Objective To analyze the correlation between the content changes of characteristic components and its antithrombotic effect in different proportions of the Danshen (Salviae Miltiorrhizae Radix et Rhizoma, SMRR)-Honghua (Carthami Flos, CF) herb pair, and optimize the compatibility ratio of SMRR and CF for promoting blood circulation and removing blood stasis. Methods Fingerprint profiles and quantitative determination methods for characteristic components were established for SMRR-CF herb pair at different ratios (1:1, 1:2, 1:3, 1:4, 1:5, 2:1, 3:1, 4:1, 5:1). The HPLC combined with a comprehensive scoring method was used to determine the optimal extraction process of the SMRR-CF herb pair. The anti-thrombotic activity of the SMRR-CF extracts at different ratios was measured using zebrafish as a model organism. The principal component analysis (PCA) method was used to integrate the content of characteristic components and the intensity of antithrombotic activity, and the optimal ratio of SMRR and CF was comprehensively evaluated from two aspects of composition and efficacy. Results The fingerprint profiles of the SMRR-CF herb pair in nine different ratios collectively identified 17 common peaks, among which nine characteristic components were characterized. These include hydroxysafflor yellow A (peak 2), luteoloside (peak 3), kaempferol-3-O-rutinoside (peak 4), quercitrin (peak 5), salvianolic acid B (peak 8), dihydrotanshinone I (peak 12), cryptotanshinone (peak 14), tanshinone I (peak 15), and tanshinone II_A (peak 17). The optimal extraction process was determined to be maceration with 10 times the amount of 70% ethanol for 1 h by reflux. The content determination showed that the characteristic components changed significantly after the compatibility of SMRR and CF compared with the single herb. When the SMRR-CF ratio was 1:5, decoction yielded the highest concentrations of salvianolic acids and tanshinones derived from SMRR. Conversely, ratios of 3:1, 4:1 and 5:1 produced the greatest amounts of hydroxysafflor yellow A, luteoloside and kaempferol-3-O-rutinoside from CF, indicating that co-decoction facilitates the extraction of bioactive constituents from both herbs. Pharmacodynamic evaluation revealed that the 3:1 ratio exhibited the strongest antithrombotic activity, achieving a thrombosis inhibition rate of 43.7%. Principal-component analysis of the “composition-effect” dataset ranked the comprehensive indices (F-values) as 3:1 > 4:1 > 5:1 > 2:1 > 1:1 > 1:5 > 1:4 > 1:2 > 1:3, confirming that the 3:1 ratio represents the optimal compatibility of the SMRR-CF pair. Conclusion In this study, chemical profiling and in vivo pharmacodynamic evaluation were integrated to elucidate the quantitative relationship between the dynamic changes of characteristic constituents and the antithrombotic efficacy of the SMRR-CF herb pair across different compatibility ratios. An optimal ratio was identified through a “composition-effect”-based comprehensive assessment. These findings not only establish a foundation for subsequent dose-effect investigations of this classic combination, but also offer a rational reference for determining prescription design in latent prescriptions.

Objective By systematically analyzing the polyphenolic composition content, astringency intensity, astringency mechanism, and their inherent correlations in Hezi (Chebulae Fructus, CF) pieces with different comminution degrees, this study aims to lay a foundation for its further development and research. Methods The CF was processed into six granule sizes (original granule, extra large granule, large granule, median granule, small granule, fine powder) for sampling. HPLC was employed to quantify the contents of chebulagic acid, gallic acid, chebulinic acid, ellagic acid, corilagin, and 1,3,6-tri-O-galloyl-β-D-glucose. The main chemical composition groups and differences of six kinds of particle size CF samples were analyzed by fingerprint. The astringency intensity of the samples was sensorially evaluated using a visual analogue scale (VAS). These data were utilized to analyze the correlations among comminution degree, polyphenolic composition, and astringency intensity. Furthermore, SDS-PAGE technology was applied to elucidate the underlying astringency mechanism through the protein interaction. Results The total content of six kinds of polyphenolic components displayed significant variation across different granule sizes in the following descending order: median granule > extra large granule > fine powder > large granule > small granule > original granule. The results of fingerprint analysis showed that the main chemical composition groups of the six samples were basically the same. Sensory evaluation revealed that different comminution degrees considerably influenced the perceived astringency intensity, which decreased sequentially as: fine powder > small granule > median granule > large granule > extra large granule > original granule. Correlation analysis identified gallic acid and corilagin as key variables linking comminution degree with astringency perception. SDS-PAGE confirmed that the astringency mechanism primarily involves binding interactions between polyphenolic components and salivary proteins, with the fine powder demonstrating the strongest protein-binding capacity. Conclusion Comminution degree serves as a critical processing parameter governing the dissolution of polyphenolic components and astringency intensity in CF. The sample with a particle size of 4.75—6.70 mm demonstrated the highest content of polyphenolic compounds, whereas the sample with a particle size of 0.15—0.18 mm elicited the most intense perception of astringency. Processing CF to a median granule size (4.75—6.70 mm) is therefore identified as the optimal strategy for balancing polyphenolic component dissolution and oral compliance. This study provides key scientific evidence to support the standardization of CF processing techniques and related product development.

Objective To investigate the relationship between the physical properties of Jinzhen Oral Liquid (金振口服液) intermediates and their total solid content (TSC) and the contents of effective components, and to establish a content prediction model, thereby providing a reference for rapid quality evaluation of intermediates. Methods Samples were collected from two types of extracts (mineral-botanical and artificial cow bezoar) and four key manufacturing steps (hot mixing, refrigeration, centrifugation, and filling). Six physical parameters (density, viscosity, surface tension, electrical conductivity, refractive index, and pH value) were measured, alongside the contents of six effective components (baicalin, wogonoside, glycyrrhizic acid, gallic acid, hyodeoxycholic acid, and cholic acid) and TSC. Correlation analysis was performed, and polynomial regression models were developed for the overall process, the mineral-botanical extract, and the artificial cow bezoar extract, respectively. Results Correlation analysis indicated that most physical parameters were significantly correlated with chemical components throughout the entire preparation process. Refractive index showed a strong correlation with TSC (r = 0.845), while density was correlated with TSC and baicalin with coefficients of 0.529 and 0.505, respectively. In mineral-botanical extracts, the correlation coefficient between refractive index and TSC was 0.525. In artificial cow bezoar extracts, refractive index was correlated with TSC, hyodeoxycholic acid, and cholic acid, with coefficients of 0.759, 0.729, and 0.593, respectively. The regression models established based on refractive index performed well in validation: the model for TSC in the full process showed a mean relative error (MRE) of 4.04% and a mean absolute error (MAE) of 1.10%. The MRE values for the baicalin and wogonoside models were both below 10%, and both passed the F-test. The MRE values for the TSC models in both mineral-botanical and artificial cow bezoar extracts were below 6%. Conclusion The refractive index of Jinzhen Oral Liquid intermediates shows significant correlation with both TSC and the contents of effective components. The refractive index method is a feasible approach for predicting TSC and the contents of baicalin and wogonoside during the manufacturing process, as well as TSC in both extracts. This study provides a novel strategy for the rapid quality assessment of Jinzhen Oral Liquid production and offers a technical basis for the practical application of online refractometry.

Objective To optimize the optimal processing technology of different processed products of Huangqin (Scutellariae Radix, SR) [fried Scutellariae Radix (fSR), Scutellariae Radix charcoal (SRc), wine Scutellariae Radix (wSR)], and explore the correlation between sensory traits such as appearance, color, odor, taste, and the main active ingredients. Method The multi-index response surface methodology (RSM) was used, with baicalin, baicalein, wogonoside, wogonin, and appearance trait scores as evaluation indicators. The analytic hierarchy process and entropy weight method were used to calculate the weight coefficients and comprehensive scores of each indicator. Further utilize central combination design (CCD)-RSM to investigate the effects of temperature and processing time (soaking time and the alcohol ratio) on the processing technology. Using electronic sensory technology (electronic eye, electronic nose, electronic tongue) to analyze the response values of samples with different processing levels, and exploring the correlation between sensory traits (color L^*, a^*, b^* value, odor sensor response value, taste sensor response value) and chemical composition through correlation analysis. Results The optimal process parameters were determined as follows: fSR at 205.4 ℃, 6.5 min, SRc at 227.4 ℃, 16 min, and wSR at 180 ℃, 5 min (moistened for 50 min, with a yellow wine dosage of 18.2%). Correlation analysis shows that different degrees of processing of SR have distinguishable sensor response patterns: odor sensors S7 and S10 and taste sensors P6 and P10—P12 in fSR have a strong positive correlation with baicalin and wogonoside; In SRc and wSR, there is a strong negative correlation between multiple sensors and baicalin, while wogonin shows a strong positive correlation with multiple sensors SRc and a strong negative correlation with fSR. The electronic eye color values (L^*, a^*, b^*) are significantly correlated with the degree of processing (ΔE^*_ab > 1.5). Conclusion Optimizing the process is stable and feasible. The quality evaluation system based on electronic sensory technology can objectively distinguish the degree of processing, and the correlation between characteristics and components revealed provides theoretical basis and innovative methods for determining the endpoint of processing and quality control.

Objective To optimize the formulation process of co-loaded nanoparticles (TBF-HQD-SAN NPs) comprising self-assembled nanoparticles of Huangqin Decoction (黄芩汤) (HQD-SAN) and terbinafine (TBF). Methods The HQD-SAN was obtained with high-speed centrifugation combined with dialysis and then TBF was further loaded to prepare TBF-HQD-SAN NPs. Based on the single-factor investigation, taking the concentration of HQD-SAN, the speed of magnetic stirring and the stirring time as the investigation factors, and the drug loading (DL) of TBF, the encapsulation efficiency and the DL of baicalin as the investigation indicators, the formulation and process of TBF-HQD-SAN NPs were optimized by using the 3-factor 3-level Box-Behnken design (BBD)-response surface methodology (RSM). The optimized TBF-HQD-SAN NPs were characterized for morphology, particle size distribution, ζ potential, and drug-loading capacity. Their saturated solubility was determined, and their antifungal activity against Trichophyton rubrum, T. mentagrophytes, and Microsporum canis was investigated. Results The TBF-HQD-SAN NPs process was optimized by BBD. The established quadratic regression model had excellent fit (all R² > 0.99), and the concentration of HQD-SAN, stirring speed, stirring time and some interaction terms had significant effects on the indicators (P < 0.05). The effect surface analysis showed that the drug loading of TBF decreased with the increase of HQD-SAN concentration and first increased and then decreased with the stirring speed/time. The encapsulation rate/drug loading of baicalin increases with the increased of HQD-SAN concentration and decreased with the extension of stirring speed/time. The optimal process for model selection was as follows: HQD-SAN concentration 5.6 mg/mL, TBF 5 mg, distilled water 4 mL, ultrasonic (50 W, 40 kHz) for 30 min, magnetic stirring at 760 r/min for 1.5 h, and filtration through 0.8 μm filter membrane. The measured values of each index in the verification experiment were close to the predicted values (RSD < 5%). The particle size of the NPs was (185.10 ± 1.73) nm, the polydispersity index (PDI) was 0.22 ± 0.01, and the ζ potential was (−15.17 ± 1.40) mV. The encapsulation efficiency and DL of TBF were (99.81 ± 0.33)% and (3.32 ± 0.09)%, respectively, while those of baicalin were (58.59 ± 1.42)% and (6.71 ± 0.15)%, respectively. The equilibrium solubility was (2.27 ± 0.09) μg/mL for TBF alone, (15.70 ± 1.66) μg/mL for the TBF-HQD-SAN physical mixture (PM), and (78.20 ± 2.22) μg/mL for the TBF-HQD-SAN NPs. The antifungal experiment showed that the antifungal activity of TBF-HQD-SAN NPs (with MIC values ranging from 0.15—0.31 µg/mL, equivalent to 4.98—10.13 ng/mL based on TBF content) was significantly superior to that of HQD-SAN (MIC value 1.56—3.13 mg/mL) and TBF alone (MIC value 0.06—0.50 µg/mL) (P < 0.05). Conclusion The BBD successfully optimized the formulation and preparation process of TBF-HQD-SAN NPs. The resulting NPs demonstrated uniform particle size, excellent drug-loading performance, and significantly enhanced antifungal efficacy, laying a foundation for further research.

Objective To investigate the effects of calcination temperature on the phase composition and chemical constituents of Shijueming (Haliotidis Concha, HC), optimize its processing methodology, and conduct comparative analysis and health risk assessment of heavy metal content in HC and calcined HC. Methods Thermal effects during processing at different calcination temperatures were examined via thermogravimetric-differential thermal analysis (TG-DTA). Primary phase transformations were analyzed using X-ray diffraction (XRD), while compositional changes were assessed by Fourier transform infrared spectroscopy (FTIR) to preliminarily screen the key factor “calcination temperature” affecting HC processing. Simultaneously, the optimal processing parameters for HC were determined using the AHP-CRITIC mixed entropy method combined with orthogonal experiments, with CaCO₃ content, crispness, and color as evaluation criteria. Calcined HC were processed according to the optimized method. Inductively coupled plasma mass spectrometry (ICP-MS) was employed to analyze 20 batches of HC and calcined HC for Pb, Cd, As, Hg, and Cu in 20 batches of HC and calcined calcined HC. Using HC powder as a reference, theoretical maximum limits for these five heavy metals and toxic elements were calculated. Daily exposure and target hazard quotient (THQ) were further assessed to evaluate health risks, providing a reference for quality and safety evaluation of HC. Results Preliminary screening of calcination temperatures indicated an optimal range of 400—600 ℃. Using the AHP-CRITIC mixed entropy method, the optimized calcination process for HC was determined as follows: Crush HC to a particle size < 1 cm, place in a muffle furnace, set the temperature to 600 ℃, and calcine for 90 min. After calcination, the organic matter content of HC decreased while calcium carbonate content increased. The calcined HC showed reduced levels of As and Hg, with minimal changes in Cu, Cd and Pb. Health risk assessment indicated that all 20 batches of HC and calcined HC posed no significant health hazards to humans. Notably, the As and Hg levels in the slices were significantly lower than those in the raw material (P < 0.05). Conclusion By integrating phase and compositional changes before and after processing, this study employed the AHP-CRITIC mixed entropy method to scientifically and comprehensively optimize the calcination process for HC. The screening results are scientifically reliable, providing new scientific evidence for quality management and clinical application of HC and calcined HC. Conducting health risk assessments of heavy metals and harmful elements in HC before and after processing provides reference for the safe clinical use of HC and other shell-derived Chinese medicinal materials.

Objective To investigate the therapeutic effects and potential mechanisms of tetramethylpyrazine (TMP) in a high humidity osteoarthritis (HHOA) rat model. Methods The HHOA rat model was established, and rats were given TMP for eight weeks. Safranin O-fast green staining was used to observe morphological changes in cartilage tissue. The levels of matrix metalloproteinase-9 (MMP-9), tissue inhibitors of metalloproteinase-1 (TIMP-1), prostaglandin-endoperoxide synthase 2 (PTGS2), glutathione peroxidase 4 (GPX4), glutathione (GSH), malondialdehyde (MDA) and superoxide dismutase (SOD) in serum were measured. Prussian blue staining was performed to detect iron deposition in cartilage. Immunohistochemistry was used to assess the expressions of GPX4 and acyl-CoA synthetase long-chain family member 4 (ACSL4) in cartilage. Serum untargeted metabolomics was performed to analyze changes in metabolites and their enriched pathways. In vitro, a ferroptosis model of chondrocytes was established and treated with main metabolite N-acetylserotonin. GSH/oxidized glutathione (GSSG) value, MDA and SOD levels were measured, the expressions of GPX4 and ACSL4 were measured by Western blotting. Results Compared with HHOA group, levels of MMP-9, PTGS2 and MDA in serum of rats in TMP group were significantly reduced (P < 0.05, 0.01), while the levels of TIMP-1, GPX4, SOD and GSH in serum were significantly increased (P < 0.05, 0.01). The pathological damage to the articular cartilage was significantly improved, and the deposition of iron ions in articular cartilage was reduced, GPX4 protein expression was increased, and ACSL4 protein expression was reduced. Metabolomics analysis showed that 2 695 differential metabolites were screened between HHOA group and sham group, with significantly more down-regulated metabolites than up-regulated metabolites; A total of 2 846 differential metabolites were screened between TMP high-dose group and HHOA group, with up-regulated metabolites being the main ones. KEGG pathway enrichment analysis results showed that the differential metabolites between HHOA group and sham group were mainly enriched in multiple metabolic pathways such as carbon metabolism and amino acid metabolism, the differential metabolites between TMP group and HHOA group were mainly enriched in multiple pathways closely related to amino acid metabolism and substance transport. Compared with sham group, the levels of γ-glutamylleucine, 3-indoolepropionic acid and N-acetylseletonin in serum of HHOA group rats were significantly reduced (P < 0.05). Compared with HHOA group, the levels of γ-glutamylleucine, 3-indoolepropionic acid and N-acetylseletonin in TMP high-dose group were significantly increased (P < 0.05). The in vitro experimental results showed that the main metabolite N-acetylserotonin significantly increased the GSH/GSSG value and SOD activity in ferroptosis model of chondrocytes (P < 0.05), decreased MDA level (P < 0.05), up-regulated GPX4 protein and down-regulated ACSL4 protein expressions (P < 0.05, 0.01). Conclusion TMP could improve the metabolomic profile of HHOA rats, regulate the tryptophan metabolism pathway represented by N-acetylserotonin, alleviate GPX4/ASCL4 axis mediated lipid peroxidation and iron ion deposition, thereby reducing ferroptosis in chondrocytes and delaying the progression of HHOA.

ObjectiveTo investigate the intervention effect and related mechanism of hyperoside on sepsis-associated acute kidney injury (SAKI) in mice. Methods A mouse model of SAKI was established by cecal ligation and puncture (CLP). Sham group, model group, dexamethasone (1 mg/kg) group, hyperoside high-, low-dose (50, 25 mg/kg) groups and hyperoside (50 mg/kg) + nuclear factor erythroid 2-related factor 2 (Nrf2) inhibitor ML385 (30 mg/kg) group were set up. After 7 d of drug intervention, the survival curve of mice was plotted. The levels of renal function, inflammatory factors and oxidative stress were detected. The pathological changes of renal tissue were observed by hematoxylin-eosin (HE) staining. TUNEL staining was used to detect apoptosis in renal tissue cells. Immunohistochemical was used to detect Nrf2 expression in renal tissue. Western blotting was used to detect the expressions of Nrf2, PTEN induced kinase 1 (Pink1) and Parkin proteins in renal tissue. Immunofluorescence was used to detect the co-localization of microtubule associated protein 1 light chain 3B (LC3B) and translocase of the outer mitochondrial membrane 20 (TOMM20) in renal tissue. Lipopolysaccharide (LPS) was used in vitro to induce injury in human tubular epithelial cells (HK-2). After intervention with hyperoside, cell viability, apoptosis and reactive oxygen species levels were detected. Mitochondrial ultrastructure was observed by transmission electron microscopy. Flow cytometry was used to detect mitochondrial membrane potential (MMP) and adenosine triphosphate (ATP) levels to evaluate mitochondrial function. Western blotting was used to detect the expressions of Nrf2, heme oxygenase-1 (HO-1) and mitochondrial autophagy proteins Pink1, Parkin, p62. Results Compared with model group, hyperoside could significantly improve the survival rate of SAKI mice (P < 0.05), alleviate weight loss and renal pathological damage (P < 0.05, 0.01), improve renal function and oxidative stress response (P < 0.05, 0.01), inhibit inflammatory factor levels (P < 0.05, 0.01), up-regulate the expressions of Nrf2, Pink1, Parkin in renal tissue (P < 0.01), and increase the co-ocalization of LC3B and TOMM20 in renal tissue. In vitro experimental results showed that hyperoside (40 μmol/L) could inhibit LPS-induced apoptosis and oxidative stress in HK-2 cells (P < 0.01), increase MMP and ATP levels (P < 0.01), up-regulate Nrf2, HO-1, Pink1, Parkin protein expressions (P < 0.01), down-regulate p62 expression (P < 0.01), promote mitochondrial autophagy, and alleviate mitochondrial damage. In addition, Nrf2 inhibitor ML385 significantly reversed the protective effect of hyperoside on CLP-induced SAKI mice (P < 0.05, 0.01), while weakening hyperoside induced mitochondrial autophagy in HK-2 cells.Conclusion Hypericin may regulate Pink1/Parkin mediated mitochondrial autophagy by activating Nrf2/HO-1 signaling pathway, alleviate mitochondrial dysfunction, alleviate oxidative stress and inflammatory response in SAKI mice, and thus exert renal protective effects.

ObjectiveTo investigate the protective effect of Rosa roxburghii fermentation broth against acetaminophen (APAP)-induced drug-induced liver injury (DILI) in mice and explore its potential mechanism based on nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) signaling pathway. Methods Taking the R. roxburghii juice as the control, the main active component contents and in vitro antioxidant capacity of R. roxburghii fermentation broth were analyzed. A total of 60 Kunming mice were randomly divided into control group, model group, N-acetylcysteine (NAC, 0.15 g/kg) group, and R. roxburghii fermentation broth low-, medium-, and high-dose (5, 10, 20 mL/kg) groups, with 10 mice in each group. All groups were consecutively administered for 7 d, 1 h after the last administration, except for the control group, all other groups received a single intraperitoneal injection of APAP (400 mg/kg) to establish the acute liver injury model. After 12 h of modeling, the mice were anesthetized and euthanized. The intact liver of mice was immediately removed and observed for any visible damage or lesions. The body weight and liver weight were measured and liver index was calculated. Activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), IL-1β in serum, as well as activity of superoxide dismutase (SOD) and levels of glutathione (GSH), malondialdehyde (MDA) in liver tissue were detected by ELISA. Hepatic pathological changes were observed by hematoxylin-eosin (HE) staining. The mRNA and protein expressions of Nrf2, heme oxygenase-1 (HO-1), SOD-1 and catalase (CAT) in liver tissue were detected by qRT-PCR and Western blotting. Neutrophil infiltration was observed by Ly6G immunofluorescence staining, and hepatocyte apoptosis was detected by TUNEL staining. Results Compared with R. roxburghii juice, the contents of total acids, total phenols and total flavonoids in R. roxburghii fermentation broth were significantly increased (P < 0.05), while the content of vitamin C was significantly decreased (P < 0.05), and the total amount of major antioxidant active ingredients were increased, and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging rate and Fe³⁺ reducing ability were significantly increased (P < 0.01). Compared with control group, model group showed obvious pathological changes such as hemorrhagic injury in liver, liver index and activities of ALT, AST, levels of inflammatory factors in serum and MDA level in liver tissue were significantly increased (P < 0.001), while GSH level and SOD activity in liver tissue were significantly decreased (P < 0.001). Inflammatory cell infiltration in liver tissue, disruption of hepatic cord structure, and increased necrotic area were observed; The mRNA and protein expression levels of Nrf2, HO-1, SOD-1 and CAT in liver tissue were significantly reduced (P < 0.001); Neutrophil infiltration and hepatocyte apoptosis in liver tissue were significantly increased (P < 0.001). Compared with model group, NAC and R. roxburghii fermentation broth all dose groups could improve the above pathological and biochemical indicators to varying degrees, including reducing visible liver damage and lesions, lowering liver index, down-regulating transaminase activity and inflammatory factor levels in serum, reducing MDA level in liver tissue, increasing GSH level and SOD activity, and up-regulating Nrf2/ARE pathway related genes and protein expressions (P < 0.05, 0.01, 0.001), while significantly inhibiting neutrophil infiltration and liver cell apoptosis (P < 0.05, 0.001). Conclusion R. roxburghii fermentation broth has a potential liver-protective effect against APAP-induced DILI in mice, and its mechanism may be related to the activation of Nrf2/ARE signaling pathway, enhancing antioxidant capacity and inhibiting inflammatory responses.

Objective To construct Ginseng polysaccharide-chitosan oligosaccharide nanoparticles (GP-NPs) and investigate whether their combination with radiotherapy could promote the differentiation of natural killer (NK) cells into highly cytotoxic subsets by activating interferon-I (IFN-I)-mediated Janus kinase 3 (JAK3)/signal transducer and activator of transcription 5 (STAT5) signaling pathway, thereby enhancing the antitumor immune response against non-small cell lung cancer (NSCLC). Methods GP-NPs were prepared via the ionic cross-linking method, and their particle size, dispersibility and stability were characterized. A subcutaneous xenograft model of A549 lung cancer cells in nude mice was established, and the mice were divided into PBS group, radiotherapy alone group and GP-NPs combined with radiotherapy group. The antitumor efficacy and mechanism of GP-NPs combined with radiotherapy were comprehensively evaluated by measuring tumor volume, detecting levels of cytokines in serum via ELISA, analyzing NK cells subsets by flow cytometry, detecting the protein expressions of JAK3/STAT5 pathway through Western blotting and performing histopathological analysis. Results GP-NPs combined with radiotherapy significantly inhibited tumor growth, and the effect was significantly better than that of the simple radiotherapy group (P < 0.01). The levels of interferon-γ (IFN-γ), interleukin-15 (IL-15), granzyme and perforin in serum of combined treatment group were significantly increased (P < 0.01). Flow cytometry showed an increase in the proportion of NK cells infiltration within the tumor (P < 0.01), and a significant increase in the proportion of mature cytotoxic subgroups (CD27^-CD11b⁺) (P < 0.01). Western blotting results showed that the combination therapy significantly activated the JAK3/STAT5 signaling pathway (P < 0.01). Organizational analysis showed that the combination therapy group had increased tumor cell apoptosis, aggravated DNA damage and enhanced NK cell infiltration, with no significant toxicity observed in major organs. Conclusion GP-NPs combined with radiotherapy promote the differentiation of NK cells into highly cytotoxic subsets and enhance their antitumor function by activating IFN-I-mediated JAK3/STAT5 signaling pathway. This combination strategy not only significantly improves the efficacy of radiotherapy but also has good biosafety, providing new experimental evidence and strategic directions for the immunocombination therapy of NSCLC.