lncRNA MEG3/miR-676对TLR4/NF-κB/NLRP3信号通路介导NVU焦亡的调控在DHCA脑损伤机制的探究

Deep hypothermic circulatory arrest (DHCA) brain injury is the most important factor influencing the prognosis of patients. Activation of NLRP3 inflammasome causes neurovascular units (NVU) pyroptosis as a key link in brain injury. Our previous studies found that TLR4/NF-κB pathway regulates the expression of Occludin and causes increased permeability-induced brain injury, but the key mechanism has not yet been elucidated. Pre-experimental studies revealed that lncRNA MEG3/miR-676 has potential sites for binding to NF-κB and NLRP3, respectively, and therefore we speculated that lncRNA MEG3/miR-676 is involved in regulating the activation of TLR4/NF-κB/NLRP3 signaling pathways, inducing Occludin degradation and NVU pyroptosis caused brain damage. This study intends to use siRNA, RIP, luciferase, and adenovirus technologies in combination with DHCA in vitro models to further validate this hypothesis and to explore the role of lncRNA MEG3/miR-676 in mediating NVU against TLR4/NF-κB/NLRP3 signaling pathways. The mechanism of focal regulation in DHCA brain injury provides a theoretical basis for finding endogenously regulated targeting molecules and developing novel brain protection strategies.

深低温停循环（DHCA）脑损伤是影响患者预后最重要的因素，激活NLRP3炎性小体引起神经血管单元（NVU）焦亡是脑损伤的关键环节。我们前期的研究发现TLR4/NF-κB通路调控Occludin表达引起通透性增加致脑损伤，但关键机制尚未阐明。预实验发现lncRNA MEG3/miR-676具有分别与NF-κB 和NLRP3结合的潜在位点，因此我们推测lncRNA MEG3/miR-676参与调控TLR4/NF-κB/ NLRP3信号通路的激活，诱导Occludin降解和NVU焦亡，最终导致脑损伤。本研究拟联合使用siRNA、RIP、荧光素酶和腺病毒等技术在DHCA体外体内模型进一步验证这一推测，旨在探究lncRNA MEG3/miR-676对TLR4/NF-κB/NLRP3信号通路介导NVU焦亡的调控在DHCA脑损伤的机制，为寻找内源性调控靶向分子、开发新颖的脑保护策略提供理论依据。

背景：在我们以前的研究中，我们发现氧-葡萄糖剥夺和复氧（OGD/R）激活了脑微血管内皮细胞（BMECs）的toll样受体4（TLR4）信号，并诱发热病和炎症。 在这项研究中，我们旨在研究(6R)-6-[N-(2-氯-4-氟苯基)氨基磺酰基]环己-1-甲酸乙酯（TAK-242）对OGD/R下BMECs的影响。.方法：分别用细胞计数试剂盒-8（CCK-8）检测、酶联免疫吸附试验（ELISA）和wb测定细胞活力、炎症因子、炎症相关的热解作用和核因子-κB（NF-κB）信号传导。 用RNA深层测序分析了长非编码RNAs（lncRNAs）和信使RNAs（mRNAs）的表达模式。 此外，用液相色谱-串联质谱法（LC-MS/MS）对lncRNAs编码的短肽进行了鉴定和定量。.结果：TAK-242促进了OGD/R细胞的活力，减少了OGD/R引起的炎症因子的分泌，并抑制了TLR4/NLRP3/Caspase-1和TLR4/NF-κB的途径。 此外，与对照组相比，AABR07000411.1、AABR070006957.1和AABR070008256.1在OGD/R细胞中的表达减少，但TAK-242在OGD/R条件下恢复了它们的表达。 AABR07000473.1、AC130862.4和LOC10254972.6被OGD/R诱导，但与OGD/R相比，在TAK-242+OGD/R细胞中被抑制。 此外，AABR07049961.1、AC127076.2、AABR07066020.1和AABR07025303.1编码的短肽在OGD/R细胞中失调，而TAK-242减轻了AABR07049961.1、AC127076.2和AABR07066020.1编码短肽的失调。.结论：TAK-242改变了OGD/R细胞中lncRNAs的表达模式，不同表达的lncRNAs可能通过竞争性内源性RNA（ceRNA）和编码短肽的机制对OGD/R损伤发挥保护作用。