Twenty-four 8- to 10-week-old male C57BL/6 mice weighing approximately 20-22 g were purchased from Hunan SJA laboratory animals Co. Ltd (China), housed in a specific-pathogen-free (SPF) animal facility at the laboratory animal center of Huazhong Agricultural University and fed a standard chow diet ad libitum. All mice used in the present study were acclimatized at least for 5 days before any experimental treatment and maintained under a 12-h light/dark cycle. Before irradiation, the mice were randomly divided into three groups as follows: (1) sham control group, that is, mice were anesthetized by intraperitoneal injection of Avertin (250 mg/kg) without irradiation, (2) vehicle group, where the mice were irradiated under anesthesia using Avertin i.p. and treated with sterile saline, and (3) FFT-treated group, the irradiated mice were gavaged with the FFT preparation at the predetermined time points. The detailed administration schedule is shown in Figure 1A . Entire experimental procedures involving mice were performed and approved by the experimental animal welfare ethics committee of Huazhong Agricultural University (license number HZAUMO-2022-0150).

A RS-2000-PRO Biological System X-ray irradiator (Rad Source Technologies, Inc., Florida, America) was used in our study. The mice were subjected to a single dose of 10 Gy total abdominal irradiation at a dose rate of 1.053 Gy/min under anesthesia using a special device equipped with the irradiator, so that the whole abdomen was irradiated while the rest of the mouse body was shield by a lead shielding. Following irradiation, the mice were returned to the animal facility, monitored daily, and evaluated for clinical scoring consisting of seven parameters such as weight loss, temperature change, physical appearance, posture, mobility, food consumption, and hydration, as previously described (Guo et al., 2020).

Fresh stool was collected from 12 gender- and age-matched donor mice from three vendors (four mice from each vendor), and then pooled together to maximize the diversity of viral profile. The fecal bacteria-free suspension was prepared according to a recent study reported by Ott et al. (Ott et al., 2017), with minor modifications. More details are presented in Additional file 1. Mice in FFT-treated group were administered with a 200 microliters aliquot of prepared suspension via oral route on the day before irradiation and daily after irradiation for 7 days. While sham and vehicle groups of mice received an equal volume of sterile vehicle solution as above.

Fourteen days after irradiation, all 24 recipient mice were euthanized by cervical dislocation, and then the small intestine, spleens, and kidneys were excised and weighted correspondingly. The entire colon segments were also harvested and measured on day 14 following irradiation.

Cytokine expression was determined by quantitative real-time polymerase chain reaction (qPCR). All experimental mice were sacrificed 14 days post radiation and the colonic segment was harvested. Total RNA was then extracted using FastPure Cell/Tissue Total RNA Isolation kit (Vazyme, Nanjing, China) according to the manufacturer’s instructions. Thereafter, reverse transcription was conducted with HiScript III RT SuperMix for qPCR (+ gDNA wiper) (Vazyme, Nanjing, China) following the manufacturer’s protocols. The qPCR was performed using ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China) in a 20 microliters reaction system according to the manufacturer’s guidelines. The primer sets used in this study are listed in Additional file 1. Gapdh was used as the internal reference gene.

For preparation of serum samples, peripheral blood was obtained from mouse orbital sinus, centrifuged at 1500 rpm for 15 min at 4°C, snap frozen in liquid nitrogen and stored at -80°C until analysis. The concentrations of target proteins in serum were determined by mouse enzyme-linked immunosorbent assay (ELISA) kits (mlbio, Shanghai, China) in accordance with the standard protocols. Absorbance of each well was measured at 450 nm with a microplate reader.

Colonic segments were harvested and fixed in 4% paraformaldehyde for at least 24 h at room temperature for histopathological evaluation. For this purpose, the fixed colonic segments were dehydrated, paraffin embedded, chopped into 4-μm sections and stained with hematoxylin and eosin (HE). The slides were evaluated by a GI pathologist blinded to study designs using a semiquantitative radiation injury score (RIS) system consisting of seven histopathology items, as described previously (Langberg et al., 1996; Demirer et al., 2006).

After irradiation at day 14 of 10 Gy TAI, mice were sacrificed under anesthesia. The ileum specimens were harvested, washed in ice-cold phosphate buffer saline (PBS), fixed in 4% paraformaldehyde overnight and chopped into 4-μm sections. Endogenous peroxidase activity was quenched with fluorescence quenching agent for 5 min and rinsed with running water for 10 min. Sections were blocked with bovine serum albumin for 30 min, then incubated with primary antibodies against γH2AX (Abcam, ab81299) overnight at 4°C and washed with PBS. After incubating with secondary antibodies in the dark for 50 min, the slides were subsequently counterstained with 4’, 6-diamidino-2-phenylindole (DAPI). All slides were observed under fluorescence microscope (Eclipse 80i, Nikon) and images were collected.

Fecal pellets were collected from each mouse before sacrificed, snap frozen in liquid nitrogen and stored at -80°C for experimentation. Fecal microbial DNA was extracted using the E.Z.N.A.^® stool DNA Kit (Omega Bio-tek, Norcross, GA, USA) according to the manufacturer’s protocols. Then, the DNA obtained was quantified (NanoDrop 2000) followed by library constructed (TruSeq™ DNA Sample Prep Kit, Illumina). The qualified libraries were amplified with a cBot TruSeq PE Cluster Kit (Illumina) and sequenced on an Illumina HiSeq 2000 platform (Truseq SBS Kit v3-HS) with pair-end 150 bp (PE150) mode, with an average sequencing depth of 86, 253,400 reads (13G raw data) per sample. All the raw sequencing data has been deposited into the NCBI public repository with the BioProject ID PRJNA1047338. The raw sequence preprocessing, quality control and optimization and taxonomic annotation are presented in Additional file 1.

Mice feces were used for metabolomics analysis. For this purpose, an ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) system (ACQUITY UPLC-Xevo TQ-S, Waters Corp., Milford, MA, USA) was used to quantify metabolites present in feces. Briefly, three to five fecal pellets from each mouse were thawed on ice-bath to diminish degradation, homogenated with zirconium oxide beads and methanol containing internal standard added to extract the metabolites, followed by centrifugation at 18,000 g for 20 min. Subsequent procedures were conducted on the Eppendorf epMotion Workstation (Eppendorf Inc., Humburg, Germany). ACQUITY UPLC BEH C18 1.7 µm VanGuard pre-column (2.1× 5 mm) and ACQUITY UPLC BEH C18 1.7 µm analytical column (2.1 × 100 mm) were employed to determine compounds to be tested. The column temperature was set at 40°C and the flow rate of mobile phase was 0.4 mL/min. The raw data files generated by UPLC-MS/MS were processed using the TMBQ software (v1.0, Metabo-Profile, Shanghai, China), which can perform a collection of data processing, interpretation, and visualization.

All data are reported as mean ± standard error of the mean (mean ± SEM). Statistical differences between two groups were analyzed using either Student T test or the Mann-Whitney U test based on data normality distribution and variance similarity, which were assessed by the Kolmogorov-Smirnov normality test with Dallal-Wilkinson-Lillie for P value. Statistical analysis was completed using GraphPad Prism software (version 8.0.1, San Diego, CA, USA). Result with P < 0.05 was defined as statistically significant.

It has been reported that the gut microbiota might be substantially altered by radiation challenge. To determine whether fecal bacteria-free filtrate plays a role in maintaining intestinal homeostasis, we performed FFT in a mouse model to unravel the potential of the sterile fecal filtrate fraction. The radiation exposure significantly reduced body weight in both vehicle- (P < 0.0001) and FFT-treated mice within the first 5 days relative to sham-irradiated mice ( Figure 1B ). Intriguingly, FFT treatment clearly erased (P < 0.0001) the body weight loss compared with vehicle group of mice ( Figure 1D ), and we also found that there was no statistically significant between the FFT and sham treatment groups at termination of the experiment ( Figure 1D ). Besides that, the clinical score was significantly higher (P < 0.05) in vehicle treatment group than in sham control group, whereas orally administered FFT markedly reversed the tendency ( Figures 1C, E ).

At termination, the abdominal organs and tissues, including the entire small intestine, colon section, spleens and kidneys, were harvested and measured to identify whether FFT treatment had a radioprotective effect against radiation caused GI injury. As shown in Figure 2A , the relative weight of small intestine was overtly decreased (P < 0.05) in vehicle treated mice than in sham controls, whereas FFT treatment remarkedly augmented (P < 0.01) the relative weight of small intestinal. In parallel, the size and relative weight of the spleens were also significantly reduced (P < 0.01) in mice subjected to 10 Gy of TAI challenge compared with sham irradiated mice; nevertheless, FFT treatment slightly abrogated (P < 0.05) the changes ( Figure 2B ). The kidney index, which refers to the percentage of wet kidney weight relative to body weight, was decreased slightly in irradiated mice with or without FFT gavage compared with sham control mice, although there was no statistical difference between the two groups ( Figure 2C ). As expected, TAI exposure resulted in shortened colorectal length, but FFT regimen significantly prolonged (P < 0.01) colorectal length ( Figures 2D, E ). Taken together, these observations suggest that FFT treatment ameliorates radiation caused GI toxicities.

To further interrogate how FFT treatment protects mice against radiation-induced GI structure and function damage, we first stained the colonic tissues of mice with HE staining to conducted overall histopathological evaluation. Histopathological analysis showed that RIS score of the FFT group was significantly lower (P < 0.001) than that of the vehicle group ( Figures 3A, B ), indicating that FFT might exert a protective effect on intestinal injury caused by radiation. Immunofluorescence staining of γ-H2AX, a marker of DNA damage, further validated that FFT treatment significantly reduced (P < 0.001) radiation induced DNA damage in intestinal cells compared with vehicle group ( Figures 3C, D ).

To better understand the mechanisms by which FFT treatment protects the intestinal tract from radiation induced dysfunction, colonic tissue was used for determination of mRNA expression using qPCR. And results suggested that FFT treatment significantly enhanced (P < 0.05) the intestinal integrity in mice subject to TAI ( Figures 3E, F ; Figure S1 ). In parallel, the relative mRNA expression levels of proinflammatory cytokines, including IL-1β (P < 0.01), TNF-α (P < 0.05) and IL-8 (P < 0.01), were dramatically reduced in FFT group compared with vehicle controls ( Figures 3G, H, K ). Notably, FFT treatment also significantly decreased (P < 0.001) the extent of fibrosis and heightened (P < 0.05) the anti-inflammatory cytokine expression ( Figures 3I, J ). Together, these findings indicate that FFT treatment contributes to the recovery of intestinal integrity and function following radiation exposure.

As previously stated, it is obvious that FFT may successfully alleviate radiation induced local intestinal dysfunction by maintaining intestinal integrity and cytokines expression equilibrium, however it is uncertain if FFT has an influence on systemic inflammatory response. Accordingly, we determined the levels of proteins involved in proinflammatory and/or anti-inflammatory response in serum samples using ELISA kits. Consistent with the qPCR results described above, the protein contents of proinflammatory cytokines in serum such as IL-1β (P < 0.01), IL-6 (P < 0.001), IL-12 (P < 0.001) and TNF-α (P < 0.01), were dramatically higher in mice only receiving radiation than those in sham controls, but lower in FFT treatment group compared with mice receiving radiation only ( Figures 4A-D ). Furthermore, FFT treatment significantly elevated (P < 0.01) the protein contents of anti-inflammatory cytokines in serum compared with vehicle group ( Figures 4E, F ). Radiation challenge can result in the activation of Nrf2 and an increase in MDA (Lu et al., 2019). Additionally, we saw that the levels of Nrf2 (P < 0.01) and MDA (P < 0.05), which have become markers of the formation and removal of reactive oxygen species, were significantly greater in the FFT treatment group compared to the vehicle group ( Figures 4G, H ). Collectively, our results obtained demonstrate that FFT plays a role in regulating radiation-challenged systemic inflammatory response.

We analyzed the structure and composition of fecal microbiome at termination, with emphasis on the effect of FFT treatment on fecal bacterial composition. In general, interventional effects were seen in both fecal bacterial and viral compositions. Specifically, the fecal bacterial composition changed significantly (P < 0.05) in vehicle group mice relative to sham controls, and the bacterial composition of FFT group mice was likewise different from (P < 0.05) the vehicle group mice ( Figure 5A ). Interestingly, the difference between the FFT and the sham group in gut microbiome was not significant (p = 0.079, Figure 5A ). Changes in fecal virome component across groups were similar to that of the bacteria. Results of analysis of similarities (Anosim) of the Bray-Curtis dissimilarity metrics showed that the fecal viral communities in the FFT group were significantly (P < 0.05) different from those in the vehicle group, whereas no significant differences were found in the viral communities of the sham compared with the vehicle group and the sham with the FFT group ( Figure 5B ). There was no significant difference in bacterial Shannon index between the FFT group and the sham group, but interestingly, the bacterial Shannon index of the FFT group was significantly lower than that of the vehicle group; however, FFT treatment did not affect the Shannon index of the viral communities ( Figures 5C, D ).

To further untangle the effect of FFT treatment on bacterial composition, fecal bacterial communities were analyzed at phylum and species levels. We observed that the Bacteroidetes, Firmicutes and Proteobacteria predominated in the gut ( Figure 5E ). In addition, compared with the sham controls, the ratio of Firmicutes to Bacteroidetes decreased in the vehicle group; nevertheless, FFT treatment abrogated the reduction of Firmicutes to Bacteroidetes ratio ( Figure 5G ), although this change did not reach a statistically significant difference between the FFT and vehicle groups. At the species level, the relative abundances of Helicobacter bilis, Helicobacter sp. MIT 03-1616, Lachnospiraceae bacterium 10-1, and Lachnospiraceae bacterium A4 in the FFT group were higher relative to the vehicle group ( Figures 5F, H ). The members of family Lachnospiraceae have showed a radioprotective role following total body radiation [6]. Taken together, it was showed that FFT treatment protects mice against radiation caused local and systemic toxicities, which was related to gut microbes.

To gain insight into the host-gut microbiota interactions, we performed UPLC-MS/MS metabolomics analysis on feces from mice with or without FFT intervention. Overall, compared with the vehicle group, the metabolic profiles of mice in the FFT group were more similar to that of the sham group. Notably, the relative abundance of short-chain fatty acids (SCFAs), which have showed well radioprotection benefits, was reduced following radiation exposure, while FFT treatment clearly enhanced the relative abundance of SCFAs ( Figure 6A ; Figure S2 ). Further, based on the supervised and unsupervised classification discrimination models, namely principal component analysis (PCA) and partial least-squares discrimination analysis (PLS-DA), we found that the metabolic profiles of FFT group were positioned between the sham and vehicle group ( Figures 6B, C ), supporting the benefit of FFT treatment for the side effects of radiotherapy.

Through the intersection of univariate statistics and multidimensional statistics, 20 significantly altered differential metabolites that more likely to become potential biomarkers were identified between the FFT group and the vehicle group. As shown in Figure 6D , the differential metabolites investigated were mainly composed of SCFAs, amino acids, carbohydrates and fatty acids. Of these, a significantly increased level of butyric acids in the FFT group was noted ( Figure 6E ). More specifically, FFT treatment overtly elevated the levels of butyric acid, citric acid, maltose/lactose and pimelic acid compared with the vehicle group, whereas the levels of amino acids metabolites (e.g., ornithine, acetylglycine, threonine and N- acetylserine), carnitines metabolites (e.g., oleylcarnitine and stearylcarnitine), and fatty acids metabolites (e.g., 10, 13-nonadecadienoic acid and 2-methy-4-pentenoic acid), were all markedly decreased. The differential metabolites enriched in the FFT group mainly clustered in the butyrate metabolism pathway and lactose degradation pathway, with an approximately 4-fold change in butyric acid level and an 8-fold change in lactose level ( Figure 6F ). Taken together, our findings suggest that FFT treatment contributes to the production of gut microbiota-derived radioprotective metabolites.