大承气汤对阳明腑实证大鼠小肠深肌层Cajal间质细胞溶酶体-线粒体轴凋亡途径干预机制

How to effectively restore gastrointestinal movement is the key problem for the treatment of Yangming fu-organ disease (YMFOD). Recent researches have confirmed that the deep muscular plexus interstitial cells of cajal (ICC-DMP) in the small intestine is the pacemaker cells of gastrointestinal motility. Preliminary study of applicant has showed that Da-Cheng-Qi Decotion (DCQD) could promote gastrointestinal motility of rats with Yangming fu-organ disease (YMFOD), by inhibiting apoptosis of small intestine deep muscular plexus interstitial cells of Cajal (ICC-DMP). It was closely related with protecting of lysosome and mitochondrial ultrastructural, but the detailed mechanism is still unclear. According to this result we put forward the hypothesis that DCQD could recover gastrointestinal motility by inhibiting lysosomal-mitochondrial axis activation of the small intestinal ICC-DMP. In this study, the rats model of YMFOD would be made and the apoptosis pathway of lysosomal-mitochondrial axis would be observed by using immune fluorescence, line analysis of fluorescence channel colocalization, fluorescence in situ hybridization, laser scanning confocal fluorescence microscope. The activation mechanism of apoptosis pathway of lysosomal-mitochondrial axis and the intervention effect of DCQD would be studied from the expression of gene, regulation of genetic transcription, expression of proteins, and physiological changes of cell organelles. This study would further reveal the mechanism of gastrointestinal motility disorder in YMFOD, and lay a theoretical foundation for the treatment of YMFOD and the further development and application of DCQD.

如何有效恢复胃肠运动是治疗阳明腑实证的关键问题。大承气汤可以显著地促进阳明腑实证患者肠道动力的恢复，而本项目前期研究显示大承气汤通过抑制小肠深肌层Cajal间质细胞（ICC-DMP）凋亡，显著修复ICC-DMP溶酶体和线粒体超微结构，据此提出大承气汤抑制小肠ICC-DMP溶酶体-线粒体轴凋亡途径激活恢复胃肠动力的假说。本研究拟应用免疫荧光多重标记、荧光通道共定位线性分析、原位杂交、共聚焦显微镜等方法，检测小肠ICC-DMP溶酶体酸碱度、膜通透性和水解酶表达，线粒体膜电位、通透性转换孔、促凋亡因子表达和凋亡调控因子表达。从基因、蛋白质和细胞器等层面，研究大承气汤对小肠ICC-DMP溶酶体-线粒体轴凋亡途径干预作用。从细胞凋亡角度揭示大承气汤促进阳明腑实证胃肠动力恢复机理。对进一步揭示阳明腑实证胃肠动力障碍发生机制有着重要意义。为阳明腑实证治疗和大承气汤进一步开发、应用奠定基础。

促进胃肠动力恢复是防治阳明腑实证的核心，近年来研究表明大承气汤可以通过抑制深肌层Cajal间质细胞（interstitial cells of Cajal in the deep muscular plexus，ICC-DMP）的凋亡恢复胃肠动力。课题以大鼠小肠为研究对象，建立阳明腑实证大鼠模型，采用透射电镜、荧光染色、TUNEL凋亡检测法、共聚焦显微镜等技术研究大承气汤抑制阳明腑实证大鼠小肠ICC-DMP溶酶体-线粒体轴凋亡途径的主要靶点，和对肠神经凋亡的干预作用。实验结果：①大承气汤可以保护ICC-DMP表型和超微结构，抑制凋亡，从而保护ICC-DMP网络样结构。②大承气汤可以保护ICC-DMP溶酶体的超微结构，维持内部pH值和膜通透性，抑制水解酶cathepsin B释放，阻断凋亡信号从溶酶体向线粒体传递。③大承气汤可以保护ICC-DMP线粒体的超微结构和Ca2+通道蛋白IP3的表达，维持跨膜电位稳定，调控线粒体通透性转换孔相关蛋白表达，抑制线粒体释放细胞色素C，阻断caspase级联反应。④大承气汤可以促进ICC-DMP细胞浆中NF-κB移位至细胞核，上调溶酶体水解酶抑制基因Spi2A的表达，阻断凋亡信号从溶酶体向线粒体的传递；还可以促进ICC-DMP细胞浆中Survivin与caspase-3的结合，抑制caspase-3的活性，从而阻断caspase级联反应。⑤大承气汤抑制大鼠小肠神经凋亡，其机制可能与保护肠神经连接、维持超微结构的稳定、促进神经生长因子的表达密切相关，从而保护肠神经-ICC-DMP网络，恢复胃肠动力。综上研究表明，大承气汤通过多个靶点阻断溶酶体-线粒体轴凋亡途径从而抑制ICC-DMP凋亡，同时保护肠神经的形态和功能，抑制肠神经凋亡，恢复阳明腑实证胃肠动力。课题研究为进一步阐明大承气汤的作用机制和推广临床应用提供了理论依据。