【作者】 崔常香；

【导师】 朱兵；

【作者基本信息】 湖北中医药大学 ， 针灸推拿学， 2014， 博士

【摘要】 目的：针刺疗法适用于临床多种疾病，其中包括消化道胃肠功能紊乱。大量的实验和临床证据表明：针刺在治疗功能性肠病方面具有独到的疗效与优势。功能性肠病临床分为腹泻与便秘两种疾病，这两种疾病在临床治疗选穴时存在用穴交叉和混乱的状况，本实验的目的旨在明确临床选穴，为临床治疗疾病准确选穴提供进一步的指导。尽管目前已经有大量关于针刺治疗肠道疾病的研究，之前也有针刺对麻醉大鼠胃运动刺激效应的相关研究，但是关于胃肠运动调节方面针刺对自主神经功能调节作用的相关研究并不多见，而关于针刺起效的传入机制以及效应方式仍在研究当中。此外，在体同时记录小鼠空肠和结肠运动的实验研究也不多见，针刺作用到底是通过何种传入机制起作用以及针刺作用的效应受体为何仍不清楚。本研究采用肠道内水囊测压的方法观察针刺曲池、上巨虚、天枢及大肠俞四穴对正常及特定基因敲除小鼠空肠和远端结肠运动的影响，从而探讨针刺作用的外周感受器传入机制和效应器官受体调控机制。材料与方法：1.实验方案C57BL/6小鼠20只，TRPV1外周感受器基因敲除（TRPV1-/-）小鼠、ASIC3外周感受器基因敲除（ASIC3-/-）小鼠及效应受体M2&3基因敲除（M2&3-/-）小鼠各10只，体重20±2g，禁食不禁水24小时后，10%乌来糖溶液（乌来糖1-1.2g/kg）腹腔麻醉。小鼠取仰卧位，腹正中线剑突下切口，在幽门下5厘米左右的空肠处和肛门上2厘米左右的远端结肠处，分别放置直径为3毫米的水囊，水囊通过导管与压力换能器相连，用Micro1401和Spike27.03数据采集软件记录肠道运动的信号。2.穴位定位和针刺上巨虚穴（ST37）：小鼠胫骨外侧偏下方6毫米处，直刺5毫米达到肌层；曲池穴（LI11）：小鼠桡骨近端肘关节外侧前方的凹陷处，直刺3毫米；天枢穴（ST25）:小鼠前正中线旁开5毫米，耻骨联合以上20毫米处，直刺2毫米；大肠俞穴（BL25）：在腰部，当第4腰椎棘突下，旁开5毫米，直刺4毫米。针刺肢体一侧单穴，进行提插捻转的操作，刺激频率为2Hz，持续60秒。3．数据采集及分析采用Spike27.03信号采集分析系统，对小鼠的肠道内压力信号进行采集与分析。实验开始后，首先记录稳定的小鼠肠道内压力曲线60秒，然后穴位针刺60秒同时观察肠道内压力曲线的变化，比较针刺前后60秒压力曲线幅值及曲线下面积平均值的变化。使用SPSS17.0统计软件对针刺前、后60秒压力曲线幅值的平均值及曲线下面积平均值进行自身配对t检验，统计资料以均数±标准差（±SD）表示，P＜0.05认为具有显著性差异，P＜0.01认为具有非常显著性差异。结果：1.针刺不同腧穴对C57BL/6小鼠及TRPV1外周感受器基因敲（TRPV1-/-）小鼠（以下简称TRPV1KO小鼠）肠道运动的影响1.1针刺不同腧穴对C57BL/6小鼠肠道运动的影响1.1.1空肠运动手针刺激LI1160秒，明显促进了C57BL/6小鼠的空肠运动。C57BL/6小鼠空肠压力曲线的平均振幅和平均曲线下面积与针刺前相比均有不同程度的增加，P＜0.05；而频率与针刺前相比则没有发生显著变化，P＞0.05。手针刺激ST3760秒，明显促进了C57BL/6小鼠的空肠运动。C57BL/6小鼠空肠运动压力曲线的平均振幅和平均曲线下面积与针刺前相比均有显著增加，P＜0.05；而其空肠运动压力曲线的平均频率与针刺前相比则没有发生明显变化，P＞0.05。手针刺激ST2560秒，明显抑制了C57BL/6小鼠的空肠运动。C57BL/6小鼠空肠运动压力曲线的平均频率、平均振幅以及平均曲线下面积与针刺前相比均有显著降低，P＜0.05。手针刺激BL2560秒，对C57BL/6小鼠的空肠运动无明显作用，空肠运动压力曲线的平均频率、平均振幅以及平均曲线下面积与针刺前相比均没有明显变化，P＜0.05。1.1.2远端结肠运动手针刺激LI1160秒，明显促进了C57BL/6小鼠的远端结肠运动。C57BL/6小鼠远端结肠压力曲线的平均振幅和平均曲线下面积与针刺前相比均有显著增加，P＜0.05；而其远端结肠运动压力曲线的平均频率与针刺前相比则没有发生明显变化，P＞0.05。手针刺激ST3760秒，明显促进了C57BL/6小鼠的远端结肠运动。C57BL/6小鼠远端结肠压力曲线的运动平均振幅和平均曲线下面积与针刺前相比均有显著增加，P＜0.05；而其远端结肠压力曲线的运动频率与针刺前相比则没有发生明显变化，P＞0.05。手针刺激ST2560秒，明显促进了C57BL/6小鼠的远端结肠运动。C57BL/6小鼠远端结肠压力曲线的运动平均振幅和平均曲线下面积与针刺前相比均有显著增加，P＜0.05；而其远端结肠压力曲线的平均频率与针刺前相比则没有发生明显变化，P＞0.05。手针刺激BL2560秒，明显促进了C57BL/6小鼠的远端结肠运动。C57BL/6小鼠远端结肠压力曲线的运动平均振幅和平均曲线下面积与针刺前相比均有显著增加，P＜0.05；而其远端结肠压力曲线的平均频率与针刺前相比则没有发生明显变化，P＞0.05。1.2针刺不同腧穴对TRPV1KO小鼠空肠及远端结肠运动的影响1.2.1空肠运动手针刺激LI1160秒，明显促进了TRPV1KO小鼠的空肠运动。TRPV1KO小鼠空肠运动压力曲线的平均振幅和平均曲线下面积与针刺前相比均有显著增加，P＜0.05；而其空肠运动压力曲线的平均频率与针刺前相比则没有发生明显变化，P＞0.05。手针刺激ST3760秒，明显促进了TRPV1KO小鼠的空肠运动。TRPV1KO小鼠空肠运动压力曲线的平均振幅和平均曲线下面积与针刺前相比均有显著增加，P＜0.05；而其空肠运动压力曲线的平均频率与针刺前相比则没有发生明显变化，P＞0.05。手针刺激ST2560秒，明显抑制了TRPV1KO小鼠的空肠运动。TRPV1KO小鼠空肠运动压力曲线的平均频率、平均振幅以及平均曲线下面积与针刺前相比均有显著降低，P＜0.05。手针刺激BL2560秒，对TRPV1KO小鼠的空肠运动无明显作用。TRPV1KO小鼠空肠运动压力曲线的平均频率、平均振幅以及平均曲线下面积与针刺前相比均没有发生明显变化，P＞0.05。1.2.2远端结肠运动手针刺激LI1160秒，明显促进了TRPV1KO小鼠的远端结肠运动。TRPV1KO小鼠远端结肠运动压力曲线的平均振幅和平均曲线下面积与针刺前相比均有显著增加，P＜0.05；而其远端结肠运动压力曲线的平均频率与针刺前相比则没有发生明显变化，P＞0.05。手针刺激ST3760秒，明显促进了TRPV1KO小鼠的远端结肠运动。TRPV1KO小鼠远端结肠运动压力曲线的平均振幅和平均曲线下面积与针刺前相比均有显著增加，P＜0.05；而其远端结肠压力曲线的运动频率与针刺前相比则没有发生明显变化，P＞0.05。手针刺激ST2560秒，明显促进了TRPV1KO小鼠的远端结肠运动。TRPV1KO小鼠远端结肠运动压力曲线的平均振幅和平均曲线下面积与针刺前相比均显著增加，P＜0.05；而其远端结肠运动压力曲线的平均频率与针刺前相比则没有发生明显变化，P＞0.05。手针刺激BL2560秒，明显促进了TRPV1KO小鼠的远端结肠运动。TRPV1KO小鼠远端结肠压力曲线的平均振幅和平均曲线下面积与针刺前相比均有显著增加，P＜0.05；而其远端结肠运动压力曲线的平均频率与针刺前相比没有发生明显变化，P＞0.05。1.3针刺不同腧穴对C57BL/6小鼠及TRPV1KO小鼠空肠及远端结肠运动的影响的比较在同种系野生小鼠C57BL/6与TRPV1KO小鼠，针刺ST37与ST25对两种小鼠空肠和远端结肠运动的影响区别较为明显。针刺LI11和ST37对TRPV1KO小鼠空肠和远端结肠运动的促进作用均低于同种野生小鼠C57BL/6（即针刺LI11和ST37时，TRPV1KO小鼠空肠及远端结肠运动压力曲线的平均振幅变化率及平均曲线下面积变化率均均低于C57BL/6小鼠相应的变化率，P＜0.05)。针刺ST25对TRPV1KO小鼠空肠运动的抑制作用也低于C57BL/6小鼠（即针刺ST25时，TRPV1KO小鼠空肠运动压力曲线的平均振幅变化率、平均曲线下面积变化率以及平均频率均低于同种系野生鼠C57BL/6小鼠相应的变化率，P＜0.05)；针刺ST25对TRPV1KO小鼠远端结肠运动的促进作用也低于C57BL/6小鼠（即针刺ST25时，TRPV1KO小鼠远端结肠运动压力曲线的平均振幅变化率及平均曲线下面积变化率均低于同种系野生鼠C57BL/6小鼠相应的变化率，P＜0.05）。针刺BL25对TRPV1KO小鼠及同种系野生鼠C57BL/6小鼠空肠运动均无明显作用（即针刺BL25时，两种实验动物的空肠运动压力曲线的平均振幅变化率及平均曲线下面积变化率均无明显差异，P＞0.05）;针刺BL25对TRPV1KO小鼠远端结肠运动的促进作用低于同种系野生鼠C57BL/6小鼠（即针刺BL25时，TRPV1KO小鼠远端结肠运动压力曲线的平均振幅变化率及平均曲线下面积变化率均低于同种系野生鼠C57BL/6小鼠相应的变化率，P＜0.05）。2针刺不同腧穴对ASIC3外周感受器基因敲除（ASIC3-/-）小鼠（以下简称ASIC3KO小鼠）空肠和远端结肠运动的影响2.1空肠运动手针刺激LI1160秒，明显促进了ASIC3KO小鼠的空肠运动。ASIC3KO小鼠空肠运动压力曲线的平均振幅和平均曲线下面积与针刺前相比均有显著增加，P＜0.05；而其空肠运动的平均频率与针刺前相比则没有发生明显变化，P＞0.05。手针刺激ST3760秒，明显促进了ASIC3KO小鼠的空肠运动。ASIC3KO小鼠空肠运动压力曲线的平均振幅和平均曲线下面积与针刺前相比均有显著增加，P＜0.05；而其空肠运动的平均频率与针刺前相比则没有发生明显变化，P＞0.05。手针刺激ST2560秒，明显抑制了ASIC3KO小鼠的空肠运动。ASIC3KO小鼠空肠运动压力曲线的平均频率、平均振幅以及平均曲线下面积与针刺前相比均有显著降低，P＜0.05。手针刺激BL2560秒，对ASIC3KO小鼠的空肠运动无明显作用。ASIC3KO小鼠的空肠运动压力曲线的平均频率、平均振幅以及平均曲线下面积与针刺前相比均没有发生明显变化，P＞0.05。2.2远端结肠运动手针刺激LI1160秒，明显促进了ASIC3KO小鼠的远端结肠运动。ASIC3KO小鼠远端结肠运动压力曲线的平均振幅和平均曲线下面积与针刺前相比均有显著增加，P＜0.05；而其远端结肠运动压力曲线的平均频率与针刺前相比则没有发生明显变化，P＞0.05。手针刺激ST3760秒，明显促进了ASIC3KO小鼠的远端结肠运动。ASIC3KO小鼠远端结肠运动压力曲线的平均振幅和平均曲线下面积与针刺前相比均有显著增加，P＜0.05；而其远端结肠压力曲线的平均频率与针刺前相比则没有发生明显变化，P＞0.05。手针刺激ST2560秒，明显促进了ASIC3KO小鼠的远端结肠运动。ASIC3KO小鼠远端结肠运动压力曲线的平均振幅和平均曲线下面积与针刺前相比均有显著增加，P＜0.05；而其远端结肠压力曲线的平均频率与针刺前相比则没有发生明显变化，P＞0.05。手针刺激BL2560秒，明显促进了ASIC3KO小鼠的远端结肠运动。ASIC3KO小鼠远端结肠运动压力曲线的平均振幅和平均曲线下面积与针刺前相比均有显著增加，P＜0.05；而其远端结肠运动压力曲线的平均频率与针刺前相比则没有发生明显变化，P＞0.05。2.3针刺不同腧穴对C57BL/6小鼠及ASIC3KO小鼠空肠及远端结肠运动的影响的比较在同种系野生小鼠C57BL/6与ASIC3KO小鼠，针刺ST37与ST25对两种小鼠空肠和远端结肠运动的影响区别较为明显。针刺LI11和ST37对ASIC3KO小鼠空肠和远端结肠运动的促进作用均低于同种系野生小鼠C57BL/6（即针刺LI11和ST37时，ASIC3KO小鼠空肠和远端结肠运动压力曲线的平均振幅变化率及平均曲线下面积变化率均低于同种系野生鼠C57BL/6小鼠相应的变化率，P＜0.05）。针刺ST25对ASIC3KO小鼠空肠运动的抑制作用也低于同种系野生小鼠C57BL/6（即针刺ST25时，ASIC3KO小鼠空肠运动压力曲线的平均振幅变化率、平均曲线下面积变化率以及平均频率变化率均低于同种系野生鼠C57BL/6小鼠相应的变化率，P＜0.05），同时对ASIC3KO小鼠远端结肠运动的促进作用也低于同种系野生小鼠C57BL/6（即针刺ST25时，ASIC3KO小鼠远端结肠运动压力曲线的平均振幅变化率及平均曲线下面积变化率均低于同种系野生小鼠C57BL/6相应的变化率，P＜0.05）。针刺BL25对ASIC3KO小鼠及同种系野生鼠C57BL/6小鼠空肠运动均无明显作用（即针刺BL25时，两种实验动物空肠运动压力曲线均无明显差异的平均振幅变化率及平均曲线下面积变化率，P＞0.05），同时对ASIC3KO小鼠远端结肠运动的促进作用低于同种系野生小鼠C57BL/6（即针刺BL25时，ASIC3KO小鼠远端结肠运动压力曲线的平均振幅变化率及平均曲线下面积变化率均低于同种系野生鼠C57BL/6小鼠相应的变化率，P＜0.05）。2.4针刺不同腧穴对C57BL/6小鼠及TRPV1KO、ASIC3KO小鼠空肠及远端结肠运动的影响的比较在同种系野生小鼠C57BL/6与TRPV1KO小鼠与ASIC3KO小鼠，针刺ST37与ST25对三种实验动物空肠及远端结肠运动的影响区别较为明显。针刺LI11和ST37对TRPV1KO小鼠及ASIC3KO小鼠空肠和远端结肠运动的促进作用均低于同种系野生小鼠C57BL/6（即针刺LI11和ST37时，TRPV1KO小鼠及ASIC3KO小鼠空肠和远端结肠运动压力曲线的平均振幅变化率及平均曲线下面积变化率均低于同种系野生鼠C57BL/6小鼠相应的变化率，P＜0.05）；在两种基因敲除动物，针刺LI11和ST37对TRPV1KO小鼠空肠及远端结肠运动的促进作用均低于ASIC3KO小鼠（即针刺LI11和ST37时，TRPV1KO小鼠空肠及远端结肠运动压力曲线的平均振幅变化率及平均曲线下面积变化率均低于ASIC3KO小鼠相应的变化率，P＜0.05）。针刺ST25对TRPV1KO小鼠及ASIC3KO小鼠空肠运动的抑制作用也低于同种系野生小鼠C57BL/6（即针刺ST25时，TRPV1KO小鼠及ASIC3KO小鼠空肠运动压力曲线的平均振幅变化率、平均曲线下面积变化率及平均频率变化率均低于同种系野生鼠C57BL/6小鼠相应的变化率，P＜0.05）；在两种基因敲除动物，针刺ST25对TRPV1KO小鼠空肠运动的抑制作用低于ASIC3KO小鼠（即针刺ST25时，TRPV1KO小鼠空肠运动压力曲线的平均频率变化率、平均振幅变化率及平均曲线下面积变化率均低于ASIC3KO小鼠相应的变化率，P＜0.05）。同时针刺ST25对TRPV1KO小鼠及ASIC3KO小鼠远端结肠运动的促进作用也低于同种系野生小鼠C57BL/6（即针刺ST25时，TRPV1KO小鼠及ASIC3KO小鼠远端结肠运动压力曲线的平均振幅变化率及平均曲线下面积变化率均低于同种系野生鼠C57BL/6小鼠相应的变化率，P＜0.05）；在两种基因敲除动物，针刺ST25对TRPV1KO小鼠远端结肠运动的促进作用低于ASIC3KO小鼠（即针刺ST25时，TRPV1KO小鼠远端结肠运动压力曲线的平均振幅变化率及平均曲线下面积变化率均低于ASIC3KO小鼠相应的变化率，P＜0.05）。针刺BL25对三种小鼠空肠运动均无明显作用（即针刺BL25时，三种实验动物空肠运动压力曲线均无明显差异的平均频率变化率、平均振幅变化率及平均曲线下面积变化率，P＞0.05），同时针刺BL25对TRPV1KO小鼠及ASIC3KO小鼠远端结肠运动的促进作用均低于同种系野生小鼠C57BL/6（即针刺BL25时，TRPV1KO小鼠及ASIC3KO小鼠远端结肠运动压力曲线平均振幅变化率及平均曲线下面积变化率均明显低于同种系野生鼠C57BL/6小鼠相应的变化率，P＜0.05）；在两种基因敲除动物，针刺BL25对TRPV1KO小鼠远端结肠运动的促进作用低于ASIC3KO小鼠（及针刺BL25时，TRPV1KO小鼠远端结肠运动压力曲线平均振幅变化率及平均曲线下面积变化率均低于ASIC3KO小鼠相应的变化率，P＜0.05）。3针刺不同腧穴对M2&3效应受体基因敲除（M2&3-/-）小鼠（以下简称M2&3KO小鼠）空肠及远端结肠运动的影响3.1空肠运动手针刺激LI1160秒，明显促进了M2&3KO小鼠的空肠运动。M2&3KO小鼠空肠运动压力曲线的平均振幅和平均曲线下面积与针刺前相比均有显著增加，P＜0.05；而其空肠运动压力曲线的平均频率与针刺前相比则没有发生明显变化，P＞0.05。手针刺激ST3760秒，明显促进了M2&3KO小鼠的空肠运动。M2&3KO小鼠空肠运动压力曲线的平均振幅和平均曲线下面积与针刺前相比均有显著增加，P＜0.05；而其空肠运动压力曲线的平均频率则没有发生明显变化，P＞0.05。手针刺激ST2560秒，明显抑制了M2&3KO小鼠的空肠运动。M2&3KO小鼠空肠运动压力曲线的平均频率、平均振幅以及平均曲线下面积与针刺前相比均有显著降低，P＜0.05。手针刺激BL2560秒，针刺对M2&3KO小鼠的空肠运动无明显作用。M2&3KO小鼠空肠运动压力曲线的平均频率、平均振幅以及平均曲线下面积与针刺前相比均没有明显变化，P＞0.05。3.2远端结肠运动手针刺激LI1160秒，明显促进了M2&3KO小鼠的远端结肠运动。M2&3KO小鼠远端结肠压力曲线的平均振幅和平均曲线下面积与针刺前相比均有显著增加，P＜0.05；而其远端结肠运动压力曲线的平均频率与针刺前相比则没有发生明显变化，P＞0.05。手针刺激ST3760秒，明显促进了M2&3KO小鼠的远端结肠运动。M2&3KO小鼠远端结肠运动压力曲线的平均振幅和平均曲线下面积与针刺前相比均有显著增加，P＜0.05；而其远端结肠运动压力曲线的平均频率与针刺前相比则没有发生明显变化，P＞0.05。手针刺激ST2560秒，明显促进了M2&3KO小鼠的远端结肠运动。M2&3KO小鼠远端结肠运动压力曲线的平均振幅和平均曲线下面积与针刺前相比均有显著增加，P＜0.05；而其远端结肠运动压力曲线的平均频率与针刺前相比则没有发生明显变化，P＞0.05。手针刺激BL2560秒，明显促进了M2&3KO小鼠的远端结肠运动。M2&3KO小鼠远端结肠运动压力曲线的平均振幅和平均曲线下面积与针刺前相比均有显著增加，P＜0.05；而其远端结肠运动压力曲线的平均频率与针刺前相比则没有发生明显变化，P＞0.05。3.3针刺不同腧穴对C57BL/6小鼠及M2&3KO小鼠空肠及远端结肠运动的影响的比较在同种系野生小鼠C57BL/6，针刺ST37与ST25对两种实验动物空肠及远端结肠运动的影响区别较为明显。针刺LI11和ST37对M2&3KO小鼠空肠和远端结肠运动的促进作用均低于同种系野生小鼠C57BL/6（即针刺LI11和ST37时，M2&3KO小鼠空肠和远端结肠运动压力曲线的平均振幅变化率及平均曲线下面积变化率均低于同种系野生鼠C57BL/6小鼠相应的变化率，P＜0.05）。针刺ST25对M2&3KO小鼠空肠运动的抑制作用也低于同种系野生小鼠C57BL/6（即针刺ST25时，M2&3KO小鼠空肠运动压力曲线的平均振幅变化率、平均曲线下面积变化率以及平均频率变化率均低于同种系野生鼠C57BL/6小鼠相应的变化率，P＜0.05）。同时对M2&3KO小鼠远端结肠运动的次进组也低于同种系野生小鼠C57BL/6（即针刺ST25时，M2&3KO小鼠远端结肠运动压力曲线的平均振幅变化率及平均曲线下面积变化率均低于同种系野生鼠C57BL/6小鼠相应的变化率，P＜0.05）。针刺BL25对M2&3KO小鼠及同种系野生小鼠C57BL/6空肠运动均无明显作用（即针刺BL25时，二种实验动物空肠运动压力曲线均无明显差异的平均振幅变化率及平均曲线下面积变化率，P＞0.05），同时对M2&3KO小鼠远端结肠运动的促进作用低于同种系野生鼠C57BL/6小鼠（即针刺BL25时，M2&3KO小鼠远端结肠运动压力曲线的平均振幅变化率及平均曲线下面积变化率均低于同种系野生鼠C57BL/6小鼠相应的变化率，P＜0.05）。结论：1ST25作为空肠的单元穴位（与空肠内脏神经支配节段相同），针刺能够通过躯体-交感神经反射通路抑制空肠运动；LI11和ST37作为空肠的集元穴位（与空肠内脏神经支配远节段腧穴），针刺能够通过激活副交感神经通路促进空肠运动。2LI11、ST37、ST25、BL25作为远端结肠的集元穴位（与远端结肠内脏神经支配远节段腧穴），针刺四个穴位均可以通过激活副交感神经神经通路促进远端结肠运动。3针刺TRPV1KO及ASIC3KO小鼠，针刺腧穴的效应方向没有改变，但针刺产生的效应大小发生了明显的变化。提示TRPV1与ASIC3离子通道可能均参与了针刺作用的外周感受器传入神经机制，其中TRPV1离子通道可能发挥了更为重要的作用。4针刺M2&3KO小鼠，针刺腧穴的效应方向也没有改变，但针刺LI11和ST37对空肠的促进作用明显减弱。提示M2&3受体可能参与了针刺作用的靶器官效应受体机制，即迷走神经胆碱能受体在针刺单元穴促进空肠运动中起主要作用。从本次研究的结果来看，针刺对于TRPV1及ASIC3基因敲除小鼠空肠及远端结肠运动的调节作用均较野生鼠有所降低，这些结果支持A类和C类纤维较A类纤维在针刺调节肠道运动中起更主要作用，这两种感受器均参与了针刺对胃肠功能的调节，即当针刺ST37及LI11时，激活TRPV1及ASIC3受体离子通道，兴奋了副交感神经，引起增强空肠运动的效应；当针刺ST25时，激活TRPV1及ASIC3受体离子通道，兴奋了交感神经，引起抑制空肠运动的效应。同时从我们的实验结果中明显可以看出于C57小鼠比较，针刺对TRPV1基因敲除小鼠空肠运动的调节效应差于ASIC3基因敲除小鼠。也说明了TRPV1离子通道在针刺调节肠道运动的神经机制中发挥着更为关键的作用。而针刺这四个穴位对TRPV1和ASIC3基因敲除小鼠远端结肠运动的作用均以兴奋性效应为主，仅以兴奋效应的强度与野生型小鼠有所不同，那么针刺对于远端结肠的调节机制是通过何种受体机制起主导作用呢？即针刺作用对远端结肠的调节机制仍需进一步研究探讨？针刺对M2&3KO小鼠肠道运动的促进作用明显低于野生鼠，提示在M2&3受体在针刺促进肠运动中共同发挥了作用。结合之前的分析，我们有理由相信针刺可能是通过激活M2&3受体，与针刺作用引起副交感神经兴奋后促进肠道释放的乙酰胆碱相结合，发挥促进肠道收缩运动的效应。同时我们还观察到针刺对M2&3KO小鼠空肠运动的抑制作用与野生鼠相比较没有明显差异，说明M2&3两种受体在抑制肠道运动即兴奋交感神经及拮抗副交感神经兴奋所产生的收缩促进作用效应中意义不大或者没有作用。而针刺这四个穴位对M2&3KO小鼠远端结肠运动的作用均以兴奋性效应为主，仅以兴奋效应的强度与野生型小鼠有所不同，那么针刺对于远端结肠的调节机制是通过何种受体机制起主导作用呢？即针刺作用对远端结肠的调节机制仍需进一步研究探讨。基于上述实验结果我们可以得出，针刺对空肠及远端结肠运动的调节作用可能是通过激活传入纤维末梢的TRPV1、ASIC3离子通道启动针刺作用的传入途径，兴奋胆碱能副交感神经，通过M2&3受体机制，引起肠道收缩以促进肠道运动；通过激活传入纤维末梢的TRPV1及ASIC3受体通道，兴奋胃肠交感神经神经，引起对肠道收缩运动的拮抗作用以产生抑制肠道运动的效应。从结果来看，TRPV1及ASIC3受体通道均参与了针刺对肠运动调节的促进和抑制效应，说明在针刺对交感及副交感神经的兴奋效应中此两种受体通道均起到了一定作用，两者比较而言TRPV1受体通道在此效应中发挥了更为重要的作用。而针刺效应产生的的神经机制中是否还有其他感受器离子通道参与仍需要进一步实验研究。针刺这四个穴位均能够加强远端结肠运动，尤其是天枢穴与其对空肠运动的作用方向不一致，这一点可能与远端结肠的神经支配较复杂且主要以兴奋肠道运动的神经效应为主有关，而在这种加强效应中何种神经支配占据主导地位仍需进一步的实验研究进行深入探讨。更多还原

【Abstract】 Object: Acupuncture has been tried against various diseases, includinggastrointestinal disorders. A large amount of experimental and clinicalevidence indicates the effectiveness of acupuncture in treating gastro-intestinal diseases. Though there were vast researches on the acupuncturetreatment of bowel diseases, previous studies have demonstrated theeffects of acupuncture stimulation on gastric motility in anesthetized rats,the evidence about the role of acupuncture on bowel motility of rodents innormal state, especially for the knockout mice, did not abound.Nevertheless, rare experiments were carried out to record the motility ofjejunum and colon of rodents in vivo, and even less studies relating to theeffects of acupuncture on that kind of bowel motility were reported athome. So the purpose of this is to investigate the effects of manual acupuncture (MA) on bowel motility in normal and autonomic nervescorrelation factor knockout mice and the mechanisms.MateriaI and Methods：1．Experimental protocols：Fufty adult mice (20mice of C57BL/6, and each10mice of TRPV1-/-mice,ASIC3-/-mice and M2/3-/-) were anaesthetized with10%urethane(intraperitoneally,1-1.2g/kg).A miniaturized solid-state pressuretransducer catheter with a water capsule (dia.3mm) in the end wasinserted into the lumen of the jejunum through the incision on theintestinal surface. Another catheter of the same type was inserted into thedistal colon through the anus. The capsules were placed1.5cm and1cmabove the incision of jejunum and the anus, respectively. This output wasacquired via a Micro1401interface and recorded using Spike2version7.03data acquisition software.2．Acupuncture procedure in anesthetized miceQuchi (LI11); located in the midpoint between the lateral end of thetransverse cubical crease and the lateral epicondyle of the humerus;Needles were inserted to a depth of5mm into the skin and underlyingmuscles at Shangjuxu (ST37) points. ST37is located at5mm lateral andlower from the anterior tubercle of the tibia in mice. Tianshu (ST25) islocated20mm above the symphysis pubis and5mm lateral from themidline in mice. Dachangshu (BL25): in the waist, under the fourthlumbar spines,5mm lateral to posterior midline. Acupuncture needleswere inserted unilaterally into acupoints and were twisted, lifted andthrust manually at a frequency of2Hz for60s in each type of mice. 3．Data acquisition and analysisEach miniature pressure transducer catheter was connected to atransducer control unit, whose output signal was subsequently amplifiedfurther using a transducer amplifier in differential mode. This output wasacquired via a Micro1401interface and recorded using Spike2version7.03data acquisition software. All the data in the present study wereexpressed as means±standard deviation (SD), and analyzed bytwo-tailed Student’s t-test or one-way ANOVA with SPSS software17.0.P-values<0.05were considered significant difference. P-values<0.01were considered very significant difference.RESULTS:1. The effect of manual acupuncture at different acupoint on bowelmotility in C57BL/6and TRPV1KO mice.1.1The effect of manual acupuncture at different acupoint on bowelmotility in C57BL/6mice.MA at LI11and ST37both promoted the motility of jejunum and distalcolon (manifested as changed intestinal pressure, precisely increasedaverage amplitude and mean area under the contractile curve) ofC57BL/6mice (P＜0.05). MA at ST25significantly inhibited themotility of jejunum (manifested as changed frequency and intestinalpressure, precisely decreased average amplitude, mean area under thecontractile curve, and the frequency) of C57BL/6mice (P＜0.05), whilesignificantly increased the distal colonic motility (manifested as changedintestinal pressure, precisely increased amplitude and mean area underthe contractile curve) of C57BL/6mice (P＜0.05). MA at BL25has notsignificantly effect at the motility of jejunum (manifested as changed frequency and intestinal pressure, precisely not significantly changedaverage amplitude, mean area under the contractile curve, and thefrequency) of C57BL/6mice (P＞0.05), while significantly increased thedistal colonic motility (manifested as changed intestinal pressure,precisely increased amplitude and mean area under the contractile curve)of C57BL/6mice (P＜0.05).1.2The effect of manual acupuncture at different acupoint on bowelmotility in TRPV1KO mice.MA at LI11and ST37both promoted the motility of jejunum and distalcolon (manifested as changed intestinal pressure, precisely increasedaverage amplitude and mean area under the contractile curve) of TRPV1KO mice (P＜0.05). MA at ST25significantly inhibited the motility ofjejunum (manifested as changed frequency and intestinal pressure,precisely decreased average amplitude, mean area under the contractilecurve, and the frequency) of TRPV1KO mice (P＜0.05), whilesignificantly increased the distal colonic motility (manifested as changedintestinal pressure, precisely increased amplitude and mean area underthe contractile curve) of TRPV1KO mice (P＜0.05). MA at BL25hasnot significantly effect at the motility of jejunum (manifested as changedfrequency and intestinal pressure, precisely not significantly changeaverage amplitude, mean area under the contractile curve, and thefrequency) of TRPV1KO mice (P＞0.05), while significantly increasedthe distal colonic motility (manifested as changed intestinal pressure,precisely increased amplitude and mean area under the contractile curve)of TRPV1KO mice (P＜0.05).The strength of the promoted motility of jejunum and distal colon thatMA at LI11and ST37of TRPV1KO mice was lower than C57BL/6mice(manifested as changed intestinal pressure, precisely increased change rate of average amplitude and increased change rate of mean area underthe contractile curve both lower than C57BL/6mice)(P＜0.05). Thestrength of the inhibited motility of jejunum that MA at ST25of TRPV1KO mice was lower than C57BL/6mice (manifested as changedfrequency and intestinal pressure, precisely decreased change rate ofaverage amplitude, decreased change rate of mean area under thecontractile curve, and the frequency all lower than C57BL/6mice)(P＜0.05), while The strength of the increased motility of the distal colonic ofTRPV1KO mice was lower than C57BL/6mice (manifested as changedintestinal pressure, precisely increased change rate of amplitude andincreased change rate of mean area under the contractile curve both lowerthan C57BL/6mice)(P＜0.05). MA at BL25has not significantly effectat the motility of jejunum (manifested as changed frequency andintestinal pressure, precisely no significant change rate of averageamplitude, change rate of mean area under the contractile curve, and thefrequency between TRPV1KO mice and C5BL/6mice) of two type miceboth (P＞0.05), while the strength of the increased motility of distalcolonic of TRPV1KO mice was lower than C57BL/6mice (manifestedas changed intestinal pressure, precisely increased change rate ofamplitude and change rate of mean area under the contractile curve bothlower than C57BL/6mice)(P＜0.05).2．The effect of MA at different acupoint on bowel motility in ASIC3KOmice.MA at LI11and ST37both promoted the motility of jejunum and distalcolon (manifested as changed intestinal pressure, precisely increasedaverage amplitude and mean area under the contractile curve) of ASIC3KO mice (P＜0.05). MA at ST25significantly inhibited the motility ofjejunum (manifested as changed frequency and intestinal pressure, precisely decreased average amplitude, mean area under the contractilecurve, and the frequency) of ASIC3KO mice (P＜0.05), whilesignificantly increased the distal colonic motility (manifested as changedintestinal pressure, precisely increased average amplitude and mean areaunder the contractile curve) of ASIC3KO mice (P＜0.05). MA at BL25has no significant effect at the motility of jejunum (manifested aschanged frequency and intestinal pressure, precisely not significantlychanged average amplitude, mean area under the contractile curve, andthe frequency) of ASIC3KO mice (P＞0.05), while significantlyincreased the distal colonic motility (manifested as changed intestinalpressure, precisely increased the average amplitude and the mean areaunder the contractile curve) of ASIC3KO mice (P＜0.05).The strength of the promoted motility of jejunum and distal colon thatMA at LI11and ST37of ASIC3KO mice was lower than C57BL/6mice(manifested as changed intestinal pressure, precisely increased changerate of average amplitude and change rate of mean area under thecontractile curve both lower than C57BL/6mice)(P＜0.05). The strengthof the inhibited motility of jejunum that MA at ST25of ASIC3KO micewas lower than C57BL/6mice (manifested as changed intestinal pressure,precisely decreased change rate of average amplitude, change rate ofmean area under the contractile curve, and the frequency all lower thanC57BL/6mice)(P＜0.05), while The strength of the increased motilityof the distal colonic of ASIC3KO mice was lower than C57BL/6mice(manifested as changed intestinal pressure, precisely increased changerate of average amplitude and change rate of mean area under thecontractile curve both lower than C57BL/6mice)(P＜0.05). MA at BL25has no significant effect at the motility of jejunum (manifested aschanged frequency and intestinal pressure, precisely not significantlychanged change rate of average amplitude, change rate of mean area under the contractile curve, and the frequency between ASIC3KO miceand C57BL/6mice) of both two type mice (P＞0.05), while the strengthof the increased motility of distal colonic of ASIC3KO mice was lowerthan C57BL/6mice (manifested as changed intestinal pressure, preciselyincreased change rate of average amplitude and change rate of mean areaunder the contractile curve both lower than C57BL/6mice)(P＜0.05).The strength of the promoted motility of jejunum and distal colon thatMA at LI11and ST37of ASIC3KO mice was lower than C57BL/6micebut higher than TRPV1KO mice(manifested as changed intestinalpressure, precisely increased change rate of average amplitude andchange rate of mean area under the contractile curve both lower thanC57BL/6mice but higher than TRPV1KO mice)(P＜0.05). The strengthof the inhibited motility of jejunum that MA at ST25of ASIC3KO micewas lower than C57BL/6mice but higher than TRPV1KO mice(manifested as changed intestinal pressure, precisely decreased changerate of average amplitude, change rate of mean area under the contractilecurve, and the frequency all lower than C57BL/6mice but higher thanTRPV1KO mice)(P＜0.05), while The strength of the increased motilityof the distal colonic of ASIC3KO mice was lower than C57BL/6micebut higher than TRPV1KO mice (manifested as changed intestinalpressure, precisely increased change rate of average amplitude andchange rate of mean area under the contractile curve both lower thanC57BL/6mice but higher than TRPV1KO mice)(P＜0.05). MA atBL25has no significant effect at the motility of jejunum (manifested aschanged frequency and intestinal pressure, precisely not significantlychanged change rate of average amplitude, change rate of mean areaunder the contractile curve, and the frequency among TRPV1KO mice、ASIC3KO mice and C57BL/6mice) of all three type mice (P＞0.05),while the strength of the increased motility of distal colonic of ASIC3KO mice was lower than C57BL/6mice but higher than TRPV1KO mice(manifested as changed intestinal pressure, precisely increased changerate of average amplitude and change rate of mean area under thecontractile curve both lower than C57BL/6mice but higher than TRPV1KO mice)(P＜0.05).3．The effect of MA at different acupoint on bowel motility in M2&3KOmice.MA at LI11and ST37both promoted the motility of jejunum and distalcolon (manifested as changed intestinal pressure, precisely increasedaverage amplitude and mean area under the contractile curve) of M2&3KO mice (P＜0.05). MA at ST25significantly inhibited the motility ofjejunum (manifested as changed frequency and intestinal pressure,precisely decreased average amplitude, mean area under the contractilecurve, and the frequency) of M2&3KO mice (P＜0.05), whilesignificantly increased the distal colonic motility (manifested as changedintestinal pressure, precisely increased average amplitude and mean areaunder the contractile curve) of M2&3KO mice (P＜0.05). MA at BL25has no significant effect at the motility of jejunum (manifested aschanged frequency and intestinal pressure, precisely not significantlychanged average amplitude, mean area under the contractile curve, andthe frequency) of M2&3KO mice (P＞0.05), while significantlyincreased the distal colonic motility (manifested as changed intestinalpressure, precisely increased average amplitude and mean area under thecontractile curve) of M2&3KO mice (P＜0.05).The strength of the promoted motility of jejunum and distal colon thatMA at LI11and ST37of M2&3KO mice was lower than C57BL/6mice(manifested as changed intestinal pressure, precisely increased changerate of average amplitude and change rate of mean area under the contractile curve both lower than C57BL/6mice)(P＜0.05). MA at ST25has not significantly different effect at the motility of jejunum(manifested as changed frequency and intestinal pressure, precisely notsignificantly different decreased change rate of average amplitude,change rate of mean area under the contractile curve, and the frequencybetween M2&3KO mice and C57BL/6mice) between two type mice (P＞0.05), while The strength of the increased motility of the distal colonicof M2&3KO mice was lower than C57BL/6mice (manifested as changedintestinal pressure, precisely increased average amplitude and mean areaunder the contractile curve both lower than C57BL/6mice)(P＜0.05).MA at BL25has not significantly effect at the motility of jejunum(manifested as not significantly changed frequency and intestinalpressure, precisely not significantly changed change rate of averageamplitude, change rate of mean area under the contractile curve, and thefrequency between M2&3KO mice and C57BL/6mice) of two type mice(P＞0.05), while the strength of the increased motility of distal colonic ofM2&3KO mice was lower than C57BL/6mice (manifested as changedintestinal pressure, precisely increased change rate of average amplitudeand change rate of average mean area under the contractile curve bothlower than C57BL/6mice)(P＜0.05).Conclusion:1．Tianshu (ST25) is the “homotopic point” for jejunum, and MA at ST25could inhibit the motility of jejunum via somato-sympathetic reflexpathway. Thus, Quchi (LI11) Shangjuxu (ST37) are the “heterotopicpoint” for jejunum, and MA at LI11and ST37could promote themotility of jejunum via increased parasympathetic tone.2.Quchi (LI11), Shangjuxu (ST37), Tianshu (ST25) and Dachangshu(BL25) are the “heterotopic point” for distal colon, and MA at LI11,ST37,ST25and BL25could promote the motility of distal colonvia increased parasympathetic tone.3．In TRPV1KO and ASIC3KO mice, MA at LI11, ST37, ST25andBL25have the same regulation of the motility of jejunum and distalcolon with C57BL/6mice. But the size of effect have the apparentdifferent. It points that TRPV1and ASIC3might be participated inthe afferent neural mechanism of MA.4．In M2&3KO mice, MA at LI11, ST37, ST25and BL25have the sameregulation of the motility of jejunum and distal colon with C57BL/6mice. But the size of effect of MA at LI11and ST37has the apparentreduce. It points that M2&3might be participated in the target’sresponse receptor mechanism of MA.So we can observe from this experiment that the effect of MA wasclosely related with acupoint’s inherent character. Both of TRPV1, andASIC3have its action in the the afferent neural mechanism of MA. AndM2&3might be participated in the target’s response receptor mechanismof MA. And why the different effect of MA at distal colon, the intrinsiccontradistinction must be investigated and research further.更多还原