PDF(2636 KB)
黄芪甲苷对新生大鼠缺氧缺血性脑损伤NLRP3炎性小体表达的影响
李娜, 穆亚平, 刘春英, 王阳, 李晓锋, 王雪薇
中国当代儿科杂志 ›› 2021, Vol. 23 ›› Issue (4) : 402-409.
PDF(2636 KB)
PDF(2636 KB)
黄芪甲苷对新生大鼠缺氧缺血性脑损伤NLRP3炎性小体表达的影响
Effect of astragaloside IV on the expression of NOD-like receptor protein 3 inflammasome in neonatal rats with hypoxic-ischemic brain damage
目的 探讨黄芪甲苷（AS-IV）对缺氧缺血性脑损伤（HIBD）新生大鼠Nod样受体蛋白3（NLRP3）炎性小体的影响。方法 将24只7日龄Sprague-Dawley大鼠随机分为假手术组、HIBD组、AS-IV治疗组（AS-IV组），每组8只。各组动物造模24 h后取脑组织进行苏木精-伊红染色、yo-PRO-1和EthD-2免疫荧光染色，以观察AS-IV在体内的脑保护作用。构建HT22细胞氧糖剥夺（OGD）模型，设置AS-IV浓度梯度（50~400 μmol/L）进行干预。采用CCK8法检测HT22细胞活力，RT-PCR和Western blot法检测不同浓度AS-IV对NLRP3、Gasdermin D蛋白（GSDMD）、半胱氨酸蛋白酶-1（Caspase-1）、白细胞介素1β（IL-1β）mRNA及其蛋白表达的影响。结果 Yo-PRO-1和EthD-2染色结果显示，与假手术组比较，HIBD组焦亡细胞增多，同时可见少量坏死细胞，AS-IV组焦亡细胞和坏死细胞均减少。HIBD组脑组织NLRP3、IL-1β、Caspase-1及GSDMD蛋白表达水平显著高于假手术组（P < 0.05）；与HIBD组比较，AS-IV组NLRP3、Caspase-1及GSDMD蛋白表达水平显著降低（P < 0.05）。HT22细胞实验结果显示，与OGD组比较，AS-IV可以抑制NLRP3、GSDMD、Caspase-1、IL-1β mRNA及其蛋白表达，200 μmol/L时治疗效果最佳（P < 0.05）。结论 AS-IV可能通过抑制NLRP3、GSDMD、Caspase-1、IL-1β的表达减轻新生大鼠HIBD。
Objective To study the effect of astragaloside IV (AS-IV) on NOD-like receptor protein 3 (NLRP3) inflammasome in neonatal rats with hypoxic-ischemic brain damage (HIBD). Methods A total of 24 Sprague-Dawley rats, aged 7 days, were randomly divided into a sham-operation group, an HIBD group, and an AS-IV treatment group, with 8 rats in each group. After 24 hours of modeling, brain tissue was collected for hematoxylin-eosin staining, yo-PRO-1 staining, and EthD-2 immunofluorescent staining in order to observe the cerebral protection effect of AS-IV in vivo. HT22 cells were used to prepare a model of oxygen-glycogen deprivation (OGD), and a concentration gradient (50-400 μmol/L) was established for AS-IV. CCK-8 assay was used to measure the viability of HT22 cells. RT-PCR and Western blot were used to observe the effect of different concentrations of AS-IV on the mRNA and protein expression of NLRP3, gasdermin D (GSDMD), caspase-1, and interleukin-1β (IL-1β). Results Yo-Pro-1 and EthD-2 staining showed that compared with the sham-operation group, the HIBD group had an increase in pyroptotic cells with a small number of necrotic cells, and the AS-IV group had reductions in both pyroptotic and necrotic cells. Compared with the sham-operation group, the HIBD group had significantly higher protein expression levels of NLRP3, IL-1β, caspase-1, and GSDMD (P < 0.05). Compared with the HIBD group, the AS-IV group had significant reductions in the protein expression levels of NLRP3, caspase-1, and GSDMD (P < 0.05). HT22 cell experiment showed that compared with the OGD group, the AS-IV group had inhibited mRNA and protein expression of NLRP3, GSDMD, caspase-1, and IL-1β, with the best therapeutic effect at the concentration of 200 μmol/L (P < 0.05). Conclusions AS-IV may alleviate HIBD in neonatal rats by inhibiting the expression of NLRP3, GSDMD, caspase-1, and IL-1β.
缺氧缺血性脑损伤 / 黄芪甲苷 / 细胞焦亡 / HT22细胞 / 新生大鼠
Hypoxic-ischemic brain damage / Astragaloside IV / Pyroptosis / HT22 cell / Neonatal rats
[1] Nonomura M, Harada S, Asada Y, et al. Combination therapy with erythropoietin, magnesium sulfate and hypothermia for hypoxic-ischemic encephalopathy:an open-label pilot study to assess the safety and feasibility[J]. BMC Pediatr, 2019, 19(1):13.
[2] Kim YE, Sung SI, Chang YS, et al. Thrombin preconditioning enhances therapeutic efficacy of human Wharton's jelly-derived mesenchymal stem cells in severe neonatal hypoxic ischemic encephalopathy[J]. Int J Mol Sci, 2019, 20(10):2477.
[3] Laptook AR, Shankaran S, Tyson JE, et al. Effect of therapeutic hypothermia initiated after 6 hours of age on death or disability among newborns with hypoxic-ischemic encephalopathy:a randomized clinical trial[J]. JAMA, 2017, 318(16):1550-1560.
[4] Shankaran S, Laptook AR, Pappas A, et al. Effect of depth and duration of cooling on death or disability at age 18 months among neonates with hypoxic-ischemic encephalopathy:a randomized clinical trial[J]. JAMA, 2017, 318(1):57-67.
[5] Jacobs SE, Morley CJ, Inder TE, et al. Whole-body hypothermia for term and near-term newborns with hypoxic-ischemic encephalopathy:a randomized controlled trial[J]. Arch Pediatr Adolesc Med, 2011, 165(8):692-700.
[6] Yang Y, Wang HN, Kouadir M, et al. Recent advances in the mechanisms of NLRP3 inflammasome activation and its inhibitors[J]. Cell Death Dis, 2019, 10(2):128.
[7] Aridas JD, Yawno T, Sutherland AE, et al. Detecting brain injury in neonatal hypoxic ischemic encephalopathy:closing the gap between experimental and clinical research[J]. Exp Neurol, 2014, 261:281-290.
[8] Paramel Varghese G, Folkersen L, Strawbridge RJ, et al. NLRP3 inflammasome expression and activation in human atherosclerosis[J]. J Am Heart Assoc, 2016, 5(5):e003031.
[9] Serdar M, Kempe K, Rizazad M, et al. Early pro-inflammatory microglia activation after inflammation-sensitized hypoxic-ischemic brain injury in neonatal rats[J]. Front Cell Neurosci, 2019, 13:237.
[10] Ystgaard MB, Sejersted Y, Løberg EM, et al. Early upregulation of NLRP3 in the brain of neonatal mice exposed to hypoxia-ischemia:no early neuroprotective effects of NLRP3 deficiency[J]. Neonatology, 2015, 108(3):211-219.
[11] 靳晓飞, 张彐宁, 周晓红, 等. 黄芪甲苷调控自噬减轻氧糖剥夺/复氧复糖PC12细胞氧化应激损伤研究[J]. 中国药理学通报, 2020, 36(1):53-58.
[12] Song MT, Ruan J, Zhang RY, et al. Astragaloside IV ameliorates neuroinflammation-induced depressive-like behaviors in mice via the PPARγ/NF-κB/NLRP3 inflammasome axis[J]. Acta Pharmacol Sin, 2018, 39(10):1559-1570.
[13] Li M, Li HY, Fang F, et al. Astragaloside IV attenuates cognitive impairments induced by transient cerebral ischemia and reperfusion in mice via anti-inflammatory mechanisms[J]. Neurosci Lett, 2017, 639:114-119.
[14] Dixon BJ, Reis C, Ho WM, et al. Neuroprotective strategies after neonatal hypoxic ischemic encephalopathy[J]. Int J Mol Sci, 2015, 16(9):22368-22401.
[15] Zhang Y, Zhang Y, Jin XF, et al. The role of Astragaloside IV against cerebral ischemia/reperfusion injury:suppression of apoptosis via promotion of P62-LC3-autophagy[J]. Molecules, 2019, 24(9):1838.
[16] Buyukakilli B, Atici A, Balli E, et al. Effects of a tumor necrosis factor-alpha inhibitor (etanercept) on the sciatic nerve in a hypoxic ischemia-induced neonatal rat model[J]. Adv Clin Exp Med, 2014, 23(5):705-713.
[17] Wang HH, Zhou XY, Li H, et al. Transient receptor potential melastatin 2 negatively regulates LPS-ATP-Induced caspase-1-dependent pyroptosis of bone marrow-derived macrophage by modulating ROS production[J]. Biomed Res Int, 2017, 2017:2975648.
[18] Rat P, Olivier E, Tanter C, et al. A fast and reproducible cell- and 96-well plate-based method for the evaluation of P2X7 receptor activation using YO-PRO-1 fluorescent dye[J]. J Biol Methods, 2017, 4(1):e64.
[19] 谢海亮, 杨镛. 细胞-动物缺血缺氧模型建立及特点[J]. 中国血管外科杂志(电子版), 2012, 4(3):184-187.
[20] Silveira RC, Procianoy RS. Hypothermia therapy for newborns with hypoxic ischemic encephalopathy[J]. J Pediatr (Rio J), 2015, 91(6 Suppl 1):S78-S83.
[21] Wang HL, Zhou QH, Xu MB, et al. Astragaloside IV for experimental focal cerebral ischemia:preclinical evidence and possible mechanisms[J]. Oxid Med Cell Longev, 2017, 2017:8424326.
[22] 李程远, 余艾霞. 黄芪注射液治疗新生儿缺氧缺血性脑病的临床效果及对血清内源性阿片肽、神经因子及炎症因子的影响[J]. 中国妇幼保健, 2019, 34(3):566-570.
[23] 关丽君, 穆亚萍, 李红, 等. 中药黄芪注射液穴位注射对脑瘫患儿肌肉电生理及影像学影响研究[J]. 时珍国医国药, 2014, 25(10):2447-2450.
[24] Hagberg H, Mallard C, Ferriero DM, et al. The role of inflammation in perinatal brain injury[J]. Nat Rev Neurol, 2015, 11(4):192-208.
[25] Li B, Concepcion K, Meng XM, et al. Brain-immune interactions in perinatal hypoxic-ischemic brain injury[J]. Prog Neurobiol, 2017, 159:50-68.
[26] Saresella M, La Rosa F, Piancone F, et al. The NLRP3 and NLRP1 inflammasomes are activated in Alzheimer's disease[J]. Mol Neurodegener, 2016, 11:23.
[27] Shao BZ, Cao Q, Liu C. Targeting NLRP3 inflammasome in the treatment of CNS diseases[J]. Front Mol Neurosci, 2018, 11:320.
[28] 唐标, 唐文静, 唐映红, 等. 黄芪甲苷减轻大鼠脑缺血再灌损伤并抑制NF-κB磷酸化及NLRP3炎症小体活化[J]. 生理学报, 2019, 71(3):424-430.
[29] Huang J, Lu WT, Doycheva DM, et al. IRE1α inhibition attenuates neuronal pyroptosis via miR-125/NLRP1 pathway in a neonatal hypoxic-ischemic encephalopathy rat model[J]. J Neuroinflammation, 2020, 17(1):152.
[30] Liao H, Wang HX, Rong XM, et al. Mesenchymal stem cells attenuate radiation-induced brain injury by inhibiting microglia pyroptosis[J]. Biomed Res Int, 2017, 2017:1948985.
[31] 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.
[32] Kuang SY, Zheng J, Yang H, et al. Structure insight of GSDMD reveals the basis of GSDMD autoinhibition in cell pyroptosis[J]. Proc Natl Acad Sci U S A, 2017, 114(40):10642-10647.
[33] Pichl T, Keller T, Hünseler C, et al. Effects of ketamine on neurogenesis, extracellular matrix homeostasis and proliferation in hypoxia-exposed HT22 murine hippocampal neurons[J]. Biomed Rep, 2020, 13(4):23.
辽宁省自然科学基金计划项目（20180550290）；沈阳市中青年科技创新人才支持计划项目（RC180035）。
