Animal

SPF C57BL/6J male mice (29 ± 0.3 g, 8 weeks) provided by the Ningxia Medical University Experimental Animal Centre were housed in controlled conditions with temperature 20 ± 2°C, relative humidity 50-60%, and a 12-hour light-dark cycle. Staff maintained aseptic procedures, regularly replenishing bedding, feed, and water, and sanitized the environment. Animal experiments were approved by the Laboratory Animal Ethics and Welfare Committee of Ningxia Medical University (IACUC-NYLAC-2023-033).

WMPP

WMPP is processed using nanoscale biomembrane filtration, followed by a 30-fold concentration, 72 °C centrifugal sterilization, 25 °C low-temperature separation, and 57 °C preheated drying, which altogether result in minimal protein denaturation and retain a high level of bioactive nutrients. Characterized by an optimal “protein golden ratio,” WMPP were obtained from Ningxia Saishang Dairy Industry Co. (Ningxia, China). Nutrition Facts of WMPP is shown in Table 1.

Table 1 Nutrition facts of WMPPExperimental design

After a week of adaptive feeding, they were completely randomized into control group (7 mice) and SIM group (36 mice). To ensure the balance of energy intake, the food supply of the control mice was based on the average daily intake of the SIM mice. The SIM mice group were subjected to subcutaneous injections of 15 mg/kg lipopolysaccharide (LPS) (L2880, Sigma-Aldrich, St. Louis, US). Subsequently, the 36 SIM mice were further randomly divided into three subgroups: SIM group, 1.5 g/kg WMPP group, and 3 g/kg WMPP group, each subgroup comprising 12 SIM mice. The selection of dose for WMPP was based on a comprehensive review of the literature and nutritional guidelines [11]. The control group were subjected to subcutaneous injections of 100 µl of physiological saline. Beginning 12 h after the LPS injection, the SIM group, 1.5 g/kg WMPP group, and 3 g/kg WMPP group received intraperitoneal antibiotic injections (imipenem, IPM; 25 mg/kg) and intravenous fluid resuscitation (intravenous injection of 100 µl 0.9% sterile physiological saline per mouse) twice daily, along with administration of a 5% dextrose in saline solution at a rate of 5 ml/kg·h, which was continued for 3 days. In the 1.5 g/kg WMPP group and the 3 g/kg WMPP group, six SIM mice in each group survived at the end of the trial. Control null mice died due to mutual biting during natural feeding. Thus, the statistics for this experiment are only from six surviving SIM mice per group. The experimental design is illustrated in Fig. 1. WMPP solutions were prepared by dissolving 3 g in 10 ml distilled water and diluted to 1.5 g/10 ml. Mice were weighed daily and quantitative injections were performed based on the body weight. The oral gavage procedure was performed utilizing a 12-gauge gavage needle in conjunction with a 1 ml syringe, ensuring that the volume administered to each mouse did not exceed 0.5 ml per. WMPP interventions were consistently administered at the same time daily, while control and SIM groups received equivalent volumes of distilled water via oral gavage throughout the rearing period. Regular measurements were taken to assess food intake, water consumption, and body weight changes.

Fig. 1

The animal treatment protocol is as follows: Mice were administered a single injection of LPS to simulate SIM, followed by gavage administration of WMPP for the prevention and alleviation of SIM. Beginning on the first day of LPS injection, daily monitoring of body weight, food intake, and water consumption was conducted until sample collection

Muscle function analysisRotarod test

The test instrument utilized was the KW-6 C Mouse Rotameter manufactured by Nanjing Calvin Biotechnology Co. Initially, the rotation speed of the instrument was set within the range of 4 to 40 r/min. Subsequently, climbing distance and latency to fall time on the Rotarod were meticulously recorded across three separate experimental trials. In the end, calculating the mean of the three times was used to statistically analyze.

Pole test

The pole device (Nanjing Calvin Biotechnology Co., Ltd.) consists of a 45 cm long, 10 mm diameter wooden pole with a 20 mm diameter ball at the top, covered in medical tape for traction. Its base is attached to a metal and positioned in a 100 cm x 50 cm white plastic box filled with bedding. Mice were timed as they moved from the top ball to the bottom of the pole in an experiment repeated three times, with the average time statistically analyzed.

Tissue collection

Mice were fasted for 8–12 h before sampling. They were then anesthetized and euthanized, and blood was collected via ocular puncture. After allowing the blood to stand at room temperature for 2 h, it was centrifuged at 3000 rpm for 10 min to separate the supernatant, which was then aliquoted and stored at -80℃. Skeletal muscles from different anatomical sites, including the right biceps brachii (BB), quadriceps (QU), gastrocnemius (GAS), tibialis anterior (TA), soleus (SOL), and extensor digitorum longus (EDL), were isolated, weighed, and frozen for immunoblotting experiments. The left GAS, TA, and SOL of the mice were also embedded for immunofluorescence staining.

Enzyme-linked immunosorbent assay

Serum samples were retrieved from the − 80℃ freezer following the protocol provided by the kit manufacturer. The levels of various biomarkers including interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), interleukin-4 (IL-4), interleukin-13 (IL-13), creatine kinase (CK), Myoglobin (MYO/MB), adenosine monophosphate (AMP), and adenosine triphosphate (ATP) were then quantified.

Biochemical indicators testing

Urea nitrogen (BUN), creatinine(Cr), albumin (ALB), total protein (TP), lactate dehydrogenase (LDH), malondialdehyde (MDA), catalase (CAT), glutathione peroxidase (GSH-PX), ROS (Nanjing Jiancheng Institute of Bioengineering, Nanjing, China), superoxide dismutase (SOD) (Beyotime Biotechnology Co., Ltd., Jiangsu, China) were assessed following the manufacturer’s protocols. Additionally, the total protein content of skeletal muscle was determined using commercial kits based on the method of Caulmers Brilliant Blue, as per the manufacturer’s instructions.

Histopathology

GAS and TA were fixed in 4% paraformaldehyde for 24 h, followed by gradual dehydration and embedding in paraffin wax. Subsequently, the paraffin blocks were sliced using a microtome to obtain sections of 5 μm thickness. These sections were then subjected to staining with hematoxylin and eosin (H&E), Oil Red O, and Masson. The CSA of muscle fibers, the proportion of Oil Red O-stained area to the total area, and the proportion of collagen fiber area to the total area were quantified using ImageJ software.

Immunohistochemistry

Frozen sections were retrieved from the − 20℃ refrigerator and allowed to equilibrate to room temperature. The sections were then washed with PBS solution for 5 min to minimize background interference. Subsequently, the sections were fixed in 4% paraformaldehyde for 30 min and washed with PBS five times for 3 min each. Following fixation, the sections were blocked with 5% BSA for 30 min. Once blocking was completed, the sections were incubated overnight at 4 °C with Succinate Dehydrogenase Complex Subunit B (SDHB) (ab14714, 1:200, Abcam), NADH Ubiquinone Oxidoreductase Subunit B8 (NDUFB8) (ab192878, 1:200, Abcam), and Ubiquinol Cytochrome C Reductase Core Protein II (UQCRC2) (ab203832, 1:200, Abcam). After incubation, the sections were washed five times with PBS for 3 min each. Subsequently, Goat Anti-Rabbit IgG (H + L) HRP (#S0001, 1:500, Affinity) was incubated at room temperature for one hour. Following incubation, the sections were washed. Subsequently, Samples were washed for 2 min, subsequently counterstained with hematoxylin for 1 min and rinsed again for 20s. Thereafter, the samples were dehydrated, transparent, and sealed for analysis.

Immunofluorescence

Frozen sections were equilibrated to room temperature and washed with PBS to minimize background interference. They were then fixed in 4% paraformaldehyde, washed with PBS, and blocked with 5% BSA. Sections were incubated overnight with Myosin Heavy Chain (MYH) antibody (sc-376157, 1:200, Santa Cruz) at 4℃, followed by washing and incubation with Goat Anti-Mouse IgG Fluor594 (#S0005, 1:500, Affinity) at 37℃. After washing to remove the secondary antibody, sections were stained with DAPI and washed again. Finally, slides were mounted for imaging under a fluorescence microscope (Echo Revolve, USA), and fluorescence intensity was measured using ImageJ software.

Western blot analysis

To evaluate protein expression, frozen muscle samples were ground, homogenized, and centrifuged to obtain the supernatant. Protein concentration was measured with a BCA assay kit. Proteins were then separated by SDS-PAGE and transferred to a PVDF membrane. After blocking, the membrane was incubated with MYH (sc-376157, 1:1000, Santa Cruz), muscle specific ring finger (MURF-1) (sc-398608, 1:1000, Santa Cruz), muscle atrophy F-box (MAFbx) also known as Atrogin-1 (sc-166806, 1:1000, Santa Cruz), phospho-mTOR (Ser2448) (sc-293133, 1:1000, Santa Cruz), mTOR (sc-517464, 1:1000, Santa Cruz), phospho-p70S6K (Thr389/Thr412) (AF3228, 1:1000, Affinity), p70S6K1 (ab32359, 1:1000, Abcam), phospho-4EBP-1 (Thr37/Thr46) (AF3830, 1:1000, Affinity), and 4EBP-1 (60246-1-Ig, 1:1000, Proteintech) overnight at 4℃. Anti-rabbit IgG, HRP-linked Antibody (#7074, 1:1000, Cell Signaling Technology) and Anti-mouse IgG, HRP-linked Antibody (#7076, 1:1000, Cell Signaling Technology)were applied the next day, and signals were detected using ECL and quantified with ImageJ software.

RNA extraction and quantitative RT-PCR

Frozen muscle samples were homogenized using Trizol reagent, followed by phase separation through chloroform addition, RNA precipitation, washing steps, and solubilization. RNA concentration was determined using an ultra-microspectrophotometer. Subsequently, a reverse transcription reaction system was prepared for cDNA synthesis, followed by PCR amplification. The expression of target genes was normalized to GAPDH expression as an internal reference, and relative quantification was performed using the (2-ΔΔCt) method. The sequences of the forward and reverse primers are listed below: TNF-a: 5’-CAGGCGGTGCCTATGTCTC-3’, 5’-CGATCACCCCGAAGTTCAGTAG-3’; IL-1β: 5’-GAAATGCCACCTTTTGACAGTG-3’, 5’-TGGATGCTCTCATCAGGACAG-3’; MURF-1: 5’-CGACATCTTCCAGGCTGCGAAT-3’, 5’‐ATCACTTCATGGCGGCACGAG-3’; MAFbx: 5’-CCATTCTACACTGGCAGCAGCA-3’, 5-ACAGGCAGGTCGGTGATCGT-3’.

Statistical analysis

The statistical analysis of all data was conducted using SPSS 24.0 software. Results were expressed as mean ± standard deviation (SD). One-way ANOVA was used to compare multiple groups of means and two-way repeated measures ANOVA was used to compare repeated observation data at different time points. Pairwise comparisons were analyzed using the LSD test. The significance level was set at α = 0.05. Band grayscale values were analyzed using Image J software, and GraphPad Prism 9.0 was employed for data visualization.