Bifidobacterium adolescentis improves lifespan and healthspan by regulating catalase activity and oxidative stress-associated metabolites

doi:10.21203/rs.3.rs-90247/v1

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Bifidobacterium adolescentis improves lifespan and healthspan by regulating catalase activity and oxidative stress-associated metabolites

https://doi.org/10.21203/rs.3.rs-90247/v1

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Microbiota-host interaction was involved in aging, while the specific bacterium was undetermined. To identify candidate bacterium with aging, we performed fecal microbiota sequencing. Less richness of gut microbial community, and a reduction of B.adolescentis abundance was observed in elderly individuals. B. adolescentis supplement improved osteoporosis and neurodegeneration in telomerase RNA component deletion (Terc^−/−) aged mice. B.adolescentis induced prolongevity and healthspan improvement in Drosophila melanogaster and C. elegans. Transgenic deletion of ctl-2 in C. elegans abolished the effect on lifespan and healthspan by B. adolescentis. The catalase activity was decreased in skeletal muscle and brain tissues of Terc^−/− mice, as well as cellular senescence in mouse embryonic fibroblasts. B. adolescentis alleviated ROS accumulation by regulation of oxidative stress-associated metabolites. These results suggest a role for B. adolescentis in improving lifespan and healthspan by regulating catalase activity and host metabolism. Supplement with commensal bacteria is a promising strategy against age related diseases.

General Microbiology

Fecal Microbiota Sequencing

Osteoporosis

Neurodegeneration

Cellular Senescence

Host Metabolism

Aging is a process and status of holistic host organs, characterized as physiological function deterioration, cellular function decline underpins the development of pathological alterations¹. Studies have been conducted to extend lifespan or induce healthy aging in different model organisms, ranging from yeast, zebra fish, worms, flies to mice^2–4. As the largest component of host microbiota, gut microbes have been proved to function in a variety of physiological and pathological processes. Recent studies demonstrated that fecal microbiota transplantation could extend lifespan and healthspan in progeria mice⁵. Studies have been performed on the elderly or centenarian populations through 16S rRNA, metagenomic or metatranscriptomic shotgun sequencing to distinguish the compositional and functional alteration of gut microbiota with aging^6–8. However, biological effect of specific commensal bacteria on aging and the potential mechanisms were unclear.

Here, we observed that the abundance of Bifidobacterium adolescentis (B. adolescentis) exhibited a significant decline in elderly populations by fecal microbiota sequencing, which was confirmed in 3500 samples from GMrepo database. B. adolescentis supplement improved age-related osteoporosis and neurodegeneration in telomerase RNA component deletion (Terc^−/−) aged mice, as well as inducing lifespan prolongation and healthspan improvement in nonmammalian organisms Drosophila melanogaster and Caenorhabditis elegans. The anti-aging effect of B. adolescentis was regulated by the activity of catalase and oxidative stress-associated metabolites.

Gut microbiota diversity and B. adolescentis abundance decreased with aging

To investigate the change of gut microbiota with aging, we performed 16S rRNA sequencing of fecal samples. 166 participants were divided into three groups according to chronological age (Supplementary Table 1). We found that alpha-diversity estimators shannon index and heip index of human gut microbiota significantly decreased from the younger to the elderly populations (Fig. 1A,B), indicating less richness of gut microbiota with aging. A remarkable reduction of Firmicutes, Actinobacteria, Saccharibacteria, together with an increment of Fusobacteria, were detected with age at phylum level (Fig. 1C). The composition of gut microbiota didn’t change with age in a linear association⁶. In addition, an increment of Bacteroidetes:Firmicutes (B:F) ratio and a shift of butyrate-producing bacteria were observed in centenarians^6,7. We observed that 45 KEGG pathways indispensable for maintaining lifespan, including 28 metabolism-associated pathways were differentially enriched with aging. Most metabolism-associated pathways were predicted to be more active in elderly individuals, such as lipopolysaccharide biosynthesis, N-glycan biosynthesis, glutathione metabolism and amino acid metabolism (Supplementary Fig. 1). Collectively, gut microbiota profiles altered with aging and might participate in the regulation of host metabolism.

We subsequently performed linear discriminant analysis (LDA) coupled with effect size measurements to screen out candidate bacteria (Fig. 1D). Notably, the B. adolescentis distribution was the most significant difference between two groups (Supplementary Table 2). Short chain fatty acids (SCFAs) were recognized as beneficial bacterial metabolites which were important for host metabolism. We found that relative abundance of bacterial taxa associated with SCFA production, such as B. adolescentis, Ruminococcaceae, Faecalibacterium prausnitzi and Eubacterium ractale, was significantly decreased in elderly individuals while B. adolescentis exhibited most prominent reduction (Fig. 1E). Furthermore,relative abundance of B. adolescentis was confirmed to be significant higher in younger individuals by qPCR assay (Fig. 1F). Similar result was obtained by analyzing GMrepo sequencing database between young and old ages (n = 2821 vs. n = 679 samples) (Fig. 1G). As reported in previous study, microbiota in aged people has displayed reduction in the abundance of several bacteria with anti-inflammatory and immunomodulatory properties, including Bifidobacterium, Akkermansia, Lactobacillus and Christensenellaceae⁹. Taken together, we concluded that the abundance of B. adolescentis was decreased with aging and B. adolescentis might play pivotal role in the regulation of healthy aging .

Oral gavage with B. adolescentis alleviated aged related osteoporosis and neurodegeneration in Terc^−/− progeroid mice

Telomerase, consisting of three main components, is essential for maintaining telomere length and plays an important role in tissue renewal and organism lifespan¹⁰. Telomerase RNA component deletion (Terc^−/−) mice with C57BL/6 background show progressive telomere shortening from first generation (G1) until the third (G3) generation, which exhibits significant phenotype of premature aging^11–13. To verify the effect of B. adolescentis on age, Terc^−/− G3 progeroid mice of 6–8 weeks age were oral gavaged with B. adolescentis for five months, while wild-type Terc^+/+ mice were gavaged with PBS as control (Fig. 2A). The body weight of Terc^−/− mice was significantly lower than those of wild-type Terc^+/+ mice, whereas B. adolescentis supplement significantly increased body weight as compared to control group (Fig. 2B). Frailty index score, which comprehensively quantified frailty in aged mice¹⁴, exhibited remarkable difference between wild-type and Terc^−/− mice gavaged with PBS. B. adolescentis supplement improved age related frailty index in Terc^−/− mice (Fig. 2C).

Aging has been characterized by multiple organic dysfunction, including musculoskeletal and neurodegenerative diseases^15,16. To assess the biological effect of B. adolescentis on bone density, we performed micro-CT scan and three-dimensional reconstruction of femora in mice. The bone volume/total volume (BV/TV) and trabecular thickness (Tb.Th) of Terc^−/− mice were significantly decreased as compared to wild-type mice. These indices in Terc^−/− mice were increased after gavaged with B. adolescentis (Fig. 2D), which suggested B. adolescentis supplement could improve osteoporosis in aged Terc^−/− mice. Previous study showed that neurodegenerative changes in brain was linked with gut microbiota¹⁷. We then assessed senescence status by comparing the morphological changes and surviving number of neurons in the CA3 region of hippocampus in mice (Fig. 2E). Nuclear deviation, cytoplasm condensed and nuclear fragmented of neurons were more prominent in Terc^−/− mice than those of control group, and the surviving number of neurons in mice gavaged with B. adolescentis also showed a significant increase compared to controls. Collectively, these results indicated that supplement with B. adolescentis improved healthspan in Terc^−/− progeroid mice.

B. adolescentis supplement improved lifespan and healthspan in D. melanogaster and C. elegans

Invertebrate organisms such as the fruit fly D. melanogaster and the nematode C. elegans with a relative short lifespan, availability of different genetic mutants and morphological and functional similarities of gut were broadly used as proof-of-concept models on microbiome-aging studies^18–20. Some bacterial strains, such as Comamonas DA1877 and Lactobacillus gasseri SBT2055, have been identified to influence the lifespan and reproduction of C. elegans by regulating series of signaling pathways^21–23. We administrated B. adolescentis and conventional food to D. melanogaster and C. elegans, and comprehensively verify the effect of B. adolescentis on the lifespan and healthspan of the two organisms. Wild-type D. melanogaster strains (both w¹¹¹⁸ and Canton-S) supplemented with B. adolescentis both showed a significant increase of lifespan (Fig. 3A,B), approximately 20% increment was observed (Supplementary Table 3). Then we tested healthspan parameters of flies on day 30. The climbing ability of female w¹¹¹⁸ supplemented with B. adolescentis was improved as compared to control group (Fig. 3C). Canton-S flies supplemented with B. adolescentis exhibited stronger creep ability than controls as well (Fig. 3D).

The lifespan of C. elegans was also significantly improved when supplied with B. adolescentis with different mixture ratios (Fig. 4A). 1:1 mixture with B. adolescentis and E. coli OP50 can significantly improve the mean maximum lifespan of C. elegans (Fig. 4B). In addition, the locomotion ability of aged worms was significantly enhanced (Fig. 4C,D). We observed that the heat stress resistance was obviously changed with B. adolescentis supplement (Fig. 4E). Healthspan was further evaluated by autofluorescence quantification of intestinal lipofuscin, which accumulated with age (Fig. 4F) The autofluorescence intensity of worms in B. adolescentis intervention group was significantly lower than that of control group (Fig. 4G). Collectively, B. adolescentis supplement could improve lifespan and healthspan in both D. melanogaster and C. elegans.

ctl-2 is essential for B. adolescentis-induced lifespan extension and healthspan improvement in C. elegans

To elucidate the mechanisms of B. adolescentis-induced lifespan improvement, genes expression involved in lifespan were evaluated in C. elegans and D. melanogaster. Expression of sod-3 and ctl-2 was significantly higher in C. elegans supplemented with B. adolescentis than control group (Fig. 5A). Similar result was observed in flies considering corresponding homologous gene sod-3 and cat (Supplementary Fig. 2A,B). To clarify the gene involved in lifespan, experiments of corresponding mutants were performed in C. elegans. Interestingly, B. adolescentis supplement could still extend the lifespan in C. elegans carried with sod-3 mutant, while the lifespan prolongation was abolished in ctl-2 mutant (Fig. 5B-D). To validate this finding, we constructed transgenic worms and detected the expression of ctl-2 directly with mCherry fluorescence. The ctl-2 expression was significantly increased with B. adolescentis supplement in aged worms (Fig. 5E,F). We then detected healthspan indicators aforementioned in ctl-2 mutant C. elegans. The enhancement of locomotion ability by B. adolescentis was blocked in ctl-2 mutant (Fig. 5G). Similarly, no significant improvement of survival time was observed in ctl-2 mutant with B. adolescentis supplement (Fig. 5H). Moreover, the autofluorescence intensity was increased in ctl-2 mutants group (Fig. 5I,J). In conclusion, B. adolescentis supplement could prolong lifespan and improve healthspan of C. elegans through the regulation of ctl-2.

B. adolescentis suppressed aged Terc^−/− G3 mice by regulating the activity of catalase and oxidative stress-associated metabolites

The activity of catalase (CAT), which was homologous to C. elegans ctl-2 gene, was subsequently detected in muscle and brain tissue of mice. Terc^−/− aged mice exhibited decreased activity of CAT compared to wild-type mice, and B. adolescentis supplement significantly enhanced the activity of CAT (Fig. 6A). In addition, B. adolescentis-gavaged Terc^−/− aged mice showed prominent increased protein expression of CAT in muscle and brain tissues (Fig. 6B,C and Supplementary Fig. 3A). Immunohistochemistry staining revealed that B. adolescentis supplement exhibited downregulation of p53, while upregulation of CAT, in cortex and hippocampus regions (Fig. 6D and Supplementary Fig. 3B). These results demonstrated that B. adolescentis supplement suppressed aged related phenotype in Terc^−/− G3 mice by regulating CAT.

To verify the effect of B. adolescentis in vitro, B. adolescentis was then administrated to culture medium in both replicative and DOX-induced senescent MEFs. B. adolescentis supplement significantly suppressed cellular senescence as shown by senescence-associated β-galactosidase staining (Fig. 6E,F and Supplementary Fig. 4A,B). In line with observation in mice, the mRNA and protein expression level of CAT was upregulated by B. adolescentis in senescent MEFs (Fig. 6G,H and Supplementary Fig. 4C).

Finally, we performed metabolomics analysis of mice feces to evaluate the effect of B. adolescentis on oxidative stress-associated metabolites as CAT was an important ROS scavenger²⁴. In accordance with our findings in brain and muscle tissues, apiin and erucic acid, which could increase the activity of CAT^25–27, were significantly enriched in the feces of B. adolescentis-gavaged Terc^−/− aged mice. Some other antioxidants also exhibited enrichment with B. adolescentis supplement, including ginsenoside Ia²⁸, 2-hydroxycinnamic acid²⁹, daidzin³⁰ and L-malic acid³¹, while ROS producer such as hypoxanthine³² was reduced. Furthermore, several metabolites with proinflammatory property or associated with cardiovascular diseases were enriched in Terc^−/− aged mice, including cholic acid³³, 9,10-DHOME³⁴, 3-dehydroxycarnitine³⁵ and 4-trimethylammoniobutanoic acid³⁶. Cosmosiin³⁷ with anti-aging potential and enterodiol³⁸ with tumor-suppression activity were enriched in B. adolescentis-gavaged Terc^−/− aged mice (Fig. 7A-C). Collectively, these results indicated that B. adolescentis supplement suppressed aged Terc^−/− mice and MEFs cellular senescence by regulating CAT and oxidative stress-associated metabolites.

Gut microbial community was changed with aging, and the abundance of B. adolescentis elicited a dramatic decline in elderly, which was supported by sequencing results in 3500 fecal samples from GMrepo database. Several bacteria have been previously identified to be related with aging and played critical role in aging prediction, such as family Ruminococcaceae, genera Alistipes, Bacteroides, Bifidobacterium, Faecalibacterium, Akkermansia, Roseburia and Eubacterium^9,39,40. Transplantation with the gut microbiota of old donor mice to young germ-free mice exhibited age-sensitive enrichment in butyrate-producing microbes⁴¹. We observed that oral gavaged with B. adolescentis induced lifespan extension and healthspan improvement in both Terc^−/− mice, and nonmammalian model organisms such as worms and flies. Transplantation with the Akkermansia muciniphila was sufficient to exert beneficial healthspan effects in two progeroid mouse models of Hutchinson–Gilford progeria syndrome⁵. These results demonstrate the association between aging and the gut microbiota, and provide experimental evidence with commensal bacteria against age-related diseases.

Gut microbiota communicates with host organs through bacterial structural product, nutrient-metabolites and complex pathways. Development of series organic dysfunction was demonstrated to be associated with gut microbiota, including neurodegenerative diseases^16,42, osteoporosis^43,44, diabetes mellitus⁴⁵, nonalcoholic fatty liver disease^46,47 and cardiovascular disease⁴⁸. Microbiota dysbiosis has been linked with disruption of intestinal barrier integrity and gut inflammation. Wang et al. demonstrated that Lactobacillus paracasei D3-5 and lipoteichoic acid from its cell wall could ameliorate aging-related leaky gut and inflammation by modulating TLR-2/MAPK/NF-kB pathway⁴⁹. High-throughput lifespan-associated screening on C. elegans uncovered that 29 E. coli mutants and bacterial metabolite polysaccharide colonic acid were involved in the prolongevity process⁵⁰. Transplant of Akkermansia muciniphila had beneficial effects in progeroid mice by reestablishing healthy microbiome through restoring secondary bile acids⁵. In present study, we observed that the accumulation of intestine lipofuscin, a lipid peroxidation product, was significantly reduced in B. adolescentis-treated C. elegans. Furthermore, B. adolescentis supplement in Terc^−/− mice regulated lipid metabolism and oxidative stress-associated metabolites. In the prediction of human gut microbiota function, endocytosis and phagocytosis were predicted to be more active in younger populations. Hence, we can hypothesize that probiotics may regulate age-related disorders through gut inflammation and host metabolism.

To gain an insight into the underlying mechanisms, we identified candidate ctl-2 gene function in Terc^−/− mice, C. elegans and senescent MEFs supplemented with B. adolescentis. Studies have shown that ctl-2 accounts for the majority of catalase activity in nematodes, and lack of ctl-2 can lead to the premature aging of nematodes⁵¹. As an important substance in living organisms, catalase is participating in the process of active oxygen metabolism. Under environmental stress, ROS resulted in cell membrane damage, DNA damage and subsequent cell senescence²⁴. Pathogenic microbes have been reported to impair intestinal cell repair and shorten host lifespan by generation of ROS in flies⁵². A recent study demonstrated that preventing ROS accumulation in gut allows survival without sleep in flies⁵³. We found that the catalase activity in muscle and brain was upregulated by supplementation with B. adolescentis in Terc^−/− mice, which was supported by the metabolomic analysis of mice feces. Metabolites facilitating the activity of CAT together with other antioxidants were enriched in B. adolescentis-gavaged Terc^−/− mice, which could remarkably reduce the intracellular ROS level. In addition, erucic acid, cosmosiin and 2-hydroxycinnamic acid have been proposed as potential therapeutic agents for Alzheimer disease by ameliorating nueroinflammation and blocking neural cell death^27,29,37. Daidzin could inhibit LPS-induced bone loss by suppressing the osteoclast differentiation⁵⁴. The enrichment of these metabolites may underlie the improvement of phenotype in Terc^−/− mice supplemented with B. adolescentis.

In conclusion, we showed that B. adolescentis exerted beneficial effects on lifespan and healthspan by the regulating of catalase activity and host metabolism. The underlying molecular mechanisms should be further explored.

Study design

The objectives of this study was to screen out candidate beneficial bacterium and elucidate its functional characteristics on aging and prolongevity. Fecal samples were collected from healthy volunteers and 16S rRNA sequencing was performed. Microbiota data obtained from GMrepo metagenomes database were used to confirm the B. adolescentis abundance in different age. Next, age-related phenotype was conducted in Terc^-/- aged mice, D. melanogaster and C. elegans with B. adolescentis supplement. Micro‐CT bone scan and staining in hippocampal CA3 region was evaluated in Terc^-/- aged mice gavaged with B. adolescentis. Lifespan was recorded and series healthspan indices were measured, which including mean survival time, frailty index score, locomotion ability and intestinal lipofuscin accumulation. For further mechanism exploration, lifespan assay on C. elegans carried with serial gene mutants were performed with B. adolescentis supplement. Lastly, expression of ctl-2 homologous gene catalase (CAT) was detected in muscle and brain tissues from Terc^-/- aged mice, as well as in senescent MEFs cells by qRT-PCR, western blot and immunohistochemistry assays. Metabolomics of mice feces were performed to analyze effect of B. adolescentis on gut microbial metabolites. Researchers were blinded to group allocation, and mice, flies and worms were randomized to groups. All procedures were conducted in compliance with institutional guidelines and were approved by the Animal Ethical Committee of Zhejiang University prior to initiating the study.

Healthy subjects and fecal samples collection

Healthy subjects aged over 18 years old were recruited at Sir Run Run Shaw Hospital (Zhejiang Province, China) from December 2016 to December 2018. Admission criteria were listed as follows: without history of digestive tract-related diseases such as intestinal adenoma and tumors; without thyroid disease or diabetes mellitus; no use of antibiotics or probiotics in the past month; BMI between 18.5 and 24.9 kg/m²; women are not in pregnant or lactation period. Fresh stool samples were collected, immediately frozen and stored at -80°C before DNA isolation. This study was approved by the Ethics Committee of Sir Run Run Shaw Hospital, College of Medicine, Zhejiang University (20161206-21). Written informed consent was obtained from all participants before enrollment.

16S rRNA sequencing

Microbial DNA from stool samples was isolated with TIANamp Stool DNA Kit (TIANGEN BIOTECH, Beijing, China) according to the manufacturer’s instructions. The V3-V4 hypervariable regions of 16S rRNA gene were amplified and purified amplicons were paired-end sequenced on an Illumina MiSeq platform according to the standard protocols by Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China). Bacterial taxonomy determination and functional predictions were performed as earlier described⁵⁵.

Online database resources

Microbiota online data were obtained from GMrepo, an metagenomes database of human gut⁵⁶. Using python software through RESTful APIs, we obtained relative abundance of B. adolescentis in samples with healthy phenotype of in all age groups from GMrepo. 3500 samples were included for final analysis.

Terc^+/-mice and aged related phenotype

Terc^+/-G0 mice (C57BL/6 background), gift from Prof. Zhangfa Song (Zhejiang University), were intercrossed to generate Terc^+/+ (wild-type C57BL/6) and Terc^-/-G1 mice. Genotype identification was conducted by agarose gel electrophoresis (Supplementary Fig. 5). Then Terc^-/-G1 mice were intercrossed to generate Terc^-/-G3 mice. 6-8-week-old wild-type C57BL/6 littermates were gavaged with PBS (WT+PBS, n=11). 6-8-week-old Terc^-/-G3 littermates were randomly assigned to PBS group (G3+PBS, n=9) and B. adolescentis group (G3+B.a, n=12). Body weight and frailty index score were recorded monthly according to previous study ¹⁴ until natural death or sacrifice at 7 months old. All mice were housed at Sir Run Run Shaw Hospital animal facility in a pathogen-free level room. The room temperature was maintained at 20-22°C with 12-h light/dark cycle. Mice were provided with food and water ad libitum.

Micro-CT imaging

The left bones of mice were isolated and the attachments of the left femora muscle were removed. Three femora samples of each group were randomly selected and scanned by micro-CT (InspeXio SMX-225 CT FPD HR; Shimadzu Co. Ltd., Kyoto, Japan) after fixation in 4% paraformaldehyde overnight. Each sample was reconstructed using micro-CT software (VGStudio MAX; Volume Graphics, Heidelberg, Germany) under the same conditions. A region of intersect (ROI) cube was taken underneath epiphyseal growth plate. The reconstruction parameters of bone volume/total volume (BV/TV) and trabecular thickness (Tb.Th) were analyzed.

Lifespan assay

For lifespan assay, flies were allowed to develop and mate for 4~6 days after eclosion. Then they were starved for 2h in an empty vial, sexually segregated and randomly assigned to intervention or control vials. Every 20 flies were flipped to a vial and transferred to new vials every 2 or 3 days. The number of dead flies were counted simultaneously. About 100 flies (5 vials) were used each assay.

For C. elegans, L4 stage (day 0) worms were cultured in OP50 plates until maturation and then transferred to OP50 or mixture plates.150 worms were distributed in 10 plates (15 worms/plate), and incubated at 20 °C. The worms were transferred to new freshly seeded plates every other day. The number of alive or dead worms were counted on every transfer day.

Survival rates were calculated as the percentage of surviving flies/worms versus the total number of flies/worms. Flies that lost and worms died as a result of getting stuck to the wall of the plate were excluded from the analysis. The evaluation of lifespan was performed at least three times.

C. eleganselegans fluorescence microscopy

Worms were synchronized and cultured as described in online supplementary materials and methods. On day 10 and 14, the lipofuscin accumulation in worm intestine and ctl-2 expression were quantified with autofluorescence and mCherry red fluorescence, respectively. Worms were randomly selected and wash twice with M9 buffer. Then they were mounted on a slide coated with 200mM sodium azide to induce anesthesia and photos were taken under blue excitation light (405~488 nm) or red excitation light (559-585 nm) with inverted laser scanning confocal microscope (Olympus IX81-FV1000, Tokyo, Japan). Fluorescence was quantified with ImageJ (National Institutes of Health, Bethesda, MD, USA). Three independent experiments were conducted with >20 worms.

C. elegans gene integration

Promoter ctl-2 and ctl-2 gDNA were PCR-amplified from N2 genomic DNA and then recombined with specific donor vector fragments using In-Fusion PCR Cloning Kit (TaKaRa, Japan). To measure the expression level of ctl-2 gene, a functional Pctl-2::ctl-2 gDNA::mCherry transgene was made by cloning the mCherry reporter sequence in frame and downstream of the ctl-2 gDNA sequence. Transgenic animals were generated by micro-injecting Pctl-2::ctl-2 gDNA::mCherry transgene (50 ng/μL) mixed with a co-injection marker Plin-44::gfp (20 ng/μL). Gene integration was performed by ultraviolet trimethylpsoralen (UV-TMP) treatment and the integrated worms were backcrossed four times with N2.

Metabolomics analysis

Metabolites from mice fecal samples were extracted according to the standard protocols by Biotree Biological Technology Co. Ltd. (Shanghai, China). LC-MS/MS analyses were performed using an UHPLC system (Vanquish, Thermo Fisher Scientific) with a UPLC BEH Amide column (2.1 mm × 100 mm, 1.7 μm) coupled to Q Exactive HFX mass spectrometer (Orbitrap MS, Thermo). The raw data were converted to the mzXML format using ProteoWizard and processed with an in-house program, which was developed using R and based on XCMS, for peak detection, extraction, alignment, and integration. Then an in-house MS2 database (BiotreeDB) was applied in metabolite annotation. The cutoff for annotation was set at 0.3. Details were described in online supplementary materials and methods.

Statistical analysis

Differences of three groups were analyzed by one-way analysis of variance (ANOVA) for data with normal distribution or Kruskal-Wallis test for data with non-normal distribution. Differences of two groups were analyzed by Student’s t-test or nonparametric tests (Wilcoxon rank sum test or Kruskal-Wallis test). For lifespan assays, log-rank (Mantel-Cox) test was performed. All statistical tests were two-tailed, and p-values <0.05 were considered statistically significant. Analysis and figures were performed using IBM SPSS Statistics V22.0 software, GraphPad Prism v7 and Image J (https://imagej.nih.gov/ij/; RRID:SCR_003070).

Data availability

All data supporting the findings of the present study are available within this article and the Supplementary Information files. Raw 16S rRNA sequencing data files are available in the Sequence Read Archive (SRA) database under the accession number PRJNA625181 (http://www.ncbi.nlm.nih.gov/bioproject/625181).

Competing interests statement

The authors declare no competing interests.

Author Contributions

SC, LW, LK, JS, XY and YX conceived the research idea and designed the study; SC, LC, YQ and JX carried out experiments; LC, LW and YZ collected clinical samples; SC, LC, YQ, JX, QG, YF, DC, TH performed data analysis; SC and LC wrote the manuscript with input from coauthors. All authors critically revised and approved the final version of the manuscript.

Acknowledgements

We thank Prof. Xin Wang and Prof. Wei Liu (Zhejiang Academy of Agricultural Sciences, China) for sharing the anaerobic operating system and guiding on bacteria culture; Prof. Zhangfa Song (Zhejiang University, China) for kindly providing Terc^+/− G0 mice; Prof. Fei Xu (Zhejiang University, China) for helpful discussion. This project was supported by the National Foundation of Natural Science of China (82072623, 31800878, 31900736) and National Key R&D Program of China (2016YFC1303200).

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Bifidobacterium adolescentis improves lifespan and healthspan by regulating catalase activity and oxidative stress-associated metabolites

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