Male Sprague–Dawley rats (6–8 weeks old) were used to minimize potential variability associated with sex-related hormonal fluctuations. Rats were randomly divided into four groups (n = 3 per group): control (Con), drug-only (Con + DQ), irradiation (IR), and irradiation plus drug (IR + DQ). For DQ treatment, rats received dasatinib (5 mg/kg) and quercetin (50 mg/kg) by oral gavage once daily for five consecutive days, starting on day 1 post irradiation. All experimental procedures were approved by the Animal Care Committee of the General Hospital of Northern Theatre Command (Approval No. 2025-52) and were conducted in strict accordance with institutional animal welfare guidelines. Rats were housed in groups under controlled environmental conditions (24 °C ± 2 °C; 12-h light/dark cycle) with free access to food and water. Before irradiation, the animals were positioned under a CT simulator, and images were transferred to a treatment planning system to delineate the target area. Right-lung irradiation was performed using 6 MV X-rays at a dose rate of 300 cGy/min, delivering a single fraction of 30 Gy. After irradiation, rats were maintained under standard conditions, and lung tissues were harvested on day 28 for subsequent analyses.

Lung tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at 4 μm thickness. Hematoxylin–eosin (HE) staining was performed to evaluate general histopathological changes, while collagen deposition was evaluated using a Masson’s trichrome staining kit according to the manufacturer’s instructions.

Frozen lung sections were thawed, fixed, permeabilized, and blocked with normal serum. Sections were incubated overnight at 4 °C with the γH2AX antibody (9718, 1:400, CST, United States). After washing with PBS, sections were incubated with fluorescently labeled secondary antibodies for 1 h at room temperature, counterstained with DAPI, and mounted with an anti-fade reagent. Images were acquired using a fluorescence microscope.

Frozen sections were thawed and washed three times with PBS (5 min each). Sections were fixed in β-galactosidase staining fixative for 15 min, washed again, and incubated with β-galactosidase staining working solution overnight at 37 °C. Observation under an optical microscope.

Lung tissues were lysed in RIPA buffer, and protein concentrations were determined using a BCA assay kit. Equal amounts of protein were separated by SDS-PAGE and transferred onto nitrocellulose membranes. Membranes were blocked with 5% skim milk for 2 h and then incubated overnight at 4 °C with primary antibodies, followed by incubation with HRP-conjugated secondary antibodies for 2 h at room temperature. Protein bands were visualized using an enhanced chemiluminescence (ECL) detection system. The following primary antibodies were used: p53 (SC126,1:500, Santa Cruz, United States), p21 (1:1000; ab109199, Abcam, United States), p16 (PA5-20379, 1:1000, Invitrogen, United States), IL-6 (GB11117, 1:1000, Servicebio, China), IL-1β (AF4006,1:1000, Affinity, China), IL-18 (E3G8R,1:1000, CST, United States), TNF-α (SC52B83,1:500, Santa Cruz, United States), MMP2 (SC13595, 1:500, Santa Cruz, United States), MMP9 (SC13520, 1:500, Santa Cruz, United States), α-SMA (19245, 1:1000, CST,United States), α-Tubulin (2125, 1:1000, CST, United States) and β-actin (GB15003, 1:5000, Servicebio, China).

Paraffin-embedded lung tissue sections were deparaffinized, rehydrated, and subjected to antigen retrieval. Endogenous peroxidase activity was quenched with hydrogen peroxide, and non-specific binding was blocked with goat serum. Sections were incubated overnight at 4 °C with the indicated primary antibodies against TGF-β (YP-Ab-15967,1:200, UpingBio, China) and MCP-1 (HY-P81171,1:200, MCE, China), followed by incubation with HRP-conjugated secondary antibodies. Immunoreactivity was visualized using a standard DAB chromogenic detection system, and images were captured under a light microscope.

Total RNA was extracted from lung tissues obtained from three independent biological replicates per group (n = 3). RNA integrity and concentration were evaluated using an Agilent 2100 Bioanalyzer. mRNA was isolated using oligo (dT) magnetic beads, and sequencing libraries were prepared following standard Illumina protocols. Library concentration was quantified using the Qubit™ system and qPCR, and fragment size distribution was verified using a bioanalyzer. After quality control, high-throughput sequencing was performed on an Illumina platform. Differentially expressed genes (DEGs) were identified using DESeq2, with |log2 fold change| ≥1 and adjusted P value <0.05. Principal component analysis (PCA) was performed to assess global expression patterns and revealed no obvious batch-driven clustering. DEGs were further analyzed by Gene Set Enrichment Analysis (GSEA), Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses.

All statistical analyses were performed using GraphPad Prism 8.0 software. Data are presented as mean ± SD. Normality was assessed using the Shapiro–Wilk test prior to parametric analyses. Comparisons between two groups were performed using an unpaired two-tailed Student's t-test. For comparisons among multiple groups, one-way ANOVA was used, followed by Tukey’s post hoc test. For body weight changes over time, two-way repeated-measures ANOVA was performed, with group and time as factors, followed by Bonferroni’s post hoc test when appropriate. A P-value of <0.05 was considered statistically significant (*P < 0.05, **P < 0.01, ***P < 0.001).

The experimental grouping and study design schematic is shown in Figure 1A. Irradiated rats exhibited a slower increase in body weight over time, which was partially reversed by DQ treatment. Consistently, the lung coefficient-an indicator of pulmonary edema-increased following irradiation, while DQ treatment markedly reduced this elevation (Figures 1B,C). The protective effects of DQ on RILI were assessed using HE and Masson’s trichrome staining. In the control group, HE staining revealed well-defined alveolar architecture, intact capillary networks, and the absence of congestion, edema, or inflammatory infiltration. In contrast, the IR group displayed severely thickened interalveolar septa, and extensive inflammatory cell infiltration. Quantitative analysis demonstrated that the histological injury score was significantly increased in the IR group (3.554 ± 0.478) compared with the control group (0.541 ± 0.085, P < 0.001). DQ treatment markedly alleviated RILI, reducing the histological score to 1.501 ± 0.495 in the IR + DQ group, corresponding to an approximately 57.8% decrease compared with the IR group (P < 0.001) (Figures 1D,F). Masson’s trichrome staining further revealed pronounced collagen accumulation after irradiation. Quantitative analysis showed that the collagen-positive area was significantly increased in the IR group (42.19 ± 1.68) compared with the control group (10.52 ± 3.02, P < 0.001). Importantly, DQ treatment significantly reduced collagen deposition to 26.46 ± 4.22 in the IR + DQ group, corresponding to an approximately 37.3% decrease relative to the IR group (P < 0.01) (Figures 1E,G). Collectively, these results demonstrated that combined DQ therapy effectively attenuates the histopathological progression of RILI in rats.

Ionizing radiation induced extensive DNA damage. To evaluate the protective effect of DQ, the expression of the DNA damage marker γH2AX was examined in lung tissues from each group using immunofluorescence staining. In the IR group, abundant γH2AX-positive nuclear foci were observed, indicating pronounced DNA damage. Quantitative analysis showed that compared with the control group, the IR group exhibited a significant increase (6.370 ± 1.772 vs. 1.000 ± 0.006, P < 0.001). DQ treatment significantly reduced γH2AX signal intensity in irradiated lungs (2.708 ± 0.254 vs. 6.370 ± 1.772, P < 0.01), corresponding to an approximately 57.5% decrease relative to the IR group (Figure 2).

Persistent DNA damage can lead to tissue dysfunction and ultimately trigger cellular senescence. SA-β-gal staining was used as a primary indicator to evaluate cellular senescence. Quantitative analysis demonstrated a marked increase in SA-β-gal–positive cells in the IR group compared with controls (33.23 ± 1.67 vs. 5.74 ± 0.74, P < 0.001). DQ treatment significantly reduced senescent cell accumulation in irradiated lungs (15.11 ± 1.68 vs. 33.23 ± 1.67, P < 0.001), corresponding to an approximately 54.5% reduction relative to the IR group (Figure 3A). These results indicate that DQ treatment effectively mitigates radiation-induced cellular senescence. Western blot analysis further demonstrated that irradiation markedly upregulated the expression of key senescence-associated proteins in lung tissues. Specifically, p53 protein levels were significantly increased in the IR group compared with controls (1.151 ± 0.023 vs.0.669 ± 0.212, P < 0.01), whereas DQ treatment significantly reduced p53 levels by approximately 32.0% (0.783 ± 0.035 vs. 1.151 ± 0.023, P < 0.05). Similarly, p21 expression was significantly elevated following irradiation (0.867 ± 0.058 vs. 0.456 ± 0.145, P < 0.01), and DQ suppressed this increase by about 33.8% (0.574 ± 0.051 vs. 0.867 ± 0.058, P < 0.05). In parallel, p16 levels were substantially increased in the IR group relative to controls (1.213 ± 0.128 vs. 0.344 ± 0.091, P < 0.001), and DQ attenuated this upregulation by roughly 38.8% (0.742 ± 0.129 vs. 1.213 ± 0.128, P < 0.01) (Figures 3B,C). Collectively, these findings indicated at the molecular level that combined DQ therapy effectively attenuates radiation-induced cellular senescence in lung tissue.

Transcriptomic sequencing was performed to further elucidate the molecular mechanisms underlying the protective effects of DQ against RILI, transcriptomic sequencing was performed to identify differentially expressed genes and signaling pathways involved in cellular senescence and apoptosis. The volcano plot of DEGs is presented in Figure 4A. GSEA identified significant differences in pathways related to cellular aging, including autophagy, apoptosis and mitophagy between the two groups (Figure 4B). These results suggested that DQ might exert its protective effects against RILI by modulating key signaling pathways involved in cellular stress and death. GO enrichment demonstrated the DEGs were predominantly enriched in oxidative phosphorylation under the biological process category, in mitochondrial-related components under the cellular component category, and in ATP synthase activity under the molecular function category (Figures 4C,D). KEGG pathway analysis further indicated that DQ treatment significantly affected multiple aging-related pathways, including the p53, MAPK, and PI3K-Akt signaling pathways, as well as mitophagy and oxidative stress responses (Figures 4E,F). These findings suggest that the senescence-clearing effects of DQ are closely associated with the regulation of apoptosis, mitochondrial metabolism, and oxidative stress pathways.

Senescent cells secrete various pro-inflammatory cytokines, chemokines, matrix metalloproteinases, and growth factors collectively known as the SASP. This phenotype has been reported to exert paracrine effects that promote senescence in neighboring normal cells (van Deursen, 2014; Hansel et al., 2020). Western blot analysis demonstrated that the expression levels of multiple SASP-associated inflammatory and fibrotic proteins were significantly increased in the IR group compared with the control group, including IL-6 (1.001 ± 0.072 vs. 0.666 ± 0.071, P < 0.05), IL-18 (0.885 ± 0.112 vs. 0.528 ± 0.047, P < 0.001), IL-1β (0.878 ± 0.009 vs. 0.262 ± 0.044, P < 0.001), TNF-α (0.938 ± 0.025 vs. 0.488 ± 0.185, P < 0.01), α-SMA (1.194 ± 0.102 vs. 0.556 ± 0.124, P < 0.01), TGF-β (1.123 ± 0.054 vs. 0.345 ± 0.051, P < 0.001), MMP2 (0.971 ± 0.038 vs. 0.602 ± 0.083, P < 0.05), and MMP9 (1.050 ± 0.052 vs. 0.706 ± 0.193, P < 0.05). Importantly, combined DQ treatment markedly attenuated the radiation-induced upregulation of these SASP components. Relative to the IR group, DQ treatment reduced IL-6 expression by approximately 30% (0.702 ± 0.092 vs. 1.001 ± 0.072, P < 0.05), IL-18 by 24% (0.673 ± 0.085 vs. 0.885 ± 0.112, P < 0.05), IL-1β by 24% (0.672 ± 0.031 vs. 0.878 ± 0.009, P < 0.05), TNF-α by 32% (0.636 ± 0.065 vs. 0.938 ± 0.025, P < 0.05), In parallel, profibrotic markers were also significantly suppressed, with α-SMA decreased by 35% (0.773 ± 0.220 vs. 1.194 ± 0.102, P < 0.05), TGF-β reduced by 37% (0.706 ± 0.131 vs. 1.123 ± 0.054, p < 0.001), Consistently, DQ treatment lowered the expression of matrix-remodeling enzymes, reducing MMP2 by 33% (0.654 ± 0.087 vs. 0.971 ± 0.038, P < 0.05), and MMP9 by 33% (0.705 ± 0.074 vs. 1.050 ± 0.052, P < 0.05). Collectively, these results indicated that ionizing radiation induced a robust SASP response in lung tissue, encompassing inflammatory, profibrotic, and extracellular matrix–remodeling components, while DQ treatment effectively suppresses this pathological SASP activation (Figure 5A) Immunohistochemical staining further corroborated these observations. Compared with controls, irradiation markedly increased TGF-β and MCP-1 expression in lung tissues (TGF-β: 14.51 ± 0.92 vs. 6.69 ± 1.19, P < 0.001; MCP-1: 18.48 ± 1.32 vs. 8.50 ± 0.68, P < 0.001). Following DQ treatment, TGF-β staining intensity was reduced by approximately 28% (10.52 ± 0.79 vs. 14.51 ± 0.92, P < 0.001), while MCP-1 expression decreased by 28% (13.32 ± 1.08 vs. 18.48 ± 1.32, P < 0.001) relative to the IR group (Figure 5B). These results further suggested that DQ eliminates radiation-induced senescent cells and mitigates SASP-associated profibrotic/proinflammatory responses in lung tissue.