| Object: Gouty arthritis seriously affects the quality of life of patients,to the family and society has brought great pressure and heavy economic burden.Juanbilijieqinfang is the agreement prescription in our hospital,the clinical application of nearly twenty years,the acute gouty arthritis patients with obvious curative effect,clinical curative effect is exact.This study intends to from the overall level and cell level to study the ability of Juanbilijieqinfang regulates TLR4-My D88 cell signaling pathway in synovial tissue and in macrophages,to prove that the mechanism of Juanbilijieqinfang in the treatment of gouty arthritis.Methods:The whole animal experiment: 48 rats were divided into A,B,C,D 4 groups,each group of 12.A represents the normal control group;B represents model group;C represents Juanbilijieqinfang treatment group;D represents a positive drug etoricoxib treatment group;A knee gouty arthritis rat model was used for intervention of Juanbilijieqinfang,with etoricoxib as the control drug group,48 h intervention respectively,Immediately after modeling,4h,12 h,24h and 48 h were measured to observe the changes of knee joint swelling in the knee joint;The changes of serum uric acid level were detected by abdominal aorta blood collection.HE staining was used to observe the pathological changes of the synovial membrane of the knee joint;Immunohistochemical method was used to detect the contents of TLR4,My D88,NF-kappa B protein in the synovial tissue of knee joint;Western-Blot method was used to detect the contents of PPAR-gamma,IKK-beta,IκB-alpha protein in the synovial tissue of knee joint in each group;The expression of TLR4 m RNA and My D88 m RNA in synovial tissue of knee joint was detected by RT-PCR;The IL-1 beta,IL-6,TNF-alpha,TGF-beta expression of in rats was detected in synovial tissue with ELISA.The correlation between the against the main effect indicators gout and the main effects of TLR4/My D88 signaling pathway was analyzed.In vitro experiments using macrophages(J774.1),Cell experiments were divided into two parts: 1.No addition of TLR4 receptor inhibitor;addition of TLR4 receptor inhibitor.Each part of the cells were randomly divided into 4 groups,group A(normal control group),B group(model group),group C(Juanbilijieqinfang group)and group D(Etoricoxib group).In this study,MSU model was added to the culture medium to simulate the macrophage model in inflammatory environment,Preparation of Juanbilijieqinfang containing serum,to explore the best intervention serum addition by MTT;Intervention 48 h with the medium containing 10% fetal bovine serum with DMEM high glucose and Juanbilijieqinfang serum.The contents of My D88,IKK-beta,I kappa B-protein in macrophages of each group were determined by Westen-blot;RT-PCR was used to detect the TLR4 m RNA,PPARgamma m RNA in macrophages after intervention;ELISA was used to detect the TNF-alpha,IL-6,IL-1 beta,and TGF-beta in macrophage culture medium after intervention;The content of NF-kappa B protein in macrophages was detected by immunofluorescence and the nuclear translocation was observed.Reasult: Juanbilijieqinfang can effectively improve the model of experimental gout arthritis in rats in general situation,reduce the joint swelling degree and the level of blood uric acid,effectively improve the pathology of synovial tissue,plays the role of anti gout.Juanbilijieqinfang can effectively inhibit the expression of TLR4 and My D88 protein in experimental rat model of gouty arthritis synovial tissues;it can also inhibit the expression of TLR4 m RNA and My D88 m RNA in synovial homogenate;It can effectively inhibit the expression of NF-kappa B,IKK-beta protein in synovial tissue of rats with experimental gouty arthritis;It can effectively enhance the expression of PPAR-gamma and I kappa B-alpha in synovial tissue of rats with experimental gouty arthritis;It can effectively enhance the expression of PPAR-gamma and I kappa B-alpha in synovial tissue of rats with experimental gouty arthritis;It can effectively improve the content of TGF-beta 1 in synovial tissue of knee joint;It can effectively inhibit the expression of TNF-alpha,IL-6,IL-1 beta and inhibit the content of these cytokines in the synovial tissue of knee joint.Correlation analysis between anti gout effect index and protein expression of cell signaling pathway,The results showed that there was a negative correlation between the expression of PPAR-gamma and I kappa B-alpha protein and the swelling index of joints(intervention 48h);There was a positive correlation between the expression of TLR4,My D88,IKK-beta and NF-kappa B protein and the swelling index of joints(intervention 48h);There was a negative correlation between the expression of PPAR-gamma and I kappa B-alpha protein and serum uric acid;There was a positive correlation between the expression of TLR4,My D88,IKK-beta and NFkappa B protein and the swelling index of joints(intervention 48h).The plasma with Juanbilijieqinfang can effectively increase the expression of PPAR-gamma m RNA and I kappa B-alpha protein in the inflammatory macrophage model;it can effectively inhibit the expression of TLR4 m RNA,My D88 protein and IKK-beta protein;It can effectively improve the content of TGF-beta 1 in the inflammatory macrophage model culture medium;It can effectively inhibit the expression of TNF-alpha,IL-6,IL-1 beta,and can inhibit the content of these cytokines in the inflammatory macrophage model culture medium.The experiment were compared with that of TAK-242(TLR4 receptor inhibitor),and the results showed that the above pathway and its upstream and downstream molecular(factor)levels(except TGF-beta)were significantly inhibited.Conclusion: Juanbilijieqinfang can effectively improve the model of experimental gout arthritis in rats in general situation,joint swelling degree,reduce the level of blood uric acid,effectively improve the pathology of synovial tissue,and then plays the role of anti gout.The expression of Juanbilijieqinfang can effectively inhibit the inflammatory signal pathway of TLR4,My D88,IKK-beta,NF-Kappa B protein in model synovial tissue and macrophages in the model;And the content of TNF-alpha,IL-6,IL-1 beta in the model and the culture medium of macrophages were reduced effectively;At the same time,it can effectively up regulate the expression of PPAR-gamma,I kappa B-alpha,and TGF-beta in synovial membrane and macrophages;There was a correlation between the index of anti gout effect(joint swelling index and serum uric acid)and TLR4/My D88 signaling pathway related indexes(TLR4,My D88,IKK-beta,I kappa B-alpha,NF-kappa B and PPARgamma protein expression).The regulation of TLR4/My D88 pathway and its upstream and downstream molecular(factor)levels in inflammatory macrophages can be partially blocked by TAK-242(TLR4 receptor inhibitor).Thus play a role in inhibiting inflammation.Juanbilijieqinfang may become innovative drugs in the treatment of gouty arthritis,it can play a regulatory role in TLR4/My D88 cell signal transduction pathways of gouty arthritis in the course of disease,is worthy of further study. |
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