电针调控NLRP3/caspase-1关键信号通路介导小胶质细胞焦亡 (pyroptosis)对脑梗死远隔损害的作用机制

The romote damages of acute cerebral infarction(ACI) aggravated nerve defect. Our team has recently found that PRG5 can downregulate the expression of LPA and RhoA proteins to inhibit the conductance of transmembrane signals and reduce the secretion of inflammatory factors. Electroacupuncture(EA) at Baihui and dazhui could regulate the expressions of RhoA and PRG5 to reduce the remote damages in substantia nigra, corpus striatum, hippocampus and brain stem. However, what about the effect of RhoA/ROS regulating NLRP3/caspase-1key signaling pathway mediated microglial pyroptosis for remote damages? How does EA to regulate NLRP3/caspase-1 signaling pathway inhibiting microglial pyroptosis to alleviate remote damages? Above scientifically new questions are still unclear. Our team will use vivo and vitro experiments to study the novel target that EA can induce remote damages of ACI by regulating NLRP3/caspase-1 signaling pathway in multi perspective: ① we will examine the expression of caspase-1, NLRP3, ASC in remote areas after siRNA-mediated silencing of NLRP3, the influence of pyroptosis in remote areas and the effect of EA would be assessed. ② we will detect the expression of IL-1β, IL-18, ROS, RhoA,and LPA, and the mechanism of EA on pyroptosis would be studied. ③ we will observe the influence of EA on cell death/nuclear shrinkage, DNA fragmentation and nerve repair in differet times of ACI remote areas, and we may provide the novel theory of EA on ACI remote damages.

急性脑梗死（ACI）远隔损害加重神经损伤。课题组近期发现PRG5阻遏LPA/RhoA抑制信号跨膜传导与小胶质细胞炎性因子分泌，电针百会、大椎穴调控RhoA/PRG5减轻黑质、纹状体、海马、脑干远隔损害。但RohA/ROS对NLRP3/caspase-1关键信号通路介导小胶质细胞焦亡致远隔损害有何影响，电针怎样调控NLRP3/caspase-1抑制细胞焦亡减轻远隔损害，这些新的科学问题不清楚。课题用体内、外实验多方位探讨电针介导NLRP3/caspase-1调控小胶质细胞焦亡为新靶点：①沉默NLRP3基因，检测inflammesome相关蛋白caspase-1、NLRP3、ASC，明确细胞焦亡对远隔损害影响及电针作用；②检测IL-1β、IL-18、ROS、RhoA，明确电针对细胞焦亡作用与机制；③观察电针不同时点对神经细胞TUNEL、DNA断裂修复作用，为电针减轻ACI远隔损害提供新理论。

急性脑梗死（ACI）继发远隔部位海马、脑干和脊髓等椎体束联系纤维发生轴突断裂、髄鞘脱失、轴索溶解、神经元丢失、破坏等远隔损害是导致其病损严重、致残率高、神经功能康复与治疗困难的重要原因。新近发现，ACI小胶质细胞分泌和释放一系列神经毒性物质和炎性因子，快速启动NLRP3/caspase-1信号通路引起级联放大式炎症反应，诱发一种新的程序性细胞死亡——焦亡(pyroptosis)，阻碍神经功能修复，导致偏瘫、失语、痴呆、吞咽障碍等后遗症，是威胁人类健康的一大医学难题。.本研究基于此探讨MCAO缺血再灌注不同时间（6h、24h、3d、7d）脑梗死灶缺血半影区与远隔损害部位小胶质细胞焦亡变化情况，明确小胶质细胞焦亡在 ACI 远隔损害中的作用及机制。研究电针干预对MCAO再灌注不同时间梗死灶远隔损害部位小胶质细胞焦亡的影响，明确电针调控NLRP3/caspase-1介导小胶质细胞焦亡的作用及机制。多方位动态观察电针对不同时间ACI 远隔损害部位小胶质细胞形态、神经纤维束与轴突再生变化的影响，明确电针调控 NLRP3/caspase-1 介导小胶质细胞焦亡对神经再生与修复的作用及机制。.研究结果显示，电针能显著降低神经功能缺损评分，减少MCAO模型的梗死脑组织体积，下调患侧远隔部位中NLRP3/caspase-1关键信号通路相关蛋白的表达，并进一步影响其介导的细胞焦亡。.其中部分关键数据发现，通过TTC染色显示EA减少缺血脑组织的梗死面积（与MCAO组比较，EA组有显著性差异（P=0.041＜0.05,）；Immunofluorescence、Immunohistochemistry、Western Blot、RT-PCR等观察到EA下调患侧CI/R大鼠海马区域RhoA、LPA、LPA-R、caspase-1、NLRP3、ASC、IL-1β等蛋白表达，此外，EA干预CI/R大鼠在不同时点患侧脑桥caspase-1、IL-1β、NLRP3、ASC、RhoA、LPA、LPA-R蛋白定性及定量的影响。通过LDH、TUNEL观察电针可以干预梗死灶远隔损害部位丘脑、纹状体、海马等不同时间点小胶质细胞焦亡的表达。.因此，从 NLRP3/caspase-1 关键信号通路介导细胞焦亡的新角度，研究电针减轻远隔损害的新靶点，对降低 ACI 残疾具有重要的社会意义。