Discussion Acupuncture Therapy KOA Rabbit Model Of Femoral Cartilage Repair Rectus Mechanism Based On The "tone Muscle Treatment Of Bone" Method

Background:Knee osteoarthritis (KOA) is the most common chronic, progressive, degenerative joint disease among patients over40years old. It is characterized by degeneration of articular cartilage, bone sclerosis, and hyperplasia manifested as progressive knee joint pain, stiffness, swelling, dysfunction of the knee joints, which may also develop into joint deformity, even loss of joint function, greatly affecting patients’ normal life and work. Globally, KOA with high rate of disability, accounts for about6%of the adult population. Its incidence in women was slightly higher than that in men at around about1.5:1~4:1. However, its etiology and pathogenesis are still unclear. There is still no cure for the disease and preventive measures.Traditional Chinese medicine has been shown to be effective for KOA, especially the acupotomy therapy in the treatment of KOA either alone or combined with other therapies. As it is easy, convenient, inexpensive and effective, it has become a mainstream therapy for this disease. At present, behavior, morphology, biomechanics, molecular biology approaches have been utilized in exploring the mechanism of acupotomy in the treatment KOA from perspectives of central analgesia, cytokine, cartilage cell apoptosis, and extracellular matrix. Studies on effects of the acupotomy therapy on soft tissue around the knee, and relation between skeletal muscle injury and articular cartilage repair have been rare.This study attempts to explore the effect and mechanism of acupotomy for KOA based on a National Natural Science Foundation Project’Intervention effect of Acupotomy on KOA ligament mechanical changes and cartilage cell signal conduction’ lead by Prof. Guo Changqing. The relationship between injury-repair of skeletal muscle and articular cartilage, or the sinew-bone balance, is the breakthrough point.Objective:To verify the theory that modulating sinew (Jin) and treating bone (Gu) could resume their balance and achieve therapeutic effects, to provide the scientific basis for a variety of therapies in traditional Chinese medicine for KOA under the guidance of the theory of muscle-sinew theory.Methods:Forty-five6-months old, health and sanitary leve, New Zealand rabbits were randomly divided into five groups (n=9in each group):normal control group, model group, acupotomy (AP) group, electro-acupuncture (EA) group and Round-sharp Acupuncture (RSA) group.8rabbits of each group were measured while the remaining one was used for validation of morphological modeling after4weeks. The left knee joint was fixed with plaster bandage in extension position to establish the rabbit model of knee joint injury with the Modified Videman method. Acupotomy, electro-acupuncture and round-sharp Acupuncture were respectively applied in AP, EA and RSA group.EA group:left "Yanglingquan (GB34) with Yinlingquan (SP9)","Neixiyan (Ex-LE04) with Waixiyan (ST35)", using rarefaction-dense and continuous wave of2/100Hz,3mA, for20min each time,3times/week,3weeks in total;AP group:the front edge of the midpoint, the starting point and the stopping point of the medial collateral ligament, lateral collateral ligament and patellar ligament. The ligament edge line and loosening direction should keep parallel,5times respectively, once a week,3weeks in total; the needle points of RAS group are same with AP group,5times respectively, once a week,3weeks in total.In the first part and the second part of the study, bilateral knee passive range (PROM) and the Lequesne MG total index was evaluated respectively at different time window after modeling. X-ray and MRI were used to assess the radiological changes of KOA rabbit knee joints. HE staining was used to observe the morphology of rectus femoris muscle, myocardial fiber rupture and abnormal cells though the ordinary optical microscope. Tidal line clearance, articular cartilage and calcified layer and deep cartilage changes were measured. Ultra structural changes of the rectus femoris were observed using transmission electron microscope while those of articular cartilage surface, by scanning electron microscope, to compare the differences between the AP, EA and RSA therapy. In the third part and the fourth part, Apoptotic index was measured using TUNEL method. Expression of Bcl-2and baxand Caspase-3proteins were assessed with western blot tests. MMP-1, so were MMP-3, MMP-13, TIMP-1mRNA by RT-PCR and TIMP-1proteins by western blot tests.Result:1. AP, EA and RSA therapy can effectively relieve articular pain and movement disorder in KOA rabbits, by reducing Lequesne MG knee arthritis index (pain, joint swelling, and deformity of knee joint) and passive motion (PROM) index behavior significantly. X-ray findings of articular cartilage of knee joint damage, joint space narrowing and osteophytosis demonstrated significant improvement as well. It was also found to have a certain improving effect on knee joint MRI synovial hyperplasia, and joint deformities. The effects were similar among three groups.2. Soft tissue and cartilage injury and repair appeared to exist at the same time around the knee joints in experimental KOA rabbits. From morphology observation, repairing effects of AP, EA and RSA therapy on skeletal muscle and cartilage were definite. Under the microscope, HE staining of the rectus femoris showed that all of the three therapies could effectively inhibit joint local inflammatory response, promoting the recovery of normal muscle fiber structure. AP group demonstrated improved effectiveness than EA and RSA group, while the latter two were similar in effects. Under transmission electron microscope, AP group, skeletal muscle fibers arrangement in EA and RSA groups appeared more orderly than those in the model group, as the M line, Z line was restored, the amount of mitochondrial bodies gathered, and presence of some of the new filament generation regeneration. The effects were similar among three groups. HE of the articular cartilage stained sections revealed that AP, EA and RSA therapy could effectively inhibit the cartilage degeneration, promote regeneration and repair of cartilage with AP group being superior to EA and RSA group, RSA group better than EA group. Under the scanning electron microscope, it seemed that three therapies could all effectively improve the surface structure of cartilage destruction with AP group being superior to EA and RSA group, and RSA group better than EA group.3. TUNEL findings showed that among the three types of intervention methods for reducing the apoptosis of skeletal muscle cells, AP group and RSA group were more effective than EA group (P<O.05). Bcl-2proteins were observed to have low level expressions in cartilage damage at4weeks after modeling and there was no significant difference between three groups (p>0.05); Expression of Bax and Caspase-3proteins, model group and EA group increased significantly (P <0.05) compared with normal control group; AP group and RSA group decreased significantly (P <0.05) compared with model group, while the results were opposite in terms of the ratio of Bcl-2/Bax. AP and RSA therapy using relaxing methods could reduce the Bax, Caspase-3protein expression, increase the ratio of Bcl-2/Bax, and inhibit the apoptosis of the rectus femoris in KOA rabbits. This showed that the treatment of KOA mechanism may be related to the inhibition of skeletal muscle lesions around the joint, mainly through the mitochondria pathway of apoptosis, the mechanism of which seemed different from E A.4. Low level expressions of MMP-1mRNA were observed in cartilage damage at4weeks after modeling, and there was no significant difference between groups (p>0.05). Expression of MMP-1protein decreased significantly (P<0.05) in model group compared with normal control group. In comparison with normal control group after modeling, TIMP-1mRNA increased significantly (P<0.05) in model, EA, AP and RSA groups, and it was much higher in EA group than that in model group (P<0.05). Expression of TIMP-1protein decreased significantly (P<0.05) in model and AP group when compared with normal control group. But it increased significantly in EA and RSA group compared with model group (P<0.05). Expression of MMP3, MMP13mRNA and proteins increased significantly (P<0.05) in EA group and model group, in comparison with normal control group while the expressions in AP group and RSA group decreased significantly (P<0.05), and MMP3mRNA level in EA and model group was much higher than that in normal control group (P <0.05) and lower in AP and RSA group compared with model group (P<0.05). AP and RSA therapy seemed to have inhibitive effects on MMP-3, MMP-13mRNA and protein expression in the articular cartilage of KOA rabbits. It suggests that the mechanism of AP and RSA therapy may be related to regulation of matrix metalloproteinase family, which is different from that of EA.Conclusion:1. Acupotomy therapy can inhibit the knee joint inflammation, synovial proliferation reaction, and reduce the degeneration of cartilage matrix and bone sclerosis, hyperplasia and other pathology, so as to improve the KOA knee joint pain, swelling and dysfunction2. Acupotomy therapy can improve the injury of the femoral rectus and cartilage tissue of KOA effectively, and can promote the regeneration and repair of muscle fiber and cartilage cells; the normal structure and function would be thus restored3. Acupotomy therapy may inhibit the rectus femoris muscle cell apoptosis, improve the extracellular matrix MMPs balance of cartilage, and promote the repair of rectus femoris and cartilage. This mechanism is different from EA.4. AP therapy probably works mainly by releasing the soft tissue around knew joints and relieving tension within the joint to restore the mechanical balance of the joint in the treatment of KOA. It is so-called modulating sinews and treating bones to achieve therapeutic effects.