| Object:Based on miRNA-146a-mediated TLR4 signaling pathway,study the effect of Juanbi Capsule on the immunoinflammatory response of fibroblast-like synoviocytes and osteoblasts in osteoarthritis,research the effect of Bushen Yiqi Fang Juanbi capsule on early osteoarthritis Molecular mechanism.Methods:(1)Twenty normal 5-month-old female infertile guinea pigs were used to establish the primary guinea pig OA model by using the biological characteristics of spontaneous OA,The fibroblast-like synovial cells and subchondral osteoblasts were isolated and cultured by trypsin-I collagenase digestion,,and the production of cell climbing tablets,The morphological characteristics of the cells were observed by microscopic observation after the HE staining.The expression of Vimentin,CD90,CD55 and CD68 in fibroblast-like synoviocytes was detected by immunohistochemistry and immunofluorescence staining and determination of Alkaline Phosphatase Activity in Cartilage Osteoblasts by Azo-Coupling Alkaline Phosphatase Staining,the fibroblastlike synovial cells and subchondral osteoblasts were isolated and identified.(2)A total of 32 female rats were randomly divided into sham operation group,model group,Juanbi capsule group and Anding Ding group.There were 4 rats in each group.The model of KOA in postmenopausal kidney model was established by the method of resection of bilateral ovaries combined with guinea pig spontaneous knee osteoarthritis in model group,Juanbi capsule group and Anding Ding group,While the sham operation group were treated with the same size and size of the adipose tissue around the ovary.Four groups of guinea pigs were followed for 30 days after the end of the operation,Among them,Juanbi capsule group were given Juanbi capsule,and Andingding group were given Andingding capsule,sham operation group and model group were given the same amount of distilled water gavage.After 5 days of continuous administration,the rats were divided into sham operation group,model group,Juanbi capsule group and Anding Ding group.The fibroblast-like synoviocytes and subchondral osteoblasts were cultured in the serum of each group.After 5 days of continuous intervention,the RNA and protein of the fibroblast-like synoviocytes and subchondral osteoblasts were extracted,Collecting the cell culture liquid at the end of each intervention,and preparing the cell supernatant by centrifugation;Elisa measured the levels of IL-1?,MMP-13 and TNF? in supernatants of fibroblast-like synovial cells and subchondral osteoblasts;The expression of micro RNA-146 a,TLR4,My D88,TRAF6,NF-?B p65 and TNF? were detected by RT-PCR.SPSS 21.0 statistical software was used to analyze the experimental data.Results:(1)Experimental separation of fibroblast-like synovial cells were mostly irregular spindle-shaped,triangular,polygonal,oval-shaped nucleus located in the central cell,morphologically observed from the characteristics of fibroblast-like cells,Immunohistochemistry and immunofluorescence staining showed that Vimentin,CD90 and CD55 were strongly positive and CD68 was negative,which confirmed that it was the fibroblast-like synovial cells needed for the experiment;The morphology of the subchondral osteoblasts was mostly spindle-shaped,triangular,and the nuclei were oval along the long axis of the cells.Morphological observation was consistent with the characteristics of osteoblasts,azo coupling method alkaline phosphatase(ALP)staining results were strong positive,confirmed that the experiment required for subchondral osteoblasts;(2)Elisa measured estrogen levels in serum of shamoperated and model guinea pigs,and the results were shown that:The mean serum concentration of E2 in guinea pigs was 76.24±3.73,and the average concentration of serum E2 in model group was 45.39±4.38,the model group was significantly lower than that in sham operation group(P<0.05),the estrogen levels in the model group were significantly lower than those in the sham operation group;The levels of IL-1?,MMP-13 and TNF? in supernatants of fibroblastlike synoviocytes were measured by Elisa.The results showed that:Compared with the sham group,the levels of IL-1?,MMP-13 and TNF? in the supernatant of the model group were significantly higher than those in the sham group(P<0.05),Compared with the model group,the levels of IL-1?,MMP-13 and TNF? in the supernatant of Juanbi capsule and Anding decoction group were significantly lower than those in the model group(P<0.05),There was no significant difference between Juanbi capsule group and Andingxian group(P>0.05);The levels of IL-1?,MMP-13 and TNF? in supernatants of osteoblasts were measured by Elisa.The results showed that:Compared with the sham group,the levels of IL-1?,MMP-13 and TNF? in the supernatant of the model group were significantly higher than those in the sham group(P<0.05);Compared with the model group,the levels of IL-1?,MMP-13 and TNF? in the supernatant of Juanbi capsule and Anding Ding group were significantly lower than those in the model group(P<0.05),there was no significant difference between Juanbi capsule group and Anding Ding group(P>0.05);(3)Fibroblast-like synovial cells by RT-PCR results show that:Compared with the sham group,the expression of TLR4,My D88,TRAF6,NF-?B p65 and TNF? in the fibroblast-like synoviocytes were up-regulated and the expression of mi RNA-146 a in the model group was signifi-cantly lower than that in the sham-operated group(P<0.05),Compared with the model group,the expression of TLR4,My D88,TRAF6,NF-?Bp65 and TNF?in fibroblast-like synoviocytes were down-regulated compared with the model group,while the expression of mi RNA-146 a was significantly up-regulated,P<0.05,The expression of TLR4,My D88 and TNF? in fibroblast-like synovial cells were significantly different between the Juanbi capsule group and the Andingxian group(P<0.05),but there was no difference in the expres-sion of TRAF6 and NF-?B p65;The results of osteoblasts RT-PCR showed that: Compared with the sham group,the expression of TLR4,My D88,TRAF6,NF-?Bp65 and TNF?in the osteoblasts of the model group were up-regulated,and the expression of mi RNA-146 a in the model group was significantly lower than that in the sham group(P<0.05),Compared with the model group,the expression of TLR4,My D88 and TNF? were down-regulated in the osteoblasts,and the expression of mi RNA-146 a was significantly upregulated(P<0.05),TRAF6,NF-?Bp65 expression of the difference was not statistically significant;Compared with model group,the expression of TLR4 and My D88 in osteoblast osteoblasts was down-regulated,and the expression of mi RNA-146 a was significantly up-regulated,P <0.05,The expression of TRAF6,NF-?B p65 and TNF? was not statistically significant,Juanbi capsules group and security will butoxy group,subchondral osteoblasts TNF?differentially expressed,P<0.05,and mi RNA-146 a,TLR4,My D88,TRAF6,NFk Bp65 expression was no significant difference,P>0.05.Conclusion:(1)The primary cells isolated from the 5-month-old female guinea pig OA model were confirmed to be fibroblast-like synoviocytes and subchondral osteoblasts;(2)Bushen Yiqi Fang Juanbi capsule can upregulated mi RNA-146 a and down-regulated the expression of TLR4,My D88,TRAF6,NF-?Bp65,TNF?,reduce the levels of IL-1?,MMP-13 and TNF?in the cells,Regulating fibroblast-like synovial cells within the inflammatory immune response,thereby inhibiting synovial inflammation immune response,play a role in the treatment of osteoarthritis;(3)Bushen Yiqi Fang Juanbi capsule can upregulated mi RNA-146 a and down-regulated the expression of TLR4,My D88 and TNF?,reduce the levels of IL-1?,MMP-13 and TNF?in the cells,regulation of subchondral osteoblasts inflammatory immune response,play a role in the treatment of osteoarthritis. |
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