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

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.