British Journal of Pharmacology

Targeting the kappa opioid receptor (KOR) system has emerged as a potential alternative to current analgesics, however, advancing the therapeutic development of KOR requires further elucidation of its intracellular signaling events and modulators. Among these intracellular modulators, Regulators of G protein signaling (RGS) proteins act as key modulators of GPCR signaling to shape nociceptive circuits and influence pain processing. Despite this, the molecular diversity of RGS proteins that shape KOR signaling and its behavioral consequences remains largely unexplored. Here we report that RGS6, a member of the R7 RGS family, is highly expressed in nociceptive areas and modulates multiple modalities of KOR-dependent anti-nociception and nocifensive behaviors. Using global single and double knockout mouse models we show that this anti-nociceptive phenotype was highly specific to RGS6 within the R7 RGS family. Further we demonstrate that the R7 RGS family displays a lack of functional redundancy in regulation of KOR signaling and behaviors. Using peripherally restricted KOR agonists, we found that KOR-RGS6 anti-nociceptive signaling displays sex differences in a site-specific manner, as females but not males displayed enhanced anti-nociceptive and blunted nocifensive behaviors. Our findings suggest that RGS6 is a highly specific modulator of KOR-dependent anti-nociceptive signaling and plays an essential role in modulating nociceptive circuits, potentially aiding in the development of novel analgesic drugs and therapeutics.

Crush syndrome (CS) is characterised by ischaemia/reperfusion-induced rhabdomyolysis, leading to systemic inflammation and high mortality. Building on our previous findings that intravenous nitric oxide (NO) donors improve survival in this condition, we investigated the therapeutic efficacy of inhaled NO delivered via a portable, controlled-release device in an experimental rat model of CS. Anaesthetised rats underwent bilateral hindlimb compression using rubber tourniquets for 5 h, followed by reperfusion. Among the various inhalation conditions tested, administration of NO (160 parts per million) for 2 h after reperfusion significantly increased survival rate from 20 to 90%. Improvements in haemodynamic parameters, biochemical markers, and histopathological findings correlated with enhanced survival outcomes. These results suggest that on-site NO inhalation therapy may serve as an effective first-line, emergency intervention for CS, particularly in disaster settings. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/710439v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@1de2a5forg.highwire.dtl.DTLVardef@b0048eorg.highwire.dtl.DTLVardef@1fb310borg.highwire.dtl.DTLVardef@50da9a_HPS_FORMAT_FIGEXP M_FIG C_FIG

There is a pressing need for effective alternatives to opioid analgesics, the development of which requires the identification of novel anti-nociceptive drug targets. Here, we have further investigated the anti-nociceptive properties of a GPR35 agonist, cromolyn, in an in vitro model of inflammatory sensitisation. We used ratiometric Ca2+ imaging of cultured sensory neurons to examine the effect of cromolyn on prostaglandin E2 (PGE2)-mediated sensitisation of the pro-nociceptive ion channel, transient receptor potential cation channel, subfamily V, member 1 (TRPV1). The sensitisation of TRPV1 by PGE2 was inhibited by cromolyn in a GPR35-dependent manner. These observations provide further evidence in support of an anti-nociceptive role for GPR35, highlighting the potential use of GPR35 agonists as analgesics.

Control of synaptic transmission efficacy by neuronal activity and neuromodulators is pivotal for brain function. Synaptic suppression by cannabinoids activating CB1 receptors has been extensively studied at the molecular and cellular levels to understand the neuronal basis for effects of cannabis intake. Here, we focused on GPR55, non-canonical type of cannabinoid receptor, which shows sensitivity to cannabidiol included in cannabis, aiming to highlight its actions on presynaptic function. Taking advantage of direct patch-clamp recordings from axon terminals of cerebellar Purkinje cells together with fluorescent imaging of vesicular exocytosis using synapto-pHluorin, we show that GPR55 suppresses synaptic transmission as CB1 receptor does, but through a distinct presynaptic modulation of release machinery. Activation of GPR55 reduced transmitter release by changing neither presynaptic action potential waveform nor Ca2+ influx, but by making a large population of Ca2+-responsive synaptic vesicles insensitive to Ca2+ influx through voltage-gated Ca2+ channels, leading to substantial reduction of the readily releasable pool of vesicles. Thus, the present study identifies a unique mechanism to suppress presynaptic transmitter release by an atypical cannabinoid receptor GPR55, which would enable subtype-specific modulation of neuronal computation by cannabinoid receptors.

The mitochondrial permeability transition pore (mPTP) is a voltage- and calcium-regulated channel located in the inner mitochondrial membrane whose activity critically influences cellular fate. While prolonged pore opening leads to mitochondrial depolarization, matrix swelling, and cell death, brief and reversible opening events, referred to as flickering, enable controlled release of calcium and reactive oxygen species and serve essential physiological functions. Emerging evidence indicates that restoring physiological mPTP flickering, rather than suppressing pore activity, may be beneficial in disorders characterized by impaired pore dynamics, including hereditary spastic paraplegia type 7 (SPG7). However, no approved therapies are currently available to promote controlled mPTP pore opening. To identify pharmacological modulators of flickering, we performed a high-content screening of 2,000 FDA and EMA-approved compounds using a validated fluorescence-based assay coupled with automated image analysis. Thirteen compounds increased both the frequency and the area of flickering events while preserving cellular and mitochondrial integrity. Validation in fibroblasts derived from two SPG7 patients and healthy controls confirmed reproducible activity across distinct genetic backgrounds. Among the prioritized candidates, berberine emerged as the most robust modulator, consistently enhancing mPTP flickering independently of SPG7 mutation status. Notably, berberine selectively increased the proportion of small-size flickering events, indicative of physiological pore activity. These findings identify berberine as a promising modulator of mPTP dynamics and support pharmacological restoration of physiological flickering as a potential therapeutic strategy for SPG7 and other disorders associated with impaired mitochondrial permeability transition pore regulation.

Despite decades of clinical implementation of medications for opioid use disorder (OUD), overdose mortality rates remain high, underscoring a critical gap in treatments that target brain mechanisms driving addiction. Mindfulness-Oriented Recovery Enhancement (MORE) has demonstrated efficacy in reducing opioid use and craving, hypothetically by restructuring the salience of drug and natural rewards. Yet, to date, MOREs neurobiological mechanisms remain unclear. In this first functional magnetic resonance imaging (fMRI) randomized controlled trial (RCT) of MORE for OUD (NCT04112186), we tested whether compared with an active psychoeducational supportive therapy (PST) control group, MORE rebalanced neural responses to drug and natural reward cues in inpatients with OUD receiving standard of care including medications. Compared with PST, eight weeks of MORE significantly reduced drug-biased activity in the dorsolateral prefrontal cortex (dlPFC) and posterior regions of the default mode network including the precuneus during downregulation of responses to drug cues relative to upregulation of responses to natural reward cues (even when controlling for passive cue viewing). The shift from drug to natural reward responses in the lateral and ventromedial PFC was associated with lower cue-induced craving exclusively in the MORE group. MORE also reduced medial PFC synchronization to naturalistic drug-related movie scenes and significantly extended abstinence duration at follow-up ([~]4 months post-treatment) relative to PST. Together, this neuroimaging RCT demonstrates that MORE normalizes function in PFC nodes of the reward, salience, and control systems, positioning MORE as a biologically-grounded adjunct to pharmacotherapy for OUD.

Metabotropic glutamate receptor 5 (mGlu5) is a class C GPCR crucial for neuronal development and synaptic transmission. mGlu5 is a potential therapeutic target in pain management and modulates pain-associated gene expression and signaling pathways. Although mGlu5 inhibitors have shown promise in treating pain, none have translated to the clinic. Up to 90% of neuronal mGlu5 expression is intracellular, although the precise locations and function of different mGlu5 intracellular pools remains unclear. Building on recent evidence showing the importance of endosome-mediated nociceptive signaling by other GPCRs, we hypothesized that endosomal pools of mGlu5 contribute to pain transmission, and that targeted inhibition of intracellular mGlu5 signaling results in superior analgesia. Using calcium mobilization assays and genetically encoded resonance energy transfer biosensors, we report that upon its activation mGlu5 recruits Gq/11 and Gs to the plasma membrane. Conversely, internalized mGlu5 in endosomes recruits only Gq/11 proteins. mGlu5 signaling is highly dependent on receptor trafficking to endosomes, with sustained nuclear ERK1/2 signaling requiring both receptor internalization and active glutamate transport into the cell. We generated pH responsive nanoparticles loaded with the mGlu5 negative allosteric modulator VU0366058 (DIPMA-VU058), enabling endosome-targeted inhibition of mGlu5. Nanoparticle encapsulation of VU0366058 enhanced inhibition of both acute and sustained nuclear ERK1/2 signaling, and significantly reduced neuronal excitability in nociceptive circuits in spinal cord slices from rats with neuropathic pain. Intrathecal administration of DIPMA-VU058 achieved superior analgesia in both inflammatory and neuropathic models of pain in mice compared to free VU0366058 and the reference compound fenobam. These studies demonstrate the importance of endosome-associated receptors for the complete mGlu5 signaling response. Furthermore, we show that manipulating the cellular distribution of an allosteric modulator can engender location-biased pharmacological effects. Together, we have revealed new and unappreciated roles for endosome-specific mGlu5 signaling and demonstrate that endosome-selective targeting may offer an alternative therapeutic approach for modulating mGlu5 activity.

IntroductionCannabinoid receptor-2 (CB2) is an emerging therapeutic target for chronic and inflammatory pain, cancer, and neurological disorders. Understanding the efficacy of CB2 ligands is crucial for future drug design and development. AimsWe aimed to establish a simple and robust system to control CB2 expression using a tetracycline-regulated mammalian expression system (T-REx), to enable application of the Black and Leff operational model to measure the operational efficacy ({tau}) of CB2 ligands. MethodsLigand-induced hyperpolarisation of AtT20 cells transfected with T-REx and human CB2 was measured by FLIPR membrane potential assay. Maximal and submaximal responses of the CB2 ligands were produced by regulating CB2 expression using tetracycline. Data were fitted to the operational model of receptor depletion to quantify the efficacy of seven ligands. Additionally, the maximal initial rate of signalling (IRmax), another putative measure of ligand efficacy, was determined. ResultsAK-F-064, CP55940 and 2-AG exhibited similar efficacy with a {tau} values of 11.4, 11 and 10.4 respectively, while anandamide (AEA) had the lowest efficacy ({tau}=1.07) among the tested agonists. The rank order of operational efficacy and IRmax was similar and was estimated as: AK-F-064 = CP55940 = 2-AG > 5F-AB-PICA = WIN55212-2 > HU-308 = AEA. ConclusionThis inducible expression system provides a reliable platform for quantifying and comparing CB2 ligand efficacy using the operational model. This approach may facilitate more precise CB2-targeted drug development and can be readily extended to other GPCR targets.

Preclinical evidence indicates that cocaine exerts acute and chronic effects on astrocyte functioning, which in turn modulate cocaine-related impacts on neural integrity and brain function. However, human evidence for astrocytic involvement in cocaine users (CU) remains limited. Glial fibrillary acidic protein (GFAP) is a marker of astrocyte activation with promising clinical utility in neurological conditions, yet its relevance in the addiction field is unclear. Hence, we investigated plasma GFAP levels in chronic CU (n=41) and cocaine-naive controls (HC; n=34) at baseline and after a 4-month follow-up. GFAP was assessed alongside plasma neurofilament light chain (NfL) levels, a marker of neuroaxonal injury previously associated with cocaine use in the same sample. Contrary to our hypothesis, we found no group differences in plasma GFAP concentrations between CU and HC. Neither cross-sectional nor longitudinal associations between GFAP levels and objective indices of cocaine use (derived from hair testing) were detected. However, exploratory analyses revealed higher plasma GFAP levels among CU with recent cocaine consumption (within the last 7 days), suggesting transient astrocytic responses following acute exposure. Additionally, GFAP and NfL were positively correlated across participants, supporting their functional association. Overall, these findings suggest that while GFAP might not be chronically elevated in CU, it may exhibit transient increases related to recent cocaine use. Further research is warranted to characterize the temporal dynamics and biological significance of these glial responses.

BackgroundSubstance use disorders (SUD) are associated with a high global burden of disease, with 5.4% of all disability-adjusted life years lost due to alcohol and illicit drugs. Highly prevalent multimorbidity includes polysubstance use, mental health conditions, and other non-communicable and infectious diseases. Where traditional treatments are insufficient alone, music therapy (MT) is highly engaging and improves motivation and reduces craving; however, its long-term effects are unknown. The present study aims to examine long-term effects of active music groups (AMG) and music listening groups (MLG) versus treatment as usual (TAU) on addiction severity, recovery, and other outcomes in people with SUD Immediate and short-term effects, as well as mechanisms of these interventions, will also be examined. MethodsIn individuals with SUD across a wide range of age, gender, socioeconomic, and cultural backgrounds, a parallel 3-arm assessor-blinded pragmatic multinational randomised controlled trial (RCT) with embedded exploratory trials and mechanistic studies will determine long-term effects of AMG and MLG versus TAU on addiction severity (primary endpoint: 1 year), recovery, and other outcomes. Embedded trials will examine immediate effects of AMG or MLG combined with individual components of TAU combined to determine the best combinations of interventions. Experimental studies will examine mechanisms using cognitive testing and brain imaging. With 600 participants in 7 countries randomised, the trial will have 80% power on the primary outcome. Patient representatives, health technology assessment (HTA) bodies, and interventionists have been involved from conception and will ensure feasibility and applicability of the intervention across Europe. DiscussionThis document describes the FALCO RCT, the main part of the FALCO project, which aims to reduce disease burden through innovative, effective, and affordable treatment, and will strengthen research and innovation expertise. Recommendations from FALCO will inform intervention delivery across Europe and beyond, leading to increased safety, effectiveness, and cost-effectiveness, and improved quality of life for individuals with SUD. Stakeholders will be involved in communicating findings across all European countries and regions and ensuring that findings are effectively implemented. Trial registrationClinicalTrials.gov, NCT07028983, registered 11th of June 2025. https://clinicaltrials.gov/study/NCT07028983

NLRP3 inflammasome is a cytosolic multi-protein complex that plays a crucial role in the immune system, responding to various exogenous and endogenous stimuli by triggering protective inflammatory responses. However, aberrant NLRP3 inflammasome activation is implicated in numerous inflammatory diseases. Therefore, the NLRP3 inflammasome is an important pharmacological target for the treatment of multiple diseases. In this context, we screened various US-FDA-approved drugs for NLRP3 inflammasome inhibition. We found that among various drugs, minoxidil hydrochloride (MXL) effectively inhibits NLRP3 inflammasome, evidenced by reduced secretion of IL-1{beta} and IL-18 in J774A.1 cells treated with MXL. The IC50 values of MXL for inhibition of IL-1{beta} and IL-18 were calculated to be 1.2 and 1.06 {micro}M, respectively. MXL was found to prevent ASC oligomerization, thereby inhibiting the NLRP3 inflammasome and leading to CASP1 cleavage. Further investigation revealed that MXL also utilizes AMPK-mediated autophagy to modulate NLRP3 inflammasome activity. Using siAMPK and bafilomycin A1, an end-stage autophagy inhibitor, we elucidated crosstalk between the NLRP3 inflammasome and autophagic pathways, which was modulated by MXL. Furthermore, we demonstrated the efficacy of MXL in two different mouse models of inflammation, involving the NLRP3 inflammasome. MXL at doses of 10 and 20 mg/kg effectively inhibited the activation of NLRP3 inflammasome by monosodium urate in the air pouch model and by ATP in the peritoneal inflammation model, as evidenced by reduced secretion of 1{beta} and IL-18 in the lavage. Our study identifies MXL as a potent NLRP3 inflammasome inhibitor, warranting further investigation as a potential therapeutic agent for inflammatory diseases.

There is growing interest in understanding how hormonal signaling pathways contribute to the pathophysiology of mood disorders, based on the premise that fluctuations in sex hormones influence mood, a relationship particularly evident in conditions such as premenstrual dysphoric disorder, prenatal depression, postpartum depression, and perimenopausal depression. Estrogen receptor alpha (ER) is predominantly localized in the nucleus, but can also be associated with the cell membrane, thus mediating a broad range of genomic and non-genomic effects through distinct intracellular pathways. By employing a combination of computational simulations and in vitro biochemical and cell-based assays, we systematically evaluated the potential binding and functional interactions of antidepressant compounds with ER. Our results provide compelling evidence that antidepressants may not only affect classical monoaminergic targets but also modulate hormone receptor activity, particularly that of ER. These findings are consistent with the hypothesis that ER plays an important role in mood regulation and highlight it as a potential therapeutic target. Moreover, this work raises the possibility that the clinical efficacy of certain antidepressants may, at least in part, derive from their capacity to influence estrogen receptor-mediated signaling. Significance statementClinical observations suggest a link between female sex hormones and mood, highlighted by the higher prevalence of depression in women and increased vulnerability to depression during hormonal fluctuations. Here, we report that structurally diverse conventional and rapid-acting antidepressants directly interact with estrogen receptor alpha (ER). This interaction is associated with rapid intracellular signaling in cellular models. These findings indicate that, alongside their conventional targets, antidepressants may also engage sex steroid receptor components and signaling. This work broadens our basic understanding of antidepressant pharmacology at the cellular level, offering an additional perspective that may inform future research into the biological mechanisms of mood disorders and suggest a framework for developing targeted therapies for hormone-associated depressive disorders.

Opioid addiction and misuse are a serious national crisis that affects public health, as well as social and economic welfare. Mortality due to opioid misuse is further exasperated by the combination of opioids with non-opioid respiratory depressants such as xylazine that are resistant to mu opioid receptor antagonists such as naloxone. This study tested the hypothesis that oxytocin can mitigate the severe opioid induced respiratory depression (OIRD) and mortality induced by high doses of fentanyl or the combination of fentanyl with xylazine. Our results show OXT can improve survival and respiratory function in both male and female rats with opioid induced respiratory depression caused by fentanyl, as well as a combination of fentanyl and xylazine. The improvement in respiratory function by OXT post fentanyl-xylazine was significantly greater than the recovery using only naloxone. Chemogenetic activation of OXT receptor positive neurons in the ventral respiratory group (VRG) provided similar benefits to that of OXT administration in reversing OIRD. These results indicate OXT is a promising therapeutic target for reversing OIRD and the respiratory depression that occurs with the combination of opioids and xylazine, a situation where naloxone is only partially effective. Additional translational benefits of OXT include it can be repurposed as it is already a FDA approved drug for other uses, has a high safety profile, and is unlikely to induce the withdrawal or reversal of analgesia that occurs with naloxone. Key PointsO_LIOxytocin (OXT) improves survival and respiratory function in both male and female rats with opioid induced respiratory depression (OIRD) caused by fentanyl C_LIO_LIOXT also reverses OIRD induced by the combination of fentanyl and xylazine C_LIO_LIThe improvement in respiratory function by OXT post fentanyl-xylazine was significantly greater than the recovery using only naloxone C_LIO_LIChemogenetic activation of OXT receptor positive neurons in the ventral respiratory group (VRG) provided similar benefits to that of OXT administration in reversing OIRD C_LIO_LIThese results indicate OXT is a promising therapeutic target for reversing OIRD and the respiratory depression that occurs with the combination of opioids and xylazine C_LI

The use of neuromodulation techniques for the treatment of alcohol use disorder is receiving increasing attention, especially non-invasive approaches, such as repetitive transcranial magnetic stimulation or transcranial direct current stimulation, while the hypothetical use of electroconvulsive therapy remains unexplored. Given our experience inducing electroconvulsive seizures (ECS) for therapeutic purposes in psychopathology rodent models, we evaluated the role of ECS on reducing the increased voluntary ethanol consumption caused by adolescent ethanol exposure in our validated preclinical model. Rats were treated in adolescence with a binge paradigm of ethanol (2 g/kg, i.p.; 3 rounds of 2 days at 48-h intervals; post-natal day, PND 29-30, PND 33-34 and PND 37-38) or saline. Following persistent withdrawal until adulthood, rats were allowed to: voluntarily drink ethanol (20%) by a two-bottle choice test, for 3 days (PND 80-82); treated with ECS (95 mA for 0.6 s, 100 Hz, pulse width 0.6 ms; ear-clip electrodes) or SHAM for 5 days (PND 86-90); re-exposed to voluntarily ethanol exposure (PND 94-96). Brains were collected on PND 97 to evaluate hippocampal markers of ethanol toxicity and/or treatment response (e.g., NeuroD, NF-L, BDNF and NF-L/BDNF ratio). Our results reproduced the increased voluntary ethanol consumption in adult rats induced by adolescent ethanol exposure and demonstrated that ECS could improve this abuse-prone response. Moreover, we suggested a possible role for BDNF in the beneficial effects induced by ECS, especially reducing the neurotoxic ratio NF-L/BDNF. Overall, we provide preclinical evidence for the potential use of ECS as an efficacious treatment for alcohol use disorder.

Opioid use disorder (OUD) is a chronic, relapsing disease driven by the reinforcing properties of opioids and perpetuated by avoidance of the negative affective states associated with the absence of the drug. Most available OUD treatments directly engage the {micro}-opioid receptor and may induce side effects that can compromise their therapeutic efficacy, thus underscoring the need for novel therapeutic alternatives. Calcitonin gene-related peptide (CGRP) is produced by a small population of neurons in the parabrachial nucleus (PBN) that has been shown to modulate itch, pain, as well as appetitive behaviors. Using a cell-specific nuclear labeling approach coupled with RNA-sequencing, we generated a baseline transcriptome of CGRPPBN neurons and confirmed expression of multiple genes associated with behavioral responses to appetitive stimuli, as well as enrichment of the {micro}-opioid receptor, suggesting that CGRPPBN neuron function may be sensitive to the presence of opioids. Indeed, cFos immunostaining showed that CGRPPBN neuron activity increases during early morphine abstinence and reduces gradually over 48 hours. Given the inhibitory effects of opioids on CGRPPBN neuron activity, we next tested whether these neurons could regulate opioid reinforcement. Using a mouse model of morphine intravenous self-administration, we found that chemogenetic inhibition of CGRPPBN neurons significantly reduced the number of morphine rewards earned in both single-dose and dose-response tests but did not affect context-induced morphine seeking after 21 days of abstinence. These results suggest that CGRPPBN neurons are sensitive to opioid administration and can regulate appetitive behaviors such as morphine-taking. Considering that CGRP signaling is regulated by opioid administration, molecular targets that regulate CGRP neurotransmission without direct -opioid receptor engagement may therefore serve as novel therapeutic avenues for the treatment of OUD. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=195 SRC="FIGDIR/small/712659v1_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@1fb9c9borg.highwire.dtl.DTLVardef@1e6ba79org.highwire.dtl.DTLVardef@dc60f5org.highwire.dtl.DTLVardef@61adaf_HPS_FORMAT_FIGEXP M_FIG C_FIG

Class B scavenger receptors BI (SR-BI) and BII (SR-BII) internalize lipoproteins but also bind and internalize bacteria. Their roles in sepsis are unknown. We overexpressed human SR-BI and BII in the liver and kidney as well as bone marrow-derived macrophages, and then performed cecal ligation and puncture (CLP) surgery. SR-BI and BII transgenic mice had significantly worse survival compared to WT mice. 24 h after CLP, liver injury markers and histological damage were prominent in both SR-BI and BII transgenic mice, whereas kidney damage was similar. Systemic inflammatory cytokines were markedly increased in SR-BI and BII transgenic mice; parallel increases were seen in liver mRNA expression, not in the kidney. The highest degree of neutrophil infiltration was observed in the liver of SR-BI. Human SR-BI and BII dramatically decreased bacterial accumulation in the liver. Green fluorescence protein-labeled E. coli were efficiently phagocytosed in hepatic macrophages of SR-BI and BII transgenic mice; phagocytosis was more prominent in SR-BII transgenic mice. Finally, human SR-BI overexpression reduced systemic HDL-C level, eliminated adrenal cortex lipid droplets, and dampened the systemic increase of corticosterone after CLP. Supplementation with glucocorticoid and mineralocorticoid improved survival in SR-BI, but not SR-BII, transgenic mice after CLP. In summary, our findings suggest human SR-BI and BII overexpression contributes to higher mortality after CLP by excessive inflammatory response due to adrenal insufficiency (SR-BI) or hyperactive phagocytosis (SR-BII) in the liver.

IntroductionInadequately controlled inflammation is a key driver of adverse cardiac remodelling after acute myocardial infarction (AMI). Central to this process is activation of the NLRP3 inflammasome-gasdermin D (GSDMD) pathway, which promotes pyroptosis and the release of the pro-inflammatory cytokine interleukin-1{beta} (IL-1{beta}), a mediator strongly associated with infarct severity and poor clinical outcomes. This study investigates whether repurposing the FDA-approved therapeutic Disulfiram, recently shown to inhibit GSDMD pore formation, could reduce inflammation and thus improve cardiac injury after AMI. Methods and ResultsCardiac ischemia-reperfusion (I/R) injury was induced in C57BL/6 mice by 60-minute ligation of the left coronary artery followed by reperfusion. Disulfiram (25 or 50 mg/kg) was administered at reperfusion and daily thereafter. Cardiac function was assessed by echocardiography, while fibrosis and inflammation were evaluated by histology, RT-PCR, immunohistochemistry and immunoblotting. Leukocyte populations in blood, spleen, bone marrow and heart were analysed by flow cytometry. In vitro, mouse bone marrow-derived macrophages (BMDMs) and PMA-differentiated THP-1 cells were treated with Disulfiram. Cytokine secretion, inflammatory gene expression and changes in cell viability (propidium iodide (PI) staining and lactate dehydrogenase (LDH) release) were measured. Disulfiram (50 mg/kg) significantly improved cardiac function 7 days post-I/R. This was accompanied by a significant reduction in cardiac fibrosis and inflammation, as reflected by a lower abundance of inflammatory cells in circulation and cardiac tissue. In LPS- and ATP/Nigericin-stimulated BMDMs and THP-1 cells, Disulfiram dose-dependently (0.1-50 {micro}M) reduced IL-1{beta} and IL-6 secretion and attenuated membrane permeability and cell lysis. ConclusionsThis study demonstrates that Disulfiram improves cardiac function post-AMI by ameliorating inflammation and fibrosis, which was associated with reductions in cytokine release from inflammatory cells in vitro. Therefore, targeting GSDMD by "repurposing" the FDA-approved drug, Disulfiram, may represent a novel way to provide cardio-protection post-AMI. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=82 SRC="FIGDIR/small/710794v1_ufig1.gif" ALT="Figure 1"> View larger version (11K): org.highwire.dtl.DTLVardef@7fd82eorg.highwire.dtl.DTLVardef@149de52org.highwire.dtl.DTLVardef@a2fe0eorg.highwire.dtl.DTLVardef@d56b4f_HPS_FORMAT_FIGEXP M_FIG C_FIG

Substance use disorder (SUD) recovery typically requires transformative change and prioritizing long-term healthy goals. Unfortunately, successful recovery is threatened by relapse rates that often exceed 50% in the first year. We previously reported on an experiential virtual reality (VR) SUD recovery intervention using personalized future self-avatars that produced emotional engagement and positive behavioral change, ie, stronger connection with the future self and future rewards and reduced craving. Here, we used fMRI to identify brain engagement to a future self experience with divergent futures. Twenty adults (14 male, 33 years old) in early SUD recovery (<1 year) interacted with age-progressed versions of themselves in two different VR future realities: an SUD Future Self and a Recovery Future Self. Vivid lifelike visual and audio animation was augmented with a personalized narrative concerning future drug use and recovery. MRI immediately followed. Participants viewed videos of their future selves in the virtual environment and were directed to contemplate what they were seeing. Viewing and contemplating the future selves elicited activation in midline default mode regions (posterior cingulate and ventromedial prefrontal cortices), visual regions including the occipital and fusiform face areas, and left middle frontal gyrus. The Recovery Future Self produced significant left occipital face area activation compared with the SUD Future Self. Midline default mode activation correlated with VR-induced increases in delayed reward preference, and also with greater trait perseverance. Using digital selves as therapeutic agents reveals an entirely novel set of possible interventions and opens exciting new frontiers in behavior change methodology. Future studies targeting decision-making and future behavior could be informed by evaluating increased midline default mode engagement, with uniquely self-focused mechanisms signaled by executive network and face area coactivation. New hope for treatment-resistant mental health conditions is offered by the nearly limitless range of therapeutic experiences enabled by immersive digital therapeutics. Plain Language SummaryHigh relapse rates in early recovery remains a serious challenge. To promote better outcomes, our team recently developed a virtual reality experience where people interacted with future versions of themselves. We used magnetic resonance imaging (MRI) to understand how the brain activated to this experience, and what brain responses were linked to positive outcomes. We worked with 20 adults in early recovery. Each person used virtual reality to interact with two different future selves: one who had returned to substance use, and one who had stayed in recovery. These digital future selves looked and sounded like the participants and were paired with a personalized story about future drug use and recovery. Right after the virtual reality session, participants brains were scanned while they watched videos of these future selves and were asked to think about what they were seeing. When people viewed and reflected on their future selves, brain areas involved in self-reflection and imagining the future became more active, along with regions that process faces. The future selves triggered brain activation in "self-focused" brain networks and in face-processing regions. Activity in key "self-focused" brain regions was linked to choosing larger, delayed rewards over smaller, immediate ones, and to lower impulsivity. These findings suggest that lifelike digital versions of peoples future selves engage brain systems that support thinking ahead, persistence, and valuing long-term outcomes. This creates a promising new avenue for immersive digital therapeutic experiences to encourage lasting behavior change in early recovery from substance use disorder.

Acute kidney injury (AKI) is a serious and common clinical syndrome that currently has no effective treatment. Emerging evidence links coagulation pathways to kidney injury, particularly through coagulation proteases. Protease-activated receptors (PARs) are a family of G-protein coupled receptors (GPCRs) that are activated by proteolytic cleavage of their N termini, exposing a tethered ligand that initiates receptor signaling. PARs have been shown to play a major role in inflammation, vascular regulation, and tissue injury. PARs play key roles in inflammation, vascular regulation, and tissue injury. Previous work from the Hamm laboratory demonstrated that PAR4 contributes to AKI progression, as PAR4 knockout mice were protected in both unilateral ureteral obstruction and ischemia-reperfusion-based models of kidney disease. In this study, we investigated the potential of a PAR4 antagonist, VU6073819, at mitigating AKI progression in an ischemia-reperfusion injury (IRI) mouse model. PAR4 antagonism not only alleviated kidney injury and inflammatory response, but it significantly improved the survival. These findings identify PAR4 as a promising therapeutic target for AKI.

Polypharmacy, the concurrent use of multiple medications, is increasingly prevalent in older people and is associated with adverse outcomes such as falls, frailty, functional and cognitive decline, and increased hospitalization and mortality. The liver, as the primary site of metabolism, is exposed to varying drug concentrations during first pass metabolism, hepatic clearance and perfusion, potentially causing alterations in liver sinusoidal endothelial cells (LSEC). LSEC are specialized endothelial cells responsible for maintaining fenestrations - dynamic, transcellular pores that facilitate the exchange of substances between the blood and liver parenchyma. Disruption of fenestrations can compromise liver function, contributing to a variety of hepatic disorders. This study investigated the effects of four commonly prescribed drugs -- metoprolol, citalopram, oxybutynin and oxycodone -- on LSEC function. We examined their impact on LSEC viability, endocytosis, and fenestration morphology at both systemic steady-state and first-pass concentrations, separately and in a polypharmacy cocktail to model clinical exposure. All treatments induced sublethal metabolic changes, but effects on LSEC functions were drug- and concentration-dependent. Citalopram and oxybutynin caused dose-dependent defenestration, whereas metoprolol and oxycodone produced mild, non-dose-dependent effects. Endocytic activity was increased with oxybutynin, metoprolol, oxycodone, and the polypharmacy cocktail, while citalopram had no effect. The polypharmacy cocktail triggered synergistic defenestration at first-pass concentrations, but not at steady-state levels. These results highlight the concentration-dependent and combinatorial effects of polypharmacy on LSECs, emphasizing the need to consider endothelial responses in drug safety and pharmacokinetic assessments, particularly in patients exposed to multiple medications.