| ObjectiveAs one of. the effective external treatment in Traditional Chinese Medcine(TCM), Moxibust ion (Mox) has advantages of the simple operation, painlessness and significantly curative effect, there was evidence that Mox shows definite clinical effectiveness in the treatment of osteoarthritis (OA)of the knee, so it is one of the distinctive Traditional Chinese Medicine therapy of preventive treatment of disease.However, the mechanisms of the therapeutic effect of Mox on knee OA are not very well understood, and there are few reports about the protective effect of Mox.Guided the methodology of the modern system scientific and advanced experimental techniques of modern medicine made use of, the model of rabbit's knee OA was established with modified with immobilized in extension position, ongoing modeling of the rabbits were imposed mild Mox for 8 weeks, the articular cartilage of knee was harvested for observed in histology and morphology, Interleukin-1β(IL-1β), Tumour necrosis factor-α(TNF-α), apoptosis rate of chondrocyte, expression of Bcl-2, Bax, p53 protein and Bcl-2, Fas messenger RNA (mRNA), therapeutic and protective effect of Mox on OA of rabbits and its mechanisms were observed in different levels of tissues, cells and molecules, in order to provide evidences of Mox on OA of the knee in clinic.Methods40 healthy Japanese White Rabbits were randmonly divided into 4 groups,10 for each group:group A, group B, group C, group D. The left hind limb of animals in group A, B, C were immobilized with plaster cast in extension position, animals in group A were treated with Mox applied on Guanyuan(CV 4), Zusanli (ST 36), Xuehai (SP 10),Neixiyan(EX-LE4),Dubi(ST 35) and Yanglingquan(GB 34) (each acupoint for 10 min once a day), animals in group B were treated with chitosan(20mg/ml,0.2ml per inject, once a week) and in group C with nothing,animals in group D were used for normal control. After 8 weeks, morphological and histological changes of each group were evaluated through X-ray photographic, naked eye, optical microscope, transmission electron microscopic(TEM) and Mankin's core by examination the change of cartilage in histology and morphology, the concentrations of IL-1βand TNF-αin joint synovial fluid were detected by ELISA, the apoptosis was detected by the terminal deoxnucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, the expressions of Bcl-2,Bax,p53 protein and Bcl-2,Fas mRNA of chondrocytes were detected by Immunohistochemical (IHC) and Real-Time PCR.Results1. X-ray:femorotibial joint space width of group A and B was normal, and no osteophytes were found, femorotibial joint space width of group C became obvious narrowed with osteophytes. Observation under naked eyes:the articular surface of group A and B was smooth and intact,but the joints of group C exhibited from oedema to cartilage ulceration and joint capsule fibrosis. Observation under the light microscope:in the group A, the articular surface was smooth and glossy with intact cells,the tidemark was integrity intact, the articular of group C became rough surface with clefts to radial zone, cloned cells and the tidemark crossed by blood vessels. Mankin score:the scores in group A and B were lower than in group C respectively(P<0.01), the scores in group A were not significantly different from in group B(P>0.05). Observation under TEM:the ultrastructure of chondrocytes in group A was similar to in group D,the membranes and nuclear membranes of chondrocytes in group A were intact with some microvilli on the surface of chondrocytes. membranes of chondrocytes in group C were incomplete with several fractures and the cells appeared to leak contents, vacuoles were also seen under the nuclear membrane with bare nuclei, changes were observed with irregular nuclei, prominent vacuoles were observed in early apoptotic cells, obliterated the cytoplasm and its organelle.2. The concentrations of IL-1βand TNF-αin joint synovial fluid were, determined using ELISA. The concentrations of IL-1βand TNF-a in group A and group B were lower than in group C respectively (P<0.01), the concentrations of IL-1βand TNF-αin group A were not significantly different from in group B (P>0.05).3. To localize the apoptotic cell death within the cartilage, we stained formalin-fixed, paraffin-embedded cartilage sections by using TUNEL assay, TUNEL staining labels fragmented DNA that was localized in the nucleus.A few of scattered TUNEL-positive cells in group A and group B was detected in the superficial zone of the articular cartilage,lots of TUNEL-positive cells in group C was detected from the superficial zone to the deep zone.We quantified apoptotic cell death rate using Apoptosis index (AI). AI for chondrocytes in group A and group B was lower than in group C respectively (P<0.01), AI for chondrocytes in group A was not significantly different from in group B (P>0.05).4. IHC analysis showed the expression of Bcl-2, Bax, p53 protein in articular cartilage.The rate of Bcl-2-positive cells in group A and group B was higher than in group C respectively(P<0.05), the rate of Bcl-2-positive cells in group A was not significantly different from in group B(P>0.05). The rate of Bax-positive cells in group A and group B was lower than in group C respectively (P<0.05), the rate of Bax-positive cells in group A was significantly different from in group B(P<0.05). Bcl-2-positive cells/Bax-positive cells in group A was higher than in group B, C, D (P<0.05).The rate of P53-positive cells in group A and group B was lower than in group C respectively (P<0.01), the rate of P53-positive cells in group A was not significantly different from in group B (P>0.05).5. The levels of Bcl-2,Fas mRNA were quantified using by Real-Time PCR. The levels of Bcl-2 mRNA in group A, B, C were 1.38±0.96 fold,1.27±1.15 fold,1.08±0.89 fold, compared with in group D respectively. The levels of Bcl-2 mRNA in group A, B were significantly different from in group C respectively (P<0.01), the levels of Bcl-2 mRNA in group A was not significantly different from in group B respectively (P>0.05).The levels of Fas mRNA in group A, B, C were 0.27±0.07 fold,0.31±1.13 fold,2.32±1.35 fold, compared with in group D respectively. the levels of Fas mRNA in group A, Bwere significantly different from in group C respectively (P<0.01), the levels of Fas mRNA in group A was not significantly different from in group B respectively (P>0.05).Conclusions1. The model of rabbit's knee OA can be successful established with modified with immobilized in extension position for 8 weeks.2.Mox on Guanyuan (CV 4), Zusanli (ST 36), Xuehai (SP 10),Neixiyan (EX-LE4),Dubi(ST 35)and Yanglingquan(GB 34) show some effectiveness in the treatment of OA of the rabbit'knee.3.Mox can decrease the joint synovial fluid contents of IL-1βand TNF-α, up-regulate the mRNA and protein expression of Bcl-2 as well as down-regulate the protein expression of Bax,P53 and the mRNA expression of Fas,thereby to inhibit the apoptosis of cartilage cells and delay the degeneration of articular cartilage changes.4.Chitosan can inhibit the apoptosis of cartilage cells and delay the degeneration of articular cartilage changes. |
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