| BackgroundKnee osteoarthritis(KOA),with knee pain and dysfunction as the main clinical manifestations,is degenerative osteoarthropathy,with an incidence rate of between 60%-70%in the population over 65 years old.Clinical studies have confirmed that acupotomology has a significant effect on KOA.Our previous experiments found that the mechanical signal of acupotomy(Apo)could alleviate cartilage matrix degradation and promote cartilage repair by activating the FAK-PI3K-AKT signaling pathway of chondrocytes.AKT signaling molecule is a key signal hub that a fects cell viability,and can regulate cell survival,proliferation,metabolism and other life activities.How the Apo signal regulates cartilage damage repair on the downstream of AKT has not been reported yet.So on the basis of the National Natural Science Foundation of China"The Effector Mechanism of tendon regulation and bone treatment with Acupotony for knee osteoarthritis and its regulation with FAK-PI3K-AKT Mechanical Signal Transduction Pathways",and starting from the glucose metabolism of chondrocytes and the regulation of chondrocyte cycle,this study continues to explore the molecular mechanism of acupotomology signal regulating the repair of KOA cartilage damage.ObjectiveIn this study,the cell glucose metabolism regulation(AKT-GSK3? pathway)and cell cycle regulation(Cyclindl-CDK4/6 complex)of injury repair were used as the entry point,we explored the molecular mechanism of Apo mechanical signals promoting repair of KOA cartilage damage on the downstream of FAK-PI3K-AKT signaling pathway,to further enrich the scientific connotation of Apo theory of" tendon regulation and bone treatment ",and provide certain experimental and theoretical basis for the clinical application of Apo in the treatment of KOA.MethodsHealthy New Zealand rabbits aged 6 months were selected and divided into normal group,model group,electroacupuncture group(E-Apu)and Apo group by random number table method.KOA rabbit model was prepared by the modified Videman immobilization method in the extended position of left hind limb,and the intervention began in the E-Apu group and the Apo group 1 week after the model was successful In the E-Apu group,acupuncture on the same side of "Xue hai-Liang qiu" and "Nei Xiyan-Wai Xiyan" was performed with frequency of 2/100hz,intensity of 3mA,20min/time,3 times/week for 3 consecutive weeks.In the Apo group,the adhesion points of the ipsilateral medial femoral muscle tendon,lateral femoral muscle tendon,rectus femoris tendon,and tendon cyst of the goose-foot tendon were selected.The longitudinal and transverse stripping of each point was performed once,and the four points were used alternately,2 points per time,2 times per week,for 3 consecutive weeks.Experiment 1:Lequesne MG Index,PROM and MRI were used to evaluate the behavioral and imaging manifestations of the knee joints in each group 1 week after modeling and intervention.Experiment 2:1 week after the intervention,the contents of markers of cartilage degradation(serum COMP and urine CTX-?),were detected by enzyme linked immunosorbent assay(Elisa).The tissue morphology and ultrastructure of the medial femoral condyle cartilage were observed by gross anatomy,light microscopy and scanning electron microscopy to evaluate the repair of articular cartilage injury in each group.Experiment 3:The mRNA and protein expression levels of AKT,GSK3?,GS,CyclinD1,CDK4 and CDK6 in cartilage were detected by Real-time PCR,Western-blot and Immunmohistochemistry,to evaluate the expression of factors related to glucose metabolism and cell cycle in rabbit chondrocytes.Res ults1.The behaviorBefore intervention,the Lequesne MG score of the model group,E-Apu group and Apo group was significantly higher than that of the normal group(P<0.01,P<0.01,P<0.01),and the PROM was significantly lower than that of the normal group(P<0.01,P<0.01,P<0.01).After the intervention,the Lequesne MG score of Apo group and E-Apu group was significantly lower than that of model group(P<0.05,P<0.05),and the PROM value of Apo group was significantly higher than that of model group(P<0.05).There was no significant difference in Lequesne MG score and PROM between Apo group and E-Apu group)(P>0.05).2.The imagingThe articular cartilage of the normal group had smooth surface,equal space,and a small amount of joint cavity effusion.In the model group,the thickness of articular cartilagebecame thinner and the hair was impetuous.The gap narrowed significantly and the bursa thickened slightly.Joint capsule dilated and fluid increased.Bone edema was found at the lbwer end of femur and the upper end of tibia,and obvious soft tissue swelling and fascial fluid were observed.In the E-Apu group,the cartilage surface was smooth,part of the articular space on the affected side was narrowed,and a small amount of effusion was observed in the articular cavity.Soft tissue swelling and fascial effusion were significantly reduced.The surface of the cartilage in the Apo group was smooth,the thickness of the cartilage was significantly thicker than that in the model group,the space was equaly wide on both sides,there was a small amount of fluid in the lumen,and there was no bone and suprapatellar tendon edema.Before intervention,the semi-quantitative MRI score of the model group,Apo group and E-Apu group was significantly higher than that of the normal group(P<0.01,P<0.01,P<0.01).After the intervention,the semi-quantitative MRI score of the E-Apu group and the Apo group was significantly lower than that of the model group(P<0.01,P<0.01).The score of Apo group was significantly lower than that of E-Apu group(P<0.05).3.The morphology3.1 General morphology:the cartilage integrity of the distal femur,proximal tibia and medial patella of the knee joint in the normal group was good,the surface was smooth and transparent,and a small amount of effusion was observed in the joint cavity.In the model group,cartilage integrity was damaged,with varying degrees of wear and tear,and cartilage thickness and transparency were reduced in section.There was a large amount of articular fluid in the articular cavity with exudation.In the E-Apu group,the cartilage integrity of the distal femur was destroyed occasionally,the color of the cartilage was more transparent,a small amount of soft tissue adhesion around the knee and articular fluid exudation were observed.In Apo group,the cartilage integrity of distal femur,proxima l tibia and medial patella was better,and the cartilage was smooth and transparent.3.2 Cartilage light microscope:the normal group had smooth surface and clear and complete structure of cartilage;In the model group,cartilage surface was dislocated,cells were disordered,and the tidal lines were blurred or double tidal lines were observed.In the E-Apu group,there were local defects in the surface layer of cartilage,slight disorder in the arrangement of cells,or pannus formation.In the Apo group,the cartilage surface was smooth,the structure was basically normal,and the cells were well arranged.Compared with the normal group,the Mankin scores of knee cartilage in the model group,Apo group and E-Apu group were significantly increased(P<0.01).Compared with the model group,the Mankin scores of Apo group and E-Apu group were significantly reduced(P<0.01).Compared with the E-Apu group,the Mankin score of the Apo group decreased(P<0.01).3.3 Cartilage electron microscopy:in the normal group,the surface of the cartilage of the medial femoral condyle of the knee joint was flat,with no uniform distribution of qualitative substances and no exposed collagen fibers.In the model group,the amorphous material on the surface of cartilage became thinner,the light layer completely disappeared,the underlying collagen fibers were exposed,and some superficial flattened chondrocytes were seen.In the E-Apu group,the rough surface of cartilage and the amorphous material on the surface seemed to dry and crack into blocks,with local peeling,collagen fibers turning up and superficial pit structure disappearing.In Apo group,the cartilage surface was smooth,the surface was covered with amorphous substance,the furrows were deep,the arrangement was regular,and a small amount of collagen fiber was exposed occasionally.4.Degradation markersCompared with the normal group,the levels of serum COMP and urine CTX-?i in the model group,E-Apu group and Apo group were significantly increased(P<0.01,P<0.01).Compared with the model group,the contents of serum COMP and urine ctx-? in the E-Apu group and acupotomology group were significantly reduced(P<0.01,P<0.01).Compared with the E-Apu group,the content of serum COMP and urine ctx-? in the Apo group was significantly reduced(P<0.05,P<0.05).5.Molecular biology5.1 Real-time PCR:Compared.with the normal group,the expression levels of AKT and GSK3? mRNA in the knee cartilage of the model group were significantly increased(P<0.01,P<0.01),and the expression levels of GS,CyclinD1,CDK4 and CDK6 mRNA were significantly decreased(P<0.01,P<0.01,P<0.01,P<0.01).Compared with the model group,the expression levels of AKT and GSK3? mRNA in the Apo group and E-Apu group were significantly decreased(P<0.01,P<0.01),and the expression levels of GS,CyclinDl,CDK4 and CDK6 mRNA were significantly increased(P<0.01,P<0.01,P<0.01,P<0.01).There were no significant differences in the expression levels of AKT,GSK3?,GS,CyclinD1,CDK4 and CDK6 mRNA between the Apo group and the E-Apu group(P>0.05).5.2 Western-blot:compared with the normal group,the expression levels of AKT and GSK3? proteins in the knee cartilage of the model group were significantly increased(P<0.01,P<0.01),and the expression levels of GS,CyclinD1,CDK4 and CDK6 proteins were significantly decreased(P<0.01,P<0.01,P<0.01,P<0.01).Compared with the model group,the expression levels of AKT and GSK3? proteins were significantly decreased in the Apo group and E-Apu group(P<0.01,P<0.01),the expression levels of GS and CDK4 proteins were significantly increased in the Apo group and E-Apu group(P<0.01,P<0.05),and the expression levels of CyclinDl,CDK4 and CDK6 proteins were significantly increased in the Apo group(P<0.01,P<0.01,P<0.01).Compared with E-Apu group,GS protein expression level was significantly increased in Apo group)(P<0.05),while AKT,GSK3?,CyclinDl,CDK4 and CDK6 protein expression levels were not significantly different between the two groups(P>0.05,P>0.05,P>0.05,P>0.05).5.3 Immunohistochemistry:compared with the normal group,the expression levels ofAKT and GSK3? proteins in the knee cartilage of the model group were significantly increased(P<0.015,P<0.01),and the expression levels of GS,CyclinD1,CDK4 and CDK6 proteins were significantly decreased(P<0.01,P<0.01,P<0.01,P<0.01).Compared with the model group,the expression levels of AKT and GSK3? proteins in the knee cartilage in the Apo group were significantly lower(P<0.01,P<0.01),and the expression levels of GS,CyclinDl,CDK4 and CDK6 proteins were significantly higher(P<0.05,P<0.05,P<0.05,P<0.05).In the E-Apu group,the expression levels of AKT and GSK3? proteins were significantly lower(P<0.01,P<0.05),and the expression levels of GS,CyclinDl and CDK4 were significantly higher(P<0.05,P<0.05,P<0.05).There were no significant differences in AKT,GSK3?,GS,CyclinDl,CDK4 and CDK6 protein expression levels between the Apo group and the E-Apu group(P>0.05,P>0.05,P>0.05,P>0.05,P>0.05,P>0.05).Conclusion1 Apo intervention can significantly reduce the local inflammatory reaction of the knee joint in KO A model,alleviate joint pain,and enhance joint activity.2 Apo intervention can significantly reduce the content of serum COMP and urine CTX-?,alleviate matrix degradation,improve cartilage surface damage,chondrocyte disorder,reduce the number and other pathological morphology,and promote repair of cartilage damage.3 Apo intervention can significantly regulate the mRNA and protein expression of AKT-GSK3? pathway and CyclinDl-CDK4/6 complex,so as to improve the glucose metabolism,proliferation and repair potential of chondrocytes.This may be one of the mechanisms by which Apo signals promote the repair of KO A cartilage injury downstream of AKT. |
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