养阴润肠方促进ICC活性维持治疗STC的机制研究

Slow transit constipation (STC) is a hot and difficult clinic problem at present. Research shows that the interstitial cells of Cajal (ICC) participate in the gastrointestinal basic electrical rhythm control and neurotransmitter signaling， which are the key factor in the basic functions of the gastrointestinal network. The specific marker of ICC is c-kit, which combines with stem cell factor (SCF) to activate the downstream signaling pathways. Their combination plays an important role in maintaining the ICC development and phenotype. Professor Zhu Bingyi used Yang-Yin-Run-Chang method as a remedy for STC. This experience-proved formula of Yang-Yin-Run-Chang has the unique composition, which has been shown efficacy in the early clinical studies. The corresponding basic research has shown that the formula can up-regulate the expression level of c-kit mRNA in constipation rat models, maintain ICC activities, and promote the colonic transit function. This study intends to establish the rat model of STC and clarify the time-effect and dose-effect relationship during the ICC activity maintaining in order to explore the therapeutic mechanism of Yang-Yin-Run-Chang formula on STC. Besides, the changes of PI3K-AKT, Ras-ERK and JAK-STAT signal pathways, which are in the SCF/c-kit pathway downstream, will be observed in the experimental cells treated by Yang-Yin-Run-Chang formula. We are supposed to interpret the internal mechanism of ICC activity maintaining, to find new therapeutic target in STC, and to provide a reference to explore the new targeted drugs for subsequent study of traditional Chinese medicine.

慢传输型便秘(STC)是临床亟待解决的热点和难点问题。研究表明，Cajal间质细胞(ICC)参与胃肠基本电节律调控和神经递质信号传导，在胃肠动力功能网络中居于核心地位。c-kit是ICC特异标志物，干细胞因子(SCF)与其结合后活化下游信号通路，对ICC发育和表型维持具有重要作用。朱秉宜教授运用养阴润肠方治疗STC，组方独特、疗效确切，前期研究初步显示该方可上调便秘大鼠c-kit mRNA表达，促进ICC活性维持，增强结肠运输功能。本研究拟通过动物实验进一步揭示该方对ICC的活性维持作用及其时效和量效关系，阐明该方治疗STC的作用机制；通过细胞实验探讨该方对SCF/c-kit途径下游的PI3K-AKT，Ras-ERK及JAK-STAT信号通路的影响，诠释该方促进ICC活性维持的内在机制，期待发现STC新的治疗靶标，为研究中药靶向药物提供借鉴。

Cajal 间质细胞(Interstitial cells of Cajal, ICC)参与胃肠基本电节律调控和神经递质信号传导，在胃肠动力功能网络中居于核心地位。慢传输型便秘(Slow transit constipation, STC)与ICC的数量和功能状态明显相关。SCF/c-kit通路对ICC发育、成熟细胞的存活、表型的维持具有重要作用。本课题分别通过大鼠和小鼠的STC模型验证养阴润肠方治疗STC的有效性，揭示本方对ICC活性的维持作用及其时效和量效关系，证实临床剂量与疗程的合理性，初步诠释本方治疗STC的作用机制；通过酶解小鼠肠道组织的方法，获得ICC细胞，经不同浓度养阴润肠方含药血清干预后，观察细胞增殖或者凋亡情况，探讨了本方对SCF/c-kit可能下游信号通路的影响，但未能取得阳性结果；课题组继而通过流式分选获得纯化的ICC，运用Ca2+荧光探针观察本方对于ICC细胞起搏功能的影响，初步诠释养阴润肠方治疗STC的部分潜在机制。.结果：①大鼠实验：养阴润肠方通过恢复结肠肌电活动和修复损伤ICC超微结构改善慢传输型便秘大鼠的结肠传输功能，观察到本方的最佳给药剂量是临床等效剂量，最佳给药时间可能为4周；②小鼠实验：养阴润肠方通过恢复SCF/c-kit通路，增加慢传输便秘小鼠结肠ICC的数量，恢复其活性，改善STC小鼠的结肠传输功能，增加小鼠的排便量和粪便含水量；③ICC增殖或凋亡：小鼠小肠组织经酶解消化得到原代“ICC”，生长曲线发现4-7天ICC处于对数期，c-kit免疫荧光鉴定后，分别用2.5%、5%、10%的含药血清干预“ICC”，与正常血清组比较，含药血清各组细胞的OD值变化不明显，含药血清对“ICC”细胞的增殖作用不明显；运用流式分析发现，正常大鼠血清干预“ICC”后，细胞凋亡增加，经不同浓度含药血清干预后，凋亡未见明显减少。由此说明含药血清对于ICC的增殖或凋亡影响较小；④胞内Ca2+的影响：分别尝试Percoll梯度密度液、磁珠分选、流式分选的方法纯化ICC，发现经流式分选可以得到纯度较高的ICC，约占总细胞的5.7%，限于培养后ICC数量较少，运用Ca2+荧光探针发现不同浓度含药血清可以增加ICC胞内Ca2+浓度。从功能学角度诠释养阴润肠方维持ICC的活性的可能作用机制。