Wen Haitao,Chen Xiaoyan,An Zheni,et al.Xingnaojing injection suppressed neuronal pyrosis after oxygen-glucose deprivation/reperfusion injury through activating Nrf2/NLRP3/GSDMD signaling pathway[J].Journal of Clinical Pediatric Surgery,2023,22(07):680-685.[doi:10.3760/cma.j.cn101785-202107045-014]
醒脑静注射液激活Nrf2/NLRP3/GSDMD信号通路抑制氧糖剥夺/再灌注损伤后神经细胞焦亡的研究
- Title:
- Xingnaojing injection suppressed neuronal pyrosis after oxygen-glucose deprivation/reperfusion injury through activating Nrf2/NLRP3/GSDMD signaling pathway
- 关键词:
- 醒脑静注射液; 焦亡; 炎症小体; 脑卒中; 核因子E2相关因子2
- Keywords:
- Xingnaojing Injection; Pyroptosis; Inflammatory Corpuscles; Cerebral Apoplexy; Nuclear Factor E2 Related Factor 2
- 摘要:
- 目的 探讨醒脑静注射液对缺糖缺氧再灌注（oxygen and glucose deprivation/reoxygenation，OGD/R）损伤诱导SH-SY5Y细胞焦亡的保护作用及机制。方法 使用不同浓度醒脑静注射液预处理SH-SY5Y细胞，确定醒脑静注射液给药浓度；将SH-SY5Y细胞随机分为对照组、OGD/R组、醒脑静注射液+OGD/R组（10 μL/mL醒脑静注射液）以及醒脑静注射液+OGD/R+核因子E2相关因子2（nuclear factor-E2 related factor 2，Nrf2）抑制剂组（10 μL/mL醒脑静注射液+2 μmol/mL ML385）共4组，每组实验进行6次（n=6），采用MTT比色法检测细胞存活率；采用酶联免疫吸附试验（enzyme-linked immunosorbentassay，ELISA）法测定细胞上清液中白介素18（interleukin 18，IL-18）和白介素-1β（interleukin 1 beta，IL-1β）表达水平；采用Western blot检测GasderminD蛋白（GSDMD-N）、NOD样受体热蛋白结构域相关蛋白3（NOD-like receptor thermal protein domain associated protein 3，NLRP3）、白介素1β前体（pro-interleukin 1，pro-IL-1β）、半胱氨酸天冬氨酸蛋白酶1（caspase-1）、Nrf2以及凋亡相关斑点样蛋白（apoptosis-associated speck-like protein containing a CARD，ASC）表达情况；采用细胞乳酸脱氢酶（lactate dehydrogenase，LDH）试剂盒法测定细胞内LDH表达变化情况，采用Hochest/PI染色法观察细胞凋亡情况。结果 与对照组比较，OGD/R组细胞存活率[（40.16±3.39）%比（101.3±1.54）%]、Nrf2表达水平[（0.47±0.09）比（1.00±0.00）]明显降低；IL-1β[（46.55±9.59）pg/mL比（9.35±3.33） pg/mL]、IL-18[（35.39±10.63） pg/mL比（7.85±3.04） pg/mL]、LDH渗出率[（0.36±0.04）%比（0.18±0.08）%]、GSDMD-N[（2.24±0.36）比（1.00±0.00）]、NLRP3[（2.15±0.23）比（1.00±0.00）]、pro-IL-1β[（3.08±0.17）比（1.00±0.00）]、caspase-1[（2.20±0.32）比（1.00±0.00）]表达水平明显升高，差异有统计学意义（P<0.001）；与OGD/R组比较，醒脑静注射液组细胞存活率[（63.69±11.28）%比（40.16±3.39）%]、Nrf2表达水平[（0.89±0.11）比（0.47±0.09）]明显升高，LDH渗出率[（0.25±0.07）%比（0.36±0.04）%]、IL-1β[（33.30±8.60） pg/mL比（46.55±9.59） pg/mL]、IL-18[（21.72±7.50） pg/mL比（35.39±10.63） pg/mL]、GSDMD-N[（1.34±0.06）比（2.24±0.36）]、NLRP3[（1.25±0.18）比（2.15±0.23）]、pro-IL-1β[（1.65±0.30）比（3.08±0.17）]、caspase-1[（1.23±0.15）比（2.20±0.32）]表达水平明显降低，差异有统计学意义（P<0.05）；与醒脑静组比较，醒脑静+Nrf2抑制剂组Nrf2蛋白表达水平[（0.38±0.12）比（0.89±0.11）]明显降低，LDH渗出率[（0.36±0.07）%比（0.25±0.07）%]、IL-1β[（53.28±6.37） pg/mL比（33.30±8.60） pg/mL]、IL-18[（49.56±8.87） pg/mL比（21.72±7.50） pg/mL]、GSDMD-N[（1.75±0.27）比（1.34±0.06）]、NLRP3[（2.22±0.17）比（1.25±0.18）]、pro-IL-1β[（3.54±0.50）比（1.65±0.30）]、caspase-1[（2.36±0.28）比（1.23±0.15）]蛋白表达水平明显升高，差异有统计学意义（P<0.05）。结论 Nrf2抑制剂可逆转醒脑静注射液对OGD/R损伤诱导SH-SY5Y细胞焦亡的保护作用；醒脑静注射液通过上调Nrf2蛋白表达，抑制OGD/R损伤诱导的SH-SY5Y细胞焦亡。
- Abstract:
- Objective To explore the protective mechanism of refreshing static injection for injury of oxygen and glucose deprivation/reoxygenation (OGD/R) induced by SH SY5Y cells.Methods SH-SY5Y cells were pretreated with Xingnaojing Injection at different concentrations to determine its dosing concentration.SH-SY5Y cells were randomized into four groups of control, OGD/R, Xingnaojing Injection + OGD/R (10 μL/mL Xingnaojing Injection) and Xingnaojing Injection+OGD/R + nuclear fact-E2 related factor 2 (Nrf2) inhibitor (10 μL/mL Xingnaojing Injection+2 μmol/mL ML385).Cell survival rate was determined by MTT colorimetry.Enzyme-linked immunosorbentassay (ELISA) was utilized for measuring the levels of interleukin 18 (IL-18) and interleuin 1 beta in cell supernatant.Western blot was employed for detecting the levels of Gasdermin D protein (GSDMD-N), NOD-like receptor thermal protein domain associated protein 3 (NLRP3), pro-interleukin 1, pro-IL-1β, caspase-1, Nrf2 and apoptosis-associated speck-like protein containing a CARD (ASC) protein.LDH expression was determined by cell lactate dehydrogenase (LDH) kit and cell apoptosis evaluated by Hochest/PI stain.Results As compared with control group, the survival rate[(40.16±3.39)% vs.(101.3±1.54)%] and the expression level of Nrf2 declined markedly in OGD/R group[(0.47±0.09) vs.(1.00±0.00)].IL-1 beta[(46.55±9.59) vs.(9.35±3.33) pg/mL], IL-18[(35.39±10.63) vs.(7.85±3.04) pg/mL] and LDH leakage rate[(0.36±0.04)% vs.(0.18±0.08)%], GSDMD-N[(2.24±0.36) vs.(1.00±0.00)], NLRP3[(2.15±0.23) vs.(1.00±0.00)], pro-IL-1 beta[(3.08±0.17) vs.(1.00±0.00)] and caspase-1[(2.20±0.32) vs.(1.00±0.00)] spiked markedly and the differences were statistically significant (P<0.001).As compared with OGD/R group, the survival rate of Xingnaojing Injection group[(63.69±11.28)% vs.(40.16±3.39)%] and the expression level of Nrf2[(0.89±0.11) vs.(0.47±0.09)] rose obviously.The levels of LDH leakage rate[(0.25±0.07)% vs.(0.36±0.04)%], IL-1 beta[(33.30±8.60) vs.(46.55±9.59) pg/ml], IL-18[(21.72±7.50) vs.(35.39±10.63) pg/mL), GSDMD-N[(1.34±0.06) vs.(2.24±0.36)], NLRP3[(1.25±0.18) vs.(2.15±0.23)], pro-IL-1 beta[(1.65±0.30) vs.(3.08±0.17)] and caspase-1[(1.23±0.15) vs.(2.20±0.32)] declined markedly and the differences were statistically significant (P<0.05).Compared with Xingnaojing group, the expression level of Nrf2 protein in Xingnaojing Injection + Nrf2 inhibitor group dropped significantly[(0.38±0.12) vs.(0.89±0.11)].The levels of LDH leakage rate[(0.36±0.07)% vs.(0.25±0.07)%], IL-1 beta[(53.28±6.37) vs.(33.30±8.60) pg/mL], IL-18[(49.56±8.87) vs.(21.72±7.50 pg/mL), GSDMD-N[(1.75±0.27) vs.(1.34±0.06)], NLRP3[(2.22±0.17) vs.(1.25±0.18)], pro-IL-1 beta[(3.54±0.50) vs.(1.65±0.30)]and caspase-1[(2.36±0.28) vs.(1.23±0.15)] spiked markedly and the differences were statistically significant (P<0.05).Conclusion Nrf2 inhibitor can reverse the protective effect of Xingnaojing Injection on OGD/R injury-induced pyroptosis of SH-SY5Y cells.And Xingnaojing Injection suppresses OGD/R injury-induced pyroptosis of SH-SY5Y cells through up-regulating Nrf2 protein expression.
参考文献/References:
[1] Zhao J, Piao XY, Wu Y, et al.Cepharanthine attenuates cerebral ischemia/reperfusion injury by reducing NLRP3 inflammasome-induced inflammation and oxidative stress via inhibiting 12/15-LOX signaling[J].Biomed Pharmacother, 2020, 127:110151.DOI:10.1016/j.biopha.2020.110151.
[2] Wei YY, Lan BD, Zheng T, et al.GSDME-mediated pyroptosis promotes the progression and associated inflammation of atherosclerosis[J].Nat Commun, 2023, 14(1):929.DOI:10.1038/s41467-023-36614-w.
[3] 吴京霓, 刘瑞敏, 许丹妮, 等.基于GC-MS技术及网络药理学探讨醒脑静注射液入脑成分抗脑缺血损伤的作用机制[J].中草药, 2021, 52(3):808-820.DOI:10.7501/j.issn.0253-2670.2021.03.024. Wu JN, Liu RM, Xu DN, et al.Anti-cerebral ischemia mechanisms of brain absorption components of Xingnaojing Injection based upon GC-MS and network pharmacology[J].Chin Tradit Herb Drugs, 2021, 52(3):808-820.DOI:10.7501/j.issn.0253-2670.2021.03.024.
[4] Zhou ZR, Zhao YH, Sun R, et al.Effects of Xingnaojing on serum high-sensitivity c-reactive protein and neuron-specific enolase in patients with acute cerebral hemorrhage:a protocol of systematic review and meta-analysis[J].Medicine (Baltimore), 2020, 99(45):e21379.DOI:10.1097/MD.0000000000021379.
[5] 王振凤, 田克姚, 刘南海.醒脑静注射液对MCAO大鼠神经功能、神经细胞炎症及氧化应激损伤的影响[J].中国现代医生, 2020, 58(36):36-39. Wang ZF, Tian KY, Liu NH.Effects of Xingnaojing Injection on nerve function, nerve cell inflammation and oxidative stress injury in MCAO rats[J].China Mod Dr, 2020, 58(36):36-39.
[6] 卢志刚, 黄家彬, 刘芸, 等.醒脑静注射液对急性脑梗死Keap1-Nrf2/ARE氧化应激通路的影响[J].广东医学, 2016, 37(20):3127-3129.DOI:10.3969/j.issn.1001-9448.2016.20.043. Lu ZG, Huang JB, Liu Y, et al.Effect of Xingnaojing Injection on the Keap1-Nrf2/ARE oxidative stress pathway in acute cerebral infarction[J].Guangdong Med J, 2016, 37(20):3127-3129.DOI:10.3969/j.issn.1001-9448.2016.20.043.
[7] 王文杰, 路娇娇, 马凯丰, 等.刺五加提取物对人神经细胞SH-SY5Y氧糖剥夺再灌注损伤的保护作用研究[J].世界科学技术-中医药现代化, 2022, 24(5):1772-1780.DOI:10.11842/wst.20211115008. Wang WJ, Lu JJ, Ma KF, et al.Protective effect of Acanthopanax senticosus extract on SH-SY5Y cells after oxygen and glucose deprivation reperfusion injury[J].Modernization of Traditional Chinese Medicine and Materia Medica-World Science and Technology, 2022, 24(5):1772-1780.DOI:10.11842/wst.20211115008.
[8] Shi M, Chen J, Liu TX, et al.Protective effects of remimazolam on cerebral ischemia/reperfusion injury in rats by inhibiting of NLRP3 inflammasome-dependent pyroptosis[J].Drug Des Devel Ther, 2022, 16:413-423.DOI:10.2147/DDDT.S344240.
[9] Yang KL, Bao TT, Zeng JS, et al.Research progress on pyroptosis-mediated immune-inflammatory response in ischemic stroke and the role of natural plant components as regulator of pyroptosis:a review[J].Biomed Pharmacother, 2023, 157:113999.DOI:10.1016/j.biopha.2022.113999.
[10] de Sá KSG, Amaral LA, Rodrigues TS, et al.Gasdermin-D activation promotes NLRP3 activation and host resistance to Leishmania infection[J].Nat Commun, 2023, 14(1):1049.DOI:10.1038/s41467-023-36626-6.
[11] Yanpiset P, Maneechote C, Sriwichaiin S, et al.Gasdermin D-mediated pyroptosis in myocardial ischemia and reperfusion injury:cumulative evidence for future cardioprotective strategies[J].Acta Pharm Sin B, 2023, 13(1):29-53.DOI:10.1016/j.apsb.2022.08.007.
[12] Sun Y, Yang X, Xu LJ, et al.The role of Nrf2 in relieving cerebral ischemia-reperfusion injury[J].Curr Neuropharmacol, 2023, 21(6):1405-1420.DOI:10.2174/1570159X21666221129100308.
[13] Liao S, Wu JN, Liu RM, et al.A novel compound DBZ ameliorates neuroinflammation in LPS-stimulated microglia and ischemic stroke rats:role of Akt(Ser473)/GSK3β(Ser9)-mediated Nrf2 activation[J].Redox Biol, 2020, 36:101644.DOI:10.1016/j.redox.2020.101644.
[14] Xiao L, Dai ZW, Tang WJ, et al.Astragaloside IV alleviates cerebral ischemia-reperfusion injury through NLRP3 inflammasome-mediated pyroptosis inhibition via activating Nrf2[J].Oxid Med Cell Longev, 2021, 2021:9925561.DOI:10.1155/2021/9925561.
备注/Memo
收稿日期:2021-07-23。
基金项目:湖南省中医药科研计划项目（2020108）；湖南省卫生健康委课题（202104040560）
通讯作者:吴水华,Email:292454021@qq.com