The Basic Research Of "Layers Adjusting External Application" On Cartilage And Synovium Mmps And Upstream NF-κB Signal Pathway In Rabbit Knee Osteoarthritis Model

[ObjectiveJBased on the clinical treatment of "Layers Adjusting External Application" (LAEA)on Knee Osteoarthritis(KOA), Observe the expression of IL-lbeta, MMP-1,MMP-13 and TIMP-1 after local application of LAEA, investigate the relationship between LAEA and the inflammation celluar signal pathway NF-κB, clarify the targeting effect and mechanism of LAEA treating KOA.[Method] Experiment 1:Thirty rabbits (weighing 2.5±0.1kg) with half male and half female were randomly divided into three groups:normal group, control group and treatment group,10 animals in each. After 1 week’s adaptive breeding, the rabbit KOA models were operated by Anterior Cruciate Ligament Transection(ACLT).6 weeks after operation, only treatment group was treated by external therapy of LAEA for two weeks, once per day. At the eighth week, all rabbits were sacrificed, synovium tissues were harvested. Expression of MMP-1 and MMP-13 were examed by Western Blot, gene expression of IL-1β、MMP-1 and MMP-13 were examed by RT-PCR.Experiment 2:Thirty rabbits (weighing 2.5±0.1kg) with half male and half female were randomly divided into three groups:normal group, control group and treatment group,10 animals in each. After 1 week’s adaptive breeding, the rabbit KOA models were operated by Anterior Cruciate Ligament Transection(ACLT).6 weeks after operation, only treatment group was treated by external therapy of LAEA for two weeks, once per day. At the eighth week, all rabbits were sacrificed, cartilage tissues were harvested. Cartilage surface histological observation was operated by HE staining. Expression of MMP-1, MMP-13 and TIMP-1 were examed by Western Blot, gene expression of IL-1β、MMP-1, MMP-13 and TIMP-1 were examed by RT-PCR.Experiment 3:Thirty rabbits (weighing 2.5±0.1kg) with half male and half female were randomly divided into three groups:normal group, control group and treatment group,10 animals in each. After 1 week’s adaptive breeding, the rabbit KOA models were operated by Anterior Cruciate Ligament Transection(ACLT).6 weeks after operation, only treatment group was treated by external therapy of LAEA for two weeks, once per day. At the eighth week, all rabbits were sacrificed, cartilage tissues were harvested. Expression of TLR-4 and NF-kB p65 were examed by Western Blot, gene expression of TLR-4、NF-κB p65 were examed by RT-PCR.[Results] Experiment 1:Expression of synovial MMP-1 and MMP-13 in the control group were higher than treatment group(P<0.05), normal group were all the lowest(P<0.05), whereas synovial MMP-1, MMP-13 mRNA expression were significantly decreased in treatment group compared with control group(P<0.05).Gene expression of IL-lbeta in normal group was lower than control group and treatment group (P<0.01), nevertheless there was no difference between control and treatment group.Experiment 2:Histological assessment demonstrated control group scored significantly higher than normal group (P<0.01) and treatment group scored significantly lower than control group (P<0.01) in accordance with Mankin Score. Expression of cartilage MMP-1, MMP-13 in the control group were higher than treatment group(P<0.01), normal group were all the lowest(P< 0.01), whereas cartilage MMP-1, MMP-13 mRNA expression were significantly decreased in treatment group compared with control group(P<0.01). Protein and gene expression of TIMP-1 in treatment group was higher than control group in cartilage(P<0.01). The protein and mRNA expression of cartilage IL-lbeta showed no difference between control and treatment group.Experiment 3:Western Blot indicated that expression of TLR-4 and NF-κB p65 in control group was higher than normal group(P<0.01), TLR-4、NF-κB p65 in treatment group was significantly lower than control group(P<0.01), cartilage NF-κB p65 showed no difference in treatment and normal group. Gene expression of TLR-4 and NF-κB p65 in control group was higher than normal group(P<0.01), NF-kB p65 gene expression in treatment group was lower than normal group(P<0.05), whereas TLR-4 gene expression in treatment group was significantly lower than normal group(P<0.01).[Conclusion]The "Layers Adjusting External Application"(LAEA) could down-regulate the protein and gene expression of MMP-1 and MMP-13 in the rabbit synovium and cartilage, meanwhile LAEA exerted the expression of TIMP-1,which recovered the dynamic balance of MMPs and TIMP-1.To some extent, LAEA may reduce the degradation of chondrocytes extracelluar matrix, protect the cartilage. Furthermore, LAEA showed certain inhibition effect of the expression of upstream NF-κB signal pathway proteins, which coincidentally matched the clinical application. In this research, LAEA failed to inhibit the protein and gene expression of IL-lbeta, however the clinical application of LAEA can significantly alleviate the local swelling and pain, thus we speculate that LAEA can relieve the symptoms of local inflammation, which predominately not through the IL-1beta-induced pathways.