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小胶质细胞焦亡在缺氧缺血性脑损伤中的作用
谭兰兰, 李梅, 冯晨希, 徐利晓, 丁欣, 孙斌, 李根, 冯星
中国当代儿科杂志 ›› 2020, Vol. 22 ›› Issue (11) : 1226-1232.
PDF(2389 KB)
PDF(2389 KB)
小胶质细胞焦亡在缺氧缺血性脑损伤中的作用
Role of microglial pyroptosis in hypoxic-ischemic brain damage
目的 初步探讨小胶质细胞焦亡在缺氧缺血性脑损伤中的作用。方法 建立体外培养大鼠小胶质细胞系氧糖剥夺再灌注（OGD/R）模型，用Western blot法检测OGD/R后0、1、3、6、12及24 h焦亡相关蛋白半胱氨酸天冬氨酸蛋白酶-l（caspase-1）、白细胞介素-1β（IL-1β）、Gasdermin D蛋白N端（GSDMD-N）的表达情况。用慢病毒构建的沉默Gasdermin D（GSDMD）序列转染小胶质细胞，使用免疫荧光和Western blot法检测GSDMD转染效率。将小胶质细胞系分为正常对照组、阴性对照组、LV-sh_GSDMD组（慢病毒沉默GSDMD），使用CCK-8和LDH试剂盒检测沉默GSDMD对OGD/R后24 h小胶质细胞活性和毒性的影响；通过Western blot法检测沉默GSDMD对OGD/R后24 h小胶质细胞中caspase-1、GSDMD-N、IL-1β含量变化的影响。结果 在OGD/R后0 h起小胶质细胞内焦亡相关蛋白caspase-1、GSDMD-N、IL-1β的表达水平即较OGD/R前发生了上调，并且在24 h达到高峰（P < 0.05）。成功地构建慢病毒沉默GSDMD转染小胶质细胞模型。OGD/R后24 h，与正常对照组相比，沉默GSDMD可提高细胞活性和降低细胞毒性（P < 0.05），降低小胶质细胞内caspase-1、GSDMD-N、IL-1β蛋白水平（P < 0.05）。结论 慢病毒沉默细胞焦亡关键底物蛋白GSDMD可减轻缺氧缺血性脑损伤，提示小胶质细胞焦亡加重缺氧缺血性脑损伤。
Objective To investigate the role of microglial pyroptosis in hypoxic-ischemic brain damage. Methods An oxygen-glucose deprivation/reoxygenation (OGD/R) model of rat microglial cells were cultured in vitro. Western blot was used to measure the expression of the pyroptosis-related proteins caspase-1, interleukin-1β (IL-1β), and N-terminal gasdermin D (GSDMD-N) at 0, 1, 3, 6, 12, and 24 hours after OGD/R. After the microglial cells were transfected with lentivirus-mediated silenced gasdermin D (GSDMD), immunofluorescence assay and Western blot were used to measure the transfection rate of GSDMD. Microglial cell lines were divided into three groups:normal control, negative control, and LV-sh_GSDMD (lentivirus-mediated GSDMD silencing). CCK-8 assay and LDH kit were used to observe the effect of GSDMD silencing on the viability and toxicity of microglial cells at 24 hours after OGD/R. Western blot was used to observe the effect of GSDMD silencing on the levels of caspase-1, GSDMD-N, and IL-1β in the microglial cells at 24 hours after OGD/R. Results The expression levels of the pyroptosis-related proteins caspase-1, GSDMD-N, and IL-1β in microglial cells were upregulated since 0 hour after OGD/R and reached the peak levels at 24 hours. A microglial cell model of lentivirus-mediated GSDMD silencing was successfully constructed. At 24 hours after OGD/R, compared with the normal control group, the GSDMD silencing group had a significant increase in the cell viability and a significant reduction in the cytotoxicity (P < 0.05), as well as significant reductions in the protein expression levels of caspase-1, GSDMD-N, and IL-1β in microglial cells (P < 0.05). Conclusions Lentivirus silencing of the key substrate protein for pyroptosis GSDMD can alleviate hypoxic-ischemic brain damage, suggesting that microglial pyroptosis aggravates hypoxic-ischemic brain damage.
缺氧缺血性脑损伤 / 细胞焦亡 / 氧糖剥夺再灌注 / 大鼠小胶质细胞
Hypoxic-ischemic brain damage / Pyroptosis / Oxygen-glucose deprivation/reoxygenation / Rat microglial cell
[1] de Vries LS, Jongmans MJ. Long-term outcome after neonatal hypoxic-ischaemic encephalopathy[J]. Arch Dis Child Fetal Neonatal Ed, 2010, 95(3):F220-F224.
[2] Perlman M, Shah PS. Hypoxic-ischemic encephalopathy:challenges in outcome and prediction[J]. J Pediatr, 2011, 158(2 Suppl):e51-e54.
[3] Hanisch UK, Kettenmann H. Microglia:active sensor and versatile effector cells in the normal and pathologic brain[J]. Nat Neurosci, 2007, 10(11):1387-1394.
[4] Adams KL, Gallo V. The diversity and disparity of the glial scar[J]. Nat Neurosci, 2018, 21(1):9-15.
[5] Donat CK, Scott G, Gentleman SM, et al. Microglial activation in traumatic brain injury[J]. Front Aging Neurosci, 2017, 9:208.
[6] Loane DJ, Kumar A. Microglia in the TBI brain:the good, the bad, and the dysregulated[J]. Exp Neurol, 2016, 275 Pt 3:316-327.
[7] Navarro V, Sanchez-Mejias E, Jimenez S, et al. Microglia in Alzheimer's disease:activated, dysfunctional or degenerative[J]. Front Aging Neurosci, 2018, 10:140.
[8] Liu F, Mccullough LD. Inflammatory responses in hypoxic ischemic encephalopathy[J]. Acta Pharmacol Sin, 2013, 34(9):1121-1130.
[9] Bergsbaken T, Fink SL, Cookson BT. Pyroptosis:host cell death and inflammation[J]. Nat Rev Microbiol, 2009, 7(2):99-109.
[10] Broz P, Dixit VM. Inflammasomes:mechanism of assembly, regulation and signalling[J]. Nat Rev Immunol, 2016, 16(7):407-420.
[11] Nyström S, Antoine DJ, Lundbäck P, et al. TLR activation regulates damage-associated molecular pattern isoforms released during pyroptosis[J]. EMBO J, 2013, 32(1):86-99.
[12] Liu X, Zhang Z, Ruan J, et al. Inflammasome-activated gasdermin D causes pyroptosis by forming membrane pores[J]. Nature, 2016, 535(7610):153-158.
[13] Huang S, Gong T, Zhang T, et al. Zhongfenggao protects brain microvascular endothelial cells from oxygen-glucose deprivation/reoxygenation-induced injury by angiogenesis[J]. Bio Pharm Bull, 2019, 42(2):222-230.
[14] Van Opdenbosch N, Lamkanfi M. Caspases in cell death, inflammation, and disease[J]. Immunity, 2019, 50(6):1352-1364.
[15] Ruan J, Wang S, Wang J. Mechanism and regulation of pyroptosis-mediated in cancer cell death[J]. Chem Biol Interact, 2020, 323:109052.
[16] Martín-Sánchez F, Martínez-García JJ, Muñoz-García M, et al. Lytic cell death induced by melittin bypasses pyroptosis but induces NLRP3 inflammasome activation and IL-1β release[J]. Cell Death Dis, 2017, 8(8):e2984.
[17] He WT, Wan H, Hu L, et al. Gasdermin D is an executor of pyroptosis and required for interleukin-1β secretion[J]. Cell Res, 2015, 25(12):1285-1298.
[18] Galluzzi L, Vitale I, Aaronson SA, et al. Molecular mechanisms of cell death:recommendations of the Nomenclature Committee on Cell Death 2018[J]. Cell Death Differ, 2018, 25(3):486-541.
[19] McKenzie BA, Mamik MK, Saito LB, et al. Caspase-1 inhibition prevents glial inflammasome activation and pyroptosis in models of multiple sclerosis[J]. Proc Natl Acad Sci U S A, 2018, 115(26):E6065-E6074.
[20] 王幽梦, 鲁利群, 屈艺. 细胞焦亡与缺氧缺血性脑损伤[J]. 实用医学杂志, 2017, 33(23):3998-4001.
江苏省研究生科研与实践创新计划项目（KYCX19_1998）；国家自然科学基金（81771625）；苏州市小儿内科临床医学中心项目（Szzx201504）。
