[Effect of acupotomy on expressions of cellular senescence markers p16Ink4a and p21Waf1/Cip1 in cartilage tissue of rabbits with knee osteoarthritis].

OBJECTIVE: To observe the effect of acupotomy on the expressions of p16Ink4a and p21Waf1/Cip1 in knee osteoarthritis (KOA) rabbits，so as to analyze whether acupotomy can treat KOA by inhibiting the cellular senescence of chondrocytes. METHODS: Twenty-four New Zealand male rabbits were randomly divided into normal, model, acupotomy and electroacupuncture (EA) groups, with 6 rabbits in each group. The KOA model was established by left hindlimb straightening fixation. After modeling, rabbits in the acupotomy group were treated with acupotomy loosening therapy on high stress points around the affected knee joints such as tendons attachment points of vastus medialis, vastus lateralis, rectus femoris, biceps femoris and pes anserine bursa, once a week for 3 weeks. In the EA group, "Xuehai"(SP10), "Liangqiu" (ST34),"Neixiyan" (EX-LE4) and "Waixiyan" (ST35) on the affected hindlimb were selected for EA treatment (3 mA, 2 Hz/100 Hz), 20 min each time, once every other day for 3 weeks. Before and after treatments, the knee Lequesne MG score and passive range of motion (PROM) of the affected knee joint were evaluated. After the treatments, the expressions of p16Ink4a and p21Waf1/Cip1 in the cartilage tissue of the affected knee joint were detected by immunohistochemistry and Western blot respectively. RESULTS: Before and after treatment, compared with the normal group, the Lequesne MG score was significantly increased (P<0.01), the PROM was significantly decreased (P<0.01) in the model group. After treatment, compared with the normal group, the positive expression and protein expression levels of p16Ink4a and p21Waf1/Cip1 were significantly increased (P<0.01) in the model group; compared with the model group, the Lequesne MG score was significantly decreased (P<0.01), the PROM was significantly increased (P<0.01), the positive expression and protein expression levels of p16Ink4a and p21Waf1/Cip1 were significantly decreased (P<0.01，P<0.05) in the acupo-tomy and EA groups; compared with the EA group, the Lequesne MG score was decreased (P<0.05), the PROM was increased (P<0.05), the positive expression and protein expression levels of p16Ink4a and p21Waf1/Cip1 were decreased (P<0.05，P<0.01) in the acupotomy group. CONCLUSION: Acupotomy intervention can down-regulate the expressions of cellular senescence markers p16Ink4a and p21Waf1/Cip1 in chondrocytes, indicating that acupotomy therapy may alleviate cartilage degeneration by inhibiting chondrocyte premature cellular senescence to treat KOA.

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

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