水苏碱调节PI3K/Akt/NF-κB信号通路对OGD/R诱导的神经元焦亡的影响
Impacts of stachydine on OGD/R-induced neuronal pyroptosis by regulating PI3K/Akt/NF-κB signaling pathway
高李 1杨祎2
作者信息
- 1. 721000 陕西省宝鸡市中心医院神经内科
- 2. 721000 陕西省宝鸡市中心医院康复医学科
- 折叠
摘要
目的 探讨水苏碱(Stachydine,STA)对氧糖剥夺再灌注(Oxygen-glucose deprivation/reoxy-genation,OGD/R)诱导的神经元焦亡及磷脂酰肌醇3-激酶/蛋白激酶B/核转录因子-κB(Phosphatidylinositol 3-kinase/protein kinase B/nuclear transciption factor-kappa B,PI3K/Akt/NF-κB)信号通路的影响机制.方法 MTT比色(MTT assay kit,MTT)法检测不同水平STA对海马神经元细胞系Ht22细胞活力的影响;将Ht22细胞进行OGD/R诱导后分为模型组(OGD/R组)、水苏碱低剂量组(STA-L组)、水苏碱中剂量组(STA-M组)、水苏碱高剂量组(STA-H组)、水苏碱高剂量+通路激活剂740Y-P组(STA-H+740Y-P组)、通路激活剂740Y-P组(740Y-P组),另设置正常培养的细胞为对照组(Control组);细胞活性检测试剂(Cell counting kit-8,CCK-8)法检测Ht22细胞增殖活性;Hoechst/碘化丙啶(Propidium iodide,PI)染色法检测细胞膜损伤;流式细胞仪检测细胞焦亡;酶联免疫吸附试验(Enzyme linked immunosorbent assay,ELISA)试剂盒检测白细胞介素(Interleukin,IL)-1 β、IL-6、IL-18 和肿瘤坏死因子-α(Tumor necrosis factor-α,TNF-α)的表达水平;吸光光度法检测细胞内超氧化物歧化酶(Superoxidedismutase,SOD)、丙二醛(Malondialdehyde,MDA)水平;2,7-二氯荧光素二乙酸酯(2',7'-Dichlorodihydrofluorescein diacetate,DCFH-DA)荧光染色检测细胞活性氧(Reactive oxygenspecies,ROS)水平;Western blot检测焦亡相关蛋白裂解半胱氨酸蛋白酶-1(Cleaved-caspase-1,C-Caspase-1)、消皮素 D(Gasdermin D,GSDMD)和 GSDMD-N 和 PI3K/Akt/NF-κB 信号通路蛋白表达水平.结果 STA水平在0.5 μmol/L以下不影响Ht22细胞活力,因此采用0.1、0.2、0.5 μmol/L STA进行后续实验.与Control组比较,OGD/R组Ht22细胞增殖活性(24、48、72 h)、SOD活性显著降低,红色荧光强度、焦亡率、焦亡蛋白C-Caspase-1,GSDMD和GSDMD-N、炎性因子、MDA,ROS及PI3K/Akt/NF-κB信号通路蛋白表达水平显著升高(P＜0.05);与OGD/R组比较,STA-L,STA-M,STA-H组Ht22细胞增殖活性、SOD活性显著上升,红色荧光强度、焦亡率、焦亡蛋白C-Caspase-1,GSDMD和GSDMD-N、炎性因子、MDA、ROS及PI3K/Akt/NF-κB信号通路蛋白表达水平显著降低(P＜0.05);STA-H+740Y-P组与OGD/R组上述指标水平无显著差异(P＞0.05);740Y-P组与OGD/R组比较,病理程度显著加重(P＜0.05),通路激活剂740Y-P可抵消STA对于神经细胞损伤的保护作用.结论 STA可以通过抑制PI3K/Akt/NF-κB信号通路来减轻OGD/R诱导的Ht22细胞焦亡.
Abstract
Objective To investigate the mechanism underlying the effects of stachydine(STA)on neu-ronal pyroptosis induced by oxygen-glucose deprivation/reoxygenation(OGD/R)and the involvement of the phosphatidylinositol 3-kinase/protein kinase B/nuclear transcription factor-κB(PI3K/Akt/NF-κB)signaling pathway.Methods The effect of different concentrations of STA on the viability of the hippocampal neuron cell line Ht22 was detected using the MTT assay.After OGD/R induction,the Ht22 cells were divided into several groups:the model group(OGD/R group),low-dose stachydine group(STA-L group),medium-dose stachydine group(STA-M group),high-dose stachydine group(STA-H group),high-dose stachydine+pathway activator 740Y-P group(H+740Y-P group),pathway activator 740Y-P group(740Y-P group),and control group consisting of normal cultured cells.The proliferation of Ht22 cells was assessed using the CCK-8 method.Cell membrane damage was evaluated using Hoechst/PI staining.Pyroptosis was measured using flow cytometry.The expression of the interleukins IL-1β,IL-6,and IL-18 and of tumor necrosis factor-α(TNF-α)was determined using ELISA kits.The levels of intracellular superoxide dismutase(SOD)and ma-londialdehyde(MDA)were measured using absorptiometry.The cellular reactive oxygen species(ROS)con-centration was assessed using DCFH-DA fluorescence staining.The expression of the pyroptosis-related pro-teins C-Caspase-1,GSDMD,and GSDMD-N,as well as the expression of PI3K/Akt/NF-κB signaling pathway proteins,was examined using Western.Results Concentrations of STA below 0.5 μmol/L did not have any impact on the viability of Ht22 cells.Therefore,for subsequent experiments,concentrations of 0.1,0.2,and 0.5 μmol/L STA were used.Compared to those in the control group,the proliferation activity(at 24 h,48 h,and 72 h)and SOD activity of Ht22 cells in the OGD/R group were significantly lower.Additionally,there were noticeable increases in red fluorescence intensity;pyroptosis rate;pyroptotic C-Caspase-1,GSDMD and GSDMD-N expression;inflammatory factor,MDA,and ROS levels;and PI3K/Akt/NF-κB signaling pathway protein expression(P＜0.05).On the other hand,compared to those in the OGD/R group,the STA-L,STA-M,and STA-H groups exhibited significant increases in proliferation and SOD activity in Ht22 cells.Moreover,there were clear decreases in red fluorescence intensity;pyroptosis rate;pyroptotic C-Caspase-1,GSDMD and GSDMD-N expression;inflammatory factor,MDA,and ROS levels;and PI3K/Akt/NF-κB sig-naling pathway protein expression(P＜0.05).The pathological severity of the 740Y-P group was significantly worse than that of the OGD/R group(P＜0.05),indicating that the pathway activator 740Y-P counteracted the protective effect of STA on nerve cell damage.Conclusion STA could attenuate OGD/R-induced Ht22 cell pyroptosis by inhibiting the PI3K/Akt/NF-κB signaling pathway.
关键词
水苏碱/氧糖剥夺再灌注/神经元焦亡/磷脂酰肌醇3-激酶/蛋白激酶B/核转录因子-κB信号通路Key words
Stachydine/OGD/R/Neuron pyroptosis/PI3K/Akt/NF-κB signaling pathway引用本文复制引用
基金项目
宝鸡市卫生健康委科研项目(2020-012)
出版年
2024
NETL
NSTL
万方数据