Gut Microbiota-Derived PGF2α Fights against Radiation-Induced Lung Toxicity through the MAPK/NF-κB Pathway

Abstract

Radiation pneumonia is a common and intractable side effect associated with radiotherapy for chest cancer and involves oxidative stress damage and inflammation, prematurely halting the remedy and reducing the life quality of patients. However, the therapeutic options for the complication have yielded disappointing results in clinical application. Here, we report an effective avenue for fighting against radiation pneumonia. Faecal microbiota transplantation (FMT) reduced radiation pneumonia, scavenged oxidative stress and improved lung function in mouse models. Local chest irradiation shifted the gut bacterial taxonomic proportions, which were preserved by FMT. The level of gut microbiota-derived PGF2α decreased following irradiation but increased after FMT. Experimental mice with PGF2α replenishment, via an oral route, exhibited accumulated PGF2α in faecal pellets, peripheral blood and lung tissues, resulting in the attenuation of inflammatory status of the lung and amelioration of lung respiratory function following local chest irradiation. PGF2α activated the FP/MAPK/NF-κB axis to promote cell proliferation and inhibit apoptosis with radiation challenge; silencing MAPK attenuated the protective effect of PGF2α on radiation-challenged lung cells. Together, our findings pave the way for the clinical treatment of radiotherapy-associated complications and underpin PGF2α as a gut microbiota-produced metabolite.

Keywords: PGF2α; gut microbiota; gut microbiota metabolites; gut-lung axis; radiation pneumonia.

Figure 1

Faecal microbiota transplantation fights against radiation pneumonia in mice: (A) The changes in body weight of experimental mice after 15 Gy TLI. (B) The lung tissues were stained with Sirius red and Masson (100× and 400×). (C) Mouse pulmonary coefficient. (Con: n = 6, TLI and FMT: n = 10). (D,E) Respiratory quotient (RQ) and VO₂ intake of mice in 24 h (n = 6). (F,G) The inflammatory factor of IL-6 and TNF-α in lung tissues from the experimental mice analysed by ELISA (Con: n = 6, TLI and FMT: n = 10). (H) The MDA level in lung tissues of each group was examined (Con: n = 6, TLI and FMT: n = 10). (* p < 0.05, ** p < 0.01 and *** p < 0.001; Student’s t-test; FMT: 200 μL Faecal dissolving fluid/mouse).

Figure 2

FMT shapes the gut microbiota configuration of mice after local chest irradiation: (A–D) Analysis of α-diversity by 16S rRNA sequencing of the gut bacteria, mainly the observed species number (A), Chao1 diversity index (B), ACE diversity index (C) and Shannon diversity index (D). (E) The heat map is colour-based on row Z-scores. The mice with the highest and lowest bacterial level are in red and blue, respectively. (Statistically significant differences are indicated: Wilcoxon rank sum test, n = 6 per group, * p < 0.05 and *** p < 0.001).

Figure 3

FMT shifts the gut microbiota structure of local chest irradiation mice: (A,B) The β diversity of enteric bacteria was compared by weighted (A) and unweighted (B) unifrac analysis. (C–E) PCoA were used to examine the alteration of intestinal bacteria taxonomic pattern; (F–H) The relative abundances of g_Parabacteroides_s_ Parabacteroides_distasonis, g_Parabacteroides_s_Parabacteroides_goldsteinii and g_Faecalibaculum_s_ Faecalibaculum_rodentium at the species level was assessed using 16S high-throughput sequencing after TLI and FMT. (Statistically significant differences are indicated: Wilcoxon rank sum test, n = 6 per group, * p < 0.05, ** p < 0.01 and *** p < 0.001).

Figure 4

FMT remoulds the gut microbiota metabolome fluctuated by local chest irradiation: (A,B) Volcano plots of identified different metabolites in the faecal pellets from mice. In the volcano plots, each point represented a metabolite; (C) Heatmap of the differences in metabolites of faecal pellets from mice in TLI and Con group; (D) Heatmap of the differences in metabolites of faecal pellets from mice in TLI group and FMT group; (E) Screening out the metabolites in specific paradigms; (F) The effects of PGF2α, Micronomicin, TMAO and L-Histidine on irradiated BEAS-2B cells were measured by clone formation assay; (G,H) The expression level of PTGS2 in tumour and peritumour was analysed; (I,J) Kaplan-Meier analysis of the overall survival rate of LUAD and LUSC patients with different expression of PTGS2. p < 0.05 by log-rank test between the patients with high and low expression of PTGS2 in LUAD.

Figure 5

The intestinal flora metabolite PGF2α improves the radiation-induced lung toxicity in mice: (A) The experimental roadmap; (B) The level of PGF2α in lung pages of mice was analysed by ELISA (n = 8); (C) Body weight of mice were compared among Con, TLI and PGF2α treatment groups; (D) The body weight of mice on the 3rd and 21st day after 15 Gy irradiation with or without PGF2α treatment (n = 8); (E,F) Tissue morphology was observed at ×100 and ×400 using tissue staining (Sirius Red and Masson staining); (G) The pulmonary coefficient in three groups of mice; (H,I) Respiratory quotient (RQ) and VO₂ intake of mice in each group in 24 h (n = 6); (J–L) The levels of TGF-β1 (J), IL-1β (K) and IL-18 (L) in the lung were measured by ELISA. (* p < 0.05, ** p < 0.01 and *** p < 0.001; Student’s t-test).

Figure 6

PGF2α activates FP/MAPK/NF-κB signalling pathway to inhibit radiation-induced lung cell apoptosis: (A) The effect of PGF2α on radiation-exposed BEAS-2B cells was assessed by clone formation assay. (B) The apoptosis of irradiated BEAS-2B cells was analysed by flow cytometry. (C) The Caspase-6 expression was examined by Western blotting in irradiated BEAS-2B cells. (D–F) The expression levels of PI3K and AKT in irradiated BEAS-2B cells with or without PGF2α treatment were measured by qRT-PCR and Western blotting. (G–J) The expression levels of MAPK, ERK and NF-κB in irradiated BEAS-2B cells with or without PGF2α treatment were measured by qRT-PCR and Western blotting. (K,L) Protein-level expression of JNK (K) and p38 (L) of lung tissue (in vivo) were measure by immunofluorescent staining. (M) Immunofluorescence showed the expression and location of NF-κB in irradiated BEAS-2B cells. (* p < 0.05, ** p < 0.01; Student’s t-test).

Figure 7

Blocking MAPK attenuates the protective effect of PGF2α on lung cells following radiation: (A) The interference efficiency of siRNA targeting MAPK was examined by Western blotting in BEAS-2B cells. (B) The apoptosis of irradiated BEAS-2B cells was analysed by flow cytometry. (C) The caspase-6 expression was examined by Western blotting in irradiated BEAS-2B cells. (D) The expression levels of JNK, p38, p-ERK and NF-κB in irradiated BEAS-2B cells were assessed by Western blotting. (E) Immunofluorescence showed the expression and location of NF-κB in irradiated BEAS-2B cells. (F) The interference efficiency of siRNA targeting MAPK was examined by Western blotting in MLE-12 cells. (G) The caspase-6 expression was examined by Western blotting in irradiated MLE-12 cells. (H) The JNK, p38, p-ERK and NF-κB expression in irradiated MLE-12 cells were examined by Western blotting. (I) Immunofluorescence showed the expression and location of NF-κB in irradiated MLE-12 cells. (J,K) Kaplan–Meier analysis of overall survival and relapse free survival rate of MAPK expression in lung cancer patients. (** p < 0.01 and *** p < 0.001; Student’s t-test).