Impact of Airway Inflammation on Mitochondria

急性呼吸道炎症的影响是由促炎细胞因子(例如，肿瘤坏死因子α)介导的。 并引发了许多呼吸道疾病。一个根本的问题是为什么有些人 个人比其他人更容易受到呼吸道炎症的负面影响。我们会 探索一种新的动态平衡机制，保护气道平滑肌细胞 炎症诱导的活性氧物种(ROS)的形成和 蛋白质展开(内质网(ER)应激)。我们认为，这种内环境平衡的失败 机制导致ROS的形成增加，从而加剧氧化和内质网应激。 总体假设：肿瘤坏死因子α诱导的ROS形成和蛋白质展开激活 HASM中的pIRE1α/XBP1s ER应激通路，启动定向的动态平衡反应 增加线粒体生物发生和线粒体体密度以降低O2 单个线粒体的消耗和ROS形成，同时仍满足 ATP需求--跨线粒体分享能量负荷。此外，减少了Mfn2 破坏线粒体与内质网的连接，从而减少线粒体钙离子内流，并 线粒体的最大呼吸量。 目的1：肿瘤坏死因子α激活pIRE1α/XBP1S ER应激通路增加线粒体 减少线粒体的氧耗和ROS的形成。 在hASM细胞中，肿瘤坏死因子α诱导pIRE1α/XBP1S ER活化的下游影响 通过导入非磷酸化的ire1α突变体来探索应激途径 质粒(DP-IRE1mRNA)或不可剪接的α(UXBP1mRNA)。此外，我们还将研究 SiRNA下调前列腺素C_1α和MFN_2过表达对肿瘤坏死因子α诱导的血管内皮细胞凋亡的影响 线粒体生物发生、线粒体体密度、耗氧量和ROS的形成。 目的2：肿瘤坏死因子α诱导的Mfn2减少破坏线粒体与内质网的连接，减少 线粒体Ca~(2+)内流，降低线粒体最大呼吸量。 在hASM细胞中，我们将检测DP-IRE1α或uXBP1 mRNA转染对HASM细胞的影响 SiRNAMfn2对肿瘤坏死因子α诱导的线粒体/内质网连接中断的抑制作用 线粒体Ca~(2+)内流，降低线粒体最大呼吸量。 目的3：肿瘤坏死因子α对pIRE1α/XBP1s ER应激通路激活的影响 下游效应可通过清除ROS和化学伴侣处理来缓解。 在hASM细胞中，ROS清除和化学伴侣处理的缓解作用 关于肿瘤坏死因子α诱导的pIRE1α/XBP1S ER应激通路的激活，将被检测。

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.