摘要
目的探讨水苏碱(Stachydine,STA)对氧糖剥夺再灌注(Oxygen-glucose deprivation/reoxygenation,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)-1β、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0.5μmol/L以下不影响Ht22细胞活力,因此采用0.1、0.2、0.5μmol/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。
Objective To investigate the mechanism underlying the effects of stachydine(STA) on neuronal 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 malondialdehyde(MDA) were measured using absorptiometry. The cellular reactive oxygen species(ROS) concentration was assessed using DCFH-DA fluorescence staining. The expression of the pyroptosis-related proteins 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 signaling 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.
作者
高李
杨祎
Gao Li;Yang Yi(Department of Neurology,Baoji Central Hospital,Baoji Shanxi 721000;不详)
出处
《卒中与神经疾病》
2024年第2期159-166,共8页
Stroke and Nervous Diseases
基金
宝鸡市卫生健康委员会科研课题(编号为2020-012)。
