Female C57BL/6J wild-type (WT) mice of 6–8 weeks (18, 19), weighing 18 – 22 g, were provided by Vital River Company (Beijing, China). All mice were housed under specific-pathogen-free conditions at the animal facility of The First Affiliated Hospital of Shandong First Medical University & Shandong Provincial Qianfoshan Hospital. The experimental animals were allowed to at least 1 week of adaptive feeding, before being utilized in the experiments. All animal care and protocols were approved by the Institutional Animal Care and Use Committee of The First Affiliated Hospital of Shandong First Medical University & Shandong Provincial Qianfoshan Hospital (No. 2022-S303), in compliance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH; Bethesda, MD, USA; publication No. 96-01). In order to investigate the effects of radiation on lung damage in mice, the mice were divided into two groups: non-radiation control group (CON) and single radiation group (RT) ( Table 1 , n=5 per group). To test the effect of NA on lung tissue after irradiation, nervonic acid was added to the above model of RILI. The mice were divided into 3 groups: non-radiation + corn oil (CON+OIL), radiation + corn oil (RT+OIL) and radiation + NA (RT+NA) groups ( Table 1 , n=5 per group, Corn oil and NA were given by gavage at a dose of 100mg/kg/every other day). The mice were observed daily and weighed weekly to ensure that the interventions were well tolerated.

Nervonic acid (NA) was provided by Shandong Academy of Agricultural Sciences, China. The chemical structure of NA was presented in Supplementary Figure 1 . Based on the previous study, NA dissolved in corn oil was administered orally to mice at a dose of 100 mg/kg by gavage (14). The initiation of nervonic acid treatment commences on the first day following radiation. Nervonic acid were given every other day, and this regimen is maintained consistently until the end of the observation period.

The mice were anesthetized with 3% isoflurane and irradiated with either a single dose of 0 Gy (non-radiation control and non-radiation + corn oil groups) or with 13 Gy over their whole thorax irradiation (WTI) using a RS 2000 pro Biological X-ray Irradiator (Rad Source Technologies, Buford, GA, USA). The mice were euthanized using a 30% volume per minute displacement rate of 100% CO₂ at 2, 4, 6 weeks after irradiation, and lung tissues were collected and processed for the analysis of RNA, protein expression levels, flow cytometry and histopathological examinations.

Total messenger RNA (mRNA) was extracted from part of freshly pulverized lung tissues using FastPure Cell/Tissue Total RNA Isolation Kit V2 (Vazyme, Nanjing, China; catalog No. RC 112- 01). The lung tissues were derived from CON group, RT group, CON+OIL group, RT+OIL group, RT+NA group. Then, HiScript III RT SuperMix for qPCR (+gDNA wiper) (Vazyme; catalog No. R323-01) was used to synthesize the complementary DNA (cDNA) according to the manufacturer’s protocol. The RT-qPCR sequences of each gene primers were as follows: β-actin, forward: 5’-GGCTGTATTCCCCTCCATCG-3’, reverse: 5’-CCAGTTGGTAACAATGCCATGT-3 ‘; tumor necrosis factor (Tnf), forward: 5’-CGGGCAGGTCTACTTTGGAG’, reverse: 5’-ACCCTGAGCCATAATCCCCT-3’; transforming growth factor beta1 (Tgfb1),forward: 5’-AGCTGCGCTTGCAGAGATTA-3’, reverse: 5’-AGCCCTGTATTCCGTCTCCT-3’; arginase1(Arg1), forward: 5’-TGTCCCTAATGACAGCTCCTT-3’, reverse: 5’-GCATCCACCCAAATGACACAT-3’; chitinase-like 3 (Chil3), forward: 5’-CTGGAATTGGTGCCCCTACAAT-3’; reverse: 5’-AGACCTCAGTGGCTCCTTCATT-3’, nitric oxide synthase 2 (Nos2), forward: 5’-CCTGCTTTGTGCGAAGTGTC-3’, reverse: 5’-CCCAAACACCAAGCTCATGC-3’. Gene expression levels were normalized to β-actin calculated by 2−ΔΔCt method (20).

Protein extraction from mice (CON+OIL group, RT+OIL group, RT+NA group) lung tissues was performed following standard protocol (21). The protein concentrations were estimated using BCA Protein Assay Kit (Solarbio, Beijing, China; catalog No. PC0020) to ensure equal loading of protein were subjected to immunoblotting. Samples were resolved on 10% sodium dodecyl sulfate (SDS) polyacrylamide gel, transferred onto a polyvinylidene difluoride (PVDF) membrane (Solarbio) and then incubated with the primary and secondary antibodies. The following primary antibodies were used: IκBα [Servicebio, Wuhan, China (CST); 1:1,000], p-IκBα (CST; 1:1,000), β-actin (Boster; 1:1,000). Finally, an enhanced chemiluminescence (ECL) reagent (Boster, catalog No. AR1173) was used to detect labeled protein bands. The density of bands was quantified by ImageJ software (version 1.62; NIH, USA).

Lung tissues were perfused (with PBS buffer) and then fixed overnight with 4% (w/v) paraformaldehyde in PBS (pH 7.2). Upon dehydration, lungs were embedded in paraffin, sectioned into 4-μm slices by paraffin microtome. Place the slices in a thermostat oven to dry them. Clear the paraffin from the slices in xylene. Then transfer the slices to ethanol (100%, 90%, 80%, 70%) and distilled water. Stain the slices in hematoxylin solution and rinse the slices under running tap water. Stain the samples in working eosin Y solution. Place the slices sequentially into 80%, 90%, 95% and 100% ethanol for dehydration. Clear the samples in xylene. Add a drop of neutral balsam onto the tissue, cover it with a coverslip, and allow it to set. After 24 hours, place them under a microscope for observation and photography.

Lung tissues with or without radiation were removed on week 2, 4, 6 after whole thorax irradiation. Lungs were manually minced with scissors, digested in a complete Roswell Park Memorial Institute (RPMI) medium containing DNase I 0.2 mg/mL, Solarbio, catalog No. D8071) and collagenase IV (1 mg/mL, Solarbio, catalog No. C8160) at 37°C in a 5% CO₂ incubator for 1.5 h. The digested segments were grounded and filtered through a 70-mm cell strainer. The cells were collected from the interphase of an 80% and 40% Percoll gradient after spinning at 2,500 rpm for 15 min at room temperature. The CD16/32 antibody (eBioscience) was used to block the Fc receptors before surface staining. In order to detect the expression and quantity changes of macrophages, lung tissues were stained with antibodies against the following markers: Fixable Dye (AF405), CD45 (FITC), Ly-6G (PerCp-Cy5.5), CD11b (APC-Cy7), CD11c(PE-Cy7), Siglec-F(APC), F4/80(PE) (22). These antibodies were incubated at 4°C for 40 min. Cells were then resuspended in phosphate-buffered saline [PBS; 0.05% bovine serum albumin (BSA)] and fixed in 4% paraformaldehyde fixative (Solarbio). All samples were assayed by FACSAria II Cell Sorter [Becton, Dickinson, and Co. (BD) Biosciences, Franklin Lakes, NJ, USA] and data analysis was performed with FlowJo Software (Tree Star, Ashland, OR, USA).

Statistical analysis was performed using GraphPad Prism 8.0 (GraphPad Software, San Diego, CA, USA) and SPSS statistical software 25.0 (IBM Corp., Armonk, NY, USA). P values were determined using two-tailed Student’s t-test for comparing two groups and one way ANOVA for comparing multiple groups (23). Data were expressed as the mean ± standard deviation (SD). Statistical significance was indicated when P<0.05.

To determine the effects of radiation on lung tissue, we established a mouse model of RIIL by exposing the whole lung to a dose of 13 Gy. ( Figure 1A ). The measurement of weight served as an indicator of the general health condition in mice. After 13 Gy local irradiation, changes in body weight between the radiation group and the non-radiation control group of mice were compared. At 1 week after irradiation, the weight of the mice in the radiation group decreased, and then began to grow slowly. After 6 weeks the weight of the mice decreased again. There were significant differences in body weight between the radiation and the control groups at 1 (p=0.0271), 2 (p=0.0004), 4 (p=0.0004), and 6 (p=0.0009) weeks post irradiation ( Figure 1B ). Compared with the control group, mice exhibited gradual hair loss and whitening in the area of exposure from 2 weeks. ( Figure 1C ).

Next, we analyzed H&E stained sections of lung tissue from mice. A 12-point scoring system were employed to quantitatively analyze the histopathological features of HE-stained pathological sections, assessing alveolar congestion, hemorrhage, neutrophils in airspace, and interstitial thickening (each item was scored from 0 to 3 based on severity). Compared to the control group, the radiation group showed significantly higher scores (24). At 2 weeks post irradiation, lung tissues showed congestion, and inflammatory cells in lung tissue increased. At 4 weeks, lung tissue congestion, hemorrhage, and more infiltration of inflammatory cells were observed, and to continue at 6 weeks as manifested by lung tissue destruction, interstitial thickening, and obviously severe infiltration of inflammatory cells ( Figure 1D ). Therefore, it was demonstrated that radiation led to damage in lung tissue.

To investigate the response of macrophages in RILI, the C57BL/6J mice were irradiated with a single dose of 13 or 0 Gy (control group) to the whole thorax and the lung tissues were analyzed at different time points using flow cytometry. Total macrophages were gated based on CD45⁺, CD11c^-, CD11b⁺, F4/80⁺ staining ( Figure 2A , the first line). The resting state of the lung is composed of AMs distributed in the alveolar cavity, whereas IMs were distributed in the interstitium. AMs were identified by a gating strategy developed by monitoring CD45⁺, Ly-6G^-, CD11b^-, CD11c⁺,Siglec-F⁺ ( Figure 2A , the third line). In contrast, IMs were marked CD45⁺, Ly-6G^-, CD11b⁺, F4/80⁺ ( Figure 2A , the second line). Macrophages, as innate immune cells, are recruited to the site of injury where they perform their phagocytic functions. Therefore, post irradiation, macrophages are recruited to the irradiated site. The direct effects of radiation on macrophages are not yet clear (11). The results demonstrated that the total proportion and count of macrophages in the radiation group were significantly higher than those in the non-radiation group at 2, 4, 6 weeks post irradiation ( Figure 2B ). Due to the specific anatomical structure of lung tissue, the dynamic changes of AMs and IMs participate in the complex intercellular signaling transduction in the microenvironment of lung injury, and play a key role in regulating the immune response and maintaining homeostasis in the lung. To further explore the changes of AMs and IMs, further gating analysis was performed using flow cytometry. The number and proportion of AMs decreased at 2 weeks in irradiated lungs, with AMs to account for 24.68% versus 5.61% in control and radiation group, respectively (p=0.0018). The number of AMs in the control group was at 0.6224×10⁵ cells, while in the radiation group, the number was at 0.06401×10⁵ cells (p=0.0058). The number and proportion of AMs at 4 weeks in irradiated lungs still showed a downward trend, though such trend was lower than that at 2 weeks (p=0.0003 and p=0.0043). The proportion and number of AMs recovered in the lungs post irradiation after 6 weeks was measured, with proportion of AM in the control and radiation group found comparable at ca. 40% (p=0.9867). In contrast, AMs count was 0.7439×10⁵ cells in the control group and 1.164×10⁵ cells in the radiation group (p=0.0372) ( Figure 3A ). However, the number and proportion of IMs showed a different pattern with an initial increase followed by a decline. 2 weeks post irradiation, the proportion of IMs was 3.24% in the control group and 10.52% in the radiation group (p < 0.0001). The number of IMs in the control group was at 1.145×10⁵ cells and 3.704×10⁵ cells in the radiation group (p=0.0012). The number(p=0.0004)and proportion(p=0.0012)of IMs at 4 weeks post irradiation was still increased. By 6 weeks post irradiation, the proportion of IMs began to decrease. The proportion of IMs in the control group was 3.14% versus 2.88% in the radiation group (p=0.7282). The amount of IMs at 6 weeks post irradiation was lower than that at 2 and 4 weeks post irradiation. At 6 weeks post irradiation, the amount of IMs in the control group was at 1.043×10⁵ cells versus 2.166×10⁵ cells in the radiation group (p=0.0030) ( Figure 3B ).

At 2 and 4 weeks post irradiation, the mRNA expression of Tnf in radiation group was significantly higher (2 weeks: p=0.0289; 4 weeks: p=0.0006) than that in control group ( Figure 3C ) ( Supplementary Figure 2 ). Similarly, at 2 weeks post irradiation, Arg1 mRNA increased, but there was no statistical significance( Supplementary Figure 2 ). At 4 weeks post irradiation, Tgfb1(p=0.0453) and Arg1 (p=0.0108) mRNA expression levels in the radiation group were significantly higher than those in the control group ( Figure 3C ) ( Supplementary Figure 2 ). The release of other factors was likewise monitored including Chil3, and Nos2. The expression of Chil3 showed a fluctuating trend, which was down-regulated at 2 weeks and up-regulated at 4 weeks after irradiation. In contrast, no significant difference in Nos2 expression was detected ( Supplementary Figure 2 ).

NA, a very long chain fatty acid belonging to omega-9, can promote the repair and regeneration of damaged tissue, thereby reducing inflammation caused by exogenous factors (25). To detect whether NA can alleviate RILI, the mice were randomly divided into three groups (non-radiation + corn oil, radiation + corn oil and radiation + NA groups, n=5 per group, 100mg/kg/every other day)( Figure 4A ). At 1 week post irradiation, the body weight of the radiation group decreased, while the weight loss of the RT+NA group was less than that in the RT+OIL group. The body weight of the mice showed gradual increase, with the body weight of the radiated group found lower than that of the non-irradiated group. The difference was particularly significant at 6 weeks after irradiation (CON+OIL group vs. RT+OIL group: p<0.0001; CON+OIL group vs. RT+NA group: p=0.0045). In contrast, there was no significant difference in body weight between RT+OIL group and RT+NA group at the early stage of radiation damage (1 week: p=0.6717; 2 weeks: p=0.6318). At 4 and 6 weeks post irradiation, weight loss was significantly reduced in the RT+NA group compared to the RT+OIL group (4 weeks: p=0.0271; 6 weeks: p=0.0071) ( Figure 4B ). With the change of time after irradiation, the hair in the exposed area gradually turned white and lost, which was particularly significant at 6 weeks. In the RT+OIL group, these changes were extremely distinct compared to those of the RT+NA group. ( Figure 4C ).

To further verify the alleviating effect of NA on RILI, we performed HE staining on lung samples. As above, the slices were scored on a 12-point scale. It was found that the changes of alveolar congestion, hemorrhage, presence of neutrophils in the airspace, level of interstitial thickening, and destruction of lung tissue structure gradually aggravated with time after irradiation. Albeit, the most severe damage to the lung tissue was observed in RT+OIL group ( Figure 4D ).

It is well established that macrophages play a key role in the pathogenesis of RILI. In order to test whether NA could affect the response of macrophages in RILI, flow cytometric analysis was performed on lung tissue from mice at 2, 4, and 6 weeks after radiation. The gating strategy was the same as described above. At 2 weeks, the proportion and count of AMs in the RT+OIL group and RT+NA group were significantly lower than those in the CON+OIL group. Compared with RT+OIL group, the proportion and quantity of AMs in RT+NA group was found higher. This trend was more significant at 4 weeks post irradiation. The proportion of AMs in RT+OIL group was 1.98% versus 14.48% in RT+NA group (p=0.0363). The number of AMs in the RT+OIL was 0.02106×10⁵ cells and was 0.1363×10⁵ cells in the RT+NA group (p=0.0216). At 6 weeks in the irradiated lungs, the number and proportion of AMs began to rise. The AMs proportion in CON+OIL group was 46.60% versus 56.92% and 36.10% in the RT+OIL and RT+NA groups, respectively. The AMs recovery of RT+NA group was lower than that of RT+OIL group. There was no statistical significance in AMs count among all groups, but it showed an upward trend ( Figure 5A ). However, the changes of IMs were as follows: After 2 weeks of radiation, the number and proportion of IMs in the radiation group were significantly higher than those in the non-irradiated group. In the radiation group, the number and proportion of IMs in RT+OIL group were significantly higher than those in RT+NA group. The trend was more pronounced at 4 weeks. The proportion of IMs in RT+OIL group was 13.08%, and that in RT+NA group was 7.57% (p < 0.0001). The IMs count of RT+OIL group was 3.635×10⁵ cells, versus lower levels of IMs count in RT+NA group at 1.388×10⁵ cells (p < 0.0001). This trend was disrupted at 6 weeks, with both the number and proportion of IMs in the radiation group showing a decreasing trend. Although there was no statistical significance between the two groups, the number and proportion of IMs in the RT+NA group showed a lower decreasing trend than that in the RT+OIL group ( Figure 5B ). The above results indicated that NA may affect the changes in the number and proportion of macrophages, especially IMs.

At 2 weeks post irradiation, there was no significant change in the levels of inflammatory factors in lung tissue. At 4 weeks post irradiation, Arg1 mRNA expression in RT+NA group was significantly decreased compared with that in RT+OIL group (p=0.0113). The mRNA expression levels of Tgfb1and Tnf also showed similar difference at 4 weeks, with the differences though not statistically significant ( Figure 5C ). Similarly, at 6 weeks post irradiation, mRNA expression levels of Nos2, Tgfb1, Arg1, Chil3 in RT+NA group were less than that in RT+OIL group, with a non-significant difference ( Supplementary Figure 3 ).