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.
BACKGROUND:The genetic variant ALDH2*2, associated with alcohol-related flushing, is a risk factor for alcohol-related health problems. The public has been largely unaware of these risks, suggesting potential for educational interventions to reduce harms among at-risk individuals. Self-efficacy and threat perception are mechanisms of health behavior change that may explain discrepancies in how people respond to health risk information. In the current pilot study, we examined the effects of a brief feedback intervention about the health risks related to flushing, ALDH2*2, and alcohol use on participants' intention to change their drinking and their past-month heaviest week drinking quantity. We also evaluated whether self-efficacy and threat perception related to intention to change drinking. METHODS:We enrolled 360 Asian American undergraduates (58% female; age 17-25). Participants were randomized to view (1) phenotype feedback about the alcohol-related cancer risk associated with flushing and ALDH2*2 (PHEN), (2) PHEN plus their own ALDH2 genotype (PHEN+GENE), or (3) an attention control session (CONTROL). Participants who completed the feedback (n = 324) reported their intention to change their drinking post-feedback and their recent alcohol consumption, self-efficacy, and threat perception at baseline and 1-, 4-, 7-, and 10-month post-feedback. RESULTS:Those in PHEN+GENE had significantly higher intention to change drinking compared with those in CONTROL regardless of ALDH2*2 status. A significant interaction was found between self-efficacy and threat perception in predicting intention to change drinking. Higher intention to change drinking did not relate to decreased heaviest week drinking quantity. CONCLUSIONS:Receiving risk feedback was associated with greater intention to change drinking among both ALDH2*2(+) and ALDH2*2(-) participants, but intention to change was not found to be associated with a decrease in heaviest week drinking quantity. Future work in larger samples will further examine self-efficacy and threat perception as potential mechanisms underlying participants' intention to change their drinking behavior. TRIAL REGISTRATION:ClinicalTrials.gov: NCT04967599.
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.
Numerous exocrine glands play key physiological roles in the body that include tearing, salivation, and lactation, as well as the control of body temperature via sweating. Malfunction of sweat glands can be deeply problematic or-in the case of anhidrosis-life-threatening. The prevalence of sweating disorders is high, affecting millions. The few available therapies are generally of limited effectiveness. Several lines of evidence point to regulation of sweating by the cannabinoid signaling system, an arrangement that would mirror cannabinoid regulation of tearing and salivation. Mice sweat in their paws via glands that closely resemble human eccrine sweat glands, including regulation by muscarinic signaling and by temperature. We applied a galvanic skin response-based assay to investigate cannabinoid regulation of sweating in awake, unanesthetized mice. The muscarinic agonist pilocarpine increased conductance while the antagonist glycopyrrolate reduced conductance, validating the model as a measure of sweating. The cannabinoid receptor agonist CP55940 substantially reduced conductance in wild-type and CB2 but not CB1 receptor knockout mice. The phytocannabinoid tetrahydrocannabinol (THC) also reduced conductance, while the non-psychoactive cannabidiol (CBD) did not. Using immunohistochemistry, we detected CB1 receptors in periglandular cholinergic axons, the anandamide-synthesizing enzyme NAPE-PLD in myoepithelial cells, and the anandamide metabolizing enzyme FAAH in acinar cells. This indicates that a local CB1/anandamide-based circuit is present in mouse walking pads. In summary, we employed a novel galvanic skin response-based assay to determine that cannabinoid CB1 receptors reduce sweating in a mouse model. This may point to a previously unappreciated effect on sweating in cannabis users.
This study examined the effects of exercise on islet fibrosis in mice with type 2 diabetes and investigated the role of irisin in the regulation of islet stellate cell (ISC) activation. Following the 16-week moderate-intensity exercise intervention, db/db mice showed reduced body weight and improved glucose tolerance and insulin sensitivity. This exercise intervention also decreased collagen-I (Col-I), fibronectin (FN), and α-smooth muscle actin (α-SMA); reduced islet fibrosis area (assessed by Masson staining); and lowered the numbers of α-SMA-positive ISCs in the pancreas. Serum irisin was increased after a single bout of moderate-intensity exercise but decreased following the 16-week exercise intervention. In vitro experiments showed that irisin suppressed ISC activation by delaying lipid droplet loss, inhibiting migration, and reducing expression of α-SMA, Col-I, and FN. Irisin also attenuated advanced glycation end products-or transforming growth factor-β (TGF-β)-induced ISC activation and extracellular matrix production and competitively bound to TGF-β receptor 2 (TGFBR2), inhibiting TGF-β/Smad pathway phosphorylation. These findings suggest that exercise may help to alleviate diabetes-related islet fibrosis, in part by suppressing ISC activation via irisin-mediated inhibition of TGF-β/Smad signaling.
Circular RNAs (circRNAs) are important regulators of signaling pathways involved in intervertebral disc degeneration (IVDD). This study investigated the role and underlying mechanism of circTMEM230 in the degeneration of endplate chondrocytes. We observed that circTMEM230 expression was significantly downregulated in chondrocytes subjected to intermittent cyclic mechanical tension (ICMT). Functional assays demonstrated that overexpression of circTMEM230 enhanced the expression of extracellular matrix (ECM)-related genes through modulation of the miR-223-3p/FOXO3/SOX9 signaling axis. Specifically, circTMEM230 acted as a molecular sponge for miR-223-3p, thereby upregulating FOXO3, which subsequently promoted SOX9 transcription. In vivo experiments further confirmed that circTMEM230 mitigated IVDD progression and regulated the expression of miR-223-3p, FOXO3, and SOX9. Additionally, expression levels of circTMEM230, miR-223-3p, FOXO3, and SOX9 were found to be correlated in endplate cartilage tissue samples from IVDD patients. These findings suggest that circTMEM230 exerts a protective role in IVDD and may serve as a promising therapeutic target for further investigation.
Fibroblast survival and dysregulated activation drive fibrotic diseases, including idiopathic pulmonary fibrosis (IPF). During physiological wound repair, fibroblasts are transiently activated to restore tissue integrity and are subsequently cleared by programmed cell death. In fibrotic disease, however, fibroblasts evade apoptosis and persist in a pathologically activated state. Although S100A4 has been implicated in fibrotic lung disease, the mechanisms by which S100A4 governs fibroblast fate and sustains profibrotic behavior remain unclear. Here, we identify S100A4 as a key regulator of apoptosis-resistant, profibrotically activated fibroblasts through engagement of extracellular signal-regulated kinase (ERK) signaling. In primary murine lung fibroblasts, S100A4 activates ERK, resulting in a coordinated program of fibroblast activation, including increased migration, extracellular matrix (ECM) contractility, stress fiber formation, and alpha-smooth muscle actin (α-SMA) induction. Functionally, S100A4 confers resistance to apoptosis induced by pro-apoptotic and oxidative stress stimuli, as evidenced by reduced cleaved caspase-3 and preserved cell viability. Pharmacological blockade of ERK signaling attenuates these responses, supporting ERK as an important downstream mediator of S100A4-driven fibroblast activation and survival programs. Extending these findings to disease-relevant contexts, bleomycin (BLM)-induced lung injury in mice induces robust fibrotic remodeling, excessive collagen deposition, and transcriptional upregulation of S100A4. Consistently, primary lung fibroblasts from IPF patients exhibit elevated S100A4 expression, enhanced ERK activation, and increased α-SMA expression, demonstrating conservation of this signaling axis across experimental models and human disease. Importantly, siRNA-mediated knockdown of S100A4 in IPF fibroblasts suppresses ERK activation and attenuates expression of key profibrotic genes, indicating that S100A4 contributes to maintaining the fibrotic program in IPF fibroblasts. Collectively, these findings define a mechanistic link between S100A4-mediated fibroblast survival and activation that drives pathological matrix remodeling and identify S100A4 and ERK as potential therapeutic targets in pulmonary fibrosis.
Previous studies showed that a T. spiralis serine proteinase (TsSPc) was identified in intestinal infective larvae (IIL) surface and excretory-secretory (ES) proteins. The in vitro experiments revealed that rTsSPc bound to intestinal epithelial cells and promoted larval invasion, but the in vivo role of rTsSPc in T. spiralis infection remains unclear. The purpose of this study was to investigate the TsSPc's function and mechanism in T. spiralis infection in mice. Immunofluorescence assay (IFA), qPCR, and Western blotting showed that rTsSPc specifically bound and co-localized with RACK1 receptor in intestinal mucosal epithelium, activated the ERK1/2 pathway, decreased the expression of the tight junctions (E-cad, Occludin, and Claudin-1), increased intestinal permeability, impaired intestinal epithelial integrity and barrier function, thereby promoted T. spiralis invasion of intestinal mucosa. The H&E and PAS staining showed that rTsSPc also caused intestinal mucosal inflammation reactions; the number and size of goblet cells in the rTsSPc group were distinctly increased, and the expression levels of inflammatory cytokines (TNF-α, IL-1β, TGF-β, and IL-10) and mucins (Muc2 and Muc5ac) were significantly elevated. These findings further verified that the in vivo binding of TsSPc to RACK1 disrupted gut epithelial integrity and mediated T. spiralis invasion of intestinal mucosa, and TsSPc may be regarded as a potential vaccine target to block T. spiralis infection.
Cervical cancer (CC) is characterized by tumor immune escape, which underlies suboptimal therapy responses and an increased recurrence risk. Despite the established role of LAT1 in cancer progression, its regulatory mechanism in the CC immune microenvironment remains elusive. LAT1 expression in cervical squamous cell carcinoma and its correlation with CD8+ T cell infiltration were assessed through the bioinformatic approach. LAT1 mRNA and protein expression were examined by qRT-PCR and Western blot, respectively. In a co-culture system with CC cells, CD8+ T cell antitumor activity was measured by lactate dehydrogenase release, ELISA, CCK-8, colony formation, and flow cytometry. The LAT1/TRIM67 interaction was identified through bioinformatics and validated by Co-IP and immunofluorescence. Potential ubiquitination substrates of TRIM67 were screened through bioinformatics. CHX chase assays combined with ubiquitination analysis were employed to verify the IRF3 degradation pathway. Finally, in vivo functional validation was conducted in a mouse xenograft model. LAT1 was overexpressed in CC tissues and cell lines. LAT1 expression negatively correlated with CD8+ T cell infiltration. LAT1 knockdown enhanced CD8+ T cell antitumor activity. Mechanistically, LAT1 suppressed CD8+ T cell antitumor function in CC by interacting with TRIM67 to promote ubiquitination and degradation of IRF3. LAT1 recruits TRIM67 to mediate IRF3 ubiquitination and degradation, ultimately suppressing CD8+ T cell function and promoting immune escape. These findings provide a theoretical basis for targeting the LAT1/TRIM67 axis to enhance immunotherapy in CC.
