经穴/时机与针刺效应相关性的研究

【作者】 李春华；

【导师】 朱江； 任晓暄； 马良宵；

【作者基本信息】 北京中医药大学 ， 针灸推拿学， 2012， 博士

【副题名】基于类痛经大鼠子宫微循环及子宫平滑肌舒缩物质实验

【摘要】 研究目的本研究以实验性类痛经模型大鼠为研究对象,以子宫微循环为切入点,选择即刻及预先两个时机介入电针,比较不同时机电针相同或相近神经节段支配的胞宫相关经穴(三阴交穴、血海穴)、非相关经穴(悬钟穴)及非经非穴对类痛经大鼠扭体反应、子宫微循环以及子宫平滑肌舒缩物质的影响,探讨电针缓解胞宫疼痛的作用机制,研究经穴与非穴、不同经脉的经穴、相同经脉的不同经穴调控胞宫效应的特异性以及介入时机对效应特异性的影响,为揭示针灸治疗原发性痛经的效应机制及经穴效应特异性影响因素提供依据,同时也为针灸临床取穴及择时提供科学指导。研究方法实验选用动情间期SD雌性大鼠192只,按照随机数字法分为即刻和预先盐水组、模型组、三阴交组、血海组、悬钟组、非穴组,每组16只。除盐水组外,其余各组大鼠均连续10天给予皮下注射苯甲酸雌二醇注射液,末次给药1h后,腹腔注射缩宫素制备类痛经大鼠模型,盐水组每日给予同等剂量的生理盐水。即刻电针组于第10d给予电针20min,共1次；预先电针组于第8d给予电针20min,每日1次,共3次；盐水组及模型组不予电针处理。实验一,即刻各组于电针同时观察大鼠的扭体反应,预先各组于末次电针后观察大鼠扭体反应。记录大鼠20min扭体潜伏期和扭体评分。实验二,即刻各组于电针同时观察大鼠电针5、10、20min时子宫微循环的变化,预先各组于末次电针5、10、20min时观察大鼠子宫微循环的变化。实验三,记录完扭体反应后,采用放射免疫法测定大鼠血浆TXB2、6-keto-PGF1α的含量；观察子宫微循环后,采用ELISA方法测定大鼠子宫ET含量,采用化学发光法测定大鼠子宫NO的含量。研究结果1不同时机电针对类痛经模型大鼠扭体反应的影响即刻组：与盐水组比较,模型组扭体潜伏期明显缩短(P<0.01),扭体评分明显增高(P<0.01)；与模型组比较,三阴交组扭体潜伏期明显延长(P<0.05),各电针组扭体评分均明显减少(P<0.01)；各电针组之间比,扭体潜伏期、扭体评分,无明显差异(P>0.05)。预先组：与盐水组比较,模型组扭体潜伏期明显缩短(P<0.01),扭体评分明显增高(P<0.05)；与模型组比较,各电针组扭体潜伏期、扭体评分无明显差异(P>0.05)各电针组之间比较,三阴交组的扭体评分较血海组、非穴组明显降低(P<0.05)2不同时机电针对类痛经模型大鼠子宫微循环的影响即刻组：与盐水组比较,模型组微血管、毛细血管(cap)粗细不均,管径收缩(P<0.01),微血管、cap条数减少(P<0.05,P<0.01),微血管、cap清晰度明显降低(P<0.01),微血管颜色明显加深(P<0.01),血流减慢或停滞(P<0.01)；与模型组比较,电针三阴交穴20mmin时,微血管、cap管径均明显扩张,微血管、cap条数均明显增多,微血管颜色明显改善(P<0.05),电针三阴交穴各时间段微血管、cap清晰度均明显改善(P<0.01,P<0.05,P<0.01,P<0.05),血流状态的差异无统计学意义(P>0.05),电针悬钟穴20min时,微血管、cap清晰度,微血管颜色均明显改善(P<0.05),电针血海穴、非穴各时间段各指标均无显著性差异(P>0.05)；各电针组之间比较,电针三阴交穴5min时较电针血海穴、悬钟穴、非穴微血管清晰度明显改善(P<0.05,P<0.01),电针三阴交穴20min时,cap管径较电针非穴明显扩张(P<0.05)。预先组：与盐水组比较,模型组微血管、毛细血管(cap)粗细不均,管径收缩(P<0.01),微血管、cap条数减少(P<0.01,P<0.05,P<0.01),微血管、cap清晰度明显降低(P<0.01),微血管颜色明显加深(P<0.01),血流减慢或停滞(P<0.01)；与模型组比较,电针三阴交穴各时间段微血管管径、cap管径(除5min外)明显扩张(P<0.01,P<0.05,P<0.01),微血管条数、cap条数(除5、10min外)明显增多(P<0.05),微血管清晰度、cap清晰度(除5min外)、微血管颜色(除5min外)明显改善,血流明显加快(P<0.01,P<0.05),电针悬钟穴各时间段微血管管径、cap管径(除5、10min外)明显扩张(P<0.05,P<0.01,P<0.05),微血管条数明显增多(P<0.05),微血管清晰度、cap清晰度(除5、10min外)、微血管颜色(除5、10min外)明显改善(P<0.01,P<0.05),电针血海穴10min时cap清晰度明显改善(P<0.05),电针非穴10、20min时微血管清晰度明显改善(P<0.05)；各电针组之间比较,电针三阴交穴、悬钟穴各时间段较电针血海穴微血管管径明显扩张(P<0.01),微血管清晰度明显改善(P<0.01,P<0.05),电针三阴交穴10min时较电针血海穴微血管条数明显增多(P<0.05),电针三阴交穴各时间段较电针血海穴血流明显加快(P<0.05),电针三阴交穴、悬钟穴各时间段较电针非穴微血管管径明显扩张(P<0.01),电针三阴交穴、悬钟穴20min时cap条数较电针非穴明显增多(P<0.05),电针三阴交穴10、20min时较电针非穴血流明显加快(P<0.05)。3不同时机电针对类痛经模型大鼠子宫平滑肌舒缩物质的影响即刻组：①血浆指标：与盐水组比较,模型组血浆TXB2的含量、TXB2/6-keto-PGF1α比值均明显升高(P<0.01)；与模型组比较,各电针组血浆TXB2的含量、TXB2/6-keto-PGF1α比值均明显降低(P<0.05,P<0.01),三阴交组血浆6-keto-PGF1α的含量明显升高(P<0.05)。②子宫指标：与盐水组比较,模型组子宫NO含量明显升高、ET/NO比值明显降低(P<0.01)；与模型组比较,各电针组(除非穴组外)子宫NO含量均明显降低(P<0.05),各电针组ET/NO比值均明显升高(P<0.01,P<0.05)。预先组：①血浆指标：与盐水组比较,模型组血浆TXB2含量、TXB2/6-keto-PGF1α比值均明显升高(P<0.05)；与模型组比较,三阴交组TXB2/6-keto-PGF1α匕值明显降低(P<0.05)。②子宫指标：与盐水组比较,模型组子宫NO含量明显升高、ET/NO比值明显降低(P<0.01)；与模型组比较,三阴交组子宫NO含量明显降低,ET/NO比值明显升高(P<0.01),悬钟组ET/NO比值亦明显升高(P<0.01)；各电针组之间比较,三阴交组子宫NO含量较血海组、非穴组明显降低,三阴交组ET/NO比值较非穴组明显升高(P<0.05)研究结论1即刻电针三阴交穴、血海穴、悬钟穴、非经非穴均可缓解模型大鼠的类痛经反应,而三阴交穴的缓解作用最为明显,初步表明了三阴交穴具有缓解类痛经反应的相对特异性。2推测即刻电针可能是通过神经反射快速调节子宫平滑肌舒缩物质,缓解子宫平滑肌的痉挛状态,从而达到缓解疼痛的目的。3预先电针三阴交穴、血海穴、悬钟穴、非经非穴对模型大鼠的类痛经反应均无统计学意义上的缓解效应,但三阴交穴的缓解作用明显优于血海穴、非经非穴。4即刻、预先电针均可改善模型大鼠的子宫微循环状态,但预先电针的作用更为显著。5不同时机介入电针,三阴交穴改善模型大鼠子宫微循环状态的作用均最为明显,且优于悬钟穴、血海穴、非经非穴。亦表明了三阴交穴具有调控胞宫微循环的相对特异性。综上可认为,穴位及介入时机不同,对胞宫的调节效应及机制不同。三阴交穴具有调控胞宫的效应特异性,该特异性是相对的；即刻电针对痛反应具有明确的缓解作用,而预先电针对子宫微循环的改善作用更为显著,表明介入时机对效应特异性具有一定影响。更多还原

【Abstract】 ObjectiveThe objective of this study was to compare the effects of electroacupuncture(EA) at uterus-relevant acupoint(Sanyinjiao SP6, Xuehai SP10), uterus-irrelevant acupoint (Xuanzhong GB39)and non-meridian point in the same or similar spinal segments, on writhing response, uterine microcirculation and diastolic materials and contractive materials of uterine smooth muscle in dysmenorrhea model rats. EA was conducted on rats instantly and beforehand, so as to investigate its mechanism on primary dysmenorrhea (PD), the specificity of acupoints in regulating function of uterus, and the effect of interventiontime on specificity of acupoints. As an experimental study, the purpose of this study is to provide experimental evidence for revealing the mechanism of EA for PD and the affecting factors of acupoint specificity. Simultaneously, this study also aimed at providing scientific guidance for selection of acupoints and determination of intervention time of EA for PD in the clinic.Methods192 diestrus SD female rats were randomly divided into instant saline(IS) group, instant model (IM) group, instant Sanyinjiao (ISP6) group, instant Xuehai(ISP10), instant Xuanzhong (IGB39) group, instant non-meridian point (INP) group and pre-saline (PS) group, pre-model (PM) group, pre-Sanyinjiao (PSP6) group, pre-Xuehai(PSP10), pre-Xuanzhong (PGB39)group, pre-non-meridian point (PNP) group according to the intervention time of EA, with 16 rats in each. Except saline group, the rats in other groups were subcutaneous injection of estradiol benzoate for 10 days continuously, and intraperitoneal injected with oxytocin 1h after the last estradiol injection to establish dysmenorrhea rat model. Rats in four instant EA groups received EA for 20 min after injection of oxytocin. On the 8th day after subcutaneous injection of estradiol benzoate, rats in four pre-EA groups received 3 treatment sessions, each of 20-min duration, once a day for 3 days, saline group and model group were not treated with EA. In the first experiment, writhing responses of rats in instant EA groups were observed with EA, while the writhing responses in pre-EA groups were observed after injection of oxytocin in the last EA session. The latency period and score of writhing were recorded for 20 minutes in each group. In the second experiment, changes of uterine microcirculation were observed by using a Cold Light Microcirculation Detector at the time points of 5min, 10min,20min with EA in instant EA groups, while uterine microcirculation in pre-EA groups were observed after injection of oxytocin in the last EA session, the study points were same as instant EA groups. In the third experiment, the content of Plasma TXB2.6-keto-PGF1αwere detected with radioimmunoassay after recording the writhing responses, the content of ET was measured by ELISA and the content of NO was measured by chemiluminescene assay after uterine microcirculation was observed. Results1 Effects of different intervention time of EA on writhing response of dysmenorrhea model ratsIn six instant groups:Compared with saline group, the latency period of writhing in model group was shortened significantly (P<0.01),and the writhing score was increased significantly (P<0.01). In comparison with model group, the latency period of writhing was lengthened significantly in ISP6 group(P<0.05), the writhing score were decreased significantly in four instant EA groups(P<0.01). There were no significant differences in latency period and score of writhing between every two of the four instant EA groups (P>0.05).In six prepare groups:Compared with saline group, the latency period of writhing in model group was shortened significantly(P<0.01), and the writhing score was increased significantly (P<0.05). In comparison with model group, there were no significant differences of the latency period and score of writhing in all instant EA groups (P>0.05).The writhing score in PSP6 group was significantly increased compared with PSP10 group and PNP group(P<0.05).2 Effects of different intervention time of EA on uterine microcirculation of dysmenorrhea model ratsIn six instant groups:Compared with saline group, diameters of microvessels and capillaries in model group were shrinked(P<0.01), numbers of microvessels and capillaries were decreased significantly(P<0.05, P<0.01), articulation indexes of microvessels and capillaries were reduced(P<0.01), the color of microvessels became deepen(P<0.01), the blood flow was slow and even stopped(P<0.01). Compared with model group, diameters of microvessels and capillaries were expanded significantly, numbers of microvessels and capillaries were increased significantly, the color of microvessels was improved at the time point 20min after EA in the ISP6 group(P<0.05), articulation indexes of microvessels and capillaries were improved significantly at the time points of 5min, 10min,20min after EA ISP6(P<0.01, P<0.05, P<0.01, P<0.05), there was no significant difference in the state of blood flow in the ISP6 group(P>0.05), articulation indexes of microvessels and capillaries, the color of microvessels were improved at 20min after EA in the IGB39 group(P<0.05), while there wasn’t significant difference in each indicator after EA on ISP10、INP(P>0.05). The articulation index of microvessels of ISP6 group was significantly improved than that of the ISP10、IGB39 and INP group at the time point 5min(P<0.05, P<0.01), the diameter of uterus capillaries of ISP6 group was significantly bigger than that of the INP group at the time point 20min(P<.05).In six prepare groups:Compared with saline group, diameters of microvessels and capillaries in model group were shrinked(P<0.01), numbers of microvessels and capillaries were decreased significantly(P<0.01, P<0.05, P<0.01), articulation indexes of microvessels and capillaries were reduced(P<0.01), the color of microvessels became deepen(P<0.01), the blood flow was slow and even stopped(P<0.01). Compared with model group, diameters of microvessels and capillaries(except 5min)were expanded significantly(P<0.01, P<0.05, P<0.01), numbers of microvessels and capillaries (except5min,10min)were increased significantly(P<0.05), articulation index of microvessels and capillaries(except 5min), the color of microvessels(except 5min) was improved and the state of blood flow sped up after EA PSP6 at the time points of 5min, 10min,20min, diameters of microvessels and capillaries(except 5min,10min)were significantly expanded(P<0.05, P<0.01, P<0.05), numbers of microvessels were increased significantly(P<0.05), articulation index of microvessels and capillaries(except 5min, lOmin). color of microvessels(except 5min,10min) were improved(P<0.01, P<0.05) after EA PGB39 at the time points of 5min, 10min,20min, articulation index of capillaries was improved after EA PSP10 at the time point 20min(P<0.05), articulation index of microvessels was improved after EA PNP at the time points of lOmin,20min(P<0.05). Diameters of microvessels were expanded significantly(P<0.01), articulation index of microvessels was improved significantly of PSP6 group, PGB39 group than that of PSP10 group at the time points of 5min, 10min, 20min(P<0.01, P<0.05), numbers of microvessels were increased significantly of PSP6 group than that of PSP10 group at the time point 10min(P<0.05), blood flow sped up of PSP6 group than that of PSP10 group at the time points of 5min, 10min,20min(P<0.05), diameters of microvessels were expanded significantly of PSP6 group, PGB39 group than that of PNP group at the time points of 5min,10min,20min(P<0.01), numbers of capillaries were increased significantly of PSP6 group. PGB39 group than that of PNP group at the time point 20min(P<0.05), blood flow sped up of PSP6 group than that of PNP group at the time points of 10min,20min(P<0.05).3 Effects of different intervention time of EA on diastolic materials and contractive materials of uterine smooth muscle of dysmenorrhea model ratsIn six instant groups:①lasma indexes:Compared with saline group, the content of plasma TXB2、and the ratio of TXB2/6-keto-PGF1αin model group were increased significantly(P<0.01). In comparison with model group, the content of plasma TXB2 and TXB2/6-keto-PGF1αin four EA groups were decreased significantly(P<0.05, P<0.01), the content of plasma 6-keto-PGF1αin PSP6 group was increased significantly(P<0.05).②terine indexes:Compared with saline group, the content of uterine NO in model group was increased significantly(P<0.01), the ratio of ET/NO was decreased significantly(P<0.01). In comparison with model group, the content of uterine NO in four EA group(except PNP group) were decreased significantly(P<0.05), the ratio of ET/NO in four EA group were increased significantly(P<0.01, P<0.05).In six prepare groups:①lasma indexes:Compared with saline group, the content of plasma TXB2 and the ratio of TXB2/6-keto-PGF1αin model group were increased significantly(P<0.05). Compared with model group, the ratio of TXB2/6-keto-PGF1αin PSP6 group was decreased significantly(P<0.05).②terine indexes:Compared with saline group, the content of uterine NO in model group was increased significantly(P<0.01), the ratio of ET/NO was decreased significantly(P<0.01). In comparison with model group, the content of uterine NO was decreased significantly and the ratio of ET/NO was increased significantly in PSP6 group(P<0.01), the ratio of ET/NO was increased significantly in PGB39 group(P<0.01).The content of uterine NO was decreased significantly of PSP6 group than that of PSP10 group and PNP group, the ratio of was increased significantly than that of PNP group(P<0.05).Conclusions1 All study acupoints (SP6, SP10, GB39) and non-meridian point with instant EA showed analgesic effect in dysmenorrhea model rats, the effect of SP6 was most evident. SP6 showed preliminarily specificity for PD.2 Regulating diastolic materials and contractive materials of uterine smooth muscle by regulating nerve reflex to relieving spasm of uterine smooth muscle and uterine pain may be the main mechanism of analgesic effect of each acupoint with instant EA.3 Nor acupoints (SP6, SP10, GB39) or non-meridian point with pre-EA showed analgesic effect in dysmenorrhea model rats. However, SP6 showed better effect than SP10 and non-meridian point.4 Both instant EA and pre-EA can improve uterine microcirculation in dysmenorrhea model rats, but pre-EA had the better effect.5 SP6 showed most significant effect to improve uterine microcirculation in dysmenorrhea model rats with instant EA and pre-EA, compared with SP10, GB39 and non-meridian point. SP6 showed preliminarily specificity for PD.In summary, this study showed EA at different acu-points and intervention time may have different regulating effect and mechanism in PD. SP6 showed specificity for PD, and the specificity was relative. Instant EA had affirmative effect in relieving uterine pain, while pre-EA had affirmative effect in improving uterine microcirculation. Intervention time of acupuncture had certain influence on specificity of acupoint.更多还原