| ObjectiveTo demonstrate the biological characteristics of cold pattern of rheumatoid arthritis(RA)by Egr2/gr3 as the core of inflammatory pathways,and collagen induced arthritis(CIA)cold syndrome animal model were intervened with Wenjing Tongluo method,to further clarify the biological mechanisms involved in RA cold syndrome,which will enrich the biological connotation of TCM syndrome differentiation of RA.Clinical researchMethods:40 cases inpatient and outpatient RA patients of Department of Rheumatology Dongfang Hospital of Beijing University of Chinese Medicine were collected during the January 2015-January 2016.20 cases were active and 20 cases were remission,including 10 cases of patients with cold pattern and 10 cases of heat pattern respectively.10 cases of healthy people as normal control group.Baseline data,routine laboratory indexes and musculoskeletal ultrasound were collected.The level of IL-17A、IFN-γ、TGF-β、IL-6、IgG in peripheral blood serum were tested with ELISA.Themorphologic expression of Egr2/Egr3、SCOS1/SOCS3 and STAT1/STAT3 in CD4+T cells was detected by immunocytochemical method.The mRNA expression of Egr2/Egr3、SCOS1/SOCS3、STAT1/STAT3 in CD4+T cells were tested by RT-qPCR.Results:The baseline data among gender and age,course of disease,drug use,X-ray classification,TCM syndrome score and health assessment questionnaire(HAQ)of the normal control group,RA cold pattern group(active and remission)and RA heat pattern group(active and remission)showed no significant difference(P>0.05),while the TCM syndrome score and HAQ of activity RA was significantly higher than that of remission(P<0.05).800 small joints were detected by MSUS,tenosynovitis,bone erosion,osteophyte formation of all groups were no significant difference(P>0.05).The classification of synovial membrane proliferation in activity cold and heat pattern of RA group was significantly higher than that in remission cold and heat pattern group(P<0.05).Routine laboratory tests of white blood cell count,neutrophil percentage,red blood cell count,hemoglobin,platelet count,C-reaction protein and liver and kidney function(ALT,AST,BUN,Cre)in the normal group,cold and heat pattern group of RA showed no significant difference(P>0.05).ESR of activity cold pattern group was significantly higher than that of other groups(P<0.05).The serum levels of IL-17A,TGF-β,IL-6 and IgG of heat pattern group were significantly higher than those of the cold pattern group except for IFN-y(P<0.05).The levels of serum cytokines in active and remission of heat pattern group were significantly higher than those in active and remission of cold pattern group(P<0.05).There was no significant difference in PBMC between cold and heat pattern group(P>0.05),while the CD4+T cells in the heat pattern group were significantly higher than those in the normal group and the cold pattern group(P<0.05).Immunocytochemical method showed that the average optical density of Egr2/Egr3 in the cold pattern group were higher than that in the heat pattern group and the normal group(P<0.05).There was no difference between the average optical density of Egr3 in cold and heat pattern group(P>0.05).The relative expression of Egr2/Egr3 and SOCS1/SOCS3 mRNA in CD4+T cells of cold pattern group was significantly higher than that of normal group and heat pattern group(P<0.05).The relative expression of STAT1 mRNA in the cold pattern group was significantly lower than that of the control group(P<0.05).The relative expression of Egr2/Egr3 and SOCS1/SOCS3 mRNA in the active and remission cold pattern group was significantly higher than that of other groups after stratified analysis(P<0.05).The relative expression of Egr3 and SOCS1 mRNA in the active cold pattern group was significantly higher than that of the active and remission heat pattern group(P<0.05).Experimental researchExperiment 1st:20 SPF female Wistar rats were randomly divided into 2 groups,one group were copied into the collagen induced arthritis(CIA)rat model,the other group combined use of animal experiment box with the CIA model,and were given the wind cold dampness stimulation starting from the initial immune for 28 d.The arthritis index(AI),foot swelling drainage volume,joint pain threshold,peripheral joint surface temperature,hemorrheology blood viscosity and blood coagulation factor of two groups were evaluated.Results:The morbidity of two group was 100%(10/10),and the CIA group was able to induce CIA in 75.00%(6/8)rats,the CIA cold syndrome group was able to induce CIA in 77.78%(7/9)rats.While the left foot drainage volume of CIA cold syndrome group was significant differences between baseline in 14d and the CIA group was in 21d(P<0.05).Double foot skin temperatures after initial immune for 14d of two groups are rising,and for 21 d begin to reduce,all time points in the group of two groups were statistically significant differences(P<0.05).The left foot surface temperature of CIA cold syndrome group in inducing 21 d was significantly lower than the CIA group(P<0.05).Foot joint pain threshold at all time points of two groups were no significantly difference(P>0.05).The reaction time of left foot by hot plate stimulated of two groups shortened and the pain threshold reduced in inducing 21d,while the pain threshold increased in inducing 28d(P<0.05).The blood viscosity values under middle shear rate(50/s、30/s)and low shear rate(1/s)of CIA cold syndrome group significantly increased(P<0.05).Thrombin time(TT)of CIA cold syndrome group decreased significantly(P<0.05).there was no statistically significant difference between the two groups on the blood viscosity under rest shear rate under and blood coagulation factors(P>0.05).Experiment 2nd:110 SPF female Wistar rats were copied into the CIA rat model of RA cold pattern except the normal group,and were divided into model group,Tripterygium wilfordii(Tp)group,methotrexate(MTX)group,Shaoyao Gancao Fuzi Decoction(SGFD)in high dose group,middle dose group and low dose grou.All groups were gastric intervention with distilled water,Tripterygium wilfordii,MTX and high dose/middle dose/low dose SGFD respectively,1 times a day and lasted for 4 weeks.The general situation of the rats,arthritis index(AI),foot swelling drainage volume and the organ index of all groups were evaluated during gastric intervention.The expression of Egr2/Egr3,SCOS1/SOCS3,STAT1/STAT3,IL-17A,IFN-γ,TGFβ and IL-6 in synovial membrane were determined by immune-ohistochemical method.The relative expression of Egr2/Egr3,SCOS1/SOCS3,STAT1/STAT3,IL-17A,IFNγ mRNA were tested by RT-qPCR.ELISA assay for the determination level of IL-17A,IFN-γ,TGF-β and IL-6 in peripheral blood serum.Results:There was no significant difference in weight,double foot drainage volume and AI score between all groups(P>0.05).The weight gradually increased,the drainage volume and AI score of all groups were decreased during the treatment,while there was no significant difference between these groups(P>0.05).After 4 weeks of intervention,the spleen index of model group and administration groups were significantly higher than that of normal group(P<0.05).The thymus index of SGFD high dose group and middle dose group were significantly lower than that of model group(P<0.05),and cardiac index of SGFD high dose group and middle dose group was higher than that in model group(P<0.05).Histopathological examination showed that the model group and treatment group had different degrees of synovial hyperplasia,inflammatory cell infiltration,bone erosion,which were significantly reduced after drug intervention compared with the model group.The immunohistochemical staining of the ankle in rats showed that the average density of Egr2/Egr3 in the treatment group increased,but the difference was not statistically significant(P>0.05).The levels of IL-17A,IFN-γ,TGF-β and IL-6 in peripheral blood of rats in model group were significantly higher than those in normal group and treatment groups(P<0.05).The levels of TGF-β and IFN-γ of the treatment groups were higher than those of normal group(P<0.05),while lower than those of model group(P<0.05).The IL-17A level of SGFD high dose group was significantly lower than that of MTX group,Tp group,SGFD middle dose group and low dose group(P<0.05).The relative expression of Egr2/Egr3 mRNA in model group and administration groups were higher than that of normal group,and the relative expression of Egr2/Egr3 mRNA in the middle dose group was significantly higher than that in the normal group(P<0.05).The relative expression of SOCS1 mRNA in the MTX group,Tp group,SGFD medium dose group was significantly higher than that of normal group(P<0.05).The relative expression of SOCS3 mRNA in model group,Tp group,SGFD high dose group and middle dose group were significantly higher than that of normal group(P<0.05).The relative expression of STAT1 mRNA in the SGFD low dose group was significantly higher than that of other groups(P<0.05).The relative expression of STAT3 mRNA in the SGFD low dose group was significantly higher than that of normal group,the model group and the MTX group(P<0.05).The relative expression of IFN-γ mRNA in the model group and the treatment group increased compared with normal group,the model group was significantly higher than the normal group(P<0.05).SGFD high dose group and small dose group IL-17A mRNA relative expression level decreased significantly compared with MTX group(P<0.05).Experiment 3rd:The expression of Egr2/Egr3,SCOS1/SOCS3,STAT1/STAT3 mRNA in CD4+T cells was determined after sorting CD4+T cells from peripheral blood of rats.Flow cytometry was used to detect the changes of CD4+IL-17A+T cells and CD4+IFN-γ+T cells after CD4+T cells stimulated by TCR.Results:The relative expression of Egr2 mRNA of MTX group was significantly higher than that of model group and other treatment groups(P<0.05).There was no significant difference in the relative expression of Egr3 mRNA in each group(P>0.05).The relative expression of SOCS1/SOCS3 mRNA of SGFD high dose group was lower than that of normal group(P<0.05).The relative expression of STAT1 mRNA was significantly higher in the MTX group(P<0.05),but the relative expression of STAT3 mRNA was not significantly different from that in the normal group,the model group and the other treatment groups(P>0.05).Rat peripheral blood CD4+IFN-γ+T cells(Th1 cell)percentage had no significant difference in the groups(P>0.05),while CD4+IL-17A+T cells(Th17 cell)percentage of Tp group increased significantly than MTX group(P<0.05),there was no significant difference among other groups(P>0.05).conclusion1、Cold pattern of RA has its own biological characteristicsCold pattern of RA has high level of Egr2/Egr3,which may increase the level of SOCS1/SOCS3,and inhibiting the expression of STAT1/STAT3,thereby reducing the serum levels of inflammatory cytokines,alleviate clinical symptoms.MSUS synovial hyperplasia classification of RA cold pattern is lower than that of the RA heat pattern,while still continued unabated synovial hyperplasia.2、Wenjing Tongluo method effect Egr2/Egr3 related indexes of RA cold patternAfter the intervention of SGFD,the representative of Wenjing Tongluo method,the expression of Egr2/Egr3 and SOCS 1/SOCS3 in synovial tissue of rats of RA cold pattern rat model upregulated,and level of cytokines in peripheral blood decreased,SGFD middle dose group has obvious performance,which echoes with the results of clinical research. |
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