糖尿病胃轻瘫发生中AMPK-mTORC1通路对自噬与凋亡的影响

Gastroparesis is one of diabetes complications. It had been shown that autophagy and apoptosis in a variety of physiological and pathological processes of the cell, and there was a certain link between the two. We previously had found that the one pathogenesis of diabetic gastroparesis was gastric smooth muscle cells in the presence of autophagy and apoptosis, and they were strengthened with the extension of the course. AMPK-mTORC1 had been shown to participate the cell autophagy and apoptosis, and the pathway was activated by high glucose-induced, but the specific mechanism of action was still unclear. It has not been reported of the literature to associate AMPK-mTORC1 pathway with autophagy with apoptosis in diabetic gastroparesis occurrence. The program will use the way of flow cytometry, confocal laser scanning microscopy, immunoprecipitation, cell transfection, RNA interference and Western Blot and other multidisciplinary experimental techniques to study diabetic rats, gastric smooth muscle cells in vitro, to explore high glucose-induced autophagy and apoptosis mechanisms occur in diabetic gastroparesis, high- glucose regulating mechanism through AMPK pathway mTORC1 and the impact of both autophagy and apoptosis. The research for this project further clarify diabetic gastroparesis pathogenesis has important theoretical significance, but also has potential applications in clinical diabetes prevention and treatment of gastroparesis.

胃轻瘫是糖尿病常见的并发症之一。研究表明，自噬与凋亡参与细胞的多种生理和病理过程，且二者存在一定的联系。我们前期发现，糖尿病胃轻瘫发病过程中胃平滑肌细胞存在自噬与凋亡，且均随病程延长而加强。AMPK-mTORC1已被证实参与细胞自噬与凋亡的发生，且该通路可受高糖诱导而活化，但是具体的作用机制至今尚不清楚。基于AMPK-mTORC1将糖尿病胃轻瘫与自噬、凋亡联合进行研究，尚未见文献报道。本研究拟利用流式细胞技术、激光共聚焦显微镜技术、免疫沉淀、细胞转染、RNA干扰及Western Blot等多学科实验技术，以糖尿病大鼠模型、体外培养胃平滑肌细胞为研究对象，探讨高糖诱导自噬和凋亡在糖尿病胃轻瘫发生中的作用机制，高糖通过AMPK途径调控mTORC1的机制以及两者对自噬和凋亡的影响；本项目的研究对于进一步阐明糖尿病胃轻瘫的发病机理具有重要的理论意义，也对临床糖尿病胃轻瘫的防治具有潜在的应用价值。

糖尿病胃轻瘫（Diabitic Gastroparesis，DGP）是常见的糖尿病消化道并发症之一，可导致机体血糖波动大、药物控制乏力，进而加速相关糖尿病急慢性并发症发生。因此探讨DGP相关发生机制及相关干预措施，对于临床探索新的治疗方案至关重要。到目前为止，DGP发病机制的研究多集中在胃的神经损伤、神经递质传导障碍及胃肠激素分泌异常等方面，而针对胃平滑肌细胞本身的研究相对较少。胃平滑肌的收缩运动是胃排空的动力来源，而有效的平滑肌细胞数量及充足的细胞能量供应则是该动力的保障。细胞自噬、内质网应激与凋亡是常见的细胞行为，三者之间关系密切。如果能够发现某种调整上述三种细胞行为的途径，影响胃平滑肌细胞凋亡与能量代谢，确定相关机制并加以干预，那么对于DGP乃至糖尿病的治疗其作用都是积极的。现已证实，AMPK与IGF-1参与自噬、凋亡、内质网应激等多种细胞行为的发生和调控，且两者关系密切。为了阐明AMPK对自噬、凋亡、内质网应激等的调控机制，本项目先后观察了DGP的发生过程中，大鼠胃平滑肌细胞凋亡与能量代谢的变化、AMPK的活化与活化方式、mTORC1的变化及mTORC2的变化，确定了高糖状态下活化的AMPK对大鼠胃平滑肌细胞凋亡与能量代谢的影响及机制，以及变化的mTORC1与mTORC2对大鼠胃平滑肌能量代谢的影响。同时，确定了IGF-1通过抑制内质网应激对高糖作用下大鼠胃平滑肌细胞自噬的抑制作用的机制，以及通过活化PI3K-Akt-PKC通路降低了细胞内Ca2+浓度，达到抑制大鼠胃平滑肌肌细胞的凋亡的机制。上述研究结论能够为进一步阐明DGP的发生机制及临床治疗新方案的探索提供科学的理论依据与实验依据。