CN114732809A - Application of butyrate in preparation of medicine for relieving skeletal muscle aging - Google Patents

Abstract

The invention discloses application of butyrate in preparation of a medicine for relieving skeletal muscle aging. Experiments show that the butyrate has the application of inhibiting the activation and the aging of the muscle satellite cells and relieving the muscle loss in the aging process of an organism, so that the butyrate can be applied to the preparation of the medicines for inhibiting the activation of the muscle satellite cells and relieving the aging of skeletal muscles.

Description

Application of butyrate in preparation of medicine for relieving skeletal muscle aging

Technical Field

The invention belongs to the fields of biotechnology and microbiology, and particularly relates to application of metabolite butyrate secreted by intestinal flora in preparation of a medicament for inhibiting muscle satellite cell activation and relieving skeletal muscle senescence.

Background

Exercise helps to improve cardiovascular function, and enhance immunity and disease resistance, and thus, improving skeletal muscle function can effectively prevent cardiovascular and metabolic diseases. Skeletal muscle accounts for about 40% of the body weight of a human body, and plays an indispensable role in supporting limb movement. Skeletal muscle is formed by proliferation, differentiation and fusion of muscle stem cells to form muscle fibers in the early stages of human development. The muscle stem cells have functions of self-replication and maintaining the number of cells stable, participating in repair and regeneration of muscle damage, and thus, the function of maintaining the number of muscle stem cells stable and regenerated is indispensable for maintaining muscle function. Human aging is mainly reflected in muscle atrophy and severe reduction of muscle regeneration capacity, which further seriously affects individual exercise capacity and further induces cardiovascular and metabolic diseases. Therefore, the development of functional foods having the effects of alleviating, delaying and even reversing muscle aging is urgently needed. The invention discloses a butyrate metabolite secreted by intestinal flora, and the butyrate metabolite has the applications of inhibiting muscle satellite cell activation and senescence and relieving muscle loss in the process of organism aging.

Disclosure of Invention

The invention aims to provide application of butyrate in preparation of medicines for inhibiting muscle satellite cell activation and relieving skeletal muscle aging.

Therefore, the invention provides the application of the butyrate in preparing the medicines for inhibiting the proliferation and activation of the muscle satellite cells and relieving the skeletal muscle aging.

It is a second object of the present invention to provide a drug for inhibiting proliferation and activation of myosatellite cells and alleviating aging of skeletal muscle, which comprises butyrate as an active ingredient.

Preferably, the butyrate salt is sodium butyrate.

Preferably, the sodium butyrate is administered to myosatellite cells at a concentration of 1mM or is administered orally to mice at a concentration of 0.5% w/v.

Preferably, the medicine also contains pharmaceutically acceptable auxiliary materials.

Experiments show that the butyrate has the application of inhibiting the activation and the aging of the muscle satellite cells and relieving the muscle loss in the aging process of an organism, so that the butyrate can be applied to the preparation of the medicines for inhibiting the activation of the muscle satellite cells and relieving the aging of skeletal muscles.

Drawings

FIG. 1: a map of the differences and metabolic pathways between metabolites in adult and senior mice;

FIG. 2 is a schematic diagram: changes in butyric acid content in feces, serum and muscle (TA: tibialis anterior and Soleus: Soleus) and effects of the microbial metabolite butyric acid on skeletal muscle and SC (myosatellite cells);

FIG. 3: changes in gut microbiota affect the satellite cell map;

FIG. 4 is a schematic view of: butyrate is independent of intestinal flora and can reverse the result graph of antibiotic-induced activation of myosatellite cells;

FIG. 5: graphs of the results of butyrate inhibition of proliferation and differentiation of myosatellite cells;

FIG. 6: graphs showing the results of butyrate inhibition of myofibroblast differentiation.

Detailed Description

The following examples are further illustrative of the present invention and are not intended to be limiting thereof.

Example 1:

the first experiment method comprises the following steps:

1. animal feeding: the project adopts C57BL/6J mice, which are purchased from the animal center of Guangzhou Chinese medicine university (animal license number: SCXK 2008-00202008A 002). All mice were housed in Specific Pathogen Free (SPF) animal facilities at the institute of microbiology, academy of sciences, guangdong, under standard laboratory conditions at 21-23 ℃ for a 12 hour light/dark cycle. C57BL/6J mice were fed rodent chow and allowed free access to water. Pairs of C57BL/6J mice were mated to generate enough mice at different age stages. Stool, serum and skeletal muscle samples were collected from C57BL/6J mice of different ages: 2 weeks old (age: 2-3 weeks old), 8 weeks old (age: 8-10 weeks old), 40 weeks old (age: 40-50 weeks old) and 80 weeks old (age: 80-90 weeks old). C57BL/6J mice were also used for monospermafiber isolation of EDL muscle and CTX-induced TA muscle regeneration. All experimental procedures were approved by the institutional animal care committee (animal use protocol number: GT-IACUC 201704071).

2. Antibiotic and butyrate treatment: in the antibiotic sequential treatment experiment (ABX), 8 week old C57BL/6J mice were given free water containing antibiotic cocktail (ampicillin and kanamycin in the first week, penicillin G and neomycin in the second week, and vancomycin and gentamicin in the third week, both at 0.3 mg/ml). Furthermore, drinking water containing ampicillin (0.3 mg/mL) and kanamycin (0.3 mg/mL) was added with or without sodium butyrate at a concentration of 0.5% (w/v).

3. Transplantation of fecal microbiota: male C57BL/6J mice 100 weeks old were used as donors. Male C57BL/6J mice, 8 to 9 weeks old, were used as adult recipients for Fecal Microbiota Transplantation (FMT) by gavage. Recipient mice were normalized using FMT and their respective donors. Prior to FMT, these adult mice were treated with 0.3mg/ml ampicillin and kanamycin in drinking water and replaced daily. After one week of antibiotic treatment, the water containing the antibiotic was changed to plain water. These antibiotic-treated mice then received FMT from an aged donor by using freshly prepared fecal supernatant prepared by dissolving fresh fecal pellets from aged donor mice in sterile Phosphate Buffered Saline (PBS) by homogenization (using sterile plastic paste and vigorous shaking). Each recipient mouse was immediately fed 300. mu.L of supernatant by sterile oral gavage to transplant the fecal microbiota. Control mice were fed 300 μ L PBS using a sterile oral gavage. Intragastric administration was repeated 3 times a week for 4 weeks before additional experiments were performed.

4. Separation of individual muscle fibers: individual muscle fibers were isolated by gently dissecting EDL muscles and digesting them in 0.2% collagenase type I for 80 minutes at 37 ℃. Digested EDL muscle was transferred to pre-warmed wash medium (DMEM supplemented with 10% FBS, 1% sodium pyruvate, 1% penicillin-streptomycin, and 2mM ethylenediaminetetraacetic acid (EDTA)). The EDL muscle is flushed until the muscle fibers are naturally released from the EDL muscle. These single muscle fibers were either transferred to horse serum-coated 24-well plates for single muscle fiber culture or fixed for immunofluorescent staining.

5. And (3) immunofluorescence staining: tibialis Anterior (TA) muscle sections or extensor longus (EDL) single muscle fibers were fixed with 4% paraformaldehyde for 10 minutes and then quenched in 50mM glycine for 10 minutes. Washed 3 times with PBS at room temperature. For Pax7 and Ki-67 staining of frozen sections, antigen retrieval in Tris-EDTA (pH9.0) at 99 ℃ was required for 1 hour. Cells were permeabilized with 0.5% Triton-X-100/PBS for 10 min at room temperature, then washed 3 times with PBS, 5 min each, treated with blocking buffer (5% goat serum and 3% Bovine Serum Albumin (BSA)) for 1 h, and diluted primary antibody with blocking buffer overnight at 4 ℃. For staining of muscle fibers, primary antibody was used at a dilution of 1: 250. For cryo-section staining, a dilution of 1:5 was used for Pax7, 1:50 for MHC, 1:100 for Ki-67, and 1:1000 for Lamin B2. The samples were then washed with PBST and incubated with appropriate Alexa Fluor-conjugated secondary antibodies (1: 500) for 1 hour at room temperature. After 3 washes with Phosphate Buffered Saline (PBST) containing tween 20 for 5 minutes each, frozen sections or individual muscle fibers were mounted with DAPI. For the EdU detection, the Click-iT reaction kit (Life Technologies) was used prior to incubation with diamidino-2-phenylindole (DAPI) according to the manufacturer's recommendations. For Pax7 and Ki-67 staining of frozen sections, the automatic background of the frozen sections was quenched with 0.5% Sudan black for 5 minutes prior to loading. Mounted sections or individual muscle fibers were observed using an LSM700 confocal microscope (zeiss).

6. Immunoblotting: C2C12 myofibroblasts were washed with PBS prior to protein isolation. After incubation in ice-cold radioimmunoprecipitation assay (RIPA) buffer (1 mM EDTA, 50mM Tris-HCl pH 7.5, 0.1% Sodium Dodecyl Sulfate (SDS), 150mM NaCl, 1% NP40, 1% sodium deoxycholate and protease inhibitor cocktail) for 10 minutes, samples were collected and sonicated on ice with a 20% amplifier for 30 s. Total protein lysates were cleared by centrifugation at 16000 Xg for 10 min at 4 ℃. The supernatant was saved for protein assay and Western blot analysis. Protein concentration was determined using a bicaprylic acid (BCA) protein assay kit (Thermo Fisher) according to the manufacturer's instructions. Protein samples were separated on 12% SDS-polyacrylamide gel electrophoresis (PAGE) and then transferred to PVDF membrane. Proteins on (polyvinylidene fluoride) PVDF membranes were blocked with 5% (w/v) skim milk at room temperature for 1 hour and incubated with primary antibody at 4 ℃ overnight and secondary antibody at RT for 1 hour. Protein bands on PVDF membrane were visualized with ECL reagent (Thermo Fisher Scientific) and images were acquired by ChemiDoc-imaging system (Bio-Rad).

7. Hippocampal XF24 metabolic analysis: measurements were performed using an XF24 analyzer (Seahorse Bioscience, North Billerica, MA, USA). For proliferation analysis, C2C12 myocytes were seeded at a density of 15,000 cells/well in 0.15% gelatin coated wells for 24 hours. For differentiation into myotubes, fully confluent myocytes were cultured in differentiation media and evaluated for differentiation into myotubes. To measure the extracellular acidification rate (ECAR), the cell culture medium was changed to hippocampal XF medium (without carbon dioxide) containing 2mM glutamine, and the cells were cultured at 37 ℃ for 1 hour, and then 10mM glucose, 2. mu.M oligomycin and 100mM 2-deoxyglucose (2-DG) were injected into each well in sequence. To measure Oxygen Consumption Rate (OCR), the cell culture medium was changed to hippocampal XF medium containing 10mM glucose, 1mM pyruvate, and 2mM glutamine, and cultured at 37 ℃ for 1 hour. mu.M oligomycin, 2. mu.M carbonyl cyanide-4- (trifluoromethoxy) phenylhydrazone (FCCP) and 1. mu.M rotenone/antimycin A were injected into each well in sequence. The hippocampal XF24 extracellular analyzer used 8 minute cycling protocol instructions (3 minutes mixing, 2 minutes resting, 3 minutes measuring).

8. MTT detection: C2C12 cells were seeded into 24-well plates and the T0 origin was examined 12 hours after cell seeding. At the checkpoint, the growth medium was changed (400 μ l per well) and 100 μ l MTT (5 mg/ml thiazole blue tetrazolium bromide solution in PBS buffer) was added to the wells. After incubation at 37 ℃ for 60 minutes, the medium was replaced with 200. mu.L of dimethyl sulfoxide (DMSO) and incubated for 5 minutes. Triplicate 50. mu.L cell lysates were measured at 562 nm.

9. 16S rRNA sequencing: stool samples were collected in 1.5 ml cryovials, immediately frozen in liquid nitrogen, and stored in a freezer at-80 ℃ until further analysis. Total DNA of Fecal bacteria was extracted using the Quick-DNA Fecal/Soil Microbe Miniprep Kit. The V3-V4 region of the 16S rRNA gene of bacteria was amplified using Q5 high fidelity DNA polymerase (NEB) with primers 341F (5 '-CCTACGGGNGGCWGCAG-3') and 805R (5 '-GACTACHVGGGTATCTAATCC-3'). Polymerase Chain Reaction (PCR) products were purified using AMPure XP (Beckman Coulter) and quantified using a Qubit dsDNA high sensitivity detection kit (ThermoFisher). Samples were pooled to create libraries and sequenced on the Illumina NovaSeq PE250 platform. Sequences were trimmed using FASTX toolkit and single-ended reads were merged according to the barcode. To obtain high quality readings, the sequence was quality controlled using the QIIME 1 (V1.91) workflow (also confirmed by the QIIME 2 workflow). Sequences that reach the 97% similarity threshold are assigned to the same OUT.

10. Metabolite analysis: metabolite concentrations of stool, serum and muscle samples were quantified by liquid chromatography coupled mass spectrometry. The Kolmogorov-Smirnov test was used to verify whether the entire data set was normally distributed. For data sets that failed the normality test, significant differences were analyzed using a nonparametric test. Differences in gut microbiota and metabolites were used for correlation analysis. Further analysis was performed using Spearman's scale correlation data with correlation coefficient greater than 0.6 and P < 0.05.

11. C2C12 myofibroblast cultures: C2C12 myocytes were purchased from ATCC (CRL-1772) and cultured at 37 ℃ with 5% CO₂And 21% of O₂Under a cell culture hood, maintained in complete Dulbecco' S modified Eagle medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS) and 1% penicillin-streptomycin (P/S). For proliferation assays, C2C12 myocytes were treated with 5

、50

1mM and 2mM acetate, propionate and butyrate treatments. For myoblast differentiation, fully confluent C2C12 myoblasts were cultured in differentiation medium (DMEM supplemented with 2% horse serum and 1% P/S) containing 1mM sodium butyrate.

Secondly, the experimental results are as follows:

1: butyrate metabolic imbalance in aging mice

In order to understand the basic mechanism of gut microbiota-mediated inhibition of SC activation and balance, the present invention analyzed metabolites in feces, serum and muscle (tibialis anterior: fast and soleus: slow) of mice of different ages. Consistent with the distribution of the gut microbiota and gene expression in muscles of different ages, the metabolite structure of adult mice is clearly different from that of older mice, as shown by the β -diversity of PCoA, particularly the difference between adult (8 years) and older (80 years) mice (fig. 1A-D). Among these metabolites, 54 TA metabolites are closely related to intestinal microbiota and fecal metabolites (fig. 1E). The invention carries out integrated analysis on the KEGG pathways of 54 TA metabolites to obtain 103 KEGG pathways. Of these pathways, 22 pathways have at least 3 differentially enriched metabolites overlapping with the pathways of the TA transcriptome, thus defining 4 KEGG pathways (fig. 1F). These pathways are associated with protein digestion and absorption, glucagon signaling pathway, carbon metabolism, and butyrate metabolism (fig. 1G-I). In these pathways, butyrate, which is associated with butyrate metabolism, comes primarily from the metabolism of the gut microbiota. Further analysis showed that butyrate levels in feces, serum and muscle (TA and Soleus) gradually decreased with aging (fig. 2A-D). To further confirm the relationship of butyrate content between skeletal muscle and feces, the present invention performed a least absolute contraction and selection operator (LASSO) regression analysis showing TA (R)²= 0.48) and SOL (R)²= 0.92) butyric acid content in muscle was all correlated well with that in feces, which further supported the effect of microbial metabolites on skeletal muscle and SC (fig. 2E-H).

2. Alteration of gut microbiota affects satellite cell homeostasis

Skeletal muscle aging is accompanied by a gradual decrease in the number of satellite cells. In addition to the known internal and external factors involved in transcription factors, immunity and nutrients, the effects of gut microbiota on skeletal muscle mass and function have recently been reported by comparing germ-free and pathogen-free mice lacking gut microbiota. However, it is still unknown whether there is a functional interaction between the gut microbiota and the muscle satellite cell microenvironment. To address the potential contribution of gut microbiota to muscle satellite cell balance, the present invention uses two different approaches to assess the potential link between gut microbiota and muscle satellite cells. The present invention explores a scheme for eliminating intestinal flora by gradually increasing antibiotic species to avoid possible antibiotic resistance. Adult C57BL/6J mice, 8 weeks old, were treated continuously with a broad spectrum antibiotic cocktail in drinking water for 3 weeks to produce conditionally sterile (GF) mice, followed by single muscle fiber isolation and immunostaining of muscle satellite cells (fig. 3A, ABX experiment). At the end of the different antibiotic treatment cycles, individual muscle fibers were isolated from EDL muscle and immunostained with Pax7 and Ki 67. Although there was no significant difference in the total number of myosatellite cells between the control group and the antibiotic-treated group, activated myosatellite cells (Pax 7)⁺/Ki67⁺) But increased dramatically (fig. 3B-D). These data indicate that dysbiosis manipulated by antibiotic therapy will cause activation of myosatellite cells within a short week of antibiotic therapy with ampicillin and kanamycin alone.

To reproduce the potential link between microbial dysregulation and muscle satellite cell balance in aged mice, the present invention performed Fecal Microbiota Transplantation (FMT) from aged to adult mice, and performed a week of antibiotic pretreatment (AKP) (fig. 3A, FMT experiment). The transfer of fecal bacteria from aged mice to adult mice resulted in a significant decrease in the number of myosatellite cells, indicating a link between changes in intestinal microenvironment and myosatellite cell balance (FIGS. 3E-F). Taken together, the results indicate that gut microbial dysregulation from aged to adult mice by antibiotic treatment or FMT leads to activation of myosatellite cells, indicating a potential link between gut microbiota/metabolites and myosatellite cell homeostasis.

3. Butyrate is independent of gut flora and can reverse antibiotic-induced activation of myosatellite cells

To further confirm the role of butyrate in myosatellite cell balance, this study was conductedThe invention assumes that butyrate can inhibit the activation of myosatellite cells independent of the gut microbiota, as the present invention has shown that oral administration of antibiotics results in the activation of myosatellite cells. The hypothesis of the present invention was to test two treatment regimens in C57BL/6J mice. One group of C57BL/6J mice was fed sodium butyrate in the drinking water, while another group was simultaneously fed antibiotic cocktail and butyrate in the drinking water (fig. 4A). To assess whether butyrate affected myosatellite cell homeostasis, individual muscle fibers were isolated from EDL muscle and immunostained with Pax7 and Ki 67. Butyrate treatment slightly increased the number of myosatellite cells, particularly after long-term treatment (21 days) (fig. 4B-C). However, the increase in the number of myosatellite cells did not result in Pax7⁺/Ki67⁺A significant increase in the percentage of myosatellite cells (fig. 4D). Butyrate treatment clearly neutralized the effect of antibiotic cocktail on myosatellite cell activation (fig. 4E). Under antibiotic cocktail treatment, a substantial amount of Pax7 was indeed observed with the present invention⁺/Ki67⁺This effect was reversed by additional butyrate supplementation of myosatellite cells (FIGS. 3B-C), antibiotic cocktail treatment (FIGS. 4F-G). The effect of butyrate on myosatellite cell homeostasis may be through metabolic pathways rather than through restoration of gut microbiota. Taken together, these data indicate that butyrate is one of the key gut microbiota metabolites that regulate muscle satellite cell balance.

4. Butyrate, but not other short chain fatty acids, inhibits proliferation and differentiation of myocytes

Short Chain Fatty Acids (SCFAs) are mainly composed of acetate, propionate and butyrate and are produced by intestinal bacteria by fermenting non-digestible fibers in the diet. The present invention is intended to demonstrate the inhibitory effect of which short chain fatty acids on myocyte proliferation/differentiation and myosatellite cell activation/proliferation. Sodium acetate, sodium propionate and sodium butyrate at various concentrations (5)

、50

1mM and 2 mM) the proliferation of C2C12 muscle cells was assessed using the MTT assay. Lower concentration compared to control group (5)

And 50

) Proliferation was not inhibited, whereas sodium butyrate at 1mM significantly inhibited proliferation of C2C12 after 48 hours of treatment (FIGS. 6A-D). EdU staining further demonstrated that 48 hours incubation with 1mM sodium butyrate resulted in a significant reduction in proliferation (FIGS. 5A-B, and 6E-F). Cell proliferation and EdU incorporation were not affected by sodium acetate and propionate salts even at 2mM (FIG. 5B, and FIGS. 6E-F). For both C2C12 myocytes and activated myosatellite cells in vitro, MyoD expression is involved in myocyte proliferation and differentiation. Treatment with butyrate at 1mM, but not 100M, significantly reduced MyoD protein levels (FIG. 6G). During myogenic differentiation, 1mM butyrate completely inhibited myogenic differentiation, and the fusion index of C2C12 myoblasts was lower (20-30% for control and 2% for experimental group) (FIGS. 5C-D). The mechanism of inhibition of myogenic differentiation by 1mM butyrate was directly related to the decrease in protein levels of MyoD, MyoG and Myosin Heavy Chain (MHC) (FIG. 6H).

To explore the effect of butyrate on myosatellite cell activation and proliferation, individual muscle fibers isolated from EDL muscle according to the invention were cultured in vitro for 72 hours and then immunostained with Pax7 and MyoD. Treatment with 1mM butyrate resulted in fewer clusters and a lower number of cells per cluster compared to control myofibers (FIGS. 5E-F). Thus, these in vitro and in vivo data indicate that only butyrate, but not acetate and propionate, inhibits cell proliferation and differentiation. With these data, the effects of butyrate on SC quiescent phase, myoblast proliferation/differentiation and newly formed myotubes during skeletal muscle regeneration were worth discussing. An intramuscular injection of CTX was performed on TA muscle to induce injury, followed by 3 doses of sodium butyrate. Bone was subsequently assessed at 5, 7, 9, 11 and 15dpi by eMyHc immunostaining of newly formed muscle fibersSkeletal muscle regeneration. Intramuscular NAB injection significantly slowed skeletal muscle regeneration, newly formed muscle fibers (eMyHc)⁺) Less, and the newly formed muscle fiber size was smaller in the sodium butyrate (NAB) treated group than in the control group (fig. 5G-H). Consistent with this observation, the data further indicate that adult C57BL/6J mice gradually decreased butyrate levels during injury/regeneration, while their serum levels did not change significantly, indicating that the internal processes of butyrate absorption and metabolism are precisely regulated (fig. 6K-M). These data indicate that in vitro and in vivo models, especially during skeletal muscle regeneration, moderate butyrate levels are critical to balance muscle satellite cell quiescence, myoblast proliferation and differentiation.

Claims (8)

1. The butyrate is applied to the preparation of the medicines for inhibiting the proliferation and the activation of the muscle satellite cells and relieving the skeletal muscle aging.

2. The use according to claim 1, wherein the butyrate is sodium butyrate or other butyrate salt.

3. The use according to claim 2, wherein the sodium butyrate is administered to myosatellite cells at a concentration of 1mM or to mice at an oral concentration of 0.5% w/v.

4. The use of claim 1, wherein the medicament further comprises a pharmaceutically acceptable excipient.

5. A drug for inhibiting the proliferation and activation of muscle satellite cells and alleviating skeletal muscle aging, characterized by comprising butyrate as an active ingredient.

6. The medicament according to claim 5, wherein the butyrate salt is sodium butyrate.

7. The medicament according to claim 6, wherein the concentration of sodium butyrate is 1mM when used for treating myosatellite cells or 0.5% w/v when used for treating mice.

8. The medicament of claim 5, further comprising a pharmaceutically acceptable excipient.

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