Xu He N, Floros Joanna, Li Lin Z, Amatya Shaili
Britton Chance Laboratory of Redox Imaging, Department of Radiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
Departments of Pediatrics and Obstetrics and Gynecology, The Pennsylvania State University College of Medicine, Hershey, PA 17033, USA.
Antioxidants (Basel). 2021 May 12;10(5):767. doi: 10.3390/antiox10050767.
Employing the optical redox imaging technique, we previously identified a significant redox shift of nicotinamide adenine dinucleotide (NAD and the reduced form NADH) in freshly isolated alveolar macrophages (AM) from ozone-exposed mice. The goal here was twofold: (a) to determine the NAD(H) redox shift in cryopreserved AM isolated from ozone-exposed mice and (b) to investigate whether there is a difference in the redox status between cryopreserved and freshly isolated AM. We found: (i) AM from ozone-exposed mice were in a more oxidized redox state compared to that from filtered air (FA)-exposed mice, consistent with the results obtained from freshly isolated mouse AM; (ii) under FA exposure, there was no significant NAD(H) redox difference between fresh AM that had been placed on ice for 2.5 h and cryopreserved AM; however, under ozone exposure, fresh AM were more oxidized than cryopreserved AM; (iii) via the use of nutrient starvation and replenishment and HO-induced oxidative stress of an AM cell line, we showed that this redox difference between cryopreserved and freshly isolated AM is likely the result of the double "hit", i.e., the ozone-induced oxidative stress plus nutrient starvation that prevented freshly isolated AM from a full recovery after being on ice for a prolonged time period. The cryopreservation technique we developed eliminates/minimizes the effects of oxidative stress and nutrient starvation on cells. This method can be adopted to preserve lung macrophages from animal models or clinical patients for further investigations.
我们先前运用光学氧化还原成像技术，在从暴露于臭氧的小鼠中新鲜分离的肺泡巨噬细胞（AM）中，鉴定出烟酰胺腺嘌呤二核苷酸（NAD和还原形式NADH）存在显著的氧化还原变化。此处的目标有两个：（a）确定从暴露于臭氧的小鼠中分离出的冷冻保存的AM中的NAD（H）氧化还原变化，以及（b）研究冷冻保存的AM和新鲜分离的AM之间的氧化还原状态是否存在差异。我们发现：（i）与暴露于过滤空气（FA）的小鼠的AM相比，暴露于臭氧的小鼠的AM处于更氧化的还原状态，这与从新鲜分离的小鼠AM中获得的结果一致；（ii）在FA暴露下，放置在冰上2.5小时的新鲜AM和冷冻保存的AM之间没有显著的NAD（H）氧化还原差异；然而，在臭氧暴露下，新鲜AM比冷冻保存的AM更氧化；（iii）通过对AM细胞系使用营养饥饿和补充以及HO诱导的氧化应激，我们表明冷冻保存的AM和新鲜分离的AM之间的这种氧化还原差异可能是双重“打击”的结果，即臭氧诱导的氧化应激加上营养饥饿，这阻止了新鲜分离的AM在长时间置于冰上后完全恢复。我们开发的冷冻保存技术消除/最小化了氧化应激和营养饥饿对细胞的影响。这种方法可用于保存动物模型或临床患者的肺巨噬细胞以供进一步研究。
