【作者】 孙娟；

【导师】 柴铁劬；

【作者基本信息】 广州中医药大学 ， 针灸推拿学， 2011， 博士

【摘要】 目的：1.观察分析艾灸神阙穴配合温针灸关元、三阴交穴治疗原发性痛经(primary dysmenorrhea,PD)的临床疗效及其镇痛作用的时效性,比较其与单纯针刺法治疗原发性痛经的疗效差别。将艾灸神阙穴配合温针灸疗法推广应用于临床,也为临床针灸治疗原发性痛经在穴位筛选及治疗方案优化等方面提供临床研究资料。2.观察艾灸神阙穴对痛经模型小鼠胸腺、脾脏、子宫的病理变化的影响,对小鼠子宫内膜前列腺素F2a (prostaglandins F2a, PGF2a)、下丘脑及血浆β-内啡肽(P-Endorphin,β-EP)的含量、小鼠外周血T淋巴细胞亚群CD4+CD8+的百分数及其比值,及子宫平滑肌细胞缝隙连接蛋白43(Connexin43,Cx43)的表达的影响,从神经-内分泌-免疫网络(Neuro-endocrine-immunity network,NEI)角度,研究探讨艾灸神阙穴单穴在治疗原发性痛经中的特异性效应。为临床针灸取穴及治疗方案的优化提供可靠的实验数据和科学依据。方法：1.采用公开实验随机对照临床研究。将90例原发性痛经患者随机分为三组,治疗组、针刺组及对照组,每组各30例。共观察四个月经周期。治疗组选穴神阙、关元、三阴交,于第一个月经周期痛经的第1天,当视觉模拟评分(Visual Analogue Scale,VAS)≥40mm时,开始用艾灸神阙穴配合温针灸治疗,在治疗前、治疗10min、治疗20min、治疗30min、治疗40min及治疗结束后30min分别对患者进行视觉模拟评分(VAS)评价其疼痛程度,观察该疗法镇痛时效性。于第二个月经周期月经来潮前3天开始治疗,1日1次,连续治疗3天至月经来潮,3天为一个疗程,一个月经周期治疗一个疗程,连续治疗三个疗程。采用回顾性症状量表(Retrospective Symptom Scale,RSS)评价艾灸神阙穴配合温针灸对原发性痛经患者痛经症状总频率及严重程度的影响,进行组内及组间比较。针刺组采用常规针刺治疗,疗程同治疗组；对照组等待进行对照。2.将48只NIH雌性小鼠随机分为三组：空白对照组、模型组及艾灸组,每组各16只。空白对照组给予灌胃生理盐水,连续12天,末次灌胃1h后腹腔注射生理盐水；模型组及艾灸组采用己烯雌酚及缩宫素建立小鼠痛经模型,每天灌胃己烯雌酚,日一次,连续12天。艾灸组于每天造模后即行艾灸神阙穴治疗,每次15min,连续12天,两组于第12天给药1h后腹腔注射缩宫素,观察各组小鼠30min内扭体反应情况及小鼠胸腺、脾脏、子宫的病理变化；采用流式细胞仪检测各组小鼠外周血T淋巴细胞亚群CD4+、CD8+百分数；采用放射免疫法检测各组小鼠子宫内膜PGF2a及下丘脑、血浆β-EP的含量；采用免疫组化法检测各组小鼠子宫平滑肌细胞Cx43的表达。结果：1.治疗组及针刺组治疗不同时刻VAS评分分别与同组治疗前及同期对照组比较,均有显著差异(P<0.01),提示艾灸神阙穴配合温针灸疗法及单纯针刺法均能明显减轻痛经患者的疼痛程度。在治疗的30min内,VAS评分不断下降,治疗结束后30minVAS评分也明显降低,但较治疗30min时VAS评分有所回升,治疗40min时与治疗30min时评分比较差异无统计学意义。治疗30min时VAS评分降低幅度最大。说明两种疗法的镇痛效果与治疗时间呈正相关(30min内),治疗30min对痛经患者镇痛效果最佳,治疗40min时可能出现了针灸耐受性。治疗组与针刺组治疗相同时刻比较,治疗10min时,VAS评分差异有统计学意义(P<0.05),治疗20min、30min、40min及治疗结束后30min,VAS评分差异显著(P<0.01),说明艾灸神阙穴配合温针灸治疗PD即刻镇痛的效果优于单纯针刺法。试验前腹痛症状RSS评分最高,其次为乏力、食欲不振、腰骶痛、易激惹等症状评分较高,说明痛经患者上述症状较明显,较多见。与治疗前比较,治疗组及针刺组三个疗程治疗后痛经症状各条目RSS评分都有下降趋势。治疗组及针刺组三个疗程治疗后的腹痛总频率评分、腹痛严重程度评分、痛经症状总频率总分及严重程度总分较治疗前及同期对照组均下降,特别是第二、第三疗程差异显著(P<0.01),而治疗组较针刺组RSS评分下降更明显,说明艾灸神阙穴配合温针灸疗法及单纯针刺法都能减少痛经症状发作的总频率,减轻痛经症状的严重程度,并且随着治疗次数的增加,其效应累积,痛经症状也随之明显减轻,艾灸神阙穴配合温针灸疗法改善痛经症状疗效明显优于单纯针刺法。2.模型组小鼠30min内的扭体均数为25.4±10.3次,与对照组比较差异显著,艾灸组小鼠30min内的扭体均数为16.8±7.9次,明显低于模型组。与空白对照组比较,模型组、艾灸组小鼠子宫内膜PGF2a含量明显升高,小鼠下丘脑及血浆中β-EP含量均明显升高,小鼠外周血T淋巴细胞亚群CD4+百分数明显降低,CD8+百分数明显升高,二者的比值降低,差异显著有统计学意义(P<0.01)；艾灸组与模型组比较,小鼠子宫内膜PGF2a含量降低(P<0.05),小鼠下丘脑及血浆中β-EP含量均高于模型组(P<0.05),小鼠外周血T淋巴细胞亚群CD4+值高于模型组(P<0.05),CD8+值略低于模型组(P>0.05),二者比值略高于模型组(P>0.05)。模型组小鼠胸腺外周皮质面积减小,胸腺细胞减少,髓质毛细血管扩张充血；脾脏淋巴小结数量减少,面积减小；子宫腺腔内充血,内膜间质充血水肿。艾灸组与模型组相比,小鼠胸腺外周皮质面积增大,胸腺细胞增多,脾脏淋巴小结数目及面积明显增多增大,子宫腺腔及内膜间质充血水肿较模型组减轻。空白对照组子宫内膜Cx43不表达,呈阴性。模型组子宫内膜细胞浆细胞膜Cx43阳性表达,呈棕黄色。艾灸组子宫内膜胞浆胞膜Cx43弱阳性表达,呈浅黄色。结论：1.艾灸神阙穴配合温针灸法能明显减少PD患者腹痛总频率、痛经症状总频率,减轻腹痛严重程度及痛经症状严重程度,并随治疗次数的增加而效应累积。该法能明显改善PD患者痛经症状,能明显减轻PD患者的疼痛程度,在治疗30分钟内,其镇痛效果与治疗时间呈正相关,治疗30min时镇痛效果最佳,治疗40min时可能出现针灸耐受性,该法疗效明显优于单纯针刺法。神阙、关元、三阴交三穴相配是治疗PD的较佳组合,三穴与冲、任、督三脉、足三阴经及肝脾肾密切相关,三穴配伍,局部与远端取穴结合,调补兼施,相互为用,治疗痛经相得益彰。而艾灸宜选用陈艾条。艾灸神阙穴配合温针灸治疗PD,临床疗效切实可靠,取穴少而精,充分发挥针刺、艾灸治疗原发性痛经的作用优势,针灸结合,优势互补,从而提高临床疗效,适于临床推广应用。2.艾灸神阙穴镇痛效果显著。艾灸神阙穴能明显改善痛经模型小鼠子宫局部的血液循环,减轻其子宫腺腔及内膜间质充血水肿,降低痛经模型小鼠子宫内膜PGF2a的表达,缓解子宫的痉挛收缩。痛经症本身可能应激性激活中枢EOP系统,艾灸神阙穴可能通过促进中枢产生释放更多的β-EP,并增加血浆β-EP的含量而发挥镇痛效应。痛经时子宫肌层内相邻的肌肉细胞间可能通过Cx43蛋白的表达,形成缝隙连接通道,以调节子宫平滑肌的收缩和代谢。而PGF2a可能诱导缝隙连接,这可能是造成子宫过度收缩的机制之一。而艾灸神阙穴能减轻痛经模型小鼠的子宫痉挛收缩,其镇痛作用可能是通过抑制PGF2a的合成和分泌,调控Cx43蛋白的缝隙连接通道实现的。3.艾灸神阙穴能调整痛经模型小鼠免疫功能。艾灸神阙穴能升高痛经模型小鼠外周血T淋巴细胞亚群中CD4+的百分数,上调CD4+与CD8+的比值,可能发挥着调整痛经模型小鼠细胞免疫功能紊乱的正向调节作用。艾灸神阙穴能减轻痛经模型小鼠胸腺脾脏的病理损伤,进而恢复和增强其免疫功能。PGF2a可能参与了痛经模型小鼠免疫功能的失调,艾灸神阙穴调节痛经模型小鼠免疫功能紊乱可能与其抑制PGF2a的合成和分泌,调节中枢及外周β-EP水平有关。4.痛经模型小鼠可能存在神经-内分泌-免疫功能紊乱,艾灸神阙穴治疗PD的作用机制可能是通过神经-内分泌-免疫网络多环节、多靶点的作用整合,调节脏腑经络、平衡阴阳、稳定机体内环境,起到镇痛免疫作用。更多还原

【Abstract】 Objective:1. To study the clinic effects of warming needle with moxibustion and Shen Que moxibustion treating primary dysmenorrhea, and it’s timely effect of analgesia. Compared its therapeutical effects with regularly acupuncture.2. To observe the effect of Shen Que moxibustion on the mice thymus gland, spleen and uterus pathologic changes, endometrium PGF2a, hypothalamus and plasmaβ-EP, peripheral blood T lymphocytes subgroup percentage and uterine smooth muscle gap junction protein 43. To analyse the function of Shen Que moxibustion treating primary dysmenorrhea by easing pain and promoting immunity, and study the mechanism of Shen Que moxibustion treating primary dysmenorrhea related to Neuro-Endocrine-Immunity network.Methods:1. In openly randomized control clinical trial,90 primary dysmenorrhea patients were recruited and ramdomized into 3 groups:intervention, acupuncture control and control.30 cases each group. Observed for 4 menstrual cycles. On the first day when dysmenorrheal symptoms occurred in the first menstrual cycle, VAS≥40mm, treated patients with warming needle with moxibustion and Shen Que moxibustion. Assessed the timely effects on the pain level by Visual Analogue Scale (VAS). Treated patients 3 days before the second menstrual cycle, qd, continued for 3 days till mensus occurred, one menstrual cycle is as a course of treatment. kept treating for 3 course. The Retrospective Symptom Scale(RSS) were filled by the patients to assess the total frequency ratings and average severity level of patients’dysmenorrheal symptoms that refers to the average level of pain and the condition changes when it did occur.2.48 NIH female mice were randomly divided into three groups:control, model and moxibustion.16 each group. For the control group, gave the mice NS for 12 days, when gave NS the last day one hour later, ip the mice with NS. And for model and moxibustion groups, The primary dysmenorrheal animal model was made by diethylstilbestrol and oxytocin. Gave moxibustion treatment the moxibustion group mice everyday after model made,15min each time, continued for 12days, when gave the last diethylstilbestrol one hour later, injected oxytocin, observed the mice twisting frequency and the mice thymus gland, spleen and uterus pathologic changes. Tested T lymphocytes subgroup percentages of CD4+ and CD8+ by flow cytometer. Tested the mice endometrium PGF2 a, hypothalamus and plasmaβ-EP by RIA and tested the mice uterine smooth muscle gap junction protein 43 by IHC.Results:1. In the intervention group and acupuncture control group, Compared the symptom RSS score between every course of treatment and that before trial and that at same time of control group, the total frequency rating and average severity rating of abdominal pain and average whole dysmenorrheal symptom are obviously decreased(P<0.01). It shows the two treatment both can relieve patients’pain level. During treating,both groups’VAS score continue decreased, but a little rise again at the time of 30 minutes after treating than that time treating for 30minutes.The VAS score decreased most obviously when treating 30 minutes. When treated for 40 minutes, maybe acupuncture tolerability occurs. It indicates that two treatments’effects for easing pain have positive correlation with treatment time in 30 minutes. And treatment for 30 minutes’effect is the best for easing pain. Compared intervention group with acupuncture control group at same term, treating for 10 minutes, the score difference is meaningful (P<0.05), score difference at other terms are obvious (P<0.01), it shows that the therapeutic effect of warming needle with moxibutsion and Shen Que moxibustion is superior than regular acupuncture. Before the trial, The abdominal pain is the most severe symptom before trial, other symptoms such as weakness, less of appetite, backaches, irritability, and depression are also severe. In the intervention group and acupuncture group, Compared the three course score with that before trial and that of control group at same term, the total frequency rating and average severity level of patients’dysmenorrheal symptoms and abdominal pain all decreased, especially in the second and third course (P<0.01). The intervention group score decreased more obviously than acupuncture group. It dedicates that two treatments both can decrease dysmenorrheal symptoms’frequency rating and average severity level, and effect accumulates along with treatment course, dysmenorrheal symptoms are also relieved, the therapeutical effect of warming needle with moxibustion and Shen Que moxibustion is obviously superior than regular acupuncture treatment.2. The twisting frequency of model group mice is obviously higher than control group, and that of moxibustion group is lower than model group. Compared with control group, the mice endometrium PGF2a,the hypothalamus and plasmaβ-EP of model and moxibustion group are obviously increased, the percentage of CD4±is obviously lower, the percentage of CD8±is obviously higher, and ratio of CD4+/CD8+ gets down(P<0.01). Compared with model group, the moxibustion group mice endometrium PGF2a decreased (P<0.05), the hypothalamus and plasmaβ-EP increased(P<0.05), and CD4+ increased (P<0.05), CD8+ is lower slightly (P>0.05), the ratio of CD4+/CD8+ get higher (P>0.05). The model mice’s thymus gland peripheral cortex area and cells reduced, medulla capillary vessel hyperemia, and spleen lymphoid nodule amount and area decreased. Uterus glandular lumen and endometrium mesenchyme hyperemia and edema. Compared with model group, the mice of moxibustion group thymus gland peripheral cortex area and cells and spleen lymphoid nodule amount and area all increased, glandular lumen and endometrium mesenchyme hyperemia and edema was relieved. Cx43 of endometrium in the control group mice is negative, it’s positive in model group, and slightly positive in moxibustion group.Conclusions:1. The treatment of warming needle with moxibustion and Shen Que moxibustion can obviously decrease the primary dysmenorrheal patients’ abdominal and dysmenorrheal symptoms’toal frequency rating and average severity level. And the effect accumulates along with treatment courses, This treatment can relieve the dysmenorrheal symptoms and pain level of primary dysmenorrheal patients.In 30 minutes of treating, its effect of easing pain positively correlates with treatment time. And It has the best easing pain effect at the time of treating 30 minutes. The effect of warming needle with moxibustion and Shen Que moxibustion is obviously superior than regular acupuncture.This treatment has practical and reliable for treating primary dysmenorrheal.Acupoints choice is refined and less, and combine the function of acupuncture and moxibustion, it’s good for clinical popularization.2. Shen Que moxibustion is effective for easing pain. It can regulate the mice’s uterus blood circulation, relieve Uterus glandular lumen and endometrium mesenchyme hyperemia and uterus spasm, decrease endometrium PGF2 a. Dysmenorrhea itself maybe can activate central nervous EOP system irritantly, moxibustion maybe promote CNS to produce and release much moreβ-EP, and also increase theβ-EP level in plasma to play the role of analgesic action. The functional mechanism of moxibustion on mice uterus spasm and pain maybe through adjusting Cx43 gap junction.3. Shen Que moxibustion can adjust dysmenorrheal model mice’s immunity function. It can up-regulate the percentage of CD4+ and the ratio of CD4+/ CD8+, maybe play the role of positively adjusting dysmenorrheal model mice’s immunity disorder. Shen Que moxibustion also can lighten the thymus gland and spleen pathological lesion to recover and promote the two organs’immunity function. PGF2a maybe related to the dysmenorrheal model mice’s immunity disorder, and Shen Que moxibustion adjusts the disorder maybe related to CNS and plasmaβ-EP expression.4. Neuro-endocrine-immunity network disorder in dysmenorrheal model mice maybe occurred. And the action mechanism of Shen Que moxibustion on primary dysmenorrheal maybe through neuro-endocrine-immunity network, adjust organs and meridians’function, balance body yin and yang, and stabilize internal environment.更多还原