Fig 1.
Schematic of fecal microbiota transplantation efficacy assessment in RILI mouse models.
Fig 2.
Protective effects of FMT on lung tissue morphology and inflammatory response in RILI mice.
(A) The timeline of experimental interventions and sampling of this study. (B) Representative Sirius Red, Masson’s trichrome, and H&E staining of lung tissues (100× and 400×). (C-H) BALF levels of IL-1β, IL-4, IL-5, IL-6, TNF-α and IFN-γ quantified by ELISA. (I-J) GSH and MDA levels of lung tissue. (K) Lung wet/dry weight ratio across groups. (L) The change of body weight of each experimental mice after 20 Gy TLI. n = 6 per group. Data are presented as mean ± SD. Statistical comparisons were performed by one-way ANOVA with Tukey’s post hoc test (C-K) or two-way ANOVA with Tukey’s post hoc test (L). * p < 0.05, ** p < 0.01, *** p < 0.001.
Fig 3.
FMT restores gut microbiota composition and intestinal function in RILI mice.
(A) Representative images of intestinal motility across groups. (B) Intestinal AB-PAS staining. (C) Ratio of intestinal weight to body weight. (D) Venn diagram of the distribution of shared and unique microbial taxa among four groups. (E) LEfSe cladogram of taxa. (F) The NMDS and (G) the PCoA plots across groups. (H) Rank-abundance curves across groups. (I-L) Alpha-diversity indices of Observed features, Chao1, Dominance and Simpson. n = 6 per group. Data are presented as mean ± SD (C) or median ± IQR (I-L). Statistical comparisons were performed using one-way ANOVA with Tukey’s post hoc test (C), or Kruskal-Wallis test with Dunn’s post hoc comparisons/Benjamini-Hochberg FDR adjustment (I-L). **p < 0.01, ***p < 0.001.
Fig 4.
FMT alleviates gut microbiota dysbiosis induced by RILI.
(A-B) Volcano plots visualizing differential taxa between indicated groups. (C-K) Relative abundance of specific bacterial taxa across groups, including Bifidobacterium, Rikenellaceae RC9, Clostridium sensu stricto 1, Turicibacter, Parabacteroides, Monoglobus, Achromobacter, Lactobacillus and Ligilactobacillus. n = 6 per group. Data are presented as mean ± SD. Statistical comparisons were performed using one-way ANOVA with Tukey’s post hoc test or Kruskal-Wallis test with Dunn’s post hoc test (C-K). *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 5.
FMT restores unsaturated fatty acid synthesis and arachidonic acid metabolism in RILI mice.
(A) Principal component analysis (PCA) of distinct metabolic profiles among groups. (B) Volcano plots showing differentially expressed metabolites between indicated comparison groups. (C) Metabolite enrichment analysis identifying significantly affected pathways in indicated comparison groups. (D) Rain cloud plot showing the metabolic profiles of unsaturated fatty acids and arachidonic acid pathway metabolites across group, including arachidonic acid, adrenic acid, arachidic acid, stearic acid, prostaglandin A2, Prostaglandin F2α, 6-Keto-prostaglandin F1α, 15-Keto prostaglandin F2α. (E) Relative quantification of the metabolites in TLI + FMT vs TLI groups. n = 6 per group. Data are presented as mean ± SD. Statistical comparisons between indicated groups were performed using two-sided unpaired Student’s t-tests (E). * p < 0.05, ** p < 0.01, *** p < 0.001.
Fig 6.
Radiation-induced disruption of ER protein processing in lung tissue and enhanced ER adaptive response by FMT (A-C) KEGG pathway enrichment analysis showing significant enrichment of protein processing in endoplasmic reticulum in differentially expressed genes across indicated comparison groups.
(D) Heatmap illustrating the differential expression of key ER protein processing-related genes associated with the adaptive ER response across groups. (E) Transmission electron microscopy images showing ER morphology across groups. (F) Western blot of ER adaptive response-related proteins, including HSPA5, PERK, IRE1α, ATF6, FAM134B, and LC3B I/II, using lung tissue from mice. n = 3 per group.
Fig 7.
Multi-omics analysis of gut microbiota, metabolite, and lung transcriptome interactions.
(A-C) Contribution of gut microbiota to the levels of metabolites with microbiota represented by red arrows and metabolites by black arrows. Acute inter-arrow angles indicate positive correlations. n = 6 per group. (D-E) Key bacterial genus and metabolite correlations in the gut-lung axis. Orange lines: positive correlations. Green lines: negative correlations. Gray lines: non-significant correlations. n = 6 per group. (G-I) Correlation network heatmaps of unsaturated fatty acid metabolites and ER-related genes across groups in different comparisons. n = 3 per group. (J–L) Scatter plots showing correlations between arachidonic acid levels and ER-related genes (Fam134b, Hspa5 and Hspa8) in mice with matched transcriptomic-metabolomic profiles. n = 3 per group.
Fig 8.
Arachidonic acid enhances DNA repair in irradiated lung epithelial cells.
(A) Cell viability and death assays indicating that AA treatment reduces radiation-induced cell death. (B) Proportions of live (blue) and dead (red) cells across treatment. (C) Western blot showing decreased γH2AX expression of DNA repair proteins in irradiated lung epithelial cells treated with AA. (D) γH2AX foci analysis showing reduced DNA double-strand breaks in AA-treated irradiated cells. n = 6 per group.
Fig 9.
Effects of arachidonic acid on ER-phagy in irradiated lung epithelial cells.
(A-B) Immunofluorescence of HSPA5 (red) and FAM134B (green) in Beas-2b and MLE-12 cells. (C-D) Quantification of fluorescence intensity for HSPA5 and FAM134B in Beas-2b and MLE-12 cell lines. n = 5 per group. (E) Western blot of HSPA5 and FAM134B expression across groups in Beas-2b and MLE-12 cell lines. n = 6 per group. (F) Representative confocal microscopy images of ER (green) and lysosome (red) distribution across experimental groups. n = 6 per group. (G) Transmission electron microscopy images depicting ER morphology across groups. n = 6 per group. Data are presented as mean ± SD. Statistical comparisons were performed by one-way ANOVA with Tukey’s post hoc test (C-D). * p < 0.05, ** p < 0.01, *** p < 0.001.
Fig 10.
Arachidonic acid upregulates FAM134B-mediated ER-phagy in a PPAR γ-depended manner.
(A) Western blot of PPARγ expression in varying concentration of AA under irradiation. (B) Molecular docking analysis between PPARγ and the fam134b promoter. (C) Immunofluorescence staining of HSPA5 (red) and FAM134B (green) in Beas-2b cells with or without following PPARγ inhibitor (T0070907) administration. (D) Representative confocal microscopy images of ER (green) and lysosome (red) distribution across groups. (E) Western blot of FAM134B expression between the T0070907-treated group and the control group. (F) Consensus DNA binding motif for PPARγ. (G) ChIP-qPCR analysis of PPARγ occupancy at the Fam134b promoter region in cells treated with 10μM T0070907 or DMSO control. (H) Dual-luciferase reporter assay of PPARγ transactivation at the Fam134b promoter. (I) Schematic mechanism of gut microbiota-derived arachidonic acid mediates PPARγ-dependent activation of FAM134B-mediated ER-phagy to ameliorate RILI. n = 3 per group. Data are presented as mean ± SD. Statistical comparisons were performed by two-way ANOVA with Tukey’s post hoc test (G-H). * p < 0.05, ** p < 0.01, *** p < 0.001.
Fig 11.
Arachidonic acid protects against radiation induced lung injury in mice by Activating PPAR γ.
(A) The timeline of experimental interventions and sampling. (B) Representative Masson’s trichrome, and H&E staining of lung tissues (100× and 400×). (C-F) BALF levels of IL-1β, IL-4, IL-6 and IFN-γ quantified by ELISA. (G, H) GSH and MDA levels of lung tissue. (I) Lung wet/dry weight ratio across groups. (J) The change of body weight of each experimental mice after 20 Gy TLI. n = 6 per group. (K) Western blot of ER adaptive response-related proteins, including HSPA5, PERK, IRE1α, ATF6, FAM134B, and LC3B I/II, using lung tissue from mice. n = 3 per group. (L) Transmission electron microscopy images showing ER morphology across groups. Data are presented as mean ± SD. Statistical comparisons were performed using one-way ANOVA with Tukey’s post hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001.