BACKGROUND:Agarwood, a precious resinous material derived from Aquilaria trees, is highly valued for its unique fragrance that mainly originates from sesquiterpenes and 2-(2-phenylethyl)chromones. However, the dynamic release behavior of these compounds during combustion remains poorly understood. Common analytical methods, such as headspace and solid-phase microextraction, are limited by their temperature range and fail to mimic true combustion conditions. An innovative pre-treatment technique, gas liquid microextraction (GLME), was employed, requiring as little as 10 mg of sample to effectively simulate agarwood combustion over the temperature range of 25-280 °C. Coupled with GC-MS, this approach enabled systematic profiling of the thermal release behavior of major agarwood varieties under combustion-like conditions. RESULTS:A hybrid analytical strategy that integrates stepwise extraction pre-treatment with a combination of untargeted and targeted screening successfully deciphered compositional changes and trace pyrolysis products. The findings elucidate the temperature-dependent changes in key aroma constituents. Odor-active components are present in relatively low abundance at ambient temperature and 100 °C. In the temperature range of 100 °C-200 °C, sesquiterpenes and aromatic compounds are the dominant chemical classes. Temperatures exceeding 200 °C markedly accelerate the generation of diverse chromone and aromatic derivatives, facilitating the detection of previously unreported thermal degradation products. SIGNIFICANCE AND NOVELTY:This study not only achieves accurate simulation of high-temperature combustion but also establishes correlations between regional origins, combustion phases and aroma signatures. These findings enhance the mechanistic understanding of the thermal behavior of agarwood, offering practical insights for authentication, quality assessment and optimized utilization in incense production and therapeutic applications.

Red ginseng is widely recognized for its anti-fatigue properties, yet the underlying mechanisms remain unclear. Ginsenoside Rg5, a major saponin in red ginseng, was investigated for its role in regulating mitochondrial bioenergetics. Notably, label-free quantitative proteomics revealed that Rg5 selectively upregulated 11 core subunits of mitochondrial respiratory chain complex I. In L-02 hepatocytes, Rg5 significantly enhanced mitochondrial function, as evidenced by increased mitochondrial membrane potential, ATP production, mitochondrial DNA copy number, as well as enzymatic activities of complexes I and II. This effect was accompanied by activation of the LKB1/PGC-1α/TFAM signaling axis, indicating stimulation of mitochondrial biogenesis. In vivo, Rg5 administration markedly prolonged exhaustion time in forced swimming and treadmill tests and improved motor coordination in the rotarod. These functional improvements were associated with elevated ATP levels in the gastrocnemius muscle, liver, and heart, along with increased expression of multiple complex I subunits and enhanced mitochondrial biogenesis. In addition, multiple fatigue-related serum biochemical parameters were significantly improved.Collectively, this study suggests that Rg5 may be associated with the upregulation of key subunits of Complex I and the activation of mitochondrial biogenesis-related processes; these changes appear to collectively lead to enhanced mitochondrial ATP production and contribute to fatigue relief. These findings identify Rg5 as a potential mitochondrial modulator of exercise endurance and support its potential as a natural intervention for fatigue management.

Baijiu is a traditional Chinese alcoholic beverages with significant dietary and medicinal value, including sauce, strong, light and rice four primary flavors. Baijiu contains an exceptionally complex mixture of volatile and non-volatile compounds that contribute to its diverse flavor profiles and potential biological activities. This study employed HS-SPME-Arrow-GC-MS and UPLC-QTOF-MS to systematically analyze four major Baijiu flavor types. We identified 122 components via GC-MS and characterized 134 compounds via UPLC-QTOF-MS, of which 32 were tentatively characterized for the first time in Baijiu. Non-targeted analysis identified 44 differential components across types, and correlation analysis with key enzymes involved in alcohol metabolism (ADH, ALDH) activation revealed that pyrazines positively correlate with enzyme activity. In vitro experiments confirmed that major pyrazines (e.g., tetramethylpyrazine, trimethylpyrazine, 2,3-dimethyl-5-ethylpyrazine) effectively promote the metabolism of ethanol and acetaldehyde. This research enriches the Baijiu component database and provides a theoretical basis for a more scientific evaluation system for its quality and health effects.

Ginsenosides are a class of natural glycosidic compounds characterized by structural diversity and significant bioactivities, mainly derived from Panax genus plants. However, there are still challenges in achieving efficient and green separation of ginsenosides from complex plant matrices. Traditional methods often suffer from high consumption of organic solvents, poor selectivity, and complex purification processes. In this study, a boronic acid-functionalized dendritic mesoporous silica nanomaterial was constructed based on the reversible covalent interaction between boronic acid ligands and the cis-diol motifs in ginsenosides. It was used as the solid phase extraction adsorbent for the separation and purification of ginsenosides. Taking the decoction of Panax notoginseng stems and leaves as an example, the adsorption performance and impurity removal efficiency of this strategy were systematically evaluated. The key extraction parameters were optimized by single-factor experiments and Box-Behnken response surface methodology. Quantitative analysis of nine major ginsenosides using HPLC-CAD demonstrated that the developed strategy outperformed traditional macroporous resin, achieving enrichment fold of 3.4-4.0, which were nearly 2.0 times higher than those of the resin, along with adsorption recoveries of 93.5-98.7% and desorption recoveries of 88.9-97.9%. The extracted sample was further analyzed by UPLC-Q-TOF-MS. 127 ginsenosides were successfully identified, and the interference signals of non-ginsenosides were markedly reduced. The greenness evaluation by Analytical GREEnness metric and Green Analytical Procedure Index indicated that the boron affinity strategy had significant advantages in terms of environmental friendliness and process sustainability. The solid phase extraction technique based on boron-affinity materials provides a promising approach for the efficient, large-scale and green preparation of ginsenosides and other cis-diol natural products.

A graphite-assisted technique was used to enhance laser-assisted ionization TLC direct analysis in real time mass spectrometry (LA-TLC-DART-MS), improving the signal responses for flavonoids, alkaloids, volatile oils, and organic acids. When applied to citrus herbs, this method identified 14 compounds and differentiated samples from different origins, demonstrating its potential for natural product quality control.

Hepatic fibrosis represents a major global public health challenge, yet effective therapeutic interventions remain limited. In this study, we synthesized 40 derivatives through systematic structural modification of l-theanine and identified compound 9a was a potent antifibrotic agent. In vitro experiments revealed that compound 9a dose-dependently inhibited TGFβ1-induced activation of hepatic stellate cells (LX-2 and mHSC). Moreover, in both rat bile duct ligation (BDL) and mouse methionine-choline-deficient high-fat diet (CDAHFD) induced liver fibrosis models, compound 9a significantly attenuated hepatic injury, fibrosis, and inflammation, demonstrating robust hepatoprotective effects. Mechanistic investigations showed that compound 9a directly interacts with Cathepsin D and promotes its degradation, thereby suppressing the expression of fibrogenic and inflammatory genes. Pharmacokinetic studies demonstrated that compound 9a undergoes metabolic conversion to yield pharmacologically active metabolites 10 and 11c. Collectively, these results highlight compound 9a as a promising l-theanine-based candidate for the treatment of hepatic fibrosis.

BackgroundObesity is a growing global health burden with rising incidence. Red ginseng (RGS), a traditional processed ginseng product, shows potential for improving metabolic parameters, though its anti-obesity mechanism is not fully understood.PurposeThis study aimed to investigate the therapeutic effects of short-term RGS administration on obesity and to elucidate its mechanism.MethodsA HFD-induced obese mouse model was employed to assess the effect of short-term RGS. To evaluate the role of the gut microbiota, antibiotic treatment and fecal microbiota transplantation were conducted. 16S rRNA sequencing and metagenomic analysis were performed to identify key bacterial species. Mass spectrometry-based proteomics was applied to identify A. muciniphila-derived proteins. The involvement of the GDF15-GFRAL axis was investigated using Gfral‒/‒ mice.ResultsShort-term RGS treatment suppressed appetite, reduced body weight, and elevated GDF15 level in DIO mice. RGS significantly enriched A. muciniphila, and depletion of A. muciniphila abolished RGS-mediated weight loss and appetite suppression. Proteomic analysis identified Amuc_1631, an A. muciniphila-derived protein, as a critical effector that promoted GDF15 secretion, and oral administration of Amuc_1631 exerts the anti-obesity effects. RGS activated the GDF15–GFRAL axis in the brainstem. Mechanistically, RGS up-regulated colonic Gdf15 transcription via the PERK-eIF2α-ATF4-CHOP axis. RGS/50 fraction is identified as the primary active component responsible for enriching A. muciniphila and elevating GDF15.ConclusionsThis study identifies a gut microbiota-dependent mechanism underlying the anti-obesity effects of RGS, centered on enrichment of A. muciniphila and its derived protein Amuc_1631, which promotes GDF15 secretion to suppress food intake via the GDF15–GFRAL axis.

Non-saponin constituents of Panax species, including amino acids, sugars, and nucleosides, have attracted increasing attention due to their nutritional relevance and potential health benefits in food-medicine homologous materials. However, their high polarity and chemical diversity pose significant challenges for comprehensive characterization, limiting their application in food quality evaluation and authenticity assessment. In this study, an integrated mass spectrometry-based workflow was developed to systematically profile water-soluble non-saponins in Panax ginseng, Panax notoginseng, and Panax quinquefolius, which are widely consumed as functional foods, dietary supplements, and traditional herbal products. An offline two-dimensional LC-MS platform was first established to improve the separation and enrichment of polar constituents, followed by feature-based molecular networking (FBMN) and machine learning-assisted structural annotation using the SIRIUS platform. A total of 201 non-saponin compounds were characterized, revealing remarkable interspecies differences in non-saponin composition. Subsequent non-targeted metabolomics identified quinic acid, raffinose, and trehalose as key species-specific markers with nutritional and quality-discriminating relevance. Furthermore, desorption electrospray ionization mass spectrometry imaging (DESI-MSI) was employed to visualize the spatial distribution of representative non-saponins, uncovering tissue-specific accumulation patterns associated with species identity. Finally, a portable nano-electrospray ionization miniature mass spectrometry (nESI-Mini MS) approach was developed for rapid species authentication, enabling high-throughput, on-site analysis with minimal sample preparation. Overall, this study provides an integrated analytical strategy for elucidating the chemical diversity, spatial distribution, and food-related quality attributes of non-saponin constituents in Panax species, offering practical tools for functional food evaluation, authenticity verification, and quality control.

Animal gallbladder-derived TCMs, such as those from pigs, bears, snakes, and chickens, are rich in constituents including bile acids, proteins, amino acids, and trace elements. They have been traditionally used for their properties in clearing heat, detoxifying, relieving cough, and improving vision. This article systematically reviewed the chemical composition and pharmacological activities of animal gallbladder-derived TCMs. It focused on elucidating the molecular mechanisms through which their active components exerted anti-inflammatory, hepatoprotective, and glucolipid metabolism-regulating effects, primarily by modulating nuclear and membrane receptors, influencing ion channels, and regulating related signaling pathways. Comparisons of similarities and differences among medicinal materials from various sources were also presented. This review aimed to provide a theoretical basis for the modern research and clinical translation of this category of TCMs.

Background Schisandrin A (Sch A), a major bioactive lignan isolated from Schisandra chinensis, exhibits hepatoprotective and metabolic regulatory activities. However, whether its metabolic effects involve context-dependent regulation of intestinal farnesoid X receptor (FXR) signaling under distinct bile acid environments remains unknown. Objective To investigate how Sch A regulates intestinal FXR to ameliorate obesity and MAFLD. Methods DIO and MAFLD mouse models were established separately with Fexaramine (Fex) as a positive control. Therapeutic effects were evaluated through histopathology, biochemistry, metabolic phenotyping, and targeted bile acid metabolomics. Mechanisms were explored using microbiota depletion, fecal microbiota transplantation, intestine-specific Fxr knockout mice, intestinal Camk2d knockdown mice, and target-binding assays. Results Sch A reduced body weight, improved glucose and lipid metabolism, alleviated hepatic steatosis, and enhanced energy expenditure in both obesity and MAFLD models, with broader effects on selected metabolic endpoints, particularly in DIO mice, compared with Fex. In DIO mice, Sch A reduced bile salt hydrolase-producing Lactobacillus, increased conjugated bile acids, inhibited intestinal FXR signaling to enhance bile acid synthesis and boost energy expenditure. In MAFLD mice, Sch A restored FXR activity in association with FXR engagement, CAMK2D-dependent phosphorylation, and nuclear translocation. Conclusion Sch A ameliorates obesity and MAFLD through bile acid context-dependent regulation of intestinal FXR, supporting further preclinical evaluation of Sch A in metabolic disease models.

Deep learning increasingly supports spectroscopic classification, but its black-box nature limits scientific interpretability and trust in the learned features. This study proposes an interpretable ATR-FTIR classification strategy, SpecResSENet, which links model predictions to chemically meaningful spectral evidence. SpecResSENet is a specialized one-dimensional convolutional neural network architecture integrated with an Activation-Weighted Grad-CAM strategy, designed to improve spectral feature localization in the discrimination of traditional agarwood categories. A total of 53 commercially valuable agarwood samples, including Guan-Xiang, Hoi-An, Sin-Chew, and the highly valued Kynam type, were analyzed by ATR-FTIR spectroscopy, yielding 2,672 technical-replicate spectra. Using a Nested Stratified Group 5-Fold Cross-Validation scheme at the biological-sample level, SpecResSENet achieved the highest mean spectrum-level accuracy among the evaluated models (92.8%), while paired fold-wise tests did not establish statistically significant superiority over closely related CNN variants. Because the preprocessing pipeline was fixed before the formal nested model comparison, these performance estimates are conditional on the predefined preprocessing input. As a supportive internal grouped-validation analysis, soft-vote aggregation across technical spectra yielded 96.2% biological-sample-level accuracy (51/53), illustrating sample-level decision aggregation within the current grouped-validation dataset. Activation-Weighted Grad-CAM, together with an Attribution-to-Band procedure, identified recurrent fingerprint-region attribution domains aligned with classification-relevant vibrational regions. The carbonyl-region attribution near 1650cm⁻¹ was consistent with reported conjugated C=O vibrational assignments in agarwood and is interpreted as spectral-domain-level evidence rather than molecule-specific marker evidence. Overall, ATR-FTIR spectroscopy combined with SpecResSENet provides a non-destructive screening framework with spectral-domain interpretability for traditional agarwood categories within the current grouped-validation setting.

Polycystic ovary syndrome (PCOS) is a multifaceted endocrine and metabolic disorder intricately associated with hyperandrogenism (HA), insulin resistance (IR), chronic inflammation, and obesity. The gut microbiota (GM) is considered a mature endocrine organ capable of exerting multiple effects by regulating bile acids (BAs) metabolism. Disruption of GM homeostasis can initiate various pathological processes, including metabolic disorders, endocrine imbalances, low-grade inflammation, and reduced insulin sensitivity, thereby providing novel avenues for research into the pathogenesis of PCOS. There is bidirectional signalling between the GM and BAs: the microbial community tightly regulates the metabolism and synthesis of BAs, while the BAs pool and its composition affect the diversity and homeostasis of intestinal microorganisms. Dysregulation of BAs metabolism mediated by the GM may constitute a crucial pathological link in the progression of PCOS. The objective of this review is to investigate the function of BAs as a signalling molecule bridging the GM and PCOS, to synthesise the current understanding of the roles of BAs and intestinal microorganisms in the pathogenesis of PCOS, and to explore new treatment strategies for PCOS further.

Meeting the food industry's demand for safe, sustainable bioactive extraction, this research proposed an innovative "Parameter-Component" correlation strategy to clarify the mechanism of green extraction, using mechanochemical-assisted extraction (MCAE) of ginsenosides as a case. Via UPLC-Q-TOF-MS/MS, 92 ginsenosides were identified from ginseng. Deconstruction of key MCAE variables (i.e., particle size, pH, temperature), combined with untargeted metabolomics, multivariate statistical analysis, and quantitative validation of critical saponins, revealed a synergistic action of mechanical forces and chemical auxiliaries. This dual intervention disrupts cell walls efficiently while minimizing the degradation of heat-labile components. Different structured saponins showed specific parameter responses. MCAE at 25-40 °C balanced prototype ginsenoside dissolution and malonylated ginsenoside retention; composite processes (MCAE-HRE, MCAE-UAE) optimized functional component yield and production sustainability. This strategy provides a scientific basis for high-quality food-relevant extracts production and a scalable mechanistic study method.

Background:Colorectal cancer (CRC) progression is profoundly shaped by the tumor immune microenvironment. Increasing evidence suggests that dysregulated immune activation and immune evasion jointly contribute to tumor progression and therapeutic resistance. This study aims to identify novel immune-related biomarkers and explore combinatorial therapeutic strategies for overcoming immunosuppression in CRC by investigating the interplay between chloride channel accessory 1 (CLCA1)-mediated immune activation and serine proteinase inhibitor B9 (SERPINB9)-driven immune escape. Methods:Integrated multi-omics analyses were performed to identify key immune-related prognostic biomarkers in CRC and to construct a prognostic immune signature. The predictive performance of the model was validated in independent cohorts. Functional assays were conducted in female C57BL/6J mice to evaluate the effects of CLCA1 on tumor growth and immune cell infiltration. Mechanistic analyses were used to investigate the regulatory relationship between CLCA1 and SERPINB9. In addition, computational drug screening, together with structural and biophysical assays, was applied to identify candidate small molecules targeting CLCA1. Results:CLCA1 was identified as a central immune-related prognostic biomarker in CRC. A 14-gene immune signature effectively stratified patients into distinct risk groups and showed robust predictive value across independent cohorts. Functional studies demonstrated that CLCA1 overexpression suppressed tumor growth and promoted the infiltration of cytotoxic immune cells. Mechanistically, CLCA1 downregulated SERPINB9, a granzyme B (GZMB) inhibitor that plays a pivotal role in immune evasion. Notably, combined CLCA1/SERPINB9 expression status provided improved prognostic stratification compared with either marker alone. Computational screening further identified phloretin as a potential CLCA1-targeting compound, which was supported by structural and biophysical validation. Conclusions:These findings identify CLCA1 as a prognostic immune modulator and a potential therapeutic target in CRC. The functional interaction between CLCA1 and SERPINB9 highlights a mechanistic axis linking immune activation and immune escape. Moreover, phloretin emerges as a candidate small molecule for modulating CLCA1, although its in vivo immunomodulatory effects and target dependency require further validation.

BACKGROUND:Agarwood, a valuable resin-infused wood derived primarily from Aquilaria species (Thymelaeaceae), is widely used in traditional medicine, incense, perfumery, and healthcare products. Its growing scarcity, along with the demand for robust quality control, has driven extensive research, supported by advances in analytical technologies. Recent efforts have focused on elucidating the scientific basis of its traditional applications and promoting sustainable utilization. OBJECTIVES:This review aims to consolidate research progress of agarwood, encompassing updates on agarwood resources, newly identified chemical constituents (2020-2026), emerging analytical strategies, and recent pharmacological findings. It further seeks to outline future research directions to address existing challenges in quality evaluation and mechanistic understanding. METHODS:A systematic retrieval of recent literatures pertaining to agarwood was conducted. Through data extraction and integrative analysis, this review synthesizes the latest research progress and identifies prevailing trends in the field. RESULTS:Recent phytochemical studies have identified over 200 new chemical constituents (2020-2026), including structurally diverse dimeric compounds. Integrated analytical strategies combining spectroscopic, chromatographic, and sensor-based techniques have greatly improved the authentication, sensory evaluation, and grading of agarwood. Additionally, a broad spectrum of pharmacological activities attributable to agarwood has been extensively corroborated. CONCLUSION:This review documents recent advancements concerning the chemical compositions, analytical strategies, and pharmacological activities of agarwood. Challenges remain in mechanisms, structure-activity relationships, and compositional variation, hindering standardization. It will be essential for future work to deepen mechanistic insights and to identify key quality markers, thereby advancing the fields of quality control and translational research for agarwood.

Background:As a debilitating syndrome, cancer cachexia (CC) manifests as ongoing weight reduction and skeletal muscle atrophy, which severely compromise patients' well-being and life expectancy, with no approved treatment available to date. Rare ginsenosides such as Rh2, Rg5, Rk1, and Rh4 have been reported to modulate Nuclear factor kappa-B (NF-κB) and Signal Transducer and Activator of Transcription 3 (STAT3) activity and attenuate inflammatory signaling pathways implicated in CC progression. Li-Ginseng powder (LGP), a specially processed Panax ginseng enriched in rare ginsenosides, including Rk1, Rk3, Rh4, Rg3, and Rg5 represents a potential therapeutic candidate for CC. Methods:The anti-cachexia effects of LGP were evaluated in a BALB/c mouse model of CC and in a cellular CC model using mouse myoblast C2C12 cells. Body weight, skeletal muscle atrophy, and histopathological analyses were performed to assess in vivo efficacy. Network pharmacology was applied to predict key regulatory pathways, and mechanistic validation was conducted using Western blotting, immunohistochemistry, and Enzyme-linked immunosorbent assay. Results:LGP treatment significantly attenuated body weight loss and skeletal muscle atrophy in CC mice. Mechanistically, LGP suppressed activation of the ubiquitin-proteasome pathway in the gastrocnemius muscle and reduced systemic and local inflammatory responses. Network pharmacology analysis identified NF-κB and STAT3 signaling as major targets of LGP, which was further confirmed in both muscle tissues and C2C12 cells. Consistently, LGP alleviated myotube atrophy and inhibited UPP, NF-κB, and STAT3 activation in vitro. Conclusion:These findings demonstrate that LGP exerts protective effects against CC by modulating muscle proteolysis and inflammation-related signaling pathways, highlighting its potential as a ginseng-based therapeutic strategy for CC.

For millennia, Dendrobium Sw., the second-largest genus of Orchidaceae, has been widely applied in traditional Chinese medicine to nourish yin, clear heat, and promote body fluid. Its stems are a rich source of phenanthrenes, which are low molecular weight polycyclic aromatic metabolites with remarkable structural diversity and broad-spectrum bioactivities. This review provides the first comprehensive and systematic analysis of 158 naturally occurring phenanthrenes isolated from 53 Dendrobium taxa (51 species, 1 variety, and 1 horticultural cultivar) based on an extensive literature survey of international and Chinese databases (PubMed, Web of Science, SciFinder, CNKI, etc.) from 1987 to 2025. The article summarizes pharmacopoeial records, classical texts, and theses. The identified metabolites include simple phenanthrenes, 9,10-dihydrophenanthrenes, diphenanthrene dimers, phenanthrenequinones, and other phenanthrene derivatives, often functionalized with hydroxyl, methoxyl, and carboxyl functionalities. Among 158 phenanthrene metabolites isolated from Dendrobium species, 64 compounds (40.5%) have been evaluated in at least one bioactivity assay, with reported activities spanning anti-tumor (cytotoxic), anti-inflammatory, antioxidant, antidiabetic, anti-fibrotic, antiplatelet, or antimicrobial endpoints. However, the majority (54/64, 84.4%) are supported solely by in vitro screening data (IC50 < 100 μM in cell-based assays), while only 8 compounds (12.5%) have validated mechanisms and a mere 4 compounds (6.2%) demonstrate in vivo efficacy in animal models. This quantitative summary reflects research intensity rather than therapeutic potential, given significant heterogeneity in assay systems and the absence of standardized activity thresholds across studies. Mechanistic studies have revealed that these compounds modulate key signaling. thereby offering therapeutic potential against cancer, diabetes mellitus, metabolic dysfunction-associated fatty liver disease (MAFLD), osteoarthritis, and thrombosis. Despite the bioactivity profile, clinical translation is limited by in vivo validation and the lack of pharmacokinetic data. Future studies should focus on systematic preclinical evaluation. Multi-omics mechanistic studies are needed to advance these natural scaffolds into clinically viable therapeutics.

3β-Hydroxysteroid dehydrogenases (3βHSDs) are key enzymes in steroid metabolism, catalyzing C3 oxidation-reduction and Δ5→Δ4 isomerization reactions that govern metabolic flux across multiple steroidogenic pathways. However, the functional diversity of 3βHSDs involved in bufadienolide metabolism in amphibians remains poorly explored. Here, we systematically characterized the 3βHSD gene family in the Asian toad (Bufo bufo gargarizans) using integrated transcriptomic, biochemical, and metabolomic analyses. Seven Bg-3βHSD genes were identified from multi-tissue transcriptomes generated under control and Pb2+ exposure conditions, and six were heterologously expressed for functional evaluation. In vitro assays revealed pronounced functional divergence among Bg-3βHSD isoforms. Bg-3βHSD1 primarily catalyzed bidirectional C3 redox reactions of C21 steroids and bile acid-related substrates, consistent with canonical steroidogenic roles. In contrast, Bg-3βHSD2 enzyme exhibited broad substrate specificity and high catalytic efficiency toward hormones, bile acids, and bufadienolides. In addition to canonical C3 redox reactions and Δ5→Δ4 isomerization, Bg-3βHSD2 also displayed additional oxidation activity at the C17 position for several steroid substrates. A third homolog, Bg-HSD3B7 (GenBank accession no. XM 044303756.1), selectively converted 7α-hydroxylated sterols, suggesting a potential role in classical bile acid metabolism. Integration of tissue-specific expression profiles with bufadienolide distribution patterns suggests that Bg-3βHSD2 may contribute to connecting classical steroid metabolism with bufadienolide biosynthesis in adrenal tissue. Together, the present study identifies Bg-3βHSD2 as an efficient and versatile steroid-transforming enzyme, expands our knowledge of functional diversity within the amphibian 3βHSD family, and provides insights into the enzymatic basis of steroid and bufadienolide metabolism in B. bufo gargarizans.

PURPOSE:Discitis is a severe suppurative infection of the intervertebral disc (IVD) posing significant clinical challenges. This study evaluated the therapeutic efficacy of ozonated water (OW) in a rat model of Staphylococcus aureus-induced discitis and elucidated its underlying mechanisms, focusing on NF-κB signaling, extracellular matrix homeostasis, and nucleus pulposus (NP) cell apoptosis. METHODS:A rat tail coccygeal IVD discitis model was established by injecting S. aureus (102 cfu) into the disc space. From day 3 post-infection, animals received transdiscal lavage with normal saline, levofloxacin (5 mg/mL), or OW (30 μg/mL) three times daily until day 9. In vitro, NP cells were stimulated with S. aureus Protein A (SpA, 1 μg/mL). Antibacterial activity, extracellular matrix metabolism, inflammatory cytokine expression, NF-κB p65 signaling, and apoptosis were evaluated by bacterial culture, Western blotting, immunohistochemistry, TUNEL assay, and flow cytometry. RESULTS:OW significantly alleviated tail swelling and IVD structural damage. OW demonstrated potent antibacterial activity against S. aureus, disrupting bacterial membrane integrity. OW attenuated extracellular matrix degradation by upregulating collagen II and aggrecan while downregulating MMP3 and MMP9, and suppressed inflammatory responses (IL-1β, IL-6, TNF-α, Cox2) by inhibiting NF-κB p65 phosphorylation and nuclear translocation. OW also reduced NP cell apoptosis, evidenced by decreased TUNEL-positive rate, elevated Bcl-2/Bax ratio, and reduced Cleaved caspase-3. CONCLUSIONS:OW attenuates S. aureus-induced discitis through direct antibacterial action, suppression of NF-κB-mediated inflammation, protection against NP cell apoptosis, and preservation of extracellular matrix integrity, highlighting its promise as a novel lavage strategy for discitis management.