Search Results (937)

Oyster health is important for aquaculture productivity and sustainability. In Thailand, the white scar oyster, Crassostrea belcheri, is being promoted for cultivation, yet its health status has not been compared between research breeding and community farming sites. This study evaluated histopathological features, ultrastructure, apoptosis, and defender against apoptotic death 1 (dad1) gene expression in sexually mature C. belcheri collected from these two sites. Gill tissues were examined by histology, transmission electron microscopy, TUNEL assay, and gene expression analysis, while organ condition was assessed using a Health Assessment Index (HAI). The proportion of TUNEL-positive cells in the gills and mantle differed significantly between sites (p < 0.05), with higher levels in oysters from the farming site. In contrast, TUNEL-positive cells in the digestive gland did not differ significantly between sites, although brown cells were observed only in the digestive gland of oysters from the breeding site, suggesting possible physiological stress. To assess the expression level of dad1 in oysters cultured under different conditions, RT-qPCR revealed no significant difference between the two sites. The breeding site also had lower temperature and salinity than the farming site. Overall, these findings suggest that site-specific environmental conditions may influence gill health and stress-related responses in C. belcheri, providing baseline information for oyster health assessment and aquaculture management. Full article

Exposure to electromagnetic fields (EMFs) at different frequencies has been reported to induce apoptotic changes in brain tissue. Apoptosis is commonly evaluated using the TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) method; however, conventional semi-quantitative scoring is subjective and may vary between observers. Therefore, this study aimed to evaluate the feasibility of transfer learning-based convolutional neural network (CNN) models for the objective and automated classification of apoptotic damage in TUNEL-stained brain sections. A total of 92 light microscopy images of TUNEL-stained rat brain tissue, obtained from experimental groups, were analyzed. Apoptotic damage was categorized into three classes (0: no, +1: slight, +2: moderate) based on semi-quantitative scoring. Pre-trained convolutional neural network models, including AlexNet, SqueezeNet, GoogLeNet, Inception-v3, and ResNet-101, were applied for image classification. All models were able to classify apoptotic damage levels, defined by the extent of TUNEL staining, from images with varying performance. The best-performing model achieved high classification accuracy and demonstrated strong agreement with manual scoring, as determined by visual assessments by experts. The models successfully distinguished between different levels of apoptotic damage observed across experimental groups. The findings suggest that transfer learning-based CNN models may provide an objective and reproducible approach for the classification of apoptotic damage in TUNEL-stained histopathological images, thereby reducing observer-dependent variability. This approach may also support histopathological evaluation in experimental models, including studies investigating EMF-induced brain injury. Full article

The present investigation was designed to assess how perinatal contact with the pyrethroid insecticide cyfluthrin (CY) influences cognitive performance in developing rat progeny and to clarify the contributing cellular events through examination of neuroinflammatory processes alongside pyroptotic and apoptotic pathways. An experimental framework involving CY administration during gestation was implemented using Sprague–Dawley (SD) dams, with subsequent monitoring of placental parameters and neonatal outcomes. Once offspring reached postnatal day twenty-one, their behavior was characterized via a battery consisting of the open field paradigm, novel object recognition task, and the Morris water navigation test. Hippocampal tissue architecture and fine structural details were visualized by employing hematoxylin–eosin (HE) staining and Nissl substance labeling. Protein and transcript abundances for pro-inflammatory mediators (TNF-α, IL-6), synaptic constituents (postsynaptic density protein-95, PSD-95; synaptophysin, SYP), and pyroptotic machinery components (NLRP3, GSDMD, Caspase-1) within hippocampal homogenates were quantified through immunoblotting and real-time quantitative PCR procedures, and the spatial distribution of these molecules was validated via immunohistochemical detection. Neuronal apoptosis was assessed by TUNEL staining. The results demonstrated that gestational CY exposure led to reduced placental weight and diameter, decreased blood sinus area in the labyrinth zone, lower offspring birth weight, and impaired catch-up growth. Behavioral tests revealed that CY-exposed offspring exhibited diminished spontaneous locomotor activity, impaired novel object recognition memory, and significant deficits in spatial learning and memory. Pathological analysis showed disorganized neuronal arrangement and reduced Nissl bodies in the hippocampal CA1 region. Compared to the control group, CY exposure markedly upregulated the protein expression of TNF-α and IL-6, downregulated PSD-95 and SYP, activated the NLRP3/GSDMD/Caspase-1-mediated pyroptotic pathway, and increased the expression of the apoptotic protein Caspase-3, culminating in a significant increase in hippocampal neuronal apoptosis. In conclusion, early-life exposure to cyfluthrin impairs cognitive function in offspring, an effect closely associated with the induction of hippocampal neuroinflammation and the activation of pyroptotic and apoptotic pathways. These findings provide novel toxicological evidence for a more comprehensive assessment of the potential health risks posed by CY exposure in human populations. Full article

Morphine addiction is driven in part by persistent reward-associated memory, yet the molecular mechanisms linking translational control to cellular stress responses remain unclear. In the present study, using a mouse morphine-induced conditioned place preference and N2a cell model, we investigated apoptosis-related alterations in the medial prefrontal cortex and the involvement of eIF4E-dependent translational regulation and BDNF/TrkB signaling. Morphine-induced conditioned place preference was associated with an increase in TUNEL-positive cells in the medial prefrontal cortex, accompanied by upregulation of Bax and downregulation of Bcl-2. In N2a cells, morphine induced apoptosis in a dose-dependent manner. Morphine also increased neuronal eIF4E expression in both mPFC tissue and N2a cells, accompanied by upregulation of BDNF and TrkB. Inhibition of the eIF4E/eIF4G interaction with 4EGI-1 significantly affected morphine-induced CPP formation and altered apoptosis-related markers and BDNF/TrkB expression. Notably, intra-mPFC administration of 4EGI-1 suppressed morphine-induced CPP without affecting anxiety-like behavior, locomotor activity, or general learning and memory performance. These findings suggest that eIF4E-dependent translational regulation is functionally associated with morphine-induced reward memory and apoptosis, potentially in association with changes in BDNF/TrkB molecular expression. This study offers novel insight into the molecular basis of morphine addiction and highlights a potentially targetable translational regulatory pathway for therapeutic intervention. Full article

Visceral adipose tissue (VAT) is a metabolically active organ that undergoes structural and functional remodeling under obesogenic conditions. Early-life stress, such as maternal separation (MS), may modulate these processes, but its depot-specific effects remain poorly characterized. This study aimed to determine whether MS modulates VAT remodeling in response to post-weaning high-fat diet (HFD) exposure in male C57BL/6 mice. Animals underwent MS during the early postnatal period (PND2–16) or remained unmanipulated (UM), and were subsequently fed either a control diet (CD) or an HFD for 16 weeks (groups: UM-CD, UM-HFD, MS-CD, MS-HFD). Visceral adipose tissue was collected and analyzed at PND133. Perigonadal (PGAT), retroperitoneal (RPAT), and mesenteric (MSAT) visceral adipose tissue deposits were analyzed by histology, Picrosirius Red staining, and immunohistochemistry for leptin and UCP-1; apoptosis was assessed by TUNEL assay. HFD induced adipocyte hypertrophy and early inflammatory changes, while MS predominantly affected stromal organization. Collagen remodeling was depot-specific: PGAT showed an adaptive pattern, RPAT exhibited a significant MS×HFD interaction, and MSAT was primarily affected by MS regardless of diet. Leptin immunoreactivity increased with HFD in UM animals but was attenuated in MS mice, particularly in MSAT. UCP-1 signal was low and heterogeneous, without clear morphological browning. Apoptosis increased in MSAT under MS-HFD conditions. These findings indicate that early-life stress programs depot-specific VAT remodeling, with MSAT emerging as particularly susceptible to obesogenic challenge. Full article

The incidence of liver injury has been increasing year by year, potentially progressing to liver fibrosis, cirrhosis, and even hepatocellular carcinoma, posing a threat to human health. Gardenia, Astragalus, and Hawthorn are commonly used natural products with dual purposes as both medicine and food. While extensive research has been conducted on their individual pharmacological activities, systematic studies on the synergistic effects and efficacy evaluation of their combined use in liver protection are still lacking. This study aimed to investigate the protective effects and underlying mechanisms of the compound Gardenia and Scutellaria extract (GACE) on CCl4-induced liver injury in mice. The composition and content of GACE were analyzed by HPLC. Serum biochemical, inflammatory, and hepatic oxidative stress indicators were detected using assay kits. Liver pathological changes were examined by HE staining, while apoptosis was observed via TUNEL staining. Transcriptomic and metabolomic sequencing were employed to analyze the potential mechanisms of CCl4 in alleviating liver injury. Finally, Western blotting was performed to validate the analytical results. The results demonstrated that CCl4 significantly upregulated the levels of pro-inflammatory cytokines IL-6, IL-12 and IL-2 (p < 0.05), downregulated the level of anti-inflammatory cytokine IL-10 (p < 0.05), and caused markedly elevated serum ALT and AST levels (p < 0.01). The hepatic lobule structure was nearly obliterated in liver tissue, with disordered arrangement of hepatic cords and numerous vacuoles appearing in hepatocytes. The TUNEL staining positive cell rate increased significantly compared with the blank group (p < 0.001). Meanwhile, CCl4 also significantly inhibited SOD and GSH-Px activities in liver tissue (p < 0.05), while markedly increasing MDA levels (p < 0.05). GACE significantly alleviated liver tissue damage and reduced hepatocyte apoptosis (p < 0.05). It markedly enhanced SOD and GSH-Px activities in liver tissue while decreasing ALT, AST, MDA, IL-6, IL-12, and IL-2 levels (p < 0.05), thereby mitigating hepatic oxidative damage and inflammatory responses. Integrated transcriptomic and metabolomic analyses revealed that the PI3K-Akt and MAPK signaling pathways play crucial roles in mediating GACE’s therapeutic effects against liver injury. Further validation demonstrated that GACE can attenuate CCl4-induced liver injury by inhibiting the PI3K-Akt and MAPK pathways. Full article

Premature ovarian insufficiency (POI) poses a serious risk to the reproductive health and psychological well-being of women. Here, the protective effects of hydroxytyrosol (HT), the primary phenolic component of olive oil, on POI were investigated. In vitro, human ovarian granulosa-like tumour cell lines (KGN cells) were challenged by D-galactose (D-gal) with or without HT. HT administration effectively alleviated KGN cell damage, decreased the number of senescence-associated β-galactosidase (SA-β-gal)-positive cells, increased superoxide dismutase (SOD) activity, reduced reactive oxygen species (ROS) and malondialdehyde (MDA) levels, enhanced the expression level of Bcl-2, inhibited the expression level of Bax, and inhibited cell apoptosis in D-gal-treated KGN cells. In vivo, HT administration reversed the decreased ovarian index, oestrous cycle disruption, and abnormal sex hormone levels observed in D-gal-induced POI mice. HT administration increased glutathione (GSH) levels, reduced the MDA levels, and attenuated apoptosis in ovarian tissues, as evidenced by a decreased number of TUNEL-positive cells, upregulated Bcl-2 expression, and downregulated Bax expression. Mechanistically, HT downregulated the expression level of Kelch-like ECH-associated protein 1 (Keap1) and enhanced the expression levels of heme oxygenase-1 (HO-1) and nuclear factor erythroid 2-related factor 2 (Nrf2) in vitro and in vivo. In conclusion, HT ameliorates D-gal-induced POI in vitro and in vivo by activating the Nrf2/HO-1 signalling pathway. Full article

Esophageal squamous cell carcinoma (ESCC) is an aggressive malignancy with a poor prognosis, largely due to therapeutic resistance and the limited availability of effective targeted therapies. Aloperine (ALO), a natural alkaloid derived from Sophora alopecuroides L., exhibits anti-cancer properties in various tumor types; however, its therapeutic potential and underlying mechanism in ESCC remain unclear. Here, we report that ALO inhibited ESCC cell proliferation and colony formation in a dose- and time-dependent manner and induced caspase-dependent apoptosis, accompanied by loss of mitochondrial membrane potential and PARP1 cleavage. Mechanistically, ALO significantly suppressed inflammatory pathways, with IL-6 identified as a critical downregulated target. ALO inhibited IL-6 production by targeting the AP-1 transcription factor complex, as evidenced by reduced cFOS expression and suppressed cJUN phosphorylation. Consequently, ALO inhibited downstream IL-6/JAK-STAT3 signaling. Functionally, exogenous IL-6 rescued ALO-induced loss of cell viability. Notably, the combination of ALO with cisplatin exerted synergistic antitumor effects. In a syngeneic mice model, the combination therapy significantly reduced tumor growth and Ki67 expression while inducing apoptosis, as shown by increased TUNEL staining and cleaved caspase-3 expression, and further suppressing the IL-6/STAT3 axis compared with either monotherapy. Together, these findings demonstrate that ALO exerts potent anti-ESCC activity by targeting the AP-1/IL-6/STAT3 signaling axis. The synergistic efficacy of ALO with cisplatin highlights its potential as a promising therapeutic agent to overcome chemoresistance and improve outcomes in ESCC patients. Full article

Due to the complex pathophysiology and serious outcomes of autoimmune myocarditis, we sought to determine whether ethanolic lemon balm extract (LBE) could attenuate disease progression and development of dilative cardiomyopathy (DCM). EAM was induced in Dark Agouti rats by immunization with porcine myosin. Fifty animals were allocated to five groups: healthy controls, untreated EAM, and EAM treated with LBE (50, 100, or 200 mg/kg) for six weeks. Hemodynamic parameters were monitored, and echocardiography assessed cardiac structure and function. Inflammatory, oxidative, fibrotic, and apoptotic markers were analyzed. Immunological profiling revealed that LBE significantly decreased proinflammatory cytokines (IL-1, IL-6, TNF-α, IL-4, IL-17) while restoring anti-inflammatory IL-10 levels (p < 0.05). Antioxidant activity was confirmed by reduced levels of O₂⁻, H₂O₂, and TBARS, accompanied by significant increases in SOD, CAT, and GSH activity (p < 0.05), and upregulation of SOD1 and SOD2 gene expression. Additionally, LBE (200 mg/kg) markedly reversed fibrotic remodeling through suppression of TGF-β expression and collagen deposition, as shown by Sirius Red staining, and mitigated apoptosis by modulating Bax/Bcl-2 balance and reducing TUNEL-positive cells. Collectively, these findings suggest that LBE exerts strong cardioprotective effects in EAM by regulating inflammatory, oxidative, fibrotic, and apoptotic pathways, thereby preventing myocarditis progression toward DCM. Full article

(1) Background: Oxidative stress (OS) has been extensively associated with male infertility, contributing to its pathophysiology in approximately 30–80% of affected individuals. Excessive reactive oxygen species (ROS) levels trigger a cascade of interconnected processes, including lipid peroxidation, DNA damage, and mitochondrial dysfunction, ultimately impairing sperm quality. (2) Methods: The present study was designed to examine the relationship between seminal oxidative stress biomarkers such as superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA) and sperm DNA integrity in men with unexplained (UMI) and idiopathic (IMI) male infertility. The study population comprised 235 participants, who were divided into three groups: fertile controls (n = 78), UMI (n = 88), and IMI (n = 69). Semen analysis was performed according to WHO criteria. Sperm DNA fragmentation index (DFI) was assessed using the TUNEL assay, while sperm decondensation index (SDI) was evaluated by aniline blue staining. Seminal OS biomarkers were measured in seminal plasma using spectrophotometric methods. (3) Results: DFI and SDI were significantly increased in infertile groups compared with fertile controls (p < 0.001), exceeding the clinical thresholds of 30% and 15%, respectively. Antioxidant enzyme activities (SOD and CAT) were significantly reduced, whereas MDA levels were significantly elevated, particularly in the IMI group (p < 0.001). Correlation analysis revealed strong positive associations between MDA and both DFI (r = 0.76, p < 0.001) and SDI (r = 0.80, p < 0.001). Additionally, MDA, DFI, and SDI were negatively correlated with semen parameters, whereas SOD and CAT were positively correlated with sperm quality (p < 0.001). (4) Conclusions: Our findings emphasize the critical role of oxidative stress and sperm DNA integrity as potential biomarkers for the diagnosis and evaluation of UMI and IMI. Full article

Haploid embryos constitute a valuable model for genetic and epigenetic studies; however, their developmental competence is reduced compared with diploid counterparts. This study evaluated whether supplementation of the culture medium with specific small molecules could improve developmental competence and outgrowth establishment of parthenogenetic haploid embryos. The effects of TGF-β inhibition (A83-01), WNT pathway modulation (CHIR99021 and IWR-1), and activin A (AA) supplementation were assessed from the morula stage onward under serum-free conditions. A83-01 treatment did not improve blastocyst formation or morphology and was associated with reduced total cell numbers relative to IVF controls. CHIR99021 supplementation increased the number of SOX2-positive cells compared with IWR-1 and vehicle-treated embryos, suggesting partial support of pluripotency; however, overall developmental progression remained inferior to diploid controls. In contrast, activin A significantly increased the proportion of haploid morulae developing into blastocysts and improved hatching rates. Nevertheless, AA supplementation did not restore CDX2-positive cell numbers or total cell counts to diploid levels. Furthermore, neither CHIR99021 nor AA affect DNA fragmentation levels, although a tendency toward increased TUNEL-positive cells was observed. Activin A treatment also failed to improve embryonic outgrowth formation. Collectively, these findings demonstrate that although activin A enhances blastocyst yield and hatching in bovine haploid embryos, modulation of TGF-β or WNT signaling alone is insufficient to restore diploid-like proliferative developmental competence. Full article

Dry eye disease (DED) is a complex ocular surface disorder characterized by tear film instability, chronic inflammation, and epithelial damage, for which current treatments remain limited. Marine-derived bioactive oligosaccharides have attracted increasing interest due to their diverse pharmacological activities and favorable safety profiles. In this study, we investigated the therapeutic potential of neoagarotetraose (NA4), a marine oligosaccharide derived from red algal agar, in a murine model of DED. DED was induced in eight-week-old female C57BL/6 mice by topical instillation of 0.2% benzalkonium chloride for seven consecutive days. NA4 was administered topically at concentrations of 125, 250, and 500 mg/L. Therapeutic outcomes were evaluated by tear secretion, corneal fluorescein staining, histopathological analysis, immunofluorescence staining for Ki67, F4/80, IL-1β, IL-6, and TNF-α, TUNEL assay for apoptosis, and ELISA for cytokine levels. NA4 treatment significantly improved tear secretion and reduced corneal fluorescein staining scores. Histological analysis revealed that NA4 preserved corneal epithelial thickness and restored conjunctival goblet cell density. Immunofluorescence analysis revealed that NA4 reversed inflammation-associated epithelial hyperproliferation and attenuated macrophage infiltration. Moreover, NA4 markedly suppressed the expression and tissue levels of IL-1β, IL-6, and TNF-α, and attenuated corneal epithelial apoptosis, with the 500 mg/L NA4 group showing no significant difference in efficacy compared to the positive control 0.1% sodium hyaluronate. These findings demonstrate that NA4, a marine-derived oligosaccharide, exerts multi-targeted protective effects against DED by improving tear film stability, preserving ocular surface integrity, suppressing inflammation, and reducing apoptosis. Our study highlights the potential of marine oligosaccharides such as NA4 as promising candidates for ocular surface disease management and supports the further exploration of marine resources for ophthalmic therapeutic applications. Full article

Background and Objectives: Green tea is known for its powerful antioxidant properties. However, the effects of green tea consumption during pregnancy on neonatal development and the mechanisms of these effects are not fully understood. The aim of this study was to investigate potential damage to atrial cardiomyocytes of newborn rat pups whose mothers received green tea during pregnancy and to elucidate the apoptotic mechanisms underlying this possible damage. Materials and Methods: Wistar albino rats (weighing 200–220 g, 10 weeks old) were used in this study. Following the confirmation of pregnancy, rats were randomly assigned to groups, and the experimental group was administered green tea by oral gavage at a dose of 50 mg/kg per day for 21 days. Atrial cardiomyocytes and mitral valve cells from newborn pups (postnatal day 1) were obtained and evaluated immunohistochemically for cytochrome c, caspase-9, and caspase-3 expression. Results: TUNEL analysis revealed a significant increase in DNA fragmentation in the green tea group, with the median number of apoptotic cells per region of interest (ROI) rising from 5.5 to 24.5 in atrial cardiomyocytes (p < 0.001), and from 2.0 to 10.0 in mitral valve cells (p < 0.05). Immunohistochemically, the control group showed faint-to-weak basal immunoreactivity of cytochrome c and caspase-3, and weak-to-moderate expression of caspase-9. In the green tea group, caspase-3 immunoreactivity was moderate, while cytochrome c and caspase-9 immunoreactivity were significantly higher. Quantitative HSCORE analysis confirmed significant elevations in atrial cardiomyocytes for cytochrome c (from 65.0 to 210.0; p < 0.001), caspase-9 (from 85.0 to 140.0; p < 0.001), and caspase-3 (from 60.0 to 120.5; p < 0.001). Similar statistically significant increases were observed across all corresponding markers in the mitral valve cells (p < 0.05). Overall, the induction of apoptosis was notably more pronounced in atrial cardiomyocytes than in mitral valve cells. Conclusions: Our findings suggest that the mechanism of potential damage in atrial cardiomyocytes of newborn rat pups is associated with mitochondria-mediated apoptosis, potentially triggered by activation of the cytochrome c, caspase-9 and caspase-3 axis. These results highlight the importance of exercising caution regarding the consumption of green tea supplements during pregnancy. Further studies are needed to correlate these preliminary neonatal observations with clinical outcomes. Full article

Background/Objectives: Aberrant DNA methylation of imprinted genes and increased sperm DNA fragmentation (SDF) have been implicated in male infertility. However, their impact on assisted reproductive technology (ART) outcomes remains unclear. This pilot study aimed to investigate SDF and methylation status of H19, IGF2, and PEG1/MEST in relation to fertilisation and embryo development following intracytoplasmic sperm injection (ICSI). Methods: Twenty male partners of women undergoing ICSI were recruited and classified according to ART outcome into viable embryos (VEs, n = 7), non-viable embryos (NVEs, n = 7), and no fertilisation (NF, n = 6). Before sperm selection, an aliquot of each seminal sample was used for semen analysis according to WHO, 2021, SDF assessment (TUNEL assay), and sperm DNA methylation analysis of H19, IGF2, and PEG1/MEST (pyrosequencing). Results: Semen parameters were above the fifth percentile. SDF was significantly lower in the VE group compared with the other groups. H19 CpG1 methylation correlated positively with viable embryos (p = 0.016), while H19 CpG2 island showed a positive correlation with sperm concentration (p = 0.028). In male/couple infertility cases, total H19 methylation correlated negatively with SDF (p = 0.050). IGF2 CpG3 island methylation correlated positively with viable embryos (p = 0.027). Total PEG1/MEST methylation was positively correlated with fertilisation events (p = 0.002) and viable embryos (p = 0.011). PEG1/MEST CpG2 island also positively correlated with sperm motility (p = 0.034), while CpG3 and CpG4 showed significant correlations with fertilisation (p < 0.001; p = 0.004). Conclusions: This pilot study shows that SDF and sperm methylation levels of H19, IGF2, and PEG1/MEST are related to ICSI outcomes, supporting that sperm molecular and epigenetic features may influence fertilisation and embryo development. Full article

Background: Oral squamous cell carcinoma (OSCC) remains a clinically challenging malignancy characterized by aggressive behavior and limited therapeutic options. Bitter taste receptors (TAS2Rs), expressed across multiple tissues and cancer types, have recently emerged as regulators of tumor biology and immune responses; however, their functional significance in OSCC remains poorly understood. Methods: Immunohistochemical analysis was performed using surgically resected human tongue OSCC specimens and a tissue microarray (TMA) cohort. In parallel, four TAS2R agonists were evaluated in SCC7 cells to assess intracellular calcium responses. RNA sequencing was conducted to analyze transcriptional changes following diphenidol treatment, and functional assays, including proliferation, migration, and apoptosis analyses, were performed in vitro. Antitumor effects were further evaluated in a syngeneic SCC7 mouse model, followed by TUNEL staining and flow cytometry to assess apoptosis and immune cell infiltration. Results: TAS2R38 expression was markedly upregulated in dysplastic and invasive OSCC lesions with predominant nuclear localization and was associated with histological grade and clinical stage, indicating an early and sustained alteration during tumor progression. Among the agonists tested, diphenidol most strongly induced IP₃-dependent intracellular Ca²⁺ elevation. RNA sequencing revealed upregulation of Il1rl1 and Lzts2. Functionally, diphenidol significantly suppressed SCC7 cell proliferation and migration and induced apoptosis in vitro. In vivo, diphenidol reduced tumor volume and weight and increased apoptotic activity. Flow cytometry demonstrated a marked reduction in tumor-infiltrating CD4⁺CD25⁺Foxp3⁺ regulatory T cells, indicating modulation of the tumor immune microenvironment. Conclusions: TAS2R activation by diphenidol suppresses tumor growth through both tumor-intrinsic mechanisms and modulation of the tumor immune microenvironment in OSCC. These findings define TAS2R-mediated calcium signaling as a novel axis linking tumor progression and immunoregulation. Given that diphenidol is a clinically approved drug with an established safety profile, our results provide a strong rationale for TAS2R-targeted drug repurposing strategies in cancer therapy. Full article