| ObjectiveIn this study,based on the previous proof of sensitization in Heding and Yanglingquan acupoints in the pathological state of rat model of knee osteoarthritis(KOA)established by monoiodoacetate(MIA).To explore the effects of electroacupuncture,hand acupuncture and warm acupuncture on matrix metalloproteinase family(MMPs),metalloproteinase inhibitor family(TIMPs)and inflammatory factors in KOA model rats from the point of view of behavior,morphology,molecular biology and histochemistry,as well as the possible mechanism of improving KOA.And compare the different effects of the three so as to provide experimental basis for clinical selection of better acupuncture methods for the treatment of KOA.MethodsA total of 50 SPF male SD rats were randomly divided into five groups:normal group(N),model group(M),warm acupuncture group(W),electroacupuncture group(D)and hand acupuncture group(S),with 10 rats in each group.1 Mold making methodThe KOA rat model was established by injecting(MIA)into the right knee joint of monoiodoacetate.The modeling period was 14 days(before the end of the model,1 rat died in S group).2 Intervention MethodsThe normal group did not receive any treatment,while the model group was only injected with MIA to make the model.After the successful establishment of the model in the intervention group,in group S was treated with acupuncture at Heding and Yanglingquan points,flat tonifying and relieving and purging manipulation,200 rpm minling 1 min,retaining needle 20 min,10 min once,D group acupuncture Heding and Yanglingquan acupoints,flat tonifying and relieving and purging manipulation,200 rpm minling 1 min,electroacupuncture,soothing wave,1 mA,2 Hz,retaining needle 20 min.In group W,acupuncture at Heding and Yanglingquan points was performed with flat tonifying and relieving and purging manipulation,200 rpm minling 1 min,needle handle was inserted into Ai Zhu,and the needle was retained for 20 min.All the above groups were intervened every other day for 28 days.3 Sampling MethodAfter pentobarbital sodium anesthesia,the venous blood of fundus plexus was taken and perfused with normal saline.6 rats in each group were randomly selected to take fresh articular cartilage of the affected side and detected by Western blot method.The knee joints of 3 randomly selected rats in each group were fixed,decalcified and embedded,and then detected by HE staining,fast green staining and immunohistochemical staining.4 Detection Methods(1)Behavioral science:the rats were scored by Lequesne MG before and after modeling,and the behavioral tests of mechanical stimulus retraction threshold,thermal stimulation retraction latency and bipedal balance difference test were performed after modeling and intervention.(2)the pathological changes of cartilage were observed by HE staining and the S afranine-O and Fast Green staining.(3)the protein expression of MMP-1,MMP-3 and TIMP-1 in cartilage tissue was detected by Western blot.(4)Immunohistochemical staining was used to detect the expression and positive expression of MMP-1?MMP-3 and TIMP-1 in cartilage.(5)the expressions of MMP-1,MMP-3,TIMP-1,IL-1? and CTX-? in serum were detected by ELISA.Results(1)the results of Lequesne MG score showed that there was no significant difference in knee joint score among all groups before modeling,but after modeling,except for group N,the scores of the other four groups were significantly higher than those before modeling(P<0.05).The differences of retraction latency and bipedal balance between groups M,W,D and S before intervention were significantly lower than those in group N(P<0.05).The results of group D,W and S had no significant difference compared with group M(P<0.05).After intervention,the levels of W,D and S groups were higher than those of M group,and except for the retraction threshold of mechanical stimulation,the value of group D was significantly higher than that of group M(P<0.05)other results were not statistically significant.(2)the results of HE staining showed that there were pathological changes in group M,W,D and S compared with group N,in which group M showed uneven surface of cartilage,obvious destruction of collagen in the whole layer of cartilage,obvious vacuoles and obvious destruction of cytoplasm.Compared with group M,the surface of articular cartilage in group W,D and S was slightly uneven,the cytoplasm was only reduced,and fibrosis appeared in each layer of collagen,and vacuoles were occasionally seen in group S.(3)Safranine-O and Fast Green staining results showed that,compared with the N group,pathological structural and morphological changes occurred in the M,W,D and S groups.Among them,in the M group,the cartilage surface was uneven,chondrocytes hyperplasia was obvious,they were arranged disorderly,clustered,staining loss was obvious,and the tides were irregular.The surface of cartilage in group W and D was basically smooth,while that in group S was not.Among them,group D has lighter color fading and more regular tidal line.(4)The results of immunohistochemical staining showed that.MMP-1:in group M was significantly higher than that in group N(P<0.05),while that in group W,D and S was significantly lower than that in group M(P<0.05),and that in group D was significantly lower than that in group W(P<0.05),and the trend was group W>group S>group D.MMP-3:in group M was significantly higher than that in group N(P<0.05),while that in group W,D,S was significantly lower than that in group M(P<0.05),and that in groupW and group S was significantly higher than that in group D(P<0.05),and the trend was S group>W group>D group.TIMP-1:in group M,W,D and S was significantly higher than that in group N(P<0.05),and the trend was group D>group S>group W>group M.compared with group M,group D and S were significantly higher than group M(P<0.05)group W and group S was significantly lower than group D(P<0.05).(5)The results of ELISA assay showed that the serum level in MMP-1:M group was significantly higher than that in N group(P<0.05),and that in D group was significantly lower than that in M group(P<0.05).MMP-3:M group was significantly higher than N group(P<0.05)D group was significantly lower than M group(P<0.05)W group was higher than D group(P<0.05).T1MP1:N,M and W groups were significantly lower than those in group D(P<0.05).IL-1? in group M was significantly higher than that in group N(P<0.05).The level of IL-1? in group D was significantly lower than that in group M(P<0.05).The level of W in group D was significantly higher than that in group D.CTX-? in group M was significantly higher than that in group N(P<0.05).CTX-? in group W and S was significantly lower than that in group M(P<0.05).The level of CTX-? in group W was significantly higher than that in group D(P<0.05).(6)The results of Western blot method showed that the cartilage tissue in MMP-1:M group was significantly higher than that in N group(P<0.05),and that in S group was significantly lower than that in M group(P<0.05).The level of MMP-3:M in group N was significantly higher than that in group N(P<0.05),and that in group S was significantly lower than that in group M(P<0.05).TIMP-1:W,D and S groups were significantly higher than those in N group(P<0.05),and W in D and S groups were significantly higher than those in M group(P<0.05).Conclusion1 Injection of monoiodoacetate into the knee joint of rats can establish a stable KOA model,which provides a basis for the study of different acupuncture methods on knee osteoarthritis.2 It is preliminarily proved by a variety of detection methods that three different acupuncture methods of hand acupuncture,electroacupuncture and warm acupuncture can improve the articular cartilage injury of KOA model rats by intervening Heding and Yanglingquan.Compared with hand acupuncture and warm acupuncture,electroacupuncture may better reduce the expression of MMP1,MMP-3 and IL-1? and increase the expression of TIMP-1,thus reducing the cleavage of CTX-? and effectively reducing the degradation of cartilage matrix.It can protect the destruction of type ? collagen and improve the behavioral function. |
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