A total of 40 specific pathogen-free (SPF) male Sprague-Dawley (SD) rats, aged 8 weeks and weighing 220 ± 20 g, were obtained from Changsha Tianqin Biotechnology Co., Ltd. (Changsha, China; license No.: SCXK (Xiang) 2019-0014). The rats were randomly and equally divided into 10 cages for rearing, with free access to food and water, a 12-hour light/dark cycle, an ambient temperature of 24˚C, and a humidity of 40% - 50%. They were acclimatized for 7 days prior to the experiment. All animal experiments were conducted in accordance with the guidelines of the Chinese Institute of Animal Research and were approved by the Ethics Committee of Youjiang Medical University for Nationalities.

Bupivacaine hydrochloride (Beijing Solarbio Science & Technology Co., Ltd., YZ-101034); Sodium pentobarbital (Shanghai Fude Chemical Co., Ltd.); Rat HGF ELISA Kit, 96T (Wuhan Huamei Biotech Co., Ltd.); Rat MyoD ELISA Kit, 96T (Shanghai Enzyme-linked Biotechnology Co., Ltd.).

Standard microplate reader (Thermo Fisher Scientific, Multiskan MK3); Electrothermal constant-temperature incubator (Huangshi Hengfeng Medical Instrument Co., Ltd., SKP-02.600); Centrifuge (Sigma, USA, 3K18). Xinsheng brand acusector apparatus G6805-I.

The 40 rats were randomly divided into five groups using a computer-generated random number sequence: normal group, model group, control group, electroacupuncture group, and BNS group. Group allocation was concealed by an independent coordinator using a coded labeling system. Rats in the model group, acusector group, and BNS group were anesthetized with 1% sodium pentobarbital (50 mg/kg) [6], and the model was established according to the following method [7]: Six points on both sides of the spine at the L3, L4, and L5 levels were selected. A disposable 4-gauge needle syringe was used to draw 0.5% bupivacaine solution. The needle was inserted into the muscle close to the spinous processes until it contacted the bony surface of the articular processes and mammillary processes. The cannula was then retracted 1 mm to confirm no blood return, indicating that the needle had reached the multifidus muscle. Subsequently, 600 μL of bupivacaine solution (100 μL × 6) was injected, with each intramuscular injection lasting no less than 3 seconds to facilitate drug absorption. A single injection completed the model establishment. The control group received an equal volume of normal saline using the same method. All procedures were performed under sterile conditions, and the model was successfully established.

Interventions were initiated 24 hours after modeling in each group. The rats were fixed on an operating table with their backs and hind limbs exposed, referencing the rat anatomical atlas.

Normal group: All rats were handled, fixed, and locally disinfected simultaneously with the other groups, but received no other treatment. Samples were collected on days 4 and 8 after modeling.

Model group: No special treatment was given after modeling. Four rats were randomly selected for sampling on day 4 after modeling, and the remaining four rats were handled, fixed, and locally disinfected simultaneously with the other groups, but received no other treatment, with final sampling on day 8 after modeling.

Control group: No special treatment was given after modeling. Four rats were randomly selected for sampling on day 4 after modeling, and the remaining four rats were handled, fixed, and locally disinfected simultaneously with the other groups, but received no other treatment, with final sampling on day 8 after modeling.

Acusector group: Intervention began on day 1 after modeling. The rats were first fixed on a specially designed holder with their backs exposed, and the L3, L4, and L5 spinal levels were locally disinfected with 75% alcohol. Huatuo brand disposable sterile acupuncture needles (0.35 × 50 mm) were then inserted into the muscle close to the spinous processes at the L3, L4, and L5 levels until the bony surface of the articular and mammillary processes was reached. After needling, the needles were connected to a Xinsheng brand acusector apparatus G6805-I, with sparse-dense waves at 10 - 26 cycles/min, a current intensity of 1 mA, for 30 minutes, once daily, for 7 consecutive days. Four rats were randomly selected for sampling on day 4 after modeling, and the remaining four rats continued the intervention, with final sampling on day 8 after modeling.

BNS group: Intervention began on day 1 after modeling. The rats were first fixed on a specially designed holder with their backs exposed, and the L3, L4, and L5 spinal levels were locally disinfected with 75% alcohol. Lejiu brand disposable blade needles (0.35 × 50 mm) were then inserted into the muscle close to the spinous processes at the L3, L4, and L5 levels until the bony surface of the articular and mammillary processes was reached, followed by rapid lifting and thrusting twice with an amplitude of 0.5 - 1.0 cm. Four rats were randomly selected for sampling on day 4 after modeling, and the remaining four rats received one additional intervention on day 4 after modeling, with final sampling on day 8 after modeling.

Rats were anesthetized intraperitoneally with 1% sodium pentobarbital (50 mg/kg). After the rats were deeply anesthetized, they were fixed on a board with their backs fully exposed. The dorsal fur was shaved, and the skin on the back was incised. The lumbar fascia, longissimus muscle, and iliocostalis muscle were dissected using tissue scissors and forceps. The lumbar multifidus muscles on both sides of the L3 - L5 spinous processes were harvested, and stored at −80˚C for later use. Tissue specimens (approximately 50 mg) were dissected from both the left and right sides of the target tissue. Each specimen was homogenized in ice-cold RIPA lysis buffer (containing protease inhibitor cocktail) at a ratio of 1:9 (weight/volume) using a tissue homogenizer. The resulting homogenates were incubated on ice for 30 minutes with intermittent vortexing/mixing, followed by centrifugation at 10,000 × g for 5 minutes at 4˚C. The clear supernatants were carefully collected for ELISA. To minimize intra-animal variability, the mean value obtained from the bilateral samples was calculated and treated as a single independent data point for each animal in the subsequent statistical analyses. The investigators performing the experiments were blinded to the sample group assignments throughout the assay procedures.

A double-antibody sandwich ELISA method was used to detect the levels of MyoD and HGF. The specific procedures were performed in accordance with the instructions provided with the kits.

SPSS 26.0 statistical software was used for data analysis. For data that followed a normal distribution and had homogeneity of variance, data from each group were expressed as mean ± standard deviation ( x ¯ ± s ). Differences among groups were analyzed using one-way analysis of variance (ANOVA), with post hoc comparisons performed using Tukey’s test. Intra-group comparisons were analyzed using independent samples t-test. Data that did not follow a normal distribution or had unequal variances were analyzed using non-parametric tests. A P-value < 0.05 was considered statistically significant.

As shown in Figure 1(a), on day 4 after modeling, there was no statistically significant difference in MyoD expression levels among the groups (F = 2.938, P = 0.056). On day 8 after modeling, there was a statistically significant difference in MyoD expression levels among the groups (F = 15.758, P < 0.0001). Further post hoc analysis using Tukey’s test revealed that, compared with the normal group, the control group showed no significant change in MyoD expression level (P > 0.05), while the model group showed a significant decrease in MyoD expression level (P < 0.0001). Compared with the model group, both the acusector group and the BNS group exhibited significantly elevated MyoD expression levels, with statistically significant differences (P < 0.0001). There was no statistically significant difference between the BNS group and the acusector group (P > 0.05).

As shown in Figure 1(b), when comparing different time points within the same group, the normal group, control group, acusector group, and BNS group all showed no significant changes in MyoD expression levels (P > 0.05). In the model group, the MyoD expression level on day 8 was significantly lower than that on day 4, with a statistically significant difference (P < 0.0001). These results indicate that following multifidus muscle injury, MyoD expression levels tend to decline. Both acusector therapy and BNS therapy can significantly inhibit the downregulation of MyoD expression after multifidus muscle injury. The specific MyoD expression levels for each group are presented in Table 1.

Figure 1. MyoD expression levels in rats of each group. (a) Comparison of MyoD expression levels among different groups at the same time point; (b) Comparison of MyoD expression levels within the same group at different time points. ns indicates no statistically significant difference. **** indicates P < 0.0001.

Table 1. MyoD expression levels of each group at different time points ( x ¯ ± s , ng/ml).

As shown in Figure 2(a), on day 4 after modeling, there was no statistically significant difference in HGF expression levels among the groups (F = 2.916, P = 0.057). On day 8 after modeling, there was a statistically significant difference in HGF expression levels among the groups (F = 18.044, P < 0.0001). Further post hoc analysis using Tukey’s test revealed that, compared with the normal group, the control group showed no significant difference in HGF expression level (P > 0.05), while the model group showed a significant decrease in HGF expression level (P < 0.0001). Compared with the model group, the acusector group showed no statistically significant difference (P > 0.05); however, the HGF level in the BNS group was significantly higher than that in both the model group and the acusector group, with statistically significant differences (P < 0.05, P < 0.01).

As shown in Figure 2(b), when comparing different time points within the same group, the normal group, control group, and BNS group all showed no significant changes in HGF expression levels (P > 0.05). In both the model group and the acusector group, the HGF expression levels on day 8 after modeling were significantly lower than those on day 4, with statistically significant differences (P < 0.01). These results indicate that following multifidus muscle injury, HGF expression levels tend to decline. BNS therapy can significantly inhibit the downregulation of HGF expression, whereas acusector therapy did not demonstrate an inhibitory effect on the decline of HGF levels. The specific HGF expression levels for each group are presented in Table 2.

Figure 2. HGF expression levels in rats of each group. (a) Comparison of HGF expression levels among different groups at the same time point; (b) Comparison of HGF expression levels within the same group at different time points. ns indicates no statistically significant difference; * indicates P < 0.05; ** indicates P < 0.01; **** indicates P < 0.0001.

Table 2. HGF expression levels in rats of each group at different time points ( x ¯ ± s , ng/ml).