针刺对不同状态肠运动的调节作用和神经机制研究
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摘要
目的:现代针灸教材和一些针灸学专著均提及针灸对机体呈现双向调节效应。该理论认为，机体或器官的功能处于亢奋的情况下，针灸治疗可以减弱亢进的功能；相反，机体或器官的功能低下时，针灸治疗可以增强它的功能活动。目前对这一理论的含义具体认识存在诸多分歧。本项研究采用压力检测技术记录大鼠空肠和远端结肠的运动，以功能性肠病中的腹泻和便秘为载体，在单元、集元理论的指导下，遵循中医针灸取穴的原则，选取曲池、上巨虚、天枢、大肠俞四个腧穴，观察针刺对不同功能状态肠运动的调节规律，探索针刺对肠运动是否存在双向性的调节作用及其科学内涵。
材料与方法：
正常大鼠：SD大鼠，雄性，体重250-300g，乌拉坦（1-1.2g/kg）腹腔麻醉。大鼠仰卧，腹正中线切口，在幽门下20cm的空肠处和肛门上5-6cm的远端结肠处，分别放置直径为0.6cm的水囊，水囊通过导管与胃肠运动换能器相连，将约0.1ml的纯净水注入空肠内的水囊，至水囊内的基础压力为100cmH2O，结肠内水囊的压力调节同空肠，用Powerlab系统记录肠运动的信号。
腹泻大鼠模型制作：0.3g生药/ml番泻叶药液，按10ml/kg的剂量灌胃，1次/d，连续灌胃2天。
便秘大鼠模型制作：0℃生理盐水，按10ml/kg的剂量灌胃，1次/d，连续灌胃5天。
药物性兴奋、抑制运动状态：
1空肠运动兴奋：M2受体激动剂Pilocarpine （3mg/kg，i.v），按0.1ml/min的速度颈静脉微量注射，空肠运动进入稳定的亢奋状态后，随机选取腧穴行针刺操作。
2空肠运动抑制：M2受体拮抗剂Methoctramine（0.5mg/kg，i.v），按0.1ml/min的速度颈静脉微量注射，空肠运动进入稳定的抑制状态后，随机选取腧穴针刺操作。
3远端结肠运动兴奋：M3受体激动剂Choline chloride（10mg/kg，i.v），按0.1ml/min的速度颈静脉微量注射，远端结肠运动进入稳定的亢奋状态后，随机选取腧穴行针刺操作。
4远端结肠运动抑制：M3受体拮抗剂4-DAMP（1mg/kg，i.v）按0.1ml/min的速度颈静脉微量注射，远端结肠运动进入稳定的抑制状态后，随机选取腧穴行针刺操作
坐骨神经纤维选择性破坏：
A类神经纤维破坏：在大鼠下肢腓骨头上1cm处做一切口，分离坐骨神经干。用微量进样器依次抽取1微升生理盐水、1微升空气、1微升0.3%蛇毒溶液，空气是用来隔开蛇毒溶液与生理盐水,将药物注射到坐骨神经的鞘膜下，30min后进行针刺操作。
C类神经纤维破坏：实验前24h，用0.3%戊巴比妥钠1ml/100g剂量作腹腔麻醉，将麻醉大鼠下肢腓骨头上1cm处做一切口，分离坐骨神经干。用浸润2%辣椒素的纱布条缠绕坐骨神经干，用塑料条包绕纱布条以避免辣椒素损伤周围的组织，缝合切口。24h后用于针刺的实验。腧穴定位和针刺操作：曲池（LI11）：桡骨近端肘关节外侧前方的凹陷处，直刺4mm；天枢（ST25）:相当于脐中旁开5mm，直刺5mm；上巨虚(ST37)：大鼠后肢足三里向下约5mm处，直刺5mm；大肠俞（BL25）：在腰部，当第4腰椎棘突下，旁开5mm，直刺4mm。针刺操作：手动行针1min，频率120次/min。
数据处理
运用Chart7软件对图形进行数据转换，使用SPSS Statistics19.0统计软件进行数据处理，采用配对t检验，计量资料以均数标准误（x±SD）表示，P<0.05认为差异有统计学意义，P<0.01认为差异有显著统计学意义。
结果：
1针刺腧穴对正常大鼠肠运动的作用
1.1针刺腧穴对正常大鼠空肠运动的作用
针刺曲池、上巨虚均提高了空肠运动幅值（P<0.05， P<0.01）。针刺天枢抑制了空肠的运动幅值和频率（P<0.01，P<0.01）。针刺大肠俞对空肠的运动频率和幅值均无明显的影响（P>0.05， P>0.05）。针刺曲池、上巨虚均促进了空肠的运动，相反，针刺天枢起到抑制作用。
1.2针刺腧穴对正常大鼠远端结肠的作用
针刺曲池使远端结肠的运动幅值上升（P<0.05），而频率无明显变化（P>0.05）。针刺上巨虚使远端结肠的运动幅值和频率均增加（P<0.01，P<0.01）。针刺天枢使远端结肠的运动幅值和频率增大（P<0.05，P<0.01）。针刺大肠俞使远端结肠的运动幅值和频率明显增加（P<0.05，P<0.05）。针刺曲池、上巨虚、天枢及大肠俞均促进了远端结肠的运动。2.针刺腧穴对腹泻大鼠肠运动的作用
2.1针刺腧穴对腹泻大鼠空肠运动的作用
针刺曲池，空肠的运动幅值明显增大(P<0.05）。针刺天枢，空肠运动的幅值和频率都明显下降（P<0.01, P<0.01）。针刺上巨虚、大肠俞对腹泻大鼠空肠运动作用不明显（P>0.05，P>0.05）。
2.2针刺腧穴对腹泻大鼠远端结肠运动的作用
针刺曲池，远端结肠的运动幅值明显升高（P<0.05）。针刺上巨虚、大肠俞，大鼠远端结肠的运动幅值和频率较基础值均显著升高（P<0.05，P<0.05）。针刺天枢，远端结肠的运动频率显著升高（P<0.01），运动幅值没有显著变化（P>0.05）。针刺上巨虚、大肠俞均促进的远端结肠的运动，针刺曲池提高运动幅值，而针刺天枢增加了运动频率。
3针刺腧穴对便秘大鼠肠运动的作用
3.1针刺腧穴对便秘大鼠空肠运动的作用
针刺曲池，空肠的运动幅值明显升高（P<0.05）。针刺上巨虚使空肠的运动幅值显著升高（P<0.01）。针刺天枢，空肠的运动幅值和频率明显下降（P<0.01，P<0.01）。针刺大肠俞对空肠的运动频率和幅值均无明显影响（P>0.05）。针刺曲池、上巨虚均可促进空肠的运动，相反，针刺天枢变现为抑制作用。
3.2针刺腧穴对便秘大鼠远端结肠运动的作用
针刺曲池使远端结肠的运动幅值提高（P<0.05），而频率无明显变化（P>0.05）。针刺上巨虚、大肠俞使远端结肠的运动幅值和频率都明显增加（P<0.05）。针刺天枢使远端结肠的运动幅值和频率均显著增加（P<0.01，P<0.01）。针刺曲池、上巨虚、天枢及大肠俞均促进了便秘大鼠远端结肠的运动。
4针刺对M受体激动剂、拮抗剂所致的亢奋和抑制状态肠运动的作用
4.1针刺腧穴对注射Pilocarpine后空肠的作用静脉注射M2受体激动剂Pilocarpine后,空肠运动处于兴奋状态。针刺曲池、上巨虚以及大肠俞对空肠的运动幅值没有明显的作用（P>0.05）。针刺天枢，空肠运动的幅值有降低的趋势，但无统计学差异（P>0.05）。
4.2针刺腧穴对注射Methoctramine后空肠运动的作用
静脉注射M2受体拮抗剂Methoctramine后，空肠运动处于抑制状态。针刺曲池使空肠运动幅值较针刺前增加（P<0.05）。针刺上巨虚使空肠的运动频率和幅值均升高（P<0.05,P<0.01）。针刺大肠俞增加了空肠运动幅值（P<0.01）。针刺天枢，空肠运动的频率和幅值均降低（P<0.01,P<0.01）。针刺上巨虚、大肠俞促进了空肠运动的恢复，针刺天枢对注射Methoctramine后空肠运动仍表现为抑制作用。
4.3针刺腧穴对注射Choline chloride后远端结肠运动的作用
静脉注射M3受体激动剂Choline chloride促进了远端结肠的运动，使其处于兴奋状态，针刺曲池、上巨虚、天枢、大肠俞对远端结肠的运动没有明显的作用（P>0.05）。
4.4针刺腧穴对注射4-DAMP后远端结肠运动的作用
静脉注射M3受体拮抗剂4-DAMP，使结肠运动处于抑制状态。针刺曲池增加了结肠运动的幅值（P<0.05），针刺上巨虚、天枢、大肠俞均增加远端结肠运动的频率和幅值（P<0.05），促进远端结肠的运动。
5躯体传入纤维在针刺调节肠运动中的作用
5.1A类纤维在针刺上巨虚对肠运动调节中的作用
在蛇毒未破坏坐骨神经的A类纤维前，针刺上巨虚促进了空肠及远端结肠的运动。A纤维破坏后，针刺上巨虚对肠运动的促进作用仍存在，只是作用的强度有所降低。说明坐骨神经的A类纤维参与针刺上巨虚对肠运动的调节。
5.2C类纤维在针刺上巨虚对肠运动调节中的作用在辣椒素未破坏坐骨神经的C类纤维前，针刺上巨虚使空肠和远端结肠运动加强。C纤维破坏后，针刺上巨虚肠运动仅有加强的趋势，没有显著的统计学差异（P>0.05）。说明坐骨神经C类神经参与针刺上巨虚调节肠运动，并起着主要的作用。
结论
（1）针刺集元穴（异神经节段的腧穴）对空肠及远端结肠运动的针刺效应表现为促进作用，是激活了副交感神经的结果。肠的运动状态影响集元穴对肠运动针刺效应的强度，但不能改变其针刺作用的方向。
（2）针刺单元穴天枢对空肠的抑制作用，是激活交感神经的结果。单元穴天枢对空肠的针刺效应不受肠运动状态影响，与其本身的腧穴特性有关。
（3）针刺天枢对腹泻及便秘的治疗，其神经动力学机制可能是针刺天枢激活交感神经抑制空肠运动，激活副交感神经促进远端结肠运动。
（4）躯体传入神经的A类纤维和C类纤维均参与针刺对肠运动的调节，其中C类神经纤维起着重要的作用。
实验结果说明，针刺双向调节概念的复杂性，在功能性肠病中，针刺双向调节效应是肠运动状态和腧穴的特性综合作用的结果。肠运动状态决定集元穴（异神经节段腧穴）针刺效应的大小，但不能改变针刺效应的方向，针刺效应的方向与腧穴的特性相关。针刺单元穴天枢对空肠运动的效应表现为抑制作用，与肠运动状态无关。提示针刺治疗肠运动障碍性疾病时，既要根据腧穴的特性也要结合肠运动的状态来选取腧穴。
Object
Bidirectional regulation of acupuncture has being mentioned insome acupuncture-moxibustion textbooks and related works.The theory is that acupuncture can down regulate the functionof organs, if it is hyperfunction, and conversely, can strengthenthe function of organ, if it is hypofunciton. But the bidirectionalregulation effect of acupuncture hasn't been studied profoundly.In the present study, the intestinal contractility was recordedwith manometric balloons put into both jejunal and distal coloniclumens in normal and also diarrhea and constipation model rats.Acupoints selected were IL11, ST37,ST25and BL25, to explore theeffect on intestinal motility of different states in normal andmodel rats respectly.We aim to investigate the bidirectionalregulation function caused by acupuncture and the underlying neuralmechanisms.
MateriaI and Methods
Sprague-Dawley rats were used，weighing between250g-300g.anesthetized with intraperitoneal injection of urethane(1.0-1.2g／kg).A median abdominal incision was made and one manometricballoon was put into the jejunum about20cm downstream from the pylorus, and another one was put in distal colon5-6cm upstream anusvia anus. The balloon was filled with about0.1ml warm water andconnect to a piece of polyethylene tube, which gave pressure atabout100mmH2O.Pressure in intestinal lumen was measured by atransducer through the thin polyethylene tube and recorded by ADInstruments.
Diarrhea rat model was made by senna solution gavage,0.3g crudedrug/ml,10ml/kg, once a day, for2days.
Constipation rat model was made by gavage0℃normal saline,10ml/kg, once a day, for5days.
Carotid vein were exposed and intubated directly connected withthe mocrosyringe for drug administration．
Pilocarpine3mg/kg was was injected intravenously at rate of0.1ml/min, and acupuncture was performed when the change ofintestinal pressure evoked by the compound was stable.
Methoctramine0.5mg/kg,was injected intravenously at rate of0.1ml/min,and acupuncture was performed when the change ofingestinal pressure declined by the compound was stabile.
Choline chloride10mg/kg,was injected intravenously at rateof0.1ml/min,and acupuncture was performed when the change ofintestinal pressure evoked by the compound was stabile.
4-DAMP1mg/kg,was injected intravenously at rate of0.1ml/min,and acupuncture was performed when the change of ingestinalpressure declined by the compound was stabile.
Sciatic nerve A-fibers was demyelinated by snake venom:Ischiadic nerve were isolated1cm above fibular head inanesthetized rats, trace sample aspirated1ul normal saline,1ulair and1ul0.3%snake venom solution in sequence. Needle Tip were penetrated into sciatic nerve membrane, and the liquid above wasinjected into the myelin sheathin avoidance of drug leakage intothe surrounding tissue and suture of incision.30min after venominjection,acupuncture stimulation were conducted
Sciatic nerve C fibers was inactivated by capsaicin: Sciaticnerve were isolated1cm above fibular head in rats anesthed by0.3%pentobarbital,the separated sciatic nerve trunks were wrapped bya cotton strip immersed in2%solution of capsaicin for24hours.
Acupoints: LI11(quchi), located in the midpoint between thelateral end of the transverse cubical crease and the lateralepicondyle of the humerus, inserting to a depth of about4mm;ST25(tianshu), on the level of the navel,2mm lateral to anteriormedian line, inserting to a depth of about5mm; ST37(shangjuxu),5mm laterl to the anterior tubercle of the tibia and15mm belowThe knee joint,inserting to a depth of about5mm; BL25(dachangshu),in the waist, under the fourth lumbar spines,5mm lateral toposterior midline, inserting to a depth of about4mm. The needlewas rotated clockwise and anti-clockwise at2Hz for60s at eachpoint.
Statistical analysis
Statistical analysis was performed with SPSS software forWindows, version19.0.All data were expressed as a mean SD. Meanvalues were compared using the paired t test to compare theintestinal motility before and after acupuncture. The differencewas considered significant at P<0.05.
Results
1Effect of acupuncture on intestinal motility in normal rats
1.1Effect of acupuncture on jejunum motility in normal rats
Acupuncture at either IL11or ST37significantly increased theamplitude of jejunum waveform （P<0.05， P<0.01）,whereas theacupuncture at ST25significantly decreased jejunum motility（P<0.01）.BL25had no significant effect on the motility ofjejunum(P>0.05).
1.2Effect of acupuncture on the distal colon motility in normalrats
Effects of single points on the distal colon motility:Acupuncture at IL11ST37, ST25or BL25accelerated diatal colonicmotility compared to that of baseline（P<0.05）.
2. Effect of acupuncture on intestinal motility in diarrhea rats.
2.1Effect of acupuncture on jejunum motility in diarrhea rats.
The mean amplitude of jejunum motility during acupuncture atIL11were significantly increased compared to baseline(P<0.05).Whereas acupuncture at ST25significantly decreased amplitude andfrequency of jejunum motility(P<0.01).There were no effect ofacupuncture at ST37、BL25on jejunum motility(P>0.05).
2.2Effect of acupuncture on the distal colon motility in diarrhearats.
Acupuncture at IL11incresed significantly the amplitude of thedistal colon (P<0.05). Both ST37and BL25acclecrated the frequencyand amplitude.The frequency but not the amplitude was increased byacupuncture at ST25(P<0.01).
3Effect of acupuncture on the intestinal motility in constipationrats.
3.1Effect of acupuncture on the jejunum motility in constipationrats.
Acupuncture at IL11and ST37increaded jejunum motility amplitude respectively (P<0.05,P<0.01).Acupuncture at ST25decreased the frequency and amplitude of jejunum (P<0.01). Therewere no obvious change during acupuncture at BL25(P>0.05).
3.2Effect of acupuncture on the distal colon motility inconstipation rats.
Acupuncture at IL11accelerated amplitude but not frequency (P<0.05). Acupuncture at ST37, BL25increased both amplitude andfrequency of colon motility (P<0.05). Acupuncture ST25acceleratedboth amplitude and frequency of colon motility（P<0.01，P<0.01）.
4. Effect of acupuncture on jejunum motility in hyperfunction andhypofunction condition
4.1Effect of acupuncture on jejunum motility after pilocarpineadministration
After M2receptor agonist Pilocarpine administration,theamplitude of jejunum increased(P<0.01), in hyperfunction. Therewere no obvious change during acupuncture atIL11,BL25,ST37, BL25(P>0.05).
4.2. Effect of acupuncture on jejunum motility after methoctramineadministration
After M2receptor antagonist Methoctramine administration,both amplitude and frequency were decreased (P<0.01, P<0.01) inhypofunction. Acupuncture at both IL11and BL25increased theamplitude (P<0.05), Acupuncture at ST37accelerated frequency andamplitude compared with that before (P<0.05, P<0.01).while, ST25decreased frequency and amplitude (P<0.01, P<0.01).
Methoctramine has inhibition on the movement of jejunumAcupuncture at IL11,ST37or BL25has antagonist like effect to thedrug,however the effect of ST25is still characterized by inhibition of the jejunum movement
4.3. Effect of acupuncture on the distal motility after Cholinechloride administrationAfter M3receptor agonist Choline chloride administration, theamplitude of colon motility increased(P<0.01),in hyperfunction.There were no obvious change during acupuncture at IL11,BL25,ST37,BL25(P>0.05).
4.4. Effect of acupuncture on the distal motility after4-DAMPadministration
After M3receptor antagonist4-DAMP administration, bothamplitude and frequency of colon motility were decreased (P<0.01,P<0.01),in hypofunction.Acupuncture at IL increased the amplitudeof colon motility （P<0.05）,Acupuncture at ST37,ST25or BL25increased both frequency and amplitude（P<0.05）.Acupuncture atIL11,ST37,ST25or BL25has antagonist effect to4-DAMP on colonmotility.
5The role of different somatic afferent fibers in acupunctureregulation of intestinal motility
5.1The role of A fibers in acupuncture regulation of intestinalmotility
The stimulation of acupuncture at ST37increased both jejunumand the disatal colon motitlity. After demyelination of Aδ fiber,the excitatory effect is less than that in normal condition.5.2The role of C fibers in acupuncture regulation intestinalmotility
After inactivation C fiber by capsaicin, acupuncture at ST37had no obvious effect on the jejunum motility and the disatal colonmotitlity. C fiber plays a major role in the regualtion of intestinal motility by acupuncture.
Conclusion
(1) Acupuncture at heterotopic acupoints has excitatoryeffects on the intestinal motility, which induced by activation ofvagal nerves.Though the effect of acupuncture is related closelyto states of intestinal motility, the direction of intestinalresponse can not been changed by acupuncture effect.
(2) Acupuncture at homotopic acupoint ST25always producesinhibition on the intestinal motility, independent of the state ofintestinal motility.
(3) The effect of acupuncture at ST25on diarrhea andconstipation demonstrates that the neural dynamic mechanism liesin the inhibition of jejunum movement through sympathetic tone andpromotion the distal colon movement through parasympathetic tone.
(4) The somatic afferent nerve fibers A and C are involved inthe regulation of acupuncture on intestinal motility, and C fibersplays an more important role.
There is complexity to understand the conception of bidirectonregulation of acupuncture. Our study shows that for functionalbowel diseases, the bilateral regulation of acupuncture reflectedsynthetically by different states of intestinal motility and thelocation of acupoints. The effect of acupuncture at heterotopicacupoints on intestinal motility is effected by intestine state,but the response direction to acupuncture can not be changed. Theinhibitory effects of acupuncture at homotopic acupoint ST25onjejnum motility is independent of bowel movement.
The present study suggests that acupoints selection principlesin treating fuctional bowel movement should be a combination of the state of the bowel movement and location of acupoints.
材料与方法：
正常大鼠：SD大鼠，雄性，体重250-300g，乌拉坦（1-1.2g/kg）腹腔麻醉。大鼠仰卧，腹正中线切口，在幽门下20cm的空肠处和肛门上5-6cm的远端结肠处，分别放置直径为0.6cm的水囊，水囊通过导管与胃肠运动换能器相连，将约0.1ml的纯净水注入空肠内的水囊，至水囊内的基础压力为100cmH2O，结肠内水囊的压力调节同空肠，用Powerlab系统记录肠运动的信号。
腹泻大鼠模型制作：0.3g生药/ml番泻叶药液，按10ml/kg的剂量灌胃，1次/d，连续灌胃2天。
便秘大鼠模型制作：0℃生理盐水，按10ml/kg的剂量灌胃，1次/d，连续灌胃5天。
药物性兴奋、抑制运动状态：
1空肠运动兴奋：M2受体激动剂Pilocarpine （3mg/kg，i.v），按0.1ml/min的速度颈静脉微量注射，空肠运动进入稳定的亢奋状态后，随机选取腧穴行针刺操作。
2空肠运动抑制：M2受体拮抗剂Methoctramine（0.5mg/kg，i.v），按0.1ml/min的速度颈静脉微量注射，空肠运动进入稳定的抑制状态后，随机选取腧穴针刺操作。
3远端结肠运动兴奋：M3受体激动剂Choline chloride（10mg/kg，i.v），按0.1ml/min的速度颈静脉微量注射，远端结肠运动进入稳定的亢奋状态后，随机选取腧穴行针刺操作。
4远端结肠运动抑制：M3受体拮抗剂4-DAMP（1mg/kg，i.v）按0.1ml/min的速度颈静脉微量注射，远端结肠运动进入稳定的抑制状态后，随机选取腧穴行针刺操作
坐骨神经纤维选择性破坏：
A类神经纤维破坏：在大鼠下肢腓骨头上1cm处做一切口，分离坐骨神经干。用微量进样器依次抽取1微升生理盐水、1微升空气、1微升0.3%蛇毒溶液，空气是用来隔开蛇毒溶液与生理盐水,将药物注射到坐骨神经的鞘膜下，30min后进行针刺操作。
C类神经纤维破坏：实验前24h，用0.3%戊巴比妥钠1ml/100g剂量作腹腔麻醉，将麻醉大鼠下肢腓骨头上1cm处做一切口，分离坐骨神经干。用浸润2%辣椒素的纱布条缠绕坐骨神经干，用塑料条包绕纱布条以避免辣椒素损伤周围的组织，缝合切口。24h后用于针刺的实验。腧穴定位和针刺操作：曲池（LI11）：桡骨近端肘关节外侧前方的凹陷处，直刺4mm；天枢（ST25）:相当于脐中旁开5mm，直刺5mm；上巨虚(ST37)：大鼠后肢足三里向下约5mm处，直刺5mm；大肠俞（BL25）：在腰部，当第4腰椎棘突下，旁开5mm，直刺4mm。针刺操作：手动行针1min，频率120次/min。
数据处理
运用Chart7软件对图形进行数据转换，使用SPSS Statistics19.0统计软件进行数据处理，采用配对t检验，计量资料以均数标准误（x±SD）表示，P<0.05认为差异有统计学意义，P<0.01认为差异有显著统计学意义。
结果：
1针刺腧穴对正常大鼠肠运动的作用
1.1针刺腧穴对正常大鼠空肠运动的作用
针刺曲池、上巨虚均提高了空肠运动幅值（P<0.05， P<0.01）。针刺天枢抑制了空肠的运动幅值和频率（P<0.01，P<0.01）。针刺大肠俞对空肠的运动频率和幅值均无明显的影响（P>0.05， P>0.05）。针刺曲池、上巨虚均促进了空肠的运动，相反，针刺天枢起到抑制作用。
1.2针刺腧穴对正常大鼠远端结肠的作用
针刺曲池使远端结肠的运动幅值上升（P<0.05），而频率无明显变化（P>0.05）。针刺上巨虚使远端结肠的运动幅值和频率均增加（P<0.01，P<0.01）。针刺天枢使远端结肠的运动幅值和频率增大（P<0.05，P<0.01）。针刺大肠俞使远端结肠的运动幅值和频率明显增加（P<0.05，P<0.05）。针刺曲池、上巨虚、天枢及大肠俞均促进了远端结肠的运动。2.针刺腧穴对腹泻大鼠肠运动的作用
2.1针刺腧穴对腹泻大鼠空肠运动的作用
针刺曲池，空肠的运动幅值明显增大(P<0.05）。针刺天枢，空肠运动的幅值和频率都明显下降（P<0.01, P<0.01）。针刺上巨虚、大肠俞对腹泻大鼠空肠运动作用不明显（P>0.05，P>0.05）。
2.2针刺腧穴对腹泻大鼠远端结肠运动的作用
针刺曲池，远端结肠的运动幅值明显升高（P<0.05）。针刺上巨虚、大肠俞，大鼠远端结肠的运动幅值和频率较基础值均显著升高（P<0.05，P<0.05）。针刺天枢，远端结肠的运动频率显著升高（P<0.01），运动幅值没有显著变化（P>0.05）。针刺上巨虚、大肠俞均促进的远端结肠的运动，针刺曲池提高运动幅值，而针刺天枢增加了运动频率。
3针刺腧穴对便秘大鼠肠运动的作用
3.1针刺腧穴对便秘大鼠空肠运动的作用
针刺曲池，空肠的运动幅值明显升高（P<0.05）。针刺上巨虚使空肠的运动幅值显著升高（P<0.01）。针刺天枢，空肠的运动幅值和频率明显下降（P<0.01，P<0.01）。针刺大肠俞对空肠的运动频率和幅值均无明显影响（P>0.05）。针刺曲池、上巨虚均可促进空肠的运动，相反，针刺天枢变现为抑制作用。
3.2针刺腧穴对便秘大鼠远端结肠运动的作用
针刺曲池使远端结肠的运动幅值提高（P<0.05），而频率无明显变化（P>0.05）。针刺上巨虚、大肠俞使远端结肠的运动幅值和频率都明显增加（P<0.05）。针刺天枢使远端结肠的运动幅值和频率均显著增加（P<0.01，P<0.01）。针刺曲池、上巨虚、天枢及大肠俞均促进了便秘大鼠远端结肠的运动。
4针刺对M受体激动剂、拮抗剂所致的亢奋和抑制状态肠运动的作用
4.1针刺腧穴对注射Pilocarpine后空肠的作用静脉注射M2受体激动剂Pilocarpine后,空肠运动处于兴奋状态。针刺曲池、上巨虚以及大肠俞对空肠的运动幅值没有明显的作用（P>0.05）。针刺天枢，空肠运动的幅值有降低的趋势，但无统计学差异（P>0.05）。
4.2针刺腧穴对注射Methoctramine后空肠运动的作用
静脉注射M2受体拮抗剂Methoctramine后，空肠运动处于抑制状态。针刺曲池使空肠运动幅值较针刺前增加（P<0.05）。针刺上巨虚使空肠的运动频率和幅值均升高（P<0.05,P<0.01）。针刺大肠俞增加了空肠运动幅值（P<0.01）。针刺天枢，空肠运动的频率和幅值均降低（P<0.01,P<0.01）。针刺上巨虚、大肠俞促进了空肠运动的恢复，针刺天枢对注射Methoctramine后空肠运动仍表现为抑制作用。
4.3针刺腧穴对注射Choline chloride后远端结肠运动的作用
静脉注射M3受体激动剂Choline chloride促进了远端结肠的运动，使其处于兴奋状态，针刺曲池、上巨虚、天枢、大肠俞对远端结肠的运动没有明显的作用（P>0.05）。
4.4针刺腧穴对注射4-DAMP后远端结肠运动的作用
静脉注射M3受体拮抗剂4-DAMP，使结肠运动处于抑制状态。针刺曲池增加了结肠运动的幅值（P<0.05），针刺上巨虚、天枢、大肠俞均增加远端结肠运动的频率和幅值（P<0.05），促进远端结肠的运动。
5躯体传入纤维在针刺调节肠运动中的作用
5.1A类纤维在针刺上巨虚对肠运动调节中的作用
在蛇毒未破坏坐骨神经的A类纤维前，针刺上巨虚促进了空肠及远端结肠的运动。A纤维破坏后，针刺上巨虚对肠运动的促进作用仍存在，只是作用的强度有所降低。说明坐骨神经的A类纤维参与针刺上巨虚对肠运动的调节。
5.2C类纤维在针刺上巨虚对肠运动调节中的作用在辣椒素未破坏坐骨神经的C类纤维前，针刺上巨虚使空肠和远端结肠运动加强。C纤维破坏后，针刺上巨虚肠运动仅有加强的趋势，没有显著的统计学差异（P>0.05）。说明坐骨神经C类神经参与针刺上巨虚调节肠运动，并起着主要的作用。
结论
（1）针刺集元穴（异神经节段的腧穴）对空肠及远端结肠运动的针刺效应表现为促进作用，是激活了副交感神经的结果。肠的运动状态影响集元穴对肠运动针刺效应的强度，但不能改变其针刺作用的方向。
（2）针刺单元穴天枢对空肠的抑制作用，是激活交感神经的结果。单元穴天枢对空肠的针刺效应不受肠运动状态影响，与其本身的腧穴特性有关。
（3）针刺天枢对腹泻及便秘的治疗，其神经动力学机制可能是针刺天枢激活交感神经抑制空肠运动，激活副交感神经促进远端结肠运动。
（4）躯体传入神经的A类纤维和C类纤维均参与针刺对肠运动的调节，其中C类神经纤维起着重要的作用。
实验结果说明，针刺双向调节概念的复杂性，在功能性肠病中，针刺双向调节效应是肠运动状态和腧穴的特性综合作用的结果。肠运动状态决定集元穴（异神经节段腧穴）针刺效应的大小，但不能改变针刺效应的方向，针刺效应的方向与腧穴的特性相关。针刺单元穴天枢对空肠运动的效应表现为抑制作用，与肠运动状态无关。提示针刺治疗肠运动障碍性疾病时，既要根据腧穴的特性也要结合肠运动的状态来选取腧穴。
Object
Bidirectional regulation of acupuncture has being mentioned insome acupuncture-moxibustion textbooks and related works.The theory is that acupuncture can down regulate the functionof organs, if it is hyperfunction, and conversely, can strengthenthe function of organ, if it is hypofunciton. But the bidirectionalregulation effect of acupuncture hasn't been studied profoundly.In the present study, the intestinal contractility was recordedwith manometric balloons put into both jejunal and distal coloniclumens in normal and also diarrhea and constipation model rats.Acupoints selected were IL11, ST37,ST25and BL25, to explore theeffect on intestinal motility of different states in normal andmodel rats respectly.We aim to investigate the bidirectionalregulation function caused by acupuncture and the underlying neuralmechanisms.
MateriaI and Methods
Sprague-Dawley rats were used，weighing between250g-300g.anesthetized with intraperitoneal injection of urethane(1.0-1.2g／kg).A median abdominal incision was made and one manometricballoon was put into the jejunum about20cm downstream from the pylorus, and another one was put in distal colon5-6cm upstream anusvia anus. The balloon was filled with about0.1ml warm water andconnect to a piece of polyethylene tube, which gave pressure atabout100mmH2O.Pressure in intestinal lumen was measured by atransducer through the thin polyethylene tube and recorded by ADInstruments.
Diarrhea rat model was made by senna solution gavage,0.3g crudedrug/ml,10ml/kg, once a day, for2days.
Constipation rat model was made by gavage0℃normal saline,10ml/kg, once a day, for5days.
Carotid vein were exposed and intubated directly connected withthe mocrosyringe for drug administration．
Pilocarpine3mg/kg was was injected intravenously at rate of0.1ml/min, and acupuncture was performed when the change ofintestinal pressure evoked by the compound was stable.
Methoctramine0.5mg/kg,was injected intravenously at rate of0.1ml/min,and acupuncture was performed when the change ofingestinal pressure declined by the compound was stabile.
Choline chloride10mg/kg,was injected intravenously at rateof0.1ml/min,and acupuncture was performed when the change ofintestinal pressure evoked by the compound was stabile.
4-DAMP1mg/kg,was injected intravenously at rate of0.1ml/min,and acupuncture was performed when the change of ingestinalpressure declined by the compound was stabile.
Sciatic nerve A-fibers was demyelinated by snake venom:Ischiadic nerve were isolated1cm above fibular head inanesthetized rats, trace sample aspirated1ul normal saline,1ulair and1ul0.3%snake venom solution in sequence. Needle Tip were penetrated into sciatic nerve membrane, and the liquid above wasinjected into the myelin sheathin avoidance of drug leakage intothe surrounding tissue and suture of incision.30min after venominjection,acupuncture stimulation were conducted
Sciatic nerve C fibers was inactivated by capsaicin: Sciaticnerve were isolated1cm above fibular head in rats anesthed by0.3%pentobarbital,the separated sciatic nerve trunks were wrapped bya cotton strip immersed in2%solution of capsaicin for24hours.
Acupoints: LI11(quchi), located in the midpoint between thelateral end of the transverse cubical crease and the lateralepicondyle of the humerus, inserting to a depth of about4mm;ST25(tianshu), on the level of the navel,2mm lateral to anteriormedian line, inserting to a depth of about5mm; ST37(shangjuxu),5mm laterl to the anterior tubercle of the tibia and15mm belowThe knee joint,inserting to a depth of about5mm; BL25(dachangshu),in the waist, under the fourth lumbar spines,5mm lateral toposterior midline, inserting to a depth of about4mm. The needlewas rotated clockwise and anti-clockwise at2Hz for60s at eachpoint.
Statistical analysis
Statistical analysis was performed with SPSS software forWindows, version19.0.All data were expressed as a mean SD. Meanvalues were compared using the paired t test to compare theintestinal motility before and after acupuncture. The differencewas considered significant at P<0.05.
Results
1Effect of acupuncture on intestinal motility in normal rats
1.1Effect of acupuncture on jejunum motility in normal rats
Acupuncture at either IL11or ST37significantly increased theamplitude of jejunum waveform （P<0.05， P<0.01）,whereas theacupuncture at ST25significantly decreased jejunum motility（P<0.01）.BL25had no significant effect on the motility ofjejunum(P>0.05).
1.2Effect of acupuncture on the distal colon motility in normalrats
Effects of single points on the distal colon motility:Acupuncture at IL11ST37, ST25or BL25accelerated diatal colonicmotility compared to that of baseline（P<0.05）.
2. Effect of acupuncture on intestinal motility in diarrhea rats.
2.1Effect of acupuncture on jejunum motility in diarrhea rats.
The mean amplitude of jejunum motility during acupuncture atIL11were significantly increased compared to baseline(P<0.05).Whereas acupuncture at ST25significantly decreased amplitude andfrequency of jejunum motility(P<0.01).There were no effect ofacupuncture at ST37、BL25on jejunum motility(P>0.05).
2.2Effect of acupuncture on the distal colon motility in diarrhearats.
Acupuncture at IL11incresed significantly the amplitude of thedistal colon (P<0.05). Both ST37and BL25acclecrated the frequencyand amplitude.The frequency but not the amplitude was increased byacupuncture at ST25(P<0.01).
3Effect of acupuncture on the intestinal motility in constipationrats.
3.1Effect of acupuncture on the jejunum motility in constipationrats.
Acupuncture at IL11and ST37increaded jejunum motility amplitude respectively (P<0.05,P<0.01).Acupuncture at ST25decreased the frequency and amplitude of jejunum (P<0.01). Therewere no obvious change during acupuncture at BL25(P>0.05).
3.2Effect of acupuncture on the distal colon motility inconstipation rats.
Acupuncture at IL11accelerated amplitude but not frequency (P<0.05). Acupuncture at ST37, BL25increased both amplitude andfrequency of colon motility (P<0.05). Acupuncture ST25acceleratedboth amplitude and frequency of colon motility（P<0.01，P<0.01）.
4. Effect of acupuncture on jejunum motility in hyperfunction andhypofunction condition
4.1Effect of acupuncture on jejunum motility after pilocarpineadministration
After M2receptor agonist Pilocarpine administration,theamplitude of jejunum increased(P<0.01), in hyperfunction. Therewere no obvious change during acupuncture atIL11,BL25,ST37, BL25(P>0.05).
4.2. Effect of acupuncture on jejunum motility after methoctramineadministration
After M2receptor antagonist Methoctramine administration,both amplitude and frequency were decreased (P<0.01, P<0.01) inhypofunction. Acupuncture at both IL11and BL25increased theamplitude (P<0.05), Acupuncture at ST37accelerated frequency andamplitude compared with that before (P<0.05, P<0.01).while, ST25decreased frequency and amplitude (P<0.01, P<0.01).
Methoctramine has inhibition on the movement of jejunumAcupuncture at IL11,ST37or BL25has antagonist like effect to thedrug,however the effect of ST25is still characterized by inhibition of the jejunum movement
4.3. Effect of acupuncture on the distal motility after Cholinechloride administrationAfter M3receptor agonist Choline chloride administration, theamplitude of colon motility increased(P<0.01),in hyperfunction.There were no obvious change during acupuncture at IL11,BL25,ST37,BL25(P>0.05).
4.4. Effect of acupuncture on the distal motility after4-DAMPadministration
After M3receptor antagonist4-DAMP administration, bothamplitude and frequency of colon motility were decreased (P<0.01,P<0.01),in hypofunction.Acupuncture at IL increased the amplitudeof colon motility （P<0.05）,Acupuncture at ST37,ST25or BL25increased both frequency and amplitude（P<0.05）.Acupuncture atIL11,ST37,ST25or BL25has antagonist effect to4-DAMP on colonmotility.
5The role of different somatic afferent fibers in acupunctureregulation of intestinal motility
5.1The role of A fibers in acupuncture regulation of intestinalmotility
The stimulation of acupuncture at ST37increased both jejunumand the disatal colon motitlity. After demyelination of Aδ fiber,the excitatory effect is less than that in normal condition.5.2The role of C fibers in acupuncture regulation intestinalmotility
After inactivation C fiber by capsaicin, acupuncture at ST37had no obvious effect on the jejunum motility and the disatal colonmotitlity. C fiber plays a major role in the regualtion of intestinal motility by acupuncture.
Conclusion
(1) Acupuncture at heterotopic acupoints has excitatoryeffects on the intestinal motility, which induced by activation ofvagal nerves.Though the effect of acupuncture is related closelyto states of intestinal motility, the direction of intestinalresponse can not been changed by acupuncture effect.
(2) Acupuncture at homotopic acupoint ST25always producesinhibition on the intestinal motility, independent of the state ofintestinal motility.
(3) The effect of acupuncture at ST25on diarrhea andconstipation demonstrates that the neural dynamic mechanism liesin the inhibition of jejunum movement through sympathetic tone andpromotion the distal colon movement through parasympathetic tone.
(4) The somatic afferent nerve fibers A and C are involved inthe regulation of acupuncture on intestinal motility, and C fibersplays an more important role.
There is complexity to understand the conception of bidirectonregulation of acupuncture. Our study shows that for functionalbowel diseases, the bilateral regulation of acupuncture reflectedsynthetically by different states of intestinal motility and thelocation of acupoints. The effect of acupuncture at heterotopicacupoints on intestinal motility is effected by intestine state,but the response direction to acupuncture can not be changed. Theinhibitory effects of acupuncture at homotopic acupoint ST25onjejnum motility is independent of bowel movement.
The present study suggests that acupoints selection principlesin treating fuctional bowel movement should be a combination of the state of the bowel movement and location of acupoints.
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