The Mechanism Of External Application Of Sanhan Huatan Reciep Via Nf-?B Signal Pathway In Rabbits With Knee Osteoarthritis

AIM The aim of this study was to investigate the protective effect and mechanism of external application of Sanhan Huatan Recipe on articular cartilage of knee osteoarthritis(KOA)in rabbit models.Methods 36 male and female 6-month-old semi-clean New Zealand rabbits were divided into normal group,model group,positive control group(Diclofenac Diethylamine Emulgel),Sanhan Huatan Recipe low-dose,middle-dose and high-dose group randomly,and every 6 rabbits in each group.Except for the normal group,the other 5 groups of rabbits were made into the models of KOA by using the modified Hulth method,and the rabbits were driven twice a day for 30 minutes in total.6 weeks after the operation,rabbits KOA model was obtained.After the successful modeling,the normal group and the model group were externally applied with a blank matrix;The positive control group was treated with Diclofenac Diethylamine Emulgel;the low-dose,the medium-dose and the high-dose of Sanhan Huatan Recipe were applied to the low-dose,the medium-dose,and the high-dose Sanhan Huatan Recipe separately.After the 6 weeks drug intervention,the femoral condyle cartilage tissue of the knee joint and the cartilage tissue of the medial tibial plateau were obtained from rabbits.HE staining and MASSON staining were used to observe the morphological changes of articular cartilage in rabbits and the ultrastructure of chondrocytes were observed by Electron Microscopy.Detecting the IL-1?,TNF-? in serum of rabbits by ELISA.The protein expression and mRNA expression of Matrix metalloproteinase-3(MMP-3),matrix metalloproteinase-13(MMP-13),a disintegrin and metalloproteinase with thrombospondin motifs-5(ADAMTS-5)and type ? Collagen(Col-2)were detected byWestern blot and RT-qPCR.The expression of the nuclear factor ?B P65(NF-?B P65)was detected by immunohistochemistry.Results Compared with the normal group,the knee cartilage and knee joint chondrocyte structure of the model group was severely damaged.The levels of serum IL-1? and TNF-? increased significantly(P<0.01),and the MMP-3,MMP-13,ADAMTS-5 proteins and mRNA expression level was significantly up-regulated(P<0.05,P<0.01),the Col-2 protein and mRNA expression level was significantly down-regulated(P<0.01),and the expression of NF-?B P65 was significantly increased(P<0.05,P<0.01).Compared with the model group,the knee cartilage and the knee joint chondrocyte structure of rabbits in the low-,medium-and high-dose groups of Sanhan Huatan Recipe were significantly improved,and the serum IL-1? and TNF-? levels were significantly reduced(P<0.01),MMP-3,MMP-13,ADAMTS-5 protein expression levels and mRNA expression levels were significantly down-regulated(P<0.05,P<0.01),the expression of NF-?B P65 was significantly reduced(P<0.05,P<0.01).And the expression level of Col-2 protein and mRNA in cartilage of knee joints of rabbits were significantly increased(P<0.05,P<0.01)in the Sanhan Huatan Recipe low-and high-dose group.Conclusion Sanhan Huatan Recipe can protect the knee joint articular cartilage tissue of rabbits and delay the occurrence and development of KOA.The mechanism maybe to reduce the expressions of IL-1?,TNF-? of serum,and to inhibit the expression of NF-?B P65,preventing the activation of NF-?B signaling pathway to down-regulate the MMP-3,MMP-13,ADAMTS-5 and up-regulate the Col-2 protein expression levels and mRNA expression levels.