Animals

Animal experiments were approved by the Animal Care and Use Committee of the Beijing University of Chinese Medicine (No. BUCM-2023032303-1119). All animal experiments were designed according to the principles of the 3Rs (Replacement, Reduction and Refinement) and were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Male Sprague–Dawley (SD) rats (6-week-old, 190–210 g) were purchased from Beijing SPF Biotechnology Co., Ltd. (Beijing, China), and housed in a pathogen-free environment with four animals per cage. The rats are fed in a 12-h light–dark cycle environment, temperature (25 ± 0.5) °C, humidity 40–50%, and had free access to diet and drinking water.

SNI model

Thirty-six rats were randomly divided into four groups (n = 9 each): blank control (CON), sham model (SHA), sciatic nerve injury model (SNI), and Tuina (TUI) group. Pathological modeling was initiated one week after acclimatization. As SNI rat model was established as previously described [4]. After anesthetized, approximately 1 cm-long incision was made in the skin along the direction of the sciatic nerve, exposing the lower edge of the piriformis. For rats in the SNI and TUI groups, special hemostatic pliers were used to clamp 5 mm from the distal end of the sciatic nerve nodule for 5 s with full force (6 N), resulting in an injury point of approximately 2 mm (Fig. 1B). For the rats in the SHA group, only the sciatic nerve was only found, sterilized, sutured, and sterilized. For the rats in CON group, only routine feeding.

Fig. 1

Tuina improved SNI rat behavioral performance and repair of nerve injury. A Animal experimental flow; B The process of modeling; a skin preparation; b anesthetization; c the position of incision; d exposure of the sciatic nerve trunk; e clamping; f suture. C Tuina intervention; a the schematic of the acupoint location, b BL 37 stimulus; c BL 57 stimulus; d GB 34 stimulus. D The angle of the inclined plate changes; E SFI; F TFI; G PFI. Results are presented as mean ± standard deviation. **P < 0.001 vs. SHA; ##P < 0.001 vs. SNI

Tuina intervention

The TUI group received “Three-Manipulation and Three-Acupoint” treatment, the procedure was as follows: The Tuina Manipulation Simulator (Self-developed machine, China invention patent number ZL202320511277.5) was set to stimulate with a force of 4 N, 60 times per minute. The stimulus rod was placed on Yinmen (BL 37), Chengshan (BL 57) and Yanglingquan (GB 34) of the surgical side, then finger pressing, plucking, and kneading manipulation were stimulated, respectively (Fig. 1C). Each method was used for 1 min at each acupoint, for a total of 9 min. Ten treatments were followed by one day rest and ten treatments were repeated; overall, twenty treatments were applied.

Grip restraint was performed in the SHA and SNI groups. The rats in the CON group were removed from the cage and returned. To reduce the animal stress response, the animals were petted and stroked for 9 min before the formal intervention every day.

Behavioral assessment

An electrically inclined plate tester was used to detect changes in hind limb muscle strength. Rats in each group were subjected to behavioral testing, and the test was performed on the day of 0th intervention (Day 15), 10th (Day 25) and 20th (Day 36) treatment. The heads of the rats were placed on the board toward the end, and the angle of the board was gradually increased after the rats calmed down. When the rats could not stay in this position for 5 s, the critical angle of the protractor was recorded, and the average of three measurements was taken.

Nerve function index

Nerve function index was collected using the DigiGait™ Imaging System and then analyzed by the DigiGait™ 15.0 analysis software (Mouse Specifics, Inc.; Quincy, MA, USA). The test was performed before modeling (Day 7), on the day of 10th (Day 25) and 20th (Day 36) treatment. The rats were acclimated to the apparatus 1 week before the experiment, and the treadmill belt was gradually accelerated to 10 cm/s.

Muscle atrophy index (MAI)

After the rats were sacrificed (Day 36), the anterior tibial and soleus muscles were removed and their wet weights were measured using an electronic balance. MAI was defined as the muscle weight divided by the body weight. Empty stomach weights of pre-dissected rats and their anterior tibial and soleus muscles (post-dissection) were measured.

Transmission electron microscopy

The anterior tibial and soleus muscles, and sciatic nerves were removed, fixed in pre-cooled 2.5% (w/v) glutaraldehyde for 3 h, and washed with 0.1 M PB. Subsequently, they were postfixed in a 1% (w/v) osmic acid solution for 1 h, washed in 0.1 M PB for 1 h, dehydrated (through a grade series of ethanol solutions), and embedded in Epon 812 epoxy resin. The segments were cut into 70 nm-thick ultrathin slices. The sections were then stained with saturated aqueous uranyl acetate (2%) and citrate and observed and analyzed by transmission electron microscopy.

Immunofluorescence

The anterior tibial muscle, soleus muscle, and sciatic nerve were fixed in 4% paraformaldehyde at 4 °C, dehydrated using a sucrose gradient, embedded in optimal cutting temperature compound, and cut into 4 μm sections. The paraffin sections were dewaxed, dehydrated, subjected to antigen retrieval, cleared of spontaneous fluorescence, and blocked with serum. Primary antibodies for CD 31 (1:500, abcam, USA) were incubated at 4 °C overnight. Sections were rinsed with phosphate-buffered saline (PBS) and incubated with secondary antibody Goat Anti-Rabbit IgG H&L (1:500, abcam, USA) at 37 °C for 30 min, and then rinsed with PBS. The sections were observed under a fluorescence microscope, and ImageJ software was used to analyze the Pearson coefficient of immunofluorescence co-localization and fluorescence intensity of each protein.

Enzyme-linked immunosorbent assay (ELISA)

Fifty milligrams of the gastrocnemius muscle and tibialis anterior muscle were weighed, and PBS was added at a weight (mg)/volume (μL) ratio of 1:10 for homogenization. The protein concentrations of the samples were determined using an ELISA kit. The test procedure was: standard dilution, sample addition, washing, color development, and reaction termination. The absorbance of each well was measured and finally calculated the concentration of forkhead box O (FoxO), p-FoxO, and insulin-like growth factor 1 (IGF-1) by drawing a standard curve.

Western blotting

The gastrocnemius, tibialis anterior muscles, and sciatic nerves were lysed in radio immunoprecipitation assay (RIPA) lysis buffer. A Bicinchoninic Acid (BCA) protein assay kit was used to measure the protein concentration. Briefly, 20 μg of total protein was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to poly (vinylidene fluoride) (PVDF) membranes at 400 mA for 30 min. Subsequently, the membranes were blocked with a blocking solution for 30 min at room temperature. The membranes were incubated overnight at 4 °C with the following primary antibodies: mouse anti-GAPDH (1:10,000, Ym3029, Immunoway), rabbit anti-p-PI3K (1:1000, AF3241, Affbiotech), rabbit anti-p-Akt (1:1000, bs-0867R, Bioss), and rabbit anti-VEGF (1:1000, bs-1665R, Bioss). After washing three times with tris-buffered saline with 0.1% Tween® 20 detergent (TBST), the secondary horseradish peroxidase (HRP)-conjugated antibodies were as follows: goat anti-rabbit IgG H&L (1:10,000, bs-0295G, Bioss) and goat anti-mouse IgG H&L (1:10,000, bs-0296G, Bioss). After washing three times with TBST, an enhanced chemiluminescence (ECL) kit was used to detect immunoactivity.

Statistical analysis

Data analysis was performed using SPSS Statistics software (version 26.0, IBM, Armonk, NY, USA). Data were presented as mean ± SD. Student’s t-test was used to compare the differences between the two groups. One-way ANOVA was used for comparisons between the groups, and the least significant difference (LSD) multiple comparison test was used for multiple comparisons. P < 0.05 was treated as statistically significant.