RIP1K调节缺血性脑中风星形胶质细胞Necroptosis中溶酶体膜不稳定性的分子机制

More recently, we found that RIP1K，the key upper stream kinase of necroptosis, induced an increase in lysosomal membrane permeability in ischemia-mediated astrocytic necroptosis. However, the molecular signaling mechanisms of RIP1K-mediated instability of lysosomal membrane remain to be revealed. In an oxygen-glucose deprivation (OGD)-induced astrocytic cell injury, the experimental results of this project showed that using gene chip technology, RNA interference (RNAi) of RIP1K up-regulated the Hsp70.1B (heat shock protein 70.1B) gene Hspa1b in ischemic astrocytes. Double immunofluorescence staining further demonstrated that RNAi RIP1K increased the lysosomal Hsp70.1B protein levels in ischemic astrocytes. Hsp70.1 is known to stabilize lysosomal membrane and to protect cells against oxidative stresses. In addition, both genetic and pharmacological inhibition (Nec-1, specific inhibitor of RIP1K) of RIP1K decreased the protein levels of autophagic marker LC3II in ischemic astrocytes. Our above results strongly suggested that RIP1K regulates the instability of lysosomal membrane via interfering with the mechanisms of lysosomal membrane stability and the mechanisms of autophagic signaling pathway, and then increases the lysosomal membrane permeability in ischemic astrocytes. Therefore, in a rat model of permanent middle cerebral artery occlusion (pMCAO) and an OGD-induced primary cultured astrocytes injury model, this project will focus on investigating the molecular signaling mechanisms of RIP1K-mediated instability of lysosomal membrane in ischemic astrocytic necroptosis, inculuding that: ①RIP1K might mediate the instability of lysosomal membrane at the lysosomal membrane level. Upon ischemia, activated RIP1K might block the dissociation of heat-shock factor 1 (HSF1) from Hsp90, leading to an reduction in translocation of HSF1 to the nuclear and the transcription of Hsp70.1B, and in turn to resulting in an decrease in the protein levels of Hsp70.1B and lysosomal Hsp70.1B, and then increasing the lysosomal membrane permeability. In addition, RIP1K might increase carbonylated lysosomal Hsp70.1B by production of ROS-mediated HNE after ischemia. Carbonylated lysosomal Hsp70.1B is the substrate of activated calpain and is much more vulnerable to the calpain cleavage. Third, RIP1K might activte calpain and increase the cleavage of carbonylated lysosomal Hsp70.1B. ② RIP1K might mediate autophagic mechanisms via activation of PARP-1/AMPK/mTOR inhibition and /or ULK1 signaling pathways. ③ We will investivate the effects of novel analogues of Nec-1 synthesized by our group on RIP1K and RIP1K-mediated necroptosis and the instability of lysosomal membrane. This project will provide novel target for ischemic stroke therapy.

新近,课题组发现necroptosis关键上游激酶RIP1K增加缺血星形胶质细胞溶酶体酶膜通透性参与necroptosis,但RIP1K调控溶酶体膜通透性机制待揭示。预实验在缺糖缺氧星形胶质细胞损伤中意外发现:基因芯片技术显示RNAi RIP1K升高Hsp70.1B基因; 基因或药理（Nec-1）抑制RIP1K增加稳定溶酶体膜的溶酶体Hsp70.1B,减少LC3II;提示RIP1K调节溶酶体膜及自噬机制而增加膜通透性。本课题在整体脑缺血及星形胶质细胞缺糖缺氧模型,采用基因芯片及RNA干扰技术等研究:RIP1K调控溶酶体膜不稳定性机制:减少溶酶体膜Hsp70.1B,增加溶酶体Hsp70.1B羰基化及calpain对羰基化Hsp70.1B剪切;RIP1K调节PARP-1/AMPK/mTOR和/或ULK1激活自噬;课题组合成Nec-1新类似物对RIP1K的作用及调控溶酶体机制。以提供药物新靶点。

本项目按研究计划顺利全部执行完成。.Receptor-interacting protein 1 kinase (RIP1K)是necroptosis 关键上游激酶。课题组前期发现RIP1K 增加缺血性星形胶质细胞溶酶体酶膜通透性参与necroptosis。本项目进一步揭示了RIP1K 调控缺血性星形胶质细胞溶酶体膜通透性的分子机制。利用大鼠局灶性大脑中动脉阻塞脑缺血模型（MCAO）及体外原代培养大鼠大脑皮层星形胶质细胞缺糖缺氧（OGD）模型，发现：.（1）RIP1K 敲低抑制OGD诱导星形胶质细胞溶酶体不稳定性。透射电镜结果显示RIP1K敲低后星形胶质细胞的细胞结构及溶酶体膜结构较pMCAO组更加完整；Annexin-V-FITC 和PI染色结果显示RIP1K敲低后缺血性星形胶质细胞凋亡和坏死较OGD组明显减少；AO染色结果亦表明RIP1K敲低后星形胶质细胞溶酶体膜完整性增加。提示：基因抑制RIP1K对缺血性星形胶质细胞溶酶体膜具有保护作用。.（2）RIP1K 敲低抑制OGD诱导星形胶质细胞溶酶体不稳定性的机制：.① RIP1K 敲低抑制OGD诱导星形胶质细胞自噬/溶酶体途径激活.② RIP1K 敲低在溶酶体膜水平抑制OGD诱导星形胶质细胞溶酶体不稳定性. RIP1K敲低抑制溶酶体膜不稳定性机制与进一步增加缺血性星形胶质细胞溶酶体上Hsp70.1B水平有关。采用基因芯片、Western Blotting、免疫组织化学等多角度发现RIP1K敲低引起缺血性星形胶质细胞溶酶体Hsp70.1B蛋白水平及其基因Hsa1b水平均显著上调。而Hsp70.1B敲低加重pMCAO大鼠的脑损伤。. 进一步研究结果发现RIP1K基因敲低对Hsp70.1B的调控的分子机制为：基因抑制RIP1K可减少缺血性星形胶质细胞细胞浆内Hsp90水平及其与Hsf1的结合，促进Hsf1入核增加，从而增加Hsf1靶基因Hsp70.1B水平，而保护缺血性星形胶质细胞溶酶体膜的稳定性。.（3） 课题组合成2个RIP1K特异性抑制剂Nec-1类似物(E)-2-(3,5–二甲氧基苯亚甲基)-环戊酮（DMBC）及5-(3’，5’-二甲氧基苯甲撑基)-2-硫代-咪唑-4-酮(DTIO)均具有明显保护缺血性脑损伤的作用，其机制与抑制RIP1K激活及cathepsin B激活及其从溶酶体释放，保护溶酶体膜有关。