| BackgroundRadiation-induced lung injuries are ineluctable and intractable adberse events-associated with radiotherapy for chest cancer,halting the remedy prematurely and reducing the life quality of patients.Among the comlications,radiation pneumonia(RP)is a disease that can easily occur.However,the therapeutic options for the radiation-induced injuries yielded disappointing results in clinical application to date,still lacking safe and effective strategies and drugs to battle against radiation-indued lung injuries.Fecal microbiota transplantation(FMT)has been applied clinically for the treatment of intestinal diseases;however,its shortcomings still exist covering side effects sucn as abdominal pain,diarrhea and unsuitability for large-scale application.Gut microbiota metabolites,such as short-chain fatty acids,have been corroborated to enter into circulatory system regulating distal organs,including brain,liver and lung,establishing "Gut-brain" axis,"Gut-liver"axis and "Gut-lung" axis etc.However,studies on metabolites of the gut microbiota and radiation lung injury are rare.AimIn the present project,we aimed to screen specific gut microbiota-derived metabolites for mitigating radiation-induced injuries and elucidate the underlying protective mechanism.MethodsA mouse model of acute lung injury was established using γ-radiation with a single local irradiation of 15 Gy to the chest of mice.After irradiation,mice were treated with FMT and prostaglandin F2α for 10 days,and lung tissues were collected on day 21 for the subsequent experiments.For the in vivo experiments,structural changes in mouse lung tissues were observed by Masson and Sirius red staining,and the respiratory metabolic cage was used to measure the amount of CO2 expired(VCO2),O2 consumed(VO2)and respiratory quotient(RQ)in the mice over 24 h.The levels of ROS was measured using malondialdehyde(MDA)kits,and the expression levels of inflammatory factors and SOD were measured using qRT-PCR and ELISA.Gut microbiota configuration and the gut microbiota metabolite profile were analyzed using 16S rRNA and non-targeted metabolomes.For the in vitro experiments,human-derived lung cells(BEAS-2B)and mouse-derived lung cells(MLE-12)were used to analyse cell proliferation by clonogenic assays.The changes in the expression of inflammatory factors,MAPK-related signalling pathways and apoptotic proteins were detected using Western Blotting,qRT-PCR,ELISA and immunofluorescence.Flow cytometry and Western Blotting were used to detect the number of apoptotic cells and apoptotic protein expression levels in each group.MAPKsilenced cell lines were constructed using siRNA fragments.ResultsChest local irradiation impaired alveoli structer and accumulated the collagen fibres,while FMT attenuated the structural lung damage.FMT also increased the RQ which was decreased by radiation exposure.Compared to the chest local irradiated mice,the levels of inflammatory factors(IL-6 and TNF-α),oxidative stress products(MDA)and superoxide dismutase(SOD)activities were reduced in the lung tissues after FMT.Further more 16S rRNA high-throughput sequencing revealed that the α-diversity of gut microbiota experienced an increase in Observed species after chest local irradiation but a decrease with FMT;Chao1,ACE and Shannon indices further validated the results.Together,the α-diversity of gut microbiota in irradiated mice with FMT was similar to that of the normal control group.Weighted unifrac analysis showed a chest local irradiation increased the β-diversity of the gut microbiota in the mice with or without FMT.However,unweghted unifrac analysis revealed a decrease in β-diversity after radiation challenge,but an increase with FMT.Principal component analysis(PCA)and weighted/unweighted principal coordinate analysis(PCoA)further showed a visible separation between the three groups.In addition,we used LC-MS/MS to analyse changes in gut microbial metabolites.Volcano plots showed that the profile of gut microbiota metabolites was altered after local chest irradiation and FMT restructured the taxonomic proportions of gut microbiota metabolites.Intriguingly,we observed that the level of the gut microbiota-derived metabolite prostaglandin F2α(PGF2α)was decreased after irradiation but increased after FMT;clone formation experiments showed that PGF2αprotected cells against radiation caused injury.PGF2α supplementation by the oral route elevated its concentration in mouse faeces,peripheral blood and lung tissue,prevented radiation-induced structural damage to alveoli and collagen accumulation,improved respiratory function(lower lung coefficients,higher RQ values)and reduced the release of inflammatory factors(IL-1,TGF-β1).In addition,clonogenesis experiments showed that PGF2α promoted the proliferation of irradiated BEAS-2B cells in a dose-dependent manner.The effect of 4μM PGF2α was suitable and this concentration was used for the next experiments.Flow cytometry showed that radiation exposure increased the number of apoptotic lung cells(from 4.8%to 11.4%);however,PGF2α treatment decreased that of apoptotic cells(from 11.4%to 4.6%)line with a decrease in Caspase-6 protein level.Decreased level of inflammatory factors(TGF-β1,IL-1 and TNF-α)was observed in the PGF2α treated group.PGF2α slightly increased the expression of F-prostaglandin receptors(FP),downregulated the expression of PI3K and AKT,up-regulated that of MAPK,ERK and its downstream gene NF-κB after irradiation.Immunofluorescence further showed that PGF2α treatment promoted the accumulation of NF-κB in the nucleus.To further verify the critical role of MAPK/NF-κB signalling in PGF2α-mediated radioprotection for lung cells,siMAPK fragments were transfected in BEAS-2B and MLE-12 cells respectively.Notably,silencing of MAPK increased the apoptotic cell number,reduced the levels of JNK,p38,ERK and NF-κB in both cell lines after irradiation and inhibited NF-κB accumulation in the nucleus.ConclusionsFecal microbiota transplantation ameliorates radiation lung injury.FMT reshapes gut microbiota configuration and metabolite profile after chest local irradiation.Gut microbiota-derived PGF2α mitigates radiation caused lung injuries in mouse models.PGF2α activates the FP/MAPK/NF-κB signaling pathway,promotes cell proliferation and inhibits radiation-induced apoptosis in lung cells.The siMAPK attenuates the protective effects of PGF2α on irradiated lung cells.Thus,PGF2α,a metabolite produced by gut microbes,plays radioprotective roles depending on the FP/MAPK/NF-κB axis,at least partly.Together,our study provides novel insight into gut microbiota-derived metabolites and suggests that gut microbiota-produced PGF2α might be employed as a potential radioprotective agent to fight against radiation-induced toxicity. |
