胆石症豚鼠nNOS神经-ICC-SMC网络损伤及电针的修复作用

Improvement of gallbladder motility is the key to the treatment of gallstones. And acupuncture is effective in regulating gallbladder motility, but the mechanism is still not clear. The Enteric nervous system (ENS)- Interstitial cells of Cajal (ICC)- Smooth muscle cells (SMC) network is the basic functional unit of gastrointerstinal motility, and similarly, the network of nerve-ICC-SMC is also found in the gallbladder. The inhibitory neurotransmitter NO is catalyzed by nNOS and regulates SMC relaxation and contraction under the mediate of ICC. It is already known that there is damage of ENS-ICC-SMC network under the condition of gastrointestinal hypomotility, and acupuncture can increase the number of reduced ICC, promote the recovery of damaged structure of ICC, regulate the activity of NOS and the release of NO, and improve the structure of ICC-SMC. Based on these, we suppose that there is nNOS nerve- ICC- SMC network damage under the condition of gallbladder hypomotility, and electro-acupuncture can repair the network damage to regulate the gallbladder motility. To verify this hypothesis, this project intends to observe gallstone in gallbladder of guinea pig, and to use apparatus and technologies such as immunohistochemistry, transmission electron microscopy, flow cytometer, Western Blot and q-PCR to observe damage of nNOS-ICC-SMC network and repair effect of electro-acupuncture, so as to provide scientific basis for electro-acupuncture for gallbladder motility.

改善胆囊动力是治疗胆结石的关键，针刺有良好的调节胆囊动力的作用，但机制未明。ENS-ICC-SMC网络是消化道动力的基本功能单位，相似地，胆囊内也存在神经-ICC-SMC网络。抑制性神经递质NO由nNOS催化生成并在ICC介导下调控SMC舒缩。胃肠道功能障碍时存在神经-ICC-SMC网络损伤，针刺能够使减少的ICC数量增多，促进ICC受损结构的恢复，调节NOS活性和NO释放，改善ICC-SMC结构。由此推测胆囊动力障碍时存在nNOS神经-ICC-SMC网络损伤，电针能修复该网络损伤达到调节胆囊动力的作用。为验证该假说，拟观察胆结石豚鼠胆囊结石情况，使用免疫组化、透射电镜、流式细胞仪、Western Blot、q-PCR等仪器和方法观察胆石症豚鼠nNOS神经-ICC-SMC网络损伤情况及电针的修复作用，为电针调节胆囊动力提供科学依据。

胆结石发病率呈上升趋势，绝大部分胆结石为胆固醇结石。胆囊运动功能减弱可导致胆汁胆固醇过饱和，并进一步加重胆囊动力障碍，促进胆结石形成。胆囊动力障碍是胆结石关键病因之一，改善胆囊动力是治疗胆结石的关键所在。临床研究证明，针灸治疗胆结石有良好的效果，对胆囊动力有良好的调节效果，但其作用机制不明。我们提出如下假说：神经-ICC-SMC网络是胆囊动力调节的基本单位，胆囊动力障碍的实质是神经-ICC-SMC网络损伤，nNOS神经-ICC-SMC网络是调节胆囊动力障碍的重要网络之一。电针能够通过修复nNOS神经-ICC-SMC网络损伤调节胆囊动力。采用豚鼠胆固醇结石模型为研究对象，揭示电针调节胆囊动力的作用机制。. 实验结果显示：（1）致石饲料喂养6周后，超声显示豚鼠胆囊壁毛糙，可见强回声团，模型造模成功。（2）电针治疗结束后，电针组豚鼠胆囊结石质量较模型组胆结石质量减轻，表明电针治疗胆结石的有效性，可减轻胆囊结石。（3）模型组胆囊ICC超微结构及nNOS神经-ICC-SMC网络出现损伤，经电针治疗后可修复胆囊ICC超微结构损伤和nNOS神经-ICC-SMC网络结构损伤。（4）电针能够改善胆结石胆囊NO受体sGC-α-1、sGC-β-1、PKG蛋白表达水平，可修复nNOS神经-ICC之间NO-sGC-cGMP-PKG通路损伤。（5）电针能够改善胆结石胆囊的缝隙连接蛋白CX43蛋白表达和mRNA表达，说明电针可修复ICC-SMC之间信号传导通路——缝隙连接和CX43损伤。. 实验结果验证了电针调节胆囊动力、减少结石生成的有效性。验证了胆囊动力障碍的实质之一是nNOS神经-ICC-SMC网络损伤的假说，阐明电针对nNOS神经-ICC-SMC网络损伤的修复是电针调节胆囊动力的机制，为电针治疗胆石症等胆囊动力障碍性疾病提供科学依据。