School of Acupuncture-Moxibustion and Tuina, Beijing University of Chinese Medicine, Beijing, 100029, China.
Acupuncture Department, Kunming Municipal Hospital of Traditional Chinese Medicine, Kunming, 650500, China.
Chin J Integr Med. 2024 Jul;30(7):633-642. doi: 10.1007/s11655-023-3553-z. Epub 2023 Jul 12.
To determine whether acupotomy ameliorates immobilization-induced muscle contracture and fibrosis via Wnt/β-catenin signaling pathway.
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.
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).
Improvements in motor function, muscle contractures, and muscle fibrosis induced by acupotomy correlates with the inhibition of Wnt/β-catenin signaling pathway.
通过 Wnt/β-连环蛋白信号通路，确定针刀是否能改善制动引起的肌肉挛缩和纤维化。
30 只 Wistar 大鼠采用随机数字表法分为 5 组（n=6），包括对照组、制动组、被动拉伸组、针刀组和针刀 3 周组。通过将右后肢跖屈固定 4 周建立比目鱼肌挛缩模型。被动拉伸组在比目鱼肌接受被动拉伸，连续 10 天，每天 10 次，每次 30 s，间隔 30 s。针刀组和针刀 3 周组在针刀治疗后 10 天内每天进行 10 次，每次 30 s，间隔 30 s，连续 10 天。此外，针刀 3 周组在 10 天治疗后允许自由行走 3 周。治疗后，测试运动范围（ROM）、步态分析[即足面积、站立/摆动和最大足面积与足面积持续时间比值（Max dA/dT）]、比目鱼肌湿重和肌肉湿重与体重比（MWW/BW）。通过苏木精-伊红染色评估比目鱼肌形态和肌纤维横截面积（CSA）。采用实时定量聚合酶链反应测量纤维化相关基因（即 Wnt1、β-连环蛋白、轴突蛋白-2、α-平滑肌肌动蛋白、纤维连接蛋白和 I 型和 III 型胶原）的表达。通过酶联免疫吸附试验测量 Wnt1、β-连环蛋白和纤维连接蛋白的浓度。采用免疫荧光法分析肌周膜和肌内膜中的 I 型和 III 型胶原。
与对照组相比，制动组的 ROM、步态功能、肌肉重量、MWW/BW 和 CSA 显著降低（均 P<0.01），而 I 型和 III 型胶原、Wnt1、β-连环蛋白、纤维连接蛋白的蛋白水平和纤维化相关基因的 mRNA 水平明显增加（均 P<0.01）。被动拉伸或针刀治疗恢复了 ROM 和步态功能，并增加了肌肉湿重、MWW/BW 和 CSA（均 P<0.05），而 Wnt1、β-连环蛋白、纤维连接蛋白、I 型和 III 型胶原的蛋白表达水平和纤维化相关基因的 mRNA 水平与制动组相比明显下降（均 P<0.05）。与被动拉伸组相比，ROM 和步态功能、MWW 显著恢复（均 P<0.05），针刀组纤维化相关基因的 mRNA 水平以及 Wnt1、β-连环蛋白、纤维连接蛋白、I 型和 III 型胶原的蛋白表达水平明显降低（均 P<0.05）。与针刀组相比，ROM、足面积、Max dA/dT 和 MWW 恢复（均 P<0.05），针刀 3 周组纤维化相关基因的 mRNA 水平以及 Wnt1、β-连环蛋白、纤维连接蛋白、I 型和 III 型胶原的蛋白水平降低（P<0.05）。
针刀改善运动功能、肌肉挛缩和肌肉纤维化与抑制 Wnt/β-连环蛋白信号通路有关。
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