Objective

2

To investigate the potential mechanism by which electroacupuncture (EA) induces macrophage polarization to promote muscle satellite cell proliferation and differentiation

accelerating the repair of acute skeletal muscle injury.

Methods

2

Forty-two SPF-grade SD rats were randomly divided into three groups: a blank group (

n

=6)

a model group (

n

=18)

and an EA group (

n

=18). The model and EA groups established acute blunt contusion model of the right gastrocnemius muscle using a self-made striking device. From day 1 after modeling

rats in the EA group received EA at "Chengshan" (BL57) and "Yanglingquan" (GB34) on the right side

using disperse-dense wave with a frequency of 2 Hz/100 Hz and a current of approximately 2 mA. The EA treatment was administered once daily for 30 minutes for 3

7

or 14 days based on the designated sampling time points. Gait analysis was performed using the Cat Walk XT

TM

system. Hematoxylin-eosin (HE) staining was used to observe the morphological changes in the gastrocnemius muscle. Masson staining was applied to evaluate collagen fiber content. Immunofluorescence was used to detect the expression of proliferating cell nuclear antigen (PCNA) in muscle satellite cells. Immunohistochemistry was used to assess the expression levels of CD68 and CD206

markers of macrophages. Serum levels of pro-inflammatory cytokines (TNF-α

IL-1β) and anti-inflammatory cytokines (IL-10

IL-13) were detected using ELISA.

Results

2

Compared with the blank group

the model group showed a significant reduction in average movement speed on days 3 and 7 after modeling (

P

<

0.05)

and a decrease in the right hind limb stride length on day 3 (

P

<

0.05). Compared with the model group

the EA group showed increased average movement speed and right hind limb stride length on day 7 (

P

<

0.05). In the blank group

the gastrocnemius muscle on the right side showed uniform and

consistent inter-fiber spacing

with neatly and regularly arranged muscle cells. In contrast

the model group exhibited enlarged inter-fiber spacing

edema

and significant infiltration of red blood cells and inflammatory cells

with progressively increasing fibrosis over time. By day 14 after modeling

the EA group showed a return to baseline levels of inflammatory cell infiltration

and the degree of fibrosis was significantly lower than that observed in the model group. Compared with the blank group

the ratio of collagen fibers in the gastrocnemius muscle of the model group increased significantly on days 3

7

and 14 after modeling (

P

<

0.05). Compared with the model group

the EA group exhibited a lower collagen fiber ratio on days 3

7

and 14 (

P

<

0.05). Compared with the blank group

PCNA positive expression in the gastrocnemius muscle of the model group was significantly increased on days 3

7

and 14 after modeling (

P

<

0.05). Compared with the model group

the EA group exhibited significantly higher PCNA positive expression on days 3 and 7 (

P

<

0.05). Compared with the blank group

the model group showed a significant increase in CD68-positive macrophage expression in the gastrocnemius muscle on day 3 after modeling (

P

<

0.05)

while CD206-positive macrophage expression increased on days 3

7

and 14 (

P

<

0.05). Compared with the model group

CD68 expression was significantly lower in the EA group on day 3 (

P

<

0.05)

whereas CD206 expression was significantly higher on days 3 and 7 (

P

<

0.05)

peaking on day 7 with CD206 expression. Compared with the blank group

serum TNF-α levels were significantly elevated in the model group on days 3 and 7 after modeling (

P

<

0.05)

while serum IL-1β levels were increased on days 3

7

and 14 (

P

<

0.05). Serum IL-10 and IL-13 levels were significantly higher on day 7 after modeling (

P

<

0.05). Compared with the model group

the EA group exhibited lower serum TNF-α level on day 3 (

P

<

0.05) and reduced serum IL-1β levels on days 3 and 7 (

P

<

0.05)

while serum IL-10 and IL-13 levels were significantly increased on day 7 (

P

<

0.05).

Conclusion

2

EA could promote the repair of acute blunt contusion-induced gastrocnemius muscle injury by regulating the proliferation and differentiation of muscle satellite cells. This process is closely related to macrophage polarization.