针刺足三里穴、曲池穴对腹泻大鼠模型结肠运动调节的中枢机制研究
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摘要
目的
经脉-脏腑相关理论是针灸学理论体系的重要内容之一，《灵枢·海论》载“夫十二经脉者，内属于府藏，外络于支节”；合穴是特定穴之一，对治疗六腑病起重要作用，《灵枢·邪气脏腑病形》载：“合治内府”。根据经脉-脏腑相关和“合治内府”理论，选取合穴和下合穴治疗六腑病是针灸临床常用的经典方法。足三里是胃的合穴和下合穴，因“大肠小肠皆属于胃”，故足三里在针灸临床治疗胃肠病证上起重要作用。曲池是手阳明大肠经的合穴，针灸临床常用于治疗大肠病证。现代针灸研究对针刺足三里，曲池(尤其是足三里)调节胃肠功能的机制探讨较为深入，特别对其作用的中枢机制探讨近年来有了很大的进展。本研究是在这些研究的基础上，探讨足三里、曲池对结肠运动调节是否与中枢神经递质有关，特别是与迷走-迷走反射的中枢的神经递质谷氨酸和r-胺基丁酸的关系，并了解足三里和曲池对结肠运动调节的差异性。目前尚未见此类研究报道。
方法
选用SD雄性大鼠，随机分为2组：正常对照组和腹泻模型组。腹泻模型采用灌肠注入乙酸的方法，而对照组应用生理盐水灌肠。所有实验在灌肠后第二天进行。灌肠第二天检测结肠髓过氧化物酶活性增加进而确认结肠炎症程度。该实验应用自行设计开发的局部灌流浴槽来记录在体大鼠结肠纵型肌运动。电针治疗分别取足阳明胃经足三里穴和手阳明大肠经曲池穴进行电针治疗30分钟来观测针刺的治疗作用。为进一步探讨针刺对腹泻治疗的作用，设计应用两次灌流方法，即用M_2受体激动剂局部结肠灌流来模拟腹泻时大量GMC(巨大移行性收缩)所造成的结肠剧烈收缩的环境，从而来观察针刺对结肠运动调节的中枢机制。为深入研究迷走-迷走反射通路，TTX应用于灌流以阻断结肠剧烈收缩时的迷走传入。用柱前衍生-电化学检测高压液相色谱(HPLC)法分析测定针刺足三里穴对脑干Glu和GABA递质水平的影响用于探索针刺的中枢作用机制。
结果
1．实验证明应用整体小动物的同时采用在体局部灌流的杂交方法来研究局部结肠运动的可行性和科学性。该实验既保留了小动物的整体结构，同时又应用了离体灌流可以精确控制研究条件的双重优势。
2．在乙酸引起结肠粘膜炎症的大鼠腹泻模型可见大便性状改变和大便次数增加，灌肠第二天检测结肠髓过氧化物酶活性增加(P＜0.01)。
3．本次实验首次报道在体炎性近端结肠活动的改变。其特点是炎症时结肠自率性收缩活动减低(p＜0.05)，但对激动剂有超敏反应(p＜0.05)，而且偶尔可见类似GMC的表现。
4．电针足三里可增加对照组和腹泻模型组结肠自率性运动的幅度和基础张力(p＜0.05)，与对照组相比电针足三里对腹泻模型组自率性运动有更强的作用(p＜0.05)，而单用电针曲池穴或非穴点则没有任何作用。
5．电针前侧脑室微量注射谷氨酸受体拮抗剂犬尿氨酸，可明显减低电针足三里的作用(p＜0.05)，而用同样方法注射人工脑脊液则没有任何作用。
6．炎性结肠模型动物的结肠对M_2受体激动剂有比正常对照组较强的收缩反应(p＜0.05)。
7．针刺足三里和曲池穴没有影响M_2受体激动剂对正常对照组和炎性结肠模型的结肠收缩反应的首次局部灌流的激动作用(P＞0.05)，但可明显减小第二次M_2受体激动剂的激动的作用(p＜0.01)，而在炎性结肠模型表现更为明显。
8．事先局部灌流TTX用以阻断结肠局部神经传导作用，可见第二次M_2受体激动剂的激动减小的作用消失(p＜0.01)。
9．用GABA受体阻断剂首先注入侧脑室，可见针刺足三里和曲池穴使第二次局部灌流M_2受体激动剂的激动作用减小的程度部分阻断(p＜0.05)，但没有能完全阻断减小第二次M_2受体激动剂的激动作用。说明还有另外的中枢传导通路参入。
10．电针针刺足三里穴可增加对照组和腹泻模型组大鼠脑干兴奋性神经递质谷氨酸浓度(P＜0.05)，应用M_2受体模拟GMC后用针刺曲池加电针足三里穴可见加对照组和腹泻模型组大鼠脑干抑制性递质γ-氨基丁酸增加(p＜0.05)。而当没有GMC刺激时，针刺曲池，足三里穴并没有见这种显著性的变化(P＞0.05)。
结论
1．该实验证明应用整体小动物的同时采用在体局部灌流的杂交方法来研究局部结肠运动的可行性和科学性。该方法可记录近端结肠自主收缩活动的同时，还可应用灌流手段来研究肌肉组织对神经介质的反应性。为针刺整体小动物实验的机理研究和经络与脏腑间的关系及作用机制研究以及与现代医学的神经递质、神经介质和体液的研究开启了一扇大门。同时从用药量考虑更是经济有效。
2．炎性近端结肠活动的特点是炎症时结肠自率性收缩活动减低但对激动剂有超敏反应，而且偶尔可见类似GMC的表现。
3．针刺手阳明大肠经的曲池穴并没有明显改变大肠运动的作用，但针刺足阳明胃经的足三里后可增加大肠运动尤其对腹泻模型动物大肠运动的作用明显。主要影响结肠运动三种不同的形式中的节率性振幅式(Phasic)收缩和基础肌收缩张力(Tonic Contraction)。如果电针足三里同时针刺曲池穴可增加单用电针足三里穴的作用。可见足三里穴的作用虽然重要，但依症配穴可增加疗效，其作用与中枢谷氨酸浓度增加有关。
4．巨大移行性收缩(Giant Migrating Contraction，GMC)引起强烈结肠收缩的刺激由肠道壁裸露的神经末梢通过传入神经到中枢而通过若干中枢传导通路来调节结肠收缩的过强反应包括增加中枢GABA递质的释放作用。结肠炎性环境可增强这一作用。
5．针刺足三里穴可增加大鼠脑干谷氨酸的浓度不需要其它的介导条件，而针刺手阳明大肠经的曲池穴没有此作用，针刺足三里穴增加大鼠脑干γ-氨基丁酸的浓度需要GMC强烈收缩刺激传入介导或易化作用，而且结肠炎症的改变可增强这种中枢的调节反应，可能与炎性反应递质或免疫因子的改变有关。这个结果可从中枢神经递质的调节来解释针刺足三里穴即可调节便秘又可治疗腹泻的双相作用。特别调节胃肠运动的迷走-迷走反射的中枢的神经核团位于脑干，推测针刺足三里调节胃肠运动与迷走-迷走反射中枢有关。
6．针刺足三里和曲池穴对结肠腹泻抑制作用可能依赖于肠道壁神经末梢通过传入信息来易化中枢对针刺足三里和曲池穴的作用，因为用TTX阻断这一传入作用而使针刺足三里和曲池穴作用消失。这一点为针刺足三里穴的双相调整作用提供了一定的理论依据。虽然本实验证实了针刺足三里和曲池穴对炎性结肠的调节作用与中枢GABA递质有关，但较为完整的机制还有待进一步研究。
Objective
The theory of meridian-organ relationships is a core in Traditional Chinese Medicine (TCM). There are more classical books of TCM emphasize the relationship of organs issues with the 12 meridians. The goal of this study was to investigate the effects of acupuncture points on colonic contraction using an inflammation rat model. There are many papers reported that "Zu San Li",ST36, it is the most effective acupuncture point to treat the both constipation patients or diarrhea patients in the TCM system. However, the mechanisms of those treatments on using acupuncture are still not clear. In this study the regulation mechanisms in central never system was investigated especially to the vago-vagal reflex related transmitters like glutamate and GABA.
Methods
There were 56 male Sprague-Dawley rats have been used in this study. The animals had been randomly divided into two groups, diarrhea model group and control group. Colitis was induced by intracolonic instillation of 4% acetic acid in diarrhea model group and the control group was using same method by intracolonic instillation of normal salt solution. The extent of inflammation was assessed by histological examination and myeloperoxidase (MPO) activity assay .The experiments were conducted in the 2nd day of intracolonic instillation. A new localized perfusion recoding method for in-vivo colonic contraction was developed in this study. Electrical acupuncture on ST36 and manually acupuncture LI11 (Qu Chi) were used in this study for either 30 min or 15 min. Also a M2 agonist induced GMC has been investigated in this study as well. For studying on effect of vagal afferent function in vago-vagal reflexes TTX has been used in the perfusion solution. Also the intra ventricle microinject has been used to apply the antagonists of glutamate or GABA, and HPLC has been used in this study to measure the concentrations of glutamate and GABA in the brain steam.
Results
1.The recording of colonic contruction in vivo with localized chamber perfusion is a new and effective way on the colonic motility researches especially for the small animal like rats.
2.In the 2nd day of intracolonic instillation, the inflamed model group revealed the diarrhea and their MPO increased the inflamed model group (p<0.01)
3.In the inflamed the proximal rat colon, the phasic contraction and tonic contraction were depressed (p<0.05) with a super sensitivity to agonist agents, and sometime the GMC contraction pattern was revealed.
4.Electrical acupuncture ST36 increased the colonic contraction for both control (P<0.05) and diarrhea (P<0.05) groups, but the effects was more stronger to diarrhea group (p<0.05) than control group. Acupuncture LI11 did not show the some results.
5.Before acupuncture ST36, microinject kynurenic acid, a glutamate antagonist, in brain ventricle can decrease (p<0.05) the effects of acupuncture ST36.
6.In diarrhea group, the reaction to M2 agonist was increased then control group (p<0.05).
7.using twice M2 agonist perfusion method to simulate the GMC attacks in diarrhea, acupuncture ST36 and LI11 did not affect the first time M2 agonist perfusion induced colonic GMC's like contractions, but decreased the following M2 agonist perfusion induced colonic contractions (P<0.05) in diarrhea group.
8.Using TTX(10~(-6)M) in the perfusion chamber blocked above effect to the 2nd M2 agonist perfusion to the diarrhea group.
9.Brain ventricle micro injection of Flucybene can partially block decreasing effects of the 2nd M2 agonist perfusion to the diarrhea group (p<0.05).
10.Acupuncture ST36 increased glutamate and after first M2 agonist perfusion using acupuncture ST36 and LI11 increased GABA in brain steam in HPLC tastings.
Conclusion
1.The recording of colonic contruction in vivo with localized chamber perfusion is a new and effective way on the colonic motility researches especial for the small animal like rats.
2.In the inflamed the proximal rat colon, the phasic contraction and tonic contraction were depressed and sometime the GMC contraction pattern was revealed.
3.Acupuncture LI11 did not affect the colonic contraction, but acupuncture ST36 increased the colonic contraction (p<0.05) especial in the diarrhea group and adding LI11 increased the effects (p<0.05), it indicated that group using acupuncture points will be more effective to the treatments.
4.The strong contraction in GMT through the GI wall sensor and vagal afferent fiber to sending the information into central never system, so that GABA increased to regulate colonic contraction and colitis can enforce this effects.
5.Acupuncture ST36 increases colonic contractions but no effect by acupuncture LI11 along. Acupuncture ST36 increases glutamate in the brain stream that does not need any other mediators, but acupuncture ST36 increases GABA in the brain steam seems require other mediators. These results might explain the acupuncture ST36 can have dual effects on the GI motilities.
6.Acupuncture ST36 and LI11 to treat diarrhea could be by the vagal afferent information from strong GMC in the colon to facilitate the central never system to initiate the regulation to the colonic motility by increasing GABA, and colitis condition might be required for this change or as a helper for this central never regulation to the colonic motility. Even this study indicates the central never system mechanisms of acupuncture, but more detail investigation is still need to fully understand the functions of acupuncture treatments to GI motility.
经脉-脏腑相关理论是针灸学理论体系的重要内容之一，《灵枢·海论》载“夫十二经脉者，内属于府藏，外络于支节”；合穴是特定穴之一，对治疗六腑病起重要作用，《灵枢·邪气脏腑病形》载：“合治内府”。根据经脉-脏腑相关和“合治内府”理论，选取合穴和下合穴治疗六腑病是针灸临床常用的经典方法。足三里是胃的合穴和下合穴，因“大肠小肠皆属于胃”，故足三里在针灸临床治疗胃肠病证上起重要作用。曲池是手阳明大肠经的合穴，针灸临床常用于治疗大肠病证。现代针灸研究对针刺足三里，曲池(尤其是足三里)调节胃肠功能的机制探讨较为深入，特别对其作用的中枢机制探讨近年来有了很大的进展。本研究是在这些研究的基础上，探讨足三里、曲池对结肠运动调节是否与中枢神经递质有关，特别是与迷走-迷走反射的中枢的神经递质谷氨酸和r-胺基丁酸的关系，并了解足三里和曲池对结肠运动调节的差异性。目前尚未见此类研究报道。
方法
选用SD雄性大鼠，随机分为2组：正常对照组和腹泻模型组。腹泻模型采用灌肠注入乙酸的方法，而对照组应用生理盐水灌肠。所有实验在灌肠后第二天进行。灌肠第二天检测结肠髓过氧化物酶活性增加进而确认结肠炎症程度。该实验应用自行设计开发的局部灌流浴槽来记录在体大鼠结肠纵型肌运动。电针治疗分别取足阳明胃经足三里穴和手阳明大肠经曲池穴进行电针治疗30分钟来观测针刺的治疗作用。为进一步探讨针刺对腹泻治疗的作用，设计应用两次灌流方法，即用M_2受体激动剂局部结肠灌流来模拟腹泻时大量GMC(巨大移行性收缩)所造成的结肠剧烈收缩的环境，从而来观察针刺对结肠运动调节的中枢机制。为深入研究迷走-迷走反射通路，TTX应用于灌流以阻断结肠剧烈收缩时的迷走传入。用柱前衍生-电化学检测高压液相色谱(HPLC)法分析测定针刺足三里穴对脑干Glu和GABA递质水平的影响用于探索针刺的中枢作用机制。
结果
1．实验证明应用整体小动物的同时采用在体局部灌流的杂交方法来研究局部结肠运动的可行性和科学性。该实验既保留了小动物的整体结构，同时又应用了离体灌流可以精确控制研究条件的双重优势。
2．在乙酸引起结肠粘膜炎症的大鼠腹泻模型可见大便性状改变和大便次数增加，灌肠第二天检测结肠髓过氧化物酶活性增加(P＜0.01)。
3．本次实验首次报道在体炎性近端结肠活动的改变。其特点是炎症时结肠自率性收缩活动减低(p＜0.05)，但对激动剂有超敏反应(p＜0.05)，而且偶尔可见类似GMC的表现。
4．电针足三里可增加对照组和腹泻模型组结肠自率性运动的幅度和基础张力(p＜0.05)，与对照组相比电针足三里对腹泻模型组自率性运动有更强的作用(p＜0.05)，而单用电针曲池穴或非穴点则没有任何作用。
5．电针前侧脑室微量注射谷氨酸受体拮抗剂犬尿氨酸，可明显减低电针足三里的作用(p＜0.05)，而用同样方法注射人工脑脊液则没有任何作用。
6．炎性结肠模型动物的结肠对M_2受体激动剂有比正常对照组较强的收缩反应(p＜0.05)。
7．针刺足三里和曲池穴没有影响M_2受体激动剂对正常对照组和炎性结肠模型的结肠收缩反应的首次局部灌流的激动作用(P＞0.05)，但可明显减小第二次M_2受体激动剂的激动的作用(p＜0.01)，而在炎性结肠模型表现更为明显。
8．事先局部灌流TTX用以阻断结肠局部神经传导作用，可见第二次M_2受体激动剂的激动减小的作用消失(p＜0.01)。
9．用GABA受体阻断剂首先注入侧脑室，可见针刺足三里和曲池穴使第二次局部灌流M_2受体激动剂的激动作用减小的程度部分阻断(p＜0.05)，但没有能完全阻断减小第二次M_2受体激动剂的激动作用。说明还有另外的中枢传导通路参入。
10．电针针刺足三里穴可增加对照组和腹泻模型组大鼠脑干兴奋性神经递质谷氨酸浓度(P＜0.05)，应用M_2受体模拟GMC后用针刺曲池加电针足三里穴可见加对照组和腹泻模型组大鼠脑干抑制性递质γ-氨基丁酸增加(p＜0.05)。而当没有GMC刺激时，针刺曲池，足三里穴并没有见这种显著性的变化(P＞0.05)。
结论
1．该实验证明应用整体小动物的同时采用在体局部灌流的杂交方法来研究局部结肠运动的可行性和科学性。该方法可记录近端结肠自主收缩活动的同时，还可应用灌流手段来研究肌肉组织对神经介质的反应性。为针刺整体小动物实验的机理研究和经络与脏腑间的关系及作用机制研究以及与现代医学的神经递质、神经介质和体液的研究开启了一扇大门。同时从用药量考虑更是经济有效。
2．炎性近端结肠活动的特点是炎症时结肠自率性收缩活动减低但对激动剂有超敏反应，而且偶尔可见类似GMC的表现。
3．针刺手阳明大肠经的曲池穴并没有明显改变大肠运动的作用，但针刺足阳明胃经的足三里后可增加大肠运动尤其对腹泻模型动物大肠运动的作用明显。主要影响结肠运动三种不同的形式中的节率性振幅式(Phasic)收缩和基础肌收缩张力(Tonic Contraction)。如果电针足三里同时针刺曲池穴可增加单用电针足三里穴的作用。可见足三里穴的作用虽然重要，但依症配穴可增加疗效，其作用与中枢谷氨酸浓度增加有关。
4．巨大移行性收缩(Giant Migrating Contraction，GMC)引起强烈结肠收缩的刺激由肠道壁裸露的神经末梢通过传入神经到中枢而通过若干中枢传导通路来调节结肠收缩的过强反应包括增加中枢GABA递质的释放作用。结肠炎性环境可增强这一作用。
5．针刺足三里穴可增加大鼠脑干谷氨酸的浓度不需要其它的介导条件，而针刺手阳明大肠经的曲池穴没有此作用，针刺足三里穴增加大鼠脑干γ-氨基丁酸的浓度需要GMC强烈收缩刺激传入介导或易化作用，而且结肠炎症的改变可增强这种中枢的调节反应，可能与炎性反应递质或免疫因子的改变有关。这个结果可从中枢神经递质的调节来解释针刺足三里穴即可调节便秘又可治疗腹泻的双相作用。特别调节胃肠运动的迷走-迷走反射的中枢的神经核团位于脑干，推测针刺足三里调节胃肠运动与迷走-迷走反射中枢有关。
6．针刺足三里和曲池穴对结肠腹泻抑制作用可能依赖于肠道壁神经末梢通过传入信息来易化中枢对针刺足三里和曲池穴的作用，因为用TTX阻断这一传入作用而使针刺足三里和曲池穴作用消失。这一点为针刺足三里穴的双相调整作用提供了一定的理论依据。虽然本实验证实了针刺足三里和曲池穴对炎性结肠的调节作用与中枢GABA递质有关，但较为完整的机制还有待进一步研究。
Objective
The theory of meridian-organ relationships is a core in Traditional Chinese Medicine (TCM). There are more classical books of TCM emphasize the relationship of organs issues with the 12 meridians. The goal of this study was to investigate the effects of acupuncture points on colonic contraction using an inflammation rat model. There are many papers reported that "Zu San Li",ST36, it is the most effective acupuncture point to treat the both constipation patients or diarrhea patients in the TCM system. However, the mechanisms of those treatments on using acupuncture are still not clear. In this study the regulation mechanisms in central never system was investigated especially to the vago-vagal reflex related transmitters like glutamate and GABA.
Methods
There were 56 male Sprague-Dawley rats have been used in this study. The animals had been randomly divided into two groups, diarrhea model group and control group. Colitis was induced by intracolonic instillation of 4% acetic acid in diarrhea model group and the control group was using same method by intracolonic instillation of normal salt solution. The extent of inflammation was assessed by histological examination and myeloperoxidase (MPO) activity assay .The experiments were conducted in the 2nd day of intracolonic instillation. A new localized perfusion recoding method for in-vivo colonic contraction was developed in this study. Electrical acupuncture on ST36 and manually acupuncture LI11 (Qu Chi) were used in this study for either 30 min or 15 min. Also a M2 agonist induced GMC has been investigated in this study as well. For studying on effect of vagal afferent function in vago-vagal reflexes TTX has been used in the perfusion solution. Also the intra ventricle microinject has been used to apply the antagonists of glutamate or GABA, and HPLC has been used in this study to measure the concentrations of glutamate and GABA in the brain steam.
Results
1.The recording of colonic contruction in vivo with localized chamber perfusion is a new and effective way on the colonic motility researches especially for the small animal like rats.
2.In the 2nd day of intracolonic instillation, the inflamed model group revealed the diarrhea and their MPO increased the inflamed model group (p<0.01)
3.In the inflamed the proximal rat colon, the phasic contraction and tonic contraction were depressed (p<0.05) with a super sensitivity to agonist agents, and sometime the GMC contraction pattern was revealed.
4.Electrical acupuncture ST36 increased the colonic contraction for both control (P<0.05) and diarrhea (P<0.05) groups, but the effects was more stronger to diarrhea group (p<0.05) than control group. Acupuncture LI11 did not show the some results.
5.Before acupuncture ST36, microinject kynurenic acid, a glutamate antagonist, in brain ventricle can decrease (p<0.05) the effects of acupuncture ST36.
6.In diarrhea group, the reaction to M2 agonist was increased then control group (p<0.05).
7.using twice M2 agonist perfusion method to simulate the GMC attacks in diarrhea, acupuncture ST36 and LI11 did not affect the first time M2 agonist perfusion induced colonic GMC's like contractions, but decreased the following M2 agonist perfusion induced colonic contractions (P<0.05) in diarrhea group.
8.Using TTX(10~(-6)M) in the perfusion chamber blocked above effect to the 2nd M2 agonist perfusion to the diarrhea group.
9.Brain ventricle micro injection of Flucybene can partially block decreasing effects of the 2nd M2 agonist perfusion to the diarrhea group (p<0.05).
10.Acupuncture ST36 increased glutamate and after first M2 agonist perfusion using acupuncture ST36 and LI11 increased GABA in brain steam in HPLC tastings.
Conclusion
1.The recording of colonic contruction in vivo with localized chamber perfusion is a new and effective way on the colonic motility researches especial for the small animal like rats.
2.In the inflamed the proximal rat colon, the phasic contraction and tonic contraction were depressed and sometime the GMC contraction pattern was revealed.
3.Acupuncture LI11 did not affect the colonic contraction, but acupuncture ST36 increased the colonic contraction (p<0.05) especial in the diarrhea group and adding LI11 increased the effects (p<0.05), it indicated that group using acupuncture points will be more effective to the treatments.
4.The strong contraction in GMT through the GI wall sensor and vagal afferent fiber to sending the information into central never system, so that GABA increased to regulate colonic contraction and colitis can enforce this effects.
5.Acupuncture ST36 increases colonic contractions but no effect by acupuncture LI11 along. Acupuncture ST36 increases glutamate in the brain stream that does not need any other mediators, but acupuncture ST36 increases GABA in the brain steam seems require other mediators. These results might explain the acupuncture ST36 can have dual effects on the GI motilities.
6.Acupuncture ST36 and LI11 to treat diarrhea could be by the vagal afferent information from strong GMC in the colon to facilitate the central never system to initiate the regulation to the colonic motility by increasing GABA, and colitis condition might be required for this change or as a helper for this central never regulation to the colonic motility. Even this study indicates the central never system mechanisms of acupuncture, but more detail investigation is still need to fully understand the functions of acupuncture treatments to GI motility.
引文
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[14]卢刚，余承高．侧脑室内注射微量血管紧张素Ⅱ对大鼠胃和十二指肠运动的影响．同济医科大学学报，1991，20(11)：25-27．
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