Experimental Study On The Animal Model Of Cold Bi And Heat Bi Syndrome For Rheumatoid Arthritis Induced By Artificial Environment

BackgroundRheumatoid arthritis(RA)is a kind of autoimmune disease with synovitis as the center of Pathology,which belongs to the category of arthralgia syndrome or traditional Chinese medicine,and is also a representative disease in arthralgia syndrome."The combination of three factors of wind,cold and dampness can cause Bi",which clearly shows the role of external environment in the pathogenesis of Bi disease.Modern research shows that temperature,humidity,wind speed and other factors play an important role in RA activity.With the development of etiology and pathogenesis research and the clinical summary of doctors in previous dynasties,wind cold dampness Bi and wind heat dampness Bi are the two main syndromes in the early stage and the active stage.The former is treated by dispelling wind and dispersing cold,removing dampness and dredging collaterals to relieve pain,the representative is Wutou Decoction(WTT),the latter is treated by clearing heat and detoxification,removing dampness and activating collaterals to stop pain,and the representative is Simiao pill(SMW).There are different opinions on the treatment plan of Bi disease in the active period of the past dynasties,which shows that the law of RA activity period may be complex and difficult to distinguish.The animal models of Cold Bi and Heat Bi are important experimental carriers to explore the etiology and pathogenesis of TCM arthralgia.At present,most of the animal models of Cold Bi or Heat Bi are made by applying environmental factors on the basis of induced arthritis model.However,whether this model can really represent the essence and pathogenesis of "Cold Bi" and "Heat Bi" syndrome type of TCM Bi.There are few reports on the pathogenic essence of the external environment,such as wind cold dampness,and heat.Therefore,it is of great significance to study the pathogenic essence of RA cold arthralgia and heat arthralgia combined with animal model and environmental factors for exploring the etiology and pathogenesis of arthralgia in TCM and studying the pharmacological action of TCM.ContentChapter 1 Effects of Artificial Environment on the Human TNFalphaTransgenic MiceObjectiveTo observe the effect of artificial wind cold dampness and wind heat dampness environment stimulation on human RA TNF-? transgenic(Tgtc)mice model,to explore the relationship between the pathogenesis of Tgtc mice and hypoxia inducible factor 1?(HIF 1?)and heat shock protein 70(HSP-70),and to establish the objective quantitative index to evaluate the animal model of Bi syndrome.Methods27 SPF grade female Tgtc mice and 27 SPF grade female FVB mice were randomly divided into blank control(BC)group,wind cold dampness(FHS)group and rheumatic fever(FSR)group with 18 mice in each group,respectively stimulated by SPF environment,artificial wind cold dampness environment and artificial wind heat dampness environment twice a day for 2 hours,a total of 64 hours.The diameter of ankle joint,deformation index of limb joint and skin color of ankle joint were observed and measured.Two weeks after the end of environmental stimulation,the blood flow imaging of laser speckle of both legs,the serum HIF 1?and HSP-70,the histopathology and immunohistochemistry of ankle joint were detected.ResultsExcept for the decrease of serum HSP-70 in FSR group,the other indexes of FVB mice were not affected by environmental factors.During the period of artificial environment stimulation,the skin color,ankle diameter and limb joint deformation index of FHS group were higher than those of BC group(P<0.05),while those of FSR group were opposite(P<0.05);After the environmental stimulation,the skin color,ankle diameter and joint deformation index of three groups in Tgtc mice tended to be the same,there was no significant difference(P>0.05).The FSR group increased,but the values were still lower than BC group(P<0.05).The expression of HIF 1? in synovium tissue was lower than that of BC group(P<0.05)The level of HIF 1? in serum and the expression of HSP-70 in synovium between the two groups had no significant difference(P>0.05);In FSR group,the SFI value,the expression of HSP-70 in synovium and serum,and the expression of HIF 1? in synovium were higher than those in BC group(P<0.05),and the expression of HIF 1? in synovium was lower than that in BC group(P<0.05).The pathology of ankle joint suggested that there were serious synovium fibroblast proliferation and a small amount of inflammatory cell infiltration in FSR group(P>0.05),but the depth of cartilage damage and the extent of cartilage surface damage in FSR group were higher than those in BC group(P<0.05)The infiltration degree of inflammatory cells in cartilage was lower than that in BC group(P<0.05).ConclusionThe stimulation of wind cold,damp heat environment can affect the syndrome of RA combined with animal model to a certain extent.Tgtc mice are cold in nature,and can be used as cold Bi model when continuously stimulated by FHS environment.Its pathogenesis is related to the up regulation of HIF 1? in synovium;FSR stimulation can up regulate HSP-70 and delay the progress of bone destruction in Tgtc mice.The pathological essence of the animal model of Cold Bi is mainly synovial fibroblast proliferation with a small amount of inflammatory cell infiltration.SFI,diameter of joint and skin color can be used as objective quantitative indexes to evaluate Bi syndrome model.Chapter 2 Effect of Wutou Decoction and Simiao Pill on the Model of Tgtc Mice ObjectiveTo explore the therapeutic effect of WWT and SMW on the mouse model of Tgtc,and to verify the cold and heat properties of the mouse model with the method of disproof.MethodsTwenty four SPF grade Tgtc mice were randomly divided into three groups:blank control(BC)group,WTT group and SMW group,with 8 mice in each group.The general condition,limb joint deformation index,ankle joint diameter and ankle joint color of each group were recorded during the Administration period.Laser speckle blood flow imaging of both legs and MRI scanning of both knee joints were performed four weeks after continuous administration of the drug The levels of serum HIF-1 ? and HSP-70,ankle histopathology and immunohistochemistry were detected.ResultsAfter WTT treatment,the increase of limb joint deformation index,ankle diameter,ankle skin red value(a value),ankle SFI value,knee joint fluid accumulation,expression of HIF 1? in synovium and microvessel density in synovium were lower than those in BC group(P<0.05),and the expression of HSP-70 in serum and synovium was higher than that in BC group(P<0.05).After SMW treatment,the ankle diameter of mice increased more than BC group(P<0.05),the limb joint deformation index and ankle SFI value were lower than BC group(P<0.05),the expression of HSP-70 in synovium was higher than BC group(P<0.05),the skin color a value of ankle joint,the amount of effusion on knee joint capsule,the content of serum HSP-70 and the microvascular density of synovium were not significantly different from BC group(P>0.05).There was no significant difference in the content of HIF 1? between the three groups(P>0.05).The pathological changes of ankle and knee joints in the three groups showed severe synovial fibroblast proliferation with a small amount of inflammatory cell infiltration(P>0.05),but the depth of cartilage damage,the extent of cartilage surface damage and the extent of inflammatory cell infiltration in the cartilage in the WTT group were lower than those in the BC group(P<0.01),there was no significant difference between SMW group and BC group,and osteolysis occurred in both groups(P>0.05).ConclusionWTT has better effect on Tgtc mice,basic properties of Tgtc mice tend to be cold.Chapter 3 Effect of Artificial Environment on the Syndrome Type of Traditional Chinese Medicine with Collagen-Induced Arthritis in RatsObjectiveTo observe the effect of artificial wind cold dampness heat on Traditional Chinese Medicine syndrome types of CIA in rats,and to explore the pathogenic essence of wind cold dampness heat environment.MethodsTwenty four SPF female SD rats were randomly divided into four groups:BC group,FHS group,FSR group and NC group,6 in each group.The BC group,FHS group and FSR group were treated with CIA,and environmental intervention was carried out after immunization.The BC group,NC group,FHS group and FSR group were treated with SPF environment,artificial FHS and FSR respectively,twice a day,2h each time,for a total of 64h.The arthritis index(AI)of each group was observed and measured in the course of study.Four weeks after environmental intervention,the skin red value(a value),X-ray of double ankle joint,serum HIF1? and HSP-70 were detected.ResultsThe AI of FSR group was faster than that of BC group to reach the peak value,the maintenance time of peak value was longer,and the decline rate was slower(P<0.05),while the AI of FHS group was the opposite(P<0.05).The a value,X-ray score,HSP-70 content in serum and synovium of FSR group were higher than that of BC group(P<0.01),while the a value and X-ray score of FHS group were no different from that of BC group(P>0.05),and the HSP-70 content in serum and synovium was lower than that of BC group(P<0.05).4 There was no significant difference in the expression of HIF1 a in serum and synovium of the rats in the FSR group(P>0.05).The pathology of ankle joint suggested that inflammatory cells were the main cell proliferation of CIA rats in the three groups,and the pathological score of FHS group was lower than that of BC group(P<0.05).The pathological pannus hyperplasia and bone destruction of FSR group were obvious,and the pathological score of FSR group was higher than that of BC group(P<0.05).ConclusionBasic properties of CIA rats tend to heat,which has a significant evolution process of heat arthralgia syndrome after FSR stimulation,and can be used as heat arthralgia model,its essence is related to the up regulation of HSP-70;after FHS stimulation,it does not show the characteristics of cold arthralgia syndrome,so it can not be used as cold arthralgia model,and its pathogenic essence needs further discussion.SummarizeWind cold dampness,wind dampness heat environment stimulation can affect the syndrome of RA combined with animal model to a certain extent.The basic property of tgtc mice is cold,and it can be used as cold arthralgia model when FHS environment is continuously stimulated.Its pathogenesis is related to the up regulation of HIF1? in synovium.Its pathological essence is mainly synovium fibroblast proliferation with a small amount of inflammatory cell infiltration.The basic property of CIA rats is heat,which can be used as the model of heat Bi when the environment of FSR is stimulated continuously.Its pathogenesis is related to the up regulation of HSP-70 in serum and synovium,and its pathological essence is inflammatory cell infiltration.Using laser speckle blood flow imaging to detect SFI value of joints and chromatic aberration to detect skin color can be used as objective quantitative indexes to evaluate biasis syndrome model.