Cajal间质细胞自噬调控在高原胃肠动力紊乱中的保护作用研究

Gastrointestinal motility disorders are common phenomenon at high altitudes and may cause greatly body harm. However, the pathogenesis is unknown. There are no effective preventions against the damage. Interstitial cells of Cajal(ICC) play a key role in the control of gastrointestinal motility and are regulated by c-Kit gene and levels of intracellular calcium. They are susceptible to the harmful factors, which may cause them structural and functional damage and result in dysfunction. Previous researches showed that gastrointestinal mucosa barrier function could be damaged at high altitude environment and induced gastrointestinal autophagy. Moderate autophagy is some kind of cellular protection. But there is uncertain that if hypoxia at high altitudes would act as an injury factor to cause excessive autophagy, which would furtherly result in abnormal expression of c-Kit, structural or functional damage in ICC, excess mitochondrial autophagy and finally lead to the plateau gastrointestinal motility disorders. Would it be possible that a moderate autophagy could reduce the severity of gastrointestinal motility disorders? To illustrate our hypothesis, rat model of high altitude is in used. Research targets include the following: analysis the expression of c-Kit gene, observation the ultrastructure and network of ICC by electron microscopy, the autophagy in mitochondrion, levels of intracellular calcium, analysis of intestinal motility function in vivo and vitro; meanwhile intervention of ICC’s function through interfering drugs and autophagy controlling are also involved. A comprehensive analysis of high altitude-induced gastrointestinal motility disorder will be done according to the above indicators. These results will give us a clear view of gastrointestinal motility disorders in plateau and show some indications in prevention or treatment of gastrointestinal motility disorders.

高原胃肠动力紊乱常见且危害大，但发病机制不祥，尚无有效防护措施。Cajal间质细胞（ICC）在胃肠道平滑肌起搏和运动中起关键作用，受c-Kit基因、细胞内钙离子等调控，易被外界因素影响而导致其损害，出现胃肠动力紊乱。研究发现：高原缺氧等因素可使胃肠粘膜受损，诱发自噬；自噬(包括线粒体自噬)是确保细胞功能稳定、存活的重要机制。但目前尚不清楚高原损伤因素所诱发的自噬是否作为损伤因素，进一步引起c-Kit表达异常、ICC结构和功能破坏、线粒体过度自噬损伤，进而导致高原胃肠动力障碍的形成。为此我们采用高原大鼠为研究对象，分析c-Kit表达变化，电镜下观察ICC超微结构、数目和网络结构，线粒体自噬状态，细胞内钙离子变化，在体和离体分析肠道运动功能；最后通过自噬干预、ICC保护等手段，观察上述指标和功能的变化。综合分析高原环境诱发胃肠动力紊乱的形成及保护机制，为高原胃肠动力紊乱的防治提供基础理论依据。

高原胃肠动力紊乱常见，但机制不祥，其受肠神经系统、Cajal间质细胞（ICC）和平滑肌共同调控。ICC在平滑肌起搏和运动中起关键作用，支配慢波；但可受因素的影响，出现动力问题。项目主要研究：高原肠动力的变化，ICC蛋白、自噬蛋白及超微结构的改变；运用自噬干预、ICC增殖、粘膜保护后指标和功能的变化；旨在揭示动力障碍机制。.一、.动力的变化 .平原大鼠小肠推动率约为68.3%，高原作用12h后降至62.9%，24h为60.2%，48h为37.9%，72h稍回升至48.6%。予以自噬干预、ICC增殖及肠粘膜保护后，各组推动率于12-48h均呈下降趋势，72h自噬抑制组降至最低，而自噬增强组回升最快。.慢波频率（cpm）直接反映ICC功能，对照组小肠慢波0h 36.6、12h 32.0、24h 30.8、48h 24.4，72h恢复至32.9。各干预组慢波于48、72h出现分歧，自噬抑制组72h最低，而增强组则明显恢复。 .二、.ICC蛋白和自噬的变化.综合Western blot和免疫荧光分析对照组c-Kit表达，呈现24h代偿增高、48h降低、72h稍回升的趋势，此与小肠推动率相似；自噬蛋白（Bclin-1、LC3b）24h增高，之后回落；HIF-1α则随时间逐步升高。免疫荧光法观察LC3b表达，也呈现24h代偿增高，48h降低，72h回升趋势。在干预组，上述指标经WB、免疫荧光分析也呈现类似24h增高，48h最低，72h回升的趋势。.三、.ICC中自噬状态.透射电镜见ICC中存在自噬体样结构，对照组于48h明显增多，自噬抑制组72h见较多自噬，自噬增强组48、72h均见较多自噬结构；结果与免疫荧光所见类似。.四、.干预结果.自噬增强可整体改善小肠的推动率，维持慢波频率，而自噬抑制则起到延缓作用；粘膜保护和补充干细胞因子的保护作用较弱。自噬抑制组于72h虽有大幅c-kit表达，但与自噬增强组相比，其HIF-α明显升高，而自噬程度不足；这也可能是小肠动力减弱的成因之一。 .五、.科学意义.高原胃肠动力紊乱机制仍极其复杂，涉及ICC功能状态、缺氧程度、自噬水平等多方面。特定环境下早期启动自噬，有助于动力功能恢复，而抑制自噬则延缓了恢复。研究结果部分揭示了高原胃肠动力紊乱的特点和机制，可为筛选抗高原胃肠动力紊乱药物提供相应理论依据。