■中暑是过度热应激的结果,导致核心温度超过40°C,并伴有一系列并发症。大脑特别容易受到热应激的损害。在我们之前的研究中,在中暑小鼠的皮层中发现了激活的小胶质细胞和增加的神经元自噬。然而,活化的小胶质细胞在热应激条件下是否能加速神经元自噬仍是未知的。在这项研究中,我们的目的是从外泌体介导的细胞间通讯的角度,探讨中暑时神经元自噬上调的潜在机制.
■在这项研究中,使用BV2和N2a细胞代替小胶质细胞和神经元,分别。通过超速离心从BV2培养上清液中提取外泌体,然后通过透射电子显微镜进行表征。纳米粒子跟踪分析和蛋白质印迹。将用/不用miR-155抑制剂预处理的N2a细胞与用/不用热应激或miR-155过表达处理的小胶质外泌体共培养,随后进行热应激处理。通过透射电镜检测自噬体和自噬相关蛋白来评估N2a细胞的自噬,免疫荧光,和西方印迹。使用实时逆转录聚合酶链反应测量miR-155在BV2和BV2外泌体以及N2a细胞中的表达。靶标结合分析通过双荧光素酶报告基因测定进行验证。
■N2a自噬响应于热应激而适度增加,并通过将外泌体转移到神经元而被BV2细胞加速。此外,我们发现神经元自噬与小胶质细胞外泌体中miR-155的含量呈正相关。抑制miR-155可部分消除N2a细胞的自噬,通过与miR-155上调的外泌体共培养而增加。机制分析证实Rheb是miR-155的功能靶点,小胶质细胞外泌体miR-155主要通过调节Rheb-mTOR信号通路加速热应激诱导的神经元自噬。
■中暑后小胶质细胞外泌体中miR-155的增加可通过转移至神经元诱导神经元自噬。miR-155通过靶向Rheb发挥这些作用,从而抑制mTOR信号的活性。因此,miR-155可能是中暑后神经元自噬干预的有希望的靶标。
UNASSIGNED: Heat stroke is the outcome of excessive heat stress, which results in core temperatures exceeding 40°C accompanied by a series of complications. The brain is particularly vulnerable to damage from heat stress. In our previous studies, both activated microglia and increased neuronal autophagy were found in the cortices of mice with heat stroke. However, whether activated microglia can accelerate neuronal autophagy under heat stress conditions is still unknown. In this study, we aimed to investigate the underlying mechanism that caused neuronal autophagy upregulation in heat stroke from the perspective of exosome-mediated intercellular communication.
UNASSIGNED: In this study, BV2 and N2a cells were used instead of microglia and neurons, respectively. Exosomes were extracted from BV2 culture supernatants by ultracentrifugation and then characterized via transmission electron microscopy, nanoparticle tracking analysis and Western blotting. N2a cells pretreated with/without miR-155 inhibitor were cocultured with microglial exosomes that were treated with/without heat stress or miR-155 overexpression and subsequently subjected to heat stress treatment. Autophagy in N2a cells was assessed by detecting autophagosomes and autophagy-related proteins through transmission electron microscopy, immunofluorescence, and Western blotting. The expression of miR-155 in BV2 and BV2 exosomes and N2a cells was measured using real-time reverse transcription polymerase chain reaction. Target binding analysis was verified via a dual-luciferase reporter assay.
UNASSIGNED: N2a autophagy moderately increased in response to heat stress and accelerated by BV2 cells through transferring exosomes to neurons. Furthermore, we found that neuronal autophagy was positively correlated with the content of miR-155 in microglial exosomes. Inhibition of miR-155 partly abolished autophagy in N2a cells, which was increased by coculture with miR-155-upregulated exosomes. Mechanistic analysis confirmed that Rheb is a functional target of miR-155 and that microglial exosomal miR-155 accelerated heat stress-induced neuronal autophagy mainly by regulating the Rheb-mTOR signaling pathway.
UNASSIGNED: Increased miR-155 in microglial exosomes after heat stroke can induce neuronal autophagy via their transfer into neurons. miR-155 exerted these effects by targeting Rheb, thus inhibiting the activity of mTOR signaling. Therefore, miR-155 could be a promising target for interventions of neuronal autophagy after heat stroke.