The Mechanism Study Of Fengshining On CIA Model With Wind-cold-dampness Bi Syndrome Via TLR4/NF-κB Signal Pathway

Objective:To build collagen-induced arthritis（CIA）mice and rats models with wind-cold-dampness bi syndrome,culture fibroblast-like synoviocytes（FLS）in synovial tissue of knee for CIA rats model,applying near-infraed fluorescence imaging technique,focus on TLR4/NF-κB signal pathway,from aspects of synovial tissue-synovial cell-signal pathway-targeted gene,study the mechanism of Fengshining in treating rheumatoid arthritis（RA）.Method:Part 1:a comparative study on CIA models of combined disease and syndrome with different exogenous pathogenic factors.Divide 110 SPF standard female rats into 4 groups,which are control group（10）,CIA model group（40）,wind-cold-dampness bi syndrome CIA group（30）,wind-dampness-heat bi syndrome CIA group（30）.The models of rheumatoid arthritis（RA）are prepared by using the wind-cold-dampness,wind-dampness-heat stimulation and combing with bovine type II collagen.Treating RA rats with curative effective experienced formulae Fengshining(18.24g·kg^-1),classical formulae Wutou Tang(4.9g·kg^-1)and Baihu Tang with Guizhi(10.15g·kg^-1)respectively.Applying formula-syndrome corresponding and verification methods,observe model rats’paw temperature,arthritis index（AI）,joint swelling degree,RF in serum,ACPA content and ankle’s histopathologic changes,define the syndrome attribution.Then determine the best rat model for Fengshining’s mechnism study.Part 2:application study of near-infrared fluorescence probe in imaging of CIA mice model with wind-cold-dampness bi syndrome.48 mice are evenly divided into 4 groups:control group,model group,positive control group,and Fengshining group.The RA mice model,with wind-cold-dampness syndrome,are prepared by using the wind-cold-dampness and bovine type II collagen.After successfully model rebuild,all mice are gavaged with corresponding drugs,once upon a time for 28 days.1 hour later,after the last gavage,give mice probe named IRDye^?800CW 2-DG by tail vein injectin,and then observe every group mice’s inflammation by current and dynamic state,and discuss the mechanism of Fengshining in treating CIA mice model with wind-cold-dampness syndrome.Part 3:the mechanism study about TLR4/NF-κB signal pathway in synovial tissue for Fengshining treating CIA rat model.54 SPF female rats randomly divided into 6 groups:control group,model group,positive control group,and 3 Fengshining group(9.12,18.24,36.48 g·kg^-1).The RA rat model,with wind-cold-dampness syndrome,are prepared by using the wind-cold-dampness and bovine type II collagen.Rats are gavaged with different drugs after successfully model rebuilt,once upon a time for 28 days.Detective RF,ACPA,inflammation cytokines IL-1βand TNF-α,anti-inflammation cytokines IL-4and IL-10,histopathologic changes of ankle and knee,synovial cell apoptosis,synovial tissue’s c-IAP1,XIAP,Caspase-3,Caspase-9 expression,TLR4,MyD88,IκB-α,NF-κB mRNA and protein expreesion with ELISA、HE、TUNEL、IHC、RT-PCR,Western Blot methods,and analyze Fengshining’s mechanism from aspect of TLR4/NF-κB signal pathway.Part 4:the mechanism of Fengshining drug serum to CIA-FLS’proliferation,apoptosis,and TLR4/NF-κB signal pathway with wind-cold-dampness syndrome.Synovial tissue of CIA model rat’s knee are cultured for fibro-synovial cells with tissue block cultivation.Then cells are cultured,passag and definition.The 3rd generation of cells are divided into 6 groups:control serum group,leflunomide serum group,Fengshining serum group（5%,10%,15%,20%）.Focus on TLR4/NF-κB signal pathway,do a further study of Fengshining serum about its mechnism,by detecting cells’proliferation and apoptosis,IL-1β,TNF-α,IL-4 and IL-10 in supernate,TLR4,MyD88,IκB-α,NF-κB mRNA and protein expression in CIA-FLS cells with CCK-8,TUNEL,ELISA,RT-PCR,Western Blot methods,from aspect of cell.Result:Part 1:compared with control group,CIA model rats’state are gradually severe,while weight increase slowly.AI and joint swelling degree increase,and RF and ACPA contents significantly increase,especially the CIA model with wind-dampness-heat bi syndrome.Except the CIA model with wind-dampness-heat bi syndrome,Fengshining group and all classical drugs’group have changed spirit state,paw temperature,RF and ACPA decrease.Jiont swelling lighten,and the movement improve.Fur,onyx form and tongue also improve.In HE stain,CIA rats from all model groups have different degree’s hyperplasia and pannus formation,and the cartilage show different degree’s degeneration and bone destruction.The therapy group have different degree’s suppression to synovial tissue hyperplasia,inflammation infiltration,pannus formation,and bone erosion.Among all therapy groups,Baihu Tang with Guizhi in CIA model,Fengshining in CIA with wind-cold-dampness syndrome group,Baihu Tang with Guizhi in CIA with wind-dampness-heat syndrome group have a better improvement.Part 2:compared with control group,model mice are in bad spirit,lazy with little movement,cowered with arch back,gather together with lcaklustre,the joint of ankle and toes swelling and transformed with long and thin onyx,dirty crissum with long and clear urine,and weight grow slowly or decrease.Compared with model group,Fengshining group is in better spirit,weight grow gradually,paw temperature recover to before The AI and ankle width decrease,as well as joint swelling degree.In near-infraed fluorescence imaging results,24 hours after tail vein injection,mice can be detected with fluorescence signal.48 hours later,2-DG has the best target effect to inflammation and the average reach the highest.There is a significantly difference,compared with control group.72 hours later,2-DG gradually expelled from the mice body,fluorescence signal become lighten.96 hours later,there is still image showing in model group.Among all the time quantum,Fengshining group has a low expression,compared with model group.Part 3:compared with control group,AI,joint swelling,RF,ACPA,IL-1β,TNF-α,spleen and thymus index,histopathologic grade are significantly higher in model group（P<0.01）.IL-4 and IL-10 level,synovial cells apoptosis are significantly lower in model group（P<0.01）.In synovial tissue,c-IAP1 and XIAP expression are significantly higher,and Caspase-3 and Caspase-9 expression are lower than model group（P<0.01）.TLR4/NF-κB signal pathway is also higher in model group compared with control group（P<0.01）.Fengshining group with different dosage,after 28 days gavage,decreases significantly in AI,joint swelling,RF,ACPA,IL-1β,TNF-α（P<0.05,P<0.01）,as well as the spleen and thymus index and histopathologic grade（P<0.05,P<0.01）.In synovial tissue,c-IAP1 and XIAP expression significantly decrease,and Caspase-3 and Caspase-9 expression increase than model group（P<0.01）.TLR4,MyD88,IκB-α,NF-κB mRNA and protein decrease in model group compared with control group（P<0.05,P<0.01）,especially Fengshining high dosage group has the best down regulation effect to TLR4/NF-κB signal pathway.Part 4:3-6 generations of CIA-FLS cells have average spindle formation.Through cell immunofluorescence definition,Vimentin has positive expression in cytoplasm.After 24hours,48 hours and 72 hours interruption,Fengshining serum groups with different dosage have suppression effect to its proliferation,and there is time dependent relation with dosage.Compared with control serum group in same time quantum,its suppression function has extremely difference（P<0.01）.After 24hours’Fengshining serum interruption in dosage of 10%,15%,20%,significantly increase the apoptosis of CIA-FLS（P<0.05,P<0.01）.In ELISA results,after 24hours’Fengshining serum interruption,the content of IL-1βand TNF-αdecrease and the content of IL-4 and IL-10 increase significantly（P<0.05,P<0.01）.In RT-PCR and Western blot results,after 24 hours’Fengshining serum interruption with different dosage,TLR4,MyD88,IκB-α,NF-κB mRNA and protein significantly decrease in cells（P<0.05,P<0.01）.Among them,20%Fengshining serum group has the best decrease effect.Conclusion:（1）Based on CIA model,combine with different exogenous pathogenic factors,we build CIA model with wind-cold-dampness bi syndrome and wind-dampness-heat bi syndrome successfully.（2）With near-infraed fluorescence imaging probe IRDye^?800CW 2-DG,we successfully observe the inflammation in target of CIA mice with wind-cold-dampness syndrome in living body.Fengshining has obvious improvement on the local inflammation of the foot palms and joints of model mice.（3）Fengshining has obvious treating effect to CIA rat with wind-cold-dampness bi syndrome,and the mechanism may be its suppression function to TLR4/NF-κB signal pathway,so as to suppress IL-1βand TNF-α,lower the spleen and thymus index,down-regulate c-IAP1 and XIAP expression,and accelerate rising of IL-4 and IL-10 and apoptosis of synovial cells,improve expression of Casepase-3 and Casepase-9.In results,it can resist inflammation and regulate immune and treat RA,and there is relative between effective and dosage.（4）Fengshining serum can suppress proliferation of CIA-FLS,accelerate spoptosis,and there is time-dosage dependence.The mechanism may be its function in suppressing TLR4/NF-κB signal pathway’s abnormal activation,so as to suppress IL-1βand TNF-α,promote IL-4 and IL-10,in a way,the synovial hyperplasia is suppressed and synovial inflammation lightened.