Impact of Airway Inflammation on Mitochondria

急性气道炎症的影响由促炎细胞因子（例如，TNFα）， 并且是许多呼吸道疾病的基础。一个基本问题是为什么有些人 个体比其他人更容易受到气道炎症的负面影响。我们将 探索一种新的稳态机制，保护气道平滑肌（hASM）细胞免受 炎症诱导的活性氧（ROS）形成的负面影响， 蛋白质解折叠（内质网（ER）应激）。我们相信这种自我平衡的失败 这一机制导致ROS形成增加，从而加剧氧化和ER应激。 总体假设：TNFα诱导的ROS形成和蛋白质去折叠激活了 pIRE 1 α/XBP 1 s在hASM中的ER应激途径，其启动了定向的稳态应答 增加线粒体生物合成和线粒体体积密度以减少O2 消费和ROS形成的个人candion，同时仍然满足增加， ATP需求-在线粒体中分担能量负荷。此外，减少的Mfn 2 破坏线粒体对ER的束缚，从而减少线粒体Ca 2+内流， 线粒体的最大呼吸能力。 目的1：TNFα激活pIRE 1 α/XBP 1 s内质网应激通路， 体积密度和减少O2消耗和ROS形成每加仑。 在hASM细胞中，TNFα诱导的pIRE 1 α/XBP 1 s ER激活的下游影响， 将使用非磷酸化IRE 1 α突变体的转染来探索应激途径 质粒（DP-IRE 1 α）或不可剪接的XBP 1（uXBP 1）mRNA。此外，我们亦会研究 siRNA敲低PGC 1 α和Mfn 2过表达对TNFα诱导的细胞凋亡的影响 线粒体生物发生、线粒体体积密度、O2消耗和ROS形成。 目的2：TNFα诱导的Mfn 2减少破坏了线粒体与ER的连接， 线粒体Ca 2+内流和降低线粒体的最大呼吸能力。 在hASM细胞中，我们将检测DP-IRE 1 α或uXBP 1 mRNA转染的影响， siRNA Mfn 2敲低对TNFα诱导的线粒体/ ER束缚的破坏，降低 线粒体Ca ~（2+）内流和线粒体最大呼吸能力降低。 目的3：探讨TNFα对pIRE 1 α/XBP 1 s ER应激通路的影响， 通过ROS清除和化学伴侣处理减轻下游效应。 在hASM细胞中，清除ROS和化学伴侣处理的缓解作用 将检查TNFα诱导的pIRE 1 α/XBP 1 s ER应激途径活化的影响。

The impact of acute airway inflammation is mediated by pro-inflammatory cytokines (e.g., TNFα), and underlies a number of respiratory diseases. A fundamental question is why are some individuals more susceptible than others to the negative impact of airway inflammation. We will explore a novel homeostatic mechanism, which protects airway smooth muscle (hASM) cells from the negative impact of inflammation-induced reactive oxygen species (ROS) formation and protein unfolding (endoplasmic reticulum (ER) stress). We believe that a failure in this homeostatic mechanism leads to increased ROS formation thereby exacerbating oxidative and ER stress. Overall Hypothesis: TNFα-induced ROS formation and protein unfolding activates the pIRE1α/XBP1s ER stress pathway in hASM, which initiates a homeostatic response directed towards increasing mitochondrial biogenesis and mitochondrial volume density to reduce O2 consumption and ROS formation by individual mitochondrion, while still meeting the increase in ATP demand – sharing the energetic load across mitochondria. Furthermore, reduced Mfn2 disrupts mitochondrial tethering to the ER, thereby decreasing mitochondrial Ca2+ influx and maximum respiratory capacity of mitochondria. Aim 1: TNFα-induced activation of pIRE1α/XBP1s ER stress pathway increases mitochondrial volume density and reduces O2 consumption and ROS formation per mitochondrion. In hASM cells, the downstream impact of TNFα-induced activation of the pIRE1α/XBP1s ER stress pathway will be explored using transfection of a non-phosphorylatable IRE1α mutant plasmid (DP-IRE1α) or an unspliceable XBP1 (uXBP1) mRNA. In addition, we will examine the effects of siRNA knockdown of PGC1α and Mfn2 overexpression on TNFα-induced changes in mitochondrial biogenesis, mitochondrial volume density, O2 consumption and ROS formation. Aim 2: TNFα-induced reduction in Mfn2 disrupts mitochondrial tethering to ER, decreases mitochondrial Ca2+ influx and reduces maximum respiratory capacity of mitochondria. In hASM cells, we will examine the impact of DP-IRE1α or uXBP1 mRNA transfection and siRNA Mfn2 knockdown on TNFα-induced disruption of mitochondrial/ ER tethering, decreased mitochondrial Ca2+ influx and reduced maximum respiratory capacity of mitochondria. Aim 3: The impact of TNFα on activation of the pIRE1α/XBP1s ER stress pathway and downstream effects are mitigated by ROS scavenging and chemical chaperone treatment. In hASM cells, the mitigating effects of ROS scavenging and chemical chaperone treatment on TNFα-induced activation of the pIRE1α/XBP1s ER stress pathway will be examined.