The Theoretical And Experimental Studies On The Correlation Between Exogenous Wind Cold Damp And Heat Evil With Blockage Syndrome

ObjectiveSix evils are very important pathogenic factor ofdeiseases.Among six evils, wind, cold, heat, dryness and fire evil,all are invisible evil.For the cause of blockage syndrome, pastdynasties doctors mostly follow the discourse that the three mixedgas of wind cold and dampness cause the blockage syndrome, and themost physicians believe that all the six evils of the exogenouspathogenic plays a larger role, sometimes even play a leading role.Recently, many scholars and researchers have done a lot of studieson cold,wet and dampness syndrome.Among of this studies,cold evilwas studied from the perspective of lung injury, dampness heatsyndrome was studied from the perspective of animal model and theessence,but the studies on the relationship between the six evilsand the blockage syndrome was obviously insufficient,especiallyfrom the perspective of etiology. Through guided by the etiologicalfactor and pathogenesis theory of traditional Chinese medicine andon the base of previous studies, this study reviewed the documentsof wind cold dampness and heat evils. According to the naturalcharacteristic of wind, cold, wet and hot, some meteorological elements such as temperature, humidity,are simulated,and then putCIA rats in this artificial environment to observe the effects ofexogenous evil on the pathogenesis of CIA,to explore therelationship between exogenous evil and the onset of rheumatoidarthritis.And through the detection of blood sedimentation, IL-17, CD44, AHR, Fas/FasL in serum and expression change of synovialCD44, to explore the immune mechanism of exogenous evil causing theblockage syndrome,to provide experiment basis for the theory ofexogenous evil causing the blockage syndrome.Methods1. Theory researchAccess to relevant information,and sum up the characteristicsof exogenous wind,cold,dampness,heat.The concept of “exoge-nous wind,cold,dampness,heat” in Chinese medicine is formed on thebasis of a long-term observation and understanding of thesephenomena. So it is difficult to make accurate, single definition,but Analyze and understand from the etiology, pathogenesis,symptoms.The wind of nature is a kind of invisible flow ofair,therefore,all of the exogenous evil which has the nature ofblown,moving,can be called wind evil.As the spring′s main gas,thewind can be seen in the other three seasons.So many diseases causedby wind are not limited to occur in spring,they also occur in otherseasons. There are many famous theories in“Huangdi Neijing”suchas “the wind is one of the most important pathogenic factors”.Traditional Chinese medicine thinks that the exogenous evil whichhas the features of cold, condensation, closes,can be known as thecold evil. Cold is the main gas of winter. In winter, if we do notpay attention to cold and get warm to petect our body, we are often susceptible to cold evil. But disease caused by cold evil is alsoseen in other seasons. There are many ways of getting cold evil,suchas getting wet in the rain, sleeping in very cold place,blowing whenyou was dripping with sweat,or drinking too cold water. All of theexogenous evil which has the nature of turbidity, viscosity,movingdownward can be called dampness evil.Dampness is long summer’s maingas.Of all the seasons,long summer has the most precipitation.Sothere are many disease caused by dampness evil in long summer. Inaddition,there are many other pathways which can cause disease bydampness evil,such as Living in damp environment, wading, gettingwet in the rain.So the formation of exogenous dampness is relatedto the season,climate and environment. All of the exogenous evilwhich has the nature of hot,can be known as the heat evil.Since the six evils is the performance of the abnormal weatherof nature.For example, wind, cold, heat, dampness, dryness or firethis six gas sometimes is too short or not too much. The abnormalweather affect the function of the body to adjust the climatechanges and then pathogens inversed and spread and becomedisease-causing pathogens.So six evils and meteorological elementsof modern meteorology will be closely linked. Meteorologicalelements are basic physical quantities and basic characterizationof atmospheric status of weather phenomena. It Mainly includes theatmospheric temperature, atmospheric pressure, air humidity, winddirection, wind speed, precipitation and solar radiation, etc. Thesudden changes of meteorological elements, directly or indirectlyaffect human life and human health, which is the content of modernmeteorological medical thought.The body’s physiological functionsand pathological changes, diagnosis and treatment of disease, prognosis and prevention health even climate change have a closerelationship with nature. Meteorological medical thought and sixqi, Yin theory are consistent. Although there is no concept ofmedical meteorological in Huangdi Neijing, but its content containsrich ideas of medical meteorological. Although these factors cannot completely equal to six evils, but after reach a certainquantity, attributable to the six evils of the category oftraditional Chinese medicine.Bi as the name of disease,is blockage syndrome,which causedby invasion of exogenous evils,such as wind,cold,dampness,heat,in-vading human body,closing meridians,leading to the blood runpoorly.And then we can feel pain,heavy,numb,swelling in the bodyjoints and muscle,even the joints cannot flex at will,and be outof shape. In past dynasties doctors’ discourse on the cause ofblockage syndrome is quite rich, in which the theory that theblockage syndrome is caused by the invasion of exogenous evils ismore important etiology and pathogenesis that many physiciansbelieve.Some diseases in rheumatism can be attributed to blockagesyndrome.Rheumatism is a group of mainly medical treatment ofdiseases of the musculoskeletal system, it includes: diffuseconnective tissue disease and soft tissue around the joints andjoints caused by different causes, including muscles, tendons,ligaments and other disease, characterized by pain, swelling,movement disorders, etc. Such as rheumatoid arthritis, ankylosingspondylitis, bone arthritis, systemic lupus erythematosus (sle)and so on, all belongs to the category of rheumatism. RA with ahigher prevalence, is very common in clinical, and RA animal experiment research is relatively mature.The collagen inducedarthritis(CIA) is recognized as ideal animal models of RA.2. Experiment study2.1Methods of experiment2.1.1Animals and grouping70specific pathogen free Wistar mice,160-l80g,female, havebeen conventionally breeded in the laboratory for7days，thenrandomly divided into5groups: normal control group,model group,wind cold dampness(WCD) group,wind dampness(WD) group and winddampness heat(WDH) group，10for normal group,15for each group ofthe rest four group.2.1.2Model making process and methods of experiment①Normal group：No treatment was given．Rats were fed underthe normal temperature and hnmidity environment4hours a day fora month which was15-20℃of temperature,50±4%hnmidity and3windlevel.②Model group：the CIA model group. According to buildingmethod, to mix fully the bovine type II collagen (CII) with the samevolume incomplete Freund ’s adjurant(IFA), then under asepticconditions, to inject the mixture under the skin,0.05mL per point.4points at rats back was injected for primary immune,3pointsinjection for strengthen immune after2weeks.The time,temperatureand hnmidity are the same with normal group.③Wind-cold-damp group:Rats were treated as the model groupwith the same immune method except that they were put intowind-cold-damp environment4hours a day for a month which was6±2℃of temperature,90±4%hnmidity and3wind level.④Wind-damp-heat group: Rats were treated as the model group with the same immune method except that they were put intowind-damp-heat environment4hours a day for a month which was33±2℃of temperature,90±4%hnmidity and3wind level.⑤Wind-damp group: Rats were treated as the model group withthe same immune method except that they were put into wind-damp-heatenvironment4hours a day for a month which was21±2℃oftemperature,90±4%hnmidity and3wind level.2.2Index detection1) General state of health in mice，such as demeanor, activity,weight，color and luster of fur and dietary, were observed.2) Assess Arthritis swelling degree by Arthritis indexintegral method.3) Synovial tissues structure were observed with hematoxylineosin(HE) staining method.4) Erythrocyte Sedimentation Rate were measured by westergrenmethod.5) IL-17expression in serum, ankle joint fluid and spleen cellculture supernatant were detected by ELISA kit.6) AHR,CD44,Fas/FasL expression in serum were detected byELISA kit.7) The expression of CD44mRNA in synovial tissues weredetected by realtime quantitative fluorescence PCR.8) The expression of AHR、Fas/FasL in synovial tissues wereassayed by immunohistochemistry.2.3Statistical analysisAll data were analyzed by SPSS19.0statistical soft ware.Experimental data were showed with mean士standard deviation. qtest and ANOVA was used to compared the differences. P<0.05level was used to indicate statistically significant difference.Results(1)General conditionThe wind cold dampness group rats nuzzled and showed lazy tomove, walk slowly, evaded capture, aet less, worked out loosestool.They had the smallest increase in weight,and their weight arelightest;Rats in wind-damp-heat group had no body hair luster, lessdynamic, malaise, defecate viscous, stinking smell; Rats inwind-damp group slightly dulled, response unresponsively,aet less,worked out soft stool; Model group actived not nimble, defecatenormally, body hair owe luster, and their diet is normal,and theirweight were equal with normal group.(2)Incidence of CIA modelAt the15thday of the experiment,the incidence of CIA in thewind cold dampness group,wind dampness heat group is higher thanmodel group and wind dampness group, the difference had statisticalsignificance.The incidence of CIA between the wind cold dampnessgroup and wind dampness heat group, had not statisticalsignificance. Compared with model group, there was no significantdifference in wind dampness group. At the end of the experiment,the incidence of CIA in the model group, wind dampness group, winddampness heat group, wind cold dampness group are66.7%,73.3%,80.0%and73.3%respectively,there were no statisticallysignificant difference between the groups. The result gave us a hintthat wind, cold, wet, heat factor on the onset of the CIA has obviouspromoting effect, and only when the wind cold dampness merger orwind dampness heat hit shows clearly the effect.(3)Pathological results Synovial cell hyperplasia can be seen in Model group,WDgroup,WDH group,WCD group. The increase in the WCD group is the mostobvious, followed WDH group, WD group was second smallest,and modelgroup minimum.①normal group: there was no inflammatory cellinfiltration, synovial cell hyperplasia in synovial tissue.②model group: synovial cell hyperplasia is not obvious.③WD group:a little synovial cell hyperplasia.④WCD group: there were obvioussynovial cells hyperplasia, layers arranged irregular synovialcells,a large number of inflammatory cells infiltration.⑤WDHgroup: there was synovial hyperplasia,2-3layers of irregularsynovial cells and inflammatory cells infiltration.(4)Arthritis index integralAt the15th day of the exrperiment, compared with normal group,the scores of the rest four sets were significantly increased;compared with model group, the WCD group,WDH group increasedsignificantly, but WD group had no significant difference; thescore of the WCD group was significantly higher than that of WDHgroup, WD group; WDH group is higher than WD group. At the30th dayof the exrperiment,the WCD group have the highest score, followedby WDH group, WD group, model group,and normal group score is zero.The CIA model copied successfully. The result gave us a hint thatwind, cold, dampness, heat, etc can increase the happining of theCIA.The effct in WCD group is the most serious.(5) ESRThe contents of ESR in serum of model group,WD group,WCD groupand WDH group were much higher than normal group.The contents ofESR in serum of WD group,WCD group and WDH group were higher thanmodel group,that of WCD group was higher than that of WD group,WDH group and model group，and that of WDH group is higher than modelgroup.There was no signifcant difference between WDH group and WDgroup. The result gave us a hint that wind, cold, dampness, heat,etc can increase the expression of ESR.The WCD group is the mostobvious.(6)IL-17expression in serum, ankle joint fluid and spleen cellculture supernatantThe concentration of IL-17of model group,WD group,WCD groupand WDH group were much higher than normal group,that of WCD groupand WDH group were much higher than model group.There was no obviousdifference between model group and WD group. The concentration ofIL-17of WD group was much lower than WCD and WDH group.Theconcentration of IL-17of WCD group was much higher than that ofother four groups.Tip:the expression of IL-17in the CIA model ratsincrease.The wind,cold,damp,heat environment can aggravate theexpression of IL-17in the CIA rats serum and arthritis. Influencedegree of the wind cold dampness environment is most obvious. AndIL-17in serum and joint fluid concentrations were positivelycorrelated.(7)AHR,CD44expression in serumCompared with normal group, serum concentration of AHR of therest of the four groups were much higher.Compared with modelgroup,the concentration of AHR of WD, WDH and WCD group were muchhigher.Compared with WD group, the concentration of AHR of WDH andWCD group were much higher. Compared with WDH group, theconcentration of AHR of WCD group was higher. Compared with the restof the four groups,the WCD group was significantly much higher.Theexpression of CD44of all groups has same result with AHR. Tip:the expression of AHR,CD44in the CIA model rats increased.Thewind,cold,damp,heat environment can aggravate the expression ofserum AHR,CD44in CIA rats.Influence degree of the wind colddampness environment is most obvious.(8)Fas/FasL expression in serumCompared with normal group, serum concentration of Fas/FasLof the rest of the four groups were much higher. Compared with modelgroup, the concentration of Fas/FasL of WD, WDH and WCD group weremuch higher.Compared with WD group, the concentration of Fas/FasLof WDH and WCD group were much higher. Compared with WDH group, theconcentration of Fas/FasL of WCD group was higher. Compared withthe rest of the four groups,the WCD group was significantly muchhigher. Tip:the expression of Fas/FasL in the CIA model ratsincreased.The wind,cold,damp,heat environment can aggravate theexpression of serum Fas/FasL in CIA rats.Influence degree of thewind cold dampness environment is most obvious.(9)The expression of CD44mRNA in synovial tissuesCompared with normal group, the expression of CD44mRNA insynovial tissues were much higher than that in rest of the fourgroups.Their difference had statistical significance. Comparedwith model group, the concentration of CD44mRNA of WD, WDH and WCDgroup were much higher.And the CD44mRNA expression in synovialtissues of WCD group was much higher than that of WDH group.(10)The expression of AHR、Fas/FasL in synovial tissuesImmunohistochemical results showed that: compared with normalcontrol group, synovial Fas expression increased in CIA rats model,and FasL expression reduced. Compared with model group, Fasexpression of WD, WDH and WCD group also increased, FasL expression decreased.Comparative differences in wind cold dampness group,wind dampness group and wind dampness heat group are statisticallysignificant.The Fas positive cells rate is highest in the wind colddampness group and FasL expression least. AHR levels in experimentgroups of synovial have significant difference. Compared withnormal group, the rest of the four groups of synovial AHR expressionwere higher; Compared with model group, wind dampness group, winddampness heat group, wind cold dampness group were much higher;Compared with wind dampness group, wind dampness heat group, windcold dampness group were much higher; Compared with wind dampnessheat group, the wind cold dampness group concentration was higher.ConclusionSummarizing the above results, we found that the invasion bywind,cold, dampness and heat evil had obvious promoting effect inthe CIA pathogenesis and this effect appeared in an early stage.Inthe early days the wind cold dampness,wind dampness heatenvironment made the incidence increase significantly.But theincidence of in wind dampness group and model group had nodifference.IL-17, CD44, AHR, Fas and FasL participated in the CIA.And exogenous pathogenic factors of wind cold dampness and heat evilcan aggravate immune response of the animal models, so thatcondition grow worse.Exogenous pathogenic factors seriously affectthe expression of ESR, IL-17, CD44, AHR, Fas and FasL, etc, andcan aggravate synovial hyperplasia of cells. The influence of windcold dampness in synovial pathological was serious than that of winddampness heat group. The influence of wind cold dampness group onESR, IL-17, CD44, AHR,Fas and FasL were more severe than that ofwind dampness heat group.These indicators to some extent, can react disease activity of RA.Anyhow, exogenous pathogenic factors of wind cold dampness andheat evil can affect the onset of the CIA and the degree of impactcondition. We believe that exogenous pathogenic factors of windcold dampness and heat evil play a very important role on theoccurrence and development of blockage syndrome, and its actionmechanism is that exogenous evils aggravate immune response bypromoting the expression of IL-17, CD44, AHR and Fas/FasL and soon many kinds of inflammatory cytokines, and make disease furtherdevelop.