
==== Front
Cell Mol Life Sci
Cell Mol Life Sci
Cellular and Molecular Life Sciences: CMLS
1420-682X
1420-9071
Springer International Publishing Cham

39261351
5435
10.1007/s00018-024-05435-5
Original Article
Vitamin B12 ameliorates gut epithelial injury via modulating the HIF-1 pathway and gut microbiota
Feng Chenxi 1
Yan Jinhua 2
Luo Ting 2
Zhang Hong 3
Zhang Hu 3
Yuan Yu 1
Chen Yi toddychan@163.com

1
http://orcid.org/0000-0001-8305-4060
Chen Haiyang chenhy82@scu.edu.cn

12
1 https://ror.org/011ashp19 grid.13291.38 0000 0001 0807 1581 Division of Gastrointestinal Surgery, Laboratory of Stem Cell and Anti-Aging Research, Frontiers Science Center for Disease-Related Molecular Network, State Key Laboratory of Respiratory Health and Multimorbidity and National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University, Chengdu, 610041 Sichuan China
2 https://ror.org/011ashp19 grid.13291.38 0000 0001 0807 1581 Center of Gerontology and Geriatrics, Laboratory of Stem Cell and Anti-Aging Research, National Clinical Research Center for Geriatrics and Frontiers Science Center for Disease-Related Molecular Network, West China Hospital, Sichuan University, Chengdu, 610041 Sichuan China
3 https://ror.org/011ashp19 grid.13291.38 0000 0001 0807 1581 Department of Gastroenterology and Hepatology and Laboratory of Inflammatory Bowel Disease, West China Hospital, Sichuan University, Chengdu, 610041 Sichuan China
11 9 2024
11 9 2024
12 2024
81 1 39730 4 2024
27 8 2024
2 9 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
Inflammatory bowel diseases (IBDs) are immune chronic diseases characterized by recurrent episodes, resulting in continuous intestinal barrier damage and intestinal microbiota dysbiosis. Safe strategies aimed at stabilizing and reducing IBDs recurrence have been vigorously pursued. Here, we constructed a recurrent intestinal injury Drosophila model and found that vitamin B12 (VB12), an essential co-factor for organism physiological functions, could effectively protect the intestine and reduce dextran sulfate sodium-induced intestinal barrier disruption. VB12 also alleviated microbial dysbiosis in the Drosophila model and inhibited the growth of gram-negative bacteria. We demonstrated that VB12 could mitigate intestinal damage by activating the hypoxia-inducible factor-1 signaling pathway in injured conditions, which was achieved by regulating the intestinal oxidation. In addition, we also validated the protective effect of VB12 in a murine acute colitis model. In summary, we offer new insights and implications for the potential supportive role of VB12 in the management of recurrent IBDs flare-ups.

Supplementary Information

The online version contains supplementary material available at 10.1007/s00018-024-05435-5.

Keywords

Gut regeneration
Intestinal inflammation diseases
Vitamin B12
Gut microbiota
Hypoxia-inducible factor-1
http://dx.doi.org/10.13039/501100001809 National Natural Science Foundation of China 92157109 Chen Haiyang http://dx.doi.org/10.13039/501100012166 National Key Research and Development Program of China 2020YFA0803602 Chen Haiyang National Clinical Research Center for Geriatrics, West China Hospital, Sichuan UniversityZ20201006 Chen Haiyang 1.3.5 project for disciplines of excellence, West China Hospital, Sichuan UniversityZYYC20024 Chen Haiyang issue-copyright-statement© Springer Nature Switzerland AG 2024
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pmcIntroduction

Inflammatory bowel diseases (IBDs) are chronic relapsing immune disorders of the intestine, characterized by chronic inflammation of the intestinal mucosa. In patients, this disease may lead to repeated damage to intestinal tissues and inflammatory injuries, which manifest as symptoms such as diarrhea, abdominal pain, and anemia [1, 2]. The incidence and prevalence of IBDs are increasing globally, and the issue of recurrent attacks has long been a challenge for patients with IBDs [3, 4]. Disrupted barrier function and the composition of intestinal microbiota have been identified as factors contributing to IBDs [5, 6]. The integrity of the gut barrier is crucial in preventing the passage of macromolecules and pathogens and is an ongoing process [7, 8]. The enhancement of intestinal barrier function in the treatment and management of colitis is also being actively researched [9–11]. For patients with IBDs, achieving and maintaining disease remission, defined as complete mucosal healing, the normalization of relevant blood markers, and the persistent disappearance of symptoms, are crucial to reducing recurrence incidence and altering the course of the disease [12, 13]. The composition of the intestinal commensal flora is frequently altered in patients with colitis, leading to the progressive worsening of symptoms and the increased frequency of disease flares [14, 15]. Therefore, maintaining a stable intestinal external environment and alleviating the problem of long-term recurrent flares for patients is critical. However, the mainstream focus of research on IBDs has been on exploring its pathogenic mechanisms and treatment options, which has resulted in a limited understanding of the mechanisms underlying recurrent IBDs and a lack of strategies to address them.

Vitamin B12 (VB12), also known as cobalamin, is an important co-factor for a class of enzymes in the human body participating in cellular mitochondrial energy metabolism and methionine metabolism [16, 17]. In certain pathological phenotypes, VB12 was shown to have neuroprotective effects on experimental pneumococcal meningitis [18] and reduce damage after renal ischemia-reperfusion in mice [19], revealing its potential in treating inflammatory diseases. Some clinical evidences suggested a correlation between IBDs and VB12, indicating that IBDs patients are more likely to have reduced VB12 levels [20–22]. Studies using human organoid cultures have found that V B12 can enhance the expression of intestinal barrier-related protein genes [17]. In clinical practice, VB12 is recommended as a supplement in the treatment of IBDs to help improve overall health and enhance the quality of life for patients [23]. However, our understanding of whether VB12 plays a role in the recurrence of IBDs and the scientific mechanisms behind it remains unclear.

As a mature genetic research model organism, Drosophila melanogaster has long been utilized in the fields of developmental and cellular biology research due to its plentiful genetic resources, simple anatomical structure, and similarity to mammalian tissues [24, 25]. The adult Drosophila gastrointestinal system has gained recognition as an invaluable model for examining inflammation within the intestines [26–28]. Exposure to dextran sulfate sodium (DSS) or bacteria Ecc15 disturbed the integrity of the intestinal barrier, enhanced the proliferation of intestinal cells, and shortened the lifespan of Drosophila [29–31]. These experimental conditions effectively emulated the pathological advancements observed in individuals with IBDs. In terms of time scale, the physiological processes of Drosophila develop rapidly, allowing for prolific breeding, making them an excellent in vivo model for drug validation over short periods. However, currently, limited research has utilized Drosophila as an experimental model for studying the recurrence of IBDs. Therefore, this study used Drosophila as the primary model to conduct experiments.

In this study, we found that VB12 could alleviate intestinal damage caused by repeated DSS stimulation using a Drosophila gut model. VB12 alleviated functional and barrier disturbances in the Drosophila gut upon injury, mitigating hyperproliferation and inflammation. Under conditions of repeated injury, VB12 enhanced the expression of intestinal junction proteins to strengthen the intestinal barrier and inhibited the abnormal increase in gram-negative bacteria in the intestine. Mechanistically, our study indicated that VB12 could ameliorate intestinal hyperplasia and barrier disruption in DSS-injured guts by promoting the activation of hypoxia-inducible factor-1 signaling pathway. We also validated the protective effect of VB12 on intestinal epithelium in a DSS-induced murine colitis model. These results provide new insight into the impact of VB12 on the integrity of the intestinal barrier and gut microbiota, establishing its scientific relevance in management strategies for suppressing and reducing IBDs recurrence.

Results

VB12 ameliorates intestinal dysfunction and prolongs the lifespan of Drosophila with gut damage

Considering the recurrent nature of IBDs [32], we generated 7% DSS-induced Drosophila gut repeated injury model to simulate recurrent colitis. To evaluate the function of VB12, we administered it to the flies after the initial injury and assessed their morality rate on the seventh day of repetitive DSS injury, as lifespan is a critical indicator of gut homeostasis regulation in Drosophila [33, 34]. Consequently, we identified VB12 as a promising treatment candidate upon the reduction in mortality due to VB12 (Fig. 1A). We treated Drosophila with 7% DSS for 3 days to induce intestinal repeated damage with or without VB12 supplementation to further determine whether VB12 had a protective impact on intestinal damage (Fig. 1B). Transmission electron microscopy was used to observe the necrosis of enterocytes (ECs) in DSS-damaged guts. The peritrophic matrix (PM) [35, 36], composed of polysaccharides and proteins, lines the Drosophila intestinal lumen to block the invasion of pathogens (Fig. 1C). In DSS-induced injury, the PM was almost eliminated, and treatment with VB12 partially preserved the structure of the PM, although the distribution of the PM became altered (Fig. 1C). Drosophila treated with DSS had shorter intestinal lengths compared to the mock group, and the concurrent administration of VB12 partially restored the intestinal lengths (Fig. 1D, E). These results showed that VB12 supplementation had a protective effect on DSS-induced damage to intestinal morphology in Drosophila. Since intestinal barrier dysfunction is a significant feature of DSS-induced injury [31, 37], we performed the Smurf assay to assess gut permeability in Drosophila. The observation that the entire body of Smurf-positive (Smurf+) flies turned blue indicated a permeabilized gut, while the Smurf-negative (Smurf−) flies appeared normal (Fig. 1F). We found that VB12 supplementation significantly prevented gut leakage induced by DSS and improved intestinal barrier function (Fig. 1G). Previous research showed that DSS-induced gut injury in Drosophila resulted in a significant decrease in midgut digestive function, including the loss of gastrointestinal acid–base balance and a decline in excretion [38]. Thus, we explored whether VB12 supplementation could also mitigate a decline in intestinal function in Drosophila with injured guts. In DSS-fed flies, VB12 decreased the ratio of Drosophila with non-eating behavior, restrained the deterioration of intestinal acid–base homeostasis (Fig. 1H–J), and restored decreases in excretion (Fig. 1K, L). We explored the potential of VB12 supplementation to extend the lifespan of Drosophila with DSS-injured guts. VB12 successfully prolonged the lifespan of flies undergoing continuous DSS or Ecc15 feeding (Fig. 1M, S1A) while demonstrating no impact on the lifespan of flies with DNA breaks induced by bleomycin (BLM) or oxidative damage induced by paraquat (PQ) (Fig. S1B, C).Fig. 1 VB12 ameliorates the intestinal dysfunction and prolongs lifespan of Drosophila with gut damage. A Quantification of the mortality rate of VB12 on the seventh day in repeatedly DSS-induced gut injuries model of Drosophila. Three independent experiments were conducted, each group included 50 flies. *p < 0.05 (Student's t-test). B Schematic diagram depicting the process of feeding VB12 in repeated 7% DSS-induced gut injuries model of Drosophila. C Images of midguts in mock group flies and repeatedly DSS-fed flies with and without VB12 supplementation by transmission electron microscopy. Green arrows indicate PM, and white arrowheads indicate EC. Scale bars: 10 μm (top) and 2 μm (bottom). D Images of midguts staining with DAPI. DAPI: blue, nuclei. Scale bars: 500 μm. E Quantification of the length of midguts VB12 in mock group flies and repeatedly DSS-fed flies with and without VB12 supplementation (n = 20, 20, 20, from left to right). F Representative images of smurf− and smurf+ flies. Scale bars: 1 mm. G Quantification of the percentage of smurf+ flies. Three independent experiments were performed, each group included 30 flies. H Representative images of the intestinal acid–base homeostasis and the non-eating intestine. Circles indicate the CCR. Scale bars: 1 mm. I, J The percentage of eating intestines (I) and acid–base balanced intestines (J) in experiment H. Three independent experiments were performed, n = 30 flies per group. K Bright-field images of excretion deposits of mock group flies and repeatedly DSS-fed flies with and without VB12 supplementation. Scale bars: 5 mm. L Quantification of the number of deposits of flies in experiment K. Three independent experiments were performed, each group included 30 flies. Significance was calculated by one-way ANOVA using the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. M Survival percentage of female w1118 flies with or without VB12 supplementation under 7% DSS treatments. Each group had 100 flies and three independent experiments were conducted. Data are represented as means ± SD. **p < 0.01 (Log-rank (Mantel-Cox) test)

VB12 alleviates damage-induced intestinal stem cell hyper-proliferation and inflammatory response after injury

Persistent flares of colitis often lead to epithelial hyper-proliferation, increasing the risk of dysplasia and colorectal cancer [5, 10]. In Drosophila, the disruption of intestinal homeostasis upon gut damage also leads to gut epithelial hyperplasia (Fig. 2A), which is demonstrated by an overt increase in the proliferation rate of intestinal stem cells (ISCs) and the accumulation of intestinal stem and progenitors (which are escargot-positive (esg+) cells) in the injured midgut of Drosophila [39]. Given that, we utilized a reporter line (esg-GFP) to assess the intestinal stem cells and progenitors in the midgut to investigate the potential impact of VB12 on intestinal hyperproliferation under repeated damage. We tested 3 concentrations of VB12 (0.1, 1, and 10 µM) and determined 1 μM as the optimal concentration, significantly reducing the number of esg-GFP+ cells in second-time DSS-exposed flies with VB12 supplementation compared to flies without VB12 supplementation (Fig. 2B, C). Phosphorylated-histone 3-positive (pH3+, labels mitotic cells) staining showed the inhibitory effect of VB12 on hyperproliferation in injured intestines (Fig. 2D, E), and EdU (5-ethynyl-2′-deoxyuridine)-labeling experiment also demonstrated consistent results (Fig. S2A-B). The DSS-induced injury also caused cells surrounded by phalloidin to transition from a single layer to multiple layers, and VB12 significantly relieved this hyperplasia (Fig. 2F). We conducted supplementary experiments by inducing damage using Ecc15 to enhance the credibility and comprehensiveness of these findings and consistently observed similar effects on esg-GFP+ and pH3+ cells (Fig. 2G, H).Fig. 2 VB12 alleviates intestinal hyperplasia and inflammation in damaged gut of Drosophila. A The hyperplasia upon gut damage. During intestinal injury, intestinal stem cells (ISCs) and enteroblasts (EBs) undergo hyperproliferation of posterior midguts in Drosophila. B Immunofluorescence images of Drosophila (esg-GFP/ +) posterior midguts of esg-GFP staining. Three concentrations of VB12 were studied: 0.1, 1, and 10 µM. esg-GFP: green, ISCs and progenitor cells; DAPI: blue, nuclei. Scale bars: 50 μm. C Quantification of the ratio of esg-GFP+ cells to DAPI+ cells per ROI (region of interest) in experiment B (n = 30, 31, 34, 30, 30 from left to right). Three independent experiments were performed, each group included ≥ 15 flies. D Representative immunofluorescence images of Drosophila (w1118) posterior midguts with pH3 staining. pH3: red, mitotic cells; DAPI: blue, nuclei. Scale bars: 50 μm. E Quantification of the number of pH3+ cells in the whole guts of flies in experiment D (n = 20, 20, 20 from left to right). Three independent experiments were performed. F Representative immunofluorescence images of Drosophila (w1118) posterior midguts of phalloidin staining in mock group and repeatedly DSS-fed group with and without VB12 supplementation. phalloidin: red; DAPI: blue. Scale bars: 50 μm. (G) Quantification of the ratio of esg-GFP+ cells to DAPI+ cells per ROI (region of interest) in Drosophila (esg-GFP/ +) posterior midguts of GFP staining. (n = 30, 34, 30, from left to right). Three independent experiments were performed, each group included ≥ 15 flies. H Quantification of the number of pH3+ cells in the whole guts of Drosophila (w1118) posterior midguts of pH3 staining in mock group and repeatedly Ecc15-fed group with and without VB12 supplementation. (n = 20, 20, 20 from left to right). Three independent experiments were performed. I Immunofluorescence images of Drosophila (upd3-lacZ) posterior midguts of upd3-lacZ staining in mock group and secondary DSS-fed group with and without VB12 supplementation. upd3-lacZ: red; DAPI: blue. Scale bars: 100 μm. J Quantitative RT–qPCR detection of upd2 and upd3 expression levels of Drosophila (w1118) posterior midguts. Three independent experiments were performed. K Quantitative RT–qPCR detection of AMP-related gene (Diptericin, Drosocin, Drsomycin, Defensin, Attacin, and Cecropin) expression levels of Drosophila (w1118) posterior midguts. Significance was calculated by one-way ANOVA of three independent experiments. Data are represented as means ± SD.*p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001

In colitis, inflammation aids in proper wound healing and recovery. However, chronic inflammation hinders normal tissue functioning and exacerbates the disease [40]. Previous studies revealed that following damaging insults to the gut of Drosophila, ECs produced Upd3 (an interleukin (IL)-6-like cytokine with an α-helical fold that is able to activate a type-I cytokine receptor) [41, 42], but sustained high levels of Upd3 impaired the health of Drosophila [42]. In this study, we observed that VB12 mitigated Upd3 over-expression in ECs after injury and rescued the transcriptional levels of upd3 and upd2 (encoding a protein with similarities to type I cytokines) [43] (Fig. 2I, J). Considering the inflammation induced by DSS, which includes the high expression of antimicrobial peptides (AMPs) that contribute to immune responses in the intestine upon secretion [38, 44], we observed that VB12 could also attenuate the increased expression of AMPs (Fig. 2K). In summary, our research demonstrated that VB12 could alleviate intestinal hyperplasia and inflammation after gut injury.

VB12 protects against the disruption of intestinal cell junctions from DSS-induced damage

Next, we sought to investigate how VB12 protects the intestine against injury. Previous research provided two primary explanations for alleviating damage. One was that resting intestinal stem cells can be activated after stress to trigger a repair process in the intestine and restore homeostasis [26, 45]. The other explanation is that the downregulation of tight junction protein expression in colitis could result in enhanced intestinal permeability [46, 47] and maintaining intercellular connections might mitigate intestinal damage [48, 49]. Organoids derived from ISCs comprehensively recapitulated the essential characteristics of intestinal regeneration [50, 51]. Since it was difficult to discriminate how VB12 exerted its effect, we verified whether VB12 contributed to intestinal tissue repair via ISC regeneration in mouse-derived intestinal organoids. VB12 had no effect on the stemness and differentiation properties of ISCs in the organoids (Fig. 3A–C).Fig. 3 VB12 protects against intestinal barrier damage by maintaining cell junction. A Bright-field images of intestinal organoids with and without VB12 supplementation on the third day of culture. Scale bars: 500 μm. B Quantification of the percentage of organoids formation with and without VB12 supplementation (n = 4 mice for each group). C Quantitative RT–qPCR detection of Lgr5 (ISCs marker), Hes1 (Intestinal absorption progenitor cells marker), and Atoh1 (Intestinal secretory progenitor cells marker) expression levels of organoids on the seventh day of culture with and without VB12 supplementation (n = 4 mice for each group). D Immunofluorescence images of intestinal organoids with and without VB12 supplementation stained with Occludin and Claudin-1. Occludin: green; Claudin-1: red; DAPI: blue. Scale bars: 50 μm. E, F Quantitation of the fluorescence intensity of Occludin (E) (n = 100, 97 cells of 30 organoids for each group) and Claudin-1 (F) (n = 112, 104 of 30 organoids for each group) from experiments D. Significance was calculated by Student's t-test of three independent experiments. Data are represented as means ± SD. ***p < 0.001, ****p < 0.0001 and ns indicates p > 0.05. G Immunofluorescence images of posterior midguts in mock group flies and repeatedly DSS-fed flies with and without VB12 supplementation stained with Arm. Arm: red; DAPI: blue. Scale bars: 50 μm. H Quantification of the fluorescence intensity of Arm from experiments G (n = 99, 99, 99 from left to right). Three independent experiments were performed, each group included ≥ 15 flies. I Immunofluorescence images of posterior midguts stained with Cora. Cora: red; DAPI: blue. Scale bars: 50 μm. J Immunofluorescence images of posterior midguts stained with Dlg. Dlg: red; DAPI: blue. Scale bars: 50 μm. K Quantification of the fluorescence intensity of Cora from experiments I (n = 103, 111, 107 from left to right). Three independent experiments were performed, each group included ≥ 15 flies, each group included ≥ 15 flies. L Quantification of the fluorescence intensity of Dlg from experiments J (n = 123, 102, 103 from left to right). Three independent experiments were performed, each group included ≥ 15 flies, each group included ≥ 15 flies. Significance was calculated by one-way ANOVA of three independent experiments, each group included ≥ 15 flies. Data are represented as means ± SD. *p < 0.05 and ****p < 0.0001. M Western blot analysis of E-cad of midguts

Previous research reported that colitis was associated with impairments of the intestinal barrier and intercellular junctions [9, 10], and VB12 had the ability to upregulate the expression of tight junction proteins in human-derived intestinal organoids [17]. We further demonstrated the impact of VB12 on tight junction proteins in mouse intestinal organoids using immunofluorescence staining (Fig. 3D–F). Subsequently, we investigated whether VB12 maintained cell junctions in injury-induced Drosophila. Our findings demonstrated that DSS destroyed the structural integrity of the adhesion junction protein Armadillo (Arm) in the Drosophila midgut and reduced the level of Arm. VB12 effectively rescued these effects (Fig. 3G, H). VB12 was also able to rescue the decrease of septate junction proteins Coracle (Cora) (Fig. 3I, K) and Drosophila discs large (Dlg) (Fig. 3J, L) [52, 53] upon DSS-induced damage. The reduced expression of E-cadherin (E-cad) was also observed in DSS-induced damage and IBDs patients [26, 54]. In this study, VB12 supplementation maintained the protein expression of E-cad in Drosophila (Fig. 3M).

Administration of VB12 significantly alters the microbiota in damaged Drosophila intestine

The intestinal microbiota plays a crucial role in regulating intestinal homeostasis. Mounting evidence suggests a strong correlation between the gut microbiota and diverse aspects of Drosophila physiology and intestinal homeostasis, with a particular emphasis on its involvement in inflammatory diseases [30, 55, 56]. Therefore, we investigated the impact of VB12 on the gut microbiota of Drosophila with gut injury. We conducted 16S ribosomal RNA gene sequencing on gut DNA extracted from flies fed DSS and compared those supplemented with VB12 to those without supplementation. Shannon and Simpson indices were utilized to assess the α-diversity of the intestinal microbiota, which revealed significant variations in the abundance of intestinal microbiota between the two groups of flies (Fig. 4A). Principal coordinate analysis and partial least-square discriminant supervised analysis were performed to assess the dissimilarities in microbiota composition between the two groups of flies. The results further supported a clear difference in microbiota composition between the VB12 and mock groups (Fig. 4B, C). The variable importance in projection (VIP) scores revealed VB12 has lessened the enrichment of some gram-negative pathogenic bacteria [57–60] like Serratia and Pseudomonas, which was previously reported to be associated with increased gut permeability (Fig. 4D, S3A–B) [61]. Furthermore, the abnormal expansion of Proteobacteria was closely associated with IBDs according to previous research, which is also restricted under VB12 treatment [62, 63]. The analysis of genus relative and absolute abundance also indicated a decrease in Serratia, and Pseudomonas in flies supplemented with VB12, which is consistent with the above analysis. (Fig. 4E, S3C).Fig. 4 VB12 alters intestinal microbial dysbiosis in damaged gut of Drosophila. A Shannon and Simpson diversity analysis of the bacterial species in gut samples from repeatedly DSS-fed flies with and without VB12 supplementation by Wilcoxon rank‐sum test. B Principal coordinate analysis (PCoA) analysis showing significant differences in the gut microbiota between mock and VB12 group from repeatedly DSS-fed flies. C Partial least-square discriminant supervised analysis (PLS-DA) analysis was generated to visualize the species abundance in samples collected from the gut microbiota of repeatedly DSS-fed flies, comparing those with and without VB12 supplementation. D The variable importance in projection (VIP) scores of PLS-DA. E Relative abundances of bacterial genus structure in gut samples from the gut microbiota of repeatedly DSS-fed flies, comparing those with and without VB12 supplementation. F–H Representative images (F) and quantification of the bacterial (G) and Gram− bacterial (H) load of midguts. The bacterial load in flies was determined by colony count. Three independent experiments were performed. I, J Representative images of microbiota with Gram staining (I) and quantification of the ratio of Gram− to Gram+ bacteria per ROI (J) in midguts of flies. Significance was calculated by one-way ANOVA of three independent experiments. Data are represented as means ± SD. **p < 0.01, ***p < 0.001 and ****p < 0.0001

According to the aforementioned results, we formulated the hypothesis that VB12 supplementation has the potential to limit G− bacteria dysbiosis in the injured gut of flies. Subsequently, we performed a bacterial culture experiment to provide further evidence. The results revealed a notable increase in the number of colony-forming units in the midgut of DSS-fed flies compared to the mock group. VB12 effectively mitigated this change (Fig. 4F, G). Significantly, supplemental VB12 inhibited the growth of G− bacteria in DSS-induced injury, as evidenced by a G− bacterial selective culture (Fig. 4F, H). Gram staining further confirmed that supplementation with VB12 significantly inhibited the increase in the ratio of G− bacteria to G+ bacteria under DSS-induced conditions (Fig. 4I, J). Together, these data provide evidence that VB12 could suppress G− bacteria increases in the intestines of flies with DSS-induced injury, thereby offering protective effects against gut injury and repair.

The relationship between VB12 and gut microbiota is associated with the oxygen consumption of intestinal cells

Previous studies demonstrated that VB12 could increase cellular oxygen consumption, which was possibly attributed to its impact on mitochondrial respiratory function [64]. The respiratory chain within mitochondria is associated with cellular oxygen consumption, and a deficiency in the respiratory chain can inhibit oxygen consumption [65, 66]. We hypothesized that the regulation of gut microbiota by VB12 may be linked to its influence on mitochondrial oxygen consumption in the intestine. Therefore, we utilized a mex-Gal4 system to knock down Cox5a, which is associated with the key complex CIV of the respiratory chain, in Drosophila ECs (Fig. 5A). The colony count of intestinal bacteria, particularly G− bacteria, in flies with mex-Gal4-driven Cox5a knockdown after repeated injury was significantly increased compared to the mock group. However, supplementation with VB12 had no effect (Fig. 5B–D). Similar results were observed by Gram staining (Fig. 5E, F). Consistent with the above results, VB12 failed to impact the changes in microbiota after injury when mt:CoII, another subunit gene involved in the function of complex CIV, was knocked down (Fig. 5G–L). Thus, these findings indicated that the effect of VB12 in regulating the gut microbiota after gut injury was dependent on mitochondrial oxidation.Fig. 5 The impact of VB12 on the damaged gut microbiota is related to mitochondrial respiration. A Quantitative RT–qPCR detection of Cox5a expression levels of posterior midguts of flies carrying mex-Gal4-driven UAS-lacZ and UAS-Cox5a-RNAi. Three independent experiments were performed. B, C Quantification of the bacterial (B) and Gram− bacterial (C) load of Drosophila (mex-Gal4 > UAS-Cox5a-RNAi) midguts in mock group and repeatedly DSS-fed group with and without VB12 supplementation. The bacterial load in flies was determined by colony count. Three independent experiments were performed. D Representative images of the bacterial (top) and Gram− bacterial (bottom) load in Drosophila (mex-Gal4 > UAS-Cox5a-RNAi) midguts from experiments B, C. E, F Representative images of microbiota with Gram staining (E) and quantification of the ratio of Gram− to Gram+ bacteria per ROI (F) in midguts of mex-Gal4 > UAS-Cox5a-RNAi flies in mock group and repeatedly DSS-fed group with and without VB12 supplementation. Three independent experiments were performed. G Quantitative RT–qPCR detection of mt:CoII expression levels of posterior midguts of flies carrying mex-Gal4-driven UAS-lacZ and UAS-mt:CoII-RNAi. Three independent experiments were performed. H, I Quantification of the bacterial (H) and Gram− bacterial (I) load of Drosophila (mex-Gal4 > UAS-mt:CoII-RNAi) midguts in mock group and repeatedly DSS-fed group with and without VB12 supplementation. Three independent experiments were performed. J Representative images of the bacterial (top) and Gram− bacterial (bottom)load in Drosophila (mex-Gal4 > UAS-mt:CoII-RNAi) midguts from experiments H, I. K, L Representative images of microbiota with Gram staining (K) and quantification of the ratio of Gram− to Gram+ bacteria per ROI (L) in midguts of mex-Gal4 > UAS-mt:CoII-RNAi flies in mock group and repeatedly DSS-fed group with and without VB12 supplementation. Significance was calculated by one-way ANOVA of three independent experiments. Data are represented as means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 and ns indicates p > 0.05

VB12-mediated protection of the intestine is associated with the modulation of the HIF-1 signaling pathway

Mitochondrial oxidative energy metabolism is changed during intestinal inflammation [9, 67], and studies showed that the HIF-1 pathway plays a positive role in the barrier function of intestinal epithelial cells [68, 69] and the management of intestinal inflammation [70–72]. We examined the expression of downstream target genes of the HIF-1 pathway [73] in Drosophila intestine and demonstrated that VB12 could restore HIF-1 activation that was inhibited in flies with DSS-induced repeated injury (Fig. 6A). We next observed intestinal homeostasis and cell junctions in DSS-treated flies with mex-Gal4-driven sima (a Drosophila homologous gene of Hif-1) overexpression. This study revealed that the overexpression of sima in ECs rescued cellular homeostasis and cell junctions in flies with DSS-induced injury (Fig. 6B, C, E, F), and VB12 did not additionally protect the damaged intestines (Fig. 6D, E, F). These results suggest that VB12 alleviated damage to Drosophila intestines by stabilizing the activation of the HIF-1 signaling pathway.Fig. 6 VB12 protects the intestines from injury via HIF-1 in Drosophila. A Quantitative RT–qPCR detection of HIF-1 pathway targeted gene (Ldh and Bnip3) expression levels of Drosophila (w1118) posterior midguts in mock group and repeatedly DSS-fed group with and without VB12 supplementation. Three independent experiments were performed. B–D Representative immunofluorescence images of posterior midguts of flies carrying mex-Gal4-driven UAS-lacZ + DSS (B), UAS-sima + DSS (C), and UAS-sima + DSS + VB12 (D), stained with DAPI, esg-GFP, and Arm. esg-GFP: green; Arm: red; DAPI: blue. Scale bars: 50 μm. E Quantification of the ratio of esg-GFP+ cells to DAPI+ cells per ROI (region of interest) in experiments B–D (n = 31, 28, 36 from left to right). Three independent experiments were performed, each group included ≥ 15 flies. F Quantification of the fluorescence intensity of Arm from experiments (B–D) (n = 108, 103, 109 from left to right). Three independent experiments were performed, each group included ≥ 15 flies. G Quantitative RT–qPCR detection of HIF-1 pathway targeted gene (Ldh and Bnip3) expression levels of posterior midguts of flies carrying mex-Gal4-driven UAS-lacZ, UAS-mt:CoII-RNAi, UAS-Cox5a-RNAi, and UAS-sima-RNAi. Three independent experiments were performed. H–L Representative immunofluorescence images of posterior midguts of flies carrying mex-Gal4-driven UAS-lacZ (H), UAS-Cox5a-RNAi (I), UAS-Cox5a-RNAi + DSS (J), UAS-Cox5a-RNAi + DSS + VB12 (K), and UAS-Cox5a-RNAi; UAS-sima + DSS (L), stained with DAPI, esg-GFP, and Arm. esg-GFP: green; Arm: red; DAPI: blue. Scale bars: 50 μm. (M) Quantification of the ratio of esg-GFP+ cells to DAPI+ cells per ROI (region of interest) in experiments (H–L) (n = 34, 34, 32, 31, 33 from left to right). Three independent experiments were performed, each group included ≥ 15 flies. N Quantification of the fluorescence intensity of Arm from experiments (H–L) (n = 114, 104, 109, 101, 101 from left to right). Three independent experiments were performed, each group included ≥ 15 flies. O The mechanism that VB12 protects the intestines upon injury. Significance was calculated by one-way ANOVA using the means ± SD.*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 and ns indicates p > 0.05

Next, considering the relationship between mitochondrial oxidative metabolism and oxygen consumption [9, 64], we hypothesized that VB12 might activate the HIF-1 pathway by promoting mitochondrial oxygen consumption and maintaining intestinal hypoxia. Our results demonstrated a significant decrease in the expression of HIF-1 pathway target genes when the CIV subunit of the respiratory chain was knocked down in ECs, consistent with the observations following sima knockdown (Fig. 6G). We next found that VB12 had no effect on the hyperproliferation or disruption of cell junctions induced by DSS upon mitochondrial respiratory chain deficiency (Fig. 6H, K, M, N). These results further underscored that the protective effects of VB12 on the gut were related to intestinal oxygen consumption. By overexpressing sima in the flies, we further confirmed that the HIF-1 pathway could mitigate DSS-induced intestinal hyperplasia and barrier dysbiosis under mitochondrial respiratory chain deficiency (Fig. 6L–N). Together, these results indicated that mitochondrial oxidative metabolism could reduce intestinal injury via the HIF-1 pathway and that VB12 exerted protective effects through this mechanism (Fig. 6O).

VB12 administration ameliorates DSS-induced colitis and maintains cell junctions and HIF-1 signaling in mice

Subsequently, we evaluated the therapeutic potential of VB12 in a murine model of colitis induced by the oral administration of 2.5% DSS (Fig. 7A). Mice treated with both DSS and VB12 simultaneously exhibited a significant reduction in colon length shortening (Fig. 7B, C) and weight loss (Fig. 7D) compared to those treated with DSS alone. Assessment of the disease activity index revealed that VB12 effectively ameliorated the intestinal damage induced by DSS (Fig. 7E), and histological analyses consistently showed that VB12 effectively attenuated DSS-induced morphological abnormalities (Fig. 7F, G). Immunofluorescence staining and immunoblotting analysis were utilized to assess the expression levels of Occludin and Claudin-1, both of which were detected in the inner lining of the intestinal epithelium with sustained expression levels in VB12-treated colitis model mice (Fig. 7H–I, S4A-B). We also examined the expression of downstream target genes of the HIF-1 pathway and observed that VB12 promoted the activation of the HIF-1 pathway in mice with DSS-induced colitis, consistent with the observations in flies (Fig. 7J). Given the impact of VB12 on gut microbiota in damaged Drosophila intestines, we preliminarily investigated its influence on intestinal microbiota in mice with acute colitis. Inoculation of DSS-treated mice with fecal microbiota revealed that colitis drove a significant expansion of intestinal microbiota, particularly gram-negative bacteria, which VB12 was able to alleviate (Fig. 7K, S4C). Through relative quantification analysis, some genera with differential abundance induced by VB12 in Drosophila also exhibit similar results in mice (Fig. S4D-E). These findings indicate that VB12 treatment holds promise as a viable approach for managing IBDs by enhancing intestinal barrier protection.Fig. 7 VB12 ameliorates DSS-induced colitis and maintains cell junctions in mice. A Schematic diagram depicting the process of feeding VB12 in 2.5% DSS-induced colitis model of mice. B, C A representative photograph of colon tissue from each group (B), and quantification of the colon length (C). Each group included 5 mice. D, E Quantification of the body weight ratio on the 7th day to the 1st day (D) and the disease activity index (DAI) (E) during the experiments were monitored. F, G Representative images of hematoxylin and eosin staining in paraffin-embedded mouse colon sections of experiments (F) and histological scores were measured (G). Scale bars: 250 μm. Each group included 5 mice. H Immunofluorescence images of colon tissue from each group stained with Occludin and Claudin-1. Occludin: green; Claudin-1: red; DAPI: blue. Scale bars: 100 μm. I Immunoblot analysis of Occludin and Claudin-1 of colon tissue from each group. Three biological replicates were conducted. J Quantitative RT–qPCR detection of HIF-1 pathway targeted gene (mLdh and mBnip3) expression levels of colon tissue from each group. Three independent experiments were performed. K Quantification of gram-negative bacterial load of murine stool. L Schematic model of the mechanism of VB12 exertion. During intestinal injury, the intestinal barrier is compromised, intercellular junction is disrupted, and ISCs undergo hyperproliferation. Vitamin B12 can inhibit gram-negative bacteria and alleviate gut damage by promoting the HIF-1 signaling pathway. Significance was calculated by one-way ANOVA using the means ± SD.*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 and ns indicates p > 0.05

Discussion

IBDs are recurrent intestinal inflammatory disorders closely associated with impaired intestinal barrier function. In individuals with IBDs, the permeability of the intestinal barrier increases, allowing bacteria, toxins, and other harmful substances to enter the circulatory system, leading to systemic inflammatory responses [6, 74]. These recurrent and persistent issues disrupt the health and quality of life of patients. Thus, inhibiting and reducing the recurrence of IBDs is crucial. In this study, we revealed the protective effect of VB12 by constructing a repeated injury model in Drosophila. Mechanistically, VB12 alleviated intestinal injury in Drosophila via the HIF-1 pathway and also played a role in modulating the composition of the intestinal microbiota in Drosophila under repeated gut injury (Fig. 7L).

As a key factor influencing IBDs, maintaining the integrity of the intestinal barrier is of great importance in reducing colitis recurrence. The integrity of the intestinal barrier is closely related to the tight junctions between epithelial cells. When the intestinal barrier is damaged, tight junctions are disrupted, leading to an increase in gaps between intestinal epithelial cells, allowing harmful substances to easily penetrate and enter the bloodstream, triggering a systemic inflammatory response [46, 75]. Our research indicated that VB12 could prevent the damage-induced disruption of cell junction proteins in Drosophila caused by repeated DSS inductions (Fig. 3). In patients with recurrent IBDs, the body’s response to intestinal damage can become dysregulated, with a risk of tumorigenesis [10, 76]. Our data indicated that VB12 inhibited ISC hyperproliferation in gut-injured Drosophila and upregulated immune cytokines and defense factor AMPs (Figs. 1, 2, 3). Overall, these effects in Drosophila may have been due to enhancements in the intestinal barrier to resist injury. Our study also confirmed that VB12 alleviated intestinal damage in a murine model of acute colitis (Fig. 7). Our research results suggest that VB12 prolonged the lifespan of flies under DSS or Ecc15 stimulation (Fig. 2). However, since VB12 may also have extraintestinal effects on other tissues, we cannot rule out the possibility that the lifespan-extending effect of VB12 was mediated by other mechanisms. Thus, further investigation is required for validation. In general, we revealed a new understanding of the mechanisms for reducing and inhibiting the recurrence of IBDs.

The composition and homeostasis of the gut microbiota are frequently disrupted in patients with IBDs, leading to a progressive worsening of symptoms and increased frequency of disease flares [14]. A healthy gut microbiota can maintain the integrity of the intestinal mucosal barrier, promote the repair and regeneration of intestinal epithelial cells, and enhance the barrier’s ability to block harmful substances and microorganisms [77–79]. However, an imbalanced gut microbiota, characterized by abnormal proliferation, reductions in bacterial populations, or changes in bacterial species, can damage intestinal barrier function [14, 80]. Here, we demonstrated that VB12 inhibited the abnormal expansion of the gut microbiota, particularly some gram-negative bacteria, with multiple DSS-induced injuries (Fig. 4). 16S rDNA amplicon sequencing analysis revealed that VB12 reduced the abundance of some pathogenic gram-negative bacteria associated with the IBDs process. Previous studies showed [64] that VB12 could enhance the oxygen consumption of intestinal cells, which also increased the survival of anaerobic bacteria. This study revealed that the changes in the microbiota underlying VB12-mediated intestinal damage were linked to mitochondrial oxidation (Fig. 5). However, current validations of whether VB12 protects against intestinal damage by inhibiting the colonization of gram-negative bacteria are limited, necessitating further investigation into specific gram-negative bacteria and their survival modes under intestinal oxidative regulation. Additionally, our analysis of intestinal microbiota has primarily focused on Drosophila, we plan to conduct a more comprehensive analysis on mammalian intestinal microbiota in the future to elucidate VB12’s effects on gut microbiota in IBDs patients.

HIF-1 is a protein that is sensitive to hypoxic environments and can regulate cellular adaptive responses to hypoxia [81]. The stability of HIF-1 is related to the prolonged low-oxygen environment in the intestinal lumen [82, 83]. DSS-induced injury can alter the oxygen environment in the intestine, leading to epithelial oxygenation [84]. The activation of the HIF-1 signaling pathway in intestinal epithelial cells and immune cells was shown to play a positive role in the damage of an intestinal inflammation model [70–72]. Our data indicated that the impact of VB12 on intestinal mitochondrial oxidative function promoted the activation of the HIF-1 signaling pathway (Fig. 6). Even with the loss of intestinal mitochondrial oxygen consumption, the overexpression of HIF-1 in Drosophila exposed to DSS reduced intestinal hyperplasia and maintained epithelial integrity. Given the pleiotropy and intricate consequences associated with the hypoxia/HIF-1-targeted pathway in host physiology [85–87], careful analysis and additional validation are imperative.

Drosophila, the primary research model in this study, has a short lifecycle, which allowed us to quickly establish a model simulating recurrent episodes of IBDs and identified VB12 for experimentation. Through their abundant genetic resources, simple anatomical structure, and significant similarity to mammalian tissues, we successfully demonstrated the protective effect of VB12 on the intestine under repeated injuries, which acted through the HIF-1 signaling pathway and gut microbiota. However, considering the differences between flies and humans, especially in terms of gut microbiota diversity, the current exploration represents only preliminary findings. Future work will involve subsequent studies in mammals and humans in later stages. Previous studies reported opposing effects of sufficient and supplemented VB12 on intestinal repair in mice (that is, after ending DSS administration and returning to a normal diet) [88]; however, this also suggests that VB12 might have potential effects through other pathways on intestinal injury, such as the methionine metabolism primarily mediated by vitamin B6 [89]. Undeniably, the sufficient VB12 conditions align with our view that VB12 administration plays a crucial role in the context of DSS-induced VB12 deficiency. Overall, our study provides preliminary work for further standardizing the use of adjunctive therapies for IBDs.

Materials and methods

Drosophila culture and stocks

All flies were kept under a 12-h light/12-h dark cycle at a constant temperature of 25 °C with the standard cornmeal diet, a mixture of 80 g of sucrose, 50 g of cornmeal, 20 g of glucose, 18.75 g of yeast, 5 g of agar, and 30 mL of propionic acid was dissolved in 1 L of water. All flies used were mated adult females between 10 and 14 days old.

The experiment utilized the following lines of Drosophila: esg-GFP/CyO, mex-Gal4, and ;esg-GFP/CyO; (provided by Allan Spradling), upd3-lacZ (provided by Zheng Guo), w1118 (BDSC, #3605), UAS-lacZ (BDSC, #8529), Cox5a RNAi line (BDSC, #27548), mt:CoII RNAi line (BDSC, #64853), sima RNAi line (BDSC, #26207), UAS-sima (BDSC, #9582).

Drosophila injury model and vitamin B12 (VB12) treatment

For repeated damage modeling, chromatography paper was cut into pieces that are 3.7 cm × 5.8 cm in size and thoroughly wetted with 7% dextran sulfate sodium (DSS) (Yeasen Biotechnology, #60316ES80, dissolved in 5% sucrose) and Ecc15 suspension (Ecc15 through overnight culture was concentrated by centrifugation and dissolved in 5% sucrose to make the final OD600 reading of 200). Flies were starved in empty tubes for 2 h and then subjected to a 3-day treatment of DSS or a 2-day treatment of Ecc15. 5% sucrose was used as a mock group in the absence of these treatments. Flies were placed on a normal diet with or without VB12 supplementation and fed for 8 days prior to exposure to secondary DSS or Ecc15 induction.

VB12 (Aladdin, #Q111273) was fully dissolved in water and mixed with the standard food medium to obtain desired concentrations (0.1, 1, 10 µM). Flies were distributed randomly into vials containing food mixed with VB12. The mock food was mixed with an equal volume of Water.

Mice husbandry and procedures

8-week-old male C57BL/6 mice were housed in groups of five animals and maintained at a controlled temperature of 23 ± 1 ℃ and a relatively humidity of 45–65% with a 12-h dark/light cycle. Before the experimental procedures, animals were adapted to the new environment for 1 week.

Mice were randomized into four groups and given ad libitum access to four types of drinking water with DSS or VB12 treatment. For DSS treatment, 2.5% DSS was dissolved in drinking water. For VB12 treatment, VB12 (1 µM) were prepared in drinking water. All mice were administered for 7 days and sacrificed at day 7.

Organoids culture

After flushing the male mouse intestines with cold phosphate buffered saline (PBS) and opening them laterally, the villi were gently scraped off using a glass slide. The remaining tissue was then cut into 1–2 cm sections, rinsed thoroughly, and incubated in 5 mM EDTA/PBS at 4 °C for 50–60 min. Following this, the crypts were harvested through mechanical shaking and filtered through a 70-µm mesh to remove villous fragments. The isolated crypts were quantified and combined with a 1:1 mixture of medium and Matrigel (Gibco, #A1413202) at a concentration of 15–25 crypts per μL for cultivation in a crypt culture medium. Unless otherwise stated, the crypts were cultured in a blend of Advanced DMEM/F12 medium (Gibco, #10,565,018) and IMDM medium (Gibco, #31,980,097), supplemented with EGF at 40 ng/ml (Peprotech, #315–09), Noggin at 200 ng/ml (Peprotech, #250–38), R-Spondin at 500 ng/ml (Peprotech, #120–38), N-acetyl-L-cysteine at 1 mM (Sigma Aldrich, #A7250), B27 at 2x (Gibco, #17,504,044), and N2 at 1x (Gibco, #17,502,001). The intestinal crypts were plated in 50 μL Matrigel droplets on flat-bottomed 24-well plates, allowed to solidify for 10–15 min in a 37 °C incubator. Subsequently, each well was supplemented with 500 μL of crypt medium and maintained at 37 °C in a humidified incubator with 5% CO2. The crypt medium was refreshed every three days. On the third day of cultivation, observe and record the organoid formation rates; stop the cultivation on the seventh day and proceed with subsequent experimental analysis.

Immunofluorescence and microscopy

The midguts of dissected flies and organoids were fixed for 30 min with 4% paraformaldehyde, and then into 0.1% PBST (PBS containing 0.1% Triton X-100 (Aladdin, # T109027)) with three times wash for 10 min each. For colon tissue of mice, the samples were fixed in 4% paraformaldehyde for 48 h, then sectioned and baked at 65 °C for 30 min. A biotransparent agent was applied for 30 min. The sections were sequentially washed in ethanol gradients (anhydrous, 95%, 90%, 80%) for 2–5 min each, followed by a 5-min rinse in double-distilled water. Microwave antigen retrieval was done using citrate, and the sections were washed twice with PBS for 5 min each. Subsequently, the samples were blocked in 0.5% BSA for 30 min under 25 ℃ and incubated overnight at 4 °C with primary antibodies diluted in 0.1% PBST. Next day, the tissues were washed with 0.1% PBST as described before and incubated with solution containing secondary antibodies and 4’, 6-diamino-2-phenylindole (DAPI) for 2 h at room temperature. Finally, guts were performed the same washing. For phalloidin staining, the samples were incubated in fluorescent probe solution (Invitrogen, #A30106) for 60 min at room temperature.

Primary antibodies and dilutions were as follows: chicken anti-GFP (1:1000, Abcam, #ab13970), rabbit anti-pH3 (1:1000, Millipore, #06-570), chicken anti-lacZ (1:1000, Abcam, #ab9361), mouse anti-Arm (1:100, DSHB, #AB_528089), mouse anti-Cora (1:50, DSHB, #AB_1161642), mouse anti-Dlg (1:100, DSHB, #AB_528203), mouse anti-Occludin (1:1000, Serviceobio, #GB111401-100), and mouse anti-Claudin-1 (1:1000, Serviceobio, #GB12032-100).

The secondary antibodies: Goat anti-chicken Alexa 488 (Invitrogen, Shanghai, China, #AB_2921070), Goat anti-chicken Alexa 568 (Invitrogen, #AB_2921072), Goat anti-mouse Alexa 568 (Invitrogen, #AB_144696), Goat anti-rabbit Alexa 488 (Invitrogen, #AB_143165), and Goat anti-rabbit Alexa 568 (Invitrogen, #AB_143157) were used at a 1:2000 dilution. The nuclei were stained using 1 µg/ml DAPI (Sigma, #D9542).

The immunofluorescence images were acquired with a Leica TCS-SP8 confocal microscope (Leica, Weztlar, Germany) and subsequently analyzed using Leica Application Suite X (Leica, Weztlar, Germany), ImageJ software (Bethesda, Rockville, MD, USA) and Adobe Illustrator.

Western blot assay

25–30 midguts of Drosophila or a small segment of murine colon tissue per group were rinsed with cold PBS and homogenized using a RIPA Lysis Buffer (MedChemExpress, #HY-K1001) supplemented with Protease Inhibitor Cocktail (MedChemExpress, #HY-K0010) and Phenylmethylsulfonyl fluoride (MedChemExpress, #HY-B0496). The samples were incubated on ice for 30 min, and the supernatant was collected by centrifugation and mixed with SDS-PAGE Sample Loading Buffer (Beyotime, #P0015F). The mixture was boiled at 95 °C for 5 min. The samples were loaded onto an 10% SDS–polyacrylamide gel, and transferred to an activated PVDF Transfer Membrane (Merck, #IPVH00010). Subsequently, the membrane was blocked with 5% skim milk and incubated overnight at 4 °C with a diluted primary antibody. Subsequently, the samples were incubated with a secondary antibody at room temperature for 2 h, and the signal was detected using an ECL reagent (Abbkine, #BMU102-CN) on the ChemiDoc XRS+ System (Bio-RAD, Hercules, USA). The following antibodies were used for Western blotting. Primary antibodies: mouse anti-E-cad (1:50, DSHB, #AB528120), mouse anti-β-Actin (1:1000, ABclonal, #AC004), rabbit anti-Occludin (1:5000, Serviceobio, #GB111401-100), and mouse anti-Claduin-1 (1:5000, Serviceobio, #GB12032-100). The secondary antibody: goat anti-mouse IgG-HRP (Beyotime, #A0216) and goat anti-rabbit IgG-HRP (Beyotime, #A0208) were used at a 1:2000 dilution.

EdU-labeling experiment

Drosophila midguts were dissected and placed into cold PBS, and 2 × of EdU working solution (Beyotime, #C0081S) was added to the plate at a final concentration of 10 µM (1×) with a 2-h incubation at room temperature for EdU labeling. Following this, the samples were fixed in 4% PFA for 15 min and washed three times with 3% BSA/PBS, with each wash lasting 5 min. Next, the guts were treated with 0.3% PBST for 10 min and then washed again with PBS containing 3% BSA for 5 min. Afterward, the click-reaction solution (Beyotime, #C0081S) was applied and incubated in the dark at room temperature for 30 min. The samples were then washed three times with 3% BSA/PBS, with each wash lasting 5 min. Finally, primary and secondary antibody incubations were carried out using standard immunofluorescence techniques.

Electron microscopy

Drosophila adults were dissected into PBS, and the guts were immediately fixed with 3% glutaraldehyde in PBS, then fixed with 1% osmium tetroxide solution. The samples were dehydrated stepwise with acetone concentrations of 30%, 50%, 70%, 80%, 90%, 95%, and 100%, with three changes of 100% acetone, and embedded in Epon-812. Guts were cut at 60–90 nm for transmission electron microscopy with a Leica ultramicrotome (Leica, Weztlar, Germany). Ultrathin sections were stained with uranyl acetate for 10–15 min, then stained with lead citrate for 1–2 min at room temperature, and observed with a transmission electron microscope.

Intestinal morphology assay

The Drosophila guts were fixed with 4% PFA for 30 min and then washed three times with 0.1% PBST. After the incubation of DAPI solution, the guts were washed and immersed in the 70% glycerol, under a Leica M205 FA stereomicroscope (Leica, Weztlar, Germany) for observation and photography, and the gut length was measured by ImageJ software. For mice, the colon tissues were collected and stretched out to measure length.

Smurf assay

Smurf assay was performed to evaluate the intestinal barrier integrity as described. 2.5% (wt/vol) brilliant blue (Aladdin, # B295002) was added to the standard food. 30 flies per vial were starved for 2 h and cultured in the prepared medium for 24 h, flies exhibiting blue dye outside the digestive tract were classified as “Smurf +”. Each group repeated three times.

Lifespan assay

For the administration of DSS, Ecc15, and PQ, pieces of chromatography paper with 7% DSS solution, Ecc15 suspension, and 20 mM PQ (dissolved in 5% sucrose) were put in the vials. For BLM administration, standard food was mixed with 5 μg/ml BLM. A total of 100 flies were collected and randomly placed into four vials with or without VB12. The dead flies were recorded every one or two days, and the vials were replaced. Repeat the experiment three times.

Bromophenol blue assay

A previously described protocol [90] conducted the bromophenol blue assay used to determine whether the acidic state of the copper cell region (CCR) in the Drosophila gut is normal. 200 µL of 2% bromophenol blue sodium (Sigma, #B5525) was added to the food medium surface, making several holes for complete absorption of the solution. 30 flies per vial were starved for 2 h and then cultured with the prepared food for 24 h. The guts were dissected and images were immediately photographed. Each group repeated three times.

Fly excretion measurement

Flies were deprived of food for 2 h and then fed with bromophenol blue-infused food for 24 h. The deposits (each blue dot represents a deposit) left by the flies on the vial walls were gathered using chromatography paper. Subsequently, the deposits on the paper were observed and counted using a Leica M205 FA stereomicroscope (Leica, Weztlar, Germany) to quantify the number of deposits.

Weight measurement and disease activity index (DAI) scoring

The disease activity index (DAI) that evaluated the severity of colitis daily is a combined score of weight loss compared to the initial weight, stool consistency, and bleeding, based on the scoring system [91]. To assess the DAI scores, measurements of body weight and detection of fecal blood were performed on the mice every other day, in order to evaluate their condition.

Histological scoring

Blinded scoring of colon sections stained with hematoxylin and eosin was conducted, following the methodology described in a previous study [92].

Nucleic acid extraction and RT-qPCR

20 adult midguts were dissected each experiment and collected into 4 ℃ diethylpyrocarbonate (DEPC)-treated water-PBS solution. For mice, a small segment of colon tissue was taken. The RNA was extracted from samples using RN07-EASYspin Fast Pure Cell/Tissue Total RNA Isolation Kit (Aidlab, #RN0702) and subsequently reverse transcribed using Evo M-MLV RT Kit (Accurate Biology, #AG11711). DNA from murine stools was extracted using Stool DNA Isolation Kit (Forgene, #DE-05713).

ChamQ Universal SYBR qPCR Master Mix (Vazyme, #Q311) was used for RT-qPCR analysis on CFX96 Touch™ Real-time PCR System (Bio-RAD, Hercules, USA). The rp49 (in Drosophila) and Gapdh (in mice) gene was used as a reference control, and the relative mRNA expression of genes was counted by the 2−ΔΔCT method. The gene primer sequences used are listed in Table S1. The specific primer pairs for Pseudomonas and Serratia were sourced from previous research [93, 94], and a universal primer for all bacteria was also utilized: 5′-GTGGTGCACGGCTGTCGTCA-3′ and 5′-ACGTCATCCACACCTTCCTC-3′ [95].

16S rRNA sequencing and analysis

Bacterial genetic material was extracted from 15 dissected female Drosophila midguts collected from DSS-fed flies, with and without VB12 supplementation. Each experimental group underwent four independent biological replicates. Total genome DNA from samples was extracted using CTAB/SDS method. DNA concentration and purity was monitored on 1% agarose gels. According to the concentration, DNA was diluted to 1 ng/μL using sterile water. 16S rRNA genes were amplified used the specific primer with the barcode. Following PCR purification with AxyPrepDNA Gel Extraction Kit (AXYGEN), Sequencing libraries were generated using NEB Next®Ultra™DNA Library Prep Kit for Illumina (NEB, Ipswich, USA) following manufacturer’s recommendations and index codes were added. The library quality was assessed on the Qubit@ 2.0 Fluorometer (Thermo Scientific, Waltham, UAS) and Agilent Bioanalyzer 2100 system. At last, the library was sequenced on an Illumina NovaSeq 6000 platform and 250 bp paired-end reads were generated. FLASH software was utilized to merge the paired-end reads.

Sequences analysis was performed by UPARSE software package using the UPARSE-OTU and UPARSE-OTUref algorithms. In-house Perl scripts were used to analyze alpha (within samples) and beta (among samples) diversity. Sequences with ≥ 97% similarity were assigned to the same OTUs. Alpha diversity was assessed using the Shannon and Simpson indices, while beta diversity was analyzed via Principal Coordinates Analysis. Partial least-square discriminant supervised analysis was employed to uncover taxonomic variations across distinct groups, using permutational multivariate analysis of variance with the Bray–Curtis distance metric to evaluate the significance of differences observed between the groups. The variable importance in projection scores were utilized to prioritize the discriminatory capabilities of different taxa among the groups. Two-tailed Wilcoxon rank sum tests were performed to identify variations in relative abundances between the two groups.

Bacterial culture and colonies analysis

For Drosophila, four female flies from each group were sanitized in 95% ethanol for 1 min, then the guts were dissected and homogenized in 1 ml of 1xPBS. For mice, collect feces from the colon in a germ-free environment and perform quantitative analysis based on fecal weight. All the samples were centrifuged for 30 s at 1000 rpm and the supernatant was taken out. Five 1/10 serial dilutions were made and plated on nutrient-agar (NA) plates or gram-negative bacteria selective medium. The bacteria were incubated on plates at 30 °C for 24 h followed by a colony count. For gram-negative bacteria selective medium, 5 g Tryptone, 2 g Yeast extract powder, 2 mg crystal violet, 1 g Non-fat milk powder, 1.6 mg Nisin and 15 g agar were mixed in 1 L water. After sterilization at 121 °C for 15 min, the medium is cooled to 45–50 °C, before adding 90-unit penicillin G for each 200 mL aliquot of medium as described previously.

Gram-staining of gut microbiota

Four female guts from each group were cut into small pieces and homogenized in 200 μL of PBS. The samples were then centrifuged at 1000 rpm for 30 s, and 50 μL of supernatant was transferred to a slide and spread thinly over a 1.5 cm diameter circle. After air-drying, the sample was gently fixed using a flame, followed by staining with crystal violet for 60 s. Excess stain was poured off, and the slide was rinsed in running water before incubating in iodine solution for 60 s. After removing excess iodine solution and rinsing the slide again, a few drops of decolorizing solution were added and rinsed off with deionized water after 20–40 s. The slide was then counterstained with basic fuchsin solution for 60 s, washed with sterile water, air-dried, and examined under a microscope. Photographs were taken from the four corners and the middle of the staining area to identify and count purple-stained gram-positive bacteria and pink-stained gram-negative bacteria. At least three independent biological replicates were performed for each group.

Statistical analysis

GraphPad Prism version 8.0 was utilized for all statistical analyses. The presented data represent the means ± SD of a minimum of three independent experiments. Statistical significance and the sample size are designated in the figures. Unless specifically stated, an ANOVA was applied for inter-group comparisons. A p-value of less than 0.05 was considered to be statistically significant in all tests. In the figures, significant differences are represented as follows: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Supplementary Information

Below is the link to the electronic supplementary material.Supplementary file1 (DOCX 1083 KB)

Acknowledgements

We thank BDSC, VDRC, and THFC for providing fly strains, and DSHB for providing the antibodies.

Author contributions

Conceptualization, H.C. and C.F.; methodology, C.F. and J.Y.; formal analysis, C.F. and T.L.; investigation, C.F., J.Y., T.L., H.Z. and H.Z.; data curation, C.F. and Y.Y.; writing-original draft preparation, C.F.; writing—review and editing, C.F., J.Y., T.L., H.Z., Y.C. and H.C.; supervision, H.C.; funding acquisition, H.C. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (92157109) (HC), the National Key Research and Development Program of China (2020YFA0803602), the National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University (Z20201006) (HC), and the 1.3.5 project for disciplines of excellence, West China Hospital, Sichuan University (ZYYC20024) (HC). The funders had no role in the study design, data collection, and analysis, decision to publish, or manuscript preparation.

Data and software availability

The ImageJ can be accessed for download at https://imagej.net/software/imagej/. GraphPad Prism version 8.0 can be accessed for download at https://www.graphpad.com/. Adobe Photoshop CC 2021 and Adobe Illustrator 2020 are both accessible for download at https://www.adobe.com/products/catalog.html. All 16 s rRNA datasets are publicly available in Sequence Read Archive (SRA) BioProject: PRJNA1074069.

Declarations

Conflict of interest

The authors have no relevant financial or non-financial interests to disclose.

Ethical approval

The animal study was approved by the Experimental Animal Ethics Committee of West China Hospital of Sichuan University (No.20231026003 and No.20231026004). The study was conducted in accordance with the local legislation and institutional requirements.

Consent to participate

Not applicable.

Consent for publication

All the authors have read and approved the final manuscript.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Chenxi Feng, Jinhua Yan and Ting Luo have contributed equally to this work.
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