祛风通络法通过调节足细胞自噬通路不同节点治疗糖尿病肾病大量蛋白尿的机制研究

The results of previous studies suggested that Qufengtongluo method was effective in treating DN massive proteinuria. However, its molecular mechanism is still not clear. Studies from our group and peer researchers have confirmed that the occurrence of massive proteinuria in DN is closely related to the accumulation of damaged organelle and degradation-needed proteins caused by the disorder of autophagy pathway (including the prohibition of formation and/or degradation of autophagosome) in podocytes. Therefore, the hypothesis is proposed: Qufengtongluo method can promote the formation of autophagosome and/or the degradation of autophagosome, improving accumulation of damaged organelle and degradation-needed proteins, which result in ameliorating the damage of podocytes and then reducing the urinary protein. First of all, the podocytes cultured with sera of massive proteinuria DN patients will be chosen to be the model in this project, aiming at exploring whether the mechanism of Qufengtongluo recipe in treating DN is related to improving the accumulation of damaged mitochondria, ubiquitinated proteins and aging-related proteins. Then, the db/db mice and podocyte model will be the research object. The chemical/biological reagents that regulate different nodes of the autophagy pathway will be used as controls. ATG proteins, the formation of autolysosome and function of lysosome will obsearved to make sure whether this recipe eliminates the damaged organelle and degradation-needed proteins through promoting the formation of autophagosome and/or the degradation of autophagosome. All in all, The mechanism of Qufengtongluo method in treating massive proteinuria of diabetic nephropathy will be uncovered from the point of podocyte autophagy.

我们研究结果提示，祛风通络法治疗糖尿病肾病（DN）大量蛋白尿疗效显著，但机制仍不明确。团队及同行研究证实，DN大量蛋白尿的产生与足细胞自噬通路障碍（自噬泡形成障碍，和/或自噬泡降解受阻）导致待降解蛋白及细胞器堆积有关。因此，提出假说：祛风通络法通过诱导足细胞自噬泡形成，和/或疏通自噬泡降解，改善待降解蛋白及细胞器堆积，进而起到减轻足细胞损伤、降低尿蛋白的作用。本研究首先以大量尿蛋白DN患者血清培养的足细胞为研究对像，探讨祛风通络方治疗DN是否通过清除受损细胞器、泛素化蛋白以及衰老相关蛋白实现；进而，以db/db小鼠和体外足细胞模型为对象，以调控自噬通路不同节点的化学/生物试剂为对照，通过观察自噬相关蛋白表达、自噬溶酶体形成情况以及溶酶体功能，探讨该方清除受损细胞器及待降解蛋白是否通过诱导足细胞自噬和/或增加自噬泡降解实现。最终从足细胞自噬的角度揭示祛风通络法治疗DN大量蛋白尿的机制。

前期研究结果提示，祛风通络法治疗糖尿病肾病（DN）大量蛋白尿疗效显著，但机制仍不明确。团队及同行研究证实，DN大量蛋白尿的产生与足细胞自噬通路障碍有关。因此，提出假说：祛风通络法通过诱导足细胞自噬泡形成，和/或疏通自噬泡降解，改善待降解蛋白及细胞器堆积，进而起到减轻足细胞损伤、降低尿蛋白的作用。本研究以DKD动物模型（db/db小鼠）和体外足细胞模型(高糖/大量蛋白尿DKD患者血清刺激)为研究对象，以雷帕霉素、3-MA、海藻糖、氯喹等干预为对照，通过WB、IF、卵清蛋白降解实验、溶酶体蛋白酶活性检测、mRFP-GFP-LC3双荧光自噬检测系统等方法观察动物肾组织和体外足细胞自噬的情况。研究结果提示，中药祛风通络方确实可以起到降低尿蛋白的作用，且与其能够调节足细胞自噬有密切的联系。进一步研究发现，无论是高糖刺激的足细胞模型还是大量蛋白尿患者血清刺激的足细胞模型，结果均显示祛风通络方能够上调LC3、ATG5、ATG7、Beclin1的表达，下调P62的表达，而对溶酶体蛋白酶活性以及卵清蛋白降解没有调节作用。以上结果提示祛风通络方确实可以通过调节足细胞自噬保护足细胞，且其作用节点主要在自噬泡的形成阶段，而对溶酶体功能没有明显的调节作用。此外，中药还可以调节线粒体自噬以及衰老相关蛋白的表达。本研究结果进一步明确祛风通络方降低DKD动物模型尿蛋白的作用，且其机制与中药调节足细胞自噬有关，其作用节点在促进自噬泡形成有关。研究结果一定程度上揭示糖尿病肾病“肾络风动”病机的科学内涵，明确祛风通络法降尿蛋白的作用机制，这对提高中医药治疗糖尿病肾病的疗效，促进中医理论进步具有重要的意义。