported the efficacy of Cannabis sativa L. (C. sativa) on NP, but no Δ9 -tetrahydrocannabinol (Δ9 -THC)-free extracts have been investigated in detail for this activity so far. The principal aim of this work is to investigate the potential pain-relieving effect of innovative cannabidiol-rich non-psychotropic C. sativa oils, with a high content of terpenes (K2), compared to the same extract devoid of terpenes (K1). Oral administration of K2 (25 mg kg-1 ) induced a rapid and long-lasting relief of pain hypersensitivity in a mice model of peripheral neuropathy. In spinal cord samples, K2 reduced mitogen-activated protein kinase (MAPKs) levels and neuroinflammatory factors. These effects were reverted by the administration of a CB2 antagonist (AM630), but not by a CB1 antagonist (AM251). Conversely, K1 showed a lower efficacy in the absence of CB1/CB2-mediated mechanisms. In LPS-stimulated murine microglial cells (BV2), K2 reduced microglia pro-inflammatory phenotype through the downregulation of histone deacetylase 1 (HDAC-1) and nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor (IKBα) and increased interleukin-10 (IL-10) expression, an important antiinflammatory cytokine. In conclusion, these results suggested that K2 oral administration attenuated NP symptoms by reducing spinal neuroinflammation and underline the important role of the synergism between cannabinoids and terpenes.

Helichrysum stoechas (L.) Moench reduces body weight gain and modulates mood disorders via inhibition of silent information regulator 1 (SIRT1) by arzanol.

The prevalence of obesity is steadily rising, making safe and more efficient anti-obesity treatments an urgent medical need. Growing evidence correlates obesity and comorbidities, including anxiety and depression, with the development of a low-grade inflammation in peripheral and central tissues. We hypothesized that attenuating neuroinflammation might reduce weight gain and improve mood. We investigated the efficacy of a methanolic extract from Helichrysum stoechas (L.) Moench (HSE), well-known for its anti-inflammatory properties, and its main constituent arzanol (AZL). HPLC-ESI-MS2 and HPLC-UV were used to characterize the extract. HSE effects on mood and feeding behavior was assessed in mice. The mechanism of action of HSE and AZL was investigated in hippocampus samples and SH-SY5Y cells by western blotting and immunofluorescence. Oral administration of HSE for 3 weeks limited weight gain with no significant decrease in food intake. HSE produced an anxiolytic-like and antidepressant-like phenotype comparable to diazepam and amitriptyline, respectively, in the absence of locomotor and cognitive impairments and induced neuroprotective effects in glutamate-exposed SH-SY5Y cells. A dose-dependent reduction of SIRT1 expression was detected in SH-SY5Y cells and in hippocampal samples from HSE-treated mice. The inhibition of the SIRT1-FoxO1 pathway was induced in the hypothalamus. Molecular docking studies proposed a mechanism of SIRT1 inhibition by AZL, confirmed by the evaluation of inhibitory effects on SIRT1 enzymatic activity. HSE limited weight gain and comorbidities through an AZL-mediated SIRT1 inhibition. These activities indicate HSE an innovative therapeutic perspective for obesity and associated mood disorders.

Novel Combination of Choline with Withania somnifera (L.) Dunal, and Bacopa monnieri (L.) Wetts Reduced Oxidative Stress in Microglia Cells, Promoting Neuroprotection.

Memory deficit is one of the major negative outcomes of chronic stress. Cholinergic system modulates memory not only through the neuronal cells, but also via interactions with non-neuronal cells, suggesting that microglia can influence synaptic function and plasticity, contributing to cognition and memory function. Withania somnifera (L.) Dunal (WS) and Bacopa monnieri (L.) Wettst (BM), are traditional herbal medicinal products used for the temporary relief of symptoms of stress. The aim of this study was to investigate whether choline (CLN) activity could be enhanced via an association with adaptogens: WS and BM extracts. First, we optimized an in vitro model of corticotropin-releasing hormone (CRH)-induced oxidative stress on microglial BV2 cells. CRH 100 nM reduced BV2 cell viability and induced morphological changes and neurotoxicity after 24 h of microglia stimulation. Moreover, it induced an increase in the production of reactive oxygen species (ROS) and dysregulated antioxidant protein (i.e., SIRT-1 and NRF-2). The association between choline and adaptogens (CBW) 10 µg/mL counteracted the effect of CRH on BV2 cells and reduced the neurotoxicity produced by BV2 CRH-conditioned medium in the SH-SY5Y cell lines. CBW 200 mg/kg produced an ameliorative effect on recognition memory in the novel object recognition test (NORT) test in mice. In conclusion, combining choline with adaptogen plant extracts might represent a promising intervention in chronic stress associated with memory disturbances through the attenuation of microglia-induced oxidative stress.

Non-psychotropic Cannabis sativa L. phytocomplex modulates microglial inflammatory response through CB2 receptors-, endocannabinoids-, and NF-κB-mediated signaling.

Cannabis sativa L. is increasingly emerging for its protective role in modulating neuroinflammation, a complex process orchestrated among others by microglia, the resident immune cells of the central nervous system. Phytocannabinoids, especially cannabidiol (CBD), terpenes, and other constituents trigger several upstream and downstream microglial intracellular pathways. Here, we investigated the molecular mechanisms of a CBD- and terpenes-enriched C. sativa extract (CSE) in an in vitro model of neuroinflammation. We evaluated the effect of CSE on the inflammatory response induced by exposure to lipopolysaccharide (LPS) in BV-2 microglial cells, compared with CBD and ß-caryophyllene (CAR), CB2 receptors (CB2r) inverse and full agonist, respectively. The LPS-induced upregulation of the pro-inflammatory cytokines IL-1ß, IL-6, and TNF-α was significantly attenuated by CSE and only partially by CBD, whereas CAR was ineffective. In BV-2 cells, these anti-inflammatory effects exerted by CSE phytocomplex were only partially dependent on CB2r modulation and they were mediated by the regulation of enzymes responsible for the endocannabinoids metabolism, by the inhibition of reactive oxygen species release and the modulation of JNK/p38 cascade with consequent NF-κB p65 nuclear translocation suppression. Our data suggest that C. sativa phytocomplex and its multitarget mechanism could represent a novel therapeutic strategy for neuroinflammatory-related diseases.

The Histamine H4 Receptor Participates in the Neuropathic Pain-Relieving Activity of the Histamine H3 Receptor Antagonist GSK189254.

Growing evidence points to the histamine system as a promising target for the management of neuropathic pain. Preclinical studies reported the efficacy of H3R antagonists in reducing pain hypersensitivity in models of neuropathic pain through an increase of histamine release within the CNS. Recently, a promising efficacy of H4R agonists as anti-neuropathic agents has been postulated. Since H3R and H4R are both localized in neuronal areas devoted to pain processing, the aim of the study is to investigate the role of H4R in the mechanism of anti-hyperalgesic action of the H3R antagonist GSK189254 in the spared nerve injury (SNI) model in mice. Oral (6 mg/kg), intrathecal (6 µg/mouse), or intra locus coeruleus (LC) (10 µg/µL) administration of GSK189254 reversed mechanical and thermal allodynia in the ipsilateral side of SNI mice. This effect was completely prevented by pretreatment with the H4R antagonist JNJ 10191584 (6 µg/mouse i.t.; (10 µg/µL intraLC). Furthermore, GSK189254 was devoid of any anti-hyperalgesic effect in H4R deficient mice, compared with wild type mice. Conversely, pretreatment with JNJ 10191584 was not able to prevent the hypophagic activity of GSK189254. In conclusion, we demonstrated the selective contribution of H4R to the H3R antagonist-induced attenuation of hypernociceptive behavior in SNI mice. These results might help identify innovative therapeutic interventions for neuropathic pain.

Combined inhibition of histone deacetylases and BET family proteins as epigenetic therapy for nerve injury-induced neuropathic pain.

Current treatments for neuropathic pain have often moderate efficacy and present unwanted effects showing the need to develop effective therapies. Accumulating evidence suggests that histone acetylation plays essential roles in chronic pain and the analgesic activity of histone deacetylases (HDACs) inhibitors is documented. Bromodomain and extra-terminal domain (BET) proteins are epigenetic readers that interact with acetylated lysine residues on histones, but little is known about their implication in neuropathic pain. Thus, the current study was aimed to investigate the effect of the combination of HDAC and BET inhibitors in the spared nerve injury (SNI) model in mice. Intranasal administration of i-BET762 (BET inhibitor) or SAHA (HDAC inhibitor) attenuated thermal and mechanical hypersensitivity and this antiallodynic activity was improved by co-administration of both drugs. Spinal cord sections of SNI mice showed an increased expression of HDAC1 and Brd4 proteins and combination produced a stronger reduction compared to each epigenetic agent alone. SAHA and i-BET762, administered alone or in combination, counteracted the SNI-induced microglia activation by inhibiting the expression of IBA1, CD11b, inducible nitric oxide synthase (iNOS), the activation of nuclear factor-κB (NF-κB) and signal transducer and activator of transcription-1 (STAT1) with comparable efficacy. Conversely, the epigenetic inhibitors showed a modest effect on spinal proinflammatory cytokines content that was significantly potentiated by their combination. Present results indicate a key role of acetylated histones and their recruitment by BET proteins on microglia-mediated spinal neuroinflammation. Targeting neuropathic pain with the combination of HDAC and BET inhibitors may represent a promising new therapeutic option.

Dual BET/HDAC inhibition to relieve neuropathic pain: Recent advances, perspectives, and future opportunities.

Despite the intense research on developing new therapies for neuropathic pain states, available treatments have limited efficacy and unfavorable safety profiles. Epigenetic alterations have a great influence on the development of cancer and neurological diseases, as well as neuropathic pain. Histone acetylation has prevailed as one of the well investigated epigenetic modifications in these diseases. Altered spinal activity of histone deacetylase (HDAC) and Bromo and Extra terminal domain (BET) have been described in neuropathic pain models and restoration of these aberrant epigenetic modifications showed pain-relieving activity. Over the last decades HDACs and BETs have been the focus of drug discovery studies, leading to the development of numerous small-molecule inhibitors. Clinical trials to evaluate their anticancer activity showed good efficacy but raised toxicity concerns that limited translation to the clinic. To maximize activity and minimize toxicity, these compounds can be applied in combination of sub-maximal doses to produce additive or synergistic interactions (combination therapy). Recently, of particular interest, dual BET/HDAC inhibitors (multi-target drugs) have been developed to assure simultaneous modulation of BET and HDAC activity by a single molecule. This review will summarize the most recent advances with these strategies, describing advantages and limitations of single drug treatment vs combination regimens. This review will also provide a focus on dual BET/HDAC drug discovery investigations as future therapeutic opportunity for human therapy of neuropathic pain.

Pinocembrin and its linolenoyl ester derivative induce wound healing activity in HaCaT cell line potentially involving a GPR120/FFA4 mediated pathway.

Wound healing represents an urgent need from the clinical point of view. Several diseases result in wound conditions which are difficult to treat, such as in the case of diabetic foot ulcer. Starting from there, the medicinal research has focused on various targets over the years, including GPCRs as new wound healing drug targets. In line with this, GPR120, known to be an attractive target in type 2 diabetes drug discovery, was studied to finalize the development of new wound healing agents. Pinocembrin (HW0) was evaluated as a suitable compound for interacting with GPR120, and was hybridized with fatty acids, which are known endogenous GPR120 ligands, to enhance the wound healing potential and GPR120 interactions. HW0 and its 7-linolenoyl derivative (HW3) were found to be innovative wound healing agents. Immunofluorescence and functional assays suggested that their activity was mediated by GPR120, and docking simulations showed that the compounds could share the same pocket occupied by the known GPR120 agonist, TUG-891.

Targeting the RNA-Binding Protein HuR as Potential Thera-Peutic Approach for Neurological Disorders: Focus on Amyo-Trophic Lateral Sclerosis (ALS), Spinal Muscle Atrophy (SMA) and Multiple Sclerosis.

The importance of precise co- and post-transcriptional processing of RNA in the regulation of gene expression has become increasingly clear. RNA-binding proteins (RBPs) are a class of proteins that bind single- or double-chain RNA, with different affinities and selectivity, thus regulating the various functions of RNA and the fate of the cells themselves. ELAV (embryonic lethal/abnormal visual system)/Hu proteins represent an important family of RBPs and play a key role in the fate of newly transcribed mRNA. ELAV proteins bind AU-rich element (ARE)-containing transcripts, which are usually present on the mRNA of proteins such as cytokines, growth factors, and other proteins involved in neuronal differentiation and maintenance. In this review, we focused on a member of ELAV/Hu proteins, HuR, and its role in the development of neurodegenerative disorders, with a particular focus on demyelinating diseases.

Phytotherapy in the Management of Diabetes: A Review.

Phytotherapy has long been a source of medicinal products and over the years there have been many attempts to use herbal medicines for the treatment of diabetes. Several medicinal plants and their preparations have been demonstrated to act at key points of glucidic metabolism. The most common mechanisms of action found include the inhibition of α-glucosidase and of AGE formation, the increase of GLUT-4 and PPARs expression and antioxidant activity. Despite the large amount of literature available, the actual clinical effectiveness of medicinal plants in controlling diabetes-related symptoms remains controversial and there is a crucial need for stronger evidence-based data. In this review, an overview of the medicinal plants, which use in the management of diabetes is supported by authoritative monographs, is provided. References to some species which are currently under increasing clinical investigation are also reported.

Novel Dual-Acting Hybrids Targeting Type-2 Cannabinoid Receptors and Cholinesterase Activity Show Neuroprotective Effects In Vitro and Amelioration of Cognitive Impairment In Vivo.

Alzheimer's disease (AD) is a neurodegenerative form of dementia characterized by the loss of synapses and a progressive decline in cognitive abilities. Among current treatments for AD, acetylcholinesterase (AChE) inhibitors have efficacy limited to symptom relief, with significant side effects and poor compliance. Pharmacological agents that modulate the activity of type-2 cannabinoid receptors (CB2R) of the endocannabinoid system by activating or blocking them have also been shown to be effective against neuroinflammation. Herein, we describe the design, synthesis, and pharmacological effects in vitro and in vivo of dual-acting compounds that inhibit AChE and butyrylcholinesterase (BChE) and target CB2R. Within the investigated series, compound 4g proved to be the most promising. It achieved IC50 values in the low micromolar to submicromolar range against both human cholinesterase isoforms while antagonizing CB2R with Ki of 31 nM. Interestingly, 4g showed neuroprotective effects on the SH-SY5Y cell line thanks to its ability to prevent oxidative stress-induced cell toxicity and reverse scopolamine-induced amnesia in the Y-maze forced alternation test in vivo.

Microglia senescence is related to neuropathic pain-associated comorbidities in the spared nerve injury model.

ABSTRACT: The increased presence of senescent cells in different neurological diseases suggests the contribution of senescence in the pathophysiology of neurodegenerative disorders. Microglia can adapt to any type of disturbance of the homeostasis of the central nervous system, and its altered activity can lead to permanent and unresolvable damage. The aim of this work was to characterize the behavioural phenotype of spared nerve injury mice and then associate it with senescence-related mechanisms. In this work, we investigated the timing of the onset of anxiety, depression, or memory decline associated with peripheral neuropathic pain and their correlation with the presence of microglial cellular senescence. Spared nerve injury mice showed a persistent pain hypersensitivity from 3 days after surgery. Twenty-eight days after nerve injury, they also developed anxiety, depression, and cognitive impairment. The appearance of these symptoms was coincident to a significant increase of senescence markers, such as ß-galactosidase and senescent-associated secretory phenotype, at the microglial level in the spinal cord and hippocampus of spared nerve injury animals. These markers were unaltered at previous time points. In murine immortalized microglial cells (BV2) stimulated with LPS 500 ng/mL for 10 days (4 hours/day) every other day, we observed an increase of ß-galactosidase and senescent-associated secretory phenotype appearance, a reduction of cell viability, and an increase of senescence-associated heterochromatin foci. Therefore, present findings could represent an important step to a better understanding of the pathophysiological cellular mechanisms in comorbidities related to neuropathic pain states.

Honokiol-Rich Magnolia officinalis Bark Extract Attenuates Trauma-Induced Neuropathic Pain.

Neuropathic pain (NP) affects about 8% of the general population. Current analgesic therapies have limited efficacy, making NP one of the most difficult to treat pain conditions. Evidence indicates that excessive oxidative stress can contribute to the onset of chronic NP and several natural antioxidant compounds have shown promising efficacy in NP models. Thus, this study aimed to investigate the pain-relieving activity of honokiol (HNK)-rich standardized extract of Magnolia officinalis Rehder & E. Wilson bark (MOE), well known for its antioxidant and anti-inflammatory properties, in the spared nerve injury (SNI) model. The molecular mechanisms and efficacy toward neuroinflammation were investigated in spinal cord samples from SNI mice and LPS-stimulated BV2 microglia cells. MOE and HNK showed antioxidant activity. MOE (30 mg/kg p.o.) produced an antiallodynic effect in SNI mice in the absence of locomotor impairment, reduced spinal p-p38, p-JNK1, iNOS, p-p65, IL-1ß, and Nrf2 overexpression, increased IL-10 and MBP levels and attenuated the Notch signaling pathway by reducing Jagged1 and NEXT. These effects were prevented by the CB1 antagonist AM251. HNK reduced the proinflammatory response of LPS-stimulated BV2 and reduced Jagged1 overexpression. MOE and HNK, by modulating oxidative and proinflammatory responses, might represent interesting candidates for NP management.

Posttranscriptional Regulation of Gene Expression Participates in the Myelin Restoration in Mouse Models of Multiple Sclerosis: Antisense Modulation of HuR and HuD ELAV RNA Binding Protein.

Neuropathic pain is the most difficult-to-treat pain syndrome in multiple sclerosis. Evidence relates neuropathic pain to demyelination, which often originates from unresolved neuroinflammation or altered immune response. Posttranscriptional regulation of gene expression might play a fundamental role in the regulation of these processes. The ELAV RNA-binding proteins HuR and HuD are involved in the promotion of inflammatory phenomena and in neuronal development and maintenance, respectively. Thus, the aim of this study was to investigate the role of HuR and HuD in demyelination-associated neuropathic pain in the mouse experimental autoimmune encephalomyelitis (EAE) model. HuR resulted overexpressed in the spinal cord of MOG35-55-EAE and PLP139-151-EAE mice and was detected in CD11b + cells. Conversely, HuD was largely downregulated in the MOG-EAE spinal cord, along with GAP43 and neurofilament H, while in PLP-EAE mice, HuD and neuronal markers remained unaltered. Intranasal antisense oligonucleotide (ASO) delivery to knockdown HuR, increased myelin basic protein expression, and Luxol Fast Blue staining in both EAE models, an indication of increased myelin content. These effects temporally coincided with attenuation of pain hypersensitivity. Anti-HuR ASO increased the expression of HuD in GAP43-expressing cells and promoted a HuD-mediated neuroprotective activity in MOG-EAE mice, while in PLP-EAE mice, HuR silencing dampened pro-inflammatory responses mediated by spinal microglia activation. In conclusion, anti-HuR ASO showed myelin protection at analgesic doses with multitarget mechanisms, and it deserves further consideration as an innovative agent to counteract demyelination in neuropathic pain states.

Sex-dependent factors of alcohol and neuroimmune mechanisms.

Excessive alcohol use disrupts neuroimmune signaling across various cell types, including neurons, microglia, and astrocytes. The present review focuses on recent, albeit limited, evidence of sex differences in biological factors that mediate neuroimmune responses to alcohol and underlying neuroimmune systems that may influence alcohol drinking behaviors. Females are more vulnerable than males to the neurotoxic and negative consequences of chronic alcohol drinking, reflected by elevations of pro-inflammatory cytokines and inflammatory mediators. Differences in cytokine, microglial, astrocytic, genomic, and transcriptomic evidence suggest females are more reactive than males to neuroinflammatory changes after chronic alcohol exposure. The growing body of evidence supports that innate immune factors modulate synaptic transmission, providing a mechanistic framework to examine sex differences across neurocircuitry. Targeting neuroimmune signaling may be a viable strategy for treating AUD, but more research is needed to understand sex-specific differences in alcohol drinking and neuroimmune mechanisms.

The Selective CB2 Agonist COR167 Reduced Symptoms in a Mice Model of Trauma-Induced Peripheral Neuropathy through HDAC-1 Inhibition.

Neuropathic pain is a chronic disabling condition with a 7-10% of prevalence in the general population that is largely undertreated. Available analgesic therapies are poorly effective and are often accompanied by numerous side effects. Growing evidence indicates cannabinoids are a valuable treatment opportunity for neuropathic pain. The endocannabinoid system is an important regulator of pain perception through the CB1 receptors, but CB1 agonists, while largely effective, are not always satisfactory pain-relieving agents in clinics because of their serious adverse effects. Recently, several CB2 agonists have shown analgesic, anti-hyperalgesic, and anti-allodynic activity in the absence of CB1-induced psychostimulant effects, offering promise in neuropathic pain management. The aim of this study was to evaluate the anti-neuropathic activity of a novel selective CB2 agonist, COR167, in a preclinical model of peripheral neuropathy, the spared nerve injury (SNI). Oral COR167, in a dose-dependent manner, attenuated mechanical allodynia and thermal hyperalgesia after acute and repeated administration, showing the absence of tolerance induction. At anti-neuropathic doses, COR167 did not show any alteration in the locomotor behavior. SNI mice showed increased microglial levels of HDAC1 protein in the ipsilateral side of the spinal cord, along with NF-kB activation. COR167 treatment prevented the HDAC1 overexpression and the NF-kB activation and increased the levels of the anti-inflammatory cytokine IL-10 through a CB2-mediated mechanism. Oral administration of COR167 shows promising therapeutic potential in the management of neuropathic pain conditions.

Zingiberene, a non-zinc-binding class I HDAC inhibitor: A novel strategy for the management of neuropathic pain.

BACKGROUND: Even though numerous Histone deacetylase inhibitors (HDACi) have been approved for the treatment of different types of cancer, and others are in clinical trials for the treatment of neurodegenerative diseases, the main problem related to the clinical use of available HDACi is their low isoform selectivity which causes undesirable effects and inevitably limits their therapeutic application. Previously, we demonstrated that a standardized Zingiber officinalis Roscoe rhizome extract (ZOE) reduced neuroinflammation through HDAC1 inhibition in a mice model of neuropathy, and this activity was related to terpenes fraction. HYPOTHESIS/PURPOSE: The aim of this work was to identify the ZOE constituent responsible for the activity on HDAC1 and to study its possible application in trauma-induced neuropathic pain. METHODS: The ability of ZOE and its terpenes fraction (ZTE) to inhibit HDAC and SIRT isoforms activity and protein expression was assessed in vitro. Then, a structure-based virtual screening approach was applied to predict which constituent could be responsible for the activity. In the next step, the activity of selected compound was tested in an in vitro model of neuroinflammation and in an in vivo model of peripheral neuropathy (SNI). RESULTS: ZTE resulted to be more potent than ZOE on HDAC1, 2, and 6 isoforms, while ZOE was more active on HDAC8. Zingiberene (ZNG) was found to be the most promising HDAC1 inhibitor, with an IC50 of 2.3 ± 0.1 µM. A non-zinc-binding mechanism of inhibition was proposed based on molecular docking. Moreover, the oral administration of ZNG reduced thermal hyperalgesia and mechanical allodynia in animals with neuropathy after 60 min from administration, and decreased HDAC-1 levels in the spinal cord microglia. CONCLUSION: We found a new non-zinc-dependent inhibitor of HDAC class I, with a therapeutic application in trauma-related neuropathic pain forms in which microglia-spinal overexpression of HDAC1 occurs. The non-zinc-binding mechanism has the potential to reduce off target effects, leading to a higher selectivity and better safety profile, compared to other HDAC inhibitors.

Chronic alcohol induced mechanical allodynia by promoting neuroinflammation: A mouse model of alcohol-evoked neuropathic pain.

BACKGROUND AND PURPOSE: Chronic pain is considered a key factor contributing to alcohol use disorder (AUD). The mechanisms responsible for chronic pain associated with chronic alcohol consumption are unknown. We evaluated the development of chronic pain in a mouse model of alcohol dependence and investigate the role of neuroinflammation. EXPERIMENTAL APPROACH: The chronic-intermittent ethanol two-bottle choice CIE-2BC paradigm generates three groups: alcohol-dependent with escalating alcohol intake, nondependent (moderate drinking) and alcohol-naïve control male and female mice. We measured mechanical allodynia during withdrawal and after the last voluntary drinking. Immunoblotting was used to evaluate the protein levels of IBA-1, CSFR, IL-6, p38 and ERK2/1 in spinal cord tissue of dependent and non-dependent animals. KEY RESULTS: We found significant escalation of drinking in the dependent group in male and female compared with the non-dependent group. The dependent group developed mechanical allodynia during 72 h of withdrawal, which was completely reversed after voluntary drinking. We observed an increased pain hypersensitivity compared with the naïve in 50% of non-dependent group. Increased IBA-1 and CSFR expression was observed in spinal cord tissue of both hypersensitivity-abstinence related and neuropathy-alcohol mice, and increased IL-6 expression and ERK1/2 activation in mice with hypersensitivity-related to abstinence, but not in mice with alcohol-evoked neuropathic pain. CONCLUSIONS AND IMPLICATIONS: The CIE-2BC model induces two distinct pain conditions specific to the type of ethanol exposure: abstinence-related hypersensitivity in dependent mice and alcohol-evoked neuropathic pain in about a half of the non-dependent mice.