ROLE OF ENDOPLASMIC RETICULUM STRESS IN IMPAIRED NEONATAL LUNG GROWTH AND BRONCHOPULMONARY DYSPLASIA. ROLE OF ENDOPLASMIC RETICULUM STRESS IN IMPAIRED NEONATAL LUNG GROWTH AND BRONCHOPULMONARY DYSPLASIA

Myeloperoxidase (MPO), oxidative stress (OS), and endoplasmic reticulum (ER) stress are all increased in the lungs of neonatal rat pups raised in hyperoxia (HOX, >90% O2), an established model of bronchopulmonary dysplasia (BPD). However, the relationship between OS, MPO, and ER stress has not been examined in HOX rat pups. To determine the relationship between OS, MPO, and ER stress in BPD we treated Sprague-Dawley neonatal rat pups with Tunicamycin (Tun) or with HOX. Tun directly induces ER stress and simplifies neonatal lung alveolarization. Previously, we showed that HOX induces a cycle of destruction that we hypothesize through increased OS, MPO, and ER stress to induce BPD. To inhibit ER stress specifically, we used tauroursodeoxycholic acid (TUDCA), a molecular chaperone. To break the cycle of destruction and reduce OS and MPO we used N-acetyl-lysyltyrosylcysteine-amide (KYC), a systems pharmacology agent. Lung structure was studied morphometrically. ER stress was detected using immunofluorescence (IF), transcriptomic, proteomic, and electron microscopic analyses. Increased ER stress was observed in the lungs of HOX rat pups and also in human BPD lungs by IF. Morphometric and proteomic studies of rat lungs showed that Tun treatment decreased lung complexity and increased ER stress and BPD severity. The fact that TUDCA improved lung complexity in Tun-treated neonatal rat pups, and decreased BPD induced by HOX provides strong support for the idea that ER stress plays a causal role in BPD. Additional support comes from data showing TUDCA decreased lung myeloid cells and MPO levels in the lungs of both Tun- and HOX-treated neonatal rat pups. These data link OS and MPO to ER stress in the mechanisms mediating BPD. KYC's effective inhibition of ER stress in the lungs of Tun-treated rat pups provides additional support for the idea that MPO-induced ER stress plays a direct causal role in BPD. Thus, ER stress appears to expand our proposed cycle of destruction. Our results suggest ER stress evolves from OS and MPO to increase neonatal lung injury and impaired neonatal lung growth and development. The encouraging effect of TUDCA indicates chemical chaperone has the potential in treating BPD. Using multiomic data to investigate the role of endoplasmic reticulum stress in the development of BPD in rat model. Overall design: Nursing dams were cared for in a 12-h dark-light cycle and had free access to chow and water. Two or four pregnant rats were used for each experiment, according to the study design. The dam and pups were cared for in either >90% oxygen chamber (HOX) or room air (NOX) from postnatal day 1 to day 10 (P1-P10). Oxygen concentrations were continuously monitored with an oxygen sensor (Reming Bioinstruments Co., Redfield, NY). Pups were caged with nursing dams while the dams were alternated between oxygen environments to diminish oxygen toxicity.

髓过氧化物酶（Myeloperoxidase, MPO）、氧化应激（oxidative stress, OS）与内质网应激（endoplasmic reticulum stress, ER stress）在高氧（hyperoxia, HOX，氧浓度>90%）环境下饲养的新生大鼠肺组织中均表达上调，而高氧模型是已确立的支气管肺发育不良（bronchopulmonary dysplasia, BPD）动物模型。但目前尚未有研究在高氧暴露新生大鼠中探究氧化应激、髓过氧化物酶与内质网应激三者间的关联。为明确支气管肺发育不良发病过程中氧化应激、髓过氧化物酶与内质网应激的关联，本研究对斯普拉格-道利（Sprague-Dawley）新生大鼠给予衣霉素（Tunicamycin, Tun）处理或高氧暴露。衣霉素可直接诱导内质网应激，同时会干扰新生大鼠肺脏的肺泡发育进程。既往研究显示，高氧暴露会引发肺损伤循环，我们推测该循环通过上调氧化应激、髓过氧化物酶与内质网应激水平，进而诱导支气管肺发育不良。为特异性抑制内质网应激，我们使用了牛磺熊去氧胆酸（tauroursodeoxycholic acid, TUDCA），一种分子伴侣；为打破该损伤循环并降低氧化应激与髓过氧化物酶水平，我们使用了N-乙酰-赖氨酰酪氨酰半胱氨酸酰胺（N-acetyl-lysyltyrosylcysteine-amide, KYC），一种系统药理学制剂。本研究通过形态计量学分析评估肺组织结构；采用免疫荧光（immunofluorescence, IF）、转录组学、蛋白质组学与电子显微镜分析检测内质网应激情况。免疫荧光检测结果显示，高氧暴露新生大鼠的肺组织与人类支气管肺发育不良肺组织中均存在内质网应激上调现象。对大鼠肺组织的形态计量与蛋白质组学研究表明，衣霉素处理会降低肺组织复杂度，同时上调内质网应激水平并加重支气管肺发育不良的严重程度。牛磺熊去氧胆酸可改善衣霉素处理新生大鼠的肺组织复杂度，并减轻高氧诱导的支气管肺发育不良，这一结果为内质网应激在支气管肺发育不良中发挥因果作用提供了强有力的证据支持。另有数据显示，牛磺熊去氧胆酸可降低衣霉素与高氧处理新生大鼠肺组织中的髓系细胞数量与髓过氧化物酶水平，进一步佐证了上述结论。这些数据将氧化应激、髓过氧化物酶与介导支气管肺发育不良的内质网应激机制关联起来。N-乙酰-赖氨酰酪氨酰半胱氨酸酰胺可有效抑制衣霉素处理大鼠肺组织中的内质网应激，这一结果进一步支持了髓过氧化物酶诱导的内质网应激在支气管肺发育不良中发挥直接因果作用的观点。综上，内质网应激似乎拓展了我们此前提出的肺损伤循环假说。本研究结果提示，内质网应激由氧化应激与髓过氧化物酶介导，进而加剧新生肺损伤并损害新生肺的生长发育。牛磺熊去氧胆酸的喜人疗效表明，化学分子伴侣在支气管肺发育不良的治疗中具有潜在应用价值。本研究以大鼠为模型，通过多组学数据探究内质网应激在支气管肺发育不良发生发展中的作用。实验整体设计如下：哺乳母鼠于12小时明暗循环环境中饲养，自由摄食标准饲料与饮水。根据实验设计，每次实验使用2或4只孕鼠。从出生后第1天至第10天（P1-P10），将母鼠与幼崽置于氧浓度>90%的高氧舱（HOX组）或正常空气环境（NOX组）中。氧浓度通过Reming生物仪器公司（Reming Bioinstruments Co., Redfield, NY）生产的氧气传感器持续监测。幼崽与哺乳母鼠同笼饲养，期间交替更换母鼠所处的氧环境，以降低母鼠的氧毒性暴露。