Objective:

2

To determine whether acupotomy ameliorates immobilization-induced muscle contracture and fibrosis via Wnt/β-catenin signaling pathway.

Methods:

2

Thirty Wistar rats were randomly divided into 5 groups (

n

=6) by a random number table

including control

immobilization

passive stretching

acupotomy

and acupotomy 3 weeks (3-w) groups. The rat model of gastrocnemius contracture was established by immobilizing the right hind limb in plantar flexion for 4 weeks. Rats in the passive stretching group received passive stretching at gastrocnemius

a daily series of 10 repetitions for 30 s each at 30-s intervals for 10 consecutive days. Rats in the acupotomy and acupotomy 3-w groups received acupotomy once and combined with passive stretching at gastrocnemius a daily series of 10 repetitions for 30 s each at 30-s intervals for 10 consecutive days. Additionally

rats in the acupotomy 3-w group were allowed to walk freely for 3 weeks after 10-day therapy. After treatment

range of motion (ROM)

gait analysis [i.e.

paw area

stance/swing and maximum ratio of paw area to paw area duration (Max dA/dT)

]

gastrocnemius wet weight and the ratio of muscle wet weight to body weight (MWW/BW) were tested. Gastrocnemius morphometric and muscle fiber cross-sectional area (CSA) were assessed by hematoxylin-eosin staining. Fibrosis-related mRNA expressions (i.e.

Wnt 1

β-catenin

axin-2

α-smooth muscle actin

fibronectin

and types Ⅰ and Ⅲ collagen) were measured using real-time quantitative polymerase c

hain reactions. Wnt 1

β-catenin and fibronectin concentrations were measured by enzyme-linked immunosorbent assay. Types Ⅰ and Ⅲ collagen in the perimysium and endomysium were analyzed using immunofluorescence.

Results:

2

Compared with the control group

ROM

gait function

muscle weight

MWW/BW and CSA were significantly decreased in the immobilization group (all

P

<

0.01)

while protein levels of types Ⅰ and Ⅲ collagen

Wnt 1

β-catenin

fibronectin and mRNA levels of fibrosis-related genes were obviously increased (all

P

<

0.01). Treatment with passive stretching or acupotomy restored ROM and gait function and increased muscle wet weight

MWW/BW and CSA (all

P

<

0.05)

while protein expression levels of Wnt 1

β-catenin

fibronectin

types Ⅰ and Ⅲ collagen and mRNA levels of fibrosis-related genes were remarkably declined compared with the immobilization group (all

P

<

0.05). Compared with passive stretching group

ROM

gait function

MWW were remarkably restored (all

P

<

0.05)

and mRNA levels of fibrosis-related genes as well as protein expression levels of Wnt 1

β-catenin

fibronectin

types Ⅰ and Ⅲ collagen in the acupotomy group were obviously decreased (all

P

<

0.05). Compared with the acupotomy group

ROM

paw area

Max dA/dT

and MWW were restored (all

P

<

0.05)

and mRNA levels of fibrosis-related genes along with protein levels of Wnt 1

β-catenin

fibronectin

types Ⅰ and Ⅲ collagen in the acupotomy 3-w group were decreased (

P

<

0.05).

Conclusion:

2

Improvements in motor function

muscle contractures

and muscle fibrosis induced by acupotomy correlates with the inhibition of Wnt/β-catenin signaling pathway.