| BackgroundKnee osteoarthritis,with the main clinical manifestations of pain and dysfunction of knee joint,is a common degenerative joint disease.Acupotomy has achieved satisfactory efficacy for KOA,biomechanical alterations of the ligaments and the mechanics signal transduction of the cartilage cells had been used as starting point for short-termmechanism research,but still it is imperative to conduct the research about long-term efficacy mechanism.As a part of the project supported by the National Natural Science Foundation of China"Effects of Acupotomy Intervention on biomechanics change of ligaments and mechanics signal transduction of the cartilage cells",this study will continue to explore the long-term efficacy mechanism of Acupotomy therapy for KOA guided by"treating bones by modulating sinews".ObjectiveThis study is based on the JINGJIN theory of Traditional Chinese Medicine,combined with the theory that the mechanical unbalance of knee joint is the pathogenesis of KOA.The morphology of cartilage was observed by methods of X-ray,optical microscope and electron microscope,mRNA and protein expression of TGF-?1,Col-I in ligament and Bcl-2,Bax,Caspase-3 in chondrocytes were tested by methods of Real-time PCR and Western blot.It is expected to verify the long-term mechanism of acupotomy therapy for KOA may be associated with the hypothesis:By loosening the adhesions of ligaments,restoring ligaments'structure and its function,which will benefit the stability and force equilibrium of knee joint,reduce undesirable stimulation for cartilage,stop from further apoptosis and degeneration of chondrocytes,and provide experimental evidence for the long-term efficacy of clinical treatment.MethodsSix-month-old New Zealand rabbits were randomly divided into the normal control group,the model group,the acupotomy group,and the electro-acupuncture group.KOA model was established by the method of fixing the left knee joint with plaster bandage in extension position.After modeling,acupotomy group and electro-acupuncture group were treated for three weeks.Acupotomy group took the lateral,collateral ligament and patellar ligament in knee joint as loose objects,with sparse,longitudinal cross stripping method for acupotomy release,1 times a week;Electro-acupuncture(2/100 Hz,3mA,20min each time)was applied to the left GB34,SP9,EX-LE4 and ST35 for rabbits in the electro-acupuncture group,3 times a week.Praxiology was evaluated by Lequesne MG before treatment and before execution,and all the animals were subjected to knee joints X-ray before execution,pathological changes of the joint cartilage were observed by optical microscope and electron microscope.Apoptosis factor Bcl-2,Bax,Caspase-3's mRNA expression of chondrocytes and TGF-?1,COL-I's mRNA expression of ligament were detected by Real-time PCR test,and Bcl-2,Bax,Caspase-3's proteins expression level of chondrocytes and TGF-?1,COL-I's proteins expression level of ligament were conducted by Western blot test.Results1 Praxiology results:After model establishment,the model group,the acupotomy group and the electro-acupuncture group praxiology scores are significant higher than normal control group(P<0.01),3 weeks after treatments,praxiology score of the EA group and the acupotomy group was significantly lower than the model group(P<0.01),and acupotomy Group had the most significant before and after treatment scoring difference(p<0.01),which shows acupotomy treatment have long-term efficacy in praxiology of KOA rabbits.2 X-ray observation:In normal control group,surface of articular cartilage is smooth,subchondral bone' s density is uniform,no significant joint narrowing.In model group,joint space narrowing,osteophytosis roughness in joint articular surfaces,high density in subchondral bone are demonstrated.After the treatment,acupotomy group and electro-acupuncture group have varying degrees of improvement.Compared with the model group,the X-ray scores of the acupotomy group and the electro-acupuncture group decreased significantly(P<0.01),and there is no statistical difference between the electro-acupuncture group and the acupotomy group(P>0,05).3 Optical microscope observation of articular cartilage:In normal control group,surface of cartilage was smooth,chondrocytes were normal and arranged regularly;Cartilages of model group were not smooth,only few chondrocytes were in the superficial zone,different layers of chondrocytes were arranged irregularly,blurred tidemark or double tidemarks could be seen.In acupotomy group,cartilages surface were smooth,basic structure was normal and chondrocytes were arranged in neat.In electro-acupuncture group,partial cartilage in the surface was defected,chondrocytes were arranged in mild disorder,pannus could be seen.After statistical analysis,there were significant difference of acupotomy group and electro-acupuncture group in comparison with the model group(P<0.01),and electro-acupuncture group had no significant difference with acupotomy group(P>0.05).4 Electron microscope observation:In terms of cartilage surface,or the distribution of material in surface,or the size of fracture and performance of collagen fiber,cartilage tissue morphology in acupotomy group is slightly better than the electro-acupuncture group.5 Real-time PCR and Western blot Test of Ligament:After model establishment,compared with the normal group,TGF-?1,Col-I' s mRNA and protein expression in the other 3 groups were both increased(P<0.01).3 weeks after treatment,TGF-?1,Col-I mRNA expression level in acupotomy group and electro-acupuncture group were lower than the model group(P<0.01,P<0.05).and TGF-?1.Col-I mRNA expression in the acupotomy group was decreased than the electro-acupuncture group(P<0.05).TGF-?1.Col-I protein levels in acupotomy group and electro-acupuncture group were lower than the model group,and TGF-?1 protein level in acupotomy group was significantly different with the model group(P<0.01).6 Real-time PCR and Western blot Test of Ligament cartilage:After modeling,Bcl-2,Bcl-2/Bax ratio mRNA expression was significantly lower than the normal group(P<0.01),Bax,Caspase-3 mRNA expression was significantly higher than the normal group(P<0.01),Bcl-2,Bax,and Bcl-2/Bax ratio protein expression didn' t significantly change(P>0.05),while Caspase-3' s protein expression compared with the normal group had significantly increased(P<0.01).After the intervention,Bcl-2,Bcl-2/Bax ratio of mRNA expression levels in acupotomy group and electro-acupuncture group was significantly increased(P<0.01),Bax,Caspase-3 mRNA expression was significantly decreased(P<0.01),protein expression of Caspase-3 in acupotomy group had no difference(P>0.05)with the normal group,and significantly better than the EA group(P<0.01).These results suggest that,after modeling,chondrocytes apoptosis appeared,the intervention of acupotomy was better at regulating apoptosis in articular chondrocytes of KOA rabbits than the electro-acupuncture method.Conclusions1 By regulating the mRNA and protein expression of TGF-?1 and COL-I in ligaments,acupotomy can regulate the ligament matrix,restore the structure and function of ligaments,thus to restore the mechanical balance of knee joint of KOA rabbits.2 By regulating chondrocytes apoptosis,acupotomy can inhibit the pathological process of knee osteoarthritis.3 By restoring structure and function of knee ligaments,in addition to improving joint mechanical balance,but also be conducive to the stability of the knee joint,thereby reducing the ndesirable stimulation for cartilage,inhibiting apoptosis repressor process and further damage may be is the long-term mechanism of acupotomy therapy for KOA. |
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