Background: Intranasal drug delivery is a critical route for both systemic and targeted therapies, particularly for drugs intended to reach the central nervous system via the olfactory region. However, current aqueous nasal sprays have limited penetration beyond the anterior nasal airway, reducing their efficacy for nose-to-brain drug delivery. Nasal powder insufflators offer a promising alternative, as they allow higher drug payloads, typically up to 50 mg. The narrower spray cone angle of some powder insufflators has the potential for improved drug penetration, making them more suitable to deliver drugs higher and deeper into the nasal airways than is possible for aqueous nasal sprays. Despite this potential, existing methods for assessing powder deposition rely on complex and expensive imaging techniques such as gamma scintigraphy or MRI, limiting their accessibility for formulation and device optimization.Methods: This study presents a novel, cost-effective fluorescent imaging method to evaluate nasal powder deposition patterns using a transparent Koken nasal cast coated with synthetic mucus. Fluorescein-dyed spray-dried powders were delivered through a capsule-based nasal powder insufflator actuated by an air pulse, and deposition patterns were visualized under UV excitation. ImageJ analysis was used to quantify fluorescent intensity and distribution across five predefined nasal regions.Results: The results demonstrated that the powder delivery device successfully bypassed the anterior nasal airway and deposited a significant proportion of the dose in the olfactory region, achieving a mean posterior upper airway deposition of 51.7%, a substantial improvement over conventional aqueous sprays. The air pulse-actuated delivery mechanism also exhibited high repeatability, with a coefficient of variation of 14.9% in olfactory region deposition.Conclusion: By providing a simple, noninvasive alternative to radiolabeled imaging, this fluorescence-based approach enables rapid evaluation of nasal powder formulations and delivery devices. Hence, the approach described in this work could enable the nasal delivery device parameters, and powder formulation parameters to be optimized for nasal powder delivery devices. This could in turn allow drug delivery efficiency to different regions of the nose-particularly the upper nasal airways-to be optimized to suit different powder formulations, and different powder delivery devices.
BACKGROUND:Acute agitation (AA) is a common symptom of psychiatric conditions such as bipolar disorder and schizophrenia, resulting in 1.7 million emergency room visits per year in the United States. Loxapine, administered by inhalation, is an approved treatment for such scenarios. A boxed warning for bronchospasm when administered via inhalation limits its use to inpatient settings, making effective treatment for AA in outpatient settings an unmet need. The objective of this study was to develop and identify a lead intranasal dry powder formulation for outpatient treatment of AA. MATERIALS AND METHODS:Seven spray-dried nasal powders (Formulations A-G) consisting of loxapine succinate (Medichem, ES) and various excipients were manufactured and filled in a Unidose Nasal Powder system (Aptar Pharma, FR). In this pharmacokinetic study with non-compartmental analysis, formulations were evaluated both in vitro and in vivo (n = 4 non-human primates [NHPs]). Formulations were characterized by yield, assay, water content, particle size distribution, emitted dose, and impactor-sized mass. RESULTS:Formulation B, comprising mannitol and hypromellose with 30% loxapine by weight, was identified as the lead candidate. In the in vivo portion of the study, all formulations were well tolerated. The plasma versus time profile for the NHP study indicated that Formulation B achieved the greatest extent of absorption (203.9 ng/mL*h) compared to the intravenous (control) formulation (231.5 ng/mL*h), each administered as a 3 mg dose. The full pharmacokinetic results for all formulations showed similar rapid absorption following dosing, although the area under the concentration-time curve and maximum concentration were lower than those observed with Formulation B. All formulations had similar apparent terminal half-lives. CONCLUSIONS:Formulation B showed clear advantages both in vitro and in vivo, supporting its suitability for further development as an outpatient treatment for AA in schizophrenia and bipolar disorder.
BACKGROUND:Pressurized metered-dose inhalers (pMDIs) are commonly used for respiratory disease treatment but contain propellants, such as hydrofluoroalkane-134a (HFA-134a), with global warming potential (GWP) that contribute to the climate emergency. To safeguard essential medicine access, it is crucial to transition to lower-GWP propellants, such as hydrofluoroolefin-1234ze (HFO-1234ze), which has 99% lower GWP than HFA-134a. In accordance with global regulatory requirements, this study was conducted to support the registration of pMDIs with HFO-1234ze. METHODS:This randomized, double-blind, multicenter, two-way crossover study assessed the effects of HFO-1234ze versus HFA-134a on mucociliary clearance (MCC) in healthy participants aged 18-60 years. Participants received six inhalations twice daily of HFO-1234ze and HFA-134a in two 7-day (+3) intervention periods separated by a 7- to 14-day washout. The primary and secondary endpoints were change from period-specific baseline in average whole lung MCC (%) through 60 minutes (MCC60) and at 3 hours (MCC3h), respectively, measured via inhalation of 99mTc-labeled colloid and gamma camera imaging. Given significant baseline variability, a post hoc analysis of the primary endpoint assessed change from average baseline instead of period-specific baseline MCC60. Additional safety and tolerability measures were assessed. RESULTS:Forty-five participants were screened; the Primary Analysis Set included 34 participants who completed both intervention periods. For the primary endpoint, change from period-specific baseline in MCC60 was negligible, with an estimated change (95% confidence interval [CI]) of -0.6% (-3.2%, 1.9%) for HFO-1234ze and 0.8% (-1.7%, 3.3%) for HFA-134a. The estimated least-squares mean difference (95% CI) between HFO-1234ze and HFA-134a was -1.4% (-5.8%, 2.9%). For the secondary endpoint, the estimated least-squares mean difference for change from period-specific baseline in MCC3h between HFO-1234ze and HFA-134a was -5.7% (-10.9%, -0.5%). There were no unexpected safety findings. CONCLUSION:There was no clinically relevant impact of HFO-1234ze versus HFA-134a on MCC in healthy participants. CLINICAL TRIAL REGISTRATION NUMBER:NCT05755932.
BACKGROUND:Pressurized metered-dose inhalers (pMDIs) rely on hydrofluoroalkane (HFA) propellants that have a high global warming potential (GWP). Reformulation with next-generation, low-GWP propellants, such as HFA-152a, offers a strategy to reduce climate impact; however, changes in propellant composition can affect aerosol characteristics and potentially alter lung deposition, requiring robust demonstration of therapeutic equivalence. METHODS:Functional respiratory imaging, combining high-resolution computed tomography and computational fluid dynamics, was used to compare the lung deposition of a fixed triple combination of beclometasone dipropionate, formoterol fumarate, and glycopyrronium bromide (BDP/FF/GB) delivered via a pMDI formulated with either HFA-134a (Reference) or HFA-152a (Test). Ten patients with chronic obstructive pulmonary disease (GOLD stages 2-4) were retrospectively selected. Patient-specific airway geometries, a standardized inhalation profile, and formulation-specific particle size distributions and plume characteristics were applied. Deposition was quantified in the intrathoracic, central + distal, and peripheral lung regions, and the (central + distal)/peripheral ([C + D]/P) deposition ratio was evaluated. RESULTS:Mean intrathoracic deposition was comparable between the Reference and Test formulations, ranging from 45.95% to 46.88% of the delivered dose (DD). Deposition in the central + distal airways accounted for 12% of DD for both formulations, whereas peripheral deposition predominated, with 33.7% of DD for the Test formulation and 34.5% of DD for the Reference formulation. The (C + D)/P ratios were similar across all active components (0.35-0.37), indicating consistent preferential deposition in the peripheral/small airways. Although inter-patient variability was observed, intra-subject comparisons showed close agreement between propellants. CONCLUSION:Reformulation of the BDP/FF/GB pMDI with the low-GWP propellant HFA-152a preserved total and regional lung deposition characteristics relative to the current HFA-134a formulation. These findings support the maintenance of deposition performance while enabling a substantial reduction in environmental impact, reinforcing the potential of HFA-152a as a next-generation propellant for carbon minimal pMDI therapies.
BACKGROUND:Asthma is characterized by variable airway obstruction, and proper inhaler choice and use are critical for effective treatment. This study aimed to identify factors influencing treatment response in newly diagnosed patients started on dry powder inhalers (DPIs) or metered-dose inhalers (MDIs). METHODS:Between March and September 2025, 177 newly diagnosed asthma patients were screened. Eighty patients with at least high-school education, correct inhaler use, and good adherence were included (40 DPI, 40 MDI). Pulmonary function, respiratory muscle strength, Asthma Control Test (ACT), and handgrip strength (HGS) were assessed at baseline and after one month. RESULTS:After one month, improvements in forced expiratory volume in 1 second (FEV1) (% predicted), maximum inspiratory pressure (MIP) and MIP (%), and maximum expiratory pressure (MEP) (%) were significantly greater in the DPI group compared with the MDI group (p = 0.03, 0.04, 0.02 respectively). In multivariate regression, among MDI users, MIP (kPa) (B = -11.305, p = 0.001), MIP (%) (B = -0.902, p = 0.003), ACT (B = 1.277, p = 0.014), and HGS (B = 0.103, p = 0.039) were independent predictors of ΔFEV1 (%). Among DPI users, MIP (kPa) (B = 11.987, p = 0.015), MIP (%) (B = 1.041, p = 0.009), MEP (kPa) (B = -6.554, p = 0.014), MEP (%) (B = -0.816, p < 0.001), and HGS (B = 0.201, p = 0.005) were significant determinants of ΔFEV1. CONCLUSION:DPIs may be preferable for patients with higher inspiratory muscle strength, whereas MDIs may be better for those with lower MIP. HGS appears to be an important factor for both inhaler control and treatment efficacy.
BACKGROUND:This study compares the usability of two levodopa dry powder inhalers. The Inbrija® inhaler involves several preparatory steps, including blister opening and capsule insertion. The Cyclops® inhaler is preloaded and disposable, designed for immediate use. Ease of use is critical, as cognitive and motor impairments may hinder the patients' ability to operate an inhaler. METHODS:Sixteen patients with Parkinson's disease experiencing predictable and recognizable off episodes were enrolled. After receiving inhalation instructions, patients performed the inhalation maneuver during their off-episode to assess the correct execution. They completed a questionnaire evaluating the ease of use for both devices. Each patient used both the Cyclops® and Inbrija® inhalers in a randomized sequence and completed the questionnaire. RESULTS:All 16 patients correctly performed the Cyclops® inhaler steps during an off episode, while only 10 patients successfully completed the steps with the Inbrija® inhaler. Twelve of the sixteen patients (75%) expressed a preference for the Cyclops®, rating it higher in terms of ease of use and hygiene. However, concerns were raised regarding the environmental sustainability of its single-use design. For the Inbrija®, patients reported difficulties with opening the peel-off blister packaging, assembling the mouthpiece, and puncturing the capsules. CONCLUSION:The Cyclops® inhaler was more user-friendly during off episodes due to its ease of use and simpler handling. Since the Cyclops® is preloaded, it requires fewer and simpler steps. The preparation steps of Inbrija® posed usability challenges, particularly with opening the peel-off blister packaging of the capsule and assembling the mouthpiece and handle.
The 5-HT2BR, a member of the G protein-coupled receptor (GPCR) family, has been implicated in various diseases, including cardiovascular conditions, fibrotic disorders, cancer, and neuropsychiatric illnesses. Despite its therapeutic potential, the 5-HT2BR remains largely underexplored due to the limited availability of subtype-selective ligands. Additionally, many drugs either exhibit off-target binding to 5-HT2BR or fail to achieve specificity for their intended receptor subtype. Here, we present three cryo-electron microscopy structures of the human 5-HT2BR in complex with the antagonist tegaserod, the inverse agonist ritanserin, and the selective antagonist RS127445, respectively. These structures reveal distinct binding modes for each ligand, and through detailed analysis, we identify residues L362 and V366 as key contributors to 5-HT2 subtype selectivity, while E363 and the ECL2 region play critical roles in 5-HT2BR subtype selectivity. Our findings offer valuable insights into the molecular mechanisms behind ligand selectivity for 5-HT2BR, laying the groundwork for the development of 5-HT2BR-selective ligands.
Spermiogenesis dysfunction is a major cause of male infertility; however, the underlying molecular mechanisms involved remain incompletely elucidated. Although transmembrane protein 67 (TMEM67), a ciliary transition zone protein implicated in ciliopathies, is highly enriched in mouse testes, its cell type-specific functional relevance in spermatogenesis is unclear. Here, we generated germ cell-specific (Stra8-Tmem67f/f) and Sertoli cell-specific (Amh-Tmem67f/f) Tmem67 knockout mice to investigate the function of TMEM67 in spermatogenesis and male fertility. Amh-Tmem67f/f mice maintained normal fertility and exhibited normal spermatogenesis, with no significant differences in testicular histology or sperm count, morphology, or motility compared with wild-type (WT) controls. However, Stra8-Tmem67f/f males were completely infertile, manifesting severe oligoasthenoteratozoospermia (OAT) characterized by a drastic reduction in sperm count, total loss of sperm motility, and global sperm malformation. Further investigations revealed that TMEM67 deletion did not impair spermatogonial proliferation or meiosis, but instead disrupted key spermiogenic events, including manchette dynamics, acrosome biogenesis, and flagellum development. Proteomic analysis indicated that TMEM67 knockout altered the expression of numerous spermiogenesis-related proteins. Furthermore, our experiments confirmed that TMEM67 deficiency led to profound perturbations in both the expression levels and subcellular localization of key spermiogenic regulators in the testis. Collectively, our findings demonstrate that TMEM67 is indispensable for spermiogenesis and male fertility, revealing its critical role in coordinating manchette function, axonemal integrity, and spermiogenesis-related protein regulation, providing novel insights into OAT pathogenesis.
An acute bout of high intensity exercise can transiently increase circulating extracellular vesicles (EVs) that possess beneficial molecular cargo. However, no studies to date have comprehensively evaluated plasma quantity, protein content, and function of EVs collected from blood after multiple bouts of endurance exercise. Here we demonstrate that 4 weeks of voluntary wheel running increases plasma EV quantity when collected immediately after the last bout of training in mice. These EVs (ExerVs) are enriched in oxidoreductases, including the antioxidant glutathione peroxidase 1 (GPX1). Repeated, systemic injections of ExerVs into sedentary recipient mice twice per week for 4 weeks did not alter mitochondrial content or function, fiber size, or fiber type, but increased capillary density and perfusion in skeletal muscle. ExerVs also stimulated tube formation and branch lengthening in vitro and improved the recovery of capillary content after a period of disuse in vivo. ExerVs isolated from GPX1-/- mice lacked the ability to stimulate vessel formation, whereas GPX1-encapsulated liposomes robustly increased capillary growth, both in vitro and in vivo. The results from this study suggest that circulating ExerVs positively impact vascular structure and function in skeletal muscle in a manner that may be dependent on GPX1.
Sepsis-associated acute respiratory distress syndrome (ARDS) is characterized by excessive inflammation and dysregulated intra-alveolar coagulation, leading to fibrin deposition and impaired alveolar function. The regulatory role of vitamin D-vitamin D receptor (VDR) signaling in epithelial coagulation responses remains incompletely defined. We investigated the role of vitamin D-VDR signaling in regulating epithelial-driven intra-alveolar coagulation in sepsis-associated lung injury using vitamin D-deficient mice, LPS-induced acute lung injury models, RLE-6TN alveolar epithelial cells, and clinical samples from patients with ARDS. Expression of VDR, NF-κB p65, tissue factor (TF), and plasminogen activator inhibitor-1 (PAI-1) was assessed using qRT-PCR, Western blotting, ELISA, and immunohistochemistry. Functional experiments included 1,25(OH)2D3 supplementation, VDR silencing, and NF-κB p65 gain- and loss-of-function approaches. Vitamin D deficiency significantly exacerbated LPS-induced lung injury, pulmonary edema, and intra-alveolar hypercoagulation, as reflected by increased TF and PAI-1 expression in lung tissue and bronchoalveolar lavage fluid. LPS challenge suppressed VDR expression and concurrently activated NF-κB signaling In Vivo and in alveolar epithelial cells. Supplementation with 1,25(OH)2D3 restored VDR expression and attenuated TF and PAI-1 induction. Conversely, VDR silencing enhanced NF-κB p65 activation and amplified epithelial procoagulant responses. Mechanistically, NF-κB p65 was required for TF and PAI-1 upregulation, and its overexpression abolished the inhibitory effects of vitamin D-VDR signaling. Clinically, patients with ARDS exhibited reduced circulating 25(OH)D levels and decreased VDR expression compared with healthy controls. Vitamin D-VDR signaling acts as an endogenous protective axis that restrains NF-κB-driven epithelial procoagulant activation in sepsis-associated ARDS. Disruption of this pathway promotes TF- and PAI-1-mediated intra-alveolar coagulation, suggesting that restoration of vitamin D-VDR signaling may represent a potential adjunctive therapeutic strategy for ARDS.
Accumulation of neutrophil extracellular traps (NETs) in ulcerative colitis (UC) is associated with impaired intestinal epithelial barrier integrity. However, little is known about how NETs affect intestinal epithelial repair. This study sheds light on the molecular mechanisms through which excess NETs cause intestinal epithelial damage in UC mice. We found that UC mice had elevated levels of circulating cell-free DNA (cfDNA), mainly from NET byproducts (e.g., NET-DNA). NET-DNA in the intestine worsened UC symptoms, while DNase I treatment to eliminate it alleviated these symptoms. RNA-seq analysis revealed significant changes in IL-22 mRNA between wild-type and peptidylarginine deiminase 4 knockout (PAD4-/-) mice. Flow cytometry results indicated that NET-DNA mainly affected IL-22 secretion by group 3 innate lymphoid cells (ILC3s), while other forms of DNA had little influence on IL-22 expression. The IL-22+ILC3s ratio was restored in both DNase I-treated and PAD4-/- mice; moreover, levels of mucin, tight junction proteins, and Ki67 were significantly increased. Co-incubating ILC3s or the mouse lymphocyte cell line MNK3 with NET-DNA decreased IL-22 levels. ILC3s expressed the NET-DNA receptor coiled-coil domain containing protein 25 (CCDC25); however, NET-DNA did not affect IL-22 secretion in shCCDC25-MNK3 cells. Additionally, inhibiting ILK-HIF-1α proteins, downstream of CCDC25, increased IL-22 production in MNK3 cells. Finally, we established an in vitro culture system using MNK3 and Caco-2 cells. The supernatant from NET-DNA-treated MNK3 cells increased FITC-dextran permeability and reduced ZO-1 expression in Caco-2 cells. Thus, CCDC25 in ILC3s responds to NET-DNA by reducing IL-22 levels in UC mice, negatively impacting mucosal healing.
Pyroptosis, as an inflammatory type of regulated cell death, is associated with the pathogenesis of various inflammatory diseases. Targeted therapy for pyroptosis has shown promise in multiple preclinical models of neurological injury and disorders. Stroke is one of the leading causes of morbidity and mortality worldwide and the top cause of disease-related death in China. Although the pyroptosis signaling pathway has been studied in cerebral ischemic diseases, its pathophysiological mechanisms in brain microvascular endothelial cells (BMECs) remain unclear. In this study, we demonstrate that pyroptosis levels in BMECs are significantly elevated under ischemia-reperfusion (I/R) conditions and are closely associated with extensive macrophage infiltration in the brain, leading to inflammatory injury. We observed that the caspase-1 signaling pathway mediates GSDMD-dependent VCAM-1 expression, promoting the adhesive interaction between reactive endothelial cells and macrophages, thereby exacerbating the inflammatory microenvironment in the brain. Furthermore, omics analysis revealed that, upon caspase-1 activation, phosphorylated PXN (p-PXN) facilitates VCAM-1-mediated adhesion upstream, amplifying the inflammatory cascade and aggravating cerebral ischemic injury. In summary, our findings highlight the potential of non-glial and non-neuronal cells in amplifying neuroinflammation, providing additional theoretical support for the treatment of ischemic brain injury.
Spinal muscular atrophy (SMA) is caused by loss of SMN protein and is increasingly recognized as a multisystem disorder involving molecular pathology beyond motor neurons. Recently, we identified dysregulated NRF2-KEAP1 signaling in SMA mice. Since NRF2 coordinates transcriptional programs that maintain cellular redox homeostasis and adaptive stress responses, we investigated whether NRF2 signaling is similarly altered in fibroblasts derived from individuals with SMA type I and whether it can be pharmacologically engaged. Compared with control fibroblasts, SMA fibroblasts displayed reduced basal expression of NRF2 target proteins, including NQO1 and xCT (SLC7A11), along with decreased levels of PGC1α. Omaveloxolone (OMAV), a pharmacological NRF2 activator approved for the treatment of Friedreich's ataxia, increased cell viability and upregulated NRF2 target proteins in both control and SMA fibroblasts. Notably, OMAV produced a modest increase in SMN protein abundance and PGC1α levels selectively in SMA cells. Together, these findings support diminished NRF2 pathway activity as a feature of SMA fibroblasts and demonstrate that OMAV activates NRF2 signaling in this human SMA cellular model, consistent with enhanced cytoprotective signaling. These results support further investigation of NRF2 activation, including OMAV, as a potential adjunctive strategy in SMA.
The aim of this study was to investigate the combined effects of a high-fiber diet supplemented with N-carbamylglutamate (NCG) (H + N) on the gut microbiota, metabolites, and transcriptome in Landrace × Yorkshire sows using a multi-omics approach. Sows were allocated to four groups in a 2 × 2 design: Low-fiber or high-fiber diets, each with or without 0.05% NCG supplementation. The H + N treatment significantly increased litter weight at weaning. Metagenomic analysis revealed H + N significantly altered gut microbiota composition and function, particularly enriching Lactobacillus at multiple taxonomic levels from order to species (including Lactobacillus sp. 910 589 175). Plasma metabolomics identified two key lipid mediators, L-α-glycerylphosphorylcholine and taurocholic acid, whose abundances were significantly elevated by H + N and positively correlated with the enriched Lactobacillus. Transcriptomic profiling showed activation of the PI3K-Akt signaling pathway in response to H + N, which was associated with observed improvement in litter weight at weaning. Collectively, the multi-omics study uncovered a novel synergistic axis wherein H + N modulated the gut microbiome (specifically Lactobacillus enrichment), which in turn shaped the lipid metabolome to activate the PI3K-Akt pathway, ultimately enhancing sow reproductive efficiency.
Although space travel is becoming more accessible, our understanding of how the space environment and microgravity (μG) affect biology, physiology, and human health remains incomplete. This study examined the effects of μG on synaptic signaling and neuromuscular aging in Caenorhabditis elegans. The D01 cohort, consisting of L4 larvae to young adults raised in μG, exhibited a downregulation of genes linked to synaptic signaling, dopamine response, locomotion, cuticle development, and mitochondrial metabolism. This was accompanied by altered synapse dynamics, reduced motility, and shorter body length. In μG, aged worms showed a reduction in collagen gene expression, increased abnormalities in motor neuron morphology, changes in synaptic vesicle dynamics, and a collapse of mitochondrial morphology in body wall muscles, highlighting exacerbated aging-like phenotypes. The gentle-touch mechanoreceptor MEC-4 was identified as a key mediator of μG-induced body length reduction and changes in extracellular matrix gene expression. mec-4 mutants did not show μG-associated body shortening. The expression of most mechanoreceptor genes, including stretch-activated channels unc-105 and del-1, was downregulated under μG conditions. Notably, the expression of tmc-1 and degt-1 mechanoreceptor genes was downregulated independently of MEC-4. Restoration of physical stimulation using culture medium with small beads in space mitigated many μG-induced neuromuscular defects and expression alterations including those in mechanoreceptor genes. These results highlight the role of mechanical stimuli in maintaining neuromuscular integrity during spaceflight and suggest that restoring tactile input could counter health risks from reduced tactile stimulation during long-term space missions.
Contrast-induced acute kidney injury (CI-AKI) poses a significant clinical challenge and contributes to a considerable healthcare burden. Ferroptosis has been increasingly recognized as an important mechanism of renal tubular epithelial cell injury in CI-AKI. Salvianolic acid B (SalB), a natural compound with anti-inflammatory and antioxidant properties, has shown protective effects in various kidney diseases. However, its role in CI-AKI-associated ferroptosis has not been fully clarified. In this study, we established a rat model of CI-AKI by subcutaneous injection of carbon tetrachloride for 6 weeks followed by iopamidol administration, and an in vitro model using iopamidol-treated HK-2 cells. Effects of tubular injury and ferroptosis were examined both in vivo and in vitro. Cycloheximide chase assay, cellular thermal shift assay, and molecular docking were used to assess the binding capacity of SalB to SIRT1. Our results showed that SalB significantly alleviated renal injury, reduced iron accumulation, oxidative stress levels, and lipid peroxidation, upregulated the expression of SLC7A11 and GPX4, and downregulated ACSL4 expression in both iopamidol-treated rat kidneys and HK-2 cells. Mechanistically, SalB targeted and bound to SIRT1, enhancing its stability, thereby promoting Nrf2 upregulation and nuclear translocation, which in turn enhanced the expression of SLC7A11 and GPX4 and attenuated ferroptosis. Silencing either SIRT1 or Nrf2 in HK-2 cells partially abrogated the protective effect of SalB. Collectively, our results support SalB as a viable treatment strategy for CI-AKI.
Preeclampsia (PE) is a major pregnancy-specific disorder driven by impaired trophoblast function, placental hypoxia, and excessive oxidative stress. Ferroptosis has recently emerged as a key contributor to placental pathology; however, the upstream regulatory mechanisms that activate ferroptosis in trophoblasts remain incompletely defined. Circular RNAs (circRNAs) have been implicated in trophoblast dysfunction, yet the ferroptosis-related circRNA network in PE is largely unexplored. This study investigated whether circCOL3A1 regulates p53-mediated ferroptosis in trophoblasts and contributes to PE pathogenesis. CircCOL3A1 expression was examined in placental tissues and HTR-8/SVneo cells. Hypoxia and hypoxia/reoxygenation models were used to mimic PE-associated stress in vitro. Loss- and gain-of-function assays, ferroptosis measurements, RNA pull-down, RIP, FISH, and Actinomycin-D stability analyses were performed to elucidate the circCOL3A1/TIAL1/p53 axis. A reduced uterine perfusion pressure (RUPP) rat model was used to evaluate the in vivo effects of circCOL3A1 knockdown. CircCOL3A1 was significantly elevated in PE placentas and hypoxia-treated trophoblasts, exhibiting cytoplasmic localization and high structural stability. CircCOL3A1 knockdown restored trophoblast migration, invasion, viability, and reduced apoptosis under hypoxia. Silencing circCOL3A1 suppressed hypoxia-induced ferroptosis by reducing ROS, MDA, and iron levels while restoring GSH and GPX4. Mechanistically, circCOL3A1 bound the RNA-binding protein TIAL1 to enhance p53 mRNA stability, thereby promoting p53-dependent ferroptosis. In vivo, circCOL3A1 silencing lowered maternal blood pressure and proteinuria, reduced placental ferroptosis, and improved fetal survival in PE rats. CircCOL3A1 drives trophoblast ferroptosis through a TIAL1-dependent stabilization of p53, contributing to PE progression. Targeting the circCOL3A1/TIAL1/p53 axis may offer a promising therapeutic strategy for preeclampsia.
Abnormal activation of Hedgehog signaling is involved in renal fibrogenesis, a key process in chronic kidney disease (CKD) progression. However, the isoform-specific roles of Gli transcription factors remain unclear. This study aimed to elucidate the contribution of Gli2 to renal fibrosis. Multiple murine models of renal fibrosis-unilateral ureteral obstruction, ischemia-reperfusion injury, and aristolochic acid nephropathy-were employed, alongside human CKD specimens. In vitro assays, including genetic silencing, overexpression, co-immunoprecipitation, immunofluorescence, promoter analysis, F-actin staining, and TGF-β/SB431542 intervention assays were used to evaluate Gli2 function and its interaction with FoxM1. This study identifies Gli2, but not Gli1 or Gli3, as the primary Hedgehog signaling mediator in fibrotic kidneys, showing its activation in both epithelial and interstitial compartments. TGF-β-induced Gli2 activation promoted fibrogenesis via tubular epithelial-mesenchymal transition (EMT) and fibroblast-to-myofibroblast differentiation (FMD), and cytoskeletal remodeling. FoxM1 is a potential dowgnstream candidate transcriptionally regulated by Gli2, with two conserved Gli-binding sites within its promoter. FoxM1 knockdown partially mitigated Gli2-driven fibrotic effects. Pharmacological Gli2 suppression with GANT61 alleviated fibrotic injury in the UUO mouse model. In conclusion, activated Gli2 is closely associated with renal tubulointerstitial fibrosis and may facilitate fibrogenesis in a FoxM1-relevant regulatory pattern. The TGF-β/Hedgehog/Gli2/FoxM1 axis represents a promising therapeutic target for combating progressive CKD.
Hirsutine, a potent drug-like indole alkaloid extracted from Uncaria rhynchophylla, exhibits several biological activities, including cardioprotective effects. However, the underlying regulatory mechanisms remain unclear. Herein, we aimed to examine the therapeutic effects of hirsutine on obesity-related cardiomyopathy and investigate the potential mechanism underlying these effects. An obesity cardiomyopathy mouse model was developed by subjecting mice to a high-fat diet (HFD) for 16 consecutive weeks, followed by an 8-week hirsutine treatment. H9c2 cardiomyocytes treated with palmitate were utilized as an in vitro model. Invasive hemodynamic parameters and left ventricular hypertrophy indices were assessed, and the expression of related signaling molecules was analyzed using western blotting, mass spectrometry, molecular docking, RNA sequencing, immunoprecipitation, histological analysis, and transmission electron microscopy, respectively. Hirsutine significantly alleviated HFD-induced cardiomyopathy in the mouse model. Notably, the therapeutic effect of hirsutine was reversed in Midivi-1-treated mice, indicating that the cardioprotective role of hirsutine is dependent on mitochondrial fission-mediated mitophagy and Parkin. Mechanically, hirsutine maintained Parkin protein stability, and the C-terminal region of 1103-1394 amino acids of leucine-rich pentatricopeptide repeat-containing protein (LRPPRC) functions as a binding motif interacting with Parkin. LRPPRC overexpression significantly enhanced Parkin protein stability, which was attenuated by deletion of the 1103-1394 amino acids of LRPPRC (LRPPRCΔ1103-1394). Collectively, these findings demonstrate that hirsutine ameliorates HFD-induced cardiomyopathy by promoting Parkin protein stability through its interaction with 1103-1394 amino acids of LRPPRC. Therefore, targeting LRPPRC may represent a promising therapeutic strategy underlying the protective effects of hirsutine in HFD-induced cardiomyopathy.
