Tougu Xiaotong Capsule Inhibitsthe Imbalance Of Cartilage Matrix Anabolism And Catabolism In Osteoarthritis By Regulating MiRNAs Expression

ObjectiveTo investigate the mechanism of Tougu Xiaotong Capsule inhibiting the imbalance of cartilage matrix anabolism and catabolism in osteoarthritis by regulating miRNAs.Methods1.Random allocation was taken to averagely di-vide the 30 2-month-old SPF grade SD male rats into the control group(10 rats,sham operation,normal saline 10 mL/kg,1 time/day orally),the model group(10 rats,modified-Hulth technique modeling,normal saline 10 mL/kg,1 time/day orally)and the TXC group(10 rats,modified-Hulth technique modeling,Tougu Xiaotong Capsule 10 mL/kg,1 time/day orally).After rats were continuously administrative by drenching for 12 weeks,then put them to death.The morphology of articular cartilage was respected by HE staining;the changes of proteoglycans in articular cartilage were observed by toluidine blue staining.The expressions of IL-lβ,TNF-α and MMP-13 in synovial tissue were identified by ELISA.2.The chondrocytes of the rat was got from 10 4-week-old SPF grade SD male rats by the mechanical type Ⅱ collagenase digestion method.The chondrocytes were cultured in vitro and identified by type Ⅱ collagen immunohistochemical staining and toluidine blue staining.The model of inflammatory chondrocytes was induced by LPS.The concentration of Tougu Xiaotong capsule was established by ELISA.To determine the final experimental group and intervention methods,chondrocytes were divided into control group,model group(10 ng/mL LPS),inhibitor group(added 10 ng/mL LPS after 10 μmol/mL SB203580 intervened 30 min),Tougu Xiaotong capsule group(10 ng/mL LPS+300 p^g/mL TXC),intervention for 8 h.ELISA was used to detect the expressions of IL-6,MMP-9,TIMP-1.QRT-PCR was used to detect the expressions of miRNA-140,miRNA-146a,miRNA-27b.The expressions of p38 MAPK,p-p38 MAPK,MMP-3,MMP-13,ADAMTS4 and ADAMTS5 were identified by Western-blot.The expressions of p-p38 MAPK,p38 MAPK,MMP-3 and ADAMTS4 were detected by immunofluorescence staining.The expressions of p38 MAPK,MMP-3,MMP-13,ADAMTS4 and ADAMTS5 were detected by QRT-PCR.Results1.The expressions of IL-1(3,TNF-a and MMP-13 in the synovial tissue of the knee of the model group were significantly higher than that of the control group(P<0.05,P<0.01),the TXC group were significantly lower than that of the model group(P<0.05,P<0.01).There were no significant differences between the TXC group and the control group(P>0.05).Toluidine blue staining showed that the control group was dark and homogeneous,and the model group was infected with a wide range.The color of the TXC group was between that of the control group and the model group.HE staining showed that the articular cartilage of the control group was complete and the chondrocytes were arranged neatly.The differentiated chondrocytes were non-founded.In the model group,we found the articular cartilage was seriously damaged and its layer was thinner than the control group.Simultaneously,the chondrocytes were exposed,arranged disorderly and appeared hypertrophy differentiation.The articular cartilage of the TXC group was intact,and the condition of the chondrocytes was between the control group and the model group.2.LPS-induced inflammatory chondrocytes were established at 10 ng/mL for 8 h and the intervention concentration of TXC was 300 μg/mL.Compared with the control group,the expressions of IL-6 and MMP-9 in the model group were increased(P<0.05,P<0.01),the expression of TIMP-1 was reduced(P<0.05).Compared with the model group,the expressions of IL-6 and MMP-9 in the TXC group were considerably decreased(P<0.05,P<0.01),and the expression of TIMP-1 was significantly increased(P<0.01).The expressions of miRNA-140 and miRNA-27b in the model group were significantly lower than that in the control group(P<0.01),and the TXC group was significantly higher than that in the model group(P<0.01).The expression of miRNA-146a in the model group was significantly higher than that in the control group(P<0.01),and the TXC group was lower than that in the model group(P<0.05).The expressions of MMP-3,MMP-13,ADAMTS4,ADAMTS5,p38 MAPK and p-p38 MAPK protein in the model group were higher than that in the control group(P<0.05,P<0.01),and the TXC group was lower than that in the model group(P<0.05).The expressions of p38 MAPK,MMP-3,MMP-13,ADAMTS4 and ADAMTS5 gene in the model group were significantly increased than that in the control group(P<0.05,P<0.01),compared with the model group,the TXC group were significantly decreased(P<0.05,P<0.01).Conclusions1.Tougu Xiaotong Capsule could regulate the expressions of miRNAs:in chondrocytes,mediate the inflammatory reaction and inhibit the expressions of cartilage matrix degradation protein,which can maintain the stability of cartilage matrix.2.Tougu Xiaotong Capsule could inhibit the degradation of chondrocyte extracellular matrix by regulating p38 MAPK signaling pathway to mediate inflammatory response.