Objective To observe the mRNA and protein expression of wingless-type MMTV integration site family member 5a (Wnt5a), glycogen synthase kinase 3 (GSK3), and β-catenin, as well as the muscle fibers and adipose tissue presented in pathological staining in the gastrocnemius muscle of white rabbits with blunt gastrocnemius contusion injuries, and provide a basis for revealing the repair mechanism of the pressing and kneading method in treating skeletal muscle injury. Methods Forty-two healthy male and female New Zealand white rabbits were selected. They were randomly divided into blank group, model 3-day group, model 7-day group, model 14-day group, press-and-knead 3-day group, press-and-knead 7-day group, and press-and-knead 14-day group, by using a random number table method, with 6 rabbits in each group. Samples of the model groups and the press-and-knead groups were taken on the 4th, 8th and 15th days after operation. The mRNA and protein expression of Wnt5a, GSK3, and β-catenin were detected by quantitative polymerase chain reaction and Western blot; the muscle tissue myofibers and adipose tissue were observed by hematoxylin and eosin (HE) staining and oil red O staining. Results The HE staining results showed that significant fibrous tissue proliferation and inflammatory cell infiltration occurred in the model 7-day group; in the model 14-day group, some muscle fibers were degenerated, necrotic, and regenerated, accompanied by fibrous tissue proliferation, slight inflammatory cell infiltration, and slight calcification; in the press-and-knead groups, obvious muscle fiber degeneration, necrosis, and regeneration, and inflammatory cell infiltration were observed, accompanied by significant fibrous tissue proliferation. The oil red O staining results showed that adipocyte deposition was visible in the model groups, which was the heaviest in the model 7-day group; in the press-and-knead groups, muscle fibers and sequences were not significantly damaged, and a small amount of adipocyte infiltration was visible in the interstitial space. There were statistically significant differences in the mRNA expression and protein expression of Wnt5a, GSK3, and β-catenin in the gastrocnemius among groups (P<0.001). Conclusions The histopathological changes of gastrocnemius muscle injury recover gradually over time, and the pressing and kneading method stimulates the mRNA expression activities of Wnt5a, GSK3, and β-catenin, which may slow down the degradation of β-catenin protein by the scaffolding protein complex (of which GSK3 is an important component), so that the protein level of β-catenin is maintained in the stable range at all times. This leads to a reduction of fatty degeneration in the gastrocnemius muscle after the intervention of pressing and kneading method, and promotes the functional repair of the injured skeletal muscle.
Objective To observe the effect of “Luo’s Roujin Technique” on the inflammatory response and joint capsule fibrosis in white rabbits with scapulohumeral periarthritis model. Methods Thirty healthy male New Zealand white rabbits were randomly divided into a control group, a model group, and a treatment group, with 10 rabbits in each group. Scapulohumeral periarthritis models were established in the model group and the treatment group, while the control group received identical restraint procedures at the same timepoints. Six rabbits in the model group and seven in the treatment group were successfully modeled. The subsequent experiment included all six successfully modeled rabbits from the model group, along with six rabbits randomly selected from each of the control and treatment groups. On the second day after successful modeling, blood samples were collected from the auricular marginal vein in all three groups. After blood collection, the treatment group began massage therapy for 21 consecutive days, while the other two groups underwent the same restraint procedure simultaneously. On Day 22, all the three groups were euthanized after blood collection from the auricular marginal vein, and the synovial tissue of the affected shoulder joint was completely collected. Hematoxylin-eosin staining was used to examine the histopathological features of the synovial tissue. Enzyme-linked immunosorbent assay was employed to measure the concentrations of interleukin (IL)-1β, IL-6, IL-17, and tumor necrosis factor-α (TNF-α). Western blot and reverse transcription polymerase chain reaction were used to assess the protein and mRNA expression levels of vascular endothelial growth factor (VEGF), connective tissue growth factor (CTGF), transforming growth factor-β1 (TGF-β1), and Smad3. Results After treatment, the control group showed no significant inflammatory cell infiltration or fibrous tissue proliferation in the synovial tissue. The model group exhibited synovial cell hyperplasia in the lining layer and inflammatory cell infiltration in the sublining layer. The treatment group displayed mild inflammatory cell infiltration in the sublining layer. Compared with the control group, the model group showed significantly increased concentrations of IL-1β, IL-6, IL-17, and TNF-α in both serum and synovial homogenate, as well as elevated protein and mRNA expression of VEGF, CTGF, TGF-β1, and Smad3 in synovial tissue (P<0.05). Compared with the model group, the treatment group exhibited significantly lower serum levels of IL-1β, IL-6, and TNF-α, as well as reduced synovial homogenate levels of IL-1β, IL-6, IL-17, and TNF-α (P<0.05); furthermore, protein expression of VEGF, CTGF, TGF-β1, and Smad3 and mRNA expression of VEGF and CTGF in synovial tissue were significantly decreased in the treatment group (P<0.05). Conclusions “Luo’s Roujin Technique” can significantly alleviate local inflammatory infiltration in the synovial tissue of rabbits with scapulohumeral periarthritis, and reduce the levels of IL-1β, IL-6, IL-17, and TNF-α in both serum and synovial tissue. The underlying mechanism may involve suppression of VEGF, CTGF, TGF-β1, and Smad3 expression, leading to attenuated inflammatory responses and inhibition of fibroblast-to-myofibroblast transition. Thereby, it mitigates fibrotic changes in the shoulder joint capsule, exerting anti-inflammatory and analgesic effects and improving joint mobility.