AMPK-Beclin-1/Vps34通路在维生素D3（Vit D)诱导足细胞自噬中的作用和机制研究

Autophagy and apoptosis are the two most important pathophysiological processes of podocytes and high level of autophagy plays an key role to maintain podocyte homeostasis. Recent researches have showed the Beclin-1/Vps34 complex was a key factor in regulation of the formation of autolysosome and the combination of BCL-2 and Beclin-1 inhibit Autophagy due to defect of Beclin-1/Vps34 complex formation. Previous studies show VitD could inhibit the apoptosis of podocytes through suppressing the expression of TRPC6 and RNAK. There exists crosstalk between autophagy and apoptosis, the activation of autophagy could inhibit apoptosis. Thus, could VitD inhibit the apoptosis of podocytes through the activation of autophagy? To confirm this propose, the autophagy process of podocytes under the stress was observed by confocal through the transfection of GFP-LC3 under the level of cell and animal, and the change of the Ca2+ Channel and Ca2+ concentration of podocytes induced by VitD were detected by Ca2+ -mark- fluorescent probes and patch clamp technique. To investigate the role of AMPK-Beclin-1/Vps34 passway in podocyte autophagy and apoptosis, the expression level of Beclin-1，Vps34 and AMPK in VitD induced podocytes were detected by gene transfection and silence. The study of this project will allow us to gain insights into the effect and mechanism of AMPK- Beclin-1/Vps34 pathway in the Vitamin D3 Induced podocyte Autophagy and provide new theoretical basis for finding drugs to promote autophagy and podocyte protection.

自噬和凋亡是足细胞最重要的两个病理生理过程。高水平的自嗜对维持足细胞稳态具有重要意义。Beclin-1/Vps34是自噬溶酶体形成关键，Bcl-2与Beclin-1结合阻碍Beclin-1/Vps34复合物形成而抑制自噬。前期研究证实VitD可以通过抑制TRPC6和RNAK的表达抑制凋亡，而凋亡信号和自噬信号存在串话作用,自噬抑制可促进凋亡。VitD对凋亡的抑制是否是通过活化自噬实现的呢？本课题以足细胞损伤的细胞、动物模型，通过confocal观察GFP-LC3转染足细胞在应激状态下自噬的变化；通过游离钙荧光探针、膜片钳技术观察VitD对细胞Ca2+通道和Ca2+浓度的影响；通过基因转染、基因沉默观察VitD对Beclin-1、Vps34、AMPK表达的影响，来探讨AMPK-Beclin-1/Vps34通路在VitD诱导足细胞自噬中的作用，为阻止足细胞损伤，寻找激活自噬的药物提供理论。

项目的背景:足细胞损伤、脱落、凋亡是导致糖尿病肾病持续进展的重要原因。高水平的自嗜对维持足细胞稳态具有重要意义，自噬障碍可导致足细胞损伤、细胞丢失并最终导致肾小球硬化。因此，调控自噬是保护足细胞和防治慢性肾脏病的一种重要的新兴策略。.主要研究内容：1、在正常足细胞和足细胞损伤模型中观察自噬的变化； 2、在体外细胞水平进一步证实，VitD 能够促进足细胞Beclin-1和Vps34的表达诱导自噬而非通过抗菌多肽HCAP-18/LL-37诱导自噬。3、证实Ca2+/CaMKK-β，AMPK信号通路是VitD诱导足细胞自噬的主要信号通路；4、用足细胞损伤的动物模型（DKD）在体内进一步证实VitD足细胞保护和自噬之间的关系。.重要结果：1、足细胞在高糖刺激下早期自噬活性增强，长期刺激自噬抑制；1α,25(OH)2D3可能是一种自噬的活化剂，通过调控自噬相关蛋白beclin-1、Vps34的水平促进自噬溶酶体的形成，延缓DKD的进展。2、足细胞在生理条件下维持基础量的凋亡和较高的自噬水平，在应急、高糖、PAN刺激下凋亡水平显著增加，自噬受到抑制。VD通过调控足细胞内钙离子水平及beclin-1/Vps34信号调控自噬。3、VitD类似物骨化三醇、帕里骨化醇及SGLT-2抑制剂达格列净可能通过调控足细胞自噬抑制糖尿病肾病的进展。.关键数据：1、 Western blot提示在高糖刺激的早期beclin-1和Vps34 表达水平逐渐升高，24h达高峰，24h后beclin-1和Vps34的表达逐渐下降（P < 0.05）；2、RT-qPCR, Western blot研显示：与对照组相比，高糖组LC3-Ⅱ及Beclin-1、Vps34表达水平显著降低。HG+VD组LC3-Ⅱ、Beclin-1、Vps34表达水平呈浓度依赖性上调，VD抑制高糖导致的自噬下调。3、加入自噬抑制剂3-MA后自噬相关蛋白LC3-Ⅱ表达水平下调，Bax、 caspase-3的表达水平显著上调（P＜0.05）4、Western印迹显示:与对照组比较，高糖组beclin-1、nephrin,podocin的表达下调；与HG组比较，HG+VD组beclin-1、nephrin,podocin的表达呈浓度依赖性上调（P＜0.05）。.科学意义：为寻找能够阻止甚至逆转DKD 持续进展的药物提供理了论基础和实验依据。