| 孙康傅,杨礼璟,付泽宇,徐百川,陈石磊,王军平.睡眠障碍对辐照后小鼠骨髓造血干细胞的影响[J].中华放射医学与防护杂志,2024,44(2):96-104 |
| 睡眠障碍对辐照后小鼠骨髓造血干细胞的影响 |
| Effects of sleep disorders on hematopoietic stem cells in bone marrow of irradiated mice |
| 投稿时间：2023-11-19 |
| DOI：10.3760/cma.j.cn112271-20231119-00177 |
| 中文关键词: 睡眠障碍 电离辐射 造血干细胞 烟酰胺腺嘌呤二核苷酸磷酸氧化酶2 半胱氨酸天冬氨酸蛋白酶-1 |
| 英文关键词:Sleep disorders Ionizing radiation Hematopoietic stem cell NOX2 Caspase-1 |
| 基金项目:国家自然科学基金面上项目(82222060,82073487);重庆市研究生教育创新计划(CYS22742) |
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| 中文摘要: |
| 目的 探讨睡眠障碍(sleep disorders,SD)对骨髓造血干细胞放射损伤的影响。方法 采用6~8周龄C57BL/6J雄性小鼠56只,以60Co γ射线对小鼠进行全身照射5.0、7.5 Gy。通过睡眠障碍仪建立SD模型,按照随机数表法,将小鼠分为对照组(Con组)、睡眠障碍组(SD组)、单纯照射组(IR组)、照后SD组(IR+SD组)、照后SD给磷酸盐缓冲盐溶液(PBS)组(IR+SD+PBS组)、照后SD给GSK2795039组(IR+SD+GSK组)、照后SD给N-乙酰半胱氨酸(NAC)组(IR+SD+NAC组),共7组,每组8只。取小鼠尾静脉血,检测5.0 Gy受照后外周血变化;并观察7.5 Gy照后小鼠生存率。采用苏木精和伊红(HE)染色观察骨髓细胞致密性与细胞数量变化。通过流式细胞术,检测骨髓造血干细胞(LSK)的数量,以及凋亡水平和细胞周期变化,分析LSK的活性氧(ROS)、线粒体来源活性氧(mtROS)等指标。采用酶标仪检测烟酰胺腺嘌呤二核苷酸磷酸(NADP+/NADPH)和谷胱甘肽(GSSG/GSH)观察LSK的氧化应激水平。采用流式细胞术,检测烟酰胺腺嘌呤二核苷酸磷酸氧化酶2(NOX2)及半胱氨酸天冬氨酸蛋白酶-1(caspase-1)的表达,分选小鼠LSK细胞,并采用聚合酶链式反应(PCR)检测NOX1-4、白介素1β(IL-1β)、白介素6(IL-6)、白介素18(IL-18)、肿瘤坏死因子α(TNF-α)炎症因子的表达。结果 与IR组相比,IR+SD组小鼠白细胞(WBC)和血小板(PLT)恢复显著减慢(t=4.39、6.37,P＜0.05),骨髓细胞计数由(2.14±0.38)×107降至(3.59±0.29)×107(t=8.55,P＜0.05),LSK细胞的G0期比例显著降低,凋亡比例显著升高(t=7.53、8.21,P＜0.05),ROS、mtROS、NADP+/NADPH水平均显著升高(t=22.99、29.47、3.77,P＜0.05)。IR情况下,SD进一步促进NOX2及Caspase-1活化及下游IL-1β、IL-6、IL-18、TNF-α炎症因子mRNA表达的升高(t=6.95、6.01、8.39、4.91、5.56,P＜0.05)。抑制NOX2-ROS可以挽救SD诱导的照后造血损伤加重,从而显著减少LSK凋亡比例以及炎症因子表达,最终加速LSK的损伤恢复(t=9.24、3.92,P＜0.05)。结论 SD促进了IR介导的骨髓造血干细胞损伤,主要通过激活NOX2-ROS-Caspase-1轴使细胞内炎症因子和ROS水平升高,促进细胞凋亡,最终抑制骨髓造血恢复。 |
| 英文摘要: |
| Objective To investigate the effects of sleep disorders (SD) on the radiation injury of hematopoietic stem cells (HSCs) in bone marrow (BM). Methods Totolly 56 C57BL/6J male mice aged 6-8 weeks were enrolled in this study. They were subjected to whole body irradiation of 60Co γ-rays with doses of 5.0 and 7.5 Gy. A SD model was established using a SD device. According to the random number table method, the mice were divided into seven groups: the control group (Con group), the SD group, the mere radiation group (IR group), the group of post-irradiation SD (IR+SD group), the group of post-irradiation SD treated with phosphate buffer solution (IR+SD+PBS group), the group of post-irradiation SD treated with GSK2795039 (IR+SD+GSK group), and the group of post-irradiation SD treated with N-acetylcysteine (IR+SD+NAC group), with in eight mice each group. The changes in the peripheral blood of the mice after 5.0 Gy irradiation were detected using the collected tail venous blood, and the survival rates of the mice after 7.5 Gy irradiation were observed. The changes in the density and count of bone marrow cells were observed using hematoxylin and eosin (HE) staining. The number of hematopoietic stem cells in bone marrow (LSK cells), as well as their apoptosis level and changes in cell cycle, were detected using flow cytometry. Furthermore, indicators of LSK, such as reactive oxygen species(ROS) and mitochondrial-derived reactive oxygen species (mtROS), were analyzed. Nicotinamide adenine dinucleotide phosphate (NADP+/NADPH) and glutathione (GSSG/GSH) were detected using an enzyme microplate reader in order to observe the oxidative stress level of LSK. Furthermore, flow cytometry was employed to sort the LSK cells from the mice, and flow cytometry was used to detect the expression of NADPH oxidase 2(NOX2) and cysteinyl aspartate specific proteinnase-1(Caspase-1), and polymerase chain reaction (PCR) was used to detect the expression of inflammatory factors such as NOX1-4, interleukin 1β (IL-1β), interleukin 6 (IL-6), interleukin 18 (IL-18), and tumor necrosis factor α (TNF-α). Results Compared to the IR group, the IR+SD group exhibited significantly slower recovery of white blood cells (WBC) and platelets (PLT) (t = 4.39, 6.37, P ＜ 0.05), the bone marrow cell count decreasing from (2.14 ± 0.38) × 107 to (3.59 ± 0.29) × 107 (t = 8.55, P ＜ 0.05), significantly decreased proportion of G0-phase LSK cells, significantly increased proportion of apoptotic cells (t = 7.53, 8.21, P ＜ 0.05), and significantly increased DCFH-DA, MitoSOX, and NADP+/NADPH (t = 22.99, 29.47, 3.77, P＜0.05). In the case of IR, SD further promoted the activation of NOX2 and led to increases in the mRNA expression of downstream inflammatory factors such as IL-1β, IL-6, IL-18, and TNF-α (t = 6.95, 6.01, 8.39, 4.91, 5.56, P ＜ 0.05). Inhibition of NOX2-ROS could prevent the SD-induced aggravation of post-irradiation hematopoietic injury. This significantly reduced the apoptotic rate of LSK cells and the expression of inflammatory factors, ultimately accelerating the hematopoietic recovery of LSK cells (t = 9.24, 3.92, P ＜ 0.05). Conclusions SD can aggravate the IR-induced injury of hematopoietic stem cells in bone marrow, primarily by activating the NOX2-ROS-Caspase-1 axis. This will increase the levels of intracellular inflammatory factors and ROS, promote cell apoptosis, and ultimately inhibit the hematopoietic recovery of bone marrow. |
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