Effect Of "Jiajian Bianji" On The Mechanical Properties Of FAK-PI3K-AKT Pathway And Quadriceps Muscle In Rabbit Articular Cartilage Of KOA Rabbits

Knee Osteoarthritis(KOA) is a retrogressive disease with joint pain, tenderness, movement dysfunction, with a hallmark of cartilage degeneration, involved with mucle, tendon, joint capsule and other soft tissues around knee joint. KOA is a leading cause of joint deformity or even disability, and seriously decline the quality of patients’ lives, and it is a significant economic burden. According to epidemiological data, radiographic KOA evidence is observed in over 60% of the population over age 65, affecting an estimated 85% of the population more than 65 years of age in our country. No cure or treatment currently exists that offers disease-or symptom-modifying effects and etiology is unclear currently. Acupotomy therapy as a new minimally invasive treatment of traditional Chinese medicine for KOA has the advantages of simple, cheap, effective, safety. In past studies, the analgesic and anti-inflammatory mechanism of acupotomy for KOA was verified. The effect of biomechanics and promoting cartilage repair of acupotomy may be a very important potential preventive and therapeutic strategy for KOA and is the main reason that the effect of acupotmy for KOA is prominent efficacy. This research is based on a National Nutural Science Foundation"The effector mechanism of Tendon Regulation and Bone Treatment with acupotomy for KOA and its relation with FAK-PI3K-AKT mechanical signal transduction pathway" leaded by Prof. Guo Changqing, attempting to explore the effect and mechanism of acupotomy for KOA and the relationship with FAK-PI3K-AKT pathway, discussing the scientificity of Tendon Regulation and Bone Treatment.Objective:With a key point of the relationship between repair of articular cartilage injury and mechanical properties changes of quadriceps femoris, to verify the molecular biology mechanism of acupotomy promoting cartilage repair and effect on mechanical properties changes of quadriceps femoris, revealing the scientificity of Tendon Regulation and Bone Treatment, to provide the experimental evidence and theoretical basis of acupotomy for KOA under the guidance of muscle-sinew theory.Method:In the first experiment,28 health and sanitary,6 months old, New Zealand rabbits were randomly divided into 4 groups(n=7 in each group):normal control group(NP), model group(MP), acupotomy group(AP), electro-acupuncture group(EA). The left knee joint was fixed in extension position to establish the KOA model. Acupomy and electro-acupuncture treatment were applied in AP and EA group respectively. In AP group, the stopping point of medial vasrus muscle tendon, lateralis vastus muscle tendon, rectus femoris muscle tendon, biceps femoris muscle tendon, pes anserinus tendon, nodule were loosed by acupotomy,2 times one week for 4 weeks. In EA group, left Liangqiu with Xuehai, Neixiyan with Waixiyan, using rarefaction-dense and continuous wave of 2/100Hz,3mA, for 20min each time,3times one week for 4 weeks. Passive range of motion(PROM) and Lequesne MG index were evaluated to assess the behavioristics changes. Safranine O-Fast green staining and Mankin index were used to assess the morphologic changes under optical microscope. Ultrastructure ohanges on surface of cartilage were assess scanning electron microscope. With these methods, the different effect of AP and EA were compared.In the second experiment,49 New Zealand rabbits were randomly divided into 7 groups(n=7 in each group):normal control group, model group, model+PF562271 group, acupotomy group, acupotomy+PF562271 group, electro-acupuncture group, electro-acupuncture+PF562271 group. Method of establishing KOA and treatment were the same to the first experiment. Inhibitor group were injected FAK inhibitoe PF562271(200μM,0.5ml) intra-articular 2h before every treatment. Expression of p-FAKs p-PI3K、p-AKT、COL-Ⅱ、 Aggrecan preoteins and mRNA were assessed with Western blot and RT-PCR. To discuss the effect of acupotomy and electro-acupuncture on catabolism and anabolism of cartilage and relationship with FAK-PI3K-AKT pathway.In the third experiment, method of establishing KOA and treatment were the same to the first experiment. Single contraction and tonic contraction of left quadriceps femoris were tested with electrophysiological methods, Young’s modulus of left quadriceps femoris were tested with SuperSonic Imagine AixPlorer SWE ultrasonic instrument, to discuss the effect of acupotomy and electro-acupuncture on mechanical property of quadriceps femoris.Results:1. Ethology results:Before treatment, compared to NP, indexes of Lequesne MG of MP, AP, EArised(P**<0.01), PROM declined(P**<0.01), and no different between MP, AP, EA; after treatment, compared to MP, Lequesne MG indexes of AP, EA declined(P##<0.01), PROM of AP declined(P#<0.05); compared to EA, Lequesne MG indexes of AP declined(P▲<0.05); PROM of AP rised(PROM of AP).2.morphology results:According to safranine O-Fast green staining and scanning electron microscope results, acupotomy and electro-acupuncture can inhibit the degradation of cartilage in different degree, and AP is better. compared to NP, Mankin index of MP, AP, EA riesd(P**<0.01); compared to MP, Mankin index of AP, EA declined(P##<0.01); compared to EA, Mankin index of AP declined(P▲<0.05).3. Western blot and Real time-PCR results:After treatment, compared to NP, p-FAK, p-PI3K protein and mRNA, p-AKT protein of MP, AP, EArised(P*<0.05 or P**<0.01); compared to MP, p-FAK, p-PI3K protein and mRNA, p-AKT protein of AP, EA rised(P#<0.05 or P#*<0.01); compared to EA, p-FAK, p-AKT protein, PI3K protein and mRNA of AP rised(P ▲<0.05); after used PF562271, p-FAK, p-PI3K protein and mRNA, p-AKT protein declined(P△<0.05 or P△ △<0.01). Compared to NP, Aggrecan, Col-Ⅱ protein and mRNA of MP, AP, EA declined (P*<0.05 or P**<0.01); compared to MP, Aggrecan, Col-Ⅱ protein and mRNA of AP, EA rised(P#<0.05 or P#<0.05); compared to EA, Aggrecan, Col-Ⅱ protein and Aggrecan mRNA of APrised(P▲▲<0.01); after used PF562271, Aggrecan, Col-Ⅱ protein and mRNA declined(P △<0.05 or P△△<0.01).4.Biomechaics results:After treatment, compared to NP, Single contraction of left quadriceps femoris of MP, EA declined(P**<0.01), tonic contraction declined(P*<0.05), Young’s modulus declined(P*<0.05); compared to MP, Single contraction of left quadriceps femoris of AP rised(P*#<0.01), tonic contraction rised(P#<0.05), Young’s modulus rised(P*<0.05); compared to EA, single contraction and tonic contraction of left quadriceps of APrised(P▲<0.05).Conclusion:1. Acupotomy can relieve pain, improve PROM and inhibit ECM degeneration.2. Acupotomy can promote anabolism of chondrocyte by activating FAK-PI3K-AKT pathway.3. Acupotomy can restore some biomechanical function of quadriceps by adjusting contraction ability and Young’s modulus.4. Acupotomy can provide the moderate mechanical stimulation for cartilage by adjusting quadriceps biomechanical properties, that is Tendon Regulation and Bone Treatment.