| Objective:osteoarthritis(OA) is a chronic degenerative disease of motion system, its etiology is complex, the joint cartilage cells, extracellular matrix(ECM), subchondral bone degradation and synthesis which losing balance caused by mechanical and biological factors. Among them, the articular cartilage ECM equilibrium synthesis and degradation of destruction is an important reason of OA cartilage degeneration. Matrix metalloproteinase (MMPs) is a kind of important degradation enzyme in all the enzymes involved in articular cartilage of ECM and basement membrane degration process, the tissue inhibitor of metalloproteinase(TIMPs) is a specific inhibitor of the MMPS, dynamic environment within the ECM remain largely dependent on MMPs/TIMPs, the two plays important role in the OA cartilage degeneration. Electro-acupuncture(EA) is a method by acupuncture stimulation will be low frequency pulse micro current into the corresponding points, and after stimulating the skin, muscle, joint capsule, ligament proprioceptor input, lend a amount of body movement and cutaneous sensory information into the central, thereby regulating muscle tension, relieve spasm, analgesic. EA is a very effective non-operation treatment method for the early metaphase of OA. But at present, the clinical research is the predominant,the mechanism research is little. In this paper by HE staining, immunohistochemical technique observing the electro-acupuncture in the treatment of rabbits knee articular cartilage after OA gross morphological changes, chondrocytes for cartilage tissue pathological and MMP-9, TIMP-1 expression changes. To discuss the EA treatment of knee osteoarthritis(KOA) mechanism on the cell, molecular level, foundation for the experimental study in the future, but also provide a theoretical basis for clinical work. Methods:(1) The research participants and intervention measures: healthy 30 New Zealand rabbits with big ear were selected, randomly divided into three groups, namely the normal group, model group and EA intervention group. The normal group received routine feeding without any intervention, the other two groups were used left knee extension fixation by plaster cast to establish the OA model. After the model group was feeding 6 weeks, used the method of injecting air into the rabbit ear vein to execution, then incision to expose the left knee joint, the medial condyle cartilage and the relevant indicators changes were observed. At the same time, the EA intervention group according to "animal science of acupuncture and moxibustion", the joint was selected four clinical common points, "Liangqiu", "Zusanli", "Xuehai", "Yanglingquan", with 30 inches of acupuncture, needle handle is connected to the G9805-C type low frequency electric pulse therapeutic apparatus, to sparse, dense wave alternating patten, the frequency of 4Hz,20Hz, pulse width 0.5ms, each time duration of 20 minutes,1 time per day, received 10 sessions of intervention. Still take the same method to execute the EA invervention group animal, and the medial femoral condyle cartilage related index changes in all aspects were observed. (2) Observation measures: ①Left knee joint of three groups’through the macroscopic observation its morphological changes. ② Cut medial condylar articular cartilage of the rabbit left femur,40g/L paraformalde-hyde solution fixed,10%EDTA decalcifying fluid decalcification, routine paraffin embedding, sectioning, after HE staining, microscopic observation on the morphology of chondrocytes, tidal line integrity, according to the Peng Taiping histopathological grading and Mankin’s scoring criteria to observe the results. ③ Immunohistochemical analysis:with rabbit anti-MMP-9, rabbit anti-TIMP-1 kit to detect the nomal group, model group and EA intervention group rabbits left knee femoral medial condyle cartilage changes in expression. (3) Statistical methods:using SPSS 15.0 statistical software, the measurement data results processing and analysis are expressed with mean±tandard devia-tion(x±s), P<0.05 had Statistical significance. Results:(1) cartilage gross morphological observation:the normal group without any intervention had no obvious articular cavity effusion, catilage surface was smooth, the appearance of cartilage surface was blue and white, color depth and flood light, no cracks, the joint suface and synovial is Ⅰ level, cartilage structure of Mankin’s score is 0. The model group used unbend fix with plaster cast for 6 weeks after knee joint cavity almost appears the joint effusion, articular surface is not smooth and slightly evenlessness, a few parts appear erosion, all of the articular cartilage surface were dark and yellowish color, gloss than nomal. EA intervention group part molding joint is still visible effusion, synovial hyperplasia, lower gloss, without obvious erosion, compared with the normal group, there are still different, no significant erosion, cartilage surface is not smooth, but compared with the model group, has been improved. The joint suface and synovial is Ⅱ level. According to the Mankin’s score at 6 weeks with modeling after EA intervention of joint cartilage structure, the difference between the two group was statistically significant(P<0.05). (2) HE staining pathological observation: the rabbit left knee with no intervention, cartilage showed smooth surface, thickness is consistent about the layer of cartilage, the cell size is accordant and regular arrangement. The surface cells were spindle-shaped, approximate horizontal arrangement, the intermediate layer cells rounded, scattered, column layer cells arranged in a column, the tidal line complete, Mankin’s score is recorded as 0, model group staining showed cartilage surface small crack, compared with the normal cartilage thickness was significantly thinner, chondrocyte number was decreasde, cell arranged irregularly, locally presenting a cluster, the tidal line disorder, EA intervention group cartilage surface is regular, the less crack,clusters of rare, chondrocytes are arranged in rows, but it is different from the normal group, chondrocyte number was significantly increased compared with the model group, the cartilage thickness, visible tidal line disturbance. The comparison in the three groups according to Mankin’s score, the results show that they were significantly different(P<0.05). (3) Immunohistochemical determination of MMP-9 and TIMP-1:the normal articular cartilage cells cytoplasm occasionally weak expression MMP-9 and TIMP-1 positive staining, model group chondrocytes in visible strong positive MMP-9 staining granules, especially highest in the deep density, mature chondrocytes in more, deep brown granules, a small number of cells visible brown TIMP-1 positive staining. EA intervention group chondrocytes showed MMP-9 positive staining, as well as a large number of TIMP-1 strong positive staining granules. MMP-9 expession in EA intervention group decreased than model group, compared with the normal group increased(P<0.05). TIMP-1 in EA intervention group was increased than the model group, compared with the normal group was high expression(P<0.05). Conclusion:(1) The experiment used gypsum extended mechanical brake on rabbit knee six weeks can copy the OA model. (2) MMP-9 and TIMP-1 basic secretion to maintain the integrity of the articular cartilage is crucial. The imbalance between MMP-9/TIMP-1 lend the corresponding with the articular cartilage ECM excessive degradation, thereby promoting cartilage degeneration. (3) EA can slow down the degeneration of rabbit KOA articular cartilage, promote the recovery of articular cartilage injury, its mechanism may be associated with a reduction in articular cartilage of MMP-9 content, augmented secretion of TIMP-1, to effectively adjust the balance between MMPs/TIMPs, prevent ECM degradation and reduce the joint injury. |
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