Transient receptor potential vanilloid 1 (TRPV1) and transient receptor potential ankyrin 1 (TRPA1) channels mediate nociceptive and neurovascular signaling and can be activated experimentally by capsaicin and cinnamaldehyde, respectively. We characterized the physicochemical stability, in vitro permeation, and human pharmacodynamic effects of topical capsaicin and cinnamaldehyde formulations. Solutions underwent six-month stability testing at 4 °C and permeation studies in Franz diffusion cells using Strat-M® and lauryl alcohol-impregnated cellulose nitrate membranes. The formulations were then applied to the forearm skin of 15 women with migraine, and dermal blood flow was measured for 90 minutes using laser speckle contrast imaging. Both formulations remained stable for six months. No active compound was detected in the receptor chamber with Strat-M®, whereas low but pharmacologically relevant permeation occurred across the cellulose nitrate membrane. Capsaicin produced a robust and sustained vasodilatory response, with dermal blood flow increasing by 264.7% at 30 minutes and peaking at 291.0% at 45 minutes; the response remained elevated at 90 minutes. Cinnamaldehyde produced a rapid but transient response, increasing dermal blood flow by 231.6% at 30 minutes and progressively returning toward baseline by 90 minutes. No local or systemic adverse events were reported. These findings support topical capsaicin and cinnamaldehyde as complementary experimental probes for studying TRPV1- and TRPA1-mediated cutaneous neurovascular responses in humans.
Psoriasis is a chronic inflammatory skin disorder characterized by dysregulated cytokine signaling and keratinocyte hyperproliferation, in which extracellular vesicles (EVs), particularly exosomes, play key roles in mediating intercellular inflammatory communication. In this study, we evaluated the pharmacological effects of calpeptin and bisindolylmaleimide I (BIM-I), inhibitors of calpain and protein kinase C (PKC) previously reported to modulate extracellular vesicle (EV)-associated pathways, in a three-dimensional (3D) human psoriatic skin model. Both inhibitors significantly reduced the expression of proinflammatory cytokines, keratinocyte activation marker and exosome-related marker, including IL-1β, TNF-α, IL-8, IFN-γ, IL-21, IL-20, CCL-20, KRT6A, CD9, and CD63 consistent with attenuation of psoriasis-associated inflammatory signaling. BIM-I additionally suppressed IL-22 and IL-6 expression and restored epidermal ultrastructural features, including desmosome density, to levels comparable to normal skin. Integrated proteomic and immunohistochemical analyses revealed distinct downstream biological responses associated with modulation of exosome-associated markers and pathways. Calpeptin treatment was associated with enrichment of endoplasmic reticulum (ER) stress–related pathways, as indicated by increased GADD153 and GRP78 expression, whereas BIM-I preferentially regulated cytoskeletal organization and keratinocyte differentiation, accompanied by reduced expression of GADD153, GRP78, and KRT16. Collectively, these findings demonstrate that pharmacological inhibition of calpain and PKC attenuates psoriatic inflammation and epidermal dysregulation and is associated with modulation of exosome-associated markers and pathways, together with differential regulation of ER stress and cytoskeletal networks.
The combination of nivolumab and ipilimumab represents a major therapeutic advance for hepatocellular carcinoma (HCC), yet its real-world hepatic safety profile remains insufficiently characterized. To address this, we conducted a disproportionality analysis using adverse event reports from the US Food and Drug Administration Adverse Event Reporting System submitted between the first quarter of 2004 and the third quarter of 2025, specifically for patients with HCC receiving this dual immunotherapy. Disproportionality signals were assessed using 4 complementary methods: the reporting odds ratio, proportional reporting ratio, Bayesian confidence propagation neural network, and multi-item gamma Poisson shrinker. A total of 128 liver adverse event reports were identified, spanning 2 primary system organ classes with the majority classified under hepatobiliary disorders, and 5 significant disproportionality signals for specific liver adverse events were detected across these methods. Collectively, these findings highlight the critical need for vigilant monitoring of hepatic function in patients with HCC treated with nivolumab/ipilimumab in routine clinical practice. SIGNIFICANCE STATEMENT: The study specifically focuses on the hepatic safety of dual immune checkpoint inhibitor therapy in patients with HCC, addressing the gap in real-world hepatic safety data, and successfully identifies 5 specific liver adverse event signals that provide clear directions for clinical monitoring of liver injury.
Despite established roles in multiple cancers, the specific function and molecular mechanism of SOX5 remain elusive in esophageal squamous cell carcinoma (ESCC). SOX5 expression in ESCC tissues, its correlation with prognosis, and its relationship with TLR4 and NLRP3 were assessed with bioinformatic methods. Expression of SOX5 and TLR4 in ESCC cells was detected by RT-qPCR. In SOX5-overexpressing or TLR4-knocking down ESCC cells, cell death was evaluated by PI staining; migration and invasion were detected using Transwell assays; cell proliferation was assessed by colony formation assay; pyroptosis was observed using optical microscope and confirmed using Annexin V/7-AAD staining; the binding of SOX5 to the TLR4 promoter was confirmed by ChIP assay; and the expression of key pyroptosis pathway proteins (GSDMD, GSDMD-N, c-CASP1, NLRP3) were analyzed by Western blot. SOX5 downregulation in ESCC tissues and cells was observed and correlated with shorter survival in ESCC patients. SOX5 expression positively correlated with the key pyroptosis pathway genes TLR4 and NLRP3. SOX5 overexpression promoted ESCC cell pyroptosis and enhanced sensitivity to cisplatin (DDP). Mechanistically, SOX5 interacted with the TLR4 promoter and upregulated key pyroptosis pathway proteins; TLR4 knockdown reduced the enhanced pyroptosis and DDP sensitivity in SOX5-overexpressing cells. SOX5 enhances DDP sensitivity in ESCC by mediating TLR4/NLRP3 expression to promote cell pyroptosis, providing novel insights for ESCC treatment.
The decarboxylated form of L-arginine, agmatine, inhibits neuroplasticity through inhibition of the NMDA receptor. We have demonstrated that central or systemic agmatine administration inhibits neuropathic pain behaviors. However, the options for pharmaceutical development of agmatine are limited. To capitalize on agmatine’s therapeutic potential, we designed a series of agmatine analogs. Systemically administered SSA1-4, agmatine, and positive control ligands were evaluated for reversal of tactile hyperalgesia in nerve-injured mice and rats as well as pre-clinical models of motor function, cardiovascular performance, and abuse liability, and the effects of SSA1-4 were compared between GluN2B knockdown and control mice. Finally, a pharmacokinetic/pharmacodynamic model was developed. Intravenous delivery of SSA1-4 reversed neuropathic pain behaviors in both the induction and maintenance phases of neuropathic pain. Similar to agmatine and ifenprodil, the effects of SSA1 and SSA2 required the presence of the GluN2B subunit-containing NMDA receptors in mice. At doses that elicit anti-hyperalgesia, SSA1-4 and agmatine lacked effects in pre-clinical models of motor dysfunction, cardiovascular function, and abuse liability. The pharmacokinetic/pharmacodynamic modeling revealed that each compound exhibited distinct pharmacokinetic/pharmacodynamic profiles and each were best described by a two-compartment model. Through strategic substitution of the agmatine molecule, we have developed and characterized a line of new chemical entities that present a pharmacological profile comparable to that of the parent compound agmatine including a wide therapeutic window without side effects common to analgesic medications.
Lipopolysaccharide (LPS) is an endotoxin that can trigger multiple types of acute organ failure by increasing the production of nitric oxide (NO), which is synthesized by inducible NO synthase (iNOS) in macrophages through increased production of tumor necrosis factor (TNF)-α, interferon gamma, and interleukin-12. We have reported that an inhibitor of MAPK/ERK kinase (MEK), which is activated by LPS, increased the mortality rate in LPS-treated mice through enhanced NO production. However, a strategy to prevent lethal NO production by MEK inhibitors, which are used as anticancer agents, remains unclear. In the present study, we examined whether prestimulation of Toll-like receptors (TLRs) influences NO production by a MEK inhibitor in the presence of LPS because LPS is a ligand of TLR4 and macrophages acquire tolerance through repeated TLR stimulation. The MEK inhibitor increased iNOS expression and NO production in LPS-stimulated mouse macrophages. While pretreatment with TLR3, TLR7/8, or TLR9 agonists abolished the increase in iNOS expression, the subsequent increase in NO production was confirmed to be suppressed by TLR3 stimulation. The MEK inhibitor also suppressed the serum levels of both NO and TNF-α in mice pretreated with the TLR3 agonist poly I:C followed by LPS administration. Both iNOS expression and NO production depended on the TNF-α concentration in mouse macrophages treated with interferon gamma and interleukin-12. These results suggest that the ability of MEK inhibitors to increase NO production in mice treated with LPS is abolished by prior TLR3 stimulation, which is associated with the suppression of TNF-α production. SIGNIFICANCE STATEMENT: A MAPK/ERK kinase inhibitor increased nitric oxide production in lipopolysaccharide-treated mice; however, prior Toll-like receptor 3 stimulation abolished this effect, accompanied by a reduction in tumor necrosis factor-α production. Prior Toll-like receptor 3 stimulation may contribute to a potential strategy to prevent lethal nitric oxide production by MAPK/ERK kinase inhibitors in mice treated with lipopolysaccharide.
Pharmacological inhibition of REV-ERBs has emerged as a potential therapeutic strategy for several diseases with unmet medical needs. Indeed, chronic treatment with SR8278, a synthetic REV-ERB antagonist, has mitigated pathology in various preclinical models of human musculoskeletal and neurological diseases, including Duchenne muscular dystrophy, epilepsy, Alzheimer disease, Parkinson disease, and frontotemporal dementia. However, SR8278, the first and most widely used REV-ERB antagonist, has poor pharmacokinetic properties, which limits its utility. The REV-ERBs (α and β) are widely expressed nuclear receptors that function as ligand-dependent transcriptional repressors, and the therapeutic potential for inhibition of these receptors remains unclear because of the lack of adequate pharmacological tools. Here, we report BE2012, a significantly improved REV-ERB antagonist with >22-fold longer half-life than SR8278. In a cell-based reporter assay, BE2012 exhibited greater potency toward REV-ERBα (EC50 = 0.38 μM) and REV-ERBβ (EC50 = 0.57 μM) compared with SR8278. Notably, in primary myoblast differentiation assays, BE2012 outperformed SR8278 in increasing the proportion of MyoG+ cells as well as differentiation and fusion indices. In a cardiotoxin-induced muscle injury model, both BE2012 and SR8278 led to increased myofiber cross-sectional area (22%-34% higher than controls). Lastly, transcriptomic profiling revealed remarkable overlap of differentially expressed genes between the 2 compounds, with oxidative phosphorylation and mitochondrial protein complex emerging as the most significantly enriched pathways for both ligands, which have been shown to accelerate regenerative myogenesis. These results establish BE2012 as a refined REV-ERB antagonist for in vivo applications and a valuable tool for deeper exploration of the therapeutic potential of inhibiting REV-ERB activity. SIGNIFICANCE STATEMENT: This study establishes BE2012 as a novel REV-ERBα/β antagonist with a 22-fold longer half-life and improved potency compared with SR8278, the only available REV-ERB antagonist whose poor bioavailability has limited its utility for in vivo use. In an acute muscle injury model in mice, BE2012 promoted regenerative myogenesis and induced a broad transcriptomic reprogramming that was highly concordant with that of SR8278, suggesting it could serve as a new gold-standard REV-ERB antagonist tool compound in the field.
Mitochondrial dysfunction is central to postcardiac arrest brain injury (PCABI); however, the underlying protein networks involved remain poorly defined. SRT1720, a silent information regulator 1 activator, exhibits potential mitochondrial protective effects, although its role and mechanisms in PCABI are unclear. Adult male Wistar rats were randomized into sham, cardiopulmonary resuscitation (CPR), and SRT1720 groups and subjected to 8-minute asphyxia-induced cardiac arrest followed by CPR, with intraperitoneal SRT1720 (5 mg/kg) given 10 minutes after resuscitation. In a functional cohort (n = 15), SRT1720 significantly restored the mitochondrial calcium retention capacity, which was markedly impaired by cardiac arrest/CPR. Complex I activity was selectively suppressed and was restored by SRT1720, whereas other respiratory complex activities remained unchanged. In a parallel proteomic cohort (n = 9), 127 differentially expressed mitochondrial proteins were identified, with metabolic pathways being the most significantly enriched. Integrated network analysis revealed a coordinated pathological signature comprising complex I deficiency (NDUFB4), oxidative stress (NDUFB11), endoplasmic reticulum stress (RPN2), and lipid dysregulation (PLA2). SRT1720 treatment was associated with the reversal of these proteomic alterations and the re-establishment of metabolic homeostasis. Collectively, this multilevel analysis identifies a mitochondrial proteomic network disrupted in PCABI. The functional and proteomic recovery observed with SRT1720 is consistent with enhanced silent information regulator 1 signaling, suggesting a potential therapeutic strategy that warrants further mechanistic validation. SIGNIFICANCE STATEMENT: This study reveals a coordinated mitochondrial protein network-characterized by complex I dysfunction, oxidative stress, endoplasmic reticulum stress, and lipid dysregulation-as a key driver of postcardiac arrest brain injury. Treatment with the silent information regulator 1 activator SRT1720 was associated with the reversal of these proteomic alterations, selective restoration of complex I activity, and recovery of mitochondrial calcium regulation, thereby re-establishing energy metabolism and metabolic homeostasis. These integrated, multilevel findings provide a mechanistic framework for targeting mitochondrial dysfunction in postcardiac arrest brain injury and support SRT1720 as a promising therapeutic candidate.
Chronic human immunodeficiency virus (HIV) infection results in a persistent state of neuroinflammation, even with combined anti-retroviral therapy. Neuroinflammation contributes to pathology termed HIV-associated neurocognitive disorder (HAND), which is exacerbated by interferon gamma (IFNγ) producing CD8+ T cells in the central nervous system. Many people living with HIV (PLHIV) self-report using Cannabis sativa (MJ) to mitigate symptoms of chronic infection and side effects of treatment. Cannabis sativa is composed of various phytocannabinoids, including Δ9-tetrahydrocannbinol (THC), which possesses immune modulating properties. This study aims to determine if Cannabis sativa use by PLHIV, and specifically THC, effects IFNγ secretion by CD8+ T cells. We found that HIV status does not influence T cell IFNγ responses, and Cannabis sativa use modestly reduces the average secretion of IFNγ by CD8+ T cells from HIV+ donors. Treatment of CD8+ T cells with THC and the selective CB2 agonist, JWH-015, reduced T cell cytokine secretion, with THC eliciting greater suppression than CBD. These results suggest that THC treatment does not directly impair IFNγ gene expression or protein production as determined by PCR and flow cytometry. However, THC treatment impaired CD8+ T cell differentiation into IFNγ competent CD45RO+ cells. These studies are of clinical relevance, as reduction of CD8+ T cell-derived IFNγ in the CNS may improve cognitive outcomes during HIV infection. Furthermore, these findings may be generalizable to other inflammatory diseases where IFNγ producing CD8+ T cells have been implicated.
Menopause is a natural biological process in women driven by estrogen deficiency and is associated with multiple post-menopausal physiological and behavioral changes. Notably, menopause increases the risk of depression. However, the precise neural mechanisms by which estrogen deficiency contributes to depressive phenotypes remain poorly understood.Estrogen deficiency is known to activate transient receptor potential melastatin 3 (TRPM3) channels and promote neuroinflammation by inducing microglial activation and upregulating pro-inflammatory cytokines. Although the paraventricular nucleus of the hypothalamus (PVN) plays a central role in integrating the physiological response to stress, the role of TRPM3 channels in this region remains unexplored. We hypothesize that estrogen deficiency during menopause activates TRPM3 channels in the PVN, promoting neuroinflammation and disrupting medial prefrontal cortex (mPFC) function, thereby contributing to depressive-like phenotypes. Using a bilateral ovariectomy rat model, we demonstrate that estrogen loss induces robust depression-like behaviors, accompanied by increased TRPM3 expression and interleukin-1β levels in the PVN, as well as reduced dendritic complexity and serotonin levels in the mPFC. In the present study, retrograde DiI tracing confirmed direct PVN-mPFC connectivity, and immunofluorescence revealed co-expression of TRPM3 and the serotonin transporter in PVN neurons, implicating TRPM3 in serotonergic regulation. Notably, intraperitoneal administration of the selective TRPM3 inhibitor, naringenin reversed behavioral deficits, attenuated neuroinflammation, restored dendritic arborization, and normalized mPFC serotonin levels. These findings indicate that TRPM3-mediated PVN neuroinflammation disrupts mPFC serotonin signaling, contributing to menopause-associated depressive phenotypes and identifying TRPM3 as a potential therapeutic target.
Epigallocatechin gallate (EGCG) exerts cardioprotective effects on acute myocardial infarction through multiple pathways. However, the clinical application of EGCG is limited because of its instability under physiological conditions and low bioavailability. In this study, poly(lactic-co-glycolic acid) was used as a drug carrier to prepare EGCG nanoparticles (NPs), then an EGCG targeting drug delivery system was designed by modifying with cardiac homing peptide. We constructed a preparation route combining the double-emulsion solvent evaporation method and the carbodiimide method. By optimizing the preparation process, the particle size of the EGCG NP was reduced, and the dispersibility was improved. In addition, the established preparation procedure uses safe and low-toxicity reagents, requires no strict temperature control or specialized instrumentation, and therefore significantly reduces the technical difficulty of large-scale industrial production. EGCG-poly(lactic-co-glycolic acid)-cardiac homing peptide had myocardial targeting and good safety in the 3‑(4,5‑dimethyl‑2‑thiazolyl)‑2,5‑diphenyl‑2H‑tetrazolium bromide (MTT) cytotoxicity test, and alleviated acute myocardial infarction by regulating apoptosis-related protein expression in a dose-dependent manner. Compared with EGCG monomer, EGCG-poly(lactic-co-glycolic acid)-cardiac homing peptide achieved a better therapeutic effect. This study provides a new strategy for promoting the application of EGCG in the clinical treatment of acute myocardial infarction. SIGNIFICANCE STATEMENT: This study develops cardiac homing peptide-modified poly(lactic-co-glycolic acid) nanoparticles to enable targeted delivery of epigallocatechin gallate for acute myocardial infarction therapy. By overcoming the inherent instability and poor bioavailability of epigallocatechin gallate, this scalable nanoplatform achieves superior cardioprotection through regulated apoptosis and exhibits excellent safety in the MTT cytotoxicity test. These findings establish a clinically translatable strategy for targeted drug delivery in the treatment of cardiovascular disease.
Natural products have inspired the discovery of several drug candidates and US Food and Drug Administration-approved drugs. However, the conventional drug development pathway has several limitations, necessitating innovative strategies. Computational pharmacology and in silico clinical trials (ISCTs) have emerged as a potential to optimize the drug discovery process. Current literature rarely discusses these 2 domains together, leaving limited guidance on how computational tools used in natural product research can complement ISCT frameworks. This review addresses this gap by summarizing key computational methods used for natural products and highlighting how their outputs can inform ISCT-based evaluation of efficacy, safety, and translational potential. Computational approaches such as molecular docking, pharmacophore modeling, quantitative structure-activity relationship analysis, absorption-distribution-metabolism-excretion-toxicity prediction, and molecular dynamics simulations have been applied in natural products-based drug discovery. The findings of these studies are promising and suggest favorable prospects for the use of computational methods in identifying, optimizing, and evaluating bioactive natural compounds. Notably, this approach minimizes the attrition rate in later clinical stages, as the most promising compounds with auspicious pharmacokinetic and pharmacodynamic profiles are chosen. ISCTs further hold considerable value in drug discovery through investigating the safety and efficacy of investigational drugs using simulated or virtual patient populations, ensuring a lower attrition rate when wet-lab clinical trials are conducted. Although the ISCTs on natural products are limited, the integration of computational pharmacology and ISCTs shows potential in the discovery of valuable natural compounds and the acceleration of drug development from natural products. Despite existing challenges in data quality and regulatory acceptance, the integration of emerging technologies such as machine learning, artificial intelligence, and hybrid platforms shows promise in advancing natural product-based therapeutics and precision medicine. Significance Statement This review brings together computational pharmacology and in silico clinical trials to address persistent challenges in natural product drug development. It highlights how early computational screening, absorption-distribution-metabolism-excretion-toxicity prediction, and dynamic modeling can inform virtual clinical evaluation, reduce attrition, and accelerate translation. The paper offers a clear and practical roadmap for improving efficiency, strengthening safety assessment, and advancing precision in the development of natural product-based drugs.
