The Journal of Pharmacology and Experimental Therapeutics

Opioid withdrawal is associated with heightened pain sensitivity, including allodynia. Although opioid-induced allodynia is well-documented in humans and animal models, the relationship between the severity of opioid withdrawal-induced allodynia and individual addiction-like behaviors remains poorly understood. To address this gap, Heterogeneous Stock rats underwent long access (12 hours/day) intravenous oxycodone self-administration, followed by measurement of mechanical sensitivity at six timepoints across three weeks of abstinence. Rats were stratified by an Addiction Index derived from individual differences in the escalation of oxycodone intake, motivation to consume oxycodone, tolerance to oxycodones analgesic effects, and acute withdrawal-induced mechanical pain sensitivity. Here, we show that oxycodone withdrawal induces significant and prolonged allodynia for up to three weeks, with High Addiction Index rats exhibiting greater intensity and longer duration of pain sensitivity than Low Addiction Index rats. Results remained consistent even when excluding allodynia from the Addiction Index, highlighting the robustness of the association between addiction-like severity and protracted allodynia. Linear regression associations revealed that self-administration behaviors, particularly oxycodone intake escalation and motivation to seek oxycodone, predicted subsequent withdrawal-induced allodynia severity. These findings demonstrate that greater addiction-like severity is associated with more intense and prolonged withdrawal-induced pain, supporting mechanical allodynia as a marker of addiction severity. These results motivate future work to define the mechanisms linking addiction severity to protracted opioid withdrawal-induced pain, with the goal of informing targeted clinical interventions for individuals most susceptible to severe abstinence-related allodynia.

The epidermal growth factor receptor (EGFR) family network comprises 4 receptors (EGFR, ERBB2, ERBB3, ERBB4) and numerous ligands, and is dysregulated in many cancers. Since anti-cancer drugs that target these receptors are cardiotoxic for some patients, it is important to understand the network in cardiac cells. Data from the Human Protein Atlas established that EGFR family members and their ligands are differentially expressed in cardiac cell types. Ligand expression was altered in human failing hearts and may contribute to disease. These ligands stimulated extracellular signal-regulated kinases 1/2 (ERK1/2) and Akt in rat cardiomyocytes but to different degrees. Afatinib (at a concentration to inhibit all EGF family receptors) was used to assess the role of the network in a mouse model of cardiac hypertrophy induced by angiotensin II (AngII). Echocardiography and segmental strain analysis demonstrated that afatinib reduced AngII-induced cardiac hypertrophy and caused cardiac dysfunction. This was associated with loss of cardiomyocyte hypertrophy, enhanced cardiac fibrosis, and reduced expression of Nrg1. NRG1 binds to ERBB4 in cardiomyocytes which homodimerizes or heterodimerises with ERBB2. The role of ERBB2 in the cardiomyocyte response to NRG1 compared with EGF was dissected using tucatinib (a selective ERBB2 inhibitor) and mRNA expression profiling. Most, but not necessarily all, of the response to NRG1 required ERBB2 signalling; most, but not all, of the response to EGF did not. Thus, the EGFR family network plays an important role in the heart. Understanding this network may identify therapeutic approaches to avoid cardiotoxicity associated with EGFR family anti-cancer drugs. Clinical perspectivesO_LIAnti-cancer drugs that target the epidermal growth factor receptor (EGFR) family are cardiotoxic for some patients; it is therefore important to understand the network in cardiac cells. C_LIO_LIThe EGFR family and their ligands are differentially expressed in cardiac cells with changes in ligand expression in heart failure; inhibition of all receptors in a mouse model of hypertrophy reduces cardiac hypertrophy and causes cardiac dysfunction with attenuation of cardiomyocyte hypertrophy and enhanced cardiac fibrosis and loss of neuregulin 1 (NRG1); in rat cardiomyocytes, NRG1 signalling to gene expression is largely mediated via ERBB2. C_LIO_LIThe EGFR family network plays an important role in the heart; understanding this network may identify therapeutic approaches to avoid cardiotoxicity associated with anti-cancer drugs targeted against it. C_LI

BackgroundNeuropathic pain is a major source of disability and distress with few pharmacological options for treatment. Opioid drugs can be effective, but high doses are needed, leading to unwanted effects. BMS-986122 is a positive allosteric modulator of the mu opioid receptor that potentiates acute opioid antinociception without increasing opioid-induced constipation, reward, or respiratory depression. Therefore, we asked if BMS-986122 could increase the effects of low-dose opioid analgesics in chronic neuropathic pain. MethodsWe employed the spared nerve injury and tibial neuroma models in rats and assessed the tactile hypersensitivity of the hind paw and site of neuroma, respectively. ResultsAdministration of low doses of (R)-methadone, morphine, or buprenorphine slightly reduced the tactile hypersensitivity of the hind paw the in spared nerve injury model. Pretreatment with BMS-986122 significantly enhanced the reversal of hypersensitivity, reaching the effect of high-dose gabapentin, a standard of care in neuropathic pain. Pretreatment with BMS-986122 similarly increased the anti-allodynic effects of low dose (R)-methadone on neuroma pain. A similar effect of (R)-methadone in the absence of BMS-986122 was only observed at a dose where respiratory distress was seen. ConclusionsThese findings show that allosteric modulators of the mu opioid receptor such as BMS-986122 can enhance opioid activity that could translate to a safe and effective treatment for chronic neuropathic pain.

Current treatments for opioid use disorder (OUD) have major barriers to access. As such, researching new potential therapies for OUD is important to public health. Previous research has implicated glucagon-like peptide-1 (GLP-1) receptor agonists in decreasing the use of addictive substances by animals. In this study, female Wistar rats (N=32) were surgically implanted with jugular catheters and trained to self-administer fentanyl at a fixed-ratio 1 (FR1) schedule of reinforcement for 21 sessions under short- (ShA; 1 hour) or long-access (LgA; 8 hours) conditions. Next, the animals received injections of semaglutide (0.1 mg/kg, s.c.) or saline (0.9% NaCl, s.c.) prior to another FR1 session. The animals underwent a progressive ratio (PR) schedule of reinforcement while receiving saline (i.v.) or fentanyl (0.625-10 {micro}g/kg/inf, i.v.) and semaglutide (0.1 mg/kg, s.c.) or saline (s.c.). Next, the animals underwent a semaglutide (0-0.1 mg/kg, s.c.) dose response procedure at FR1 and a single dose of fentanyl (2.5 {micro}g/kg/inf, i.v.). Following drug discontinuation, spontaneous locomotor activity and withdrawal-like symptoms were measured. Semaglutide dose-dependently decreased fentanyl rewards under ShA and LgA conditions (p<0.05). Under a PR, semaglutide significantly decreased breakpoint (p<0.05), suggesting semaglutide decreases motivation to self-administer fentanyl. Semaglutide-treated ShA animals displayed significantly less withdrawal-like behavior (p<0.05) but not LgA animals. Overall, these findings suggest semaglutide may modulate motivation to seek opioid reward and could be useful in the development of pharmacotherapies to address OUD.

Perinatal opioid exposure is a prevalent clinical concern linked to respiratory instability and adverse infant outcomes. The opioid buprenorphine is prescribed as a medication for opioid use disorder during pregnancy and used to treat neonatal opioid withdrawal syndrome, yet its direct effects on neonatal control of breathing have not been examined. Here, we asked how acute buprenorphine exposure affects breathing at rest, and during chemoreceptor stimulation. Using dual-chamber head-out plethysmography, we measured pulmonary ventilation rate ([V]I) and metabolic rate in awake male and female Sprague-Dawley neonatal rats on postnatal days 4-5 (P4-5) during eupnea and a hypoxic-hypercapnic (HH) challenge. The effects of buprenorphine and two opioid receptor antagonists, naloxone hydrochloride, or peripherally restricted naloxone methiodide, were assessed using a repeated measures design. [V]I during eupnea and HH were markedly depressed following buprenorphine administration. Buprenorphine reduced [V]O2 and [V]CO2 and produced ventilatory equivalents for O2 and CO2 consistent with frank hypoventilation, driven by reduced breathing frequency and tidal volume (VT). When administered after buprenorphine, neither naloxone hydrochloride nor naloxone methiodide could rescue the buprenorphine-mediated hypoventilation in eupnea or during HH. In contrast, pre-treatment with either naloxone hydrochloride or naloxone methiodide attenuated buprenorphine-induced hypoventilation by preserving VT. These findings demonstrate that neonatal protective chemoreceptor reflexes are depressed by buprenorphine and suggest that pre-treatment with a peripheral opioid receptor antagonist could mitigate buprenorphine-induced hypoventilation without inducing opioid withdrawal. Key PointsO_LIAcute buprenorphine exposure significantly depressed pulmonary ventilation rate ([V]I) during eupnea and hypoxic hypercapnia (HH) in awake neonatal rats. C_LIO_LIBuprenorphine-induced hypoventilation was driven by reduced tidal volume (VT) and breathing frequency. C_LIO_LIBuprenorphine also reduced oxygen consumption ([V]O2) and carbon dioxide production ([V]CO2). C_LIO_LINaloxone given after buprenorphine failed to reverse hypoventilation. C_LIO_LIIn contrast, pre-treatment with either naloxone hydrochloride or peripherally restricted naloxone methiodide mitigated buprenorphine-induced hypoventilation by preserving VT. C_LI

The poor prognosis of brain tumors, including IDH-wild-type glioblastoma (GB), as well as brain and leptomeningeal metastases, is partly related to the blood-brain barrier (BBB), which limits the delivery of hydrophilic anticancer drugs to the tumor site and surrounding brain parenchyma. Early studies using vital dyes demonstrated that intracranial injection could bypass the BBB in cats. We confirmed that, in guinea pigs, the vital dye Bleu Patente V diffused efficiently into the brain after a bolus intracranial injection, whereas the brain remained unstained after intravenous administration. Similarly, brain concentrations of the hydrophilic anticancer drug gemcitabine were significantly higher following intracranial injection than after intravenous administration. Consistent with these findings, Bleu Patente penetrated deeply into the cerebral cortex of sheep after a 24-hour intraventricular infusion. At the end of a 24-hour intraventricular infusion of 20 mg gemcitabine in sheep, mean gemcitabine concentrations reached 1,415 {micro}g/L in cerebrospinal fluid and 850 {micro}g/kg in brain tissue. These concentrations exceeded the IC90 values of gemcitabine for A172, U87-MG, and U118-MG human glioblastoma cell lines, as determined in vitro after 24 hours of incubation. We hypothesize that Bleu Patente dye and gemcitabine circumvent the blood-brain barrier (BBB) by utilizing the glymphatic system. Tolerance of a single 24-hour intraventricular infusion of gemcitabine at doses of 5, 10, and 20 mg was good. Taken together, these encouraging preclinical results support the resumption of Phase I clinical trials evaluating intraventricular infusion of gemcitabine in patients with refractory primary or secondary brain tumors.

BackgroundEvidence suggests steeper accelerating opioid-related overdose, and non-medical use rates, in middle aged men in recent years compared with younger cohorts. Little is known about whether this is driven by age-related differences in the effects of opioids compared with socio-cultural factors driving non-medical consumption. Rodent models can be useful for dissociating biological from psychosocial factors, however, only minimal evidence exists on the effects of opioids in middle-age rats. ObjectiveTo determine if the anti-nociceptive and rewarding effects of opioids differ between adult and middle-age rats. MethodsFemale and male Wistar rats were obtained in early adulthood and examined across 4 to 11 months of age for nociceptive responses to heroin (0-1.56 mg/kg, s.c.) using a warm-water tail withdrawal assay. Subgroups (N=8 per group) were initiated on intravenous self-administration (IVSA) of heroin at either 5 months or 12 months of age. ResultsAnti-nociceptive effects of heroin did not differ across age. Female rats that initiated IVSA in early adulthood or middle-age obtained significantly more infusions of heroin than male rats of the same age during acquisition, and in dose-substitution under a FR1 schedule. Male, but not female, rats that initiated IVSA in middle age self-administered less heroin then rats that initiated in early adulthood; this was observed in acquisition and in dose-substitution. DiscussionThis study shows that opioid reward is diminished in middle aged male rats. It also found that middle age rats can be used effectively to model opioid-related outcomes, including drug seeking using the IVSA procedure.

TRPA1 is a non-selective cation channel that plays a crucial role in several pain and inflammatory conditions. Agents reducing membrane cholesterol decrease TRPA1 activation, but it remains unclear how cholesterol-lowering medications affect TRPA1 function. Given that TRPA1 is activated by a wide variety of chemicals, we explored whether statins have acute effects on this channel. We found that five commonly used statins activate human and mouse TRPA1 in a reversible and concentration-dependent manner. The effective concentrations were above the micromolar range, in the order: simvastatin {approx} lovastatin < fluvastatin < atorvastatin < pravastatin. Statin-induced activation was not correlated to changes in membrane order, nor mediated by N-terminal cysteine residues contributing to electrophilic compound agonism. Molecular docking calculations and the functional characterization of single-point mutants revealed two separate putative binding sites, one situated close to the kink of transmembrane segment 5 (TM5) and the other at the interface between TM4 and TM5. The mTRPA1 inhibitor A-967079 largely abrogated the response to the electrophilic agonist allyl isothiocyanate, but had weaker and varied effects across different statins and menthol. Mutation T877L strongly altered the effect of A-967079, also in an agonist-dependent manner, suggesting competitive binding between this antagonist and the non-electrophilic agonists. The identification of two distinct agonist binding sites may help explaining how TRPA1 is able to respond to a large variety of non-electrophilic compounds, while the finding of competitive interactions at one of these sites may help guide the development of agonist-specific antagonists of therapeutic relevance.

Crush syndrome (CS) is a serious medical condition characterized by damage to the muscle cells due to pressure and is associated with high mortality, even in patients receiving fluid therapy. We focused on adrenaline (Adr), a standard medication administered by medical teams dispatched during disasters. Adr is readily available for use in disaster scenarios owing to its inclusion in standard emergency kits. The effectiveness of Adr in the treatment of CS remains a subject of ongoing debate. This study aimed to evaluate the impact of Adr on acute complications, such as heart failure, shock, and renal failure, and explore whether its influence on inflammatory pathways is correlated with improved survival in rats with CS. The CS model involved subjecting anesthetized rats to bilateral hindlimb compression using a rubber tourniquet for 5 h. Subsequently, the rats were randomly divided into eight groups. Under continuous monitoring and recording of the arterial blood pressure, blood and tissue samples were collected for biochemical analyses at designated time points before and after reperfusion. The survival rate, vital signs, and blood gas parameters were higher in the CS group than in the sham group. They were improved in the Adr-treated group (0.01 or 0.01 mg/kg), which was not significantly different from that in the CS group, despite the improvement in shock and kidney dysfunction. In conclusion, intramuscular Adr provides immediate hemodynamic stabilization and renal protection during the early stages of CS. However, its use requires careful dose titration; low doses may promote the systemic release of lethal toxins, whereas high doses may worsen metabolic acidosis. These findings highlight the importance of combining Adr with other therapies, such as fluid resuscitation, to manage systemic toxemia inherent in CS.

BACKGROUNGBisphenol A (BPA) has been linked to hypertension and disturbances in lipid metabolism; however, limited evidence is available regarding its association with hypertensive intracerebral hemorrhage (ICH). METHODSA multicenter, retrospective case-control study was conducted involving 129 participants, including individuals from an ICH group and healthy controls. Standard assays were employed to assess serum thyroid function, lipid profiles, serum fatty acid-binding [x]protein 4 (FABP4), oxidative stress markers, gap junction proteins, Wnt/{beta}-catenin signaling pathway activity, and expression changes of S100A8-mediated inflammatory cytokines involved in gut-brain interactions. Correlation analyses using Pearson and Spearman methods revealed that both BPA exposure and low T3 levels were significantly associated with elevated diastolic blood pressure, altered lipid metabolism, gut microbiota composition, and microglial activation. RESULTSGender-based disparities in lipid metabolism were identified. Changes in {beta}3-adrenergic receptor and neuromodulin-1 expression appear to influence fat regulation and attenuate oxidative stress responses. Subsequently, increased expression of gap junction proteins and activation of the Wnt/{beta}-catenin signaling pathway contribute to metabolic reprogramming and alterations in biochemical kinetics. Gut microbiota analysis demonstrated that, compared to controls, the ICH group exhibited significant dysbiosis and reduced alpha diversity. Further correlation analyses indicated that BPA levels were positively associated with FABP4 and oxidative stress markers, while S100A8 showed a strong dependence on microglial expression. CONCLUSIONThe interplay between lipid metabolism dysfunction and pro-inflammatory cytokines enhances vascular vulnerability. Collectively, BPA exposure, oxidative stress, and microglia-mediated neuroinflammation are significantly associated with an elevated risk of hypertensive ICH. China Clinical Trial Registry registration noticeFrom: China Clinical Trials Registry <chictr@vip.qq.com>+To:guopingwang60a<guopingwang60a@163.com> yunyanshuangfei <yunyanshuangfei@126.com> FUNDINGThis work was supported by the Natural Science Foundation of Shanxi Province (grant no. 201701D121177) Key informationGender-specific differences were observed in lipid metabolism and oxidative stress parameters; BPA exposure was shown to induce lipid metabolic disturbances, promote excessive production of oxidative stress byproducts, and consequently elevate oxidative stress responses; BPA was associated with stress-induced alterations in thyroid hormone function, further exacerbating dysregulation of lipid metabolism and oxidative stress; Fatty acid binding protein 4 (FABP4), a key adipokine implicated in metabolic disorders and adipose tissue inflammation, exhibited a significant positive correlation with serum BPA levels, whereas low levels of triiodothyronine (T3) were negatively correlated with FABP4. These findings suggest that serum FABP4 may serve as a biochemical marker for chronic low-grade adipose tissue inflammation and metabolic dysfunction; Gap junction proteins and the Wnt/{beta}-catenin signaling pathway may contribute to microglial activation and mediate neuroinflammatory responses, nerve injury, and secondary pathological processes in obesity-related cerebral hemorrhage.

Rhodiola rosea is a traditional medicinal plant often classified as an adaptogen, with reported effects in supporting the bodys response to physical, environmental, and emotional stressors. The present study investigated the antioxidant properties of Rhodiola rosea extract and its major chemical constituents to provide insight into their potential mechanisms of action. Through in vitro biochemical assays, we demonstrated that Rhodiola rosea extract has the capacity to reduce hydrogen peroxide (H2O2) levels. Among its primary chemical components, rosavin significantly decreased H2O2, whereas salidroside had no effect. Neither compound affected superoxide levels. Structural analysis revealed that the intact phenylpropanoid glycoside architecture of rosavin is required for activity, as its individual components, arabinose and rosin, showed no inhibitory effect. Further investigation demonstrated that rosavin attenuates H2O2-mediated oxidation of thiol groups, supporting a role in cellular redox regulation. In cultured human cells, rosavin mitigated reductions in cell viability induced by exposure to H2O2, indicating cytoprotective effects under oxidative stress conditions. Finally, in an in vivo model, administration of SARS-CoV-2 spike protein increased circulating levels of H2O2, which were subsequently reduced following rosavin treatment. Collectively, these findings identify rosavin as a structurally dependent antioxidant component of Rhodiola rosea that modulates H2O2-associated oxidative stress and supports further investigation of phenylpropanoid glycosides as adaptogens.

Manganese neurotoxicity, arising from environmental overexposure or inherited transporter disorders due to pathogenic variants in SLC30A10 and SLC39A14, leads to manganism, a debilitating Parkinsonian movement disorder. Alhtough chelation therapy can partially reverse neuropathology, current clinical practice relies on intravenous CaNa2EDTA, which is burdensome and poorly suited for long-term use. Consequently, there remains a significant unmet need for more effective, orally bioavailable chelators. This study aimed to establish and validate a pipeline for identifying and assessing novel ligands that attenuate manganese neurotoxicity and support preclinical translational development. Based on the structural features of manganese-based MRI contrast agents, we selected two chelators, N-picolyl-N,N',N'-trans-1,2-cyclohexylenediaminetriacetic acid (H3PyC3A) and ethylenediaminetetraacetic acid-benzothiazole aniline (H4EDTA-BTA), and their methyl ester derivatives, Me3PyC3A and Me4EDTA-BTA. These were evaluated in vivo using zebrafish (slc39a14U801/U801) and mouse (Slc30a10KO/KO) models of manganese overload. H3PyC3A and Me3PyC3A demonstrated greater manganese-mobilizing efficacy than CaNa2EDTA, improving locomotor behavior in slc39a14U801/U801 zebrafish. In Slc30a10KO/KO mice, intravenous administration confirmed selective in vivo chelation of excess manganese over physiological concentrations of zinc and copper. Although oral bioavailability was low (<1%), long-term oral administration of H3PyC3A modestly reduced liver and brain Mn accumulation, suggesting an added benefit of oral administration via gastrointestinal chelation. This integrated in vitro to in vivo pipeline provides a robust and scaleable approach for the development of next-generation Mn chelators. Slc39a14U801 loss-of-function zebrafish enable high throughput identification of candidate compounds while Slc30a10KO/KO mice offer a clinically relevant disease model for pharmacokinetic profiling and proof-of-concept validation.

Background and objectiveHighrisk nonmuscleinvasive bladder cancer (HRNMIBC) is treated with transurethral resection and intravesical BCG instillations, yet {approx}50% recur and 20% progress to invasive disease. Although molecular subtyping, e.g., BCG-response-subtype (BRS), is associated with progression risk and may aid risk stratification, yet is costly and time-consuming. Intratumoral heterogeneity complicates accurate subtyping. To address these challenges, we developed a deep-learning model that predicts BRS from routine hematoxylin-eosin-stained images. We verified the models area-by-area predictions against tissue-level gene-expression maps. Methods and participantsHematoxylin-eosin-stained images from 231 HR-NMIBC patients with known BRS were used to develop a deep-learning model through cross-validation, then validated in 83 independent samples. The models spatial predictions were assessed using spatial transcriptomics to map gene expression to tissue locations in five HR-NMIBC tumors. Outcome measurements and statistical analysisDiscriminative ability for BRS3 vs. BRS1/2 was measured by AUC. Spatial alignment was assessed by calculating Pearson and Spearman correlation coefficients between model predictions and BRS fractions; significance was assessed through permutation analysis. Key findings and limitationsThe trained algorithm achieved AUC of 0.79 (development) and 0.71 (external) to detect BRS3 vs BRS1/2. Tile-level correlation between model output and molecular labels was significant (Pearson r = 0.33-0.44; p [&le;] 0.002). Limitations include retrospective sampling and limited spatial transcriptomic cases. Conclusions and clinical implicationsOur trained algorithm showed potential to stratify HRNMIBC patients by clinically relevant BCGresponse subtypes using routine hematoxylin-eosin-stained images and showed predicted spatial heterogeneity comparable to molecular profiling. Prospective validation is required before any clinical implementation. Patient summaryStandard pathology images contain hidden details related to tumors molecular subtype. We trained an AI model to read these routine images and identify specific bladder cancer subtypes associated with poor response to BCG therapy. This approach may help reveal molecular subtype-associated information from routine pathology images, without additional laboratory procedures.

In the terminal segment of the seminiferous tubules, SOX17 expression in the rete testis (RT) epithelium plays a crucial role in the formation of the Sertoli valve (SV), as revealed by phenotypic analyses of RT-specific Sox17 conditional knockout (cKO) mouse testes. In these RT-specific Sox17 cKO testes, SV disruption leads to the backflow of RT fluid into the seminiferous tubules, resulting in defective spermiogenesis and male infertility. Although valve deformation in the Sox17 cKO testes is likely caused indirectly by impaired downstream actions of Sox17 in the RT, the mechanisms by which SOX17 in RT influences SV formation in the seminiferous tubules remain unclear. To address this, we generated a novel AMH-Sox17 transgenic (Tg) mouse line carrying a human AMH promoter-driven Sox17 cDNA cassette. We analyzed the phenotypes of the Sertoli valve and spermatogenesis in AMH-Sox17 Tg mice, as well as in RT-specific Sox17 cKO; AMH-Sox17 Tg double mutant mice. Ectopic SOX17 (SOX17+) expression in Sertoli cells resulted in excessive Sertoli valve structures with acetylated tubulin bundles in the terminal segment of the AMH-Sox17 Tg testes, along with enhanced WNT4/RSPO1 signaling, suggesting the enhanced valve formation of ectopic SOX17+ Sertoli cells by themselves. Moreover, the AMH-Sox17 Tg could partially rescue the SV deformation and infertility in RT-specific Sox17 cKO mice, leading to proper SV formation, normal spermiogenesis and a partial recovery of male fertility in AMH-Sox17 Tg; RT-specific Sox17 cKO double mutant mice. These findings genetically demonstrate that ectopic SOX17+ Sertoli cells can compensate for SOX17 paracrine signaling in the RT, underscoring a key shared downstream pathway between RT and SV. Summary statementThe paracrine actions downstream of ectopic SOX17 expression in the Sertoli cells not only promote the valve formation, but also partially rescue the defective spermiogenesis of the rete testis-specific Sox17-null mice.

ObjectiveTo 1) determine the expression and distribution of all PDE4 isozymes (A-D) along the length of the anterior urethra, 2) culture fibroblasts and epithelial cells from healthy and strictured urethras, 3) investigate an in vitro model of anterior urethral stricture disease (aUSD), and 4) assess the therapeutic potential of phosphodiesterase-4 (PDE4) inhibitors and testosterone compared to paclitaxel. MethodsThe presence and relative abundance of PDE4 isozymes (A-D) was confirmed using immunohistochemistry on 5 male cadaveric urethras. Human urethral fibroblasts (FBs) were cultured from healthy control urethras of patients undergoing vaginoplasty (n=3) and from idiopathic bulbar urethral strictures (L2S1E2) of patients undergoing urethroplasty (n=3). Epithelial cells (ECs) were cultured from a healthy control urethra and two urethral strictures. To investigate a model of aUSD, Control FBs were stimulated with TGF{beta}1 and compared to Stricture FBs on assays of cell proliferation and expression of genes relevant to aUSD pathophysiology. To test therapeutics, Stricture FBs were treated with the PDE4 inhibitor, roflumilast, testosterone (T), or paclitaxel and compared to Control FBs on the previously mentioned assays and cell viability. ResultsPDE4- A, B, and D were detected along the length of the urethra. Expression levels did not differ between urethral regions. TGF{beta}1 altered proliferation and gene expression in a dose-dependent manner. Roflumilast and T preserved cell viability and proliferation and decreased expression of genes positively associated with auSD. ConclusionUrethral FBs and ECs can be cultured from healthy and strictured surgical specimens, enabling in vitro research. PDE4 inhibitors and T may be non-cytotoxic alternatives or additions to paclitaxel for aUSD. HighlightsO_LIPDE4 isozymes A, B, and D are expressed in adult anterior urethras C_LIO_LIPDE4 is expressed equally from proximal bulbar to meatal urethra C_LIO_LIEpithelial cells and fibroblasts can be cultured from healthy and stricture urethra C_LIO_LITGF{beta}1 may not be an optimal method to model aUSD in vitro C_LIO_LIUnlike paclitaxel, roflumilast and testosterone are not toxic to urethral cells C_LI

The chemical constituents and cytoprotective potential of Cyathea podophylla, a Vietnamese fern, remain poorly investigated. This study aimed to isolate its compounds and evaluate their in vitro cytoprotective activity against 6-hydroxydopamine (6-OHDA)-induced toxicity in F11 cells. Compounds were chromatographically isolated and structurally characterized using NMR and HR-ESI-MS. Seven compounds were identified: five phenolics (trans-cinnamic acid, (E)-4-(3,4-dihydroxyphenyl)but-3-en-2-one, p-coumaric acid, 3,4-dihydroxybenzoic acid, 4-O-acetyl-caffeic acid), 5-hydroxymethylfurfural, and butyl-{beta}-D-fructofuranoside. Six of these are newly reported for the Cyathea genus. In MTT assays, butyl-{beta}-D-fructofuranoside exhibited the strongest cytoprotective effect (69.6% cell protection at 10 {micro}M, p < 0.001), followed by (E)-4-(3,4-dihydroxyphenyl)but-3-en-2-one (39.2% at 10 {micro}M). The remaining compounds lacked significant activity. These findings expand the phytochemical profile of Cyathea podophylla and provide preliminary evidence of its cytoprotective properties against 6-OHDA-induced injury, warranting further mechanistic and in vivo validation.

BackgroundTriple-negative breast cancer (TNBC) is an aggressive subtype lacking well-defined molecular targets, leaving chemotherapy as the primary treatment despite drug resistance, systemic toxicity, and high recurrence rates. Therefore, the development of effective and less toxic therapeutic agents is essential. This study investigated the anti-cancer potential of gloriosine, a bioactive alkaloid with antiproliferative activity and low toxicity toward normal breast cells. MethodsPotential targets of gloriosine were predicted using SwissTargetPrediction, TargetNet, and PharmMapper, and overlapping genes related to TNBC and glutamine metabolism were selected. Protein-protein interaction networks, Gene Ontology, and KEGG pathway enrichment analyses were performed. Molecular docking evaluated binding affinity, followed by in vitro validation using cell viability, colony formation, and wound healing assays. ROS levels were measured by DCFDA and GSH assays, and ferroptosis was assessed by Western blot and FerroOrange staining in MDA{square}MB{square}231 cells. ResultsA total of 100 potential targets were identified, with 60 overlapping with TNBC and glutamine metabolism-related genes. Key targets included SRC, EGFR, mTOR, and HSP90AA1. Enrichment analyses indicated involvement in cancer progression, metabolic regulation, and resistance pathways, including central carbon metabolism, EGFR inhibitor resistance, and ErbB signaling. Gloriosine showed strong binding affinity toward hub targets. Experimental studies confirmed concentration-dependent inhibition of cell proliferation and migration. Mechanistically, gloriosine suppressed glutamine metabolism via GLS1 downregulation and induced ferroptosis, evidenced by increased ROS, glutathione depletion, GPX4 downregulation, and elevated intracellular iron levels. ConclusionsGloriosine exerts significant anti-cancer effects in TNBC through multi-target modulation and induction of ferroptosis, highlighting its potential as a promising therapeutic candidate. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/725321v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@ce0ebcorg.highwire.dtl.DTLVardef@29603borg.highwire.dtl.DTLVardef@6d0025org.highwire.dtl.DTLVardef@249700_HPS_FORMAT_FIGEXP M_FIG C_FIG Flow chart of the network pharmacological and in vitro study of gloriosine

Glycyrrhiza uralensis is a widely used medicinal plant present in more than 70% of Kampo formulations in Japan owing to its diverse pharmacological activities, including immunomodulatory, antitumor, and antioxidant effects. Isoliquiritigenin (ILG), a major chalcone constituent of G. uralensis, exhibits anti-inflammatory activity; however, its molecular mechanism remains unclear. Here, we employed an activity-based protein profiling approach to identify the molecular targets of ILG. Given that the ,{beta}-unsaturated carbonyl moiety of ILG can covalently react with reactive cysteine residues via nucleophilic addition, we used an iodoacetamide-based probe to globally profile cysteine-reactive proteomes. The comparative analysis between ILG- and vehicle-treated RAW 264.7 macrophages identified cysteine 65 (Cys65) of lipocalin-type prostaglandin D2 synthase (L-PGDS) as a potential covalent target. ILG treatment did not alter L-PGDS expression levels, indicating that reduced probe labeling reflects direct covalent competition rather than changes in expression. Consistently, ILG significantly suppressed prostaglandin D2 (PGD2) production, comparable to the selective L-PGDS inhibitor AT-56. Although both ILG and AT-56 reduced interleukin-6 expression, ILG exerted a stronger inhibitory effect. Our results demonstrate that covalent inhibition of L-PGDS and subsequent suppression of PGD2 production represent a key mechanism underlying the anti-inflammatory activity of ILG.

Post-stroke inflammation contributes to poor outcomes in both clinical and experimental studies. Interleukin-6 (IL-6) is a key inflammatory mediator in ischemic stroke, and higher circulating IL-6 levels are associated with greater stroke severity and worse clinical outcomes. Targeting IL-6 signaling therefore represents a potential therapeutic strategy. We tested whether inhibition of IL-6 signaling with the IL-6 receptor (IL-6R) blocking antibody tocilizumab (TCZ) improves recovery after experimental stroke. Aged mice (18-20 months) underwent 60 minutes of middle cerebral artery occlusion. TCZ (20 mg/kg) was administered 5 hours after ischemia onset, and behavioral outcomes were assessed weekly for 5 weeks. Delayed TCZ treatment improved long-term functional recovery in aged male mice but not in aged females. To explore this difference, we measured circulating soluble IL-6R (sIL-6R) levels in mice and patients with ischemic stroke. Females exhibited significantly higher post-stroke sIL-6R levels. Increasing the TCZ dose to 100 mg/kg restored efficacy in aged female mice and improved long-term outcomes. These findings support a role for IL-6R pathway modulation in improving recovery after experimental stroke and suggest that therapeutic response may differ by sex and target availability, potentially related to differences in circulating sIL-6R after ischemic injury.

The hippocampus is essential for memory consolidation, a process mediated by high-frequency oscillations known as ripples during non-rapid eye movement (NREM) sleep. Ramelteon, a selective MT1/MT2 receptor agonist, has been reported to possess cognitive-enhancing properties; however, its impact on the fine-scale dynamics of hippocampal ripples remains unclear. We performed chronic local field potential recordings from the dorsal hippocampus and prefrontal cortex in mice. Following the intraperitoneal administration of either vehicle or ramelteon, we evaluated sleep architecture and characterized ripple properties, including occurrence rate, amplitude, instantaneous frequency, and duration during NREM sleep. Ramelteon administration significantly increased NREM sleep occupancy. Notably, we found that ramelteon significantly enhanced both the occurrence rate and amplitude of hippocampal ripples compared to the control group. While a slight increase in intra-ripple frequency was observed, other structural features, such as ripple duration and asymmetry index, remained unaffected. Our findings demonstrate that ramelteon facilitates hippocampal ripple dynamics by increasing their occurrence and synchrony during NREM sleep. Given the critical role of ripples in memory consolidation, these neurophysiological changes may underlie the procognitive effects of ramelteon. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=55 SRC="FIGDIR/small/723673v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@c798c7org.highwire.dtl.DTLVardef@1ff616eorg.highwire.dtl.DTLVardef@1557dc8org.highwire.dtl.DTLVardef@1b4e89e_HPS_FORMAT_FIGEXP M_FIG C_FIG