外感风寒湿热与痹证相关性的理论和实验研究
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摘要
目的
六淫是自然界气候异常变化产生的致病邪气，包括风、寒、暑、湿、燥、火六种自然界反常的气候变化，它们多从肌表或口鼻侵入人体而发病，属于外感病的病因。六淫致病说是中医病因学的重要内容之一。对于痹证感受六淫邪气的病因病机，历代医家对其作了许多详细的记载，认为风寒湿热等外邪是痹证发生的外在因素，也是重要的发病因素。近年来关于寒、湿及热证的现代研究有很多，其中寒邪多从伤肺的角度去研究，湿热证多从动物模型及证型实质的角度去研究，而从病因学的角度系统研究外邪与痹证发病机理的较少。本课题拟在中医学病因理论的指导下，在已完成的研究基础上，对风、寒、湿、热等外邪进行理论回顾，依据外风、寒、湿、热的自然特性，调控温度、湿度及时间等作用因素，模拟风寒湿热等外邪，对胶原诱导关节炎大鼠进行刺激，观察外邪对CIA发病及病情的影响，探讨外邪与类风湿关节炎发病之间的关系；检测血沉、血清IL-17、CD44、AHR、Fas/FasL及滑膜CD44水平，分析这些指标之间的相关性，探索AHR、CD44参与类风湿关节炎发病的可能机制，从而探讨外邪致痹可能的免疫机制，为外邪致痹理论提供实验依据。
方法
1.理论研究
查阅相关资料，归纳总结外风、寒、湿、热的各项特性。中医学中“风、寒、湿、热”等概念，是对这些现象的长期观察和认识的基础上，基于取类比象的思维而形成的。因此很难作出精确的、单一的定义，而应该从病因、病机、症状等不同方面分析和理解。自然界的风是一种无形的流动的气流，因此，自然界中具有风之轻扬开泄、善动不居特性的外邪，称为风邪。风为春季的主气，但四季皆有，故风邪引起疾病虽以春季为多，但不限于春季，其他季节也可发生。风邪是最重要的致病因素，《内经》将其视为六淫之首。风邪四季皆能伤人，外感为病，常以风为先导，风邪常率他邪合而伤人，且风邪伤人往往兼它邪为患，它邪犯人，常借风而侵袭，乘虚而入。中医学认为，自然界中具有寒冷、凝结、收引特性的外邪，称为寒邪。寒乃冬季之主气，若在气温较低的冬季，人体不注意防寒保暖，则常易感受寒邪。但寒邪为病也可见于其它季节，如气温骤降、淋雨涉水、汗出当风、空调过凉及贪凉露宿，或过饮寒凉之物，均为感受寒邪的途径。自然界中具有水湿之重浊，粘滞，趋下特性的外邪称为湿邪。湿为长夏的主气，长夏乃夏秋之交，此时阳热下降，水气上腾，氤氲熏蒸，湿气充斥，为一年之中湿气最盛的季节，故长夏多湿病。此外居处潮湿，以水为事，淋雨涉水等均为湿邪致病的途径。热邪指自然界中具有火之炎热特性的外邪。热邪为病称为外热病。热邪属阳，多为外感，可见于任何季节，但夏季多见。一般与其他诸邪相兼为患，例如风热、湿热、暑热等,其中湿热致病尤为常见。因此外风、湿、热、寒等的形成与季节、气候及环境密切相关。
既然六淫是自然界气候反常（风、寒、暑、湿、燥、火六气太过不及或非其时而有其气）并影响到人体的调节适应机能及病原体的孽生传播而成为致病的邪气,那么六淫与现代气象学中的气象要素必是紧密相连的。“六淫”所概括的气象要素包括大气温度、湿度和气流方向及速度。“六淫”中的寒、热（火）、暑是对大气温度两极的定性描述,而湿、燥是对大气水汽含量的定性概括,风是对空气流动的概述。从现代气象学理解“六淫“的含义，应包含两层意思：一是风寒暑湿燥火这六种气候（实际只有风、温度及湿度这3种气象要素）和气候因素（某地区的平均天气状况及其变化特征）本身对人体的影响；二是这些气象要素和气候因素太过、不及或非时之候对人体产生不良影响而致病（包括诱发、加重、发生）。
痹作为病名，即痹证，是因风、寒、湿、热等外邪杂合侵袭人体，闭阻经络，导致气血运行不畅所发生的肢体关节肌肉疼痛、重着、麻木、肿胀、屈伸不利，甚则关节肿大变形，或累及脏腑的一类病证。历代医家对痹证的病因论述颇为丰富，其中感受外邪致痹是诸多医家认为较为重要病因病机。
西医风湿病中的部分疾病可归属于中医痹证的范畴。风湿病是一组以内科治疗为主的肌肉骨骼系统疾病，它包括：弥漫性结缔组织病及各种病因引起的关节、关节周围软组织，包括肌肉、肌腱、韧带等的疾病，表现为疼痛、肿胀、运动障碍等。如类风湿关节炎、强直性脊柱炎、骨关节炎、痛风等风湿病可归属到痹证范畴。其中RA患病率较高、临床较为常见，而且RA动物实验研究也较为成熟，目前RA动物模型中公认较为理想的是胶原诱导关节炎(collagen-induced arthritis，CIA)。
2.实验研究
2．1实验方法
2.1.1实验动物及分组
SPF级雌性Wistar大鼠70只，5周龄，体重160-180g。常规饲养一周后随机分为5组：①正常组，②模型组，③风寒湿组，④风湿热组，⑤风湿组，正常组10只，其余四组各15只。
2.1.2造模过程及实验方法
①正常组：不做任何处理，常温常湿下喂养，相对湿度（RH）：50±4%，温度（T）：21±2℃，风力0级。
②模型组：即胶原诱导关节炎（collagen-induced arthritis，CIA）模型,根据造模方法:将牛II型胶原（II collagen，CII）与等体积的不完全弗氏佐剂(Incomplete Freund's Adjurant,IFA)充分乳化，在无菌条件下，按每点皮下注射乳剂0.05mL，初次免疫在大鼠背上任选4点，加强免疫于2周后同法3点注射；常温常湿下喂养。
③风寒湿组：免疫方法同模型组，并在接受免疫的第一天开始，每天在人工气候箱内接受风寒湿刺激1次，每次持续4小时，连续30天。相对湿度（RH）：90±4%，温度（T）：6±2℃，风力3级。
④风湿热组：免疫方法同模型组，并在接受免疫的第一天开始，每天在人工气候箱内接受风湿热刺激1次，每次持续4小时，连续30天。相对湿度（RH）：90±4%，温度（T）：33±2℃，风力3级。
⑤风湿组：免疫方法同模型组，并在接受免疫的第一天开始，每天在人工气候箱内接受风湿刺激1次，每次持续4小时，连续30天。相对湿度（RH）：90±4%，温度（T）：21±2℃，风力3级。
2.2指标检测
2.2.1一般观察项目
观察大鼠精神状态、活动情况、饮食饮水量、毛色、皮肤关节、体重及二便等情况。
2.2.2关节炎指数测定
采用关节炎指数积分法评定。
2.2.3大鼠滑膜病理学观察
采用苏木素伊红(HE)染色法，光镜观察。
2.2.4血沉的检测
采用魏氏法测定。
2.2.5血清、踝关节液及脾细胞培养上清IL-17的检测
采用ELISA试剂盒检测。
2.2.6血清AHR的检测
采用ELISA试剂盒检测。
2.2.7血清CD44的检测
采用ELISA试剂盒检测。
2.2.8血清Fas/FasL的检测
采用ELISA试剂盒检测。
2.2.9滑膜CD44表达的检测
采用定时定量荧光PCR检测。
2.2.10滑膜AHR、Fas/FasL
免疫组化法检测。
2.3统计学处理
应用SPSS19.0软件进行统计学处理。符合正态分布的计量资料以均数±标准差(x s)表示，采用单因素方差分析,组间两两比较采用q检验、t检验、卡方检验。P<0.05为差异有统计学意义。
结果
（1）一般情况
风寒湿组大鼠萎靡不振，蜷卧懒动，行动迟缓，反应迟钝，抓捕时逃避，食量减少，大便稀溏，体重增幅最小，体重最轻；风湿热组体毛无光泽，少动，萎靡，大便粘滞，臭秽难闻；风湿组稍显呆滞，反应迟钝，饮食量减少，大便软；模型组活动欠灵活，大便基本正常，体毛欠光泽，饮食量基本正常，体重与模型组相当。
（2）CIA模型发病率情况
实验15天时，风寒湿组、风湿热组发病率比模型组、风湿组要高，差异有统计学意义，风寒湿组与风湿组之间比较，发病率没有显著差异，模型组与风湿组比较亦无显著差异。实验结束时，模型组、风湿组、风湿热组、风寒湿组发病率分别为66.7%、73.3%、80.0%、86.0%，组间差异无统计学意义。提示风、寒、湿、热因素对CIA的发病有明显的促进作用，且只有风寒湿合并或风湿热合并侵袭时这种作用才显现明显。
（3）病理结果
滑膜病理切片显示模型组、风湿组、风湿热组、风寒湿组滑膜细胞增生按此顺序依次增多，以风寒湿组最为明显。①正常组：滑膜组织无炎性细胞浸润，滑膜细胞无增生。②模型组：滑膜细胞增生不明显。③风湿组：可见滑膜细胞少许增生。④风寒湿组：滑膜细胞增生明显，可见数层排列不规则的滑膜细胞，大量炎性细胞浸润。⑤风湿热组：可见滑膜组织增生，可见2～3层排列不规则的滑膜细胞及炎性细胞浸润。
（4）关节炎指数的测定情况
结果显示在实验15天时，与正常组比较，其余四组得分均明显升高；与模型组比较，风寒湿组、风湿热组明显升高，但风湿组差异不明显；风寒湿组得分明显高于风湿热组、风湿组；风湿热组高于风湿组。实验结束时测得风寒湿组得分最高，其次是风湿热组，风湿组次之，模型组最低,正常组得分为零。说明CIA模型复制成功。提示风、寒、湿、热等外邪能加重CIA的发病，以风寒湿邪合并侵袭时程度最为严重。
（5）实验各组血沉检测结果
与正常组对比，其余四组血沉均明显升高；与模型组比较，风湿组、风寒湿组、风湿热组血沉均较高；风寒湿组明显高于风湿组、风湿热组；风湿组与模型组差异不明显。提示风寒湿热都能加重血沉的表达，风寒湿环境更明显。
（6）实验各组血清、关节液及脾细胞培养上清中IL-17的检测结果
与正常组相比，其余四组浓度均明显要高；与模型组相比，风寒湿组、风湿热组浓度要高，但模型组与风湿组比较差异不显著；风湿组与风寒湿组、风湿热组比较，浓度较低；风寒湿组水平明显高于其余四组。且相关性分析显示，血清与关节液中IL-17的浓度显著相关。提示：CIA模型大鼠血清及关节液IL-17表达升高，风湿、风寒湿、风湿热环境能加重CIA大鼠血清及关节炎IL-17的表达，影响程度以风寒湿环境最为明显。且血清及关节液中IL-17浓度呈正相关。
（7）实验各组血清AHR、CD44的检测结果
与正常组比较，其余四组血清AHR浓度要高；与模型组比较，风湿组、风湿热组、风寒湿组要高；与风湿组比较，风湿热组、风寒湿组浓度较高；与风湿热组比较，风寒湿组浓度较高；风寒湿组与其余四组比较差异显著。各组血清CD44的表达情况与AHR的表达情况相同。提示：CIA模型大鼠血清AHR、CD44表达升高，风湿、风寒湿、风湿热环境能加重CIA大鼠血清AHR、CD44的表达，影响程度以风寒湿环境最为明显。
（8）实验各组血清Fas/FasL的检测结果
与正常组比较，其余四组血清Fas、FasL浓度要高；与模型组比较，风湿组、风湿热组、风寒湿组要高；与风湿组比较，风湿热组、风寒湿组浓度较高；与风湿热组比较，风寒湿组浓度较高。提示：CIA模型大鼠血清Fas、FasL表达升高，风湿、风寒湿、风湿热环境能加重CIA大鼠血清Fas、FasL的表达，影响程度以风寒湿环境最为明显。
（9）滑膜CD44表达的检测结果
模型组、风寒湿组、风湿热组、风湿组的滑膜CD44表达量与正常组相比，明显升高；风湿组、风湿热组、风寒湿组与模型组比较，表达量升高；风湿热组、风寒湿组与风湿组比较，表达量升高；风寒湿组表达率比风湿热组要高。
（10）滑膜AHR、Fas/FasL的检测结果
免疫组化结果显示：与正常对照组比较，CIA大鼠模型滑膜中Fas表达增加，而FasL表达减少。与模型组比较，风寒湿组、风湿组、风湿热组的Fas表达亦增加，FasL表达减少；风寒湿组、风湿组及风湿热组之间两两比较差异亦有统计学意义，其中风寒湿组Fas阳性细胞率最高，FasL表达最少。实验各组滑膜中AHR水平表达有显著性差异。与正常组比较，其余四组滑膜AHR表达要高；与模型组比较，风湿组、风湿热组、风寒湿组要高；与风湿组比较，风湿热组、风寒湿组较高；与风湿热组比较，风寒湿组浓度较高。
结论
综合上述结果，发现风寒湿热等外邪对CIA发病有明显的促进作用，这种作用在早期阶段就显现出来，在早期即能看到风寒湿、风湿热环境使发病率有明显提高，而风湿组发病率与模型组相当；IL-17、CD44、AHR、Fas、FasL等参与了CIA的发病；风寒湿热等外邪能加重动物模型的免疫反应，从而使病情加重；外邪因素严重影响ESR、IL-17、CD44、AHR、Fas、FasL等的表达，并能加重滑膜细胞的增生；风寒湿组对滑膜病理学影响比风湿热组严重，风寒湿组对ESR、IL-17、CD44、AHR、Fas、FasL的影响也比风湿热组更严重。提示这些指标在一定程度上能反应RA病情的活动性。
总之，外感风寒湿热等外邪能影响CIA的发病并影响病情程度。我们认为外感风寒湿热等外邪对痹证的发生和发展起了较为重要的作用，其作用机制是外邪促进了IL-17、CD44、AHR、Fas/FasL等多种炎性细胞因子的表达，使得免疫反应加重，导致病情进一步发展。
Objective
Six 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.
Methods
1. Theory research
Access 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 study
2.1Methods of experiment
2.1.1Animals and grouping
70specific 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 experiment
①Normal 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 detection
1) 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 analysis
All 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 condition
The 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 model
At 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 integral
At 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) ESR
The 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 supernatant
The 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 serum
Compared 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 serum
Compared 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 tissues
Compared 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 tissues
Immunohistochemical 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.
Conclusion
Summarizing 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.
六淫是自然界气候异常变化产生的致病邪气，包括风、寒、暑、湿、燥、火六种自然界反常的气候变化，它们多从肌表或口鼻侵入人体而发病，属于外感病的病因。六淫致病说是中医病因学的重要内容之一。对于痹证感受六淫邪气的病因病机，历代医家对其作了许多详细的记载，认为风寒湿热等外邪是痹证发生的外在因素，也是重要的发病因素。近年来关于寒、湿及热证的现代研究有很多，其中寒邪多从伤肺的角度去研究，湿热证多从动物模型及证型实质的角度去研究，而从病因学的角度系统研究外邪与痹证发病机理的较少。本课题拟在中医学病因理论的指导下，在已完成的研究基础上，对风、寒、湿、热等外邪进行理论回顾，依据外风、寒、湿、热的自然特性，调控温度、湿度及时间等作用因素，模拟风寒湿热等外邪，对胶原诱导关节炎大鼠进行刺激，观察外邪对CIA发病及病情的影响，探讨外邪与类风湿关节炎发病之间的关系；检测血沉、血清IL-17、CD44、AHR、Fas/FasL及滑膜CD44水平，分析这些指标之间的相关性，探索AHR、CD44参与类风湿关节炎发病的可能机制，从而探讨外邪致痹可能的免疫机制，为外邪致痹理论提供实验依据。
方法
1.理论研究
查阅相关资料，归纳总结外风、寒、湿、热的各项特性。中医学中“风、寒、湿、热”等概念，是对这些现象的长期观察和认识的基础上，基于取类比象的思维而形成的。因此很难作出精确的、单一的定义，而应该从病因、病机、症状等不同方面分析和理解。自然界的风是一种无形的流动的气流，因此，自然界中具有风之轻扬开泄、善动不居特性的外邪，称为风邪。风为春季的主气，但四季皆有，故风邪引起疾病虽以春季为多，但不限于春季，其他季节也可发生。风邪是最重要的致病因素，《内经》将其视为六淫之首。风邪四季皆能伤人，外感为病，常以风为先导，风邪常率他邪合而伤人，且风邪伤人往往兼它邪为患，它邪犯人，常借风而侵袭，乘虚而入。中医学认为，自然界中具有寒冷、凝结、收引特性的外邪，称为寒邪。寒乃冬季之主气，若在气温较低的冬季，人体不注意防寒保暖，则常易感受寒邪。但寒邪为病也可见于其它季节，如气温骤降、淋雨涉水、汗出当风、空调过凉及贪凉露宿，或过饮寒凉之物，均为感受寒邪的途径。自然界中具有水湿之重浊，粘滞，趋下特性的外邪称为湿邪。湿为长夏的主气，长夏乃夏秋之交，此时阳热下降，水气上腾，氤氲熏蒸，湿气充斥，为一年之中湿气最盛的季节，故长夏多湿病。此外居处潮湿，以水为事，淋雨涉水等均为湿邪致病的途径。热邪指自然界中具有火之炎热特性的外邪。热邪为病称为外热病。热邪属阳，多为外感，可见于任何季节，但夏季多见。一般与其他诸邪相兼为患，例如风热、湿热、暑热等,其中湿热致病尤为常见。因此外风、湿、热、寒等的形成与季节、气候及环境密切相关。
既然六淫是自然界气候反常（风、寒、暑、湿、燥、火六气太过不及或非其时而有其气）并影响到人体的调节适应机能及病原体的孽生传播而成为致病的邪气,那么六淫与现代气象学中的气象要素必是紧密相连的。“六淫”所概括的气象要素包括大气温度、湿度和气流方向及速度。“六淫”中的寒、热（火）、暑是对大气温度两极的定性描述,而湿、燥是对大气水汽含量的定性概括,风是对空气流动的概述。从现代气象学理解“六淫“的含义，应包含两层意思：一是风寒暑湿燥火这六种气候（实际只有风、温度及湿度这3种气象要素）和气候因素（某地区的平均天气状况及其变化特征）本身对人体的影响；二是这些气象要素和气候因素太过、不及或非时之候对人体产生不良影响而致病（包括诱发、加重、发生）。
痹作为病名，即痹证，是因风、寒、湿、热等外邪杂合侵袭人体，闭阻经络，导致气血运行不畅所发生的肢体关节肌肉疼痛、重着、麻木、肿胀、屈伸不利，甚则关节肿大变形，或累及脏腑的一类病证。历代医家对痹证的病因论述颇为丰富，其中感受外邪致痹是诸多医家认为较为重要病因病机。
西医风湿病中的部分疾病可归属于中医痹证的范畴。风湿病是一组以内科治疗为主的肌肉骨骼系统疾病，它包括：弥漫性结缔组织病及各种病因引起的关节、关节周围软组织，包括肌肉、肌腱、韧带等的疾病，表现为疼痛、肿胀、运动障碍等。如类风湿关节炎、强直性脊柱炎、骨关节炎、痛风等风湿病可归属到痹证范畴。其中RA患病率较高、临床较为常见，而且RA动物实验研究也较为成熟，目前RA动物模型中公认较为理想的是胶原诱导关节炎(collagen-induced arthritis，CIA)。
2.实验研究
2．1实验方法
2.1.1实验动物及分组
SPF级雌性Wistar大鼠70只，5周龄，体重160-180g。常规饲养一周后随机分为5组：①正常组，②模型组，③风寒湿组，④风湿热组，⑤风湿组，正常组10只，其余四组各15只。
2.1.2造模过程及实验方法
①正常组：不做任何处理，常温常湿下喂养，相对湿度（RH）：50±4%，温度（T）：21±2℃，风力0级。
②模型组：即胶原诱导关节炎（collagen-induced arthritis，CIA）模型,根据造模方法:将牛II型胶原（II collagen，CII）与等体积的不完全弗氏佐剂(Incomplete Freund's Adjurant,IFA)充分乳化，在无菌条件下，按每点皮下注射乳剂0.05mL，初次免疫在大鼠背上任选4点，加强免疫于2周后同法3点注射；常温常湿下喂养。
③风寒湿组：免疫方法同模型组，并在接受免疫的第一天开始，每天在人工气候箱内接受风寒湿刺激1次，每次持续4小时，连续30天。相对湿度（RH）：90±4%，温度（T）：6±2℃，风力3级。
④风湿热组：免疫方法同模型组，并在接受免疫的第一天开始，每天在人工气候箱内接受风湿热刺激1次，每次持续4小时，连续30天。相对湿度（RH）：90±4%，温度（T）：33±2℃，风力3级。
⑤风湿组：免疫方法同模型组，并在接受免疫的第一天开始，每天在人工气候箱内接受风湿刺激1次，每次持续4小时，连续30天。相对湿度（RH）：90±4%，温度（T）：21±2℃，风力3级。
2.2指标检测
2.2.1一般观察项目
观察大鼠精神状态、活动情况、饮食饮水量、毛色、皮肤关节、体重及二便等情况。
2.2.2关节炎指数测定
采用关节炎指数积分法评定。
2.2.3大鼠滑膜病理学观察
采用苏木素伊红(HE)染色法，光镜观察。
2.2.4血沉的检测
采用魏氏法测定。
2.2.5血清、踝关节液及脾细胞培养上清IL-17的检测
采用ELISA试剂盒检测。
2.2.6血清AHR的检测
采用ELISA试剂盒检测。
2.2.7血清CD44的检测
采用ELISA试剂盒检测。
2.2.8血清Fas/FasL的检测
采用ELISA试剂盒检测。
2.2.9滑膜CD44表达的检测
采用定时定量荧光PCR检测。
2.2.10滑膜AHR、Fas/FasL
免疫组化法检测。
2.3统计学处理
应用SPSS19.0软件进行统计学处理。符合正态分布的计量资料以均数±标准差(x s)表示，采用单因素方差分析,组间两两比较采用q检验、t检验、卡方检验。P<0.05为差异有统计学意义。
结果
（1）一般情况
风寒湿组大鼠萎靡不振，蜷卧懒动，行动迟缓，反应迟钝，抓捕时逃避，食量减少，大便稀溏，体重增幅最小，体重最轻；风湿热组体毛无光泽，少动，萎靡，大便粘滞，臭秽难闻；风湿组稍显呆滞，反应迟钝，饮食量减少，大便软；模型组活动欠灵活，大便基本正常，体毛欠光泽，饮食量基本正常，体重与模型组相当。
（2）CIA模型发病率情况
实验15天时，风寒湿组、风湿热组发病率比模型组、风湿组要高，差异有统计学意义，风寒湿组与风湿组之间比较，发病率没有显著差异，模型组与风湿组比较亦无显著差异。实验结束时，模型组、风湿组、风湿热组、风寒湿组发病率分别为66.7%、73.3%、80.0%、86.0%，组间差异无统计学意义。提示风、寒、湿、热因素对CIA的发病有明显的促进作用，且只有风寒湿合并或风湿热合并侵袭时这种作用才显现明显。
（3）病理结果
滑膜病理切片显示模型组、风湿组、风湿热组、风寒湿组滑膜细胞增生按此顺序依次增多，以风寒湿组最为明显。①正常组：滑膜组织无炎性细胞浸润，滑膜细胞无增生。②模型组：滑膜细胞增生不明显。③风湿组：可见滑膜细胞少许增生。④风寒湿组：滑膜细胞增生明显，可见数层排列不规则的滑膜细胞，大量炎性细胞浸润。⑤风湿热组：可见滑膜组织增生，可见2～3层排列不规则的滑膜细胞及炎性细胞浸润。
（4）关节炎指数的测定情况
结果显示在实验15天时，与正常组比较，其余四组得分均明显升高；与模型组比较，风寒湿组、风湿热组明显升高，但风湿组差异不明显；风寒湿组得分明显高于风湿热组、风湿组；风湿热组高于风湿组。实验结束时测得风寒湿组得分最高，其次是风湿热组，风湿组次之，模型组最低,正常组得分为零。说明CIA模型复制成功。提示风、寒、湿、热等外邪能加重CIA的发病，以风寒湿邪合并侵袭时程度最为严重。
（5）实验各组血沉检测结果
与正常组对比，其余四组血沉均明显升高；与模型组比较，风湿组、风寒湿组、风湿热组血沉均较高；风寒湿组明显高于风湿组、风湿热组；风湿组与模型组差异不明显。提示风寒湿热都能加重血沉的表达，风寒湿环境更明显。
（6）实验各组血清、关节液及脾细胞培养上清中IL-17的检测结果
与正常组相比，其余四组浓度均明显要高；与模型组相比，风寒湿组、风湿热组浓度要高，但模型组与风湿组比较差异不显著；风湿组与风寒湿组、风湿热组比较，浓度较低；风寒湿组水平明显高于其余四组。且相关性分析显示，血清与关节液中IL-17的浓度显著相关。提示：CIA模型大鼠血清及关节液IL-17表达升高，风湿、风寒湿、风湿热环境能加重CIA大鼠血清及关节炎IL-17的表达，影响程度以风寒湿环境最为明显。且血清及关节液中IL-17浓度呈正相关。
（7）实验各组血清AHR、CD44的检测结果
与正常组比较，其余四组血清AHR浓度要高；与模型组比较，风湿组、风湿热组、风寒湿组要高；与风湿组比较，风湿热组、风寒湿组浓度较高；与风湿热组比较，风寒湿组浓度较高；风寒湿组与其余四组比较差异显著。各组血清CD44的表达情况与AHR的表达情况相同。提示：CIA模型大鼠血清AHR、CD44表达升高，风湿、风寒湿、风湿热环境能加重CIA大鼠血清AHR、CD44的表达，影响程度以风寒湿环境最为明显。
（8）实验各组血清Fas/FasL的检测结果
与正常组比较，其余四组血清Fas、FasL浓度要高；与模型组比较，风湿组、风湿热组、风寒湿组要高；与风湿组比较，风湿热组、风寒湿组浓度较高；与风湿热组比较，风寒湿组浓度较高。提示：CIA模型大鼠血清Fas、FasL表达升高，风湿、风寒湿、风湿热环境能加重CIA大鼠血清Fas、FasL的表达，影响程度以风寒湿环境最为明显。
（9）滑膜CD44表达的检测结果
模型组、风寒湿组、风湿热组、风湿组的滑膜CD44表达量与正常组相比，明显升高；风湿组、风湿热组、风寒湿组与模型组比较，表达量升高；风湿热组、风寒湿组与风湿组比较，表达量升高；风寒湿组表达率比风湿热组要高。
（10）滑膜AHR、Fas/FasL的检测结果
免疫组化结果显示：与正常对照组比较，CIA大鼠模型滑膜中Fas表达增加，而FasL表达减少。与模型组比较，风寒湿组、风湿组、风湿热组的Fas表达亦增加，FasL表达减少；风寒湿组、风湿组及风湿热组之间两两比较差异亦有统计学意义，其中风寒湿组Fas阳性细胞率最高，FasL表达最少。实验各组滑膜中AHR水平表达有显著性差异。与正常组比较，其余四组滑膜AHR表达要高；与模型组比较，风湿组、风湿热组、风寒湿组要高；与风湿组比较，风湿热组、风寒湿组较高；与风湿热组比较，风寒湿组浓度较高。
结论
综合上述结果，发现风寒湿热等外邪对CIA发病有明显的促进作用，这种作用在早期阶段就显现出来，在早期即能看到风寒湿、风湿热环境使发病率有明显提高，而风湿组发病率与模型组相当；IL-17、CD44、AHR、Fas、FasL等参与了CIA的发病；风寒湿热等外邪能加重动物模型的免疫反应，从而使病情加重；外邪因素严重影响ESR、IL-17、CD44、AHR、Fas、FasL等的表达，并能加重滑膜细胞的增生；风寒湿组对滑膜病理学影响比风湿热组严重，风寒湿组对ESR、IL-17、CD44、AHR、Fas、FasL的影响也比风湿热组更严重。提示这些指标在一定程度上能反应RA病情的活动性。
总之，外感风寒湿热等外邪能影响CIA的发病并影响病情程度。我们认为外感风寒湿热等外邪对痹证的发生和发展起了较为重要的作用，其作用机制是外邪促进了IL-17、CD44、AHR、Fas/FasL等多种炎性细胞因子的表达，使得免疫反应加重，导致病情进一步发展。
Objective
Six 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.
Methods
1. Theory research
Access 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 study
2.1Methods of experiment
2.1.1Animals and grouping
70specific 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 experiment
①Normal 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 detection
1) 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 analysis
All 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 condition
The 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 model
At 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 integral
At 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) ESR
The 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 supernatant
The 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 serum
Compared 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 serum
Compared 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 tissues
Compared 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 tissues
Immunohistochemical 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.
Conclusion
Summarizing 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.
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