Experimental Study And Preliminary Clinical Observation On The Effect Of Acupuncture On Treating Primary Dysmenorrhea

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.Partâ… : The Experimental Studies on Acupuncture in TreatingPrimary Dysmenorrhea1.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 AcupointObjective: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.PGE₂ 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:â‘ The 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).â‘¡The 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).â‘¢The 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, PGE₂ 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, PGE₂ 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 PGE₂, 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 DysmenorrheaObjective: To observe the effect of acupuncture on expression of estrogen(E₂), 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 E₂, 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:â‘ The 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).â‘¡Contrasted with normal group, the serum levels of E₂ 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 E₂ in acupuncture group and ELI groupwere markly descend(P<0.05), while the serum level of P was obviously ascend(P<0.05).â‘¢Contrasted 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 E₂, the mRNA and protein expression level of ERin endometrium, increasing the serum of P, the mRNA and protein expression level of PR inendometrium Partâ…¡: Preliminary Clinical Observation on the Effect of Acupuncture inTreating Primary DysmenorrheaObjective:To understand preliminarilyâ‘ The effect of acupuncture treatment of primary dysmenorrhea.â‘¡To investigate the relationship between deqi and the effect of acupuncture;â‘¢To investigate the relationship between manipulation and the effect of acupuncture;â‘£To 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:â‘ 2 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%,â‘¡Deqi 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),â‘¢There was a significant correlation betweenmanipulation and the difference value of the pain VAS score before and afteracupuncture(P<0.05).â‘£There 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.