European Journal of Pharmacology

AimTo identify the challenges that hinder the successful translation of anticonvulsants from plant origins, from preclinical research to clinical application. DesignThis review was conducted using the Joanna Briggs Institute (JBI) guidance for a scoping review. Data SourcesThe following bibliographic databases were searched between November 11th and 20th, 2023: PubMed, Scopus, Google Scholar and ClinicalTrials.gov. The search in Google Scholar was done via a third-party application called Harzings Publish or Perish, where the search results limit was set at 1000 relevant articles. After full-text review, the reference lists of the included articles were examined to identify additional sources. Review MethodsThe Population, Intervention, Comparison, Outcome, Time, Setting (PICOTS) framework was employed in developing the eligibility criteria. The articles were uploaded to Rayyan.ai for title and abstract screening, as well as full-text article review. Data was extracted and synthesised from included studies using a table. ResultsThirty-seven articles met the eligibility criteria. From these articles, six distinct categories of challenges were identified: (1) methodologic issues; (2) insufficient evidence to support the use of herbal drugs in clinical settings; (3) financial disincentives; (4) challenges in obtaining natural products or its active principles; (5) poor pharmacokinetics; clinical trials and regulatory challenges. ConclusionUnderstanding and effectively addressing these challenges will ensure that more plant-based anticonvulsants are successfully translated into clinical practice, thereby enhancing the treatment of epilepsy. This review also made recommendations to tackle some of these challenges that plant anticonvulsants face in the drug development process.

Arctigenin is a bioactive dibenzylbutyrolactone-type lignan exhibiting various pharmacological activities. The neuroprotective effects of arctigenin were demonstrated to be mediated via inhibition of AMPA/KA type glutamate receptors in the somatosensory cortex of the rat brain. The aim of this study was to compare the effects of arctigenin with matairesinol and trachelogenin on synaptic activity in ex vivo rat brain slices. Arctigenin, matairesinol and trachelogenin were isolated from Arctium lappa, Centaurea scabiosa and Cirsium arvense, respectively, and applied on brain slices via perfusion medium at the concentration range of 0.5-40 M. The effects of the lignans were examined in the CA1 hippocampus and the somatosensory cortex by recording electrically evoked field potentials. Arctigenin and trachelogenin caused a significant dose-dependent decrease in the amplitude of hippocampal population spikes (POPS) and the slope of excitatory postsynaptic potentials (EPSPs), whereas matairesinol (1 M and 10 M) decreased EPSP slope but had no effect on POPS amplitude. Trachelogenin effect (0.5 M, 10 M, 20 M) was comparable to arctigenin (1 M, 20 M, 40 M) (p > 0.05). In the neocortex, arctigenin (10 M, 20 M) and trachelogenin (10 M) significantly decreased the amplitude of evoked potential early component, while matairesinol (1 M and 10 M) had no significant effect (p>0.05). The results suggest that trachelogenin and arctigenin act via inhibition of AMPA/KA receptors in the brain and trachelogenin has a higher potency than arctigenin. Thus, trachelogenin and arctigenin could serve as lead compounds in the development of alternative neuroprotective drugs.

BackgroundThe etiology of essential tremor (ET) remains unclear but may involve abnormal firing of Purkinje cells, which receive excitatory inputs from granule cells in the cerebellum. Since 6 subunit-containing GABAA receptors (6GABAARs) are abundantly expressed in granule cells, we validated a hypothesis that 6GABAAR-selective positive allosteric modulators (PAMs) are promising pharmacological interventions for ET therapy. MethodsEmploying the harmaline-induced ET model in male ICR mice, we evaluated the possible anti-tremor effects of four 6GABAAR-selective PAMs, the pyrazoloquinolinones Compound 6 and LAU-463 and their respective deuterated derivatives. Propranolol, a clinical anti-tremor agent, was employed as positive control. To investigate the involvement of cerebellar 6GABAARs in the antitremor effect of intraperitoneal (i.p.) Compound 6, furosemide, an 6GABAAR antagonist, was intracerebellarly (i.cb.) co-administered with Compound 6. The burrowing activity, an indicator of wellbeing in rodents, was measured concurrently. ResultsHarmaline (10-30 mg/kg, s.c.) induced action tremor in ICR mice dose-dependently and markedly reduced their burrowing activity. Compound 6 (3 and 10 mg/kg, i.p.) significantly attenuated harmaline (20 mg/kg)-induced action tremor and burrowing activity impairment. Propranolol (20 mg/kg, i.p.) diminished tremor but failed to restore the burrowing activity in harmaline-treated mice. Importantly, both anti-tremor and burrowing activity restorative effects of Compound 6 (10 mg/kg, i.p.) was significantly reversed by co-administration of i.cb. furosemide at a dose (10 nmol/0.5 l) having no effect per se. All four 6GABAAR PAMs exhibited a similar therapeutic efficacy. Conclusion6GABAAR-selective PAMs significantly attenuated action tremor and restored physical well-being in a mouse model mimicking ET by acting in the cerebellum. Thus, 6GABAAR-selective PAMs may be potential therapeutic agents for ET.

RationaleMescaline is a classical psychedelic compound with a phenylethylamine structure that primarily acts on serotonin 5-HT2A/C receptors, but also binds to 5-HT1A and 5-HT2B receptors. Despite being the first psychedelic ever isolated and synthesized, the precise role of different serotonin receptor subtypes in its behavioral pharmacology is not fully understood. ObjectivesIn this study, we aimed to investigate how selective antagonists of 5-HT2A, 5-HT2B, 5-HT2C, and 5-HT1A receptors affect the behavioral changes induced by subcutaneous administration of mescaline (at doses of 10, 20, and 100 mg/kg) in rats. MethodsWe used adult male Wistar rats in all our experiments. We evaluated locomotor activity using the open field test, and assessed sensorimotor gating deficits by measuring prepulse inhibition (PPI) of acoustic startle reaction (ASR). ResultsWhile the highest dose of mescaline induced hyperlocomotion, which almost all the other antagonists reversed, the PPI deficits were selectively normalized by the 5-HT2A antagonist. The 5-HT2C antagonist partially reversed the small decrease in locomotor activity induced by lower doses of mescaline. ConclusionOur findings suggest that mescaline-induced changes in behavior are primarily mediated by the 5-HT2A receptor subtype, with less pronounced contributions from the 5-HT2C receptor. The other antagonists had limited effects.

Pharmacological targeting of ion channels represents a crucial avenue for pain management. Among these, the Kv7 family of ion channels plays a significant role controlling neuronal excitability and the generation and propagation of pain-related nerve impulses, thus mitigating excessive electrical signaling and curtailing the exaggerated transmission of pain signals. Pain management strategies often involve a multimodal approach, combining various medications with distinct mechanisms of action to achieve optimal outcomes. Eugenol possesses a spectrum of biological activities, including analgesic and anti-inflammatory properties. When used in conjunction with the anti-inflammatory drug diclofenac, eugenol demonstrates enhanced analgesic efficacy in animal models. We investigated the effects of diclofenac and eugenol on Kv7 and TRPV1 ion channels, both agents act as Kv7 activators, whereas diclofenac inhibits the TRPV1 current, and eugenol reduce the capsaicin-activated current presumably competing for the same binding site. Eugenol shows a time dependent biphasic effect on acid-activated TRPV1 current, first activating and then a slow decay of the current. When eugenol and diclofenac are used together, they limit the extent of depolarization of cells expressing Kv7 and TRPV1. Our results shed light on the combined effectiveness of eugenol and diclofenac in the treatment of acute pain.

Neuropathic pain, a debilitating condition with unmet medical needs, can be charactarised as hyperexcitability of nociceptive neurons caused by dysfunction of ion channels. Voltage-gated potassium channel type 7 (Kv7), responsible for maintaining neuronal resting membrane potential and thus neuronal exitability, resides under tight control of G protein-coupled receptors (GPCR). Calcium-sensing receptor (CaSR) is a GPCR that is known to regulate activity of numerous ion channels, but whether CaSR could control Kv7 channel function has been unexplored until now. Our results demonstrate that CaSR is expressed in recombinant cell models, human induced pluripotent stem cell (hiPSC)-derived nociceptive-like neurons and mouse dorsal root ganglia neurons, and its activation induced depolarisation via Kv7.2/7.3 channel inhibition. The CaSR-Kv7.2/7.3 channel crosslink was mediated via the Gi/o protein/adenylate cyclase/cyclic adenosine monophosphate/protein kinase A signalling cascade. Suppression of CaSR function rescued hiPSC-derived nociceptive-like neurons from algogenic cocktail-induced hyperexcitability. To conclude, this study demonstrates that CaSR-Kv7.2/7.3 channel crosslink via the Gi/o protein signalling pathway effectively regulates neuronal excitability, providing a feasible pharmacological target for neuronal hyperexcitability management in neuropathic pain.

Background and purposeSodium butyrate (SB) is a major product of gut microbiota with signaling activity in the human body. However, the toxic effect of SB remains largely unknown. This issue is addressed in current study. Experimental approachSB (0.3 - 2.5 g/kg) was administrated through a single peritoneal injection in mice. The core body temperature and mitochondrial function in the brain hypothalamus were monitored. Pharmacodynamics, targeted metabolomics, electron microscope, oxygen consumption rate and gene knockdown were employed to dissect the mechanism for the toxic effect. Key resultsThe temperature was reduced by SB (1.2 -2.5 g/kg) in a dose-dependent manner in mice for 2-4 hr. In the brain, the effect was associated with SB elevation and neurotransmitter (Glutamate and GABA) reduction. The mitochondria exhibited a transient volume expansion and crista loss in the hypothalamic neurons. ADP/ATP ratio was increased with accumulation of intermediate metabolites in the glycolysis, TCA cycle and pentose phosphate pathways. The mitochondrial protein, adenine nucleotide transporter (ANT), was activated for proton transportation leading to a transient potential collapse by proton leak. The SB activity was attenuated by ANT inhibition from gene knockdown or pharmacological blocker. The temperature drop was attenuated by i.p. injection of norepinephrine. The HDAC inhibitors, such as SAHA and pyruvate, did not exhibit the same effect. Conclusion and implicationsSuper-dosed SB generated an immediate and reversible toxic effect for inhibition of body temperature through transient mitochondrial reprogramming in the brain. The mechanism was quick activation of ANT proteins for the proton leak in mitochondria.

Background and PurposeEpilepsy is a neurological condition characterized by recurring seizures and neuronal hyperexcitability. Cell-based high-throughput screening applications have been essential for drug development and discovering novel biological processes. However, cell-based screens do not provide information on how drug-targeted pathways are integrated into a whole animal. Our objective was to develop and evaluate a screening application using zebrafish larvae to identify signalling mechanisms that modulate neural activity. Experimental ApproachWe developed an in vivo automated high-content screening assay using zebrafish larvae expressing the calcium sensor CaMPARI (calcium-modulated photoactivatable ratiometric integrator) in neurons. This assay can quantify neural activity of multiple individual larvae per well in a 96-well format. We quantified neural activity in 8725 individual larvae, in response to 1292 different drugs to identify molecules that protect against convulsant-induced neuronal hyperexcitability. Key ResultsThe assay was effective at identifying drugs that target diverse neurotransmitter signalling systems. While some commonly used anti-convulsants (e.g. phenytoin, carbamazepine, valproic acid) had poor activity in the assay, Kv7 potassium channel activators were consistently effective (ICA-069673, ICA-27243, ICA-110381, retigabine, and ML213). Many compounds approved for treatment of other conditions, including amitriptyline (depression), cyclobenzaprine (muscle spasm), clomipramine (obsessive-compulsive disorder) and ganaxolone (seizures), also strongly suppressed excitability in the assay. Conclusion and ImplicationsNeuronal CaMPARI expression in zebrafish larvae is a powerful tool for plate-based compound library screening to identify drugs that suppress hyperexcitability in vivo. Bullet Point SummaryO_ST_ABSWhat is already knownC_ST_ABSO_LICaMPARI is an integrative Ca2+ sensor that can be used to identify active neurons. C_LIO_LIKv7 activators (retigabine, ML213, and ICA-069673) are effective at reducing convulsant-induced (4-AP) neuronal hyperexcitability. C_LI What this study addsO_LIAn automated in vivo high-content drug screening assay to quantify neural activity. C_LIO_LIA series of drug targets that influence convulsant-induced hyperexcitability. C_LI Clinical significanceO_LIOur new tool will help identify novel compounds and signalling mechanisms that could be pursued as therapeutic targets for diseases involving electrical hyperexcitability. C_LI

Diabetes Mellitus (DM) is a rapidly increasing disease around the world. It is known that DM is associated with numerous complications which affect life quality by its debilitating nature. DM is associated with cognitive impairment and neurodegeneration, partly driven by neuroinflammation and disrupted neuronal signalling. Incretin-based treatments have recently been suggested to exert potential effects on the central nervous system in diabetic patients. However, the triple agonist of GIP/GLP-1/GCG Retatrutides effects on cognition under diabetic conditions remain unexplored. This study aims to reveal whether impaired cognitive performance, such as learning and memory, is ameliorated by Retatrutide treatment in diabetic rats, together with associated metabolic, inflammatory and histological changes. Male Sprague-Dawley rats were allocated to four groups: control (C), streptozotocin-induced diabetic (STZ), streptozotocin-induced diabetic rats treated with Retatrutide (STZR), and sham rats treated with Retatrutide alone (R). DM was induced by streptozotocin injections. Spatial learning and memory were assessed using the Morris Water Maze and Passive Avoidance tests. Metabolic parameters were monitored, while neuroinflammatory markers (IL-1{beta}, TNF-), neurotrophic-related gene expression (BDNF, CREB, AKT), Tau protein levels, and histopathological changes in the cortex and hippocampus were evaluated using molecular, biochemical, and histological analyses. Streptozotocin-induced diabetes resulted in persistent hyperglycaemia, total body weight loss, impaired learning and memory. Retatrutide treatment reduced blood glucose levels without achieving a full euglycaemia or preventing weight loss. Behavioural tests showed that Retatrutide treatment preserved spatial learning and short-term memory compared to untreated animals. These effects were accompanied by attenuation of neuroinflammatory responses, particularly reduced TNF- levels, trends toward preserved neurotrophic-related transcriptional profiles, and partial maintenance of cortical and hippocampal structural integrity. Retatrutide alone did not enhance cognitive performance beyond control levels. These findings support the hypothesis that triple agonists may exert beneficial effects on cognitive performance under diabetic conditions. Retatrutide alleviates DM-associated cognitive impairment in streptozotocin-induced diabetic rats and is associated with reduced neural inflammatory burden and protected neuroanatomical structure. The observed cognitive benefits appear to extend beyond metabolic regulation alone. Further studies in models more closely reflecting type 2 diabetes are warranted to clarify the underlying mechanisms and translational relevance.

Neuropathological mechanisms triggering manic syndrome or manic episodes in bipolar disorder remain poorly characterised, as the research progress is severely limited by the paucity of appropriate animal models. Here we developed a novel manic mice model by combining a series of chronic unpredictable rhythm disturbances (CURD), which include disruption of circadian rhythm, sleep deprivation, exposure to cone light, with subsequent interference of followed spotlight, stroboscopic illumination, high temperature stress, noise disturbance and foot shock. To validate this novel manic model, we used multiple behavioural and cell biology approaches comparing the CURD-model with healthy controls and depressed mice. The depression model was created by an exposure to an improved chronic unpredictable mild stress, which we defined as chronic unpredictable mild restraint (CUMR). A novel manic mice model induced by environmental stressors and free from genetic or pharmacological interventions will benefit research into pathological mechanisms of mania.

The human serotonin 1A (5-HT1A/HTR1A) receptor is a central target in the treatment of anxiety and mood disorders. However, ligand efficacy is sensitive to receptor conformation. Both Buspirone and Tandospirone are clinically relevant 5-HT1A agonists. This comparative in silico analysis was performed to examine their binding behavior and toxicity profiles. Molecular docking was performed on the serotonin-bound HTR1A receptor (PDB ID: 7E2Y) using SwissDock, and the toxicological predictions were generated with ProTox 3.0. Both ligands exhibited the greatest binding affinity for Chain A of the receptor (-6.14 kcal/mol for Buspirone and -5.88 kcal/mol for Tandospirone), suggesting a preferred receptor conformation that may mediate therapeutic efficacy. Buspirone demonstrated greater binding stability than Tandospirone across models. Both compounds demonstrated weak or unstable interactions for chains G and R. Predicted toxicity profiles revealed high probabilities of neurotoxicity and respiratory toxicity for both ligands, with blood-brain barrier penetration probabilities of 0.99 (Buspirone) and 1.00 (Tandospirone). Additionally, Tandospirone showed potential immunotoxic effects (probability 0.73). These findings demonstrated that the receptor conformation in ligand binding enhances efficacy in drug design while reducing CNS-related adverse effects. Overall, this comparative study provides a starting point that may inform the design of next-generation serotonergic therapeutics for mood and anxiety disorders.

This study focuses on RU-1205, a new kappa-opioid agonist exhibiting analgesic effect without causing dysphoric or aversive reactions. It is assumed that the absence of dysphoric or aversive effects can be attributed to functional selectivity or it might be due to an additional mechanism of action that involves blocking the p38 mitogen-activated protein kinase (MAPK). The aimof this study was the experimental identification of the mechanisms of action of RU-1205 associated with inhibition of MAPK p38 and functional selectivity at kappa-opioid receptors. Materials and methodsRats weighing 260-280 g were implanted with chronic cortical and deep electrodes. LFP activity was recorded after intracerebroventricular administration of well-studied reference substances: the selective kappa-opioid agonist U-50488 at a dose of 100 g; the MAPK p38 blocker SB203580 at a dose of 1 g; and the investigational compound RU-1205 at 350 g. The weighted phase lag index (WPLI) was calculated. Subsequently, machine learning techniques were employed to reduce dimensionality and extract connectivity features using the principal component analysis method. Finally, signal classification was conducted using models based on Gaussian processes. By applying the patch-clamp technique in the whole-cell configuration, the spike activity of pyramidal neurons in the basolateral amygdala was studied. The neurons were identified by their accommodation properties. After local perfusion of the test compounds, 3 dose-response curves were obtained for: (1) U-50488 at concentrations ranging from 0.001 to 10 M; (2) combinations of U-50488 (0.001-10 M) and RU-1205 (10 M); and (3) combinations of U-50488 (0.01-10 M) and RU-1205 (100 M). ResultsThe developed models were able to classify the compound RU-1205 as a <<non-inhibitor>> of MAPK p38 with a probability of 0.89. The results obtained were confirmed in patch clamp experiments on acute brain slices, where it was demonstrated that U-50488 statistically significantly increases the spike activity of pyramidal neurons in the basolateral amygdala (p <0.05) and RU-1205 interacts with U-50488, suppressing its effect on the spike activity of neurons. ConclusionsThe findings suggest that compound RU-1205 displays properties consistent with a functional kappa opioid receptor agonist and does not have a significant effect on MAPK p38. The study demonstrates the possibility of integrating electrophysiological measurements and advanced data analysis methods for a deep understanding of neuronal mechanisms of drug action and underscores the potential for further research in this area.

Binge alcohol consumption elicits robust sympathoexcitation and excitatory neuronal output. However, the central mechanism that mediates these effects remains elusive. We investigated the effects of ethanol metabolism within the central nucleus of the amygdala (CeA) on sympathoexcitation, and elucidated the role of acetate in these excitatory responses. In vivo arterial blood pressure, heart rate and sympathetic nerve activity responses to CeA microinjected ethanol or acetate with appropriate inhibitors/antagonists were tracked. In vitro whole-cell electrophysiology recording responses to acetate in CeA neurons with axon projecting to the rostral ventrolateral medulla (CeA-RVLM) were investigated, and cytosolic calcium responses in primary neuronal cultures were quantified. We demonstrate that in Sprague Dawley rats, local brain metabolism of ethanol in the CeA to acetic acid/acetate elicits sympathoexcitatory responses in vivo through activation of NMDA receptor (NMDAR). Alcohol dehydrogenase or aldehyde dehydrogenase inhibition using fomepizole or cyanamide and NMDAR antagonism using AP5 or memantine blunted these effects. Whole-cell patch-clamp recordings in brain slices containing autonomic CeA-RVLM neurons revealed a dose-dependent increase in neuronal excitability in response to acetate. NMDAR antagonists suppressed the acetate-induced increase in CeA-RVLM neuronal excitability, and memantine suppressed the direct activation of NMDAR-mediated inward currents by acetate in brain slices. We observed that acetate increased cytosolic Ca2+ in a time-dependent manner in primary neuronal cell cultures. The acetate enhancement of calcium signaling was abolished by memantine. These findings suggest that within the CeA, ethanol is sympathoexcitatory through local brain metabolism, which generates acetic acid/acetate leading to activation of NMDAR. NEW AND NOTEWORTHYBrain ethanol metabolism to acetic acid (vinegar)/acetate causes activation of N-methyl-D-aspartate receptors (NMDARs) in the central nucleus of the amygdala and elicits sympathoexcitatory responses. This excitatory mechanism is opposite to the inhibitory effects of ethanol at NMDAR. Understanding the active compounds that arise from ethanol metabolism, and the molecular mechanisms by which they influence alcohol reward and cardiovascular function, may be beneficial in developing targeted intervention strategies for both alcohol use disorder and its cardiovascular sequelae. Graphical Figure O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=172 SRC="FIGDIR/small/212597v4_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@18d8ec9org.highwire.dtl.DTLVardef@996b70org.highwire.dtl.DTLVardef@5cbecaorg.highwire.dtl.DTLVardef@f1cd4b_HPS_FORMAT_FIGEXP M_FIG Proposed mechanisms for ethanol and acetate induced increases in sympathoexcitation within the central nucleus of the amygdala (CeA). Abbreviations: Acetic acid (HOAc), acetate (-OAc), ADH (alcohol dehydrogenase), ALDH (aldehyde dehydrogenase), BBB (blood brain barrier), FOM (fomepizole), CYAN (cyanamide), CYP450 (cytochrome P450), IML (intermediolateral nucleus), RVLM (rostral ventrolateral medulla), SNA (sympathetic nerve activity). C_FIG

IntroductionFentanyl and non-pharmaceutical fentanyl use have been the leading causes of opioid-induced death worldwide. Being 50 times stronger than heroin and 100 times stronger than morphine, fentanyl is a potent opioid with overdoses causing over 250,000 deaths since 2018 in the US alone. The treatment of fentanyl addiction is a complex process and a clinical challenge. There is a dire need to find other innovative and alternative modalities in the fight against fentanyl crisis. Increasing evidence suggests a correlation between neuroinflammation and symptoms of drug abuse, opening up the possibility of immunoregulatory agents as therapy for fentanyl addiction as well as a other opioid-induced addiction. Cannabidiol (CBD) is a non-opioid, relatively safe, non-psychoactive phyto-cannabinoid produced by cannabis plants. Importantly, recent reports have documented benefits of CBD in the treatment and management of complications related to opioid withdrawal. We investigated if inhaled CBD could reverse the fentanyl addiction and whether the CBD treatment could ameliorate the addiction symptoms by regulating neuroinflammatory signals and re-establishing the homeostasis in CNS. MethodWe used a fentanyl-induced conditioned place preference (CPP) model in mouse to test whether inhaled CBD could reverse the fentanyl addiction and ameliorate the adversarial symptoms. By employing a combination of flow cytometry as well as behavioral tests, we further assessed the impact of fentanyl addiction on cells and neuroinflammatory signals in CNS and we measured the effects of CBD in the treatment of addiction symptoms and inflammatory signals. ResultsOur findings suggest that CBD inhalation could be used effectively in the treatment of fentanyl addiction. CBD mitigated the excessive fentanyl-induced neuroinflammatory responses and decreased cellular stress and senescence. Conclusioninhaled CBD could alleviate the fentanyl addiction and regulate neuroinflammatory responses. This novel approach is non-invasive, accessible, effective, and warrants further, translational and research.

This study investigates the therapeutic effectiveness of intranasal dantrolene nanoparticles pretreatment to inhibit lipopolysaccharide (LPS)-induced pathological inflammation and synapse destruction and depressive and anxiety behavior in mice. B6SJLF1/J adult mice were pretreated with intranasal dantrolene nanoparticles (dantrolene: 5mg/kg), daily, Monday to Friday, 5 days per week, for 4 weeks. Then, mice were treated with intraperitoneal injection of LPS (5mg/kg) for one time. Behavioral tests for depression and anxiety were performed 24 hours after a one-time LPS injection. Biomarkers for pyroptosis-related inflammation cytokines (IL-1{beta} and IL-18) in blood and brains were measured using enzyme-linked immunosorbent assay (ELISA) and immunoblotting, respectively. The changes of primary proteins activation inflammatory pyroptosis (NLRP3: NLR family pyrin domain containing 3, Caspase-1, N-GSDMD: N terminal protein gasdermin D) and synapse proteins (PSD-95 and synpatin-1) in brains were measured using immunoblotting. Intranasal dantrolene nanoparticles robustly inhibited LPS-induced depression and anxiety behavior. Intranasal dantrolene nanoparticles significantly inhibited LPS-induced pathological elevation of IL-1{beta} and IL-18 in the blood and brain and inhibited LPS induced activation of pyroptosis. Intranasal dantrolene nanoparticles significantly ameliorated decrease of PSD-95 and synpatin-1 proteins in brains. Thus, intranasal dantrolene nanoparticles has demonstrated neuroprotection against inflammation mediated depression and anxiety behaviors and should be studied furthermore as a future effective drug treatment of major depression disorder or anxiety psychiatric disorder.

Myocardial damage caused by the newly emerged coronavirus (SARS-CoV-2) infection is one of key determinants of COVID-19 severity and mortality. SARS-CoV-2 entry to host cells are initiated by binding with its receptor, angiotensin converting enzyme (ACE) 2, and the ACE2 abundance is thought to reflect the susceptibility to infection. Here, we found that clomipramine, a tricyclic antidepressant, potently inhibits SARS-CoV-2 infection and metabolic disorder in human iPS-derived cardiomyocytes. Among 13 approved drugs that we have previously identified as potential inhibitor of doxorubicin-induced cardiotoxicity, clomipramine showed the best potency to inhibit SARS-CoV-2 spike glycoprotein pseudovirus-stimulated ACE2 internalization. Indeed, SARS-CoV-2 infection to human iPS-derived cardiomyocytes (iPS-CMs) and TMPRSS2-expressing VeroE6 cells were dramatically suppressed even after treatment with clomipramine. Furthermore, the combined use of clomipramine and remdesivir was revealed to synergistically suppress SARS-CoV-2 infection. Our results will provide the potentiality of clomipramine for the breakthrough treatment of severe COVID-19.

ObjectiveElectroacupuncture has demonstrated beneficial effects in post-stroke motor dysfunction, yet the molecular mechanisms underlying its therapeutic efficacy remain incompletely understood. This study aims to investigate whether electroacupuncture promotes post-stroke motor function recovery by modulating the interaction between miR-124-3p and the NRG1/ErbB4 signaling pathway, specifically exploring whether miR-124-3p directly targets NRG1 to regulate neural plasticity in a focal cerebral ischemia rat model. MethodsThe ischemic stroke model was established by middle cerebral artery occlusion/reperfusion (MCAO/R) in adult rats. These rats were randomly divided into sham, model, electroacupuncture and model plus miR-124-3p inhibitor groups. The model group, electroacupuncture group and model plus miR-124-3p inhibitor group received EA intervention 24 h after modelling for 7 consecutive days. Behavioural function was assessed by Zea Longa score and mechanical pain rating. Hippocampal damage was detected by HE staining and neuronal apoptosis was observed by TUNEL staining. IL-1{beta} and IL-18 levels were measured by ELISA. PCR and Western blotting were used to detect the expression of miR-124-3p and inflammatory pathway proteins. The interaction between miR-124-3p and TLR4 was verified by dual-luciferase reporter assay. ResultsElectroacupuncture improved motor function in rat model of MCAO/R, as evidenced by improved Zea Longa scores and decreased mechanical withdrawal thresholds. Electroacupuncture significantly attenuated neuronal damage and also inhibited the inflammatory response by decreasing IL-18 and IL-1{beta} levels (P < 0.001). Notably, electroacupuncture upregulated miR-124-3p expression (P < 0.0001) and activated the NRG1/ErbB4 signaling pathway in the hippocampus. When miR-124-3p was inhibited, NRG1 protein expression decreased while GABA expression tended to increase. Dual-luciferase reporter assays confirmed that miR-124-3p directly targets the 3UTR of NRG1 mRNA and regulates its expression at the translational level. These findings suggest that electroacupuncture may alleviate neuronal and axonal damage by modulating miR-124-3p/NRG1/ErbB4 signalling and regulating GABA release. ConclusionElectroacupuncture can ameliorate motor dysfunction induced after brain I/R injury by targeting and modulating the NRG1-ErbB4 signaling pathway via miR-124-3p. These data are expected to provide new insights into the mechanisms of electroacupuncture for the prevention of potential targets for the recovery of motor dysfunction after stroke.

It is widely said that healthy intestinal environment takes essential role for better mental condition. One of the known dietary nutrients which maintains intestinal environment is the dietary fiber. Recent study showed that maintaining intestinal environment by dietary fiber succeeded to alleviate the psychiatric disorder symptoms in animals. However, such effects have only been reported with soluble fiber, which is highly fermentable and promotes short-chain fatty acid (SCFA) production, and not with insoluble fiber. Therefore, we aimed to verify whether insoluble fiber, such as cellulose, can alter emotion via changes in the gut. We divided mice into two groups and fed either standard diet (SD, contains both insoluble and soluble dietary fibers) or cellulose rich diet (CRD, contains cellulose alone as the dietary fibers). The CRD-fed mice displayed 1) the increased the anxiety-like behavior accompanied with 2) the modified amygdalar dopamine signaling. We further found the decreased intestinal SCFA levels along with intestinal permeability, dysmotility and hypersensitivity in CRD-fed mice. These behavioral and physiological effect of CRD has been completely abolished in vagotomized mice, indicating the direct link between intestinal environment exacerbation to the emotion through gut-brain axis. Additionally, the opioid antagonist abolished the CRD-induced anxiety, suggesting the involvement of opioidergic system to the anxiety which may evoked by increased amygdalar dopamine levels. Altogether, our findings suggest that consumption of cellulose alone as the dietary fiber may evoke intestinal abnormalities which fires the vagus nerve then opiodergic system and amygdalar dopamine upregulation, resulting in the enhancement of anxiety. Graphical Abstract: Possible mechanism of CRD-induced anxiety unveiled by current studyOur study clarified that long-consumption of cellulose-rich food (CRD) will lead decrease of SCFAs which may cause the intestinal disability, including decreased motility and increased intestinal permeability as well as upregulation of TRPA1 and SGLT1. These physiological modifications resulted as the intestinal hypersensitivity, which possibly overstimulate the vagal transmission which may activate endogenous opioidergic systems such as enkephalin (Enk) at the nucleus tractus solitarii (NTS). The activation of opioidergic system may suppress the GABAergic neuron in ventral tegmental area (VTA), resulting in the excess release of dopamine and further receptor modification in amygdala (Amyg), which might in the end cause the characteristic anxiety. The figure was created with BioRender.com. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/593082v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@1a0c4aborg.highwire.dtl.DTLVardef@1cfefceorg.highwire.dtl.DTLVardef@88cfc0org.highwire.dtl.DTLVardef@1ff5b0a_HPS_FORMAT_FIGEXP M_FIG C_FIG

There is growing interest in co-administering know analgesics for pain management, to reduce side effects and maximize therapeutic effects by pharmacological synergism, defined as supra-additive effects to biological stimuli. This work aimed to evaluate, using isobolgraphic analysis, synergistic effects of three antinociceptive substances-- anandamide (AEA), a cannabinoid CB1 receptor agonist; xylazine (XYL), an adrenergic 2-receptor agonist; and DAMGO, an {micro}-opioid receptor agonist--administered in binary doses in a prostaglandin E2 (PGE2)-induced peripheral pain model. Hyperalgesia was induced in Swiss male mice, and subsequently, animals were treated with binary agonist combinations administered to the hind paw. Mechanical nociceptive thresholds were measured using an algesimetric task, and the results obtained were compared with additive predicted effects. For AEA+XYL and AEA+DAMGO combinations, the observed effects were significantly greater than those predicted by Loewes additivity principles at all tested effect levels (10%, 30%, and 50% maximum possible effect, MPE). DAMGO+XYL combination showed significant synergistic effects at 10% and 30% MPE but not at 50% MPE. Confirming these findings, combination indexes (CI) for AEA+XYL and AEA+DAMGO were less than 1, indicating synergism, while CI for DAMGO+XYL was near 1, indicating additivity. Notably, single-system antagonism with either AM251, a CB1 antagonist, yohimbine, an 2C-receptor antagonist or naloxone, pan-opioid receptor antagonist, could prevent synergy or any analgesia at all for AEA+XYL and AEA+DAMGO. Furthermore, the binary agonist combinations did not produce systemic effects, sedation, or motor impairments. The results suggest synergistic antinociceptive effects for AEA+XYL and AEA+DAMGO, which are dependent on concomitant agonism upon known metabotropic receptors.

Withdrawal StatementThe authors have withdrawn their manuscript owing to the inability to complete the revisions or follow up on the manuscript at this time, due to personal circumstances. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author.