BACKGROUND:Atherosclerosis, driven by macrophage foam cell formation and lipid accumulation, remains inadequately addressed by current pharmacotherapies, necessitating novel natural therapeutic strategies. Nimbolide (NIM), a limonoid derived from Azadirachta indica, possesses established anti-inflammatory properties, yet its efficacy against atherosclerosis and underlying mechanisms remain elusive. PURPOSE:The study was designed to assess the impact of NIM on treating atherosclerosis, focusing on cholesterol efflux and lipophagy. STUDY DESIGN:The study combined both in vivo atherosclerotic mouse modeling and in vitro macrophage foam cell studies. METHODS:Apolipoprotein E-deficient (ApoE-/-) mice fed a high-fat diet received NIM to assess therapeutic effect. Oxidized low-density lipoprotein was used to induce macrophage foam cell formation, and effects of NIM on cholesterol efflux and lipophagy were assessed by western blot, immunofluorescence and electron microscopy. Cellular thermal shift assay (CETSA), surface plasmon resonance (SPR), and co-immunoprecipitation (Co-IP) were employed to investigate molecular interactions. A TFEB-knockdown mouse model was used to evaluate the mediating role of transcription factor EB (TFEB). RESULTS:NIM administration dose-dependently attenuated aortic plaque burden, improved serum lipid profiles, and reduced vascular inflammation in ApoE-/- mice. In macrophages, NIM enhanced cholesterol efflux via upregulation of ATP-binding cassette transporter A1/G1 and activated lipophagy through TFEB nuclear translocation. Mechanistically, NIM directly bound 14-3-3 epsilon and competitively displaced TFEB, thereby promoting TFEB dephosphorylation and autophagy-lysosomal activation. TFEB knockdown abolished NIM-mediated lipophagy and atheroprotection both in vitro and in vivo. CONCLUSION:NIM exerts anti-atherosclerotic effects by disrupting the 14-3-3 epsilon/TFEB interaction to enhance macrophage cholesterol clearance and lipophagy, identifying this axis as a promising therapeutic target for atherosclerosis.
Dysregulated arginine metabolism promotes hepatic stellate cell (HSC) activation and liver fibrosis. However, the mechanisms underlying arginine hosmeostasis in hepatic fibrogenesis and therapeutic targeting remain unclear. Using Multicellular hepatic spheroid (MCHS) models recapitulating in vivo liver fibrosis, we investigated the effects of arginine deprivation or supplementation. Genetic and pharmacological (ABRL8301, a 2-methoxyestradiol derivative) inhibition of hypoxia-inducible factor 1 alpha (HIF-1α) were assessed for effects on argininosuccinate synthetase 1 (ASS1) expression and antifibrotic efficacy in vitro and in vivo. Holotomography and quantitative imaging approaches characterized cellular and molecular fibrogenic changes.Arginine deprivation increased MCHS compactness and α-smooth muscle actin (α-SMA) expression, whereas L-arginine supplementation reversed effects. Considering pivotal roles of ASS1 and arginase 1 (Arg1) in arginine biosynthesis and catabolism, respectively, ASS1 overexpression suppressed mesenchymal markers, whereas Arg1 overexpression aggravated fibrotic signatures. HIF-1α knockdown and ABRL8301 treatment upregulated ASS1, reduced epithelial-mesenchymal transition (EMT) and fibrotic markers, and enhanced arginine biosynthesis in MCHSs. Holotomography analysis confirmed ABRL8301 attenuated TGFβ1-induced cell elongation and α-SMA fiber accumulation in HSCs and fibroblasts. In CCl₄-mouse liver fibrosis models, ABRL8301 significantly reduced collagen deposition, necrosis, and fibrosis. Combination sorafenib and ABRL8301 therapy decreased MCHS compactness, increased apoptosis, and achieved superior tumor control in HCC xenografts.Restoring arginine metabolic homeostasis through HIF-1α inhibition-mediated ASS1 upregulation suppresses HSC activation and liver fibrosis. ABRL8301 acts as a pharmacologic inducer of ASS1 and arginine biosynthesis, inhibiting EMT and fibrotic progression while enhancing anticancer therapy. These findings support ABRL8301 as a promising antifibrotic and tumor-sensitizing agent in hepatic fibrosis.
BACKGROUND:Atherosclerosis (AS) and depression are major global health burdens with high comorbidity, yet effective treatments are limited. While ginsenoside Rb1 (GRb1) has shown promise in alleviating both conditions individually and in comorbidity, its precise mechanisms of action remain unclear. PURPOSE:This study investigated whether GRb1 alleviates AS co-depression by targeting LPCAT3 and modulating the ACSL4/LPCAT3/ALOX15 ferroptosis pathway. METHODS:In vivo, an AS co-depression model was established in ApoE-/- mice through a combination of high-fat diet feeding and restraint stress. Following intragastric administration of GRb1, adeno-associated virus (AAV)-mediated knockdown of LPCAT3 was employed to validate its target. The anti-atherosclerotic efficacy was assessed by measuring serum lipid profiles, plasma inflammatory cytokines, and evaluating pathological tissue sections. Antidepressant-like effects were evaluated through behavioral tests. Western blotting, molecular docking, and transmission electron microscopy (TEM) were utilized to investigate the anti-ferroptotic effects and associated molecular signaling pathways. In vitro, primary mouse hippocampal neurons were stimulated with lipopolysaccharide (LPS) to mimic in vivo stress. Lentivirus-mediated LPCAT3 knockdown combined with GRb1 treatment was applied, allowing for parallel evaluation of pharmacological efficacy and validation of the underlying mechanisms. RESULTS:GRb1 ameliorated AS co-depression in vivo, downregulating the expression of ACSL4, LPCAT3, and ALOX15 proteins, while promoting the expression of SLC7A11 and GPX4, thereby enhancing anti-oxidant capacity and inhibiting ferroptosis. Hippocampal LPCAT3 knockdown normalized the expression of the above pathway proteins, alleviated oxidative stress, and suppressed ferroptosis. Additionally, in vitro experiments demonstrated that GRb1 exerted cytoprotective effects, including anti‑apoptotic and anti‑ferroptotic activities. Following LPCAT3 knockdown, the expression of ferroptosis‑related proteins was more effectively restored, anti-oxidant capacity was enhanced, and mitochondrial morphology was improved. CONCLUSION:Ferroptosis represents a central mechanism underlying the comorbidity of AS and depression. GRb1 exerts its therapeutic effects by targeting LPCAT3 and modulating the ACSL4/LPCAT3/ALOX15 ferroptosis pathway to attenuate inflammation, oxidative stress, and mitochondrial dysfunction. This study identifies LPCAT3 as a specific target of GRb1 and establishes ferroptosis inhibition as a promising therapeutic strategy for AS co-depression.
Background Although the use of Traditional, Complementary, and Integrative Medicine (TCIM) interventions to treat coronavirus disease 2019 (COVID-19) is common, the effectiveness of these interventions is unclear. Objective To compare different TCIM interventions for the treatment of mild/moderate acute COVID-19. Data sources EMBASE (Elsevier), Medline (OVID), VHL TCIM database, Cochrane Central, ClinicalTrials.gov, ICTRP, as of April 29th, 2025, as well as eight Chinese and four Korean databases. Study selection Randomized controlled trials comparing a TCIM intervention with another, the standard of care (SoC), placebo, or conventional medicine in participants with suspected, probable, or confirmed mild/moderate acute COVID-19. Data Extraction Reviewers worked in pairs and independently extracted data and assessed the risk of bias and rated the certainty of the evidence for each outcome. Results One hundred fifty-five trials evaluating 142 TCIM therapies were eligible for review, of which 69 (45%) trials enrolling 18,627 patients met criteria for analysis, contributing at least 80 participants or 20 events to one intervention. All the following estimates come from direct evidence, as networks were sparse. Compared with SoC or placebo, high to moderate certainty evidence showed that three TCIM add-on interventions probably reduced time to symptom resolution: Lianhua Qingwen (mean difference [MD] -2.82 days, 95% confidence interval -3.14 to -2.50, high certainty); Kovir capsule (MD -3.14 days, -3.67 to -2.61, moderate certainty); and honey and Nigella sativa (MD -3.00 days, -4.12 to -1.88, moderate certainty). Using conversion to severe cases as a proxy for hospitalization, Andrographis paniculata extract probably reduced hospital admission (risk difference -15 per 1000, -24.9 to 12.3, moderate certainty) for patients at moderate risk of hospitalization. No compelling evidence supports that any TCIM intervention reduced hospital admissions for patients at low or high risk of hospitalization, or reduced mortality and mechanical ventilation requirement across all patients. High to moderate certainty evidence showed that Lianhua Qingwen, Kovir capsule, COROPROTECT kit, and Shen Cao Gan Jiang likely did not increase adverse events leading to drug discontinuation; evidence for other TCIM interventions was insufficient to inform adverse events leading to discontinuation. High to moderate certainty evidence supported that Lianhua Qingwen was effective in resolving cough (MD -1.62 days, -1.80 to -1.45, high certainty) and probably effective in resolving fatigue (MD -1.41 days, -1.73 to -1.08, moderate certainty), honey and Nigella sativa was probably effective in resolving fever (MD -4.00 days, -4.72 to -3.28, moderate certainty), and Jinghua Jiedu was probably effective in resolving cough (MD -1.72 days, -2.23 to -1.21, moderate certainty). Other TCIM interventions were either ineffective, or of uncertain effectiveness. Conclusion Adding Lianhua Qingwen, Kovir capsule, or honey and Nigella sativa to SoC likely reduces symptomatic duration of mild/moderate acute COVID-19. Andrographis paniculata extract probably decreases hospital admissions among patients at moderate risk of hospitalization. Replication of positive findings is warranted. Registration PROSPERO (CRD42024517321)
Background SARS-CoV-2 continues to exert a profound impact on global health by inducing inflammatory and metabolic dysregulation. Purpose Our study investigates the therapeutic potential of polyherbal formulation, Ayush-64, in mitigating SARS-CoV-2 Spike-induced immunometabolic modulations. The COVID-19 illness has long-lasting effects in terms of multi-organ effects, particularly in the nervous system. Study design With post-COVID complications, we assess the efficiency of this phytocompound at transcript and protein levels by utilizing in vitro transfection models with wildtype and delta mutants of Spike protein in lung (A549) and neural (IMR-32+LN-229) co-culture systems. Methods To identify specific metabolic and structural modulations, we also made assessments through LC-MS and Raman spectroscopy. Results Our findings demonstrate that Ayush-64 restores ATP production, enhances fatty acid oxidation, and restores cofactors’ levels such as biotin and pyridoxal phosphate (PLP), all of which are hijacked upon viral infections. Notably, the Delta variant induces greater inflammation, yet Ayush-64 effectively restores such immunomodulations. Key phytochemicals, such as betulin, sweroside, picroside, and amarogentin, contribute to the stabilization of lipid membranes and the reduction in inflammatory signaling with Ayush-64 exhibiting targeted metabolic recovery in both lung and neural microenvironments. Conclusion Therefore, our results underscore Ayush-64’s role in re-establishing immunometabolic balance and enhancing neural resilience, highlighting utilization of formulation against SARS-CoV-2 viral infection.
Background Breast cancer is the most common malignancy in women. Triple-negative breast cancer (TNBC), due to a lack of effective targets, has a particularly poor prognosis. The efficacy of paclitaxel, a core chemotherapeutic drug, is often limited by multidrug resistance in tumor cells. Flavonoids, natural polyphenols with multi-pathway intervention properties, show significant potential for reversing this resistance, yet their clinical use is hindered by low bioavailability. Thus, understanding how Flavonoids overcome resistance and combining them with advanced delivery systems are key to developing new TNBC therapies. Purpose This review systematically outlines the multi-dimensional mechanisms of paclitaxel resistance in TNBC and elucidates how Flavonoids reverse this resistance by regulating ferroptosis, key signaling pathways, epigenetic programs, and the immune microenvironment. It further discusses advanced delivery systems to facilitate clinical translation, providing a theoretical basis for developing effective, low-toxicity chemosensitization strategies against TNBC. Methods We systematically reviewed PubMed (PubMed), Web of Science (https://www.webofscience.com/), and ScienceDirect (http://www.sciencedirect.com/) databases, spanning relevant studies from 2005 to 2026. Article selection was performed in strict accordance with the PRISMA guidelines. Furthermore, manual screening and citation tracking were implemented based on pre-defined inclusion and exclusion criteria to ensure the accuracy and comprehensiveness of the study. Results TNBC resistance to paclitaxel is driven by ferroptosis evasion, pro-survival signaling, epigenetic reprogramming, and a “cold” tumor microenvironment. Flavonoids counteract this by inducing ferroptosis via the glutathione-glutathione peroxidase 4 (GSH-GPX4)/Kelch-like ECH-associated protein 1 (KEAP1)–nuclear factor erythroid 2-related factor 2 (NRF2) axis inhibiting signaling networks, remodeling epigenetics, and immunomodulating the TME, thereby synergizing with paclitaxel. Additionally, advanced delivery systems, including prodrug self-assembly, metal–organic framework (MOF)-based carriers, and biomimetic targeting, significantly improve the bioavailability, targeting, and safety of the treatment. Conclusion Complex TNBC paclitaxel resistance necessitates multi-target approaches. Flavonoids systematically dismantle resistance barriers and synergize with paclitaxel, while advanced delivery systems overcome pharmacokinetic limitations. Integrating these strategies offers a promising therapeutic solution, though further in vivo and clinical validation is required to confirm efficacy and safety.
Solanum nigrum L. is a widely distributed ethnomedicinal plant with significant antitumor potential, primarily attributed to its abundant steroidal saponins and steroidal glycoalkaloids. However, compositional variability and safety concerns have historically hindered its clinical translation. In this study, we developed a standardized fraction of S. nigrum berries specifically enriched with steroidal saponins and glycoalkaloids (S-SNB), and systematically evaluated its chemical composition, therapeutic potential against non-small cell lung cancer (NSCLC), underly mechanisms, and safety profile. Compared with the conventional aqueous extract, S-SNB exhibited markedly enhanced tumor suppression in A549 and PC-9 xenograft models at substantially lower crude herb equivalent doses. Phytochemical characterization and bioactivity-guided analyses revealed solasonine, solamargine, macrostemonoside B, and solanigroside J as core bioactive constituents, with the latter reported here for their anti-NSCLC activity for the first time. From a mechanistic perspective, proteome-wide target profiling via limited proteolysis–mass spectrometry (LiP-MS) identified glycolytic pathway as a primary node of S-SNB, which was validated by functional metabolic assays showing suppressed glycolytic flux both in vitro and in vivo. Integrated biophysical, biochemical, and computational analyses further converged on PKM2 as a central target of the major active constituents, revealing a non-classical mode of regulation involving coordinated multi-component interactions rather than direct catalytic inhibition. Collectively, these findings demonstrate that enrichment of steroidal saponins and glycoalkaloids enhances both pharmacological potency and mechanistic clarity of S. nigrum against NSCLC, and establishes S-SNB as a chemically defined, safety-validated, and mechanism-informed botanical preparation, providing a robust scientific foundation for its development as an adjunctive oncology therapy.
Background Diabetic kidney disease (DKD) has become a leading cause of end-stage renal disease globally. Metabolic dysregulation is a core characteristic of DKD, with metabolites acting as both disease biomarkers and drivers of pathological progression. Thus, exploring new therapeutic strategies centered on metabolism holds promise for the treatment of DKD. Purpose This study aimed to characterize spatially resolved metabolic reprogramming in DKD, elucidate the mechanisms of uridine-mediated renoprotection, and identify therapeutic compounds targeting uridine metabolism. Methods Clinical metabolomics of 228 participants (62 healthy individuals and 166 DKD patients) and desorption electrospray ionization mass spectrometry imaging (DESI-MSI) were integrated to profile metabolic alterations in DKD. DKD mouse models were used to evaluate the effects of uridine supplementation. Multi-omics analysis (transcriptomics, proteomics, and subcellular mapping) deciphered molecular mechanisms. High-throughput screening of 1880 natural products was performed to identify modulators of enzymes involved in uridine metabolism. Results DESI-MSI revealed pronounced disruption of pyrimidine metabolism characterized by a marked reduction in renal cortical uridine levels in DKD. Uridine supplementation significantly ameliorated glucolipid metabolic disturbances and renal injury in DKD mice. We identified a dual-synergistic mechanism by which uridine exerts renoprotective effects: restoring mitochondrial bioenergetics while inhibiting the EGR1-P300-ETS1 signaling axis. High-throughput screening identified schisandrin B (SchB) as a potential modulator of uridine metabolism, with binding free energies of -8.5 and -8.2 kcal/mol to DHODH and UMPS, respectively. SchB increased uridine levels and ameliorated DKD pathology via the same mechanism. Conclusion The findings highlight uridine as a key metabolic regulator in DKD and offer a novel precision treatment strategy targeting metabolic modulation, demonstrating promising potential for the treatment of DKD.
Background Ulcerative colitis (UC) is a type of inflammatory bowel disease. Macrophage polarization is crucial in the development of UC. Oxypalmatine (OPAL) is an isoquinoline alkaloid that can be isolated from various plant, including Phellodendron amurense and Sinomenium acutum. It is also an oxidative metabolite derived from the hepatic biotransformation of palmatine (PAL), which is one of the active constituents of Coptis chinensis that demonstrates anti-inflammatory and antioxidant properties. Purpose This study aimed to explore the anti-inflammatory properties of OPAL and the mechanisms involved. Methods Efficacy of OPAL and its impact on macrophage polarization were assessed in a dextran sodium sulfate (DSS)-induced mouse model of colitis. Necessity of macrophage in the alleviation by OPAL was confirmed with a macrophage-depletion model. Transcriptomics analysis was performed on the colon to identify the corresponding signaling pathways. In an in vitro model of THP-1-derived macrophage, the effect of OPAL was assessed in terms of cell energy metabolism, cytokines production, and epithelium damage. Target of OPAL was explored by limited proteolysis-mass spectrometry (LiP-MS) analysis and siRNA-mediated knockdown. Results OPAL significantly ameliorated DSS-induced colitis by reducing colon shortening, weight loss and intestinal barrier damage, which relied on the inhibition on M1 polarization as confirmed by flowcytometry and macrophage-depletion. In vitro, OPAL suppressed the inflammatory response in THP-1-derived macrophages, and reprogrammed the energy metabolism profile by reducing the glycolysis and enhancing basal respiration and maximal respiration. Mechanistically, OPAL inhibited M1 polarization via suppressing the JAK-STAT signaling pathway, which mainly relied on binding to RAB8A protein to suppress STAT1 phosphorylation and the consequent HK2 expression. Conclusion OPAL ameliorated UC by suppressing M1 macrophage polarization via the RAB8A/STAT1 axis, highlighting a promising treatment strategy for UC management.
Background Ultraviolet (UV) radiation causes skin photoaging by inducing reactive oxygen species (ROS) accumulation, extracellular matrix remodeling, and lipid metabolic dysregulation, which may progress to actinic keratosis and invasive squamous cell carcinoma. Plant-derived exosome-like nanoparticles (PDNPs) are promising anti-aging strategies, but current studies mainly focus on PDNPs-associated miRNAs, whereas the roles and targets of enriched secondary metabolites remain unclear. This study aimed to elucidate the key metabolite and mechanism underlying the anti-photoaging activity of aloe rind-derived nanoparticles (rADNPs). Methods Aloe gel-derived nanoparticles (gADNPs) were used as controls to evaluate rADNPs in UV-induced photoaging models. Widely targeted plant metabolomics and transcriptomics were integrated to characterize the metabolic fingerprint of rADNPs and identify metabolite-target interactions. Molecular docking, molecular dynamics simulations, gene knockdown, and targeted fatty acid metabolomics were further performed to validate the key regulatory axis. Luteolin-7-O-rutinoside-engineered rADNPs (L7R-rADNPs) were constructed and assessed in dermal fibroblasts and HaCaT cells. Results rADNPs showed stronger anti-photoaging effects than gADNPs. Integrated analyses identified luteolin-7-O-rutinoside (L7R) as a key enriched metabolite in rADNPs. Mechanistic analyses showed that L7R targeted fatty acid desaturase 1 (Fads1), restoring UV-induced polyunsaturated fatty acid (PUFAs) depletion, rebalancing fatty acid metabolism, and enhancing endogenous antioxidant defenses. L7R-rADNPs further enhanced protection against UV-induced oxidative stress and photoaging phenotypes, with consistent validation in HaCaT cells. Conclusion rADNPs attenuate skin photoaging by delivering L7R, which restores Fads1-mediated fatty acid metabolism and strengthens antioxidant defense. These findings reveal a metabolite-driven mechanism underlying rADNPs anti-photoaging activity and provide new insights into studies of PDNPs for disease intervention.
Background The occurrence of muscle atrophy in chronic kidney disease (CKD) is a prevalent complication with serious consequences but lacks effective treatment. Modulating the gut microbiota offers a promising new therapeutic approach. Shenshuai Yingyang Jiaonang (SSYYJN) is a clinically validated prescription of traditional Chinese medicine for muscle atrophy in CKD, yet the molecular basis for its therapeutic action requires elucidation. Purpose To evaluate the therapeutic efficacy of SSYYJN against CKD-induced muscle atrophy, investigate the mechanism from the perspective of the gut microbiota, and explore potential strategies for enhancing the treatment efficacy of SSYYJN. Methods A rat model of CKD with concomitant muscle atrophy was established by 5/6 nephrectomy. 16S rDNA sequencing and fecal microbiota transplantation (FMT) experiments were conducted to elucidate the gut microbiota’s role in SSYYJN efficacy. Untargeted metabolomics profiling and the pharmacological network analysis were conducted to investigate the potential mechanism of Faecalibacterium prausnitzii (FP) probiotics on SSYYJN. The regulatory mechanism of SSYYJN in CKD-associated muscle atrophy was validated through in vitro C2C12 cell experiments. Results In patients with CKD-associated protein-energy wasting (PEW), effective SSYYJN treatment improved mid-arm muscle circumference, hand grip strength, mid-arm circumference, and serum albumin. Moreover, post-hoc microbiome analysis revealed that the abundance of FP was higher in treatment-responsive patients. In a CKD rat model, SSYYJN conferred protection against renal injury, malnutrition, and muscle atrophy, this therapeutic effect was related to the gut microbiota modulation. Of note, a higher abundance of FP was also observed in SSYYJN-treated CKD rats. Further analyses suggested that FP was associated with increased levels of carnosol and may enhance EGFR/PI3K/AKT signaling, thereby potentiating the therapeutic effect of SSYYJN against CKD-induced muscle atrophy. Conclusion These findings suggest a gut microbiota-dependent mechanism underlying the action of SSYYJN against muscle atrophy in CKD. FP may be linked to the effects of SSYYJN, potentially involving the generation of carnosol and the upregulation of the EGFR/PI3K/AKT pathway, representing a targeted therapeutic strategy.
BACKGROUND:Medicinal plants remain a primary reservoir of specialized metabolites for addressing fungal resistance. Combining plant-derived compounds with conventional antifungal drugs represents a promising strategy to improve antifungal efficacy while reducing fungal virulence. PURPOSE:This study evaluated the antifungal activity, mechanism of action, anti-virulence properties, and in vivo efficacy of a ternary mixture composed of Zuccagnia punctata hexane extract, Gaillardia megapotamica methanol extract, and itraconazole. METHODS:Building on previous optimizations, antifungal activity was assessed by broth microdilution against clinically relevant fungi. Mechanistic studies included ergosterol binding and sorbitol protection assays. Anti-virulence effects were evaluated in Candida albicans by analyzing adhesion, morphogenesis, lytic enzyme secretion, and biofilm formation. In vivo efficacy was investigated using the Caenorhabditis elegans infection model. RESULTS:The ternary mixture exhibited potent antifungal activity against a broad spectrum of fungi, including Candida, Cryptococcus, Aspergillus, and dermatophytes, outperforming its individual components. Mechanistic assays suggested that antifungal activity was mainly associated with the G. megapotamica extract through interaction with ergosterol in the fungal membrane without affecting the cell wall. The mixture also significantly inhibited key virulence traits of C. albicans and increased the survival of infected C. elegans. CONCLUSIONS:These findings demonstrate that combining plant-derived extracts with itraconazole represents a promising multi-target antifungal strategy. The results support the exploration of ethnopharmacologically relevant plants as sources of synergistic partners capable of enhancing antifungal efficacy while attenuating fungal virulence.
BACKGROUND:Penicillin-binding protein 2a (PBP2a) is a major determinant of β-lactam resistance in methicillin-resistant Staphylococcus aureus (MRSA). Natural PBP2a-binding compounds may serve as antibacterial adjuvants. PURPOSE:To identify PBP2a-binding constituents from Artemisia scoparia and evaluate their anti-MRSA and β-lactam-potentiating activities. STUDY DESIGN:A target-oriented workflow combining virtual screening, SPR-based molecular fishing, kinetic analysis, antibacterial synergy testing, and quantitative proteomics was established. METHODS:Forty-four constituents were virtually screened, and recombinant PBP2a was immobilized on a CM5 sensor chip by EDC/NHS-mediated amine coupling for subsequent SPR-based molecular fishing coupled with LC-MS. Selected standards were analyzed using a 1:1 Langmuir kinetic model and molecular dynamics simulations. Luteolin was further evaluated by lysate-based thermal stability analysis, MIC determination, checkerboard assays, and DIA proteomics. RESULTS:Thirty-three PBP2a-binding constituents were identified. Luteolin, chlorogenic acid, cynarin, and rutin showed comparatively stronger binding and formed stable complexes with PBP2a in molecular simulations. Luteolin inhibited MRSA with an MIC of 4 μg/mL and increased PBP2a thermal stability in an MRSA-derived protein environment. At 1 μg/mL, luteolin reduced the MICs of penicillin G and amoxicillin from 512 to 64 μg/mL, with FICI values of 0.375. Proteomics identified 363 differentially expressed proteins mainly associated with carbon metabolism, RNA degradation, RNA polymerase, and oxidative phosphorylation. CONCLUSION:Luteolin is a potential PBP2a-targeting compound with β-lactam-potentiating activity against MRSA. Its effects may involve PBP2a interaction and broader metabolic and regulatory perturbations, although direct inhibition of PBP2a enzymatic activity remains unconfirmed.
Background Pancreatic ductal adenocarcinoma (PDAC) harbors TP53 mutations at high frequency, yet therapeutic strategies that specifically target mutant p53 remain limited. Purpose This study aimed to identify S100A6, a calcium-binding protein frequently upregulated in TP53-mutant PDAC, as a critical regulator of mutant p53 stability and tumor progression, and to explore potential S100A6-targeting agents for therapeutic intervention. Methods We integrated computer-assisted drug screening with transcriptomics, acetylation omics, and molecular biology techniques to identify Agrimol B (AgrB), a bioactive compound derived from the traditional Chinese herb Agrimonia pilosa Ledeb., as a potential S100A6-targeting agent. Results High S100A6 expression was closely associated with poor prognosis in patients with TP53-mutant PDAC, whereas S100A6 depletion markedly suppressed PDAC cell growth and metastatic potential. Mechanistically, AgrB enhanced the interaction between S100A6 and the deacetylase HDAC2, leading to reduced acetylation of mutant p53 at lysine 382. This disruption activated autophagy-dependent cell death and thereby inhibited PDAC progression. Conclusion Our findings reveal an S100A6-HDAC2-mutant p53 acetylation axis that regulates TP53-mutant pancreatic tumorigenesis, providing mechanistic evidence supporting S100A6 as a therapeutic vulnerability and highlighting AgrB as a promising natural-product-derived candidate for further development against this aggressive malignancy.
BACKGROUND:Diabetic kidney disease (DKD) is a major cause of chronic kidney disease (CKD) worldwide, characterised by tubular injury, inflammation, and fibrosis. Increasing evidence suggests that mechanotransduction and innate immune activation contribute to DKD progression. However, the mechanistic link between mechanosensitive ion channels, cGAS-STING signalling, and pyroptosis remains incompletely understood. PURPOSE:This study aimed to investigate whether the modified Shen-Yan-Fang-Shuai formula (M-SYFSF) attenuates DKD and to explore its potential regulatory effects on Piezo1-mediated Ca²⁺ signalling, cGAS-STING activation, and tubular pyroptosis. METHODS:DKD was induced in rats by unilateral nephrectomy combined with streptozotocin injection, with valsartan as a positive control, and HK-2 cells were stimulated with advanced glycation end products (AGEs). Renal function, histopathology and fibrosis were assessed by biochemical assays and histological staining. Renal transcriptome sequencing, network pharmacology and analysis of public human renal transcriptomic datasets were used to identify candidate pathways. Western blotting, immunohistochemistry, immunofluorescence, qRT-PCR, Fluo-4 AM calcium imaging, ELISA, serum LDH activity and renal caspase-1 activity assays were performed to evaluate Piezo1 expression, cGAS-STING signalling and pyroptosis. Piezo1 knockdown and pharmacological modulation were used to assess pathway ordering, and Co-IP was used to examine the cGAS-STING association. Molecular docking and molecular dynamics simulation were performed as exploratory, hypothesis-generating analyses. RESULTS:DKD rats exhibited renal dysfunction, increased fibrosis, elevated Piezo1 expression, increased intracellular Ca²⁺ levels, activation of cGAS-STING signalling and upregulation of pyroptosis-associated proteins. Renal transcriptome sequencing and network pharmacology converged on mechanotransduction- and AGE-RAGE-associated pathways, and Piezo1 upregulation was further confirmed in db/db and HFD + STZ mice and in human tubulointerstitial datasets. M-SYFSF treatment improved renal function, attenuated fibrosis, reduced Piezo1 expression and intracellular Ca²⁺ accumulation, and suppressed cGAS-STING signalling and downstream inflammatory responses. Piezo1 knockdown attenuated STING and GSDMD-N upregulation, which was partially restored by Yoda1, whereas the STING inhibitor H-151 suppressed both STING and pyroptotic responses. Co-IP analysis showed an AGEs-induced cGAS-STING association that was reduced by M-SYFSF. CONCLUSION:M-SYFSF attenuated experimental DKD in association with reduced Piezo1-associated Ca²⁺ signalling, cGAS-STING activation and tubular pyroptosis-associated responses, and Piezo1 upregulation was reproducible across three rodent models and in human renal tissue. The data support an upstream contribution of Piezo1 under AGEs stress but do not establish the genetic necessity of STING, the proposed mitochondrial DNA intermediate, or the occurrence of fully executed pyroptosis.
BACKGROUND:Tsaoko Fructus, a traditional Chinese medicinal herb, has long been used to alleviate gastritis and enteritis. Nevertheless, the active constituents and underlying anti-inflammatory mechanisms remain insufficiently characterized. PURPOSE:This study aims to optimize a polyphenol-rich fraction (3CB) from Tsaoko Fructus, evaluate its effects against ulcerative colitis (UC), and reveal the underlying mechanisms of action. METHODS:The preparation of 3CB was optimized using response surface methodology (RSM), and its major constituents were identified by LC-PDA-MS analysis. A murine UC model was established by administering dextran sulfate sodium (DSS). To evaluate the effects of 3CB on UC mice, metagenomic sequencing of the intestinal microbiome and RNA sequencing of colon tissues were conducted. The anti-inflammatory activity of 3CB and its principal constituents was further verified by quantitative real-time PCR (qPCR), Enzyme linked immunosorbent assay (ELISA), Western blotting, immunohistochemical staining, and histopathological analysis. Network pharmacology, molecular docking, and surface plasmon resonance (SPR) assays were employed to elucidate the molecular mechanisms underlying the anti-inflammatory effects of 3CB. RESULTS:3CB significantly alleviated UC symptoms in DSS-induced mice, reshaped the gut microbiota with reducing pathogenic Pseudomonadota and Deferribacterota while enriching beneficial Bacteroidota, and restored microbial amino sugar and nucleotide sugar metabolism pathways of intestinal flora. Additionally, 3CB preserved colonic oxidative phosphorylation, protected the mucus barrier, and suppressed inflammatory cell infiltration and the expression of cytokines. Seven major polyphenols were identified in 3CB, with epicatechin (3) and epiafzelechin (6) being the most abundant. Mechanistic investigation revealed that the anti-inflammatory effect of 3CB was partially dependent on the JNK1-modulated MAPK signaling pathway. JNK1 was identified as a direct target of 3CB, with epiafzelechin (6) exhibiting a high binding affinity (Kd = 10.4 μM). CONCLUSION:3CB ameliorates UC potentially through modulation of gut microbiota, protection of the mucus barrier, and JNK1-targeted anti-inflammatory effects, highlighting its potential as a protective intervention for inflammatory bowel disease (IBD).
