摘要
目的
依据中医基础理论和参考相关文献,采用复合因素建立慢性疲劳综合征(CFS)大鼠模型。从理论上深入探讨及阐释气络与CFS的关系;通过实验研究,验证气络与CFS相关的假设;并探索补气通络方改善CFS的作用机制。
材料和方法
整理有关CFS的国内外文献,按照中医理论归纳CFS与中医脏腑经络气血阴阳的关系。在综合分析气络与NEI(神经内分泌免疫网络)、CFS相互关系的基础上,通过逻辑推理,首次提出“CFS与气络相关”的假说;并针对气络“虚、滞”的病机特点,结合临床实际,筛选药物组成补气通络方,以实验的方法,观察补气通络方对CFS的作用机制,从而验证该假说的科学性和正确性。
清洁级Wistar雄性大鼠50只,随机分成正常组(10只)、造模组(40只),造模组动物采用复合因素造模。造模过程:①运动造模制作160cm×80cm×60cm的注水鼠箱,水深30cm,水温4℃左右。每日上午取40只大鼠,冷水(4℃)游泳,每次30min;连续21天②房劳造模每晚让造模大鼠与发情期雌鼠合笼(每次合笼都新换一批雌鼠);连续21天。③应激造模每日下午用尖端缠有纱布的止血钳夹其中一只大鼠尾部,让每只大鼠被夹的机会均等,令其与其它大鼠撕打,模仿人类生存环境不佳,竞争激烈,精神压力大的情况;夹尾以不破皮、不流血为度,每次刺激2小时。定期更换夹尾大鼠,使造模结束时每只大鼠夹尾次数基本相同。连续21天。
造模结束后,比较正常组和模型组动物体重、皮毛及其色泽、饮水量、进食量、活动量、精神状态等一般表现的差异,测定各组大鼠体力情况及疲劳程度。然后将造模组重新分为模型组(B)、补气通络方组(C)和补气组(D)及通络组(E)每组10只,与未造模的正常组(A)一起使参加实验的动物总数达到5组,共50只。重新分组后,A、B两组灌胃生理盐水2ml,C、D、E三组分别灌胃补气通络方、补气方、通络万的水煎液,连续灌胃14天。补气通络方由人参、黄芪、枳壳、川芎、地龙等组成,补气方由人参、黄芪组成,通络方由枳壳、川芎、地龙组成,三方均按人与动物体表面积折算法算得大鼠等效剂量为每公斤体重50g生药。用5倍于药材的冷水浸泡20分钟,然后用武火煎至水沸,改用文火煎煮40分钟,滤出药液加冷水适量,煎至水沸,改用文火煎煮30分钟,滤出药渣,将两次煎取药液混匀浓缩成每毫升含生药2g的药液,3种药液均置冰箱0-4℃保存,每次灌胃2ml。
灌胃结束后,检测各组动物下丘脑单胺类神经递质的含量、肝糖原(LG)、肌糖原含量、血清超氧化物歧化酶活性、丙二醛含量、IL-1、β-EP、CRHmRNA等指标,探讨补气通络方对CFS动物模型的疗效,及产生疗效的作用机制。
结果
造模结束后,模型组动物体重减轻,进食量、饮水量明显减少(与正常组比较,p<0.05),鼠尾悬挂试验中,6分钟内不动时间明显增多(与正常组比较,p<0.01)。旷场实验中,6分钟内,模型大鼠穿格次数显著降低(P<0.05),理毛时间极显著增高(P<0.05);力竭游泳试验测试中,6分钟内,模型大鼠力竭时间显著降低(P<0.01),下丘脑单胺类神经递质含量检测结果:模型组动物下丘脑DA含量明显降低(p<0.01),5-HT含量明显增高(p<0.01);治疗后补气通络方组、补气组、通络组DA含量较模型组有明显升高(p<0.01),但三者差异无统计学意义(p>0.05)。5-HT含量较模型组有明显降低(p<0.01),说明三组都有明显疗效,但补气通络方组与通络方组无显著性差异(p>0.05),补气通络方组疗效优于补气组(p<0.05)。肝糖原含量模型组明显低于正常组(p<0.01)、通络组含量高于模型组,但二者差异无统计学意义(p>0.05),补气通络方组高于补气组(p<0.05);说明补气通络方组疗效优于补气组;肌糖原含量正常组高于模型组(p<0.01),治疗后,补气通络方组、补气组、通络组肌糖原含量高于模型组(p<0.01),但三者差异无统计学意义(P>0.05)。SOD检测结果中,模型组动物SOD活性明显低于其他组,说明CFS动物体内存在着清除自由基能力降低的现象。补气通络方组、补气组、通络组SOD活性有所提高,说明补气通络方、补气方、通络方均具有一定的抗氧化能力,可以拮抗因为过度疲劳、睡眠障碍和精神紧张所带来的氧自由基损害。并且三组在提高SOD活性方面无明显差异(P>0.05)。MDA检测结果中,模型组MDA明显高于正常组,说明模型组动物体内组织、器官遭受自由基损害非常突出。与模型组比较,补气通络方组MDA含量明显降低(P<0.05),说明补气通络方有阻断自由基毒性反应的作用;而补气组与通络组MDA含量无显著性差异(P>0.05),说明此二组均无阻断自由基毒性反应的作用。垂体β-内啡肽(β-EP)含量检测中,模型组动物垂体内β-EP含量明显高于正常组(P<0.01),说明复合因素造模法严重影响了β-EP的代谢,使其合成增加或降解减慢。补气通络方组、补气组、通络组阳性细胞数及阳性细胞灰度值明显低于模型组(P<0.05),接近正常组(P>0.05),提示三组能显著降低垂体内β-EP,对解除慢性疲劳有一定作用;补气通络方组阳性细胞数及阳性细胞灰度值与补气组、通络组有显著性差异(P<0.05),证明补气通络方组疗效优于补气组和通络组。外周血IL-1含量检测中,模型组动物外周血IL-1含量明显高于正常组(P<0.01),说明复合因素造模法严重影响了IL-1的代谢,使其合成增加或降解减慢。治疗后,补气通络方组、补气组、通络组动物外周血IL-1含量均低于模型组(P<0.01),说明三组均可以抑制IL-1的活性,增加其降解。而补气通络方组IL-1含量与补气组、通络组有显著性差异(P<0.05),说明补气通络方疗效优于补气组与通络组。下丘脑CRHmRNA表达情况检测中,正常组动物CRHmRNA表达情况最低,其半定量检测结果明显低于模型组(P<0.01)。说明采用复合因素造模可以降低动物CRHmRNA的表达,这可能是模型动物运动能力下降的原因之一。经用补气通络方、补气方、通络方治疗后,造模动物CRHmRNA的表达均明显低于模型组(P<0.01),说明三组治疗方法均有降低CRHmRNA表达的作用,可能是下调动物下丘脑CRHmRNA的表达,抑制了HPA轴功能,从而减轻CFS的疲劳、烦躁、失眠等表现。在疗效上,补气通络方组CRHmRNA表达结果与补气组、通络组比较有显著差异(P<0.05),说明补气通络方疗效优于补气组与通络组。与其它实验结果相参照,说明补气通络方、补气方、通络方产生的疗效并不一致,补气通络方疗效最好,补气方与通络方疗效的综合指标则大致相当。
结论
采用形劳、房劳、应激刺激等复合因素制造的CFS大鼠模型,综合考虑了CFS的病因和症状。从造模后动物的进食量、饮水量、疲劳程度等方面的症状看,该模型的外在症状与CFS相符,说明该造模方法具有一定科学性。
经过补气通络方治疗,模型动物的外在症状和实验指标均有改善,说明补气通络方可以有效治疗CFS。补气通络方对模型动物的作用机制主要是对NEI网络的调节。一是影响肝糖原、肌糖原等与疲劳有关的生化指标,二是影响下丘脑DA、5-HT及垂体β-EP等与中枢性疲劳和精神情志有关的神经递质代谢,三是对影响机体代谢的有关酶类代谢,如可以提高血清SOD活性,SOD可以减轻自由基对机体的破坏,四是对免疫功能的调节,如补气通络方可以降低外周血IL-1含量,而IL-1与CFS具有相关性,五是调节了内分泌功能,模型组动物海马CRHmRNA表达明显高于正常组,揭示CFS动物下丘脑CRHmRNA的转录增强,从而上调了HPA轴功能,导致CFS的疲劳、烦躁、失眠等表现。综合上述实验指标,我们可以得出如下结论:一.采用游泳运动、房劳和夹尾刺激可以较好地复制CFS模型,二.补气通络方主要通过对NEI网络的调节来治疗CFS,三.CFS与中医气络相关。
从气络着手,治疗CFS具有深入研究的价值,本实验为今后开展从气络论治CFS的研究奠定了坚实的基础,同时,对于深入研究气络的本质具有重大的指导价值。
object
Based on the theory of Chinese medicine and literature records,the complicated factors used to build up chronic fatigue syndrome(CFS) rat model.In theory,study deeply of CFS pathogenesis oftraditional Chinese medicine to explain the relationship between the Qi collatetal channel and the CFS;through experimental research,test and verify the assumption that the Qi collateral channel related with the CFS,and explore the mechanism which reinforcing Qi and smoothing collateral channel improve the CFS.
Materials and methods
sort out systemly the relevant literature of the CFS,according to the theory of tradional Chinese medicine,tiding up the relationship of CFS with organs,the yin and yang,qi and blood,in-depth study of its causes,disease place,disease nature,and put forward boldly "CFS associated with the Qi collateral channel" hypothesis;according to the Pathogenesis nature of the Qi collateral channel for weakness,stagnation,by clinical practice,we pick out drug components of the prescription of reinforcing Qi and smoothing the collateral channel.Through the way of animal experiments,we observe the effect that prescription of reinforcing Qi and smoothing the collateral channel treat CFS,we can verify that the hypothesis is Sciencial and rational.
Clean level 50 male Wistar rats were randomly divided into normal groups (10),model group(40).the modelgroup is adopted complicated factors to build up model.Modeling process:①movement build up model:make a rat-box of 160cmx80cmx60cm,the water depth of 30cm,the water temperature of about 4℃.40 rats are checked out daily,and swim in the cold water(4℃),30min each time for 21 days②sexy make model:modelrats mated with rut female rats each night,for 21 days.③stimulate make model:we nip one rat tail by hemostat which tip is enwraped by gauze,making the rat fight with each other.The living environment simulating human being's is bad,competition is fierce,not breaking skin and not bleeding as a limit,at the end of model,The number of each niped tail rat is basically the same,for 21 days,for 2 hours every time.
Making model is over,we compared with the body weight,hair color,drinking water,food intake,activity and mental state such as the the differences of general performance in the determination of physical fatigue status of the normal group and rats model.And then divided model animal into the model group(B),the reinforcing Qi and smoothing collateral channel group(C) and the reingforcing Qi Group(D) and the smoothing collateral channel group(E),10 each group.Altogether with the normal group,the total number reach 50.After re-grouping,A group and B group are filled 2ml 0.9%sod.chloride to the stomach,C group,D group and E group are respectively filled with reinforcing Qi and smoothing collateral channel prescription,reinforcing Qiprescription,and dredging collateral channel prescription,for 14 days.
The Reinforcing Qi and smoothing collateral channel prescription is consist of ginseng,astragalus,Aurantii,such as lung,The Reinforcing Qi prescription is consist of ginseng,astragalus.
The smoothing collateral channel is consist of Aurantii and Long, according animal body surface area conversion,The equivalent rat dosages of person are converted,rats equivalent dosage are every kilogram of 50g.The weight ofcold water is 5 times than the drugs,and is immersed for 20 minutes.after the water is boiled,we use slow fire for 40 minutes.
The liquid mixtures will be condensed to every milliter of 2g crude drug.3 kinds of drugs are saved in refrigerators at 0-4℃,and each oral 2ml.
After the delivery,testing the content of the hypothalamus monoamine neurotransmitter,liver glycogen(LG),muscle glycogen,MDA,IL-1,β-EP, the levels of serum SOD activity,and other indicators of animal model to explore the relevant indicators of the animal model,using RT-PCR detected the expression of CRHmRNA,and such as indicators to observe the vitality of hypothalamus CRHmRNA expression bythe reinforcing Qi and smoothing collacteral channel treating the CFS model rat.
Results of the above tested clearly indicate that reinforcing Qi and smoothing collacter channel have a effect on CFS rat model,and the micro-mechanism of reinforcing Qi and smoothing collacter channel treating CFS animal model.
The results
After the making model is over,the weight of model group animals reduce,the amount of water and food intake reducing significantly(with the normal group,p<0.05),tail suspension test,fxed time is increased in 6 minutes,(compared with the normal group,p<0.01).Open-field experiments, The time of the rats cliping hair a significant reduction in 6 minutes,in the number of moving through cells(P<0.05),the rational gross time is very higher significantly(P<0.05);exhaustive swimming test,the rat model of strength Dried up significantly and the time of swimming lower in 6 minutes (P<0.01),
The test results of Hypothalamus monoamine neurotransmitter content: animal model of hypothalamic DA group were significantly lower(p<0.01),5-HT content was significantly higher(p<0.01);after treatment DA content of reinforcing Qi and smoothing collacteral channel Group, reinforcing Qi Group,the smoothing collacteral channel than the model group,andhave significantly higher(p<0.01),but the three group was no significant difference(p>0.05).5-HT content is significantly low -er(p<0.01)than the model group,three groups have clear effects,but compared with the smoothing the collacteral channel prescription,the reinforceing Qi and smoothing collacteral channel prescription was no sig -nifycant difference (p>0.05),The effect of the reinforcing Qi and smo -othing collacteral channel prescription and the smoothing the coll act -eral channel prescription is better than reinforcing Qi Group(p<0.05).The content of Liver glycogen test:normal group was significantly lower than the model group(p<0.01),The treatment group was higher than the model group(p<0.05),but there are no significant difference among the three groups (p>0.05),the reinforcing Qi and smoothing collacteral channel prescription is higher than the reinforcing Qi Group(p<0.05);that show that the effect of the reinforcing Qi and smoothing collacteral channel prescription is better than the reinforcing Qi Group;in the muscle glycogen content test:the normal model group is higher(p<0.01),After treatment,the treatment muscle glycogen content is higher than the model group(p<0.01),but there are no significant difference among the three groups(P>0.05).SOD test results:The SOD content of animal model group is significantly lower than other groups,there are no significant difference in raising SOD activity among the reinforcing Qi and smoothing collacteral channel prescription,the reinforcing Qi prescription,andthe smoothing collacteral channel prescription(P>0.05). MDA test results:the MDA content of model group was significantly higher than the normal group,that show that the damage ofthe model group animals, organs from free-oxygen radical is the Very prominent.Compared with the model group,the MDA content of the reinforcing Qi and smoothing the collacteral channel group are significantly lower(P<0.05),that show that the reinforcing Qi and smoothing collacteral channel prescription have the role of preventing free-oxygen radical toxicity,in MDA content,there are no significant difference(P>0.05)between the reinforcing Qi act and smoothing collacteral channel act,that makes it clear that the two groups can not block the free radical toxicity.Pituitaryβ-endorphin(β-EP) content detection: pituitaryβ-EP content of the model group of animals is significantly higher than the normal group(P<0.01),which show that the means of complex factors making model have seriously effect on the metabolism ofβ-EP, Synthesis of its increase or the degradation slow down.The number of positive cells of the reinforcing Qi and smoothing collacteral channel group,the reinforcing Qi group,and the smoothing collacteral channel group are significantly lower than the model group(P<0.05),close to the normal group (P>0.05),the three groups could promptly reduceβ-EP in the pituitary,and have a role in lighting the chronic fatigue syndrome;the number of positive cells and the gray value of the reinforcing Qi and smoothing collacteral channel group compare with the reinforcing Qi Group and the smoothing collacteral channel group,there are significant different(P<0.05),the reinforcing Qi and smoothing collacteral channel group proved more effective than the reinforcing Qi Group and the smoothing collacteral channel group.The IL-1 content of peripheral blood test:the content of peripheral blood IL-1 of animal model is significantly higher than the normal group(P<0.01),which indicate the means of complex factors making model have seriously effect on the metabolism of IL-1,and its can increase the synthesis of IL-1 or Slow down degradation of it.After treatment,the three group of peripheral IL-1 level was lower than the model group(P<0.01),the three groups can inhibit the activity of IL-1 and increase its degradation.The IL-1 content of the reinforcing Qi and smooth collacteral channel group compare with the reinforcing Qi group and the smooth collacteral channel group,there are significant difference(P<0.05),which show the effects of the reinforcing Qi and smooth collacteral channel group than the reinforcing Qi group and the smooth collacteral channel group.
Hypothalamus CRHmRNA expression detection,the CRHmRNA expression of the normal group is lowest,the result of the semi-quantitative test of it is significant lower than the model group(P<0.01).the complex factors can reduce the CRHmRNA expression of animal model,which may be one of the reasons for the decline ofthe ability of model animal movement.
after the treatment,the CRHmRNA expression of the treatment group is significantly lower than the model group(P<0.01),which show the three groups can reduce the expression of CRHmRNA,which may be decrease the CRHmRNA expression of Hypothalamus of animals,and inhibit the function of the HPA axis,so as to lighten the fatigue of CFS,irritability,insomnia and so on.In the relationship of effect,the results of the CRHmRNA expression of the reinforcing Qi and smooth collacteral channel group compare with the reinforcing Qi group and the smooth collacteral channel group,there are significant difference(P<0.05),the effects of the reinforcing Qi and smooth collacteral channel group than the reinforcing Qi group and the smooth collacteral channel group.And in the light of other experimental results,we know that the mechanism of the effect of the three group are different,the effect of the reinforcing Qi and smooth collacteral channel group is the best, and the effect of the reinforcing Qi group and the smooth collacteral channel group are nearly uniform.
Conclusion
By complex factors,we manufacture rat model of CFS,and considere the causes and manifestations of the CFS.From the model of the animal after eating,drinking water,after making model,we observe the degree of fatigue,water intake,food intake,such as performances,the model's performance is uniform to CFS,which shows that the methods has a certain rationality.
After the treatment of the reinforcing Qi and smooth collacteral channel prescription,the external symptoms and micro-mechanism of animal model have improved,which show that the reinforcing Qi and smooth collacteral channel prescription can prevent and treat CFS,and have a complicated mechanisms,It can regulate the neuroendocrine and immune network.
Based on the above experimental results,we can conclude that the making modelsuch as swimming,labor room and stimulation can be used to copy the CFS model;CFS relate with Qi collacteral channel;the reinforcing Qi and smooth collacteral channel prescription can improve the clinical symptoms of CFS;the treatment mean from Qi collacteral channel theory has an the value of in-depth study,and has the foundation for future research of CFS.At the same time,there is great guiding value in in-depth study of the nature of the Qi collacteral channel.
引文
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