To Investigate The Mechanism Of Tongluo Zhitong Recipe Regulating Synovial Inflammation In Knee Osteoarthritis Based On Toll-like Receptor-mediated Inflammatory Network Control

Knee osteoarthritis(KOA)is one of the most common diseases among the elderly,the main reason leading to pain and disability.Inflammation mediated by the Toll-like receptors(TLRs)network is considered in the development of synovitis and articular cartilage inflammation activation and catabolism activities positive role,leading to the progress of KOA.We hope to systematically study the apoptosis of cartilage and synovial membrane under the influence of inflammation network during KOA process,the distribution of the inflammatory cells,expression of TLR4 in All kinds of cells,the variety of important inflammatory factors,to enrich the current theory of inflammation,and further estimate the possible mechanism Tongluo Zhitong Formula used to the synovial inflammation of knee osteoarthritis.It is of great practical significance to explore the pathogenesis of KOA and the development of new therapeutic targets and strategies.Section 1 Study on the correlation between synovial inflammation and TLRs and pathogenesis of KOAExperiment 1 Study on the Conditions and relationships between apoptosis of cartilage and synovial cells in knee osteoarthritisObjective:To discuss the role and relationship of cartilage and synovial cells in knee osteoarthritis(KOA),by the detection of the apoptosis of them in the different course of KOA.Methods:We used 72 male Wistar rats,and we divided them into 6 groups randomly,normal group(N),the false surgical group(F)and OA model group(2W,4W,8W and 12W),each group had 12 rats.The KOA model of the right knee joint was prepared by using the classic Hulth modeling method.N group weren't treated,while F group were opened the joint cavity,do not make model.model groups 4W,8W and 12W(mild,moderate and severe)were used for sampling at specified time points.TUNEL method was used to detect the apoptosis of synovial membrane and chondrocytes in each group,and then the variation was analyzed.Results:There was no significant difference between the apoptosis rate of cartilage and synovium in the false surgical group and the normal group(P>0.05),indicating the success of the model.And in the process of disease,cartilage and synovial membrane revealed a different trend.In the comparison of cartilage apoptosis,the apoptosis rate of the N group,mild,moderate and severe group presents significantly higher in turn(P<0.01).In the comparison of synovial cell apoptosis,there is no significant difference in apoptosis rate between the mild model group and normal group(P>0.05),moderate model group was significantly higher than mild model group(P<0.01),while in severe model group appeared to decreasing trend.In addition,synovial cells of rats in mild group appear to obvious hyperplasia under the microscopic.Conclusion:Excessive apoptosis and synovial inflammation are accompanied by the whole process of cartilage damage and degradation,while the synovial excessive apoptosis appeared in the middle and late stage of the course.Inference:early stage Inflammatory mediators released by synovial inflammatory reactions may trigger the excessive apoptosis of cartilage.Prompting:Synovial inflammation in the KOA inherent immune inflammatory factor network may play a leading role.Early clinical intervention in synovitis can slow the progress of KOA and help protecting cartilage.Experiment 2 Study on the distribution of synovial inflammatory cells in knee osteoarthritis and the site of innate immunity mediated by TLRsObjective:To investigate the pathogenesis of KOA and the role of innate immunity in pathogenesis,the distribution of fibroblast,immune cells in knee osteoarthritis(KOA)and the expression range of TLR4 were observed.Methods:We used 24 male Wistar rats,and we divided them into two groups randomly:normal group(N)and modle(4W)group,each group had 12 rats.The classic Hulth modeling method was applied to prepare the KOA animal model for the right knee joint of the rat model group,and after the model was made for 4W,they were used for sampling.Main application of double immunofluorescence analysis of synovial cell markers and TLR4 common dyeing were used to observe the distribution of the fiber cell and immune cell infiltration in KOA synovial tissue and the expression of TLR4 range.Results:Six kinds of specific markers,Vimentin,CD68,CD3,CD20,CD38,CD15 respectively represents:synovial fibroblasts,macrophages and T cells,B cells,plasma cells,neutrophils,its proportion respectively in the N group:81.43%,12.95%,4.26%,0.47%,0.08%and 0.11%;In the KOA group the proportions were 62.17%,24.27%,8.94%,1.97%,2.14%and 0.51%.In the KOA group,the proportion of immune cells in all cells increased significantly from 18.57%to 37.83%.And among the ratio between the immune cells in contrast,in KOA group and N group the overwhelming majority were macrophages,T cells proportion was higher,and the proportion of B cells and neutrophils in the KOA group reduced significantly,and plasma cells reduce the most(from 5.67%to 0.38%).In TLR4 staining,TLR4 expression was found in all the above six cells,and it was significantly more than normal groups in the KOA group.Conclusion:In synovitis,inflammatory cells increased obviously,in which macrophages occupied the t majority and T cells proportion are higher,while the proportion of B cells and neutrophils in synovitis decreased significantly,and plasma cells reduced the most.In KOA synovitis,the site of innate immunity mediated by TLRs including:synovial fibroblasts,macrophages and T cells,B cells,plasma cells,and neutrophils,and when KOA occurs,TLR4 expression were significantly active in the site above mentioned.Section 2 Study on the role of inflammatory networks mediated by TLRs in KOAExperiment 1 Study on the distribution and variation of synovial inflammatory cytokines IL-1? and TNFa in the process of knee osteoarthritisObjective:The expression of the important inflammatory cytokines IL-1? and TNF? in the synovial membrane during the process of knee osteoarthritis(KOA)and their relative ratio was discussed.Methods:We used 72 male Wistar rats,and we divided them into 6 groups randomly:normal group(N),false surgical group(F)and OA model group(2W,4W,8W and 12W),each group had 12 rats.The classic Hulth modeling method was used to make the modle.N group weren't treated,and F group were only opened the joint cavity.In the end of 2W,4W,8W and 12W,KOA group rats were used for the sampling.The relative expression of IL-1? and TNF-? in each group of synovial samples was detected by western blot method.Results:The expression of TNF-? and IL-1? during the course of 2w(early stage),4w(mild),8 w(moderate)KOA model,presents significant increasing trend,and maintained at a certain concentration in 8w and 12w,while in 12w declined slightly.In addition,after careful analysis we can see that the expression.Western blot detection method was used to detect the protein expression of IL-? and TNF-? in each group of synovial samples,and then the variation was analyzed.of IL-1?in 2w-4w increased obviously,and the expression of TNF-? was slightly lagginger than interleukin,there was a marked increase in 8w moderate KOA model group.Conclusion:Experiment shows that IL-1? and TNF-? in the process of the development and progression in KOA were accompanied by,related to each other and synchronous change.While IL-1? has a leading role,provide data support for the whole cytokine network research,which laid a foundation for exploring the pathogenesis of KOA,offered a new way and targets for the drug therapy,provided the basis for further implementation of the new KOA treatment strategies.Experiment 2 Study on the correlation between the expression of important inflammatory factors in synovial membrane in knee osteoarthritisObjective:To investigate the expression and correlation of the important inflammatory cytokines IL-1?,TNF-? and MMP-13 in knee osteoarthritis(KOA)process.Methods:We used 72 male Wistar rats,and we divided them into 6 groups randomly:normal group(N),false surgical group(F)and OA model group(2W,4W,8W and 12W),each group had 12 rats.The classic Hulth modeling method was used to make the modle.N group weren't treated,and F group were only opened the joint cavity.In the end of 2W,4W,8W and 12W,KOA group rats were used for the sampling.First,total RNA and reverse transcription were extracted,and the relative expression of IL-1?,TNF-? and MMP-13 in each group of synovial samples was detected by real-time PCR,and then the correlation was analyzed.Results:Three inflammatory cytokines gene expression in F group and N group did not see significant difference(P>0.05),and mRNA and GAPDH relative expression of IL-1?,TNF-? and MMP-13 in each modle group were significantly higher than in N group(P<0.05),in 2w,4w and 8w model group three kinds of inflammatory factor expression presents increasing trend in turn,and in week 12w in model group they appeared different degree of decline trend.IL-1? was highly correlated with TNF-?,with a correlation coefficient of 0.86 and a significant probability of 0.00;IL-1? is also highly correlated with MMP-13,with a correlation coefficient of 0.89 and a significant probability of 0.00.Conclusion:It was found that IL-1? and TNF-? and MMP-13 had the similar expression trend in the whole process of KOA,and had a high correlation,and provided data support for the whole of cytokine network research,so as to lay a good foundation to explore the pathogenesis of KOA,offers a new way and targets for the drug therapy,provide the basis for further implementation of the new KOA treatment strategies.Section 3 The influence of the sovereign medicinal of tongluozhitong throughInflammatory network mediated by TLRs on inflammation of OA synovial membraneExperiment 1 The experimental study on the synovial fibroblast cell cycle intervention by sovereign medicinal of tongluozhitong decoction external treatmentObjective:To investigate the influence of concentration of sovereign medicinal discretion of cassia twig,radix paeoniae alba water decoction on the cell cycle of fibroblast-like synovial cells(FLS),as well as to explore sovereign medicinal of cassia twig,radix paeoniae alba's therapeutic effect and target on synovial tissue inflammation of knee osteoarthritis(KOA).Methods:Under the intervention of cassia twig and radix paeoniae alba water decoction,using flow cytometry(FCM)to detect the cell cycle of FLS,to observe and analyze cassia twig and radix paeoniae alba's influence on the growth of FLS.Results:Cassia twig and radix paeoniae alba of high concentration and low concentration can significantly shorten S phase for DNA replication(P<0.01),prolong the G1,G2(P<0.05,while in G2 stage cassia twig of high concentration P<0.01;In G1 phase radix paeoniae alba of the low concentration of was P>0.05),indicating that cassia twig and radix paeoniae alba of high concentration and low concentration can significantly block DNA replication,thus inhibiting the proliferation of FLS.In addition,both cassia twig and radix paeoniae alba were dependent on the concentration of the liquid,both of which had a higher effect in higher concentration than in low concentration.Conclusion:cassia twig and radix paeoniae alba of high and low concentrations can significantly inhibited the overproliferation of FLS,among them,cassia twig and radix paeoniae alba decoction of high concentration were more effective.By inhibiting the FLS excessive proliferation to treat KOA.it is one of the pathways to treat KOA with drug,that is to say interventing FLS' excessive proliferation is the new target of KOA therapy,but the mechanism remains to be further research.Experiment 2 The study on the important inflammatory cytokines in knee osteoarthritis intervened by sovereign medicinal of tongluozhitong decoction external treatmentObjective:To investigate the influence of concentration of sovereign medicinal discretion of cassia twig,radix paeoniae alba water decoction on the KOA synovial inflammation cells,as well as to observe the change of the inflammatory cytokines interleukins(Interleukin,IL)-1 beta,tumor necrosis factor alpha(necrosis factor alpha,TNF alpha),matrix metalloproteinases(matrix metalloproteinase 13,MMP-13)and NO,to explore the mechanism of action of cassia twig and radix paeoniae alba,reveal the ways and targets of drug,so as to lay a good foundation to find the pathogenesis of tongluozhitong decoction external treatment.Methods:The researchers collected abandoned synovial tissue cleared from the knee joint in the operation,trained again after by collagenase(type I)digestion,gathered liquid supernatant,under intervention of high and low concentrations cassia twig and radix paeoniae alba water decoction,detected the concentration of supernatant fluid after the intervention using enzyme-linked immunosorbent method.Results:Cassia twig and radix paeoniae alba of high and low concentrations significantly decreased the release of IL-1?,TNF-?,TMMP13 and NO(P<0.05).High concentrations of both drugs were significantly better than low concentrations(P<0.05).It is indicated that both drugs are dependent on the high and low levels of concentration.Conclusion:Both the high and low concentrations of sovereign medicinal of tongluozhitong decoction external treatment(cassia twig,radix paeoniae alba)can interfer with the important inflammatory factors in KOA significantly.It is shown that cassia twig,radix paeoniae alba can effectively reduce the destruction of articular cartilage by interfering with the secretion of inflammatory factors in KOA,so as to achieve the purpose of treatment of KOA.