CN113521111B - Application of Bifidobacterium adolescentis in delaying aging - Google Patents

Abstract

The present invention provides the application of Bifidobacterium adolescentis in delaying aging. Through in vivo and in vitro experiments, it is clarified that Bifidobacterium adolescentis can delay cell aging, regulate sod-3 genes and catalase CAT genes in Drosophila, and regulate Caenorhabditis elegans. Endogenous ctl-2 gene regulation, mouse catalase CAT gene regulation, mouse endogenous p53 gene regulation, mouse oxidative stress metabolite regulation, intestinal aging in mice, especially improvements in related phenotypes It has effects in other aspects, and has the prospect of being used in the preparation of biological aging-delaying drugs and the use of catalase as an aging-delaying target in the preparation of biological aging-delaying drugs.

Description

Application of Bifidobacterium adolescentis in delaying aging

Technical Field

The invention belongs to the field of biotechnology, and in particular relates to the application of Bifidobacterium adolescentis in delaying aging.

Background Art

With the rapid growth of the elderly population, aging has attracted widespread social attention around the world. According to forecasts, by 2050, the global population aged 60 and over will increase to 2 billion, accounting for 21% of the total population. As a populous country in the world, my country is also aging rapidly, and it is estimated that by 2100, the elderly population will reach 31.09% of the total population. The aging population is generally accompanied by a decline in physical health, a decrease in the body's resistance to multiple systemic diseases, and a significant increase in the incidence of age-related diseases. Therefore, individuals, families, and society will face a heavy burden of health care. At present, in addition to lifestyle adjustments, there is still no recognized simple, efficient, and cost-effective measure to delay aging. Based on this situation, it is of great significance to study the mechanism of aging and actively seek intervention measures to delay aging.

Strictly speaking, aging is not a disease, but a process and state of the entire body's organs, characterized by the deterioration of physiological functions and the decline of cell functions, which is the basis for the development of pathological changes. Traditional intervention measures for aging include genetic modification, lifestyle and environmental risk control. In recent years, intestinal flora has been shown to play an important regulatory role in the occurrence and development of various diseases. As the largest component of the body's microbiome, intestinal flora can not only directly affect the function of the intestine, but also regulate the physiological functions of extra-intestinal organs through complex pathways such as the brain-gut axis, brain-liver axis, and brain-kidney axis. Population studies of different cohorts at home and abroad have confirmed that there are significant differences in the composition and abundance of intestinal flora in different age groups.

Summary of the invention

The first object of the present invention is to provide an application of a Bifidobacterium adolescentis composition in delaying the aging of an organism in view of the deficiencies in the prior art.

While adopting the above technical solutions, the present invention may also adopt or combine the following technical solutions:

As a preferred technical solution of the present invention: the Bifidobacterium adolescentis composition comprises Bifidobacterium adolescentis and auxiliary materials.

As a preferred technical solution of the present invention: the auxiliary materials are PBS buffer or sucrose solution and standard corn flour or M9 buffer.

The second purpose of the present invention is to provide an application of Bifidobacterium adolescentis in delaying cell aging in view of the deficiencies in the prior art.

The third object of the present invention is to provide an application of Bifidobacterium adolescentis in regulating the sod-3 gene and catalase CAT gene in Drosophila in view of the deficiencies in the prior art.

The fourth object of the present invention is to provide an application of Bifidobacterium adolescentis in regulating the ctl-2 gene in Caenorhabditis elegans in view of the deficiencies in the prior art.

The fifth object of the present invention is to provide an application of Bifidobacterium adolescentis in regulating the catalase CAT gene in mice in view of the deficiencies in the prior art.

The sixth object of the present invention is to provide an application of Bifidobacterium adolescentis in regulating p53 gene in mice in view of the deficiencies in the prior art.

The seventh object of the present invention is to provide an application of Bifidobacterium adolescentis in regulating oxidative stress metabolites in mice in view of the deficiencies in the prior art.

The eighth object of the present invention is to provide the use of Bifidobacterium adolescentis in improving the aging of the intestinal system of mice, especially in improving the phenotype related to the aging of the intestinal system of mice, in view of the deficiencies in the prior art.

The ninth objective of the present invention is to provide a use of Bifidobacterium adolescentis in preparing a drug for delaying aging of an organism in view of the deficiencies in the prior art.

Another object of the present invention is to provide the use of catalase as an aging-delaying target in the preparation of an aging-delaying drug for an organism, in view of the deficiencies in the prior art.

The present invention provides an application of Bifidobacterium adolescentis in delaying aging. Through in vivo and in vitro experiments, it is clarified that Bifidobacterium adolescentis has effects on delaying cell aging, regulating the sod-3 gene and catalase CAT gene in Drosophila, regulating the ctl-2 gene in Caenorhabditis elegans, regulating the catalase CAT gene in mice, regulating the p53 gene in mice, regulating oxidative stress metabolites in mice, improving intestinal aging in mice, especially related phenotypes, and has the prospect of use in preparing drugs for delaying aging of organisms and use of catalase as a target for delaying aging in preparing drugs for delaying aging of organisms.

BRIEF DESCRIPTION OF THE DRAWINGS

In Figure 1 : A and B are survival curves of wild-type fruit flies w ¹¹¹⁸ (A) and Canton-S (B), respectively (Ba is the group supplemented with Bifidobacterium adolescentis; NC is the control group with 2.5% sucrose solution, with about 100 fruit flies in each group);

C and D are: Comparison of the tube climbing ability of wild-type fruit flies w ¹¹¹⁸ (C) and wild-type fruit flies Canton-S (D) on the 30th day (Ba is the group supplemented with Bifidobacterium adolescentis; NC is the control group with 2.5% sucrose solution, with about 60 fruit flies in each group);

Data are expressed as mean ± standard error and analyzed by t test. ^** p <0.01; ^*** p <0.001; ns, not significant.

In Figure 2 : A and B are respectively: the survival curve (A) and the average maximum lifespan (B) of wild-type Caenorhabditis elegans N2 fed with Escherichia coli OP50, Escherichia coli OP50 and Bifidobacterium adolescentis mixed in a ratio of 1:1 or 1:2, with 150 nematodes in each group;

C: Quantitative analysis of the motility of wild-type Caenorhabditis elegans N2. The worms were fed with E. coli OP50 or a 1:1 mixture of E. coli OP50 and Bifidobacterium adolescentis for analysis on the 2nd, 8th, and 16th days, with 50 worms in each group.

D is: grading of the motility of wild-type Caenorhabditis elegans N2, which were fed with E. coli OP50 or a 1:1 mixture of E. coli OP50 and Bifidobacterium adolescentis and analyzed on the 8th and 16th days. Grade A: represents spontaneous movement or vigorous movement in response to stimulation; Grade B: does not move unless stimulated or seems to have uncoordinated movements; Grade C: only moves the head and/or tail when stimulated; Grade D: dead; 50 nematodes in each group;

E is the average survival time of wild-type Caenorhabditis elegans N2 under heat stress. The nematodes were fed with Escherichia coli OP50 or a 1:1 mixture of Escherichia coli OP50 and Bifidobacterium adolescentis, and the heat stress resistance was analyzed on the 4th, 8th and 12th days, with 30 nematodes in each group;

F and G are: autofluorescence image (F) and quantitative comparison (G) of intestinal lipofuscin deposition, where: each group has more than 20 nematodes; scale bar: 100 μm.

Data are expressed as mean ± standard error and analyzed by t test. ^** p <0.01; ^*** p <0.001; ns, not significant.

In Figure 3: A is: Terc ^-/- G0 mice (C57BL/6) were cross-hybridized to produce Terc ^+/+ (wild-type C57BL/6) and Terc ^-/- G1 mice. The obtained Terc ^-/- G1 mice were cross-hybridized to produce Terc ^-/- G3 mice, and divided into three groups for further study. 6-8 week old wild-type C57BL/6 were set as the control group (WT+PBS, n=11) and gavaged with PBS; Terc ^-/- G3 mice were randomly divided into PBS gavage group (G3+PBS, n=9) and Bifidobacterium adolescentis group (G3+Ba, n=12). Gavage once every other day until natural death or sacrifice at 7 months of age;

B and C are: relative value curves of body weight (B) and frailty index (C);

D is: computed tomography (CT) and micro-CT reconstructed bone block images. Bone volume/total volume (BV/TV) and trabecular thickness (Tb.Th) were analyzed using vgstudiomax software, n=3;

E: Hematoxylin and eosin staining of the hippocampal CA3 region. The figure shows the number of surviving neurons in the hippocampal CA3 region of each group. Scale bar, 100 μm.

Data are expressed as mean ± standard error and subjected to one-way ANOVA. ^* p<0.05; ^** p<0.01; ^*** p<0.001;

^**** p<0.0001; ns, not significant.

Figure 4: A and B are: senescence-associated β-galactosidase (SA-β-gal) staining (A) and the corresponding percentage of SA-β-gal-positive cells (B) of replicatively senescent MEFs cells supplemented with PBS (NC) or Bifidobacterium adolescentis (B.a) at passage 4 (P4) and passage 12 (P12), respectively. Scale bar: 100 μm;

C and D are representative images of SA-β-gal staining of wild-type MEFs supplemented with PBS (NC), DOX-induced senescent MEFs supplemented with PBS (D-sen) and Bifidobacterium adolescentis (D-sen+Ba) (C) and the corresponding percentage of SA-β-gal positive cells (D), respectively. Scale bar: 100 μm. Data are expressed as mean ± standard error and analyzed by t test; ^* p<0.05; ^** p<0.01; DOX, doxorubicin;

E: Real-time quantitative PCR expression determination and comparison of CAT in the 4th passage (P4) and 12th passage (P12) replicative senescent MEFs cells;

F: Comparison of the protein expression of CAT in the 4th (P4) and 12th (P12) passages of replicative senescent MEFs cells and DOX-induced senescent MEFs cells (added with PBS (D-sen) or Bifidobacterium adolescentis (D-sen+B.a)) by immunoblotting experiments;

G: Real-time fluorescence quantitative PCR detection of CAT expression in DOX-induced senescent MEFs supplemented with PBS (D-sen) and Bifidobacterium adolescentis (D-sen+B.a);

Data are expressed as mean ± standard error and analyzed by t test. ^** p <0.01; ^*** p <0.0001; ns, not significant; DOX, doxorubicin.

In Figure 5: A and B respectively show the relative mRNA expressions of sod-3 and CAT in male and female fruit flies fed with Bifidobacterium adolescentis (B.a) or 2.5% sucrose solution (NC).

Data are expressed as mean ± standard error and analyzed by t test. ^** p <0.01; ^*** p <0.0001; ns, not significant.

In FIG6 : A is the relative expression of aging-related gene mRNA levels after nematodes were fed with Escherichia coli OP50 or a 1:1 mixture of Escherichia coli OP50 and Bifidobacterium adolescentis;

B and C are respectively: Comparison of the survival curves of the nematode sod-3 mutant (B) and ctl-2 mutant (C) with the wild-type N2, where: 150 nematodes in each group;

D: Comparison of the average maximum lifespan of wild-type nematodes N2 and nematode ctl-2 mutant after nematodes were fed with Escherichia coli OP50 or a 1:1 mixture of Escherichia coli OP50 and Bifidobacterium adolescentis, respectively;

E and F are: mCherry fluorescence contrast and ctl-2 expression (E) and quantitative comparison (F) of wild-type nematodes N2 on days 10 and 14 after feeding nematodes with E. coli OP50 or a 1:1 mixture of E. coli OP50 and Bifidobacterium adolescentis, respectively. Each group has more than 20 nematodes, scale bar: 100 μm;

G: Quantitative analysis of the motility of wild-type nematodes N2 and nematode ctl-2 mutants on the 2nd and 8th days after nematodes were fed with E. coli OP50 or a 1:1 mixture of E. coli OP50 and Bifidobacterium adolescentis, respectively, where: 20 nematodes in each group;

H is: the average survival time of wild-type nematodes N2 and nematode ctl-2 mutant under heat stress was compared on the 4th, 8th and 12th days after nematodes were fed with Escherichia coli OP50 or a 1:1 mixture of Escherichia coli OP50 and Bifidobacterium adolescentis, 30 nematodes in each group;

I and J are: autofluorescence images (I) and quantitative comparison (J) of intestinal lipofuscin deposition on the 14th day after nematodes were fed with Escherichia coli OP50 or a 1:1 mixture of Escherichia coli OP50 and Bifidobacterium adolescentis, respectively, where: each group has more than 20 nematodes, scale bar: 100 μm.

Data are expressed as mean ± standard error and analyzed by t-test. ^* p<0.05; ^** p<0.01; ^**** p<0.0001; ns, not significant.

FIG7 : A: The activity of CAT in the muscle and brain homogenates of 7-month-old wild-type mice (WT+PBS, n=10), Terc ^-/- G3 PBS-administered mice (G3+PBS, n=6), and Terc ^-/- G3 Bifidobacterium adolescentis-administered mice (G3+Ba, n=9) was measured and compared;

B: Real-time quantitative PCR was used to detect the relative expression of CAT in the muscle tissue of 7-month-old mice in three groups; n = 10, 6, 9;

C and D are respectively: comparison of the immunoblotting experiments of CAT protein expression in mouse muscle (B) and brain (C) tissues;

E: Immunohistochemical staining of CAT in the CA3 region, DG region and cerebral cortex of the hippocampus. Scale bar: 50 μm.

Figure 8: Representative images of p53 immunohistochemical staining in the hippocampal DG region of the three groups, scale bar, 200 μm. The black frame magnified image is shown in the figure below. Scale bar, 50 μm. WT+PBS indicates wild-type C57BL/6 mice were gavaged with PBS; G3+PBS indicates Terc-/-G3 mice were gavaged with PBS; G3+B.a indicates Terc-/-G3 mice were gavaged with Bifidobacterium adolescentis.

Data are expressed as mean ± standard error and analyzed by t test. ^** p <0.01; ^*** p < 0.0001.

Figure 9: A is a volcano plot analysis of intestinal metabolites in Terc ^-/- G3 mice after oral administration of PBS (n=7) or Bifidobacterium adolescentis (n=9). The marked data points show that the abundance of metabolites differs by two times or more. The red box indicates the enrichment of metabolites in the feces of mice gavaged with Bifidobacterium adolescentis, and the blue box indicates the enrichment of metabolites in the feces of mice gavaged with PBS.

B: Hierarchical cluster analysis heat map. Blue or red boxes indicate that the fold change of metabolite abundance is less than or greater than the mean. G3+PBS means Terc ^-/- G3 mice were gavaged with PBS, n=7; G3+Ba means Terc ^-/- G3 mice were gavaged with Bifidobacterium adolescentis, n=9.

C is: Total ion chromatogram (TIC) of the quality control (QC) sample in positive and negative ion modes. 1, ginsenoside Ia; 2, cholic acid; 3, hypoxanthine; 4, 4-trimethylaminobutyric acid; 5, enterodiol; 6, apigenin; 7, 3-dehydroxycarnitine; 8, erucic acid; 9, 9, 10-DHOME; 10, cosmosin; 11, daidzin; 12, 2-hydroxycinnamic acid; 13, L-malic acid. Metabolites with higher concentrations in Terc ^-/- G3 mice gavaged with Bifidobacterium adolescentis are marked in red; metabolites with higher concentrations in Terc ^-/- G3 mice gavaged with PBS are marked in blue.

In Figure 10: A is a diagram showing the morphological changes in the small intestine of mice aged 7 months after continuous intervention;

B: Measure and compare the length of small intestine (cm) of mice with continuous intervention at 7 months old;

C: HE microscopic structural diagram of the morphological changes of the small intestinal mucosa layer (villus height, villus width, crypt depth) of mice with continuous intervention for 7 months;

D is: Statistical graph of the morphological changes of the small intestinal mucosal layer (villus height, villus width, crypt depth) of mice with continuous intervention at 7 months of age (n=6-9/group);

E: Microscopic structural diagram of the changes in the number of goblet cells in the small intestine epithelium of mice aged 7 months after continuous intervention (PAS staining);

F is: Statistical graph of changes in the number of goblet cells in the small intestine epithelium of mice aged 7 months after continuous intervention (PAS staining) (n=6-9/group);

G is: immunofluorescence expression of small intestinal barrier proteins (ZO-1, Occlodin, Muc2) in mice with continuous intervention at 7 months of age (n=6-9/group);

H is: the mRNA expression of small intestinal senescence-associated secretory phenotype (SASP) in mice with continuous intervention at 7 months of age (n=3-4/group);

I represents: the expression levels of small intestinal aging-related genes (p21, p53) and immunohistochemistry (p21, p53) in mice aged 7 months after continuous intervention (n=6-9/group).

WT+PBS indicates wild-type mice were intragastrically administered with PBS, G3+PBS indicates Terc ^-/- G3 mice were intragastrically administered with PBS, and G3+Ba indicates Terc ^-/- G3 mice were intragastrically administered with Bifidobacterium adolescentis. Scale bar: 100 μm.

In Figure 11: A is a graph showing changes in the colon mucosal structure (HE staining) and goblet cell number (PAS staining) of mice aged 7 months after continuous intervention, n=6-9/group;

B: mRNA expression of colon aging-related genes (p21, p53) in mice treated with continuous intervention at 7 months of age (n=4-7/group);

C: the expression of colon aging-related proteins (p21, p53) in mice aged 7 months after continuous intervention (n=4-7/group);

D is: the expression level of colon stem cell-related protein (Lgr5 Ascl2) in mice with continuous intervention at 7 months of age (n=4-7/group).

WT+PBS indicates that wild-type mice were intragastrically administered with PBS, G3+PBS indicates that Terc ^-/- G3 mice were intragastrically administered with PBS, and G3+Ba indicates that Terc ^-/- G3 mice were intragastrically administered with Bifidobacterium adolescentis.

DETAILED DESCRIPTION

The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.

1. The present invention investigates the application of Bifidobacterium adolescentis (B.a) composition in delaying aging of organisms (specifically model organisms such as fruit flies, nematodes and mice).

1.1 Climbing is a kind of motor ability and an important indicator of healthy life span. The present invention adds Bifidobacterium adolescentis to the standard food of fruit flies, draws the survival curve of fruit flies and detects the climbing ability of fruit flies at specific time points. Through a series of experiments, the delaying effect of Bifidobacterium adolescentis on fruit fly aging was investigated, and it was verified that exogenous supplementation of Bifidobacterium adolescentis (Ba) can promote the longevity of fruit flies and improve the healthy life span indicators of fruit flies. In wild-type fruit flies ^w1118 and Canton-S, the median survival time, average life span and average maximum life span of the Bifidobacterium adolescentis intervention group were significantly higher than those of the control group, as shown in Figures 1A and 1B, with male fruit flies on the left and female fruit flies on the right; on the 30th day, the climbing ability of the Bifidobacterium adolescentis intervention group was also significantly stronger than that of the control group, as shown in Figures 1C and 1D.

1.2 The present invention adds Bifidobacterium adolescentis to the uracil-deficient Escherichia coli (E. coli OP50), a regular food of nematodes, to draw the survival curve of nematodes and detect various health life indicators of nematodes at specific time points. Through a series of experiments, the delaying effect of Bifidobacterium adolescentis on nematode aging was investigated, and it was verified that exogenous supplementation of Bifidobacterium adolescentis can promote the longevity of nematodes and improve the nematodes' motor ability, heat stress resistance, and intestinal lipofuscin deposition and other health life indicators. The specific process is as follows: After feeding wild-type Caenorhabditis elegans N2 with Escherichia coli OP50, Escherichia coli OP50 and Bifidobacterium adolescentis mixed in a ratio of 1:1 or 1:2, the average lifespan and average maximum lifespan of nematodes in the Bifidobacterium adolescentis intervention group were significantly higher than those in the control group, as shown in Figures 2A and 2B; wild-type Caenorhabditis elegans N2 was fed with Escherichia coli OP50 or a 1:1 mixture of Escherichia coli OP50 and Bifidobacterium adolescentis, and its motor ability was quantitatively analyzed on the 2nd, 8th and 16th days (as shown in Figure 2C 2D), and the ability to resist heat stress was analyzed on the 4th, 8th and 12th days (as shown in FIG2E). The results showed that at a specific time point, the motility and heat stress resistance of the nematodes in the Bifidobacterium adolescentis intervention group were stronger than those in the control group; in addition, the autofluorescence image of intestinal lipofuscin deposition (as shown in FIG2F) and quantitative comparison (as shown in FIG2G) also showed that the intestinal lipofuscin deposition of the nematodes in the Bifidobacterium adolescentis intervention group was significantly less than that in the control group.

1.3 The present invention adds Bifidobacterium adolescentis to the drinking water of mice for free drinking or oral gavage, and sets up a PBS control group at the same time, draws the survival curve of mice, and records the changes of mouse weight and systemic aging score at fixed time nodes. Through a series of experiments, the delaying effect of Bifidobacterium adolescentis on mouse aging was investigated, and it was verified that exogenous supplementation of Bifidobacterium adolescentis can improve mouse aging scores and aging indicators. The method is as follows: As shown in Figure 3A, Terc-/-G0 mice (C57BL/6) were cross-hybridized to produce Terc+/+ (wild-type C57BL/6) and Terc-/-G1 mice, and the obtained Terc-/-G1 mice were cross-hybridized to produce Terc-/-G3 mice, and divided into three groups for further study. 6-8 week old wild-type C57BL/6 was set as the control group (WT+PBS, n=11, oral gavage PBS), and 6-8 week old Terc-/-G3 mice were randomly divided into PBS oral gavage group (G3+PBS, n=9) and Bifidobacterium adolescentis group (G3+B.a, n=12). Oral gavage was performed every other day until natural death or sacrifice at 7 months of age. The body weight of Terc-/- mice was significantly lower than that of wild-type Terc+/+ mice, while the body weight of mice in the Bifidobacterium adolescentis group increased significantly compared with the control group, as shown in Figure 3B. The frailty index score, which comprehensively quantifies the degree of frailty in aged mice, showed significant differences between wild-type and PBS-fed Terc-/- mice, and Bifidobacterium adolescentis improved the age-related frailty index of Terc-/- mice, as shown in Figure 3C. Micro-CT scanning and three-dimensional reconstruction were performed on the femurs of mice. Compared with wild-type mice, the bone volume/total volume (BV/TV) and trabecular thickness (Tb.Th) of Terc-/- mice were significantly reduced. After gavage of Terc-/- mice with Bifidobacterium adolescentis, the above indicators were significantly improved, as shown in Figure 3D, indicating that supplementation with Bifidobacterium adolescentis has an improving effect on osteoporosis in Terc-/- mice. By comparing the morphological changes and survival numbers of neurons in the CA3 region of the mouse hippocampus to evaluate the aging state (as shown in Figure 3E), it was found that compared with the control group, the nuclear displacement, cytoplasmic condensation and nuclear fragmentation of Terc-/- mice were more obvious, and the number of surviving neurons in mice gavaged with Bifidobacterium adolescentis also increased significantly. These results indicate that exogenous addition of Bifidobacterium adolescentis can improve the health lifespan of terc-/- premature mice.

2. The present invention investigates the application of Bifidobacterium adolescentis in delaying cell aging. The present invention investigates the delaying effect of Bifidobacterium adolescentis on the replicative aging and doxorubicin-induced aging of mouse embryonic fibroblasts through in vitro cell experiments. The specific method is as follows: Bifidobacterium adolescentis co-cultured with mouse fibroblasts with replicative aging and doxorubicin-induced aging, respectively, and then aging-related β-galactosidase (SA-β-gal) staining is performed to detect the degree of cell aging. The results show that Bifidobacterium adolescentis can significantly reduce the degree of replicative aging (as shown in Figures 4A and 4B) and doxorubicin-induced aging (as shown in Figures 4C and 4D) of mouse fibroblasts, and can significantly increase the mRNA and protein expression levels of catalase CAT in mouse fibroblasts aged under the two induction modes (as shown in Figures 4E-4G), confirming that exogenous supplementation of Bifidobacterium adolescentis can delay the aging of mouse embryonic fibroblasts.

3. The present invention investigates the application of Bifidobacterium adolescentis in regulating the superoxide dismutase sod-3 gene and catalase CAT gene in Drosophila. In order to clarify the mechanism of Bifidobacterium adolescentis-induced lifespan improvement, the present invention investigates the expression of lifespan-related genes in Drosophila ^w1118 . Compared with the control group, the expression of sod-3 and CAT in Drosophila ^w1118 supplemented with Bifidobacterium adolescentis was significantly higher than that in the control group (as shown in Figures 5A and 5B). Sod-3 and CAT are crucial to the extension of lifespan and improvement of health of Drosophila induced by Bifidobacterium adolescentis (Ba).

4. The present invention investigates the application of ctl-2 gene regulation in Caenorhabditis elegans. By screening aging-related genes, it was found that the catalase (CAT) gene plays an important role in delaying aging. Catalase, also known as catalase CAT, is a terminal oxidase widely present in animals, plants, and microorganisms. As an important substance in an organism, the most important function of the catalyst is to participate in the reactive oxygen metabolism process. Under adverse conditions such as environmental stress, the free radicals in the organism increase, and the cell membrane produces peroxidation, resulting in cell membrane damage and damage. CAT plays an important role in reducing and preventing the formation of hydroxyl radicals. In nematodes, CAT is mainly divided into three types, ctl-1, ctl-2, and ctl-3, which are homologous genes of CAT. Ctl-2 accounts for the majority in nematodes. The present invention uses a nematode model to screen and clarify a catalase (CAT) gene as a targeting gene of Bifidobacterium adolescentis in delaying the aging of organisms. The mechanism of Bifidobacterium adolescentis in delaying aging in Caenorhabditis elegans is described. The method is as follows:

Nematodes from the Bifidobacterium adolescentis intervention group and the OP50 control group were collected, and RNA was extracted and then qPCR was used to screen out the oxidative stress-related target genes sod-3 and ctl-2 that were highly expressed in the Bifidobacterium adolescentis intervention group at the mRNA level (as shown in Figure 6A). Subsequent Bifidobacterium adolescentis intervention experiments were further conducted on the sod-3 and ctl-2 mutants of nematodes, and it was found that Bifidobacterium adolescentis could still extend the lifespan of the sod-3 mutant, but had no obvious effect on extending the lifespan of the ctl-2 mutant (as shown in Figures 6B and 6C). At the same time, Bifidobacterium adolescentis could increase the average maximum lifespan of the wild-type nematode N2, but had no obvious improvement on the ctl-2 mutant nematode, as shown in Figure 6D, thus clarifying the potential regulatory role of ctl-2.

The Pctl-2:ctl-2gDNA:mCherry plasmid was constructed and integrated into the genome of the nematode through UV-TMP after microinjection. The red fluorescence of mCherry under a fluorescent confocal microscope was used to visually reflect the expression of ctl-2. The results showed that the expression of ctl-2 in the nematode in the Bifidobacterium adolescentis intervention group was significantly higher than that in the control group on the 14th day, as shown in Figures 6E and 6F.

Bifidobacterium adolescentis intervention was performed on the nematode ctl-2 mutant, and various health lifespan related indicators of the intervention group and the control group were detected at specific time points (same as 1.2). Wild-type Caenorhabditis elegans N2 and ctl-2 mutant nematodes were fed with Escherichia coli OP50 or a 1:1 mixture of Escherichia coli OP50 and Bifidobacterium adolescentis, and their motility was quantitatively analyzed on the 2nd and 8th days, as shown in Figure 6G; their heat stress resistance was analyzed on the 4th, 8th and 12th days, as shown in Figure 6H. The results showed that at specific time points, the Bifidobacterium adolescentis intervention group could improve the motility and heat stress resistance of wild-type Caenorhabditis elegans N2; in addition, the autofluorescence image of intestinal lipofuscin deposition (as shown in Figure 6I) and quantitative comparison (as shown in Figure 6J) also showed that in wild-type Caenorhabditis elegans N2, the intestinal lipofuscin deposition of the nematodes in the Bifidobacterium adolescentis intervention group was significantly less than that of the control group. However, the effect of Bifidobacterium adolescentis on improving health lifespan was completely blocked in the ctl-2 mutant (as shown in Figure 6G-J). The above clearly defines the potential regulatory role of ctl-2, indicating that ctl-2 is crucial in the regulation of Bifidobacterium adolescentis on the lifespan and health lifespan of C. elegans.

5. The present invention investigates the application of Bifidobacterium adolescentis in regulating the catalase CAT gene in mice. The method is as follows: The mouse modeling process is the same as described in 1.3, and the specific process is detailed in the text. After 7-month-old wild-type mice (WT+PBS, n=10), Terc ^-/- G3 PBS gavage mice (G3+PBS, n=6) and Terc ^-/- G3 Bifidobacterium adolescentis gavage mice (G3+Ba, n=9) were killed, the muscle tissue and cerebral cortex of each group of mice were collected, the activity of catalase was detected by a kit, and the expression of ctl-2 homologous gene CAT was detected by qPCR, western blot and immunohistochemistry. The results showed that after supplementation with Bifidobacterium adolescentis, the catalase activity in muscle tissue and cerebral cortex of Terc ^-/- mice was significantly increased (as shown in Figure 7A), and the expression of CAT gene at mRNA and protein levels was also significantly increased (as shown in Figures 7B-7D). At the same time, in the CA3 region, DG region and cerebral cortex region of the hippocampus, the CAT expression of Terc ^-/- mice was also significantly increased after intervention with Bifidobacterium adolescentis (as shown in Figure 7E). Therefore, the present invention also clarifies that the CAT gene plays an important role in delaying the aging of mice as a targeted gene of Bifidobacterium adolescentis, and explains the mechanism by which Bifidobacterium adolescentis delays the aging of mice by regulating the CAT gene.

6. The present invention investigates the application of Bifidobacterium adolescentis in regulating the p53 gene in mice. The p53 immunohistochemical staining was performed on the hippocampal DG region of three groups of experimental mice (wild-type C57BL/6 mice (oral administration of PBS), Terc ^-/- G3 mice (oral administration of PBS), and Terc ^-/- G3 mice (oral administration of Bifidobacterium adolescentis). The results showed that supplementation with Bifidobacterium adolescentis can downregulate the expression of p53 in the brain tissue of mice (as shown in Figure 8).

7. The present invention investigates the application of Bifidobacterium adolescentis in regulating oxidative stress metabolites in mice. The fecal samples of Terc ^-/- G3 mice after intragastric administration of PBS (n=7) and Bifidobacterium adolescentis (n=9) were subjected to LC-MS/MS detection by UPLC-QE-MS system to analyze the intestinal metabolites of the two groups of mice. Combining the detection results of LC-MS/MS, the metabolomics analysis of mouse feces was performed, and a volcano map (as shown in Figure 9A) and a hierarchical cluster analysis heat map (as shown in Figure 9B) were drawn to evaluate the effect of Bifidobacterium adolescentis on oxidative stress-related metabolites. Figure 9C shows the total ion chromatogram of mouse metabolites. It can be seen from the figure that in the feces of Terc-/- aging mice gavaged with Bifidobacterium adolescentis, the content of metabolites that can increase CAT activity is high, such as apigenin and erucic acid, which are significantly increased, and the content of some other antioxidants, including ginsenoside Ia, 2-hydroxycinnamic acid, soybean glycoside and L-malic acid, is also significantly increased; while the producers of reactive oxygen species, such as hypoxanthine, are correspondingly reduced. In addition, cosmosin with anti-aging potential and enterodiol with tumor inhibitory activity are also enriched in large quantities. In the intestinal metabolites of Terc-/- aging mice gavaged with PBS in the control, the content of several pro-inflammatory or cardiovascular disease-related metabolites is significantly increased, including bile acid, 9,10-DHOME, 3-dehydroxycarnitine and 4-trimethylaminobutyric acid. These results indicate that Bifidobacterium adolescentis inhibits the aging of Terc-/- mice by regulating CAT and host oxidative stress-related metabolites, thereby having a beneficial effect on lifespan and health.

8. The present invention investigates the application of Bifidobacterium adolescentis in improving intestinal aging in telomerase knockout mice. The model mice and the control group were continuously gavaged until they were seven months old, and then small intestinal samples were taken from each group. The aging-related indicators were measured at the gene level and protein level, and it was found that Bifidobacterium adolescentis had the effect of improving intestinal aging in mice.

After aging occurs, the expression levels of a series of related molecules in aging cells will show characteristic changes, especially the related molecules of tumor suppressor molecular network and cell cycle inhibition pathway. In the process of cell aging, the p53-p21 pathway and the p16-Rb pathway are the most important signal pathways mediating most cell aging phenomena, and are also the main components of the tumor suppressor molecular network. The expression of p53 gene and p21 gene in aging cells increases sharply, so they become important markers for detecting cell aging. The tight junction protein structure is the structural basis of the intestinal epithelial barrier. It is composed of a variety of tight junction proteins. As a tight structure between cells, it can prevent exogenous toxins from leaking into the surrounding tissues and regulate substances passing through the epithelial layer. Studies have shown that aging can cause changes in the structure, distribution and quantity of intestinal mucosal barrier proteins. In view of the important changes in the intestinal mucosal barrier during the aging process of the body, the differences in the performance of tight junction proteins and aging-related classical genes in the aging-related intestinal aging, the present invention focuses on the above related indicators and focuses on the improvement of the intestinal aging phenotype of telomerase knockout mice by Bifidobacterium adolescentis.

Previous studies have shown that the length of the small intestine decreases to a certain extent with age. The present invention collects and measures the intestinal tissues of 7-month-old wild-type mice (WT+PBS), Terc ^-/- G3 PBS gavage mice (G3+PBS) and Terc ^-/- G3 adolescent Bifidobacterium gavage mice (G3+Ba) that have been continuously intervened (as shown in Figures 10A and 10B). By measuring the length of the small intestine, it was found that the length of the telomerase knockout mice was significantly shortened, while in the experimental group intervened by Bifidobacterium adolescentis, the state of shortened small intestine length was alleviated (as shown in Figure 10B). Further, the small intestinal tissue was fixed, embedded, sliced, and HE and PAS stained. The HE staining results showed that compared with the normal control group, the number of villi, villus length, and villus width of the small intestinal mucosal layer of telomerase knockout mice were significantly reduced, and the number of crypts per unit area was also significantly reduced (as shown in Figure 10C); the number of goblet cells indicated by PAS staining was also significantly reduced (as shown in Figure 10E). By comparing the small intestinal mucosa of mice in the adolescent bifidobacterium gavage group, it was found that adolescent bifidobacterium can improve the changes in mucosal morphology during intestinal aging. The statistical results are shown in Figures 10D and 10F. The intestinal mucosal barrier is also an important component of the intestine. By performing ZO-1 and Occludin protein immunohistochemical staining on paraffin sections, the results showed that compared with the control group, the intestinal barrier protein of telomerase knockout mice was significantly reduced, and under the intervention of adolescent bifidobacterium, the expression of barrier-related proteins was significantly increased, as shown in Figure 10G. On the other hand, the content of mucin (Muc2) in the mouse intestine was also significantly reduced in telomerase knockout mice, and also significantly increased after the intervention of adolescent bifidobacterium. Mucin is secreted by goblet cells, and the amount of goblet cells indicated by PAS is significantly higher, as shown in Figure 10E, which further explains the reason for the increased expression of Muc2.

In the aging process, long-term exogenous aging-inducing factors (DNA damage, exogenous metabolites, etc.) can synergistically act on cells, leading to cell aging through classic p16INK4a/Rb, p53/p21 pathways or other pathways. These may lead to widespread changes in gene expression, resulting in aging-related growth stagnation and morphological changes. The present invention further detects typical related genes, as shown in Figures 10H and 10I. Compared with the normal control group, the typical genes of the aging-related secretory phenotype (SASP) of telomerase knockout mice were significantly highly expressed, and further immunohistochemistry suggested that the protein levels of p21, p53, etc. were also significantly increased. It shows that the intestinal tract of telomerase knockout mice indicates an obvious aging state. In the telomerase knockout mouse group after intervention with Bifidobacterium adolescentis, most of the highly expressed factors of SASP were improved and reduced, and the results of immunohistochemistry and qPCR experiments also suggested a significant downward trend of p21 and p53 at the transcriptional level and protein level, suggesting that Bifidobacterium adolescentis can play a role in improving the aging of the mouse intestine by reducing the expression levels of SASP and aging-related genes p53, p21, etc.

Similarly, as shown in Figure 11, compared with the normal group, the morphological structure of the colon mucosa of the telomerase knockout group mice also showed aging changes, and the content of goblet cells represented by PAS staining was also significantly reduced. Similarly, after the intervention of Bifidobacterium adolescentis, the structural disorder of the colon mucosa was improved, and the number of goblet cells was also increased (Figure 11A). Further detection of colon tissue aging-related genes found that the expression levels of p21 and p53 in colon tissue were significantly increased in telomerase knockout mice, indicating that the colon of telomerase mice does exist in an aging state. Further observation of the immunohistochemical protein levels of p21 and p53 after the intervention of Bifidobacterium adolescentis found that they were significantly reduced, suggesting that Bifidobacterium adolescentis can improve the state of intestinal aging in mice by reducing the expression of colon aging-related genes p53 and p21, as shown in Figures 11B and C.

In order to further explore the mechanism of improvement of colon aging phenotype, the present invention also detected the changes in the classic genes Lgr5 and Ascl2 related to intestinal stem cells, as shown in Figure 11D. Compared with the control group, the gene expression of intestinal stem cells in telomerase knockout mice was significantly reduced, while after intervention with Bifidobacterium adolescentis, the expression levels of colon stem cell related genes Lgr5 and Ascl2 rebounded, suggesting that Bifidobacterium adolescentis can improve the intestinal aging of mice by increasing the expression of colon stem cell related genes, as shown in Figure 11D.

The specific experimental process is as follows:

RNA sample extraction

Take a homogenizer tube, add 1mL of Trizol Reagent, and pre-cool on ice. Take 100mg of tissue and add it to the homogenizer tube. Grind thoroughly with a homogenizer until no tissue chunks are visible. After sample pretreatment, centrifuge at 12000rpm for 10min to take the supernatant. Add 250μL of chloroform, invert the centrifuge tube for 15s, mix thoroughly, let stand for 3min, centrifuge at 12000rpm for 10min at 4℃, and transfer 400μL of supernatant to a new centrifuge tube. Add 0.8 times the volume of isopropanol, invert and mix. Place at -20℃ for 15min. Centrifuge at 12000rpm for 10min at 4℃. The white precipitate at the bottom of the tube is RNA. Aspirate the liquid and add 1.5mL of 75% ethanol to wash the precipitate. Centrifuge at 12000rpm for 5min at 4℃ and aspirate the liquid. Place the centrifuge tube on a clean bench and blow for 3min, add 15μL of RNase-free water to dissolve the RNA, and incubate at 55℃ for 5min. Use Nanodrop 2000 to detect RNA concentration and purity: After the instrument is blanked and zeroed, take 2.5 μL of the RNA solution to be tested on the detection base, put down the sample arm, and use the software on the computer to start absorbance detection.

Real-time quantitative polymerase chain reaction (qRT-PCR)

Total RNA from mice, fruit flies, nematodes and cells was extracted using Trizol (Ambion, USA). cDNA was synthesized using primescriptertase (TaKaRa, Japan). qRT-PCR was performed using SYBR-Green-Mix (TaKaRa, Japan) on a Roche LightCycler480 (Roche, Mannheim, Germany) PCR instrument. The relative expression of mRNA was determined using the 2 ^-ΔΔCT method. The primer sequences are shown in Table 1 below.

Table 1

Online database resources

Using python software (relying on RESTful APIs technology), the relative abundance of Bifidobacterium adolescentis in 3,500 healthy samples of different age groups was obtained and analyzed from the human intestinal metagenomic database GMrepo.

Cultivation and preparation of Bifidobacterium adolescentis and Escherichia coli OP50

Bifidobacterium adolescentis bio-67127 (ATCC 15703) was purchased from Beijing Biobw Biotechnology Co., Ltd. and cultured in a modified fortified Clostridium medium (ATCC medium 2107) at 37°C in an anaerobic atmosphere (80% _N2 , 10% _CO2 , 10% _H2 ). Escherichia coli OP50 was cultured overnight in Luria Bertani (LB) liquid medium at 37°C with shaking.

In the mouse experiment, Bifidobacterium adolescentis was heat-inactivated in a 95°C water bath for 15 minutes and adjusted to a final concentration of 10 ⁹ CFU/mL. Every other day, each mouse was gavaged with 200 μL of sterile PBS or pretreated Bifidobacterium adolescentis.

In the cell co-culture experiment, mouse embryonic fibroblasts were co-cultured with heat-killed Bifidobacterium adolescentis slurry at a ratio of 1:100.

In the fruit fly intervention experiment, Bifidobacterium adolescentis was first resuspended in 2.5% sucrose solution to 10 ¹⁰ CFU/mL and then inactivated by heating. The Bifidobacterium adolescentis suspension was mixed with standard corn flour to a final concentration of 10 ⁹ CFU/mL in the intervention group, and the same volume of 2.5% sucrose solution was mixed with standard corn flour in the control group.

In the Caenorhabditis elegans aging intervention experiment, the culture medium of Bifidobacterium adolescentis and Escherichia coli OP50 were centrifuged and the supernatant was discarded, the precipitate was washed with M9 buffer, the precipitate was obtained by centrifugation again and resuspended with M9 buffer to adjust the final concentration to 0.04 mg/mL (wet weight), and finally heat-inactivated. The intervention group was fed with a mixture of Escherichia coli OP50 and Bifidobacterium adolescentis, and the control group was fed with Escherichia coli OP50.

Drosophila strains and growth

The two wild fruit fly strains were w ¹¹¹⁸ and Canton-S. They were raised on standard cornmeal medium at 25°C with a 12-hour light/dark cycle, and the medium was based on the Nutri fly Bloomington formula: 600 mL of the medium contained 3.72 g agar, 35.28 g cornmeal, 35.28 g inactivated dry yeast, 16 mL 10% methyl paraben (solvent was 85% ethanol solution), 36 mL fruit juice, and 2.9 mL 99% propionic acid.

C. elegans strains and synchronization

Wild-type Caenorhabditis elegans N2 and mutant nematodes sod-3(tm760) and ctl-2(ok1137) were purchased from the Caenorhabditis Genetics Center (CGC). Before intervention, nematodes were selected in nematode growth medium (NGM) containing E. coli OP50. Hermaphroditic nematodes in the egg-laying stage were bleached in sodium hypochlorite solution to obtain fertilized eggs. Fertilized nematode eggs were cultured on NGM plates containing E. coli OP50 until they developed to the L4 stage, completing synchronization.

Life span determination

Life span determination method: The same batch of fruit flies completed development and mating 4 to 6 days after eclosion, and then transferred to an empty culture tube for starvation for 2 hours, and then randomly assigned to the intervention group or the control group according to gender. Every 20 fruit flies were divided into a small tube and transferred to a new small tube every 2 to 3 days. The number of dead fruit flies was recorded at the same time. Each group used about 100 fruit flies (5 tubes) for each experiment.

In the C. elegans experiment, nematodes at the L4 stage (day 0) were cultured in NGM plates containing E. coli OP50 until they became adults, and then transferred to plates containing OP50 or mixed bacterial liquid. 150 nematodes were distributed in 10 plates (15 nematodes/plate) and cultured at 20°C. They were transferred to new plates every other day, and the number of survivors, deaths, and culls was recorded.

Survival was calculated as the percentage of surviving flies or nematodes to the total number of flies or nematodes. Nematodes that escaped to the plate wall and cover and dried to death or burrowed into the agar, and flies lost during the tube transfer process should be excluded from the statistics. Lifespan experiments were performed at least three times.

Drosophila tube climbing test

Three tubes of 30-day-old fruit flies (about 15 to 20 flies in each tube) were transferred to three tubes connected vertically by two empty tubes. After gently tapping the bottom, the number of fruit flies that crawled beyond the 8-cm mark within 10 seconds was measured to calculate the climbing index (compared with the total number in the test tube). Each group of experiments was repeated 3 times.

Assessment of nematode motility

1200 synchronized adults were transferred to 20 control plates and 20 intervention plates, cultured at 20°C and transferred every other day. The movement ability of nematodes can be evaluated by two indicators: movement speed and movement trajectory. On the 2nd, 8th and 16th days, 50 nematodes were randomly selected from each group, and the number of sinusoidal waves they crawled through within 30 seconds was recorded. The criteria for movement trajectory analysis are: spontaneous movement without touch stimulation, recorded as A; movement only after touch stimulation, recorded as B; only swinging the head and tail after touch stimulation, recorded as C; and no movement at all, recorded as D.

C. elegans acute heat stress test

On the 4th, 8th and 12th days, 30 nematodes were randomly selected from each group and transferred from 20°C to 37°C. Their reactions were measured every two hours, the number of deaths was counted, and then the average survival time was calculated. Each experiment was repeated 3 times.

Nematode fluorescence microscopy test

The nematodes were synchronized and cultured as described above. On the 10th and/or 14th day, the accumulation of lipofuscin in the intestine and the expression of ctl-2 were quantitatively detected by autofluorescence and mCherry red fluorescence, respectively. The specific method is as follows: the worms were randomly selected and washed twice with M9 buffer. Then they were placed on a 1% agarose gel, anesthetized by 200mM sodium azide, and photographed with an inverted laser scanning confocal microscope (Olympus IX81-FV1000, Tokyo, Japan) under blue excitation light (405-488nm) or red excitation light (559-585nm). The fluorescence intensity was quantified using ImageJ software (NationalInstitutes of Health, Bethesda, MD, USA). The experiment was repeated three times independently, with more than 20 nematodes in each group each time.

Caenorhabditis elegans gene integration

The ctl-2 promoter and ctl-2gDNA were amplified from N2 genomic DNA and then recombined with a specific donor vector fragment using a fusion PCR cloning kit (TaKaRa, Japan). In order to detect the expression level of the ctl-2 gene, the mCherry sequence was cloned downstream of the ctl-2gDNA sequence to construct a functional Pctl-2::ctl-2gDNA::mCherry transgenic plasmid. The Pctl-2::ctl-2gDNA::mCherry transgenic plasmid (50ng/μL) was mixed with Plin-44::gfp (20ng/μL) and injected into N2 nematodes by microinjection. The gene was then integrated using ultraviolet-trimethylolpropane (UV-TMP). The integrated worms were backcrossed with N2 4 times to obtain nematodes that stably expressed fluorescence.

Cell culture and aging

Mouse embryonic fibroblasts (MEFs) were obtained using hybrid mice lacking telomerase RNA components (Terc+/-) and cultured in a 37°C, 5% CO2 incubator. In the replicative aging experiment, the cells were cultured to the 12th generation (p12) with or without the addition of (bacteria: cells = 100:1) of Bifidobacterium adolescentis. In the induced aging experiment, 40nM doxorubicin (SIGMA-ALDRICH, Germany) was added to the culture medium for 3 consecutive days, and then cultured with fresh complete medium for three consecutive days. Two induction cycles were performed, and the whole cycle lasted 12 days. Then the aged cells were co-cultured with Bifidobacterium adolescentis for 6 days. Next, the aging-related β-galactosidase (SA-β-gal) staining test was performed.

Western blot assay

Total protein was extracted with RIPA lysis buffer and quantified with BCA protein detection kit. Proteins were separated by polyacrylamide gel electrophoresis and electrotransferred to polyvinylidene fluoride membrane. The membrane was blocked with 5% skim milk for 2 hours at room temperature and then incubated with catalase primary antibody (Catalog No. 21260-1-AP, Proteintech) at 4°C overnight. The membrane was washed and incubated with anti-rabbit IgG antibody for 2 hours at room temperature, and the signal was detected with ECL kit. β-Actin was used as an internal reference gene.

Statistical analysis

The differences among the three groups were analyzed by one-way ANOVA for normally distributed data and by Kruskal-Wallis (K-W) test for non-normally distributed data. The differences between the two groups were analyzed by t-test or non-parametric test (Wilcoxon rank sumtest or Kruskal-Wallis test). The lifespan test was performed by log-rank (Mantel-Cox) test. All statistical tests were two-tailed, and p < 0.05 was considered statistically significant.

Data analysis and graphs were performed using IBM SPSS Statistics v22.0 software, GraphPad Prism v7 software, and ImageJ software (https://imagej.nih.gov/ij/; RRID:SCR_003070).

Mouse experimental analysis

Terc ^+/- G0 (background C57BL/6) mice were donated by Professor Song Zhangfa of Zhejiang University and cross-hybridized to obtain Terc ^+/+ (wild type) and Terc ^-/- G1 mice. Terc ^-/- G1 mice were then cross-hybridized to obtain Terc ^-/- G3 mice. Starting from 6-8 weeks, mice from the same littermates in different groups were gavaged. Wild-type C57BL/6 group mice (WT+PBS, n=11) and Terc-/-G3PBS gavage group (G3+PBS, n=9) were gavaged with PBS. Terc-/-G3 Ba gavage group (G3+Ba, n=12) was gavaged with adolescent bifidobacteria resuspension (PBS resuspension). Gavage was performed every other day, 200ul per mouse each time. Body weight and systemic aging index score were recorded once a month until natural death or the mice were killed at the age of seven months.

All mice were housed in a sterile animal room. The room was maintained at 20-22°C with a day-night cycle every twelve hours. Mice had free access to food or water. All procedures were performed in accordance with institutional guidelines and approved by the Animal Ethics Committee of Zhejiang University before the start of the study. After sacrifice, skeletal muscle and brain tissues were collected for mRNA and protein quantification, histological evaluation, immunohistochemistry, and catalase activity detection. The left femur was isolated and subjected to micro-computed tomography (micro-CT). All tissue samples were stored at -80°C for further analysis, except for samples used for histological evaluation, immunohistochemistry, and micro-CT scanning (fixed in 4% paraformaldehyde).

Micro-computed tomography and result analysis

After euthanasia of mice, all left bones were separated and the excess of left femoral muscle was removed. Three femoral samples were randomly selected from each group and scanned by micro-CT (InspeXio SMX-225CT FPD HR; Shimadzu Co. Ltd., Kyoto, Japan) after fixation at 225 kV acceleration voltage and 4 μm resolution. Under the same conditions, each sample was reconstructed using micro-CT software (VGStudio MAX; Volume Graphics, Heidelberg, Germany). A 1mm*1mm*1mm intersection (ROI) cubic region was intercepted below the epiphyseal growth plate. The reconstruction parameters of bone volume/total volume (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N) and trabecular separation (Tb.Sp) were analyzed.

Hematoxylin-eosin staining (H&E staining) and immunohistochemistry

After euthanasia, the brain tissue was fixed with 4% paraformaldehyde at room temperature and then embedded in paraffin for sectioning. 3-μm-thick coronal sections were dewaxed in xylene, hydrated with a series of graded alcohols, and then stained with H&E to evaluate morphological changes. The CA3 region of the hippocampus was magnified 100 times and 400 times, and the number of surviving neurons in the CA3 region of the hippocampus was statistically compared.

After paraffin-embedded brain tissue was dewaxed, hydrated, antigen repaired, and endogenous peroxidase activity eliminated, the tissue was blocked with 3% BSA and incubated with primary and secondary antibodies. Finally, it was stained with DAB, rinsed thoroughly, counterstained, dehydrated, and mounted with neutral glue. Images were scanned with a Pannoramic scanner (Pannoramic DESK, 3D HISTECH, Hungary) and observed and counted with CaseviewerC.v2.3.

PAS staining experiment

Paraffin sections were dewaxed to water: the sections were placed in xylene I for 20 min-xylene II for 20 min-anhydrous ethanol I for 5 min-anhydrous ethanol II for 5 min-75% alcohol for 5 min, washed with tap water, and the sections were stained in PAS staining solution B for 10-15 min, washed with tap water, and washed twice with distilled water; the sections were stained in PAS staining solution A for 25-30 min, protected from light, and rinsed with running water for 5 min; then the sections were stained in PAS staining solution C for 30 s, washed with tap water, differentiated with hydrochloric acid aqueous solution, washed with tap water, blued with ammonia water, and rinsed with running water. Then the sections were dehydrated and sealed: the sections were placed in anhydrous ethanol I for 5 min-anhydrous ethanol II for 5 min-anhydrous ethanol III for 5 min-xylene for 5 min-xylene II for 5 min to make them transparent, and sealed with neutral gum. Finally, the goblet cell walls were purple-red and the nuclei were light blue under a microscope.

Immunofluorescence (IF)

Dewax the paraffin sections to water: put the sections into xylene I for 15 minutes, xylene II for 15 minutes, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, 85% alcohol for 5 minutes, 75% alcohol for 5 minutes, and distilled water for washing. Then perform antigen repair: place the tissue sections in a repair box filled with EDTA antigen repair buffer (pH8.0) in a microwave oven for antigen repair. Boil on medium heat for 8 minutes, stop the fire for 8 minutes, and then turn to medium-low heat for 7 minutes. During this process, excessive evaporation of the buffer should be prevented, and the sections should not be dried. After natural cooling, place the slides in PBS (pH7.4) and shake on a decolorizing shaker for 3 times, each time for 5 minutes. After the sections are slightly dried, use a tissue pen to draw circles around the tissue (to prevent the antibody from flowing away), dry PBS, add BSA, and block for 30 minutes. (If the primary antibody is from goat, use 10% donkey serum to block, and if the primary antibody is from other sources, use 3% BSA to block) to draw circles for serum blocking. Then add the primary antibody: gently shake off the blocking solution, add the primary antibody prepared in a certain proportion of PBS on the slices, and incubate the slices flat in a humidified box at 4°C overnight. (Add a small amount of water in the humidified box to prevent the antibody from evaporating). Add the secondary antibody: Place the slide in PBS (pH 7.4) and shake on a decolorizing shaker for 3 times, 5 minutes each time. After the slices are slightly dried, add the secondary antibody of the corresponding species to the primary antibody in the circle to cover the tissue, and incubate at room temperature in the dark for 50 minutes. Finally, counterstain the cell nucleus with DAPI: Place the slide in PBS (pH7.4) and shake on a decolorizing shaker for 3 times, 5 minutes each time. After the slices are slightly dried, add the DAPI staining solution in the circle and incubate at room temperature in the dark for 10 minutes. Quench the tissue autofluorescence: Place the slide in PBS (pH7.4) and shake on a decolorizing shaker for 3 times, 5 minutes each time. Add the autofluorescence quencher in the circle for 5 minutes and rinse with running water for 10 minutes. After the slices are slightly dried, seal them with anti-fluorescence quenching sealing agent. The sections were observed and images were collected under a fluorescence microscope.

Immunohistochemistry (IHC) experiments

Dewax the paraffin sections to water: sequentially place the sections in xylene I for 15 min-xylene II for 15 min-xylene III for 15 min-anhydrous ethanol I for 5 min-anhydrous ethanol II for 5 min-85% alcohol for 5 min-75% alcohol for 5 min-distilled water. Place the tissue sections in a repair box filled with citric acid antigen repair buffer (PH6.0) in a microwave oven for antigen repair, boil on medium heat for 8 min, stop the heat for 8 min, keep warm, and then turn to medium-low heat for 7 min. During this process, excessive evaporation of the buffer should be prevented, and the sections should not be dried. After natural cooling, place the slides in PBS (PH7.4) and shake and wash on a decolorizing shaker 3 times, each time for 5 min for antigen repair, place the sections in 3% hydrogen peroxide solution, incubate at room temperature in the dark for 25 min, place the slides in PBS (PH7.4) and shake and wash on a decolorizing shaker 3 times, each time for 5 min. Then use serum blocking: add 3% BSA in the tissue circle to evenly cover the tissue, and block at room temperature for 30 min. Gently shake off the blocking solution, add the primary antibody prepared in a certain proportion of PBS to the slices, and lay the slices flat in a humidified box and incubate overnight at 4°C. (Add a small amount of water to the humidified box to prevent the antibody from evaporating). Place the slides in PBS (PH7.4) and shake on a decolorizing shaker to wash 3 times, 5 minutes each time. After the slices are slightly dried, add the secondary antibody (HRP labeled) of the corresponding species to the primary antibody in the circle to cover the tissue and incubate at room temperature for 50 minutes. Finally, DAB color development and counterstaining of cell nuclei: hematoxylin counterstaining for about 3 minutes, washing with tap water, hematoxylin differentiation solution differentiation for a few seconds, rinsing with tap water, hematoxylin blueing solution blueing, and washing with running water. Finally, dehydrate and seal the slices: put the slices into 75% alcohol for 5 minutes--85% alcohol for 5 minutes--anhydrous ethanol I for 5 minutes--anhydrous ethanol II for 5 minutes--n-butanol for 5 minutes--xylene I for 5 minutes to dehydrate and make them transparent, take the slices out of xylene and dry them slightly, and seal the slices with neutral gum. Observe under a microscope and collect and analyze images.

Catalase (CAT) activity assay

Skeletal muscle and brain tissues were homogenized with ice-cold PBS (25% w/v), and the catalase activity in the supernatant was detected by visible light method (catalase CAT assay kit, Nanjing Jiancheng Bioengineering Institute).

UHLC-QE-MS System

1. Metabolite extraction

Weigh 25 mg of sample, add 500 μL of extract (methanol: acetonitrile: water = 2:2:1 (V/V/V), containing isotope labeled internal standard mixture), vortex mix for 30 seconds; grind at 35 Hz for 4 minutes, ultrasonic for 5 minutes (ice water bath); repeat steps 2 and 3 times; stand at -40℃ for 1 hour; centrifuge the sample at 4℃, 12000 rpm for 15 minutes; take the supernatant into the injection bottle for detection. Take an equal amount of supernatant from all samples and mix them into QC samples for detection.

2. LC-MS/MS analysis

The target compounds were separated by chromatography on a Vanquish (Thermo Fisher Scientific) ultra-high performance liquid chromatograph using a Waters ACQUITY UPLC BEH Amide (2.1 mm × 100 mm, 1.7 μm) liquid chromatography column. Phase A of the liquid chromatography was an aqueous phase containing 25 mmol/L ammonium acetate and 25 mmol/L ammonia water, and phase B was acetonitrile. Gradient elution was used: 0-0.5 min, 95% B; 0.5-7 min, 95%-65% B; 7-8 min, 65%-40% B; 8-9 min, 40% B; 9-9.1 min, 40%-95% B; 9.1-12 min, 95% B. Mobile phase flow rate: 0.5 mL/min, column temperature: 30 °C, sample tray temperature: 4 °C, injection volume: 2 μL.

The Thermo Q Exactive HFX mass spectrometer can perform primary and secondary mass spectrometry data acquisition under the control of the control software (Xcalibur, Thermo). The detailed parameters are as follows: Sheath gas flow rate: 50Arb, Aux gas flow rate: 10Arb, Capillary temperature: 320℃, Full ms resolution: 60000, MS/MSresolution: 7500, Collision energy: 10/30/60 in NCE mode, Spray Voltage: 3.5kV (positive) or -3.2kV (negative).

3. Data preprocessing and annotation

The raw data were converted into mzXML format using ProteoWizard software, and then processed using a self-written R package (with XCMS as the kernel) for peak identification, peak extraction, peak alignment, and integration. They were then matched with the self-built secondary mass spectrometry database of BiotreeDB (V2.1) for substance annotation, and the cutoff value of the algorithm score was set to 0.3.

The above-mentioned specific implementation methods are used to explain the present invention and are only preferred embodiments of the present invention, rather than limiting the present invention. Any modifications, equivalent substitutions, improvements, etc. made to the present invention within the spirit of the present invention and the protection scope of the claims shall fall within the protection scope of the present invention.

Claims (1)

1. The application of Bifidobacterium adolescentis in the preparation of age-promoting products for improving the intestinal system of mice, characterized in that: the Bifidobacterium adolescentis is bio-67127, ATCC 15703.