Daytime-restricted feeding reshapes the gut microbiota to achieve intestinal radioprotection through creatine-mediated ferroptosis inhibition

doi:10.21203/rs.3.rs-4817068/v1

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Daytime-restricted feeding reshapes the gut microbiota to achieve intestinal radioprotection through creatine-mediated ferroptosis inhibition

https://doi.org/10.21203/rs.3.rs-4817068/v1

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Ionizing radiation-induced intestinal injury (IRIII), a catastrophic disease that occurs after tumor radiotherapy or accidental nuclear radiation, is lack of sufficient medical countermeasures currently. The gut microbial composition affects the intestinal radiosensitivity, and regulation of the gut microbiota through dietary adjustments is a potential solution for mitigating IRIII. Time-restricted feeding (TRF) is an emerging behavioral nutrition intervention with pleiotropic health benefits. Whether this dietary pattern influences the pathogenesis of IRIII remains vague. We evaluated the impact of TRF on intestinal radiosensitivity in this study and discovered that only daytime TRF (DTRF), not nighttime TRF, could ameliorate intestinal damage in mice that received a high dose of IR. Faecal metagenomic and metabolomic studies revealed that the intestinal creatine level was increased by approximate 9 times by DTRF, to which the Bifidobacterium pseudolongum enrichment contribute. Further investigations showed that creatine enable to activate the energy sensor AMP-activated protein kinase in irradiated enterocytes and induce phosphorylation of the downstream effector acetyl-CoA carboxylase, resulting in reduced production of unsaturated fatty acidsand reduced ferroptosis after IR. The administration of creatine mitigated IRIII and reduced bacteraemia and proinflammatory responses. Blockade of creatine import compromised the ferroptosis inhibition and mitigation of DTRF on IRIII. Our study demonstrates a radioprotective dietary mode functioning by reshaping the gut microbiota to suppress IR-induced ferroptosis, thereby providing effective countermeasures for IRIII prevention.

Health sciences/Gastroenterology/Gastrointestinal diseases/Dysbiosis

Biological sciences/Microbiology/Microbial communities/Microbiome

Ionizing radiation-induced intestinal injury

Gut microbiota

Ferroptosis

Creatine

Time-restricted feeding

Since the discovery of X-rays by Roentgen in 1895, nuclear energy and technology have been widely applied in industry, health care and agriculture. In addition to the benefits of ionizing radiation (IR), its potential threat to the public has also been increasingly recognized, particularly after the occurrence of two level 7 (major accident) nuclear power plant accidents, Chernobyl and Fukushima, which caused hundreds of thousands of people to be exposed to excess IR¹. The mammalian intestine is one of the most sensitive tissues to IR. If given a high dose of IR, people may suffer from lethal intestinal injury that primarily presents as the loss of intestinal barrier integrity and severe endogenous infections^{2, 3}. IR induced intestinal injury (IRIII) is also a common complication in patients with abdominal and pelvic malignant tumors after radiotherapy ⁴, which seriously affects patient's quality of life. Currently, no drugs are approved by the FDA to treat IRIII, which necessitates the development of effective countermeasures.

The pathogenesis of IRIII provides an important basis for the research on prevention strategies. The human intestine is colonized by 100 trillion microorganisms that are in a dynamic equilibrium state under physiological conditions⁵. Advances in microbiome research have revealed that gut microbiota dysbiosis plays a crucial role in the pathogenesis of IRIII^{6, 7}. In patients with IR-induced enteritis, gut microbial diversity is reduced, and the observed microbiome disturbances are closely related to symptoms such as diarrhoea, fatigue, and systemic inflammatory responses⁸. Similar results have been reported in mice with IRIII³. Compared with wild-type mice, germ-free mice are more resistant to IRIII⁹. Additionally, oral supplementation of high-abundance enterobacteria, Lachnospiraceae and Enterococcaceae, from mice that survive IRIII induces radioprotection⁷, which indicates the occurrence of microbial regulation of intestinal radiosensitivity.

Alterations in dietary habits affect the composition of the gut microbiota¹⁰. Time-restricted feeding (TRF) is an intermittent fasting dietary pattern that limits food consumption to a specific time window (usually 8 ~ 12 h) rather than requiring fasting days. Owing to unlimited energy intake during eating periods and pleiotropic health benefits, including improvements in sleep, glucoregulation, blood pressure, liver triglycerides, plasma lipids, and cardiac function^{11, 12, 13, 14}, TRF has received substantial attention in recent years. Mechanistically, an increasing number of studies have indicated that gut microbiota remodelling contributes to the benefits of TRF^{15, 16}, suggesting that TRF may influence intestinal radiosensitivity. Notably, applying TRF during the daytime (the rest period in mice; DTRF) or the nighttime (NTRF) influences the potency of the induced effects on longevity and running endurance^{17, 18}. Whether DTRF and NTRF have different impacts on the pathogenesis of IRIII also warrants investigation.

In addition to the gut microbiota dysbiosis, ferroptosis in enterocytes is also an important cause driving the occurrence of IRIII. Ferroptosis is a type of programmed cell death caused by excessive production of lipid peroxides in an Fe-dependent manner¹⁹. Mounting evidence suggests that ferroptosis initiation contributes to IR-induced cytotoxicity^{20, 21, 22}. Accordingly, increased ferroptotic flux sensitizes cancer cells to radiotherapy²², whereas inhibiting IR-induced ferroptosis confers radioprotection²¹. The gut microbiota has regulatory effects on ferroptosis through its microbial composition and metabolites²³. Although gut microbiota regulation has been found to mitigate IRIII^{3, 6, 7}, whether this radioprotective effect correlates to ferroptosis inhibition is unclear.

Focusing on the “diet–gut microbiota–host triangle”²⁴, our study starts with an evaluation of the impact of TRF on intestinal radiosensitivity, followed by a multiomics analysis to probe the underlying microbial and metabolic factors involved. We found that DTRF enabled to reshape gut microbiota and increase intestinal creatine (Cr) levels. Cr, an arginine (Arg) metabolite that could be upregulated by Bifidobacterium pseudolongum in the gut, was identified as a ferroptosis inhibitor by reducing the production of unsaturated fatty acids after IR. Our findings demonstrate that DTRF can regulate the gut microbiota to achieve intestinal radioprotection through Cr-mediated ferroptosis inhibition and provide promising solutions for IRIII prevention.

DTRF ameliorates the intestinal injury caused by IR

To investigate the influence of TRF on intestinal radiosensitivity, male mice subjected to DTRF (ZT2-10), NTRF (ZT14-22), and ad libitum feeding (ALF) for two weeks were evaluated using a total abdominal irradiation (TAI) model induced by 11 Gy γ-ray (Fig. 1a). In line with the ability of TRF to prevent weight gain in mice fed a high-fat diet²⁵, DTRF and NTRF both decreased body weight compared with ALF (Extended Data Fig. 1). However, NTRF only slightly improved the survival of irradiated mice from 60–70%, whereas the mice treated with DTRF were all alive (Fig. 1b). Detection of the body weight indicated that DTRF outperformed NTRF in reducing the weight loss caused by TAI (Fig. 1c). Similarly, obvious intestinal symptoms such as diarrhoea and bloody stools were observed in mice subjected to ALF on Day 5 after TAI, whereas these symptoms were notably mitigated by DTRF, rather than NTRF, as indicated by a decreased disease activity index (DAI) score (Fig. 1d). These findings indicate that only DTRF, not NTRF, can mitigate IRIII.

B. pseudolongum enrichment contributes to the radioprotection of DTRF

To gain insight into the radioprotective effect of DTRF, we next performed a dirty-cage experiment and observed that the survival of mice subjected to ALF living in cages with faeces from mice subjected to DTRF after TAI was improved from 60–80% (Fig. 2a). Additionally, FMT to transfer the microbiota from mice subjected to DTRF to mice subjected to ALF increased mouse survival from 60–90%, along with reduced weight loss (Fig. 2b) and intestinal symptoms after TAI (Fig. 2c), which suggests that remodelling of the gut microbiota contributes to reduced intestinal toxicity. 16S rRNA sequencing was then employed to analyse the impact of DTRF on the faecal microbiota. The values of two α diversity indices, the Shannon and Simpson indices, were significantly greater in mice subjected to DTRF than in those subjected to ALF (Fig. 2d). The nonparametric test ANOSIM revealed a P value of 0.0049 (R = 0.74), which indicated greater intergroup differences than within-group differences. The species annotation revealed that DTRF increased the abundance of Bacteroidota from 37.87–55.37% but decreased the Firmicutes abundance from 47.1–29.52% (Extended Data Table 1). A t test revealed that Bifidobacteriales was the only bacterium whose abundance changed (upregulated by DTRF) significantly with a P value lower than 0.01 (P = 0.002, Fig. 2e). Improvements in microbial α diversity, the Bacteroidota/Firmicutes ratio, and the abundance of Bifidobacterium were also detected in mice subjected to DTRF compared with mice subjected to ALF after TAI (Extended Data Fig. 2).

Furthermore, a metagenomic study and linear discriminant analysis (LDA) effect size (LEfSe, P < 0.05, LDA > 4) analysis were carried out and revealed the differential abundance of Bifidobacteriales between mice subjected to ALF and DTRF (Fig. 2f). Specifically, DTRF increased the contents of Bifidobacterium pseudolongum (B. pseudolongum) and Bifidobacterium animalis at the species level (Fig. 2g). Bacteria such as Duncaniella dubosii, Bacteroides sp. CAG 927, Muribaculum intestinale, and Alistipes muris also increased in abundance after DTRF. Notably, B. pseudolongum was one of the bacteria with the most significantly altered abundance in mice subjected to DTRF compared with mice subjected to ALF (P = 0.0039, Extended Data Table 2). Oral administration of B. pseudolongum improved the survival of irradiated mice from 60–80% (Fig. 2a) and markedly relieved the weight loss and intestinal symptoms caused by TAI (Fig. 2b,c). It is plausible that the increased abundance of B. pseudolongum contributes to the radioprotective effect of DTRF.

DTRF induces an increase in Cr content in the gut

Changes in microbiome composition influence the production of gut microbiota metabolites that can affect host physiology. To identify the differentially abundant metabolites in the gut of mice subjected to DTRF, we next conducted a metabolomics study. A total of 169 metabolites (117 with increased abundance, 52 with decreased abundance) exhibited significant alterations (FC > 2 or FC < 0.5, and P < 0.05, VIP > 1.0) in the faeces of mice subjected to DTRF compared with those of mice subjected to ALF (Fig. 3a and Extended Data File 1), as detected in negative ion mode. Arg and proline metabolism was determined to be the sole signalling pathway significantly (P = 0.018) enriched via Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis (Fig. 3b). Moreover, no significant change was observed for the pathways enriched in the results obtained in positive ion mode (Extended Data Table 3). Metabolomic profiling revealed that the level of Cr, a product of Arg metabolism that is beneficial for improving muscle strength²⁶, was 8.28-fold greater in mice subjected to DTRF than in mice subjected to ALF (Fig. 3c). This finding was corroborated by a coupled enzyme reaction in which the faecal Cr level was detected to increase by 9.24 times after DTRF (Fig. 3d). Consistently, DTRF outperformed ALF in increasing mouse grip-force strength (Fig. 3e) and faecal Arg contents (Fig. 3f).

As Cr functions to accelerate ATP regeneration during energy expenditure²⁷, and because the microbial function alteration triggered by DTRF is concentrated on energy production and conversion (Fig. 2h), as indicated by Evolutionary genealogy of genes: Non-supervised Orthologous Groups (eggNOG) enrichment in a metagenomic study, we further asked whether the increase in Cr levels was attributed to the enrichment of bacteria in mice subjected to DTRF. To investigate this possibility, we examined the faecal Cr concentration in mice subjected to ALF that were orally administered B. pseudolongum. In accordance with the effect of Arg supplementation, B. pseudolongum colonization markedly increased the Cr level in vivo (Fig. 3g). An increase in Arg levels was also detected in mice subjected to ALF after probiotic treatment (Extended Data Fig. 3). These data suggest that the enrichment of radioprotective B. pseudolongum in mice subjected to DTRF increases the Cr content in the gut.

Cr mitigates IR-induced intestinal epithelial injury in vitro and in vivo

To investigate whether Cr accounts for the observed radioprotection, we preincubated rat intestinal epithelial IEC-6 cells with Cr and found that Cr could dramatically improve cell survival 24 h after 8-Gy γ-ray IR (Fig. 4a). Furthermore, we added 1% Cr to the drinking water and repeated the TAI experiment in mice subjected to ALF. The results showed that Cr supplementation mitigated the intestinal symptoms caused by TAI (Fig. 4b) and improved mouse survival from 60–90% (Fig. 4c). Measurement of small intestine length revealed evident tissue shrinkage in mice subjected to ALF after TAI (Fig. 4d), which corresponded with ileal histopathological alterations, including villous shedding (Fig. 4e), shortened villous length (Fig. 4f), and reduced crypt counts (Fig. 4g). Inspiringly, Cr administration improved all pathological changes caused by IR. As intestinal mucosal destruction enables the invasion of endogenous pathogens and toxins into the circulation, we detected abundant bacteria (Extended Data Fig. 4) and a high level of endotoxin (LPS, Fig. 4h) in the blood on Day 3 after TAI in mice. Owing to the maintenance of barrier integrity, Cr treatment markedly dampened bacterial and LPS translocation, resulting in a prominent reduction in the levels of proinflammatory factors, including IL-6 and IL-1β (Fig. 4i). The above results indicate that Cr administration can mitigate IRIII and reduce bacteraemia and proinflammatory responses.

Cr suppresses IR-induced ferroptosis by activating AMPK under energy stress

Because Cr is an important component of the energy shuttle system that buffers adenosine triphosphate (ATP) and adenosine diphosphate (ADP) levels, we next inferred that the radioprotective effect of Cr might correlate to energy metabolism. Determination of the ATP/ADP ratio supplied that IR caused an energy stress in irradiated IEC-6 cells, whereas this energy deficiency was mitigated by Cr (Fig. 5a). Along with energy fluctuations, the status of energy-sensing effectors in irradiated cells might be affected. Our data-independent acquisition (DIA) proteomics study revealed that 96 proteins (FC > 1.5 or FC < 0.67, P < 0.05) exhibited altered levels in irradiated IEC-6 cells after treatment with 25 µg/mL Cr (Extended Data File 2). AMPK signalling was determined to be the most significantly enriched pathway (P = 0.0018) via KEGG analysis of the upregulated proteins (Fig. 5b). Western blotting revealed that Cr increased the phosphorylation of AMPKα (p-AMPKα) in the cells after IR (Fig. 5c). The downstream effector acetyl-CoA carboxylase (ACC), which mediates the synthesis of malonyl-CoA, also presented increased phosphorylation after Cr treatment.

Since the activation of AMPK/ACC signalling decreases the synthesis of polyunsaturated fatty acids (PUFAs) and inhibits the initiation of ferroptosis²⁸, a form of programmed cell death characterized by cytoplasmic Fe²⁺ accumulation and disrupted cell membranes caused by lipid peroxidation²⁹, and because ferroptosis is an important cause of IRIII³⁰, we next speculated that Cr might improve the survival of irradiated enterocytes via suppression of ferroptosis. To verify this hypothesis, we performed C11-BODIPY581/591 staining and discovered increased lipid oxidation in IEC-6 cells after IR, whereas this peroxidation was attenuated by 25 µg/mL Cr (Fig. 5d and Extended Data Fig. 5). Determination of the content of malondialdehyde (MDA), a product of cellular lipid peroxidation, also confirmed the ability of Cr to mitigate lipid peroxidation (Fig. 5e), and reduced production of unsaturated fatty acids that provide the substrate for lipid peroxidation during ferroptosis might contribute to this effect (Fig. 5f). On the other hand, Fe²⁺ accumulation, as indicated by FerroOrange staining (Extended Data Fig. 6), and the total Fe content in the irradiated cells were both decreased by Cr (Fig. 5g). Further experiments revealed that incubating IEC-6 cells with 5 µM erastin, an acknowledged ferroptosis activator, caused approximately 40% death, whereas cell survival was markedly improved by Cr (Fig. 5h). Additionally, the application of 0.5 µM compound C (CC), a small molecule inhibitor of AMPK, counteracted the ability of Cr to suppress the activation of AMPK/ACC signalling, unsaturated fatty acid production, lipid peroxidation, and Fe accumulation (Fig. 5c-g), resulting in an abrogation of the radioprotective effect of Cr (Fig. 5i). These findings demonstrate that Cr administration has an inhibitory effect on IR-induced ferroptosis and that this process is dependent on AMPK activation.

Cr contributes to the ferroptosis inhibition and radioprotection of DTRF

To investigate whether Cr-mediated ferroptosis inhibition contributes to the radioprotection of DTRF, we further performed the mouse TAI experiment and evaluated the influence of 3-Guanidinopropionic acid (RGX-202), an oral small-molecule solute carrier family 6 member 8 (SLC6A8) transporter inhibitor that suppresses creatine import in vivo³¹. Consistent with the findings in the irradiated cells treated with Cr, DTRF decreased the levels of unsaturated fatty acid production (Fig. 6a), lipid peroxidation (Fig. 6b) and Fe accumulation (Fig. 6c) in mice that received TAI. Nevertheless, these improvements elicited by DTRF were abrogated by the oral administration of RGX-202. Measurement of the length of the small intestine revealed a reduction in tissue shrinkage in irradiated mice after DTRF (Extended Data Fig. 7), which was supported by histopathological analysis, in which the villous shedding and shortening (Fig. 6d) and loss of intestinal crypts (Fig. 6e) induced by IR were abated by DTRF. However, RGX-202 administration impaired the ability of DTRF to maintain intestinal length and mucosal barrier integrity after TAI (Fig. 6d,e and Extended Data Fig. 7). Therefore, Cr is crucial for DTRF to inhibit IR-induced ferroptosis and mitigate IRIII.

The gut microbiota dysbiosis and ferroptosis in intestinal epithelial cells (IECs) are important causes driving the pathogenesis of IRIII^{6, 8, 30}. It has been discovered that regulation of the gut microbiota can reduce IR-induced intestinal toxicity^{3, 7}. TRF is an emerging behavioral nutrition approach affecting the gut microbiota composition^{15, 16}. Whether this dietary pattern influences the intestinal radiosensitivity is unclear. Furthermore, the gut microbiota has regulatory effects on ferroptosis²³, raising a possibility that ferroptosis inhibition may contribute to the radioprotection of gut microbiota regulation. Herein, we evaluated the impact of TRF on intestinal radiosensitivity and demonstrated that DTRF could reshape the gut microbiota to mitigate IRIII by suppressing ferroptosis.

Our metagenomic study revealed that B. pseudolongum, a predominant bifidobacterial species in various mammals³², was associated with the radioprotective effect of DTRF. B. pseudolongum has long been known to ameliorate severe radiation injury³³, but the underlying mechanism remains vague. We discovered that B. pseudolongum enabled to increase the enteric Arg content and facilitate the production of Cr, which protected IECs from lethal IR. As known, endogenous Arg can be synthesized de novo through the consumption of glutamate and acetyl-CoA in many microorganisms³⁴. Since B. pseudolongum generates acetate that can be transformed into acetyl-CoA³⁵, we propose that this bacterium may promote Arg production by supplying extra acetyl-CoA. Intriguingly, because increased intestinal Arg levels facilitate the colonization of B. pseudolongum³⁶, there seems to be a positive feedback loop related to Arg metabolism after DTRF, accounting for the observed increase in Cr abundance. Cr is a nitrogenous organic compound that promotes improvements in muscle and exercise performance²⁷. The finding that B. pseudolongum increased enteric Cr levels corroborated the predominant shift in microbial function to energy production and conversion in mice subjected to DTRF and underpinned the efficacy of Bifidobacterium supplementation in restoring age-related muscle loss³⁷.

The study is the first, to our knowledge, to identify Cr as a ferroptosis inhibitor to achieve radioprotection. Mechanistically, Cr activated AMPK and induced the phosphorylation of downstream ACC, resulting in reduced biosynthesis of unsaturated fatty acids, which provides the substrates for lipid peroxidation during ferroptosis¹⁹. A similar mechanism was revealed for the suppression of ferroptosis by energy stress²⁸. AMPK inhibition abrogated the alleviation of IR-induced ferroptosis by Cr, which supports the role of AMPK activation in IRIII mitigation³⁰. Moreover, we found that Cr could markedly improve the survival of cells exposed to ferroptosis inducer erastin, possibly due to that AMPK activation negatively regulates ferroptosis induced by erastin³⁸.

AMPK is a pivotal sensor of cellular energy status. In the presence of decreasing cellular energy levels, AMPK activation switches on catabolism to generate ATP while downregulating anabolism to reduce ATP consumption³⁹. IR induced energy stress in IECs. However, neither we nor the authors of a recent study reported the activation of AMPK in IECs after IR³⁰, which suggested the occurrence of energy metabolism maladaptation. The ability of Cr to increase AMPK activation after IRIII indicated the ability of this metabolite to facilitate metabolic adaptation under energy stress. Cr is taken up by IECs through the SLC6A8 transporter and is transformed to phosphorylcreatine (PCr) under the catalysis of Cr kinase. An increased ratio of cellular Cr to PCr induces the activation of AMPK⁴⁰. Because SLC6A8 expression is upregulated by IR⁴¹, more Cr might be transported to the cytoplasm, which may explain why Cr triggered more marked AMPK activation in IECs after irradiation injury than in normal cells.

Cr is widely used by healthy individuals and patients with neurological disorders. It possesses a diverse set of regulatory effects beyond the effects of a typical energy supplement. A pioneering study revealed that Cr could increase energy availability in T cells partially through AMPK activation to eliminate cancer cells⁴². As small intestinal injury is a primary obstacle for patients with abdominal and pelvic tumours who are undergoing high-dose radiotherapy, Cr supplementation may protect the small intestine from IR while enhancing the efficacy of tumour radiotherapy in these patients. Notably, emerging studies have indicated a role for Cr in facilitating cancer progression and metastasis^{31, 43}, which highlights the delicate balance of Cr in cancers⁴⁴ and raises questions about the impacts of Cr supplementation on the outcome of radiotherapy. Currently, there is no clear correlation between enteric Cr levels and tumour metastasis after radiotherapy. Considering the promising radioprotective effect of Cr on normal intestinal tissues, further investigations to identify reasonable Cr supplementation schemes to reduce IR-induced intestinal toxicity without compromising the prognosis of tumour radiotherapy are needed. Alternatively, inhibiting Cr uptake by cancer cells through tumour-targeting technology may be effective⁴⁵. Additionally, since circadian reprogramming explains the time-of-day effect of TRF⁴⁶, whether the increased Cr levels in mice subjected to DTRF are dictated by circadian regulation also requires further exploration.

Overall, our findings show that DTRF can enrich the probiotic B. pseudolongum and increase the level of enteric Cr to reduce IR-induced intestinal toxicity. Under energy stress caused by IR, Cr promotes metabolic adaptation by activating AMPK, which induces phosphorylation of the downstream effector ACC, resulting in reduced lipid peroxidation and reduced ferroptosis (Fig. 6f). Given that the effective measures for mitigating IRIII are lacking, we believe that in addition to DTRF, B. pseudolongum and Cr supplementation may be important tools for radioprotection. Considering the ferroptosis inhibition of Cr, it may also has application values in the treatment of ferroptosis-related diseases, such as ischemia/reperfusion, Alzheimer’s disease, and Parkinson’s disease⁴⁷.

Total abdominal irradiation mouse experiment

Six- to eight-week-old male C57BL/6J mice weighing 18–20 g were purchased from Beijing Vital River Laboratory Animal Technology Co., Inc. (Beijing, China). The mice were housed in a specific pathogen-free facility at the Institute of Combined Injury of PLA, Army Medical University (AMU), and were maintained under controlled conditions (ambient temperature: 22 ± 2°C, air humidity: 40–70%) with a 12-h light‒dark cycle. After one week of adaptive feeding, the mice were randomly divided into 6 groups (ALF, ALF + IR, DTRF, DTRF + IR, NTRF, and NTRF + IR; n = 10 per group). The mice subjected to ALF had continuous access to a standard diet and drinking water. In the DTFR group, the mice were fed only during the Zeitgeber time (ZT) 2–10 period, while water intake was unlimited throughout the day. For the NTRF mice, feed was provided during the ZT14-22 period, and a sufficient water supply was provided. Mice subjected to ALF, DTRF, or NTRF for two weeks were anaesthetized with 150 µL of 1% pentobarbital sodium and placed in the supine position, followed by TAI using a ⁶⁰Co source of 11 Gy γ-rays at a dosage rate of 0.48 Gy/min³.

For the dirty cage experiment, ten mice subjected to DTRF for one week were transferred to the cages of mice subjected to ALF (n = 10) at ZT2. This hybrid cage feeding continued until ZT10, when the mice subjected to DTRF were returned to their previous cages. The mice subjected to ALF were subjected to TAI after 7 days of hybrid cage feeding. To evaluate the radioprotective effect of B. pseudolongum, a total of 2×10⁸ CFU of this bacterium was delivered to ten mice subjected to ALF through oral administration. Probiotic supplementation was performed for 7 consecutive days before TAI, followed by the addition of B. pseudolongum for another 3 days. Additionally, to evaluate the role of Cr supplementation in TAI, forty mice subjected to ALF were divided into 4 groups (sham, Cr, IR, and Cr + IR; n = 10 per group). Cr (1%; C3630, Sigma-Aldrich, Merck, Shanghai, China) was added to the drinking water 4 days before TAI and continued for 7 days. The mice in the sham group were treated with sterile water. The mice were cared for and treated in accordance with the recommendations of the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85e23 Rev. 1985). The animal experiments were approved by the Institutional Review Board of AMU (approval no. AMUWEC20230331). After TAI, mouse survival was monitored for 30 days, and body weight was determined on Days 0, 1, 3, 5, 7, 9, and 11. DAI scores were determined as previously described on Day 5 post-irradiation⁴⁸.

Histopathologic analysis

Another 54 mice randomly divided into 9 groups (ALF, ALF + IR, DTRF, DTRF + IR, and DTRF + IR + RGX-202; sham, Cr, IR, and Cr + IR; n = 6 per group) were used to repeat the TAI experiment. RGX-202 (800 mg/kg; HY-W015828, MedChemExpress, Shanghai, China) was orally administrated 4 days before TAI and continued for 7 days (q.d.). The mice were euthanized 3 days after TAI via the injection of an excessive amount of pentobarbital sodium, and their small intestine tissues were obtained via surgery. Ileum tissues were fixed in Carnoy's fluid (G1120, Servicebio, Wuhan, China) at 4°C for 48 h, processed with gradient concentrations of ethanol for dehydration, and embedded in paraffin. The samples were sectioned at a 3 µm thickness and observed after haematoxylin and eosin (H&E) staining, after which the ileal villous height and the number of crypts were determined.

Faecal microbiota transplantation

Faecal samples from mice subjected to DTRF were collected after two weeks and resuspended in sterile PBS (1 g/10 mL). The supernatant was obtained after centrifugation at 3000 rpm for 30 s, followed by high-speed centrifugation (12 000 rpm) at 4°C for 5 min to prepare the microbiota pellets, which were further resuspended in 2.5 mL of sterile PBS and delivered to mice subjected to ALF by oral administration (q.d.). The mice were treated with antibiotics containing ampicillin (1 mg/mL; A600064-0025, BBI Life Science, Shanghai, China), metronidazole (1 mg/mL; A6000633-0025, BBI Life Science), neomycin (0.5 mg/mL; A610366-0025, BBI Life Science), and vancomycin (0.5 mg/mL; V105495, Aladdin, Shanghai, China) to eliminate intestinal bacteria 3 days before transfer of the DTRF faecal microbiota. After 7 days of FMT (q.d.), the mice were subjected to TAI, and FMT was further performed for another 3 days. Mouse survival, body weight, and DAI scores were monitored as described for the TAI mouse experiment.

16S rRNA gene amplicon sequencing analyses

Fresh faeces from mice treated with ALF or DTRF (n = 10) for two weeks were obtained and stored at -80°C until use. Faecal samples were also collected from mice subjected to ALF and DTRF (n = 6) 48 h after TAI. DNA from the stool was extracted via a TIANGEN magnetic soil and stool DNA kit (4992736, Beijing, China). The polymerase chain reaction (PCR) products were purified with a Qiagen Gel Extraction Kit (28706, Germany). 16S ribosomal RNA (rRNA) V4 was amplified using specific primers and Phusion High-Fidelity PCR Master Mix (M0531L, New England Biolabs, Beijing, China). The TIANGEN Universal DNA Purification Kit (DP214) was used to purify the PCR products. Library construction, sequencing, amplicon sequence variant (ASV) denoising and species annotation were performed by Novogene (Beijing, China). The sequencing libraries were generated via the NEBNext® Ultra™ II FS DNA Library Prep kit (E6177S, New England Biolabs) and evaluated via a Qubit@ 2.0 fluorometer (Thermo Fisher Scientific, Shanghai, China). High-throughput gene sequencing was conducted on a Illumina NovaSeq6000 platform, and 250 bp paired-end reads were obtained, which were further merged via FLASH (version 1.2.11, http://ccb.jhu.edu/software/FLASH/) after the barcode and primer sequences were removed. The raw tags were strictly filtered with fastp (version 0.23.1, https://github.com/OpenGene/fastp#) to generate high-quality clean tags. After comparison with the reference database (Gold database, http://drive5.com/uchime/uchime_download.html) through the UCHIME algorithm (http://www.drive5.com/usearch/manual/uchime_algo.html), the chimaera sequences were selected and removed. Quantitative Insights Into Microbial Ecology 2 (QIIME2) employs Divisive Amplicon Denoising Algorithm (DADA2) in the construction of ASVs⁴⁹. Species information for each ASV was obtained by comparing the obtained ASVs with the database via the classify-sklearn module in QIIME2. Bacterial diversity analysis was performed on the NovoMagic platform (https://magic.novogene.com).

Metagenomic analysis

Fresh faecal samples from mice subjected to ALF or DTRF (n = 6) for two weeks were collected and stored at -80°C until use. DNA from the faeces, which was extracted as described for bacterial diversity analysis, was broken into short fragments. Before size selection, these fragments were end-polished, subjected to the addition of poly A tails, and ligated with full-length adapters for Illumina sequencing. An Agilent (Beijing, China) 2100 bioanalyzer was used to evaluate the insert size of the library. The Q‒PCR method was used to quantify the effective concentration of the library accurately (effective concentration > 3 nM). The qualified libraries were pooled and sequenced on the Illumina platform PE150 at Novogene (Beijing, China). The raw data were processed with fastp (version 0.23.1) to generate clean data, which were blasted against the mouse genome (GRCm38) to filter out reads that may have originated from the host. The clean data were further assembled via MEGAHIT software with the following parameter settings: --presets meta-large (--end-to-end, --sensitive, -I 200, -X 400)^{50, 51}. ORF prediction for scaftigs (≥ 500 bp) was performed for each sample via MetaGeneMark (http://topaz.gatech.edu/GeneMark/). CD-HIT software (http://www.bioinformatics.org/cd-hit/) was used to eliminate redundancy and collect the nonredundant initial gene catalogue. To calculate the number of reads of the genes in each sample alignment, the clean data were aligned to the initial gene catalogue via Bowtie2⁵². After genes with < = 2 reads in each sample were filtered out, the final gene catalogue for subsequent analysis was obtained. For species annotation, DIAMOND software (https://github.com/bbuchfink/diamond/) was employed for alignment of unigene sequences with the Micro_NR database. The species annotation information of the sequence was determined via the LCA algorithm, and diversity analysis was performed on the NovoMagic platform (https://magic.novogene.com). LEfSe software was used for LEfSe analysis (LDA score of 4 by default). Functional annotations were performed using DIAMOND software to align unigenes with those in the eggNOG database (http://eggnogdb.embl.de/#/app/home).

Untargeted metabolomics

Faecal samples (100 mg for each) from mice treated with ALF or DTRF (n = 6) for two weeks were ground with liquid nitrogen, followed by resuspension in 500 µL of prechilled 80% methanol. After coincubation on ice for 5 min, the homogenate was centrifuged at 15,000 × g and 4°C for 20 min. The supernatant was diluted with liquid chromatography‒mass spectrometry (LC‒MS)-grade water (1.15333.2500, Merck) to generate a solution containing 53% methanol and centrifuged for further purification. Ultrahigh-performance liquid chromatography‒tandem mass spectrometry (UHPLC‒MS/MS) analysis of the purified supernatant was then conducted via a Vanquish UHPLC system (Thermo Fisher) coupled with an Orbitrap Q ExactiveTM HF mass spectrometer (Thermo Fisher) at Novogene (Beijing, China). A Hypersil Gold column (C18, 100×2.1 mm, 1.9 µm; Thermo Fisher) and a 12-min linear gradient at a flow rate of 0.2 mL/min were applied for sample analysis. Eluents A and B were 0.1% formic acid in water and methanol, respectively. Mass spectrometry was performed in positive/negative polarity mode with a spray voltage of 3.5 kV, capillary temperature of 320°C, S-lens RF level of 60, and Aux gas heater temperature of 350°C. The raw data were processed with Compound Discoverer 3.3 (CD3.3, Thermo Fisher) to perform peak alignment and selection and metabolite quantification. Statistical analyses were performed using R (v.R-3.4.3), Python (Python, v.2.7.6) and CentOS (v.6.6) software. The obtained metabolites were annotated using the KEGG database (https://www.genome.jp/kegg/pathway.html), HMDB (https://hmdb.ca/metabolites) and LIPIDMaps database (http://www.lipidmaps.org/). Metabolites with VIP > 1, P value < 0.05 and FC ≥ 2 or FC ≤ 0.5, were considered to be significantly altered. The functions of the identified metabolites and metabolic pathways were investigated via the Kyoto Encyclopedia of Genes and Genomes (KEGG, https://www.genome.jp/kegg/) database.

Cr and Arg determination

Fresh faeces from mice subjected to ALF or DTRF (n = 6) for two weeks, and from mice subjected to ALF received oral administration of B. pseudolongum for 10 days (n = 6), were collected and homogenized in sterile PBS (20 mg/500 µL). The supernatant was obtained after centrifugation at 15,000 × g and 4°C for 10 min. Cr contents were determined using a Cr assay kit (MAK079, Sigma Aldrich, Shanghai, China). Fifty microlitres of each sample was coincubated with a reaction mixture containing Cr assay buffer, creatinase, the Cr enzyme, and the Cr probe (50 µL in total) at 37°C for 60 min. Absorbance at 570 nm was then detected using a Molecular Devices M2e microplate reader (Sunnyvale, CA, USA). For Arg determination, the amount of the supernatant derived from the faecal homogenate was measured using an Arg content assay kit (BC5635, Solarbio) according to the manufacturer’s instructions.

Mouse grip-force strength determination

The grip force of mice subjected to ALF and DTRF (n = 6) was assessed with a 47200 Grip Strength Meter (Ugo Basile, Italy). The mice were placed on a metal grid so that their paws were gripping the wire mesh grid, which equipped with a force transducer. Subsequently, the mice were pulled by the tail with increasing force until they release their claws. The maximum grip force was recorded for each mouse after 6 assessments. The same person conducted this experiment.

Evaluation of bacteraemia and endotoxaemia

Fresh blood from the hearts of mice subjected to TAI in the presence or absence of Cr supplementation was diluted 10 times in sterile PBS. One hundred microlitres of the sample was used to evenly coat Mueller Hinton agar and cultured in a 37°C constant-temperature incubator. Bacterial colonies were counted after 24 h. For evaluation of endotoxaemia, the blood samples were centrifuged at 3000 rpm for 15 min to collect the serum, one hundred microlitres of which was further coincubated with 100 µL of tachypleus amebocyte lysate (EC80545, Xiamen Bioendo Technology, Fujian Province, China) at 37°C for 10 min. Afterwards, one hundred microlitres of the chromogenic matrix solution was added and incubated for 6 min. Hydrochloric acid solution was added to stop the reaction. The absorbance of the mixture at 545 nm was finally measured after the addition of the azo reagent using a microplate reader. IL-6 and IL-1β levels in the serum were determined via enzyme-linked immunosorbent assay (ELISA), in which two Beyotime ELISA kits (PI301 and PI326) were used according to the manufacturer’s instructions.

Cell experiments

Rat intestinal epithelial IEC-6 cells (CRL-1592) were obtained from the American Type Culture Collection (ATCC) and cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Thermo Fisher) supplemented with 10% foetal bovine serum (FBS; Gibco). The cells were grown at 5% CO₂ and 37°C, and the number of passages was no more than 8 during the experiment. IEC-6 cells were seeded into sterile 96-well plates at a density of 5 000 CFU/well and cultured overnight. IR was performed using a ⁶⁰Co source of 8 Gy γ-rays at a dose rate of 0.48 Gy/min. Cr was added 6 h before IR, and 5 µM erastin (S7242, Selleck Chemicals, USA) was added 4 h before Cr was added. Cell viability was assessed 24 h after IR using a cell counting kit-8 (Dojindo, Shanghai, China). To evaluate the influence of AMPK inhibition on the radioprotective effect of Cr, CC (0.5 µM; BML-275, MedChemExpress) was added 1 h before Cr supplementation. These experiments were conducted in duplicate and repeated three times.

Lipids from IEC-6 cells subjected to 8-Gy γ-ray irradiation, in the presence or absence of Cr (25 µg/mL) or 0.5 µM CC 6 h before IR, were extracted using a lipid extraction kit (ab211044, Abcam). Unsaturated fatty acids were measured with a lipid assay kit (ab242305, Abcam). A fluorescent ratio probe for lipid oxidation, C11-BODIPY581/591 (HY-D1301, MedChemExpress), was applied to detect lipid peroxidation. The intracellular Fe²⁺ concentration was probed with 10 µM FerroOrange (F374, Dojindo). Images were captured with a Zeiss LSM 780 NLO confocal microscope. Quantitative analysis was conducted using ZEN 2011 (blue edition). The MDA levels in the cells and ileal tissues were measured via the classic thiobarbituric acid (TBA) method (M496, Dojindo). The total iron content was determined via a colorimetric method (I291, Dojindo). These assays were conducted in duplicate and repeated three times.

ATP/ADP ratio detection

IEC-6 cells were seeded into a sterile 96-well plate at a density of 1000 CFU/well, cultured overnight at 5% CO₂ and 37°C, and irradiated with an 8 Gy γ-ray at a dose rate of 0.48 Gy/min. Cr (25 µg/mL) was added 6 h before IR. The ATP/ADP ratio in IEC-6 cells in the absence or presence of IR and Cr after 24 h was detected via a luminescence kit from Dojindo (A552). This assay was conducted in triplicate and repeated three times.

Proteomics analysis

Proteins from IEC-6 cells in the presence or absence of 25 µg/mL Cr were extracted 24 h after irradiation using a dissolved buffer containing 8 mM urea and 100 mM triethylammonium bicarbonate (TEAB; T7408, Sigma-Aldrich). The supernatant was obtained by centrifugation at 12,000 × g and 4°C. Dithiothreitol (DTT, D9163, Sigma-Aldrich; 1 M) was then added, and the mixture was incubated at 56°C for 1 h, followed by incubation in an ice bath for 2 min. Anhydrous iodoacetamide (IAM, I6125, Sigma-Aldrich) was further added, and the mixture was incubated at room temperature in the dark for 1 h. The protein samples were quantified and digested with trypsin. Formic acid (FA) was added to adjust the solution pH to less than 3.0. The supernatant was desalted, washed with a cleanout solution containing 0.1% FA and 3% acetonitrile (ACN) three times, and eluted with a solution containing 0.1% FA and 70% ACN.

The samples were then analysed using the Vanquish™ Neo UHPLC system (Thermo Fisher) equipped with a C18 precolumn (174500, 5 mm×300 µm, 5 µm; Thermo Fisher) and a C18 analytical column (ES906, 150 µm×15 cm, 2 µm; Thermo Fisher). Mass spectrometry was conducted at Novogene Co., Ltd. (Beijing, CHN) using a Thermo Orbitrap astral mass spectrometer equipped with an easy spray ion source. The ion spray voltage was set to 1.9 kV, and the mass spectrum was collected in data-dependent collection mode. The full scan range of the primary mass spectrum was 380–980 (m/z), and the resolution of the primary mass spectrum was 240000 (200 m/z). The secondary m/z collection range was 150 to 2000. The raw data obtained by mass spectrometry were analysed using DIA-NN software ⁵³. Peptide segments and proteins with false discovery rates (FDRs) above 1% were removed. Proteins with P values < 0.05 and FC > 1.5 or FC < 0.67 were considered to be significantly altered. The functional enrichment of the differentially expressed proteins was analysed via the KEGG.

Western blotting

IEC-6 cells were collected and lysed for protein extraction. Twenty-five micrograms of each sample were resolved via 10% sodium dodecyl sulfate‒polyacrylamide gel electrophoresis. A primary rabbit polyclonal antibody (1:1000; 2532S, Cell Signaling Technology (CST), USA) and a goat antirabbit secondary antibody (1:1000; A0208, Beyotime) were used to detect AMPKα. The p-AMPK level was determined via the use of a primary rabbit polyclonal antibody (1:1000; 2535S, CST). A primary rabbit polyclonal antibody (1:1000; 3662S, CST) was used to detect ACC. The p-ACC was determined using a rabbit antibody (1:1000, 10108-T60, 3661S, CST). A BeyoECL Plus chemiluminescence kit (P0018S, Beyotime) and a Bio-Rad ChemiDoc™ MP Imaging System (Hercules, CA, USA) were used to visualize the protein bands. Tubulin was detected with a mouse monoclonal antibody (1:1000; AF2831, Beyotime) as a reference. These experiments were conducted in duplicate and repeated three times.

Statistical analysis

The significance of differences was analysed with SPSS 25.0 via one-way analysis of variance (ANOVA) for multiple comparisons or an unpaired two-tailed Student's t test for comparison between two groups. A P value < 0.05 was considered to indicate statistical significance.

Data availability

All data generated or analyzed during this study are included in the published article and its extended data files. Raw microbial sequencing data are deposited at the NCBI Sequence Read Archive (SRA) under the accession numbers: PRJNA1133968 (16S rRNA, before TAI), PRJNA1134280 (16S rRNA, after TAI), and PRJNA1134918 (Metagenomic).

Acknowledgements

This work was supported by grants from the Independent Research Project of State Key Laboratory of Trauma and Chemical Poisoning (No: SKLYQ202101) and the Chongqing Talent Program (CQYC20220510803).

Author contributions

C.W. and J.W. conceived and designed the study. Y.H. and G.Z. raised the mice and performed the TAI experiment. Y.H. and X.O. conducted the dirty-cage and TAI experiments. Y.H. and Y.C. carried out the faecal 16S rRNA and metagenomic sequencing and analysed the data. X.O., S.W., and C.L. prepared the samples for histopathologic analysis. C.L. and Y.J. determined the bacteraemia and proinflammatory responses in mice. Y.H., G.Z., and T.X. conducted the untargeted metabolomics and analysed the data. G.Z. determined the faecal Cr and Arg contents. Y.H. evaluated the mouse grip-force strength. Y.H. and J.G. performed the cell experiments. G.Z. and Y.H. (Yongwu He) conducted the proteomics and analysed the data. S.H. drew the schematic diagram. J.Z. and C.W. drafted the original paper. All authors contributed to the revising and approval of the final paper.

Competing interests

The authors declare no competing interests.

Additional information

Extended data is available for this paper at.

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Daytime-restricted feeding reshapes the gut microbiota to achieve intestinal radioprotection through creatine-mediated ferroptosis inhibition

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Abstract

Figures

Introduction

Results

DTRF ameliorates the intestinal injury caused by IR

DTRF induces an increase in Cr content in the gut

Cr suppresses IR-induced ferroptosis by activating AMPK under energy stress

Cr contributes to the ferroptosis inhibition and radioprotection of DTRF

Discussion

Online Methods

Total abdominal irradiation mouse experiment

Histopathologic analysis

Faecal microbiota transplantation

16S rRNA gene amplicon sequencing analyses

Metagenomic analysis

Untargeted metabolomics

Cr and Arg determination

Mouse grip-force strength determination

Evaluation of bacteraemia and endotoxaemia

Cell experiments

ATP/ADP ratio detection

Proteomics analysis

Western blotting

Statistical analysis

Declarations

References

Additional Declarations

Supplementary Files

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