Department of Emergency Medicine, Korea University College of Medicine, Seoul, Korea.
J Surg Res. 2012 Nov;178(1):401-8. doi: 10.1016/j.jss.2012.01.055. Epub 2012 Mar 23.
The function of polymorphonuclear (PMN) cells can be influenced by the choice of resuscitation fluids in hemorrhagic shock. Widespread interest in the use of hypertonic solutions for resuscitation has led to extensive investigation of their immune-modulating properties. Hypertonic saline (HTS) is known to modulate immune reactions, preventing the multiorgan failure mediated by immune reactions in trauma and hemorrhagic shock. PMN cells play a key role in such immune-mediated inflammatory processes, and HTS is believed to affect these PMN cells. However, how these events influence the actual event of apoptosis has not yet been described. Thus, in the present study, we aimed to investigate the differences in the apoptosis of PMN cells when exposed to isotonic and hypertonic environments and the temporal relations between the interval of administration of HTS after the stimulation of PMN cells.
Whole blood was sampled from healthy volunteers, and the PMN cells were isolated. The isolated layer of PMN cells was washed twice with phosphate-buffered saline to yield the PMN cells. The number of cells was kept uniform, and an overall survival rate greater than 95% was maintained. After stimulation of the isolated PMN cells with N-formyl-methionyl-leucyl-phenylalanine, the PMN cells were allocated into 3 study groups (i.e., 1 isotonic group and 2 hypertonic groups with an osmolarity of 160 mM and 180 mM each). The extent of apoptosis was investigated in each group after culturing the PMN cells for 0, 1, 3, 6, 12, 15, 18, and 24 h. Depending on whether the PMN cells were stimulated with N-formyl-methionyl-leucyl-phenylalanine, they were also divided into stimulated and nonstimulated groups. In the stimulated group, the hypertonic environment was fostered immediately (HTS 0 h) and 6 h (HTS 6 h) after stimulation, which was accomplished after allocating the cells into an isotonic group (140 mM) and a hypertonic group (180 mM), according to the concentration of the culture medium. The PMN cells were then cultured at 37°C for 15 h with 5% carbon dioxide incubation. Each PMN suspension was labeled with Annexin V-fluorescein isothiocyanate and propidium iodide. Each sample underwent immediate flow cytometric analysis. PMN cells with high propidium iodide uptake were considered nonviable (necrotic). Among the viable PMN cells, those with no Annexin V uptake were considered normal and those with Annexin V uptake were considered apoptotic.
Decreased apoptosis was observed in the PMN cells stimulated with N-formyl-methionyl-leucyl-phenylalanine. Increased apoptosis was observed in the stimulated PMN cells incubated in hypertonic condition compared with the cells incubated in isotonic condition. Early HTS administration demonstrated increased apoptosis compared with late administration.
HTS treatment resulted in increased PMN apoptosis and an anti-inflammatory effect. Decreased apoptosis (prolonged lifespan) has been implicated in neutrophil-mediated tissue damage. HTS, by increasing the apoptosis of PMN cells, attenuates the postinjury inflammatory response. Also, early treatment with HTS was more efficient than delayed treatment.
在失血性休克中，选择复苏液会影响多形核（PMN）细胞的功能。人们广泛关注高渗溶液在复苏中的应用，这促使人们对其免疫调节特性进行了广泛的研究。高渗盐水（HTS）可调节免疫反应，防止创伤和失血性休克中免疫反应引起的多器官衰竭。PMN 细胞在这种免疫介导的炎症过程中起关键作用，HTS 被认为会影响这些 PMN 细胞。然而，这些事件如何影响细胞凋亡的实际过程尚未描述。因此，在本研究中，我们旨在研究在暴露于等渗和高渗环境时 PMN 细胞凋亡的差异，以及在 PMN 细胞刺激后给予 HTS 的时间间隔与细胞凋亡之间的时间关系。
从健康志愿者中抽取全血，并分离 PMN 细胞。用磷酸盐缓冲盐水（PBS）洗涤分离的 PMN 细胞两次，以获得 PMN 细胞。保持细胞数量均匀，存活率大于 95%。用 N-甲酰基-甲硫氨酸-亮氨酸-苯丙氨酸刺激分离的 PMN 细胞后，将 PMN 细胞分为 3 个研究组（即 1 个等渗组和 2 个渗透压分别为 160mM 和 180mM 的高渗组）。在培养 PMN 细胞 0、1、3、6、12、15、18 和 24 小时后，在每组中研究细胞凋亡的程度。根据是否用 N-甲酰基-甲硫氨酸-亮氨酸-苯丙氨酸刺激 PMN 细胞，将它们也分为刺激组和非刺激组。在刺激组中，在刺激后立即（HTS 0h）和 6 小时（HTS 6h）提供高渗环境，将细胞分配到等渗组（140mM）和高渗组（180mM）后完成，根据培养基的浓度。然后，将 PMN 细胞在 37°C 下孵育 15 小时，5%二氧化碳培养。将每个 PMN 悬浮液用 Annexin V-荧光素异硫氰酸酯和碘化丙啶标记。每个样本立即进行流式细胞术分析。摄取高碘化丙啶的 PMN 细胞被认为是不可存活的（坏死的）。在存活的 PMN 细胞中，未摄取 Annexin V 的细胞被认为是正常的，而摄取 Annexin V 的细胞被认为是凋亡的。
用 N-甲酰基-甲硫氨酸-亮氨酸-苯丙氨酸刺激的 PMN 细胞观察到细胞凋亡减少。与等渗条件下孵育的细胞相比，在高渗条件下孵育的刺激 PMN 细胞观察到细胞凋亡增加。与晚期给药相比，早期 HTS 给药显示出增加的细胞凋亡。
HTS 治疗导致 PMN 细胞凋亡增加和抗炎作用。PMN 介导的组织损伤中涉及细胞凋亡减少（延长寿命）。HTS 通过增加 PMN 细胞的凋亡，减轻创伤后的炎症反应。此外，早期治疗 HTS 比延迟治疗更有效。
