Repair Effects Of Close-to-bone Needing Combined With Electroacpuncture On Extracellular Matrix Of Cartilage In Rabbits With Knee Osteoarthritis

ObjectiveTo observe the effectiveness of short trust needling(STN,close-to-bone needing)combined with electroacupuneture(EA)in regulating expressions of propeptide(PIICP),C-telopeptide of type II collagen(CTX-II)and cartilage collagen type II/discoidin domain receptor/ matrix metalloproteinase 13(collagen type II/DDR2/MMP13)signaling pathway in rabbits with knee osteoarthritis(KOA),and to explore the possible action mechanism of this method on repair of extracellular matrix of knee cartilage.MethodsForty New Zealand white rabbits were randomly assigned into a normal group(10 rabbits)and a model establishing group(30 rabbits).The Hulth-Telhag technique was applied to establish the model of KOA,X-ray and Lequesne MG were used for outcome assessment.The rabbits with successful modeling were randomly assigned into a model group,a close-to-bone needing group,a regular acupuncture group,10 rabbits in each one.The rabbits in the close-to-bone needing group were treated with close-to-bone needing and EA;the rabbits in the regular acupuncture group were treated with regular acupuncture and EA."Neixiyan"(EX-LE4),"Dubi"(ST 35),"Yinlingquan"(SP 9),"Zusanli"(ST 36)and "Liangqiu"(ST 34)were selected in the two groups.The intervention was given for 20 min,once a day;the intervention of 5 days made 1 session,2 days as the interval and totally 4 sessions were given.Rabbits in normal and model group were immobilized without any treatment.After treatment,Lequesene MG was used for therapeutic evaluation.Then,lateral meniscus and cartilage in the left knee joints were cut out.HE staining was used to observe the pathological change of knee;transmission electron microscopy(TEM)was used to observe the cell structure of knee joint cartilage;western blotting method was applied to evaluate the expressions of DDR2,MMP13 and collagen type II;the activity of collagen type II,DDR2 and MMP13 was assessed by immunohistochemistry method;the mRNA expression of DDR2 and MMP13 was determined by reverse transcription-polymerase chain reaction(RT-PCR);the contents of serum PIICP and CTX-II were assayed by ELISA.Results1.As shown by the X-ray examination,rabbits in the Normal group had no significant pathological changes,and Narrowing of medial articular space,rough articular surface,osteophyte formation at the articular margin were found in the KOA rabbit.2.As shown by the HE staining results: compared with Normal group,Model group showed chondrocyte arrangement disorder with clusters,severer cell nucleus splitting and degeneration,many vacuoles;STN+EA group showed relatively regular arrangement of chondrocytes with clusters,fewer abnormal vacuoles,even cytoplasm and the cellular nucleus being in the center.Similar results were found in the EA group.As shown by the TEM staining results: compared with Normal group,Model group showed pyknosis of chondrocytes with partial dissolution and apoptosis,irregular shape,peri-cellular halo,vague karyotheca and cytoplasm organelles;STN+EA group showed abundant cellular organelles,basically normal cellular nuclei,relativelt uniform chromatin,plenty of rough endoplasmic reticulum with a few of lipid droplets;EA group showed uniform chromatin,abundant organelles with a few of lipid droplets.3.As shown by the Lequesne MG scores: 6 weeks after modeling,the rabbits in Model group,STN +EA group and EA group all showed a swollen knee with local ache reactions,and ranges of motion were between 15 and 45 scope.Compared with Normal group,the Lequesne MG scores of other 3 groups were considerably up-regulated(P<0.01);10 weeks after modeling,ache reactions of the rabbits in STN+EA group and EA group were relieved,ranges of motion were between 45 and 90 scope;Compared with 6 weeks after modeling,the Lequesne MG scores STN+EA group and EA group were notably down-regulated(P<0.01).4.As shown by WB,ELISA,RT-PCR and immunohistochemistry results: Compared with the Normal group,the levels of collagen type II and PIICP were significantly reduced in the other groups(all P<0.01),while the activity and mRNA expressions of DDR2,MMP 13 and CTX-II were notably increased(all P<0.01).Compared with the Model group,the expressions of collagen type II and PIICP in the STN+EA group and EA group were increased(all P<0.01),while the activity and mRNA expression of DDR2,MMP 13 and CTX-II were reduced(all P<0.01).Compared with the EA group,the activity and mRNA expression of MMP 13,DDR2 and CTX-II in the STN+EA group were reduced(all P<0.01),while the activity expression of collagen type II and PIICP were increased(all P<0.01).ConclusionThe KOA models were created successfully.The two acupuncture methods both made modulation of cartilage cells,played a role in inhibiting the degradation and in promoting the composition of extracellular matrix and thus accelerate the repair of cartilage.Compared with simple electroacpuncture,STN+EA had better effects on KOA.The mechanism may be associated with the blocking effect of collagen typeII /DDR2/MMP13 signaling pathway.