| Purpose:In this experiment,an animal model of knee osteoarthritis(KOA)was prepared by the knee joint immobilization method.After acupuncture at the the focal points of tendon,the changes in the structure and function of the quadriceps femoris in KOA rats were observed,and the changes in the biomarkers related to the regeneration and repair of the quadriceps femoris were also observed.Furthermore,based on the Wnt/β-catenin pathway,explore the mechanism of acupuncture at the focal points of tendon to promote the regeneration and repair of the quadriceps femoris in KOA rats,and provide experimental evidence for the clinical application of the meridian sinew therapy in the treatment of KOA.Material and method:44 SPF male SD rats(6 weeks old,weight 180-220g)were randomly divided into normal control group(normal group),KOA model group(model group),needle focal point of tendon group(focal point of tendon group),needle acupoint group(acupoint group),11 rats in each group(1 used for model evaluation,10 used for indicator detection).KOA models were prepared by knee joint immobilization method in model group,focal point of tendon group and acupoint group.After the completion of the model,the rats in the normal group and the model group were fixed with a fixator for 20 minutes a day for 14 consecutive days.The rats in the focal point of tendon group were fixed in a fixator daily,analogous to the location of the points of musculature lesion in the "Chinese Study of Meridians and Tendons" and the palpation of the sinew around the knee joint to mark the following position on the right hind limb of the rats:The rectus femoris tendon on the anterior side of the thigh,the rectus femoris tendon on the groin,and between the semitendinosus and the long head of the biceps femoris at the back of the gluteus,which is equivalent to the isc-hial tuberosity.Then perform acupuncture intervention on the above 3focal poits of tendon,one time for 20 minutes,and continuous intervention for 14 days.The rats in the acupoint group were fixed in a fixator daily,and the Dubi,Zusanli and Yanglingquan acupoints of the right hind limb were acupunctured for 20 minutes each time for 14 consecutive days.In terms of morphology,the leg circumference and the mass of the quadriceps femoris of the right hind limbs of the rats in each group were measured,and the morphological structure of the quadriceps femoris of the right hind limbs of each group was observed by the HE staining method.In terms of behavior,the passive range of motion and the modified Lequesne MG index of the right hind limbs of each group of rats were measured.And the gait analysis technology was used to measure gait parameters such as paw print area,maximum contact area,maximum pressure,swing speed and standing time of rats in each group.In terms of molecular biology,immunohistochemistry was used to observe the protein expression of Pax7,MyoD,MyoG and MyHC1 in the quadriceps femoris of the right hind limbs of rats in each group;RT-PCR technology was used to detect the expression of Pax7,MyoD,MyoG,MyHC1 mRNA in the quadriceps femoris of the right hind limbs of rats in each group;Western blot technology was used to detect the Wnt,GSK3β,β-catenin,Cyclin D1 protein expression and the phosphorylation level of GSK3β and β-catenin protein in the quadriceps femoris of the right hind limbs of rats in each group;RT-PCR method was used to detect the the expression of Wnt,GSK3β,βcatenin and Cyclin D1 mRNA in quadriceps femoris of the right hind limbs of rats in each group.The experimental data was statistically analyzed using SPSS 22.0software.The data was expressed as mean±standard deviation((?)±s).The data between groups was compared by one-way analysis of variance(one-way ANOVA)to compare the differences between the groups,and P<0.05 as a significant difference,which is statistically significant.Results:1.Changes in the circumference of the right hind limb of the rats in each group:The leg circumference of the KOA rats prepared by the knee joint immobilization method decreased significantly(P<0.05);After intervention,compared with the model group,the thigh circumference of rats in the focal point of tendon group and the acupoint group increased significantly(P<0.05).2.Comparison of the mass of the quadriceps femoris of the right hind limbs of rats in each group: Compared with the normal group,the absolute wet weight of the model group was significantly reduced(P<0.05);Compared with the model group,the absolute wet weight of the focal point of tendon group and the acupoint group was significantly increased(P<0.05),and the increase degree of the focal point of tendon group was significantly higher than that of the acupoint group(P<0.05).Compared with the normal group,the maintenance rate of muscle wet weight in the model group was significantly reduced(P<0.05);Compared with the model group,the maintenance rate of muscle wet weight in the focal point of tendon group and the acupoint group was significantly increased(P<0.05).Compared with the normal group,the wet-weight-to-weight ratio of the model group was significantly reduced(P<0.05);Compared with the model group,the ratio of muscle wet weight to weight in the focal point of tendon group and the acupoint group was significantly increased(P<0.05).3.Comparison of changes in gait parameters of the right hind limbs of rats in each group:(1)Comparison of changes in the paw print area of the right hind limb of rats in each group:The paw print area of the right hind of the KOA rats prepared by the knee joint immobilization method decreased significantly(P<0.05).After intervention,compared with the normal group,the paw print area of the model group was significantly reduced(P<0.05);Compared with the model group,the paw print area of the right hind limb of rats in the focal point of tendon group and the acupoint group was significantly increased(P<0.05).(2)Comparison of changes in the maximum contact area of the right hindlimb of rats in each group:The maximum contact area of the right hind of the KOA rats prepared by the knee joint immobilization method decreased significantly(P<0.05).After intervention,compared with the normal group,the maximum contact area of the model group was significantly reduced(P<0.05);Compared with the model group,the maximum contact area of the focal point of tendon group and the acupoint group was significantly increased(P<0.05).(3)Comparison of changes in the maximum pressure of the right hindlimb of rats in each group:The maximum pressure of the right hind of the KOA rats prepared by the knee joint immobilization method decreased significantly(P<0.05).After intervention,compared with the normal group,the maximum pressure of the model group was significantly reduced(P<0.05);Compared with the model group,the maximum pressure of the focal point of tendon group and the acupoint group was significantly increased(P<0.05),and the degree of increase of the focal point of tendon group was significantly higher than that of the acupoint group(P<0.05).(4)Comparison of changes in swing speed of right hind limbs of rats in each group: The swing speed of the right hind of the KOA rats prepared by the knee joint immobilization method decreased significantly(P<0.05).After intervention,compared with the normal group,the swing speed of the model group was significantly reduced(P<0.05);Compared with the model group,the swing speed of the focal point of tendon group and the acupoint group increased significantly(P<0.05),and the increase degree of the focal point of tendon group was significantly higher than that of the acupoint group(P<0.05).(5)Comparison of changes in standing time of right hind limbs of rats in each group: The standing time of the right hind of the KOA rats prepared by the knee joint immobilization method decreased significantly(P<0.05);The comparison between the groups after treatment showed that compared with the normal group,the standing time of the right hind limb of the model group was significantly reduced(P<0.05);Compared with the model group,the standing time of the focal point of tendon group and the acupoint group increased significantly(P<0.05).4.Comparison of changes in passive range of knee joint motion of the right hind limb joints of rats in each group:The passive range of knee joint motion of the right hind of the KOA rats prepared by the knee joint immobilization method decreased significantly(P<0.05).After intervention,compared with the normal group,the passive range of knee joint motion of the model group was significantly decreased(P<0.05);Compared with the model group,the passive range of knee joint motion in the focal point of tendon group and the acupoint group was significantly increased(P<0.05),and the increase degree of the focal point of tendon group was significantly higher than that of the acupoint group(P<0.05).5.Comparison of modified Lequesne MG index of rats in each group:The modified Lequesne MG index of KOA rats prepared by knee immobilization method increased significantly(P<0.05).After intervention,compared with the normal group,the modified Lequesne MG index of the model group was significantly increased(P<0.05);Compared with the model group,the modified Lequesne MG index of the focal point of tendon group and the acupoint group was significantly decreased(P<0.05).6.Analysis of HE staining results of quadriceps femoris of right hind limbs of rats in each group:The muscle fiber structure of the normal group is complete and neatly arranged;The morphology of muscle fibers in the model group was changed,and the structure was destroyed;The muscle fiber structure in the focal point of tendon group is relatively complete,and regenerated muscle fibers can be seen;The muscle fiber structure in the acupoint group recovered to a certain extent,but there was a relatively large gap between the muscle fibers.7.Immunohistochemical results of molecular markers for regeneration and repair of quadriceps femoris muscle in the right hind limbs of rats in each group:7.1 The morphological results showed that the immunoreactive staining results of Pax7,MyoD,MyoG,MyHC1 of the quadriceps femoris of the right hind limbs of rats in each group were all brown.Among them,the immunopositive reaction of Pax7,MyoD and MyoG is expressed in the nucleus of the muscle fiber,and the immunopositive reaction of MyHC1 is expressed in the cytoplasm of the muscle fiber.7.2 Result of the comparison of the integral optical density value:(1)Comparison of Pax7 integral optical density value: Compared with the normal group,the Pax7 integral optical density value of the model group increased significantly(P<0.05);Compared with the model group,the integrated optical density value of the focal point of tendon group and the acupoint group was significantly increased(P<0.05),and the increase degree of the focal point of tendon group was significantly higher than that of the acupoint group(P<0.05).(2)Comparison of MyoD integral optical density value: Compared with the normal group,the MyoD integral optical density value of the model group increased significantly(P<0.05);Compared with the model group,the MyoD integral optical density value of the focal point of tendon group and the acupoint group was significantly increased(P<0.05),and the increase degree of the focal point of tendon group was significantly higher than that of the acupoint group(P<0.05).(3)Comparison of MyoG integral optical density value:Compared with the normal group,the MyoG integral optical density value of the model group increased significantly(P<0.05);Compared with the model group,the integrated optical density value of the focal point of tendon group and the acupoint group was significantly increased(P<0.05).(4)Comparison of MyHC1 integral optical density value: Compared with the normal group,the MyHC1 integral optical density value of the model group significantly decreased(P<0.05);Compared with the model group,the MyHC1 integral optical density value of the focal point of tendon group and the acupoint group was significantly increased(P<0.05).8.The mRNA expression levels of Pax7,MyoD,MyoG,MyHC1 in the quadriceps femoris of the right hind limbs of rats in each group:(1)Pax7 mRNA expression level: Compared with the normal group,the mRNA expression level of the model group was significantly increased(P<0.05);Compared with the model group,the mRNA expression level of the focal point of tendon group was significantly increased(P<0.05).(2)MyoD mRNA expression level: Compared with the normal group,the mRNA expression level of the model group increased significantly(P<0.05);Compared with the model group,the mRNA expression level of the focal point of tendon group and the acupoint group was significantly increased(P<0.05),and the increase degree of the focal point of tendon group was significantly higher than that of the acupoint group(P<0.05).(3)MyoG mRNA expression level: Compared with the normal group,the mRNA expression level of the model group increased significantly(P<0.05);Compared with the model group,the mRNA expression level of the focal point of tendon group and the acupoint group was significantly increased(P<0.05).(4)MyHC1 mRNA expression level: Compared with the normal group,the mRNA expression level of the model group was significantly reduced(P<0.05);Compared with the model group,the mRNA expression level of the focal point of tendon group and the acupoint group was significantly increased(P<0.05).9.The mRNA expression levels of Wnt,GSK3β,β-catenin,and Cyclin D1 in the quadriceps femoris of the right hind limbs of rats in each group:(1)Wnt mRNA expression level: Compared with the normal group,the mRNA expression level of the model group was significantly increased(P<0.05);Compared with the model group,the mRNA expression level of the focal point of tendon group and the acupoint group was significantly increased(P<0.05),and the increase degree of the focal point of tendon group was significantly higher than that of the acupoint group(P<0.05).(2)GSK3β mRNA expression level: Compared with the normal group,the mRNA expression level of the model group was significantly decreased(P<0.05);Compared with the model group,the mRNA expression level of the focal point of tendon group and the acupoint group was significantly decreased(P<0.05),and the decrease degree of the focal point of tendon group was more significant(P<0.05).(3)β-catenin mRNA expression level: Compared with the normal group,the mRNA expression level of the model group significantly increased(P<0.05);Compared with the model group,the mRNA expression level of the focal point of tendon group and the acupoint group was significantly increased(P<0.05),and the increase degree of the focal point of tendon group was significantly higher than that of the acupoint group(P<0.05).(4)Cyclin D1 mRNA expression level: Compared with the normal group,the mRNA expression level of the model group was significantly increased(P<0.05);Compared with the model group,the mRNA expression level of the focal point of tendon group and the acupoint group was significantly increased(P<0.05),and the increase degree of the focal point of tendon group was significantly higher than that of the acupoint group(P<0.05).10.The relative protein content of Wnt,GSK3β,β-catenin,Cyclin D1 and the phosphorylation level of GSK3β,β-catenin protein in the quadriceps femoris muscle of the right hind limb of each group of rats:(1)wnt/β-actin: Compared with the normal group,the relative protein expression of the model group was significantly increased(P<0.05);Compared with the model group,the relative protein expression of the focal point of tendon group and the acupoint group was significantly increased(P<0.05),and the increase degree of the focal point of tendon group was significantly higher than that of the acupoint group(P<0.05);(2)p-GSK3β/GSK3β: Compared with the normal group,the p-GSK3β/GSK3β of the model group was increased significantly(P<0.05);Compared with the model group,the p-GSK3β/GSK3β of the focal point of tendon group was increased significantly(P<0.05);(3)p-β-catenin/β-catenin: Compared with the normal group,the p-β-catenin/β-catenin of the model group was decreased significantly(P<0.05);Compared with the model group,the p-β-catenin/β-catenin of the focal point of tendon group and the acupoint group was decreased significantly(P<0.05),and the degree of decrease in focal point of tendon group was more significant(P<0.05);(4)Cyclin D1/β-actin: Compared with the normal group,the relative protein expression of the model group was increased significantly(P<0.05);Compared with the model group,the relative protein expression of the focal point of tendon group and the acupoint group was significantly increased(P<0.05),and the increase degree of the focal point of tendon group was significantly higher than that of the acupoint group(P<0.05).Conclusion:1.Acupuncture at the focal points of tendon can reshape the morphological structure of muscle fibers,increase the leg circumference and muscle mass of the affected hind limbs of KOA rats,thereby improving the pathological morphology of the quadriceps femoris of KOA rats.2.Acupuncture at focal points of tendon can effectively increase the passive range of motion of the affected knee joint in KOA rats,correct the abnormal gait of the affected hind limbs,reduce the score of knee osteoarthritis,and thereby improving functional abnormalities such as restricted movement of the knee joint in KOA rats.3.Acupuncture at focal points of tendon can significantly increase the expression of biomarkers such as Pax7,MyoD,MyoG,and MyHC1 in the quadriceps femoris muscle of the affected hind limbs of the KOA rats,and promote the proliferation and differentiation of skeletal muscle stem cells to achieve regeneration and repair of damaged skeletal muscle.4.Acupuncture at focal points of tendon can regulate the expression of genes and proteins related to the Wnt/β-catenin pathway.The mechanism of regeneration and repair of skeletal muscle stem cells mediated by this pathway may be one of the ways of acupuncture meridian sinew to improve the morphological structure and function of the quadriceps femoris in KOA rats. |
PDF Full Text Request