摘要
目的 探讨瑞香素通过抑制硫氧还蛋白结合蛋白(Thioredoxin-interacting protein, TXNIP)/核苷酸结合寡聚化结构域样受体蛋白3(Nucleotide-binding oligomerization domain-like receptor protein 3,NLRP3)信号通路来对氧糖剥夺/复氧(Oxygen-glucose deprivation/Reoxygenation, OOGD/R)诱导的海马神经元炎症反应和凋亡的影响。方法 对体外培养的小鼠海马神经元HT-22做OGD/R处理诱导建立细胞损伤模型,采用细胞计数试剂盒-8(Cell counting Kit-8,CCK-8)实验检测0、5、10、20、40、80μmol/L的瑞香素对其细胞活力的影响,筛选出最佳作用水平;将体外培养的HT-22细胞随机分为对照组、模型组、瑞香素(40μmol/L)组、TXNIP空载(转染TXNIP空载质粒)组、TXNIP过表达(转染TXNIP过表达质粒)组、瑞香素(40μmol/L)+TXNIP过表达组,以瑞香素和质粒提前处理12 h后进行OGD/R诱导建立细胞损伤模型;采用二苯甲亚胺、三氯化氢2′-(4-羟基苯基)-5-(4-甲基-1-哌嗪基)-2,5′-二-1H-苯并咪唑(2′-(4-Hydroxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5′-bi-1H-benzimidazole, trihydrochloride, Hoechst)33258染色及流式细胞实验检测各组HT-22细胞凋亡情况;采用试剂盒检测各组HT-22细胞线粒体膜电位;应用酶标仪测量各组HT-22细胞肿瘤坏死因子-α(Tumor necrosis factor-α,TNF-α)、白细胞介素(Interleukin, IL)-6、IL-1β、IL-18、超氧化物歧化酶(Superoxide Dismutase, SOD)、过氧化氢酶(Catalase, CAT)、活性氧(Reactive oxygen species, ROS)及乳酸脱氢酶(Lactate dehydrogenase, LDH)释放水平;采用免疫印迹实验检测各组HT-22细胞TXNIP/NLRP3通路蛋白表达水平。结果 选择40μmol/L瑞香素处理OGD/R诱导HT-22细胞进行后续实验;与对照组比较,模型组HT-22细胞凋亡率、细胞TNF-α,IL-6,IL-1β,IL-18及ROS水平、细胞LDH释放水平、细胞TXNIP,NLRP3及天冬氨酸特异性半胱氨酸蛋白酶1(Cysteine-containing aspartate-specific proteases-1,Caspase-1)蛋白表达水平明显升高(P<0.05),线粒体膜电位、细胞SOD及CAT水平明显降低(P<0.05)。分别与模型组、瑞香素+TXNIP过表达组比较,瑞香素组HT-22细胞凋亡率、细胞TNF-α,IL-6,IL-1β,IL-18及ROS水平、细胞LDH释放水平、细胞TXNIP,NLRP3及Caspase-1蛋白表达水平均降低(P<0.05),线粒体膜电位、细胞SOD及CAT水平均升高(P<0.05);TXNIP过表达组HT-22细胞凋亡率、细胞TNF-α,IL-6,IL-1β,IL-18及ROS水平、细胞LDH释放水平、细胞TXNIP,NLRP3及Caspase-1蛋白表达水平均升高(P<0.05),线粒体膜电位、细胞SOD及CAT水平均降低(P<0.05);TXNIP空载组HT-22细胞各指标水平均无明显变化(P>0.05)。结论 瑞香素可通过抑制TXNIP/NLRP3信号通路来降低OGD/R诱导的ROS和炎性因子过量表达,抑制炎症和氧化应激反应,缓解海马神经元线粒体损伤,减轻神经元凋亡。
Objective To explore the effect of daphnetin on oxygen-glucose deprivation/reoxygenation(OGD/R)-induced inflammatory response and apoptosis in hippocampal neurons by inhibiting thioredoxin-interacting protein(TXNIP)/nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3) signaling pathway. Methods The mouse hippocampal neuron HT-22 cultured in vitro was treated with OGD/R to induce a cell injury model, and the effects of daphnetin at 0, 5, 10, 20, 40, 80 μmol/L on cell viability were detected by CCK-8 assay, and the optimal concentration was screened.The HT-22 cells cultured in vitro were randomly divided into control group, model group, daphnetin(40 μmol/L) group, TXNIP empty(transfected with TXNIP empty plasmid) group, and TXNIP overexpression(transfected with TXNIP overexpression plasmid) group, and daphnetin(40 μmol/L) + TXNIP overexpression group. The cell injury model was established by OGD/R induction after pre-treatment with daphnetin and plasmid for 12 h. Hoechst 33258 staining and flow cytometry were performed to detect the apoptosis of HT-22 cells in each group. The mitochondrial membrane potential of HT-22 cells in each group was detected by commercial kits.The microplate reader was used to measure the release levels of tumor necrosis factor-α(TNF-α), interleukin(IL)-6, IL-1β, IL-18, superoxide dismutase(SOD), superoxide dismutase(SOD), peroxidation Hydrogenase(CAT), reactive oxygen species(ROS) and lactate dehydrogenase(LDH).Western blotting was performed to detect the expression levels of TXNIP/NLRP3 pathway protein in HT-22 cells in each group. Results 40 μmol/L Daphnetin was selected to treat OGD/R-induced HT-22 cells for subsequent experiments. Compared with the control group, the apoptosis rate, the levels of TNF-α, IL-6, IL-1β, IL-18 and ROS, the release level of LDH, the protein expression levels of TXNIP, NLRP3 and caspase-1 of the HT-22 cells were significantly increased in the model group(P<0.05), and the mitochondrial membrane potential, levels of cellular SOD and CAT were significantly decreased(P<0.05). Compared with the model group and the daphnetin + TXNIP overexpression group, the apoptosis rate of HT-22 cells, the levels of TNF-α, IL-6, IL-1β, IL-18 and ROS, the release level of LDH in the cells, the protein expression levels of TXNIP, NLRP3 and caspase-1 of the HT-22 cells were significantly decreased in the daphnetin group(P<0.05), and the mitochondrial membrane potential, levels of cellular SOD and CAT were significantly increased(P<0.05);the apoptosis rate of HT-22 cells, the levels of TNF-α, IL-6, IL-1β, IL-18 and ROS, the release level of LDH in the cells, the protein expression levels of TXNIP, NLRP3 and cysteine-containing aspartate-specific proteases 1(caspase-1) in the cells were significantly increased in the TXNIP overexpressiongroup(P<0.05), and the mitochondrial membrane potential, levels of cellular SOD and CAT were significantly decreased(P<0.05). There was no significant change in all indexes of HT-22 cells in TXNIP empty group(P>0.05). Conclusion Daphnetin can reduce the overexpressions of ROS and inflammatory factors induced by OGD/R by inhibiting the TXNIP/NLRP3 signaling pathway, inhibiting inflammation and oxidative stress, alleviating mitochondrial damage in hippocampal neurons, and reducing neuronal apoptosis.
作者
赵敏
谢丽华
逯琴
Zhao Min;Xie Lihua;Lu Qin(Department of Neurology,Linfen People's Hospital,Linfen Shanxi 041000)
出处
《卒中与神经疾病》
2022年第6期503-510,共8页
Stroke and Nervous Diseases
关键词
瑞香素
硫氧还蛋白结合蛋白/核苷酸结合寡聚化结构域样受体蛋白3
氧糖剥夺/复氧
海马神经元
炎症
凋亡
Daphnetin
Thioredoxin-interacting protein/nucleotide-binding oligomerization domain-like receptor protein 3
Oxygen-glucose deprivation/reoxygenation
Hippocampal neurons
Inflammation
Apoptosis
作者简介
通信作者:逯琴。
