清风胶囊治疗阿片类药物依赖稽延性戒断症状的实验研究及中医理论探讨
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摘要
背景
阿片类物质依赖稽延性戒断症状,属于中医学断瘾后诸症范畴,是指病人在脱毒后还遗留一系列的症状,如倦怠烦躁,周身疼痛,顽固失眠,心情抑郁,渴求焦虑等,是导致脱毒者复吸的因素之一。目前西药治疗主要有阿片类制剂的替代递减疗法、α2受体激动剂的治疗和镇静催眠药治疗等;其特点是作用迅速、疗效确切,但戒毒西药毒副作用大,本身易成瘾,不能很好地控制脱毒后的稽延性戒断症状。中医药治疗较为安全,毒副作用小,本身无成瘾性。中药扶正驱邪,整体调理,标本兼治,低毒无瘾的作用特点是西药无法比拟的。目前我国已有多种中药戒毒新药制剂被批准生产,用于临床治疗。中医药戒毒治疗目前仍属于缓解急性戒断症状这一类,对防治复吸以及治疗稽延性症状方面,研究甚少,还没有疗效确切的药物,目前尚无主要用于治疗阿片类稽延性戒断症状,防止复吸的中药制剂上市。清风胶囊经过初步的药效学实验及临床疗效评估,表明本方具有显著的镇静催眠,镇痛制瘾作用,对于吸毒者脱毒后的烦躁失眠,渴求焦虑,周身疼痛等主要的稽延症状群具有针对性治疗效果,能有效控制脱毒后的稽延性戒断症状;同时能减轻吸毒者对毒品的精神依赖,防止复吸。清风胶囊用于戒毒治疗的研究至今已有数年,以往的研究主要着重于药理学及临床疗效等方面。作为中药新药,清风胶囊临床用于治疗阿片类戒断综合征有明显的疗效,药理学方面的研究揭示了该药部分的作用及作用机制,但其中医理论方面的作用机制研究尚未有具体涉及。
目的
本课题以探讨清风胶囊治疗稽延性戒断症状的中医机理为目的,在原有实验及临床研究基础上,进一步探讨其功效及作用机制,以进一步完善相关方面的研究。
方法
1.理论研究:(1)整理中医药有关戒毒的文献,理解中医学如何认识阿片类物质依赖稽延性戒断症状,如何进行辨证治疗及治疗的关键;(2)剖析清风胶囊的中药药物组成,结合相关中医学理论知识,从中医学角度理解清风胶囊的功效及各主药的作用。
2.实验研究:选取相关实验指标,通过建立动物模型,观察清风胶囊对吗啡依赖动物反复染毒后各指标的影响,结合相关理论,解释清风胶囊的功效:(1)按剂量递增法建立吗啡依赖大鼠模型,各组大鼠除空白对照组,给予吗啡从每次10mg/kg(sc)逐次递增至每次80mg/kg(sc),连续注射吗啡5d,空白对照组皮下注射等体积生理盐水;各组大鼠于末次给吗啡后2小时,腹腔注射纳洛酮2.0mg/kg进行催促戒断;停药14天后,按上述方法重复染毒一遍。模型建立后各组大鼠灌胃给予相应药物5天,观察清风胶囊对吗啡依赖大鼠反复染毒后体重、进食量,及全血粘度、血浆粘度、红细胞数、血红蛋白量、红细胞比积等指标的影响。(2)按剂量递增法建立吗啡依赖小鼠模型,给予吗啡从每次25mg/kg(sc)逐次递增至每次150mg/kg(sc),连续注射吗啡5d,空白对照组皮下注射等体积生理盐水;各组小鼠于末次给吗啡后2小时,腹腔注射纳洛酮4mg/kg进行催促戒断;停止给药7天,于停药第8天重复上述方法再次注射吗啡3d及戒断(此步骤重复操作2遍)。模型建立后各组小鼠灌胃给予相应药物5天,观察清风胶囊对吗啡依赖小鼠反复染毒后自主活动及游泳时间的影响。(3)按剂量递增法建立吗啡依赖小鼠模型(方法同上)。模型建立后各组小鼠灌胃给予相应药物5天,观察清风胶囊对吗啡依赖小鼠反复染毒后血液中c-fos基因表达的影响。(4)实验结果用SPSS11.5统计软件分析,方法用单因素方差分析。
结果
1.理论研究:(1)稽延性戒断症状在中医学中被称之为“断瘾后诸症”。中医学认为它是由于长期吸食阿片烟毒后,机体气血津液严重耗损,正气衰竭;戒断后,余毒未去,脏腑功能受损后一时难以恢复,故而出现一系列的戒断后症状。断瘾后诸症以烟毒余邪稽留,经气血脉瘀遏,气血津液受损,脏腑阴阳失和为主要病机。断瘾后诸症的主要特征是毒瘀蕴结,正气不足。有四种常见证型:毒瘀蕴结,气血亏虚型;毒瘀蕴结,气阴不足型:毒瘀蕴结,阴虚火旺型:毒瘀蕴结,阴阳两虚型。根据正虚与邪实的实际情况辨证施治,是中医药治疗断瘾后诸症的精髓所在。总的来说,扶正祛邪是中医学在戒毒治疗及断瘾后康复中一以贯之的原则,扶助正气主要从益气养血、益气养阴、滋补阴液、阴阳并补等几方面论治;祛邪的具体治法有利尿解毒、清热解毒、泻下烟毒、涌吐烟毒、以毒攻毒等。断瘾后的调补尤以补益气血,恢复脾胃功能、巩固后天之本为主。(2)清风胶囊由青风藤、人参等中药组成。其中青风藤味苦性平,归肝、脾经,具有祛风湿、通经络、镇痛、利小便之功效。其主要的有效成分青藤碱具有与吗啡相似的化学结构,但在分子构型及基团的排列上均不同,因而形成青风藤具有抗吗啡作用但无成瘾性的功效。人参味甘、微苦,性平,归脾、肺、心经。功可大补元气,复脉固脱,补脾益肺,生津,安神,补气生血,补气助阳,益气生津,益气滋阴。适于成瘾者在戒毒期和断瘾后的虚弱证候。其主要成分人参皂苷有抗吗啡耐受及成瘾作用,同时人参具有镇静、抗惊厥作用,并有提高肠输送功能、改善消化道症状的作用。从中医学角度来看,青风藤有祛瘀逐邪的作用,人参起补益扶本的作用,两者结合使用可起到祛除毒瘀,补益气血的功效,从而对“断瘾后”正虚邪留的机体起到治疗作用,此法亦顺应了中医学认为断瘾后诸症的治疗以扶助正气与祛除余毒为主的治疗原则。
2.实验研究
(1)药物对吗啡依赖大鼠进食和体重的影响
给药后各组大鼠的进食量有不同程度的增加,用增加值进行统计学分析,方差不齐(P=0.000),且Dunnett's T3法比较未见显著性差异,结果无统计学意义。
给药后大鼠体重增加,用给药第3天、末次给药后24h体重增加值分别进行统计学分析,各组间有显著性差异(分别为F=16.930,P=0.000和F=13.673,P=0.000);给药第3天、末次给药后24h吗啡模型组大鼠体重增加值均比空白对照组小(均为P=0.000),清风胶囊高剂量组大鼠体重增加值均比吗啡模型组大(分别为P=0.004,P=0.002);给药第3天清风胶囊中剂量组大鼠体重增加值与吗啡模型组比较无显著性差异(P=0.061),末次给药后24h清风胶囊中剂量组大鼠体重增加值比吗啡模型组大(P=0.010);给药第3天、末次给药后24h清风胶囊低剂量组大鼠体重增加值与吗啡模型组比较均无显著性差异(分别为P=0.216,P=0.336),十全大补丸组大鼠体重增加值与吗啡模型组比较均无显著性差异(分别为P=0.883,P=0.968)。
(2)药物对吗啡依赖大鼠血液粘度的影响
大鼠全血粘度的检测结果表明,切变率1/s各组间全血粘度有显著性差异(F=2.558,P=0.042),吗啡模型组大鼠比空白对照组高(P=0.005),清风胶囊高、中、低剂量组大鼠均比吗啡模型组低(分别为P=0.005,P=0.016,P=0.021),十全大补丸组大鼠与吗啡模型组比较无显著性差异(P=0.061)。切变率5/s各组间全血粘度有显著性差异(F=2.702,P=0.034),吗啡模型组大鼠比空白对照组高(P=0.010),清风胶囊高、中、低剂量组大鼠均比吗啡模型组低(分别为P=0.006,P=0.026,P=0.005),十全大补丸组大鼠与吗啡模型组比较无显著性差异(P=0.149)。切变率30/s、150/s各组间全血粘度无显著性差异(分别为F=1.624,P=0.176和F=1.390,P=0.249)。
大鼠血浆粘度检测结果表明,各组间有显著性差异(F=3.165,P=0.017),吗啡模型组大鼠血浆粘度比空白对照组高(P=0.032),清风胶囊高剂量组大鼠比吗啡模型组低(P=0.029),清风胶囊中、低剂量组和十全大补丸组分别与吗啡模型组比较均无显著性差异(分别为P=0.824,P=0.194,P=0.428)。
(3)药物对大鼠红细胞数、血红蛋白量、红细胞比积的影响:实验结果经统计学分析表明,各组大鼠红细胞数、血红蛋白量、红细胞比积均未表现出显著性差异(分别为F=1.604,P=0.186:F=1.179,P=0.340和F=1.088,P=0.385)。
(4)药物对吗啡依赖小鼠自主活动次数的影响:吗啡依赖小鼠在给药治疗前后自主活动次数有明显变化,以给药后比给药前活动次数增加值进行统计学分析,各组间有显著性差异(F=3.994,P=0.005),吗啡模型组小鼠自主活动增加值比空白对照组低(P=0.041),清风胶囊高、中、低剂量组小鼠自主活动增加值均比吗啡模型组高(分别为P=0.000,P=0.001,P=0.008),十全大补丸组小鼠自主活动增加值与吗啡模型组比较无显著性差异(P=0.182)。
(5)药物对吗啡依赖小鼠游泳时间的影响:给药治疗后,以各组小鼠游泳时间进行统计学比较,方差不齐(P=0.008),且Dunnett's T3法比较未见显著性差异,结果无统计学意义。
(6)药物对吗啡依赖小鼠血液中c-fos基因表达的影响:在镜下分别观察每张血涂片左、中、右三个视野,以三个视野的平均阳性细胞数进行统计学分析,各组间有显著性差异(F=4.346,P=0.003),吗啡模型组平均阳性细胞数比空白对照组高(P=0.016),清风胶囊高、中、低剂量组平均阳性细胞数均比吗啡模型组低(分别为P=0.000,P=0.005,P=0.018),十全大补丸组平均阳性细胞数与吗啡模型组比较无显著性差异(P=0.163)。
结论
1.从中医学角度来看,进食量和体重是反映机体脾胃气血功能的指标,实验结果表明清风胶囊有促进吗啡依赖戒断后大鼠进食和体重增加的作用,说明该药增强了脾胃功能,促进了机体对食物的纳入运化和气血的化生。中医学认为气为血帅,气能行血,气虚则行血功能减弱,导致血液运行不利,血液粘滞瘀留;大鼠在成瘾戒断过程中损伤元气,余毒瘀留,导致气血虚弱,气血瘀滞,本实验结果表明清风胶囊有降低吗啡依赖戒断后大鼠全血粘度和血浆粘度的作用,说明该药有补气行血,活血祛瘀,促进气血运行的作用。红细胞数和血红蛋白量是反映血液功能状态的指标,实验结果无统计学意义,药物对该指标的影响尚待进一步的验证。
2.中医学认为气有推动、温煦、固摄、防御、气化等功能;血有濡养脏腑、经络、组织等功能;机体在正气虚损,气血亏虚时,机体的生理机能、脏腑组织的机能活动相应会有明显的下降,表现为活力下降,喜静,不喜动,甚至嗜睡等。吗啡依赖小鼠在反复染毒后,出进食量减少,嗜睡,活力减退,活动量减少,喜静伏等表现。清风胶囊治疗可使染毒小鼠自主活动次数增加,提高小鼠活力,说明该药起到了扶助正气、补益气血、增强身体机能的作用。游泳时间是体现小鼠抗疲劳及耐受能力的一个指标,实验中的设置是为检验药物扶正益本的功效,可看到清风胶囊治疗组游泳时间比吗啡模型组游泳时间长,但实验结果无统计学意义,药物对该指标的影响尚待进一步的验证。
3.小鼠在吗啡依赖后血液中c-fos基因表达比正常小鼠活跃,给清风胶囊治疗可降低染毒小鼠血液中c-fos基因表达。有研究认为,血瘀证与c-fos基因表达存在一定的联系。清风胶囊的治疗在改善血瘀证的同时也校正了染毒小鼠c-fos基因的表达。结合该观点及实验研究结果,可以认为血瘀证与c-fos基因表达存在一定的联系。从中医学角度来看,清风胶囊对染毒后小鼠血液中c-fos基因表达的矫正作用反映在宏观上即是其对机体血瘀、气滞等病理现象的改善,可以认为清风胶囊具有行气活血的功能,这也是其治疗稽延性戒断症状的中医机理之一。该实验是在原有的宏观观察基础上,进一步深入到微观分子水平的研究,是对原有研究的进一步完善,从宏观到微观,使内外联系,揭示机体在微观研究层次上基因的变化与宏观辨证气虚、血瘀等证型的内在联系,从而为中医学宏观辨证论治提供分子生物学水平上的研究依据。
总结理论论证及实验观察结果,表明清风胶囊具有祛除毒瘀、补益气血、扶本逐邪的功效。
Background
The protracted withdrawal symptoms of opiate dependence is included in the syndrome after drug abstinence in TCM theory. The protracted withdrawal symptoms is the series of sustained symptoms following the acute abstinence syndrome, such as tireness, impatience, pain round the body, persistent insomnia, depression, desire for drug, anxiety, and so on. It is one of the factors of relapse. At present, western medicine treats protracted withdrawal symptoms with opioid agents,α2 receptor agonist or sedative hypnotic therapy and so on. These therapies possess a rapid and exact efficacy, with many side effects, and easily to cause an addiction as well. TCM treatment for protracted withdrawal symptoms is safe therapy with few side effects, and no addiction itself. TCM has the characteristic of strengthening healthy qi to eliminate pathogens, whole regulation, dealing with both root cause and symptoms, a low toxicity and no addiction. At present there are various prescription preparations of Chinese medicine treatment for opiate dependence which have been approved production for the clinical use, but these preparations are mostly used for the acute withdrawal symptoms, and no for protracted withdrawal symptoms. Based on the previous pharmacological studies and clinical efficacy assessments, Qingfeng capsule, a new prescription preparations, was showed obviously sedative and hypnotic actions, analgesic and prevention of addiction. It could treat the protracted withdrawal symptoms, such as impatience, insomnia, desire for medicine, anxiety, pain round the body. The preliminary researches on Qingfeng capsules were mostly focused on pharmacological and clinical effects. The clinical effects of Qingfeng for protracted withdrawal symptoms is obvious and the researches in pharmacology have revealed parts of the function mechanism, but the mechanism of Qingfeng based on TCM theory have not been involved .
Objective
The aim of this study is to explore and develop TCM theory on protracted withdrawal symptoms of opiate dependence, and to perfect mechanism of Qingfeng capsules.
Methods
1. Theoretical researches: (1) Arranging literature of TCM to know how TCM understand protracted withdrawal symptoms of opiate dependence, and how to carry out treatment and the key of therapy. (2) Analyzing Qingfeng capsules' components of Chinese drug with Chinese medicine theory to understand how the various components of drug work, and the efficacy of Qingfeng capsules.
2. Experimental researches: Establishing the repeated morphine-dependent and withdrawal animal model, to investigate the effects of Qingfeng capsules to each indicator related that had been select for the experiment, and explaining the efficacy of Qingfeng capsules with related theories: (1) The model of morphine-dependent rats was established by dose-increasing administration. The rats were given morphine from 10mg/kg (sc) to 80mg/kg (sc) each time for 5 days, the rats of control group were given the same volume of saline. Withdrawal syndrome was precipitated by naloxone (2.0mg/kg) 2 hour after the last time morphine was given. The process above was repeated after stopped morphine administration for 14 days. Qingfeng capsules was given(ig) for 5 days. The bodyweight and food intake of the rats were observed and the erythrocyte count, hemoglobin level and blood viscosity in rats were examined. (2) The model of morphine-dependent mice was established by dose-increasing administration. The rats were given morphine from 25mg/kg (sc) to 150mg/kg (sc) each time for 5 days, the rats in control group were given the same volume of saline. Withdrawal syndrome was precipitated by naloxone (4.0mg/kg) 2 hour after the last time morphine was given. The process above was repeated two times after stopped morphine administration for 7 days. Qingfeng capsules was given(ig) for 5 days. The locomotor activity and swimming time of the mice were observed. (3) Establishing the model and giving Qingfeng capsules by the methods above, to observe c-fos gene expression in the blood of the mice. (4) The Experiment results were analyzed by SPSS11.5 statistical software, the method was single-factor analysis of variance.
Results
1. Theoretical researches
(1) The protracted withdrawal symptoms was named as symptoms after abstinence in TCM theory. TCM considers that after long-time use of opioid, Qi-Blood and body fluid are seriously depletive in the body. After withdrawal from opioid, a series of withdrawal symptoms appear due to the damage to the viscera in the body. The pathogenesis of the symptoms after abstinence is due to withdrawal of pathogenic factor, Qi-Blood and body fluid are depletive, yin and yang become estranged, and viscera is damaged in the body. The characteristics of symptoms after abstinence are stagnant toxin and lack of healthy qi. There are four common types of protracted withdrawal symptoms: stagnant toxin and deficiency of qi and blood, stagnant toxin and deficiency of qi and yin, stagnant toxin and flaming of fire from yin deficiency, stagnant toxin and deficiency of yin and yang. About treatment for protracted withdrawal symptoms, according to the deficiency of healthy energy and sthenia of evil in the body of addicts, strengthening healthy qi to eliminate pathogens is the main principle in TCM treatment and rehabilitation on addicts. Strengthening healthy qi and body resistance is mainly replenishing qi and blood, replenishing qi and yin, nourishing yin and fluid and replenishing yin and yang. Eliminating pathogenic factors is mainly from diuresis, heat-clearing and detoxifying, purgation of smoke toxicity, emesis of smoke toxicity, and so on. After abstinence of drug, it's important to replenish qi and blood, restore function of the spleen and stomach, and consolidate the acquired constitution.
(2) Qingfeng capsules are composed of Caulis Sinomenii, Ginseng and other Chinese drugs. Caulis Sinomenii is bitter in flavour and flat in property, the channel-tropism of medicine is mostly liver and spleen, and Caulis Sinomenii possesses the efficacy of dispelling wind, dredging collaterals, analgesia and inducing urination. Sinomenine is a main active ingredients from Caulis Sinomenii, has a similar chemical structure with morphine, but they are different in the molecular structure and the groups arrangement, so that Sinomenine possesses anti-morphine without the addictive effect. Ginseng is sweet in flavour and slight bitter in property, the channel-tropism of medicine is mostly spleen, lung and heart. Ginseng possesses the efficacy of replenishing primordial qi, restoring pulse and collapse syndrome, strengthening spleen and lung, promoting body fluid, tranquilization, replenishing qi to strengthen blood, yang, body fluid and yin. It could be applied to deficiency syndrome of acute withdrawal symptoms or protracted withdrawal symptoms. Ginsenosides are main ingredients from ginseng, possess the effects of resisting morphine addiction. Ginseng also possesses the effects of sedative and anticonvulsant, and it can increase intestinal transport functions, improve the gastrointestinal symptoms. Base on TCM theory Caulis Sinomenii is mainly used for removing stasis and eliminating pathogenic factor in the body, ginseng mainly for strengthening body resistance. The mechanism of the prescription is just according to the principle of treatment and rehabilitation after withdrawal in TCM theory.
2. Experimental researches
(1) The effects of the Qingfeng capsules on food intake and bodyweight in the morphine-dependent rats: After given Qingfeng capsules (ig), the rats in each groups had increased in food intake to a certain extent. Trough analyzing the increases of food intake with statistical methods, we found that the variance was irregular(P = 0.000), and Dunnett's T3 showed that there was no significant difference in the different groups. The bodyweight of the rats were increased after administration, through analyzing the increases on third day and sixth day with statistical methods, we found that there were significant differences in the groups(F = 16.930, P = 0.000 and F = 13.673, P = 0.000). The bodyweight increases on third day and sixth day were both less in morphine model group than control group(P = 0.000), and the index was more in Qingfeng capsule high dose group than morphine model group (P= 0.004, P= 0.002). The bodyweight increases on third day had on significant difference between Qingfeng capsule middle dose group and morphine model group(P= 0.061). On third day and sixth day the bodyweight increases both had no significant difference between Qingfeng capsule low dose group and morphine model group (P= 0.216, P= 0.336). The index on third day and sixth day both had no significant difference between Shiquandabuwan group and morphine model group ( P= 0.883, P= 0.968).
(2) The effects of Qingfeng capsules on blood viscosity in the morphine-dependent rats: the results of whole blood viscosity showed that in shear rate of 1s~(-1), there were significant differences between the groups(F= 2.558, P= 0.042), the whole blood viscosity were higher in morphine model group than that in control group(P= 0.005), the index in Qingfeng capsule high, middle and low dose group were all lower than that in morphine model group(P= 0.005, P= 0.016, P= 0.021), and there were no significant difference between Shiquandabuwan group and morphine model group(P= 0.061). In shear rate of 5s~(-1), there were significant differences in the groups(F= 2.702, P= 0.034), the whole blood viscosity were higher in morphine model group than blank control group(P= 0.010), the index in Qingfeng capsule high, middle and low dose group were all lower than morphine model group(P=0.006, P= 0.026, P= 0.005), and there were no significant difference between Shiquandabuwan group and morphine model group(P= 0.149). In shear rate of 30s~(-1) and 150s~(-1) there were no significant difference between the groups(F= 1.624, P= 0.176 and F= 1.390, P= 0.249).
The results of plasma viscosity showed that there were significant differences between the groups(F= 3.165, P= 0.017), the index were higher in morphine model group than that in control group(P= 0.032), and were lower in Qingfeng capsule high dose group than in morphine model group(P= 0.029). There were no significant difference between morphine model group and Qingfeng capsule middle or low dose group, or Shiquandabuwan group(P= 0.824, P= 0.194, P= 0.428).
(3) The effects of Qingfeng capsules on erythrocyte count, hemoglobin level and hematocrit in the morphine-dependent rats: The results showed that in comparison with the above indices there were no significant difference between the groups(F= 1.604, P= 0.186; F= 1.179, P= 0.340 and F= 1.088, P= 0.385).
(4) The effects of Qingfeng capsules on locomotor activities in the morphine-dependent mice: The numbers of locomotor activity changed after the mice were given drug. Through analyzing the numbers of increase with statistical methods, we found that there were significant differences between the groups(F= 3.994, P= 0.005), the numbers increased were lower in morphine model group than that in control group(P= 0.041). The locomotor activity were higher in Qingfeng capsule high, middle and low dose group than that in morphine model group(P= 0.000, P= 0.001, P= 0.008). There were no significant difference between morphine model group and Shiquandabuwan group(P= 0.182).
(5) The effects of Qingfeng capsules on swimming time in the morphine-dependent mice: The swimming time after the mice were given Qingfeng capsules were analyzed with statistical methods. The variance was irregular(P= 0.008), and Dunnett's T3 showed that there was no significant difference along the groups.
(6) The effects of Qingfeng capsules on c-fos gene expression of blood in the morphine-dependent mice: Observing each blood smear from left to right for three positions under the microscope, to count the numbers of positive cells for the three positions and get a meanvalue number. Trough analyzing the meanvalue numbers of all blood smear with statistical methods, we found that there were significant differences between the groups(F= 4.346, P= 0.003). The number were higher in morphine model group than in control group (P= 0.016), and were higher in Qingfeng capsule high, middle and low dose group than that in morphine model group(P= 0.000, P= 0.005, P= 0.018). There were no significant difference between morphine model group and Shiquandabuwan group(P= 0.163).
Conclusion
1. The present study showed that Qingfeng capsules could increase the body-weight and food intake in repeated morphine-dependence and withdrawal rats. Based on TCM theory, food intake and bodyweight is a reflection on the functions of the spleen and stomach, and the results of the experiment suggested that Qingfeng capsules has increased the spleen and stomach function so that promoted digestion and absorption of food and strengthened Qi and blood in the body. TCM theory considers that Qi work to keep blood moving in veins and collaterals, the deficiency of Qi would cause blood stasis. After repeated morphine-dependence and withdrawal, the primordial qi of rats were damaged. Because of remaining pathogenic factor in the body, it leaded to a deficiency of Qi and blood stasis. Qingfeng capsules could reduce the whole blood viscosity and plasma viscosity of the rats, it suggest that the prescription preparation possesses the actions of replenishing qi, activating blood circulation and removing stasis and pathogenic factor. Erythrocyte count and hemoglobin level are designed to reflect the function of blood, but the results wasn't statistically significant, the impact of the medicine to them would need further to study.
2. Qi possesses the actions of promoting, warming, consolidation, defense, gasification and so on. Blood possesses the actions of nourishing viscera, collaterals and organization, and so on. When the body is hurt, it would cause the deficiency of Qi and blood, and lead to a deficiency of the body function and decreases of vitality and movement, even drowsiness. In the study, after repeated morphine-dependence and withdrawal, the mice showed the number of locomotor activity reduced obviously. Qingfeng capsules could increase the number locomotor activity of mice, it suggest that the preparation possessed the actions of strengthening body resistance, replenishing Qi and blood, and promote vitality of the body. Swimming time of the mice was designed to reflect the function of anti-fatigue and tolerance, but the results wasn't statistically significant, the reason need further to study.
3. After repeated morphine-dependence and withdrawal, mice showed a more active c-fos gene expression in blood than that in normal group. The c-fos gene expression of mice were low after treatment with Qingfeng capsules. Many researches showed that blood stasis is connected with c-fos gene expression in the body. The treatment with Qingfeng capsules could improve the blood stasis, it also corrected the c-fos gene expression. Considering with all above, we come to a conclusion that blood stasis is connected with c-fos gene expression. Qingfeng capsules could regulate the c-fos gene expression in morphine-dependent mice, at the same time it reflected macroscopically on the body that it improve the symptom of blood stasis and qi stagnation. Qingfeng capsules possess the efficacy of promoting qi and activating blood circulation, this is one of the reasons why it could be used to treat protracted withdrawal symptoms. From the experiment of c-fos gene expression, we got a research of microcosmic level basing on the original macro observation, it perfected our researches further more, and revealed the connection between Qi deficiency, blood stasis syndrome and levels of gene changes from macro to micro, so as to provide molecular biology researches for diagnosis and treatment in TCM.
In conclusion, through theoretical and experimental researches above, we can know that Qingfeng capsules possess the efficacy of removing stasis, replenishing Qi and blood, strengthening and restoring effects and eliminating pathogenic factor in the body, and could treat protracted withdrawal syndrome.
阿片类物质依赖稽延性戒断症状,属于中医学断瘾后诸症范畴,是指病人在脱毒后还遗留一系列的症状,如倦怠烦躁,周身疼痛,顽固失眠,心情抑郁,渴求焦虑等,是导致脱毒者复吸的因素之一。目前西药治疗主要有阿片类制剂的替代递减疗法、α2受体激动剂的治疗和镇静催眠药治疗等;其特点是作用迅速、疗效确切,但戒毒西药毒副作用大,本身易成瘾,不能很好地控制脱毒后的稽延性戒断症状。中医药治疗较为安全,毒副作用小,本身无成瘾性。中药扶正驱邪,整体调理,标本兼治,低毒无瘾的作用特点是西药无法比拟的。目前我国已有多种中药戒毒新药制剂被批准生产,用于临床治疗。中医药戒毒治疗目前仍属于缓解急性戒断症状这一类,对防治复吸以及治疗稽延性症状方面,研究甚少,还没有疗效确切的药物,目前尚无主要用于治疗阿片类稽延性戒断症状,防止复吸的中药制剂上市。清风胶囊经过初步的药效学实验及临床疗效评估,表明本方具有显著的镇静催眠,镇痛制瘾作用,对于吸毒者脱毒后的烦躁失眠,渴求焦虑,周身疼痛等主要的稽延症状群具有针对性治疗效果,能有效控制脱毒后的稽延性戒断症状;同时能减轻吸毒者对毒品的精神依赖,防止复吸。清风胶囊用于戒毒治疗的研究至今已有数年,以往的研究主要着重于药理学及临床疗效等方面。作为中药新药,清风胶囊临床用于治疗阿片类戒断综合征有明显的疗效,药理学方面的研究揭示了该药部分的作用及作用机制,但其中医理论方面的作用机制研究尚未有具体涉及。
目的
本课题以探讨清风胶囊治疗稽延性戒断症状的中医机理为目的,在原有实验及临床研究基础上,进一步探讨其功效及作用机制,以进一步完善相关方面的研究。
方法
1.理论研究:(1)整理中医药有关戒毒的文献,理解中医学如何认识阿片类物质依赖稽延性戒断症状,如何进行辨证治疗及治疗的关键;(2)剖析清风胶囊的中药药物组成,结合相关中医学理论知识,从中医学角度理解清风胶囊的功效及各主药的作用。
2.实验研究:选取相关实验指标,通过建立动物模型,观察清风胶囊对吗啡依赖动物反复染毒后各指标的影响,结合相关理论,解释清风胶囊的功效:(1)按剂量递增法建立吗啡依赖大鼠模型,各组大鼠除空白对照组,给予吗啡从每次10mg/kg(sc)逐次递增至每次80mg/kg(sc),连续注射吗啡5d,空白对照组皮下注射等体积生理盐水;各组大鼠于末次给吗啡后2小时,腹腔注射纳洛酮2.0mg/kg进行催促戒断;停药14天后,按上述方法重复染毒一遍。模型建立后各组大鼠灌胃给予相应药物5天,观察清风胶囊对吗啡依赖大鼠反复染毒后体重、进食量,及全血粘度、血浆粘度、红细胞数、血红蛋白量、红细胞比积等指标的影响。(2)按剂量递增法建立吗啡依赖小鼠模型,给予吗啡从每次25mg/kg(sc)逐次递增至每次150mg/kg(sc),连续注射吗啡5d,空白对照组皮下注射等体积生理盐水;各组小鼠于末次给吗啡后2小时,腹腔注射纳洛酮4mg/kg进行催促戒断;停止给药7天,于停药第8天重复上述方法再次注射吗啡3d及戒断(此步骤重复操作2遍)。模型建立后各组小鼠灌胃给予相应药物5天,观察清风胶囊对吗啡依赖小鼠反复染毒后自主活动及游泳时间的影响。(3)按剂量递增法建立吗啡依赖小鼠模型(方法同上)。模型建立后各组小鼠灌胃给予相应药物5天,观察清风胶囊对吗啡依赖小鼠反复染毒后血液中c-fos基因表达的影响。(4)实验结果用SPSS11.5统计软件分析,方法用单因素方差分析。
结果
1.理论研究:(1)稽延性戒断症状在中医学中被称之为“断瘾后诸症”。中医学认为它是由于长期吸食阿片烟毒后,机体气血津液严重耗损,正气衰竭;戒断后,余毒未去,脏腑功能受损后一时难以恢复,故而出现一系列的戒断后症状。断瘾后诸症以烟毒余邪稽留,经气血脉瘀遏,气血津液受损,脏腑阴阳失和为主要病机。断瘾后诸症的主要特征是毒瘀蕴结,正气不足。有四种常见证型:毒瘀蕴结,气血亏虚型;毒瘀蕴结,气阴不足型:毒瘀蕴结,阴虚火旺型:毒瘀蕴结,阴阳两虚型。根据正虚与邪实的实际情况辨证施治,是中医药治疗断瘾后诸症的精髓所在。总的来说,扶正祛邪是中医学在戒毒治疗及断瘾后康复中一以贯之的原则,扶助正气主要从益气养血、益气养阴、滋补阴液、阴阳并补等几方面论治;祛邪的具体治法有利尿解毒、清热解毒、泻下烟毒、涌吐烟毒、以毒攻毒等。断瘾后的调补尤以补益气血,恢复脾胃功能、巩固后天之本为主。(2)清风胶囊由青风藤、人参等中药组成。其中青风藤味苦性平,归肝、脾经,具有祛风湿、通经络、镇痛、利小便之功效。其主要的有效成分青藤碱具有与吗啡相似的化学结构,但在分子构型及基团的排列上均不同,因而形成青风藤具有抗吗啡作用但无成瘾性的功效。人参味甘、微苦,性平,归脾、肺、心经。功可大补元气,复脉固脱,补脾益肺,生津,安神,补气生血,补气助阳,益气生津,益气滋阴。适于成瘾者在戒毒期和断瘾后的虚弱证候。其主要成分人参皂苷有抗吗啡耐受及成瘾作用,同时人参具有镇静、抗惊厥作用,并有提高肠输送功能、改善消化道症状的作用。从中医学角度来看,青风藤有祛瘀逐邪的作用,人参起补益扶本的作用,两者结合使用可起到祛除毒瘀,补益气血的功效,从而对“断瘾后”正虚邪留的机体起到治疗作用,此法亦顺应了中医学认为断瘾后诸症的治疗以扶助正气与祛除余毒为主的治疗原则。
2.实验研究
(1)药物对吗啡依赖大鼠进食和体重的影响
给药后各组大鼠的进食量有不同程度的增加,用增加值进行统计学分析,方差不齐(P=0.000),且Dunnett's T3法比较未见显著性差异,结果无统计学意义。
给药后大鼠体重增加,用给药第3天、末次给药后24h体重增加值分别进行统计学分析,各组间有显著性差异(分别为F=16.930,P=0.000和F=13.673,P=0.000);给药第3天、末次给药后24h吗啡模型组大鼠体重增加值均比空白对照组小(均为P=0.000),清风胶囊高剂量组大鼠体重增加值均比吗啡模型组大(分别为P=0.004,P=0.002);给药第3天清风胶囊中剂量组大鼠体重增加值与吗啡模型组比较无显著性差异(P=0.061),末次给药后24h清风胶囊中剂量组大鼠体重增加值比吗啡模型组大(P=0.010);给药第3天、末次给药后24h清风胶囊低剂量组大鼠体重增加值与吗啡模型组比较均无显著性差异(分别为P=0.216,P=0.336),十全大补丸组大鼠体重增加值与吗啡模型组比较均无显著性差异(分别为P=0.883,P=0.968)。
(2)药物对吗啡依赖大鼠血液粘度的影响
大鼠全血粘度的检测结果表明,切变率1/s各组间全血粘度有显著性差异(F=2.558,P=0.042),吗啡模型组大鼠比空白对照组高(P=0.005),清风胶囊高、中、低剂量组大鼠均比吗啡模型组低(分别为P=0.005,P=0.016,P=0.021),十全大补丸组大鼠与吗啡模型组比较无显著性差异(P=0.061)。切变率5/s各组间全血粘度有显著性差异(F=2.702,P=0.034),吗啡模型组大鼠比空白对照组高(P=0.010),清风胶囊高、中、低剂量组大鼠均比吗啡模型组低(分别为P=0.006,P=0.026,P=0.005),十全大补丸组大鼠与吗啡模型组比较无显著性差异(P=0.149)。切变率30/s、150/s各组间全血粘度无显著性差异(分别为F=1.624,P=0.176和F=1.390,P=0.249)。
大鼠血浆粘度检测结果表明,各组间有显著性差异(F=3.165,P=0.017),吗啡模型组大鼠血浆粘度比空白对照组高(P=0.032),清风胶囊高剂量组大鼠比吗啡模型组低(P=0.029),清风胶囊中、低剂量组和十全大补丸组分别与吗啡模型组比较均无显著性差异(分别为P=0.824,P=0.194,P=0.428)。
(3)药物对大鼠红细胞数、血红蛋白量、红细胞比积的影响:实验结果经统计学分析表明,各组大鼠红细胞数、血红蛋白量、红细胞比积均未表现出显著性差异(分别为F=1.604,P=0.186:F=1.179,P=0.340和F=1.088,P=0.385)。
(4)药物对吗啡依赖小鼠自主活动次数的影响:吗啡依赖小鼠在给药治疗前后自主活动次数有明显变化,以给药后比给药前活动次数增加值进行统计学分析,各组间有显著性差异(F=3.994,P=0.005),吗啡模型组小鼠自主活动增加值比空白对照组低(P=0.041),清风胶囊高、中、低剂量组小鼠自主活动增加值均比吗啡模型组高(分别为P=0.000,P=0.001,P=0.008),十全大补丸组小鼠自主活动增加值与吗啡模型组比较无显著性差异(P=0.182)。
(5)药物对吗啡依赖小鼠游泳时间的影响:给药治疗后,以各组小鼠游泳时间进行统计学比较,方差不齐(P=0.008),且Dunnett's T3法比较未见显著性差异,结果无统计学意义。
(6)药物对吗啡依赖小鼠血液中c-fos基因表达的影响:在镜下分别观察每张血涂片左、中、右三个视野,以三个视野的平均阳性细胞数进行统计学分析,各组间有显著性差异(F=4.346,P=0.003),吗啡模型组平均阳性细胞数比空白对照组高(P=0.016),清风胶囊高、中、低剂量组平均阳性细胞数均比吗啡模型组低(分别为P=0.000,P=0.005,P=0.018),十全大补丸组平均阳性细胞数与吗啡模型组比较无显著性差异(P=0.163)。
结论
1.从中医学角度来看,进食量和体重是反映机体脾胃气血功能的指标,实验结果表明清风胶囊有促进吗啡依赖戒断后大鼠进食和体重增加的作用,说明该药增强了脾胃功能,促进了机体对食物的纳入运化和气血的化生。中医学认为气为血帅,气能行血,气虚则行血功能减弱,导致血液运行不利,血液粘滞瘀留;大鼠在成瘾戒断过程中损伤元气,余毒瘀留,导致气血虚弱,气血瘀滞,本实验结果表明清风胶囊有降低吗啡依赖戒断后大鼠全血粘度和血浆粘度的作用,说明该药有补气行血,活血祛瘀,促进气血运行的作用。红细胞数和血红蛋白量是反映血液功能状态的指标,实验结果无统计学意义,药物对该指标的影响尚待进一步的验证。
2.中医学认为气有推动、温煦、固摄、防御、气化等功能;血有濡养脏腑、经络、组织等功能;机体在正气虚损,气血亏虚时,机体的生理机能、脏腑组织的机能活动相应会有明显的下降,表现为活力下降,喜静,不喜动,甚至嗜睡等。吗啡依赖小鼠在反复染毒后,出进食量减少,嗜睡,活力减退,活动量减少,喜静伏等表现。清风胶囊治疗可使染毒小鼠自主活动次数增加,提高小鼠活力,说明该药起到了扶助正气、补益气血、增强身体机能的作用。游泳时间是体现小鼠抗疲劳及耐受能力的一个指标,实验中的设置是为检验药物扶正益本的功效,可看到清风胶囊治疗组游泳时间比吗啡模型组游泳时间长,但实验结果无统计学意义,药物对该指标的影响尚待进一步的验证。
3.小鼠在吗啡依赖后血液中c-fos基因表达比正常小鼠活跃,给清风胶囊治疗可降低染毒小鼠血液中c-fos基因表达。有研究认为,血瘀证与c-fos基因表达存在一定的联系。清风胶囊的治疗在改善血瘀证的同时也校正了染毒小鼠c-fos基因的表达。结合该观点及实验研究结果,可以认为血瘀证与c-fos基因表达存在一定的联系。从中医学角度来看,清风胶囊对染毒后小鼠血液中c-fos基因表达的矫正作用反映在宏观上即是其对机体血瘀、气滞等病理现象的改善,可以认为清风胶囊具有行气活血的功能,这也是其治疗稽延性戒断症状的中医机理之一。该实验是在原有的宏观观察基础上,进一步深入到微观分子水平的研究,是对原有研究的进一步完善,从宏观到微观,使内外联系,揭示机体在微观研究层次上基因的变化与宏观辨证气虚、血瘀等证型的内在联系,从而为中医学宏观辨证论治提供分子生物学水平上的研究依据。
总结理论论证及实验观察结果,表明清风胶囊具有祛除毒瘀、补益气血、扶本逐邪的功效。
Background
The protracted withdrawal symptoms of opiate dependence is included in the syndrome after drug abstinence in TCM theory. The protracted withdrawal symptoms is the series of sustained symptoms following the acute abstinence syndrome, such as tireness, impatience, pain round the body, persistent insomnia, depression, desire for drug, anxiety, and so on. It is one of the factors of relapse. At present, western medicine treats protracted withdrawal symptoms with opioid agents,α2 receptor agonist or sedative hypnotic therapy and so on. These therapies possess a rapid and exact efficacy, with many side effects, and easily to cause an addiction as well. TCM treatment for protracted withdrawal symptoms is safe therapy with few side effects, and no addiction itself. TCM has the characteristic of strengthening healthy qi to eliminate pathogens, whole regulation, dealing with both root cause and symptoms, a low toxicity and no addiction. At present there are various prescription preparations of Chinese medicine treatment for opiate dependence which have been approved production for the clinical use, but these preparations are mostly used for the acute withdrawal symptoms, and no for protracted withdrawal symptoms. Based on the previous pharmacological studies and clinical efficacy assessments, Qingfeng capsule, a new prescription preparations, was showed obviously sedative and hypnotic actions, analgesic and prevention of addiction. It could treat the protracted withdrawal symptoms, such as impatience, insomnia, desire for medicine, anxiety, pain round the body. The preliminary researches on Qingfeng capsules were mostly focused on pharmacological and clinical effects. The clinical effects of Qingfeng for protracted withdrawal symptoms is obvious and the researches in pharmacology have revealed parts of the function mechanism, but the mechanism of Qingfeng based on TCM theory have not been involved .
Objective
The aim of this study is to explore and develop TCM theory on protracted withdrawal symptoms of opiate dependence, and to perfect mechanism of Qingfeng capsules.
Methods
1. Theoretical researches: (1) Arranging literature of TCM to know how TCM understand protracted withdrawal symptoms of opiate dependence, and how to carry out treatment and the key of therapy. (2) Analyzing Qingfeng capsules' components of Chinese drug with Chinese medicine theory to understand how the various components of drug work, and the efficacy of Qingfeng capsules.
2. Experimental researches: Establishing the repeated morphine-dependent and withdrawal animal model, to investigate the effects of Qingfeng capsules to each indicator related that had been select for the experiment, and explaining the efficacy of Qingfeng capsules with related theories: (1) The model of morphine-dependent rats was established by dose-increasing administration. The rats were given morphine from 10mg/kg (sc) to 80mg/kg (sc) each time for 5 days, the rats of control group were given the same volume of saline. Withdrawal syndrome was precipitated by naloxone (2.0mg/kg) 2 hour after the last time morphine was given. The process above was repeated after stopped morphine administration for 14 days. Qingfeng capsules was given(ig) for 5 days. The bodyweight and food intake of the rats were observed and the erythrocyte count, hemoglobin level and blood viscosity in rats were examined. (2) The model of morphine-dependent mice was established by dose-increasing administration. The rats were given morphine from 25mg/kg (sc) to 150mg/kg (sc) each time for 5 days, the rats in control group were given the same volume of saline. Withdrawal syndrome was precipitated by naloxone (4.0mg/kg) 2 hour after the last time morphine was given. The process above was repeated two times after stopped morphine administration for 7 days. Qingfeng capsules was given(ig) for 5 days. The locomotor activity and swimming time of the mice were observed. (3) Establishing the model and giving Qingfeng capsules by the methods above, to observe c-fos gene expression in the blood of the mice. (4) The Experiment results were analyzed by SPSS11.5 statistical software, the method was single-factor analysis of variance.
Results
1. Theoretical researches
(1) The protracted withdrawal symptoms was named as symptoms after abstinence in TCM theory. TCM considers that after long-time use of opioid, Qi-Blood and body fluid are seriously depletive in the body. After withdrawal from opioid, a series of withdrawal symptoms appear due to the damage to the viscera in the body. The pathogenesis of the symptoms after abstinence is due to withdrawal of pathogenic factor, Qi-Blood and body fluid are depletive, yin and yang become estranged, and viscera is damaged in the body. The characteristics of symptoms after abstinence are stagnant toxin and lack of healthy qi. There are four common types of protracted withdrawal symptoms: stagnant toxin and deficiency of qi and blood, stagnant toxin and deficiency of qi and yin, stagnant toxin and flaming of fire from yin deficiency, stagnant toxin and deficiency of yin and yang. About treatment for protracted withdrawal symptoms, according to the deficiency of healthy energy and sthenia of evil in the body of addicts, strengthening healthy qi to eliminate pathogens is the main principle in TCM treatment and rehabilitation on addicts. Strengthening healthy qi and body resistance is mainly replenishing qi and blood, replenishing qi and yin, nourishing yin and fluid and replenishing yin and yang. Eliminating pathogenic factors is mainly from diuresis, heat-clearing and detoxifying, purgation of smoke toxicity, emesis of smoke toxicity, and so on. After abstinence of drug, it's important to replenish qi and blood, restore function of the spleen and stomach, and consolidate the acquired constitution.
(2) Qingfeng capsules are composed of Caulis Sinomenii, Ginseng and other Chinese drugs. Caulis Sinomenii is bitter in flavour and flat in property, the channel-tropism of medicine is mostly liver and spleen, and Caulis Sinomenii possesses the efficacy of dispelling wind, dredging collaterals, analgesia and inducing urination. Sinomenine is a main active ingredients from Caulis Sinomenii, has a similar chemical structure with morphine, but they are different in the molecular structure and the groups arrangement, so that Sinomenine possesses anti-morphine without the addictive effect. Ginseng is sweet in flavour and slight bitter in property, the channel-tropism of medicine is mostly spleen, lung and heart. Ginseng possesses the efficacy of replenishing primordial qi, restoring pulse and collapse syndrome, strengthening spleen and lung, promoting body fluid, tranquilization, replenishing qi to strengthen blood, yang, body fluid and yin. It could be applied to deficiency syndrome of acute withdrawal symptoms or protracted withdrawal symptoms. Ginsenosides are main ingredients from ginseng, possess the effects of resisting morphine addiction. Ginseng also possesses the effects of sedative and anticonvulsant, and it can increase intestinal transport functions, improve the gastrointestinal symptoms. Base on TCM theory Caulis Sinomenii is mainly used for removing stasis and eliminating pathogenic factor in the body, ginseng mainly for strengthening body resistance. The mechanism of the prescription is just according to the principle of treatment and rehabilitation after withdrawal in TCM theory.
2. Experimental researches
(1) The effects of the Qingfeng capsules on food intake and bodyweight in the morphine-dependent rats: After given Qingfeng capsules (ig), the rats in each groups had increased in food intake to a certain extent. Trough analyzing the increases of food intake with statistical methods, we found that the variance was irregular(P = 0.000), and Dunnett's T3 showed that there was no significant difference in the different groups. The bodyweight of the rats were increased after administration, through analyzing the increases on third day and sixth day with statistical methods, we found that there were significant differences in the groups(F = 16.930, P = 0.000 and F = 13.673, P = 0.000). The bodyweight increases on third day and sixth day were both less in morphine model group than control group(P = 0.000), and the index was more in Qingfeng capsule high dose group than morphine model group (P= 0.004, P= 0.002). The bodyweight increases on third day had on significant difference between Qingfeng capsule middle dose group and morphine model group(P= 0.061). On third day and sixth day the bodyweight increases both had no significant difference between Qingfeng capsule low dose group and morphine model group (P= 0.216, P= 0.336). The index on third day and sixth day both had no significant difference between Shiquandabuwan group and morphine model group ( P= 0.883, P= 0.968).
(2) The effects of Qingfeng capsules on blood viscosity in the morphine-dependent rats: the results of whole blood viscosity showed that in shear rate of 1s~(-1), there were significant differences between the groups(F= 2.558, P= 0.042), the whole blood viscosity were higher in morphine model group than that in control group(P= 0.005), the index in Qingfeng capsule high, middle and low dose group were all lower than that in morphine model group(P= 0.005, P= 0.016, P= 0.021), and there were no significant difference between Shiquandabuwan group and morphine model group(P= 0.061). In shear rate of 5s~(-1), there were significant differences in the groups(F= 2.702, P= 0.034), the whole blood viscosity were higher in morphine model group than blank control group(P= 0.010), the index in Qingfeng capsule high, middle and low dose group were all lower than morphine model group(P=0.006, P= 0.026, P= 0.005), and there were no significant difference between Shiquandabuwan group and morphine model group(P= 0.149). In shear rate of 30s~(-1) and 150s~(-1) there were no significant difference between the groups(F= 1.624, P= 0.176 and F= 1.390, P= 0.249).
The results of plasma viscosity showed that there were significant differences between the groups(F= 3.165, P= 0.017), the index were higher in morphine model group than that in control group(P= 0.032), and were lower in Qingfeng capsule high dose group than in morphine model group(P= 0.029). There were no significant difference between morphine model group and Qingfeng capsule middle or low dose group, or Shiquandabuwan group(P= 0.824, P= 0.194, P= 0.428).
(3) The effects of Qingfeng capsules on erythrocyte count, hemoglobin level and hematocrit in the morphine-dependent rats: The results showed that in comparison with the above indices there were no significant difference between the groups(F= 1.604, P= 0.186; F= 1.179, P= 0.340 and F= 1.088, P= 0.385).
(4) The effects of Qingfeng capsules on locomotor activities in the morphine-dependent mice: The numbers of locomotor activity changed after the mice were given drug. Through analyzing the numbers of increase with statistical methods, we found that there were significant differences between the groups(F= 3.994, P= 0.005), the numbers increased were lower in morphine model group than that in control group(P= 0.041). The locomotor activity were higher in Qingfeng capsule high, middle and low dose group than that in morphine model group(P= 0.000, P= 0.001, P= 0.008). There were no significant difference between morphine model group and Shiquandabuwan group(P= 0.182).
(5) The effects of Qingfeng capsules on swimming time in the morphine-dependent mice: The swimming time after the mice were given Qingfeng capsules were analyzed with statistical methods. The variance was irregular(P= 0.008), and Dunnett's T3 showed that there was no significant difference along the groups.
(6) The effects of Qingfeng capsules on c-fos gene expression of blood in the morphine-dependent mice: Observing each blood smear from left to right for three positions under the microscope, to count the numbers of positive cells for the three positions and get a meanvalue number. Trough analyzing the meanvalue numbers of all blood smear with statistical methods, we found that there were significant differences between the groups(F= 4.346, P= 0.003). The number were higher in morphine model group than in control group (P= 0.016), and were higher in Qingfeng capsule high, middle and low dose group than that in morphine model group(P= 0.000, P= 0.005, P= 0.018). There were no significant difference between morphine model group and Shiquandabuwan group(P= 0.163).
Conclusion
1. The present study showed that Qingfeng capsules could increase the body-weight and food intake in repeated morphine-dependence and withdrawal rats. Based on TCM theory, food intake and bodyweight is a reflection on the functions of the spleen and stomach, and the results of the experiment suggested that Qingfeng capsules has increased the spleen and stomach function so that promoted digestion and absorption of food and strengthened Qi and blood in the body. TCM theory considers that Qi work to keep blood moving in veins and collaterals, the deficiency of Qi would cause blood stasis. After repeated morphine-dependence and withdrawal, the primordial qi of rats were damaged. Because of remaining pathogenic factor in the body, it leaded to a deficiency of Qi and blood stasis. Qingfeng capsules could reduce the whole blood viscosity and plasma viscosity of the rats, it suggest that the prescription preparation possesses the actions of replenishing qi, activating blood circulation and removing stasis and pathogenic factor. Erythrocyte count and hemoglobin level are designed to reflect the function of blood, but the results wasn't statistically significant, the impact of the medicine to them would need further to study.
2. Qi possesses the actions of promoting, warming, consolidation, defense, gasification and so on. Blood possesses the actions of nourishing viscera, collaterals and organization, and so on. When the body is hurt, it would cause the deficiency of Qi and blood, and lead to a deficiency of the body function and decreases of vitality and movement, even drowsiness. In the study, after repeated morphine-dependence and withdrawal, the mice showed the number of locomotor activity reduced obviously. Qingfeng capsules could increase the number locomotor activity of mice, it suggest that the preparation possessed the actions of strengthening body resistance, replenishing Qi and blood, and promote vitality of the body. Swimming time of the mice was designed to reflect the function of anti-fatigue and tolerance, but the results wasn't statistically significant, the reason need further to study.
3. After repeated morphine-dependence and withdrawal, mice showed a more active c-fos gene expression in blood than that in normal group. The c-fos gene expression of mice were low after treatment with Qingfeng capsules. Many researches showed that blood stasis is connected with c-fos gene expression in the body. The treatment with Qingfeng capsules could improve the blood stasis, it also corrected the c-fos gene expression. Considering with all above, we come to a conclusion that blood stasis is connected with c-fos gene expression. Qingfeng capsules could regulate the c-fos gene expression in morphine-dependent mice, at the same time it reflected macroscopically on the body that it improve the symptom of blood stasis and qi stagnation. Qingfeng capsules possess the efficacy of promoting qi and activating blood circulation, this is one of the reasons why it could be used to treat protracted withdrawal symptoms. From the experiment of c-fos gene expression, we got a research of microcosmic level basing on the original macro observation, it perfected our researches further more, and revealed the connection between Qi deficiency, blood stasis syndrome and levels of gene changes from macro to micro, so as to provide molecular biology researches for diagnosis and treatment in TCM.
In conclusion, through theoretical and experimental researches above, we can know that Qingfeng capsules possess the efficacy of removing stasis, replenishing Qi and blood, strengthening and restoring effects and eliminating pathogenic factor in the body, and could treat protracted withdrawal syndrome.
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