Milk samples assortment
Bovine milk assortment: We obtained unpasteurized milk from wholesome Hustin dairy cows inside 60 days after giving start on the Beixing farm (Heilongjiang province, China). Chilly chain transportation instantly transported the milk tanks to the pattern library [9].
Human milk assortment: From October 2021 to Could 2022, we recruited 10 lactating moms on the Division of Obstetrics and Gynecology, Tongji Hospital, Tongji medical faculty, Huazhong College of Science and Know-how, China. All moms had wholesome, full-term vaginal births and have been 29 ± 1.58 years previous with out different ailments. All moms additionally produced an extra quantity of milk for his or her infants. Earlier than taking part within the research, the donors have been knowledgeable of the aim of the analysis and offered knowledgeable consent. Meantime, contemplating actuality, feasibility, and a few authority tips, once they acquired care within the first 3 weeks postpartum within the scientific division [28,29,30,31], we collected 200 mL of mature milk from every mom (Donors briefly info: DeskS1), which have been instantly saved at 4 °C and saved on this state throughout transportation to the pattern library. The analysis was authorized by the Medical Ethics Committee of Tongji Medical Faculty of Huazhong College of Science and Know-how (IRB Quantity: S237) and registered on the China Scientific Check Registration Heart (Registration Quantity: ChicTR2100052827). To research the consequences of HMEs or BMEs on skeletal muscle, we combined freshly collected human milk or bovine milk samples in equal volumes, making certain similarity among the many volunteers’ backgrounds.
Preparation of EVs by differential ultracentrifugation
HME and BME have been remoted from milk samples utilizing differential centrifugation, referring to established protocols [32, 33]. Briefly, the milk pattern was initially centrifuged at 13,000 × g for 30 min at 4 °C (Allegra 64R, Beckman Coulter, United States), and the ensuing supernatant was collected to take away fat, cells, and particles. The supernatant was centrifuged at 100,000 × g for 60 min at 4 °C utilizing a SW41Ti rotor and an ultracentrifuge (Optima XE-100, Beckman Coulter, United States). The decrease slush portion, together with the pellet, was discarded to remove bigger particles and micro-vesicles. Lastly, the remaining supernatant was centrifuged at 145,000 × g for 90 min at 4 °C to discard it, and the EVs pellet obtained was re-suspended in phosphate-buffered saline (PBS, 0.01 M) and subjected to a different spherical of ultracentrifugation and washing. After eradicating residual sediment via centrifugation at 10,000 × g for five min at 4 °C, the ensuing EVs suspension was sterilized by way of filtration with a 0.22 μm filter. The overall protein content material of EVs was quantified, adjusted to six mg/mL, divided into equal aliquots, and saved at -80 °C till use (avoiding repeated freezing and thawing).
EVs affirmation
Transmission electron microscopy (TEM)
The profile or morphology of HME and BME have been examined utilizing TEM (FEI Tecnai 12, Philips, Holland). Briefly, 10 µL of the pattern was pipetted dropwise onto a copper grid and allowed to accept 1 min. The suspension was then eliminated with filter paper earlier than including 10 µL of uranyl acetate dropwise onto the copper mesh for an additional minute. The grid was dried at room temperature for a number of minutes earlier than imaging with TEM at 100 kV to visualise the EVs.
Nanoparticle monitoring evaluation
HME and BME samples have been pre-diluted 1:100 in phosphate-buffered saline (PBS, 0.01 M) and loaded right into a 1 mL syringe. The measurements have been carried out utilizing a nanoparticle monitoring analyzer (NTA; NanoSight NS300, Malvern, UK), throughout which the samples have been analyzed below scattered mild indicators whereas being pumped at a syringe pump velocity of 75 µL/min. Three 60-second movies have been recorded, with 30 frames per second, and analyzed utilizing the NTA 3.3 Dev Construct 3.3.104 software program. This software program tracked and analyzed the Brownian movement of every particle to find out their hydrodynamic radius and focus.
Western blotting for EVs protein markers
To detect EVs protein markers CD81 and TSG101 and all procedures referred to earlier publications [34], we loaded 20 µg of protein samples onto a 12% SDS-PAGE gel and separated them. Subsequently, we transferred the proteins onto a 0.45 μm nitrocellulose (NC) membrane (Millipore, HATF00010, USA) and blocked it for an hour utilizing 5% nonfat milk in TBST. The membrane was then incubated in a single day at 4 °C with a major antibody at a 1:1000 dilution. Main antibodies of CD81 (ab109201) and TSG101 (ab133586) have been obtained from Abcam (Cambridge, UK). We incubated the membrane with a horseradish peroxidase-conjugated secondary antibody to detect the proteins for an hour. Lastly, we analyzed the pictures collected by GeneSnap (Syngene, Cambridge, UK) and GeneTools (Syngene, GeneTools 4.0, Cambridge, UK) after placing the NC membrane sprinkled with ECL coloring resolution into the gel imaging analyzer (Syngene, UK).
Cell strains and coverings
Cell tradition situations
C2C12 cells have been obtained from The American Kind Tradition Assortment (ATCC, Manassas, USA), and cells have been cultured in 6-well plates containing 3 mL of the medium at a density of three × 105 cells per nicely below 5% CO2 and 37 °C. The expansion medium comprised 10% (v/v) fetal bovine serum (GIBCO), 2 mm glutamine (GIBCO), 100 U/mL penicillin, and 100 µg/mL Dulbecco’s modified Eagle’s medium (DMEM; GIBCO). After the cells reached 70–80% confluence, the medium was changed with a differentiation medium consisting of two% (v/v) horse serum (GIBCO), 2 mm glutamine, 100 U/mL penicillin, and 100 µg/mL DMEM. The differentiation medium was refreshed each 24 h for 7 days. Myotube formation was monitored day by day utilizing an inverted microscope.
BME and HME therapies
Following the differentiation of C2C12 cells, we added 5 × 108 EVs to every nicely of a 6-well plate and incubated the cells for twenty-four h in an EVs-Free medium [35]. After the incubation interval, we collected the cells and re-suspended them in PBS containing a protease inhibitor cocktail (B1400) and phosphatase inhibitor cocktail (B15001) from Bimake (Houston, USA). The cells have been positioned on ice and sonicated utilizing an amplitude of 30% for 10 s, with this course of repeated 3 times. We collected the cell supernatant by way of centrifugation at 3000 rpm for 10 min and quantified the entire protein within the supernatant utilizing the BCA methodology. The supernatant was saved at -20 °C for subsequent protein blot evaluation.
C2C12 cells uptake EVs
In our research, we employed the purple fluorescent dye DiI (1,1-dioctadecyl-3,3,3,3- tetramethylindocarbocyanine perchlorate, G1705, Servicebio, Wuhan, China) to label EVs, the inexperienced fluorescent dye DiO (3,3’-dioctadecyloxacarbocyanine perchlorate, G1704, Servicebio, Wuhan, China) to visualise C2C12 cell membranes, and the blue fluorescent dye Hoechst-33,342 (G1127, Servicebio, Wuhan, China) to mark cell nuclei. These dyes permit for environment friendly and correct monitoring of the mobile uptake of EVs. Particularly, BME or HME was labeled with a 1:1000 working resolution of DiI, and cell membranes have been labeled utilizing a 1:1000 working resolution of DiO, and cell nuclei have been labeled utilizing a ten µg/mL Hoechst 33,342 working resolution, respectively. Extra fluorescent dyes have been washed away after incubating at 37 °C for 30 min in the dead of night. To research mobile uptake, labeled BME or HME particles (5 × 108) have been added to every nicely containing C2C12 cells in a 6-well plate, and their internalization was examined by inverted fluorescence microscopy. In the meantime, all experimental procedures have been carried out in keeping with the producer’s directions.
Jenner-Giemsa staining and determine evaluation
On this research, we employed Jenner-Giemsa staining to investigate myotubes in vitro. First, C2C12 cells have been washed with PBS, fastened with 100% methanol for five min and dried at room temperature for 10 min. Subsequently, cells have been incubated for five min with diluted Jenner dye in distilled water, adopted by 5 minutes’ publicity to Giemsa dye diluted in 1:5 distilled water. After washing with distilled water, myotubes have been visualized utilizing an inverted microscope and analyzed with Picture J (Model 1.52i, Nationwide Institutes of Well being, Bethesda, MD, United States). We quantified muscle fibers of round 30 myotubes per nicely (whole 180 myotubes per image).
In accordance with the earlier analysis [36], the myotubes have been analyzed in an unbiased method after being dyed with Jenner-Giemsa. The RGB photos have been transformed into grey knowledge utilizing Picture J, which have been then offered in grey tones. The picture histogram evaluation was obtained by deciding on “Evaluation/Discount”. The X-axis of the picture represents the vary of 255 Gy tones (0 = black, 255 = white), whereas the Y-axis represents the pixel variety of every coloration. For every picture, the histogram of the myotubes exhibited a big proportion of pixels attributed to the darkest tone. The common pixels attributed to every picture have been calculated for the typical pixel of the tone 0-X between every situation to quantify the density of the myotubes. The darkest threshold (X) was arbitrarily chosen, however it trusted the form of the histogram. The form might differ from experiment to experiment and may embody essentially the most completely different tone intervals between situations. It’s important to notice that every one the pictures in a single experiment should be used with the identical threshold.
Cell viability assay
In accordance with the producer’s protocol, the cell viability assay was carried out with Cell Counting Package-8 (CCK-8; Dojindo, Japan). C2C12 cells have been seeded in a 96-well plate at a density of 1 × 104 cells per nicely. Earlier than assessing cell viability utilizing the CCK-8 assay, cells have been handled with completely different concentrations of Rapamycin (3.125, 6.25, 12.5, 25, 50, and 100 nM) for twenty-four h. Subsequently, the tradition medium within the 96-well plate was changed with 100 µL of tradition medium containing CCK-8 and incubated at 37 °C for 1 h. The absorbance at 450 nm was measured utilizing a microplate reader. Cell viability was expressed as a share relative to the management (untreated) cells.
Animals care, remedy, and research design
Animals’ housing and remedy
Male C57BL/6J mice, aged 21 days (3 weeks), have been acquired from Life River Experimental Animal Know-how Firm (Beijing, China). The mice have been housed in a selected pathogen-free animal laboratory, maintained below a 12-hour mild/darkish cycle with regulated air flow (air change price of 18 occasions per hour). They offered advert libitum entry to meals and water. The Institutional Animal Care and Use Committee (IACUC Quantity: 3030) of Tongji Medical Faculty of Huazhong College of Science and Know-how authorized the experimental protocol. It was performed following the rules for experimental animal care and use established by the Nationwide Well being Analysis Institute.
Experiment design
Following adaptive feeding for one week, mice have been randomly divided into three teams (weighing: 15.525 ± 0.709 g): a management group, an HME group and a BME group (N = 12/group). The HME and BME teams acquired injections of HME and BME into each their quadriceps. To make sure constant dosing, we injected 80 µL of extracellular vesicles suspension (equal to five × 109 vesicles/40 µL per quadriceps injection web site) into the bilateral quadriceps of the mice each three days for a complete of 30 consecutive days [35]. The management group acquired PBS injections following the identical strategies. After the experiment, we euthanized the mice by cervical dislocation and dissected them instantly. We collected the quadriceps and saved them at -80 °C for subsequent index willpower.
H&E staining
The quadriceps have been fastened in 4% paraformaldehyde for not less than 24 h, embedded in paraffin, and sliced into 4-µm sections utilizing a Leica microtome (Solms, Germany). H&E staining was utilized for morphological evaluation. The Olympus IX-71 microscope (Tokyo, Japan) noticed the quadriceps sections.
Muscle fibers cross-sectional space (CSA) measurements
In accordance with our revealed research [37], six completely different areas of the quadriceps muscle have been imaged at 200× magnification utilizing H&E-stained sections. For every picture, 50 consecutive muscle fibers have been manually traced to acquire a mean of 300 fibers per muscle. Muscle fibers CSA values have been calculated utilizing Picture J software program (Model 1.52i, Nationwide Institutes of Well being, Bethesda, MD, USA) by figuring out the realm and variety of traced fibers.
Collagen evaluation
Paraffin-embedded tissue sections have been deparaffinized and hydrated on glass slides. Subsequently, the slides have been immersed in an answer of Sirius Pink dye for 1 h, adopted by a 30-second rinsing with working water and dehydration utilizing ethanol. After clearing the slides with a xylene-based clearing agent, they have been mounted and pictures have been captured. The Picture-Professional Plus software program was utilized to calculate the realm fraction occupied by collagen fibers. Moreover, a polarizing microscope (Eclipse Ci-L, Nikon, Japan) was employed to look at the slides and visualize collagen birefringence. Collagen I used to be recognized as purple to orange fibers, whereas collagen III was recognized as inexperienced fibers.
Grip check
Grip energy was measured utilizing a customized equipment designed and manufactured by Yiyang Know-how Growth Co., Ltd. (XR-YLS-13 A, Jinan, China). Mice have been gently held by the tail and allowed to know the steel wire mesh of the grip energy meter. In accordance with a relative research, the mice have been then gently pulled again till they launched the mesh. In the course of the first week, second and a half week, and fourth week of BME and HME intervention intervals, grip energy was measured in every mouse six occasions [38].
Coordination-fatigue check: rotarod check
In accordance with revealed analysis [39], we employed a rotary fatigue instrument (XR-6 C, Xinruan info and know-how Ltd, Shanghai, China) to coach and check small animals. The “General rod efficiency” (ORP) was used to find out the realm below the rotor’s time and rotation velocity curve, thereby scoring and summarizing the general efficiency of the check topics. In the course of the preliminary week of the experiment, the small animals have been skilled and familiarized with rotary abilities and devices. Subsequently, ORP was measured on the second and fourth week of the BME and HME intervention interval, with every mouse receiving three repeated measurements.
Biochemical index measurement for serum samples
Mouse serum samples have been positioned at 4 °C and centrifuged at 3000 rpm for 15 min. Then, to evaluate the impression of HME and BME on the physiological capabilities of animals, we employed a totally automated biochemical instrument (Shenzhen Learn Life Know-how, Shenzhen, China) to measure ranges of creatine kinase, uric acid, urea nitrogen, whole protein, calcium, alanine aminotransferase, and aspartate aminotransferase within the serum.
Western blotting
To extract whole protein from C2C12 cells and mouse quadriceps, we loaded 40 µg of protein samples onto a ten% SDS-PAGE gel and transferred the protein to a nitrate cellulose membrane. Our experimental strategies are referred to revealed research [40]. We used major antibodies towards AMPKα (#5831, 1:1000, Cell Signaling Know-how, MA, USA), p-AMPKα (thr172) (#2535, 1:1000, Cell Signaling Know-how, MA, USA), SIRT1 (#9475, 1:1000, Cell Signaling Know-how, MA, USA), PGC-lα (ab54481, 1:1000, Abcam, UK), AKT (#4691, 1:1000, Cell Signaling Know-how, MA, USA), p-AKT (#4060, 1:1000, Cell Signaling Know-how, MA, USA), mTOR (#2983, 1:1000, Cell Signaling Know-how, MA, USA), p-mTOR (#5536, 1:1000, Cell Signaling Know-how, MA, USA), p70S6 Kinase (#34,475, 1:1000, Cell Signaling Know-how, MA, USA), Phospho-p70S6 Kinase (#9205, 1:1000, Cell Signaling Know-how, MA, USA), 4EBP1 (#9644, 1:1000, Cell Signaling Know-how, MA, USA), p-4EBP1 (#2855, 1:1000, Cell Signaling Know-how, MA, USA), Myog (ab1835, 1:250, Abcam, UK, noticed band measurement on this research: 55KD), Myf5 (ab125301, 1:1000, Abcam, UK), Myod1 (A16218, 1:1000, ABclonal, Wuhan, China), and GAPDH (#5174, 1:10000, Cell Signaling Know-how, MA, USA). We used secondary HRP-linked antibodies (#7076, 1:10000, Cell Signaling Know-how, MA, USA) and Lumigen ECL Extremely (Lumigen, MI, USA) detection reagents to visualise the proteins.
Focused metabolomics evaluation for HME, BME and quadriceps
On this part, earlier publications referred to our pattern preparation, extraction, and LC-ESI-MS/MS system situations [41, 42].
HME and BME samples preparation and extraction
To arrange the pattern for focused metabolomics evaluation of amino acid and its’ metabolites, the frozen pattern was thawed on ice and 0.4 mL of it was freeze-dried. The freeze-dried pattern was then combined with 500 µL of 80% methanol/water pre-cooled at -20 °C. The combination was vortexed for two min at 2500 r/min. The pattern was then frozen in liquid nitrogen for five min and left on ice for five min earlier than being vortexed once more for two min. This step was repeated 3 times. The pattern was then centrifuged at 12,000 r/min for 10 min at 4 °C. After centrifugation, 300 µL of the supernatant was taken and sonicated at -20 °C for 30 min. The supernatant was then centrifuged at 12,000 r/min for 10 min at 4 °C. Lastly, 200 µL of the supernatant was transferred via a Protein Precipitation Plate for additional LC-MS evaluation. This methodology ensures the environment friendly preparation of the pattern for LC-MS evaluation whereas minimizing the chance of contamination or lack of analytes.
Quadriceps samples preparation and extraction
After thawing and homogenizing the muscle samples, 0.05 g was combined with 500 µL of 70% methanol/water and vortexed for 3 min at 2500 r/min. The combination was then centrifuged at 12,000 r/min for 10 min at 4 °C. The supernatant (300 µL) was transferred to a brand new centrifuge tube and saved at -20 °C for 30 min earlier than being centrifuged at 12,000 r/min for 10 min at 4 °C. Lastly, 200 µL of supernatant was transferred via a protein precipitation plate for LC-MS evaluation.
UPLC Circumstances
An LC-ESI-MS/MS system (UPLC, ExionLC AD, https://sciex.com.cn/; MS, QTRAP® 6500 + System, https://sciex.com/) was used to investigate the pattern extracts. The HPLC column used was ACQUITY BEH Amide (i.d.2.1 × 100 mm, 1.7 μm) and the solvent system consisted of water with 2 mM ammonium acetate and 0.04% formic acid (A) and acetonitrile with 2 mM ammonium acetate and 0.04% formic acid (B). The gradient was initiated at 90% B (0-1.2 min), decreased to 60% B (9 min), 40% B (10–11 min) and at last ramped again to 90% B (11.01-15 min). The stream price was set at 0.4 mL/min and the temperature was maintained at 40 °C. The injection quantity used was 2 µL.
ESI-MS/MS situations
The AB 6500 + QTRAP® LC-MS/MS System has an ESI Turbo Ion-Spray interface and may function in constructive or damaging ion modes. It’s managed by Analyst 1.6 software program (AB Sciex). The ion supply is a turbo spray with a supply temperature of 550 °C. The ion spray voltage is about to 5500 V for constructive mode and − 4500 V for damaging mode. Curtain gasoline is about to 35.0 psi. We optimized DP and CE for every MRM transition. We monitored a selected set of MRM transitions for every interval primarily based on the amino acid eluted inside that interval.
Statistical evaluation
The preprocessed amino acids and their metabolites dataset was imported into Metabo-Analyst for principal part evaluation (PCA), orthogonal partial least squares discriminant evaluation (OPLS-DA), and volcano plot evaluation. Metabolite species that differed considerably between teams have been screened utilizing the factors of variable significance in projection (VIP) > 1, p < 0.05, and fold change (FC) > 2 (up-regulated) or FC < 0.5 (down-regulated). Pathway enrichment evaluation and metabolite annotation have been carried out utilizing the Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathways database. Statistical evaluation and knowledge processing have been performed utilizing Prism software program (GraphPad Prism 8.0). All outcomes are offered as imply ± commonplace error (x ± SE), and all knowledge have been generated from not less than three impartial experiments. A number of teams have been in contrast utilizing one-way evaluation of variance (ANOVA), and significance was outlined as p < 0.05. Extra particulars for “Focused metabolomics evaluation” confirmed in “Supplies and strategies S1”.
