| ObjectiveTo study the expression of related genes and proteins in synovial fluid,synovium and cartilage of rabbit knee osteoarthritis model treated with Miaoyao fumigation therapy,and to explore the mechanism of regulating SDF-1/CXCR4 signaling pathway by vascular endothelial growth factor in osteoarthritis.Methods1.Thirty-six New Zealand white rabbits were randomly divided into normal group(group A)and model B-F group(6 rabbits in each group).In group A,0.2 ml sterile saline was injected into the left knee joint cavity;in group B-E,0.2 ml sodium iodide acetate solution was injected into the left knee joint cavity at doses of 2.5,5,7.5 and 10 mg/kg,respectively.In group F,the same dosage was injected as in group E,and 30 min/d driving exercise was used twice as the intervention factor.The pathological changes of cartilage in each group were observed by HE staining and histopathological score of osteoarthritis(Mankin score).2.(1)Experimental group:50 healthy adult New Zealand white rabbits,male and female,weighing(2.0±0.2)kg,were randomly divided into 5 groups,10 in each group,which were normal group,model group and positive control group.Early intervention group,Miao medicine fumigation group.(2)Modeling:In addition to the normal group,the other 4 groups were injected with sodium iodide in the knee joint cavity to establish a model of knee osteoarthritis.(3)experimental intervention:on the second day after modeling,the early intervention group was given Miao medicine fumigation treatment with fumigation preparation for 8 weeks.Two weeks after model establishment,the positive control group was given Futalin ointment for external treatment for 6 weeks.The Miao medicine fumigation group was treated with Miao medicine fumigated liquid for 6 weeks.(4)experimental materials:After the treatment,the animals were killed by air injection through the ear vein,and the samples were collected.(5)observation indicators:a.visually observe the general changes of the synovial membrane and cartilage.b.using hematoxylin and eosin staining method,the pathological changes of synovial membrane and cartilage were observed under light microscope;c.immunohistochemical method was used to detect the positive expression of IL-1 βand TNF-α in synovial tissue;d.Elisa detection method was used to detect the expression of MMP-3,MMP-9,MMP-13 and VEGF in serum,and the expression of SDF-1 in synovial fluid was detected.e.Real-time quantitative PCR was used to detect the expression of MMP-3 mRNA,MMP-9 mRNA,MMP-13 mRNA and VEGF mRNA in cartilage tissue.f.Western blotting detection of Col-2 and aggrecan expression in cartilage,expression of NF k B,P38 MAKP and PTGER2 in synovial tissue.Result1.Compared with group A,there was no significant difference in group B and C(P>0.05),but there was significant difference in group D,E and F(P<0.05).There was no significant difference between group F and group E(P>0.05).2.(1)visual observation:a.Synovial membrane:Compared with the normal group,the other 4 groups showed different degrees of synovial hyperplasia,the model group was the heaviest,and the positive control group,the early intervention group and the Miao medicine fumigation group were lighter than the model group;b.Cartilage:Compared with the normal group,the other 4 groups showed different degrees of cartilage damage,and the model group was the heaviest.The positive control group,the early intervention group and the miaoyao fumigation group improved compared with the model group.(2)HE staining of synovium and cartilage:Compared with the normal group,the scores of the other four groups were higher,with statistical significance(P<0.05);compared with the model group,the scores of the positive control group,the early intervention group and the Miao fumigation group were lower,with statistical significance(P<0.05);there was no difference in the scores between the positive control group,the early intervention group and the Miao fumigation group(P<0.05).The difference was not statistically significant(P>0.05).(3)immunohistochemistry results:Compared with the normal group,the IOD values of IL-1 β and TNF-α in synovial tissue were increased,the difference was statistically significant(P<0.05).Compared with the model group,the IOD values of positive control group,early intervention group and Miao fumigation group were lower,with significant difference(P<0.05).There was no significant difference among the positive control group,early intervention group and Miao fumigation group(P>0.05).(4)Elisa results:Compared with the normal group,the serum levels of MMP-3,MMP-9,MMP-13 and vascular endothelial growth factor in the other four groups were significantly higher(P<0.05).Compared with model group,the serum levels of MMP-3,MMP-9,MMP-13 and VEGF in positive control group,early intervention group and Miao fumigation group were significantly lower(P<0.05).There was no significant difference among the positive control group,early intervention group and Miao fumigation group(P>0.05).Compared with the normal group,the content of SDF-1 in the synovial fluid of the other four groups increased significantly(P<0.05).Compared with the model group,the SDF-1 content in the synovial fluid of the three treatment groups decreased significantly(P<0.05).There was no significant difference between the 3 groups in the treatment group(P>0.05).(5)RT-PCR test results:Compared with the normal group,the expression of MMP-3,MMP-9,MMP-13 and VEGF in cartilage tissue of the other four groups increased significantly(P<0.05).Compared with the model group,MMP-3,MMP-9,MMP-13 and VEG in cartilage tissue of the three groups were significantly lower(P<0.05).There was no significant difference between the 3 groups in the treatment group(P>0.05).(6)WB test results:Compared with the normal group,the expression of type Ⅱ collagen and aggregation protein polyglycoprotein in cartilage tissue of the other four groups decreased,with significant difference(P<0.05).Compared with the model group,the expression of type II collagen and aggregation protein polyglycoprotein in cartilage tissue of the three groups increased,with significant difference(P<0.05),and the expression of type II collagen and aggregation protein polyglycoprotein in cartilage tissue of the three There was no significant difference in the expression of type I collagen and aggregation protein polyglycoprotein between groups(P>0.05).Conclusion1.The drug concentration of group D was 7.5mg/kg,which was the minimum effective drug dose for establishing OA model.The method of establishing the model was simple and less invasive,and could ensure the stability of knee joint and the survival rate of experimental animals.2.(1)SDF-1 can induce articular cartilage degeneration in rabbits with osteoarthritis.(2)Miao medicine fumigation therapy can delay the synovitis and cartilage degeneration of osteoarthritis models in rabbits.(3)There was no significant difference in the treatment of osteoarthritis in rabbits between the Miao medicine fumigation group and the control group.Miao medicine fumigation therapy can be chosen to replace the external use of Votalin ointment in clinic,because there is a treatment cycle in the clinical,can not be used for a long time,and long-term use has obvious side effects.(4)Miao medicine fumigation therapy may inhibit the expression of cytokines such as MMP-3,MMP-9,MMP-13,VEGF and SDF-1 by blocking the binding of SDF-1 to its receptor CXCR4,thus delaying the development of osteoarthritis and playing a therapeutic role. |
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