Effects of Acupotomy on Immobilization-Induced Gastrocnemius Contracture and Fibrosis in Rats via Wnt/β-Catenin Signaling

Abstract

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

Methods: 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 chain 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: 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 was 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: Improvements in motor function, muscle contractures, and muscle fibrosis induced by acupotomy correlates with the inhibition of Wnt/β-catenin signaling pathway.

Keywords: Wnt/β-catenin signaling; acupotomy; immobilization; muscle contracture; muscle fibrosis.