针刺治疗原发性痛经的实验研究与初步临床观察
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摘要
痛经是指女性月经期或行经前后，周期性出现小腹疼痛或痛引腰骶，甚至剧痛晕厥者，属祖国医学“经行腹痛”范畴。临床上分原发性和继发性两种。原发性痛经(Primary Dysmenorrhea，PD)又称功能性痛经，是指女性生殖器官无明显器质性病变的痛经，为妇科常见病，多发病。据1980年的抽样调查，我国的痛经发病率为33.19％，其中PD占36.06％，严重影响工作者占13.55％。近年来，针灸治疗原发性痛经取得了可喜的进展，针灸治疗PD的临床报道颇多，且疗效肯定、无任何毒副作用，说明针灸治疗PD具有一定的优势和发展潜力。但目前对针刺治疗原发性痛经的作用机制尚缺乏确凿有力的实验依据。
RNA干扰(RNA interference，RNAi)作为一种新的反义基因工具，较以往的重组反义DNA、反义寡聚脱氧核糖核苷酸的效率更高，细胞内少许siRNA分子就能发挥显著基因下调作用。实验研究表明，在大鼠、猴子、狗、马、羊猪等体表存在很多高电导点(低电阻点)，有报道说这些低电阻点与缝隙连接的高密度分布有关。这些研究提示缝隙连接通讯可能参与经穴的信号传导。本课题前期研究工作表明，缝隙连接蛋白(Connexin 43，Cx43)在经线及穴位处的表达显著高于非经穴处，且针刺能明显增加Cx43在穴位处的表达，那么沉默穴位局部Cx43的表达，对经络信号的传导有无影响，是值得深入研究的课题。为进一步探讨Cx43与穴位-经络-针刺效应的相关性，以及针刺治疗原发性痛经的作用机制，本实验通过应用RNAi技术沉默针刺穴位局部Cx43基因的表达，以催产素所致大鼠子宫剧烈收缩(模拟痛经症)为模型，观察针刺对原发性痛经大鼠的镇痛机制。我们设计了第一部分实验方案：
第一部分针刺治疗原发性痛经的实验研究
第一节针刺对痛经大鼠前列腺素、催产素受体、加压素受体、β-内啡肽的影响及沉默穴位局部Cx43的表达
目的：
1．观察针刺对痛经大鼠前列腺素、催产素受体、加压素受体及β-内啡肽的影响，探讨针刺治疗原发性痛经的作用机制；
2．探讨沉默穴位局部Cx43的表达与针刺效应的关系。
方法：
将50只SD雌性大鼠分为正常组(N)、模型组(M)、针刺组(A)、针刺+干扰组(A+I)、针刺+干扰对照组(A+IC)，以催产素建立大鼠原发性痛经模型，应用RNAi技术沉默穴位局部Cx43的表达。针刺组采用针刺治疗，取关元，双侧地机、三阴交(直刺0.5～1.0寸，每5 min顺时针和逆时针捻转30 s，平补平泻，持续30 min)，在灌服已烯雌酚的第5天开始治疗，每日1次，连续7天。A+I组和A+IC组，在用已烯雌酚的第5、7、9、11天于针刺穴位处，分别局部高压注射干扰质粒Plasmid-Connexin43-shRNA和阴性对照质粒Plasmid-Control-shRNA，20μg／每穴，其余处理与A组相同。末次给予已烯雌酚1 h后，腹腔注射催产素，观察各组大鼠的扭体反应潜伏期和30 min内扭体数。分别应用放免法和ELISA检测血浆中前列腺素E_2(Prostaglandin E_2，PGE_2)，前列腺素F_(2α)(Prostaglandin F_(2α)，PGF_(2α))的含量；RT-PCR和免疫组化方法检测大鼠子宫内膜的催产素受体(Oxytocin Receptor，OTR)、加压素受体(Vasopressin Receptor，VPR)mRNA和蛋白的表达情况。放免法检测各组大鼠下丘脑、垂体、卵巢及血浆β-内啡肽(β-endorphin，β-EP)的含量。
结果：
①N组大鼠无扭体，M组大鼠潜伏期较短，扭体次数较多，两组之间差异有非常显著的统计学意义(P＜0.01)。②A+I组穴位处的Cx43 mRNA和蛋白表达低于N组(P＜0.05)；而A+IC组与N组比较，差异无统计学意义(P＞0.05)。③A组和A+IC组的大鼠扭体次数、血浆PGF_(2α)水平、子宫内膜OTR、VPR mRNA及蛋白水平明显低于M组，扭体潜伏期、血浆PGE_2水平、下丘脑、垂体、卵巢及血浆β-EP含量明显高于M组，差异有统计学意义(P＜0.05)；而A+I组与M组相比，以上各项指标无明显差异(P＞0.05)；A+I组的大鼠扭体次数、血浆PGF_(2α)水平、子宫内膜OTR、VPR mRNA及蛋白水平明显高于A组，扭体潜伏期、血浆PGE_2水平、下丘脑、垂体、卵巢及血浆β-EP含量低于A组，差异有统计学意义(P＜0.05)；A+IC组与A组比较，以上各项指标无统计学差异(P＞0.05)。
结论：
1．针刺治疗原发性痛经可能与降低PGF_(2α)，同时促进PGE_2合成，降低痛经大鼠子宫内膜OTR、VPR mRNA及蛋白表达水平，提高下丘脑-垂体-卵巢和血浆的β-EP含量有关。
2．RNAi沉默穴位Cx43表达后，针刺效应被抑制，表明Cx43在针刺效应中发挥着重要作用。
第二节针刺对痛经大鼠雌激素、孕激素水平及其受体表达的影响
目的：
观察针刺对原发性痛经大鼠雌激素(Estrogen，E_2)、孕激素(Progesterone，P)水平及其受体表达的影响，探讨针刺治疗原发性痛经的作用机制。
方法：
40只雌性大鼠随机分为正常组、模型组、针刺组、益母草膏组各10只，采用已烯雌酚(3 mg／kg／d)建立大鼠痛经模型。针刺组治疗同第一部分第一节，益母草膏组采用益母草膏(80 g／kg／d)灌胃治疗，2组均在灌服已烯雌酚的第5天开始治疗，每日1次，连续7天。末次给予已烯雌酚1 h、后，腹腔注射催产素，观察各组大鼠的扭体反应潜伏期和30min内扭体数；放免法检测各组大鼠血清E_2、P的水平；RT-PCR检测各组大鼠子宫内膜雌激素受体(Estrogen Receptor，ER)及孕激素受体(Progesterone Receptor，PR)mRNA水平。免疫组织化学法检测各组大鼠子宫内膜ER、PR的蛋白水平。
结果：
①模型组的大鼠扭体次数高于正常组，潜伏期低于正常组；针刺组和益母草膏组的大鼠扭体次数明显低于模型组，潜伏期高于模型组(P＜0.05)；②与正常组相比，模型组大鼠血清E_2含量均显著增加(P＜0.05)，P含量显著降低(P＜0.05)；而针刺组和益母草膏组与正常组相比，无统计学差异(P＞0.05)。针刺组和益母草膏组E_2含量较模型组显著降低(P＜0.05)，P含量较模型组显著增加(P＜0.05)。③与正常组相比，模型组大鼠子宫内膜ER mRNA和蛋白表达水平显著增加(P＜0.05)，PR mRNA和蛋白表达水平显著降低(P＜0.05)；针刺组和益母草膏组子宫内膜ER mRNA和蛋白表达水平与模型组相比，显著降低(P＜0.05)；PR mRNA和蛋白表达水平较模型组显著升高(P＜0.05)。
结论：
针刺治疗原发性痛经可能与针刺降低痛经大鼠血清E_2、子宫内膜ER mRNA和蛋白表达水平，增加血清P，子宫内膜PR mRNA和蛋白表达水平有关。
第二部分针刺治疗原发性痛经的初步临床观察
目的：
初步了解：
1．针刺治疗原发性痛经的疗效观察；
2．探讨得气与针刺疗效之间的关系；
3．探讨手法与针刺疗效之间的关系；
4．探讨患者的心理因素与针刺疗效之间的关系。
方法：
30例原发性痛经患者，随机分为手法组和非手法组各15例。以直观模拟标尺法(Visual Analogue Scale，VAS)让患者自我评价自己对针刺疗效的信任度、紧张度、痛阈值，得气程度，痛经程度。焦虑自评量表及抑郁自评量表测量患者的焦虑及抑郁程度，艾森克个性问卷(Eysenck Personality Questionnaire，EPQ)及卡特尔16种人格因素问卷(Cattell′s 16 Personality Factor Questionnaire，16 PF)测量患者的个性特征。记录针刺治疗前后患者的疼痛程度VAS分数差值作为疗效评价的指标。信任度、紧张度、患者的焦虑及抑郁程度、EPQ和16 PF结果作为心理因素评价的指标。
结果：
①手法组治愈2人，显效2人，有效7人，无效4人，有效率为73.33％；非手法组有效4人，无效11人，有效率为26.67％。②得气与针刺治疗痛经前后程度VAS差值呈正相关(R=2.798，P=0.017)。③手法与针刺治疗痛经前后程度VAS差值有明显相关性(P＜0.05)。④患者的焦虑与痛经疼痛程度VAS差值之间呈负相关性(R=-0.02，P=0.037)，EPQ中患者的情绪稳定性与痛经疼痛程度VAS差值之间呈负相关性(R=-0.095，P=0.031)。
结论：
研究结果初步表明：针刺能有效治疗原发性痛经，得气、手法与针刺疗效有明显相关性，患者的焦虑、情绪稳定性影响了针刺疗效。此结果作为初步临床研究，还需要大量样本进一步的证实。
Dysmenorrhea refers that women have the symptoms of abdomen pain, painspreading lumbo-sacrum, even syncope during menstruation or pre- and post menses,which belongs to "the abdominal pain" category in TCM.Clinically, it is classified into twogeneral types: primary and secondaryd dysmenorrrhea.Primary dysmenorrhea, which isalso called functional dysmenorrhea, is the department of gynaecology common disease,and frequently-occurring disease.Women ofter present as abdomen pain with the menstrualcycle, but the female reproductive organs without apparent organic change.According tothe sampling survey in 1980, the disease rate of dysmenorrhea is 33.19% in domestci,among which PD is about 36.06%, the seriously affecting work accounts 13.55%.Inrecent years, it is achieved a great progress that acupuncture treatment of primarydysmenorrhea.There are many clinical reports that acupuncture treatment of PD has madedefinite curative effect without any side effects, which indicated it has certainadvantages and development potential.But there is a lack of convincing analgesicmechanisms of experiment basis at the moment.
RNA interference (RNAi) as a kind of new antonymous gene tool has a higherefficacy than departed recombined DNA and oligodeoxynucleotide.It plays significantly down regulation effect when a bit siRNA in the cell.Our preparatory, work showed,that the, expressions of the Gap junctions Connexin protein (Cx43) in the meridian andacupoints were significantly higher than, that in non-meridian and non-acupoints, andacupuncture can obviously increase the expression of Cx43 in the meridians.We supposedthat if we silencing the expression of Cx43 in local acupoint, the conduction signals ofmeridian have influence, which is a worthing research topic.For further discussion thecorrelation between Cx43 with acupoint-meridian-acupuncture effect, and the mechanismof acupuncture treatment of primary dysmenorrhea, this experiment applied with RNAitechnology silencing Cx43, and using rat'uterine intense contraction (simulation model ofdysmenorrhea disease) caused by oxytocin as model, in order to observe the analgesiamechanism of primary dysmenorrhea treated with acupuncture.We design the first part ofthe experiment plan, as following.
PartⅠ: The Experimental Studies on Acupuncture in TreatingPrimary Dysmenorrhea
1.The Effect of Acupuncture on the Expression of Prostaglandin,Oxytocin Receptor, Vasopression Receptor andβ-EP in Rats withPrimary Dysmenorrhea and Silencing Cx43 Expression of Local Acupoint
Objective:
1.To observe the effect of acupuncture on the expression of prostaglandin, oxytocinreceptor, vasopression receptor andβ-EP in rats with primary dysmenorrhea and Localsilencing Cx43, and in order to investigate the mechanism of acupuncture in treatingprimary dysmenorrhea.
2.To investigate the relationship between silencing Cx43 expression of partial acupoint andthe effect of acupuncture
Methods:
We used diethylstibestrol to result in the model of primary dysmenorrhea, and RNAinterference technology to silence the expression of Cx43 in acupoint.50 SD female ratswere divided into five groups, nomal group, model group, acupuncture group, acupunctureand interference group(A+I), acupuncture and interference control group (A+IC).For ratsin acupuncture group, sterilized disposable stainless steel needles were perpendicularlyinserted into RN4, bilateral SP8 and SP6 at 0.5～1.0 cun.The inserted needles were rotatedclockwise and anti-clockwise 30 s with mild reinforcing and attenuating every 5 mins for30 mins.Acupuncture group was received treatment once daily from the on fifth day ofperfusing diethylstilbestrol for consecutive days.Rats in A+I group and A+IC group werepartially high-pressure injected with the interference plasmid and the negative controlplasmid respectively, on fifth, seventh, ninth, eleventh day of perfusing diethylstilbestrol.20μg of the plasmid per acupoint was used.Then, after 1 hour perfusing via gastrogavageon the twelfth day, the five groups were injected with oxytocin(14 U/kg) viaintraperitoneum.The latency period and the incidence rate of writhing body within 30 minswere observed.Then treatment was the same as acupuncture.PGE_2 and PGF_(2α) of plasmawere detected by radioimmunity and ELISA, respectively.RT-PCR andimmunohistochemistry were used to observe the oxytocin and vasopressin mRNA andprotein expression in each group.The contents ofβ-EP in hypothalamus, pituitary, ovaryand plasma were detected by radioimmuassay.
Results:
①The latency period of writhing body was shorter and the incidence rate of writhing bodywas more in model group, compared with those of normal group(P<0.05).②The level ofCx43 mRNA and protein of acupoint in A+I group were markedly lower than those ofnomal group(P<0.05).There was no significant difference in the level of Cx43 mRNA andprotein between A+IC group and normal group(P>0.05).③The incidence rate of writhingbody, PGF_(2α), the level of oxytocin receptor and vaspression receptor mRNA and protein in uterus in acupuncture group and A+IC group were lower than those of model group.Thelatency period of writhing body, PGE_2 and the contents ofβ-EP in hypothalamus, pituitary,ovary and plasma in acupuncture group and A+IC group were higher than those of modelgroup.The differences were statistically significant(P<0.05).There were not significantdifferences about the above-mentioned indices between A+I group and modelgroup(P>0,05).The incidence rate of writhing body, PGF_(2α), the level of oxytocin receptorand vaspression receptor mRNA and protein in uterus in A+I group were higher than thoseof acupuncture group.The latency period of writhing body, PGE_2 and the contents ofβ-EPin hypothalamus, pituitary, ovary and plasma in A+I group were lower than those ofacupuncture group.There were no significant differences about the above-mentionedindices between A+IC group and acupuncture group(P>0.05).
Conclusion:
1.The mechanisms of acupuncture treating with primary dysmenorrhea might be concernedwith the two-way regulating prostaglandin synthetic system, decreasing PGF_(2α), whileproviding the synthesis of PGE_2, decreasing the expression level of OTR, VPR mRNA andprotein in dysmenorrheci rats' endometrium, increasing the contents ofβ-EP inhypothalamus, pituitary, ovary and plasma.
2.Silencing Cx43 expression of partial acupoint could inhibit effectively the effects ofacupuncture, which suggested Cx43 play an important role in the effects of acupuncture.
2.The Effect of Acupuncture on the Expression of Estrogen, Progestogenand its Receptors in Rats with Primary Dysmenorrhea
Objective:
To observe the effect of acupuncture on expression of estrogen(E_2), progestogen(P) and itsreseptors in rats with primary dysmenorrhea, and in order to investigate the mechanism ofacupuncture in treating primary dysmenorrhea.
Methods:
40 female rats were evenly randomized into control group, model group, acupuncture groupand Extractum Leonuri Inspissatum(ELI) group, and primary dysmenorrhea model ratswere established by perfusing diethylstilbestrol(3 mg/kg/d).For rats in acupuncture group,sterilized disposable stainless steel needles were perpendicularly inserted into RN4,bilateral SP8 and SP6 at 0.5～1.0 cun.The inserted needles were rotated clockwise andanti-clockwise 30 s with mild reinforcing and attenuating every 5 mins for 30 mins.ELIgroup was received perfusion via gastrogavage with ELI(80 g/kg/d).Acupuncture groupand ELI group were received treatment once daily from the on fifth day of perfusingdiethylstilbestrol for consecutive days.Then, after 1 hour perfusing via gastrogavage on thetwelfth day, the four groups were injected with oxytocin(14 U/kg) via intraperitoneum.Thelatency period and the incidence rate of writhing body within 30 mins were observed.Theserum levels of E_2, and P were detected by RIA, and the mRNA expression of estrogenreceptor(ER) and progesterone receptor(PR) in endometria were determined using RT-PCR.The protein levels of ER and PR in the uterus were detected by immunochemistry in eachgroup.
Results:
①The latency period of writhing body was shorter and the incidence rate of writhing bodywas more in model group, compared with those of normal group(P<0.05).The latencyperiod of writhing body was longer and the incidence rate of writhing body was less inacupuncture group and ELI group, compared with those of model group(P<0.05).②Contrasted with normal group, the serum levels of E_2 in model group were marklyascend(P<0.05), while the serum level of P was obviously descend(P<0.05).There was nosignificant difference between acupuncture group, ELI group and normal group(P>0.05). Contrasted with model group, the serum levels of E_2 in acupuncture group and ELI groupwere markly descend(P<0.05), while the serum level of P was obviously ascend(P<0.05).③Contrasted with normal group, the mRNA and protein expression levels of ER in modelgroup markly ascend(P<0.05), while the mRNA and protein expression level of PR wasobviously descend(P<0.05).Contrasted with model group, the mRNA and proteinexpression levels of ER in acupuncture group and ELI group were markly descend(P<0.05),while the mRNA and protein expression level of PR was obviously ascend(P<0.05).
Conclusion:
Acupuncture works to relieve the sysmptom of uterine smooth muscle cramping might beconcerned with decreasing the serum of E_2, the mRNA and protein expression level of ERin endometrium, increasing the serum of P, the mRNA and protein expression level of PR inendometrium
PartⅡ: Preliminary Clinical Observation on the Effect of Acupuncture inTreating Primary Dysmenorrhea
Objective:
To understand preliminarily
①The effect of acupuncture treatment of primary dysmenorrhea.
②To investigate the relationship between deqi and the effect of acupuncture;
③To investigate the relationship between manipulation and the effect of acupuncture;
④To investigate the relationship between the patients' psychological factor and the effectof acupuncture.
Methods:
Thirty patients with primary dysmenorrhea were randomly divided into manipulation groupand non-manipulation group, each group of 15 cases.The credibility for the therapeuticeffects of acupuncture, nervousness, threshold of pain, deqi, the severity of pain wereevaluated by the patient with visual analogue scale (VAS).All subjects completed a seriesof standardized questionnaires for psychological variables before treatment.The degree ofthe patients' anxiety and depression were evaluted by Self-Rating Anxiety Scale(SAS).Thepersonality of the patients was evaluted by Eysenck Personality Questionnaire(EPQ) and 16Personality Factor Questionnaire(16 PF).The pain VAS scores before and after acupuncturewere recorded as the index for assessment of therapeutic effect.The scores of the credibilityfor the therapeutic effects of acupuncture, nervousness, the patients' anxiety and depression,the results of EPQ and 16 PF were recorded as the evaluation index of the psychologicalfactors.
Results:
①2 patients got cure, 2 patients marked effectiveness, 7 patients effectiveness and 4patients failure in manipulation group, so the effective rate was 73.33%.4 patients effectiveness and 11 patients failure in non-manipulation group, so the effective rate was26.67%,②Deqi exerted an influence on acupuncture therapeutic, which represents Deqihas positively correlated with the difference value of the pain VAS score before and afteracupuncture(R=2.798, P=0.017),③There was a significant correlation betweenmanipulation and the difference value of the pain VAS score before and afteracupuncture(P<0.05).④There is a negative correlation between the patients'anxiety(R=-0.02, P=0.037) and the emotional stability in EPQ(R=-0.095, P=0.031) withthe difference value of the pain VAS score before and after acupuncture.
Conclusion:
The results preliminarily indicated that acupuncture was an effective method for primarydysmenorrhea, It was found the effect of acupuncture was effectively related to deqi andmanipulation.The patient's anxiety and emotional stability has affected the effect ofacupuncture.This results served as preliminary clinical research, which need a largenumber of samples to be confirmed.
RNA干扰(RNA interference，RNAi)作为一种新的反义基因工具，较以往的重组反义DNA、反义寡聚脱氧核糖核苷酸的效率更高，细胞内少许siRNA分子就能发挥显著基因下调作用。实验研究表明，在大鼠、猴子、狗、马、羊猪等体表存在很多高电导点(低电阻点)，有报道说这些低电阻点与缝隙连接的高密度分布有关。这些研究提示缝隙连接通讯可能参与经穴的信号传导。本课题前期研究工作表明，缝隙连接蛋白(Connexin 43，Cx43)在经线及穴位处的表达显著高于非经穴处，且针刺能明显增加Cx43在穴位处的表达，那么沉默穴位局部Cx43的表达，对经络信号的传导有无影响，是值得深入研究的课题。为进一步探讨Cx43与穴位-经络-针刺效应的相关性，以及针刺治疗原发性痛经的作用机制，本实验通过应用RNAi技术沉默针刺穴位局部Cx43基因的表达，以催产素所致大鼠子宫剧烈收缩(模拟痛经症)为模型，观察针刺对原发性痛经大鼠的镇痛机制。我们设计了第一部分实验方案：
第一部分针刺治疗原发性痛经的实验研究
第一节针刺对痛经大鼠前列腺素、催产素受体、加压素受体、β-内啡肽的影响及沉默穴位局部Cx43的表达
目的：
1．观察针刺对痛经大鼠前列腺素、催产素受体、加压素受体及β-内啡肽的影响，探讨针刺治疗原发性痛经的作用机制；
2．探讨沉默穴位局部Cx43的表达与针刺效应的关系。
方法：
将50只SD雌性大鼠分为正常组(N)、模型组(M)、针刺组(A)、针刺+干扰组(A+I)、针刺+干扰对照组(A+IC)，以催产素建立大鼠原发性痛经模型，应用RNAi技术沉默穴位局部Cx43的表达。针刺组采用针刺治疗，取关元，双侧地机、三阴交(直刺0.5～1.0寸，每5 min顺时针和逆时针捻转30 s，平补平泻，持续30 min)，在灌服已烯雌酚的第5天开始治疗，每日1次，连续7天。A+I组和A+IC组，在用已烯雌酚的第5、7、9、11天于针刺穴位处，分别局部高压注射干扰质粒Plasmid-Connexin43-shRNA和阴性对照质粒Plasmid-Control-shRNA，20μg／每穴，其余处理与A组相同。末次给予已烯雌酚1 h后，腹腔注射催产素，观察各组大鼠的扭体反应潜伏期和30 min内扭体数。分别应用放免法和ELISA检测血浆中前列腺素E_2(Prostaglandin E_2，PGE_2)，前列腺素F_(2α)(Prostaglandin F_(2α)，PGF_(2α))的含量；RT-PCR和免疫组化方法检测大鼠子宫内膜的催产素受体(Oxytocin Receptor，OTR)、加压素受体(Vasopressin Receptor，VPR)mRNA和蛋白的表达情况。放免法检测各组大鼠下丘脑、垂体、卵巢及血浆β-内啡肽(β-endorphin，β-EP)的含量。
结果：
①N组大鼠无扭体，M组大鼠潜伏期较短，扭体次数较多，两组之间差异有非常显著的统计学意义(P＜0.01)。②A+I组穴位处的Cx43 mRNA和蛋白表达低于N组(P＜0.05)；而A+IC组与N组比较，差异无统计学意义(P＞0.05)。③A组和A+IC组的大鼠扭体次数、血浆PGF_(2α)水平、子宫内膜OTR、VPR mRNA及蛋白水平明显低于M组，扭体潜伏期、血浆PGE_2水平、下丘脑、垂体、卵巢及血浆β-EP含量明显高于M组，差异有统计学意义(P＜0.05)；而A+I组与M组相比，以上各项指标无明显差异(P＞0.05)；A+I组的大鼠扭体次数、血浆PGF_(2α)水平、子宫内膜OTR、VPR mRNA及蛋白水平明显高于A组，扭体潜伏期、血浆PGE_2水平、下丘脑、垂体、卵巢及血浆β-EP含量低于A组，差异有统计学意义(P＜0.05)；A+IC组与A组比较，以上各项指标无统计学差异(P＞0.05)。
结论：
1．针刺治疗原发性痛经可能与降低PGF_(2α)，同时促进PGE_2合成，降低痛经大鼠子宫内膜OTR、VPR mRNA及蛋白表达水平，提高下丘脑-垂体-卵巢和血浆的β-EP含量有关。
2．RNAi沉默穴位Cx43表达后，针刺效应被抑制，表明Cx43在针刺效应中发挥着重要作用。
第二节针刺对痛经大鼠雌激素、孕激素水平及其受体表达的影响
目的：
观察针刺对原发性痛经大鼠雌激素(Estrogen，E_2)、孕激素(Progesterone，P)水平及其受体表达的影响，探讨针刺治疗原发性痛经的作用机制。
方法：
40只雌性大鼠随机分为正常组、模型组、针刺组、益母草膏组各10只，采用已烯雌酚(3 mg／kg／d)建立大鼠痛经模型。针刺组治疗同第一部分第一节，益母草膏组采用益母草膏(80 g／kg／d)灌胃治疗，2组均在灌服已烯雌酚的第5天开始治疗，每日1次，连续7天。末次给予已烯雌酚1 h、后，腹腔注射催产素，观察各组大鼠的扭体反应潜伏期和30min内扭体数；放免法检测各组大鼠血清E_2、P的水平；RT-PCR检测各组大鼠子宫内膜雌激素受体(Estrogen Receptor，ER)及孕激素受体(Progesterone Receptor，PR)mRNA水平。免疫组织化学法检测各组大鼠子宫内膜ER、PR的蛋白水平。
结果：
①模型组的大鼠扭体次数高于正常组，潜伏期低于正常组；针刺组和益母草膏组的大鼠扭体次数明显低于模型组，潜伏期高于模型组(P＜0.05)；②与正常组相比，模型组大鼠血清E_2含量均显著增加(P＜0.05)，P含量显著降低(P＜0.05)；而针刺组和益母草膏组与正常组相比，无统计学差异(P＞0.05)。针刺组和益母草膏组E_2含量较模型组显著降低(P＜0.05)，P含量较模型组显著增加(P＜0.05)。③与正常组相比，模型组大鼠子宫内膜ER mRNA和蛋白表达水平显著增加(P＜0.05)，PR mRNA和蛋白表达水平显著降低(P＜0.05)；针刺组和益母草膏组子宫内膜ER mRNA和蛋白表达水平与模型组相比，显著降低(P＜0.05)；PR mRNA和蛋白表达水平较模型组显著升高(P＜0.05)。
结论：
针刺治疗原发性痛经可能与针刺降低痛经大鼠血清E_2、子宫内膜ER mRNA和蛋白表达水平，增加血清P，子宫内膜PR mRNA和蛋白表达水平有关。
第二部分针刺治疗原发性痛经的初步临床观察
目的：
初步了解：
1．针刺治疗原发性痛经的疗效观察；
2．探讨得气与针刺疗效之间的关系；
3．探讨手法与针刺疗效之间的关系；
4．探讨患者的心理因素与针刺疗效之间的关系。
方法：
30例原发性痛经患者，随机分为手法组和非手法组各15例。以直观模拟标尺法(Visual Analogue Scale，VAS)让患者自我评价自己对针刺疗效的信任度、紧张度、痛阈值，得气程度，痛经程度。焦虑自评量表及抑郁自评量表测量患者的焦虑及抑郁程度，艾森克个性问卷(Eysenck Personality Questionnaire，EPQ)及卡特尔16种人格因素问卷(Cattell′s 16 Personality Factor Questionnaire，16 PF)测量患者的个性特征。记录针刺治疗前后患者的疼痛程度VAS分数差值作为疗效评价的指标。信任度、紧张度、患者的焦虑及抑郁程度、EPQ和16 PF结果作为心理因素评价的指标。
结果：
①手法组治愈2人，显效2人，有效7人，无效4人，有效率为73.33％；非手法组有效4人，无效11人，有效率为26.67％。②得气与针刺治疗痛经前后程度VAS差值呈正相关(R=2.798，P=0.017)。③手法与针刺治疗痛经前后程度VAS差值有明显相关性(P＜0.05)。④患者的焦虑与痛经疼痛程度VAS差值之间呈负相关性(R=-0.02，P=0.037)，EPQ中患者的情绪稳定性与痛经疼痛程度VAS差值之间呈负相关性(R=-0.095，P=0.031)。
结论：
研究结果初步表明：针刺能有效治疗原发性痛经，得气、手法与针刺疗效有明显相关性，患者的焦虑、情绪稳定性影响了针刺疗效。此结果作为初步临床研究，还需要大量样本进一步的证实。
Dysmenorrhea refers that women have the symptoms of abdomen pain, painspreading lumbo-sacrum, even syncope during menstruation or pre- and post menses,which belongs to "the abdominal pain" category in TCM.Clinically, it is classified into twogeneral types: primary and secondaryd dysmenorrrhea.Primary dysmenorrhea, which isalso called functional dysmenorrhea, is the department of gynaecology common disease,and frequently-occurring disease.Women ofter present as abdomen pain with the menstrualcycle, but the female reproductive organs without apparent organic change.According tothe sampling survey in 1980, the disease rate of dysmenorrhea is 33.19% in domestci,among which PD is about 36.06%, the seriously affecting work accounts 13.55%.Inrecent years, it is achieved a great progress that acupuncture treatment of primarydysmenorrhea.There are many clinical reports that acupuncture treatment of PD has madedefinite curative effect without any side effects, which indicated it has certainadvantages and development potential.But there is a lack of convincing analgesicmechanisms of experiment basis at the moment.
RNA interference (RNAi) as a kind of new antonymous gene tool has a higherefficacy than departed recombined DNA and oligodeoxynucleotide.It plays significantly down regulation effect when a bit siRNA in the cell.Our preparatory, work showed,that the, expressions of the Gap junctions Connexin protein (Cx43) in the meridian andacupoints were significantly higher than, that in non-meridian and non-acupoints, andacupuncture can obviously increase the expression of Cx43 in the meridians.We supposedthat if we silencing the expression of Cx43 in local acupoint, the conduction signals ofmeridian have influence, which is a worthing research topic.For further discussion thecorrelation between Cx43 with acupoint-meridian-acupuncture effect, and the mechanismof acupuncture treatment of primary dysmenorrhea, this experiment applied with RNAitechnology silencing Cx43, and using rat'uterine intense contraction (simulation model ofdysmenorrhea disease) caused by oxytocin as model, in order to observe the analgesiamechanism of primary dysmenorrhea treated with acupuncture.We design the first part ofthe experiment plan, as following.
PartⅠ: The Experimental Studies on Acupuncture in TreatingPrimary Dysmenorrhea
1.The Effect of Acupuncture on the Expression of Prostaglandin,Oxytocin Receptor, Vasopression Receptor andβ-EP in Rats withPrimary Dysmenorrhea and Silencing Cx43 Expression of Local Acupoint
Objective:
1.To observe the effect of acupuncture on the expression of prostaglandin, oxytocinreceptor, vasopression receptor andβ-EP in rats with primary dysmenorrhea and Localsilencing Cx43, and in order to investigate the mechanism of acupuncture in treatingprimary dysmenorrhea.
2.To investigate the relationship between silencing Cx43 expression of partial acupoint andthe effect of acupuncture
Methods:
We used diethylstibestrol to result in the model of primary dysmenorrhea, and RNAinterference technology to silence the expression of Cx43 in acupoint.50 SD female ratswere divided into five groups, nomal group, model group, acupuncture group, acupunctureand interference group(A+I), acupuncture and interference control group (A+IC).For ratsin acupuncture group, sterilized disposable stainless steel needles were perpendicularlyinserted into RN4, bilateral SP8 and SP6 at 0.5～1.0 cun.The inserted needles were rotatedclockwise and anti-clockwise 30 s with mild reinforcing and attenuating every 5 mins for30 mins.Acupuncture group was received treatment once daily from the on fifth day ofperfusing diethylstilbestrol for consecutive days.Rats in A+I group and A+IC group werepartially high-pressure injected with the interference plasmid and the negative controlplasmid respectively, on fifth, seventh, ninth, eleventh day of perfusing diethylstilbestrol.20μg of the plasmid per acupoint was used.Then, after 1 hour perfusing via gastrogavageon the twelfth day, the five groups were injected with oxytocin(14 U/kg) viaintraperitoneum.The latency period and the incidence rate of writhing body within 30 minswere observed.Then treatment was the same as acupuncture.PGE_2 and PGF_(2α) of plasmawere detected by radioimmunity and ELISA, respectively.RT-PCR andimmunohistochemistry were used to observe the oxytocin and vasopressin mRNA andprotein expression in each group.The contents ofβ-EP in hypothalamus, pituitary, ovaryand plasma were detected by radioimmuassay.
Results:
①The latency period of writhing body was shorter and the incidence rate of writhing bodywas more in model group, compared with those of normal group(P<0.05).②The level ofCx43 mRNA and protein of acupoint in A+I group were markedly lower than those ofnomal group(P<0.05).There was no significant difference in the level of Cx43 mRNA andprotein between A+IC group and normal group(P>0.05).③The incidence rate of writhingbody, PGF_(2α), the level of oxytocin receptor and vaspression receptor mRNA and protein in uterus in acupuncture group and A+IC group were lower than those of model group.Thelatency period of writhing body, PGE_2 and the contents ofβ-EP in hypothalamus, pituitary,ovary and plasma in acupuncture group and A+IC group were higher than those of modelgroup.The differences were statistically significant(P<0.05).There were not significantdifferences about the above-mentioned indices between A+I group and modelgroup(P>0,05).The incidence rate of writhing body, PGF_(2α), the level of oxytocin receptorand vaspression receptor mRNA and protein in uterus in A+I group were higher than thoseof acupuncture group.The latency period of writhing body, PGE_2 and the contents ofβ-EPin hypothalamus, pituitary, ovary and plasma in A+I group were lower than those ofacupuncture group.There were no significant differences about the above-mentionedindices between A+IC group and acupuncture group(P>0.05).
Conclusion:
1.The mechanisms of acupuncture treating with primary dysmenorrhea might be concernedwith the two-way regulating prostaglandin synthetic system, decreasing PGF_(2α), whileproviding the synthesis of PGE_2, decreasing the expression level of OTR, VPR mRNA andprotein in dysmenorrheci rats' endometrium, increasing the contents ofβ-EP inhypothalamus, pituitary, ovary and plasma.
2.Silencing Cx43 expression of partial acupoint could inhibit effectively the effects ofacupuncture, which suggested Cx43 play an important role in the effects of acupuncture.
2.The Effect of Acupuncture on the Expression of Estrogen, Progestogenand its Receptors in Rats with Primary Dysmenorrhea
Objective:
To observe the effect of acupuncture on expression of estrogen(E_2), progestogen(P) and itsreseptors in rats with primary dysmenorrhea, and in order to investigate the mechanism ofacupuncture in treating primary dysmenorrhea.
Methods:
40 female rats were evenly randomized into control group, model group, acupuncture groupand Extractum Leonuri Inspissatum(ELI) group, and primary dysmenorrhea model ratswere established by perfusing diethylstilbestrol(3 mg/kg/d).For rats in acupuncture group,sterilized disposable stainless steel needles were perpendicularly inserted into RN4,bilateral SP8 and SP6 at 0.5～1.0 cun.The inserted needles were rotated clockwise andanti-clockwise 30 s with mild reinforcing and attenuating every 5 mins for 30 mins.ELIgroup was received perfusion via gastrogavage with ELI(80 g/kg/d).Acupuncture groupand ELI group were received treatment once daily from the on fifth day of perfusingdiethylstilbestrol for consecutive days.Then, after 1 hour perfusing via gastrogavage on thetwelfth day, the four groups were injected with oxytocin(14 U/kg) via intraperitoneum.Thelatency period and the incidence rate of writhing body within 30 mins were observed.Theserum levels of E_2, and P were detected by RIA, and the mRNA expression of estrogenreceptor(ER) and progesterone receptor(PR) in endometria were determined using RT-PCR.The protein levels of ER and PR in the uterus were detected by immunochemistry in eachgroup.
Results:
①The latency period of writhing body was shorter and the incidence rate of writhing bodywas more in model group, compared with those of normal group(P<0.05).The latencyperiod of writhing body was longer and the incidence rate of writhing body was less inacupuncture group and ELI group, compared with those of model group(P<0.05).②Contrasted with normal group, the serum levels of E_2 in model group were marklyascend(P<0.05), while the serum level of P was obviously descend(P<0.05).There was nosignificant difference between acupuncture group, ELI group and normal group(P>0.05). Contrasted with model group, the serum levels of E_2 in acupuncture group and ELI groupwere markly descend(P<0.05), while the serum level of P was obviously ascend(P<0.05).③Contrasted with normal group, the mRNA and protein expression levels of ER in modelgroup markly ascend(P<0.05), while the mRNA and protein expression level of PR wasobviously descend(P<0.05).Contrasted with model group, the mRNA and proteinexpression levels of ER in acupuncture group and ELI group were markly descend(P<0.05),while the mRNA and protein expression level of PR was obviously ascend(P<0.05).
Conclusion:
Acupuncture works to relieve the sysmptom of uterine smooth muscle cramping might beconcerned with decreasing the serum of E_2, the mRNA and protein expression level of ERin endometrium, increasing the serum of P, the mRNA and protein expression level of PR inendometrium
PartⅡ: Preliminary Clinical Observation on the Effect of Acupuncture inTreating Primary Dysmenorrhea
Objective:
To understand preliminarily
①The effect of acupuncture treatment of primary dysmenorrhea.
②To investigate the relationship between deqi and the effect of acupuncture;
③To investigate the relationship between manipulation and the effect of acupuncture;
④To investigate the relationship between the patients' psychological factor and the effectof acupuncture.
Methods:
Thirty patients with primary dysmenorrhea were randomly divided into manipulation groupand non-manipulation group, each group of 15 cases.The credibility for the therapeuticeffects of acupuncture, nervousness, threshold of pain, deqi, the severity of pain wereevaluated by the patient with visual analogue scale (VAS).All subjects completed a seriesof standardized questionnaires for psychological variables before treatment.The degree ofthe patients' anxiety and depression were evaluted by Self-Rating Anxiety Scale(SAS).Thepersonality of the patients was evaluted by Eysenck Personality Questionnaire(EPQ) and 16Personality Factor Questionnaire(16 PF).The pain VAS scores before and after acupuncturewere recorded as the index for assessment of therapeutic effect.The scores of the credibilityfor the therapeutic effects of acupuncture, nervousness, the patients' anxiety and depression,the results of EPQ and 16 PF were recorded as the evaluation index of the psychologicalfactors.
Results:
①2 patients got cure, 2 patients marked effectiveness, 7 patients effectiveness and 4patients failure in manipulation group, so the effective rate was 73.33%.4 patients effectiveness and 11 patients failure in non-manipulation group, so the effective rate was26.67%,②Deqi exerted an influence on acupuncture therapeutic, which represents Deqihas positively correlated with the difference value of the pain VAS score before and afteracupuncture(R=2.798, P=0.017),③There was a significant correlation betweenmanipulation and the difference value of the pain VAS score before and afteracupuncture(P<0.05).④There is a negative correlation between the patients'anxiety(R=-0.02, P=0.037) and the emotional stability in EPQ(R=-0.095, P=0.031) withthe difference value of the pain VAS score before and after acupuncture.
Conclusion:
The results preliminarily indicated that acupuncture was an effective method for primarydysmenorrhea, It was found the effect of acupuncture was effectively related to deqi andmanipulation.The patient's anxiety and emotional stability has affected the effect ofacupuncture.This results served as preliminary clinical research, which need a largenumber of samples to be confirmed.
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