Animal Model Of Achilles Tendinopathy Treated By Acupuncture At Foot Shaoyang Meridian

Objective:Achilles tendinopathy（AT）is a chronic overuse injury,which is mainly manifested as local swelling and pain of the Achilles tendon and functional limitation.The incidence of AT is increasing day by day,which not only seriously affects the quality of life of patients,but also causes a huge economic burden to individuals and society,so the prevention and treatment of AT is still an urgent problem to be solved in the field of sports medicine.In this study,the rat AT model was established by collagenase injection,and based on the theory of"Shaoyang main bone",AT rats were treated by acupuncture at the points of the Foot Shaoyang Meridian,and the changes of the effects of acupuncture at the points of the Foot Shaoyang Meridian on the Achilles tendon and its function of AT rats were observed from different angles such as gait behavior,biomechanics,histomorphology and molecular biology to explore the efficacy and mechanism of acupuncture on AT rats based on the theory of"Shaoyang main bone"and further enrich and develop the scientific connotation of the theory of"Shaoyang dominates bone".Methods:The study consists of 2 parts.Part I:20 male SD rats were randomly divided into normal group（Normal,n=10）and model group（Model,n=10）.Collagenase injection was used to establish the model.Four weeks after collagenase injection,samples were collected and the differences in the gross morphology,gait behavior,biomechanical properties,histological structure and molecular biology of Achilles tendon between normal rats and AT rats were observed to verify whether the model was successful.Part II:Forty-eight male SD rats were randomly divided into model control group（Control,n=16）,acupuncture 2-week group（Acu-2w,n=16）,and acupuncture 4-week group（Acu-4w,n=16）.The model was also established by collagenase injection.After the model was successfully established,acupuncture intervention was performed according to the requirements of intervention time（no intervention was performed in the control group）.After the completion of intervention,samples were taken and the effects of different acupuncture intervention time on the gross morphology,gait behavior,biomechanical properties,Achilles tendon tissue structure and molecular biological composition of Achilles tendon in AT rats were observed.Test indexes and methods:（1）The appearance changes of Achilles tendon in each group were observed macroscopically.（2）HE staining,Masson staining and Sirius red staining were used to observe the changes of collagen fiber structure and tenocytes in rat Achilles tendon under light microscope.（3）Footprint analysis was used to observe the changes of gait behavior in rats.（4）INSTRON,E3000 electronic dynamic and static fatigue tester was used to determine the maximum load,maximum displacement and stiffness of Achilles tendon.（5）Western blot and RT-q PCR were used to detect the expression of TGF-β₁,TIMP-1 and MMP-1 protein and m RNA in Achilles tendon.Excel software was used for data collection,and statistical software SPSS 20.0 was used for statistical analysis of experimental data,with P<0.05 considered statistically significant.RESULTS:（1）Compared with the Normal group,the Achilles tendon in the Model group showed different degrees of atrophy,severe tissue proliferation,and obvious adhesion to the surrounding tissues;Walking cycle,mean stride length of right foot,stride deviation degree and Achilles Functional Index（AFI）decreased（P<0.05）;The maximum tensile displacement breaking force,maximum tensile displacement and stiffness of Achilles tendon were significantly lower than those of normal Achilles tendon（P<0.01）;Under the light microscope,HE staining showed that collagen fibers in the Achilles tendon tissue of the Model group were columnar,and showed nuclear rounding,fiber disarrangement,partial rupture,and neovascularization;The protein content and m RNA expression of TGF-β₁ and MMP-1 in the Achilles tendon of the Model group were significantly increased compared with those of the Normal group（P<0.05）,and the protein content and m RNA expression of TIMP-1 were significantly decreased compared with those of the Normal group（P<0.05）.（2）After acupuncture at Foot Shaoyang Meridian,compared with Control group,with the increase of intervention time,the proliferation and adhesion of gross tissue of Achilles tendon were improved;walking cycle,average stride amplitude and stride deviation of right foot were reduced,AFI value was increased（P<0.05）;the maximum tensile displacement rupture force,maximum tensile displacement and stiffness of Achilles tendon were increased（P<0.05）;HE staining,Masson staining and Sirius red staining under light microscope showed that the number of collagen fibers increased,the arrangement tended to be neat,fibroblasts gradually decreased,tenocytes tended to mature and increased;the protein and m RNA expression levels of TGF-β₁ and TIMP-1 increased（P<0.05）,while the protein and m RNA expression levels of MMP-1 showed a downward trend（P<0.05）,and the expression levels of MMP-1 and TIMP-1 tended to be balanced.CONCLUSION:Based on the theory of"Shaoyang main bone",acupuncture at the points of Foot-Shaoyang meridian can improve the gait behavior of AT rats,regulate the metabolic function of collagen in rat Achilles tendon,promote collagen secretion,and then restore the structural arrangement order of tendon fibers,which is conducive to the improvement of biomechanical properties of Achilles tendon,and regulate the anabolism of MMP-1 and TIMP-1 by up-regulating the expression of TGF-β1,and then improve the metabolic function of collagen in rat Achilles tendon and promote the recovery of its Achilles tendon function.