延迟亚低温保护氧糖剥夺-复氧复糖新生幼鼠脑白质损伤的机制研究

doi:10.11852/zgetbjzz2022-0036

摘要/Abstract

摘要： 目的探讨延迟亚低温保护氧糖剥夺-复氧复糖新生幼鼠脑白质损伤的效果及可能机制。方法建立40只新生幼鼠全脑灌流和氧糖剥夺模型,随机均分为4组每组10只,分别进行亚低温32 ℃延迟0 h、24 h、48 h、72 h干预。用Western Blot检测髓鞘碱性蛋白(MBP)的蛋白表达情况;用免疫荧光鉴定小胶质细胞的表达情况;用ELISA测定TNF-α、IL-6浓度情况。结果在亚低温32 ℃延迟24 h、48 h和72 h的MBP蛋白表达均显著高于对照组0 h(F=478.007,t=-18.180、22.940、-29.670,P＜0.001),可发现蛋白表达量与延迟亚低温时长呈显著正相关;随着延迟亚低温时间的延长,小胶质细胞活化程度受到抑制,24 h、48 h和72 h的Iba-1阳性细胞比例均显著低于对照组0 h(F=99.892,t=5.879、9.295、18.760,P＜0.001),可发现荧光表达增高与延迟亚低温时间呈显著负相关;TNF-α、IL-6浓度均较对照组0 h显著降低(TNF-α:F=454.197,t= 2.184、15.300、15.000,P＜0.05;IL-6:F=123.995,t=5.650、13.120、13.910,P＜0.001),可发现IL-6浓度与延迟亚低温时间呈负相关。结论延迟亚低温能保护氧糖剥夺-复氧复糖新生幼鼠脑白质损伤,其机制可能与抑制小胶质细胞活化和下调炎症反应有关。

关键词: 新生幼鼠, 亚低温, 小胶质细胞, 炎症因子, 脑白质损伤, 髓鞘碱性蛋白

Abstract: Objective To analyze the protective effect and possible mechanism of delayed mild hypothermia on white matter injury in oxygen-glucose deprivation and restoration newborn rats. Methods The models of whole brain perfusion and oxygen glucose deprivation were established in 40 neonatal rat pups. Forty neonatal rats were randomly divided into four groups and treated with mild hypothermia (32 ℃) for 0 h, 24 h, 48 h, 72 h, with 10 rats in each group. The expression of myelin basic protein (MBP) was detected by Western Blot.The expression of microglia was identified by immunofluorescence.The concentrations of TNF-α and IL-6 were determined by ELISA. Results The level of MBP protein expression at 32 ℃ for 24 h, 48 h and 72 h was significantly higher than that at 0 h in the control group (F=478.007, t=-18.180、22.940,-29.670,P＜0.001), and there was a significant positive correlation between the protein expression level and the duration of delayed mild hypothermia.With the extension of delayed mild hypothermia time, the activation degree of microglia was inhibited. The number of Iba-1 positive cells at 24 h, 48 h and 72 h was significantly lower than that at 0 h in control group (F=99.892, t=5.879, 9.295, 18.760,P＜0.001), showing that increased fluorescence expression level was negatively correlated with delayed mild hypothermia time. The concentrations of TNF-α and IL-6 were significantly lower than those of the control group at 0 h (TNF-α:F=454.197, t=2.184, 15.300, 15.000, P＜0.05;IL-6:F=123.995, t＝5.650,13.120, 13.910, P＜0.001), indicating that the concentrations of IL-6 was negatively correlated with the time of delayed hypothermia. Conclusion Delayed mild hypothermia can protect the white matter injury of oxygen-glucose deprivation and reoxygen-glucose newborn mice, and its mechanism may be related to inhibiting microglial cell activation and down-regulating inflammatory response.

Key words: newborn mice, mild hypothermia, microglial cell, inflammatory factor, white matter injury, myelin basic protein

中图分类号:

R722.1

梁穗新, 提运幸, 黄骏荣, 李秀红, 周雯嘉. 延迟亚低温保护氧糖剥夺-复氧复糖新生幼鼠脑白质损伤的机制研究[J]. 中国儿童保健杂志, 2022, 30(10): 1083-1087.

LIANG Sui-xin, TI Yun-xing, HUANG Jun-rong, LI Xiu-hong, ZHOU Wen-jia. Protective effect and mechanism of delayed mild hypothermia on white matter injury inoxygen-glucose deprivation and restoration newborn rats[J]. Chinese Journal of Child Health Care, 2022, 30(10): 1083-1087.

参考文献

[1] 王永存,沈亚,刘晓,等. 磁共振多模态检查技术对新生儿缺血缺氧性脑白质损伤的应用价值[J]. 影像研究与医学应用,2021,5(5):204-209.
[2] 潘维伟,童笑梅,李在玲. 母乳喂养与人工喂养对早产儿脑白质损伤预后的影响[J]. 中国儿童保健杂志,2021,29(8):820-823.
[3] 司冉冉,宋红,宋焕清,等. 早产儿脑白质损伤早期相关高危因素[J]. 中国实用神经疾病杂志,2022,25(1):49-52.
[4] 王赫,陈凤收,马虹. 鞘内注射miR-125b mimic抑制小胶质细胞NLRP3炎症体激活减轻缺血再灌注损伤后的炎症反应[J].中国医师杂志, 2021,23(4):553-557.
[5] 陈翔,李广站,王玉琴, 等. 载脂蛋白E和Ⅱ型髓系细胞受体在脂多糖诱导的小胶质细胞炎症反应中的作用[J]. 临床神经病学杂志, 2021,34(1):46-49.
[6] Huang J, Liu G, Shi B, et al. Inhibition of microglial activation by minocycline reduced preoligodendrocyte injury in a neonatal rat brain slice model[J]. J Thorac Cardiovasc Surg, 2018,156(6):2271-2280.
[7] Zou L, Yuan H, Liu Q, et al. Potential protective effects of bilirubin following the treatment of neonatal hypoxic-ischemic encephalopathy with hypothermia therapy[J]. Biosci Rep, 2019,39(6). doi:10.1042/BSR20182332.
[8] Falck M, Osredkar D, Maes E, et al. Hypothermia is neuroprotective after severe hypoxic-Ischaemic brain injury in neonatal rats pre-exposed to PAM3CSK4[J]. Dev Neurosci, 2018,40(3):189-197.
[9] 沈佩婷,黄金华,温晓红,等. 振幅整合脑电图对晚期早产儿脑白质损伤的早期诊断价值及与预后评估的关系研究[J]. 中国儿童保健杂志,2019,27(6):602-605.
[10] Panfoli I, Candiano G, Malova M, et al. Oxidative stress as a primary risk factor for brain damage in preterm newborns[J]. Front Pediatr,2018,7(6):369.
[11] 贾飞勇. 新生儿脑损伤的预后与康复[J]. 中国儿童保健杂志,2020, 28(11):1185-1187,1192.
[12] 王智英,霍海花,吕红艳,等. 脐血NF-κB及MBP检测对早产儿脑白质损伤的早期诊断及其与预后的关系[J]. 川北医学院学报,2017,32(4):518-521.
[13] 薛萌,程涛,陈丽薇,等. 亚低温治疗对机械取栓术后急性大面积半球脑梗死病人神经功能及血清NSE、S100-β蛋白水平的影响[J]. 中西医结合心脑血管病杂志,2021,19(22):3972-3975.
[14] Liu L, Li X, Wang Y, et al. Impacts of mild hypothermia on LPS-mediated TLR4/NF-κB signaling pathway in microglia[J]. J Biosci Med,2019,7:86-97.
[15] Chu X, Cao L, Yu Z, et al. Hydrogen-rich saline promotes microglia M2 polarization and complement-mediated synapse loss to restore behavioral deficits following hypoxia-ischemic in neonatal mice via AMPK activation[J]. J Neuroinflammation,2019,16(1):104.
[16] Pan QB, Jiang KQ, Ye K, et al. Mild hypothermia combined with isoflurane post-treatment alleviates cerebral ischemai reperfusion injury in rats through inhibition of STAT3 activation[J]. Int J Clin Exp Med,2016,9(12):23312-23320.
[17] Tu Y, Guo C, Song F, et al. Mild hypothermia alleviates diabetes aggravated cerebral ischemic injury via activating autophagy and inhibiting pyroptosis[J]. Brain Res Bull, 2019,150:1-12.
[18] Liu X, Wu D, Wen S, et al. Mild therapeutic hypothermia protects against cerebral ischemia/reperfusion injury by inhibiting miR-15b expression in rats[J]. Brain Circ,2017, 3(4):219-226.
[19] Liu X, Wen S, Zhao S, et al. Mild therpeutic hypothermia protects the brain from ischemia/reperfusion injury through upregulation of iaspp[J]. Aging Dis,2018,9(3):401-411.
[20] Wang CF, Zhao CC, He Y, et al. Mild hypothermia reduces endoplasmic reticulum stress-induced apoptosis and improves neuronal functions after severe traumatic brain injury[J]. Brain Behav, 2019, 9(4):e1248.
[21] Li Q, Chen L, Liu X, et al. Pterostilbene inhibits amyloid-β-induced neuroinflammation in a microglia cell line by inactivating the NLRP3/caspase-1 inflammasome pathway[J]. J Cell Biochem,2018,119(8):7053-7062.
[22] Hwang JH, Kumar VR, Kang SY, et al. Effects of flower buds extract of tussilago farfara on focal cerebral ischemia in rats and inflammatory response in BV2 microglia[J]. Chin J Integr Med,2018,24(11):844-852.
[23] Wang Q, Lv C, Sun Y, et al. The role of alpha-lipoic acid in the pathomechanism of acute ischemic stroke[J]. Cell Physiol Biochem,2018,48(1):42-53.
[24] 田智琛,张宣,尹晓娟. 亚低温治疗新生儿缺氧缺血性脑病的研究进展[J].中国儿童保健杂志,2021,29(3):292-295.
[25] 邓天国. 颅内血肿冲洗中应用亚低温冲洗液对神经功能、氧化应激的影响[J].海南医学院学报,2018,24(22):1975-1983.
[26] 孙艳娟,吴玉华. 颅脑超声联合血清NSE、S100β及IL-6检测在早产儿脑白质损伤诊断中的应用[J].宁夏医科大学学报, 2021,43(8):787-791.
[27] 杨雪珂,张晓丽娜,陈澍雨,等. 天麻素对M1型小胶质细胞中TNF-α和CD86表达的影响[J].神经解剖学杂志,2022,38(1):28-32.