淫羊藿苷对神经元细胞氧化应激损伤的保护作用及机制研究

Protective effect and mechanism of Icariin on oxidative stress injury in neurons

目的探索淫羊藿苷对于神经元细胞氧化损伤的保护作用机制,从而为其用于认知障碍的治疗提供基础药理学依据。方法利用谷氨酸(glutamate)诱导HT22细胞构建氧化应激损伤模型。将HT22细胞分为对照组(正常培养细胞)、模型组(谷氨酸诱导损伤模型)和低、中、高剂量实验组(分别以5、10、20μmol·L^(-1)淫羊藿苷预处理后建模)。用细胞计数试剂盒-8(CCK-8)法检测细胞增殖情况;用乳酸脱氢酶(LDH)法检测细胞毒性;用流式细胞仪检测细胞中活性氧(ROS)水平;用生化试剂盒检测细胞中超氧化物歧化酶(SOD)水平;用蛋白质印迹法检测Kelch样环氧氯丙烷相关蛋白-1(Keap1)、核因子E2相关因子2(Nrf2)蛋白的表达水平;用实时荧光定量聚合酶链反应法检测mRNA的表达水平。结果对照组、模型组和低、中、高剂量实验组的细胞活力分别为(100.00±1.31)%、(66.38±2.44)%、(72.07±4.95)%、(82.41±3.57)%和(87.97±4.98)%;LDH细胞毒性分别为(0.48±0.52)%、(18.82±2.09)%、(15.32±1.17)%、(10.37±1.39)%和(6.51±0.87)%;ROS水平分别为(14.23±1.13)%、(41.74±1.60)%、(35.69±1.08)%、(33.28±1.69)%和(30.32±2.03)%;SOD水平分别为(54.84±1.17)、(37.95±1.13)、(48.02±1.28)、(50.56±1.34)和(52.55±1.04)U·mg^(-1);Keap1蛋白表达水平分别为0.36±0.01、0.52±0.03、0.46±0.04、0.39±0.09和0.35±0.12;Nrf2蛋白表达水平分别为0.29±0.02、0.13±0.08、0.18±0.03、0.21±0.11和0.26±0.04;过氧化氢酶(CAT)mRNA表达水平分别为1.01±0.08、0.81±0.06、0.90±0.04、1.05±0.15和1.33±0.26;SOD mRNA表达水平分别为1.09±0.12、0.83±0.03、0.86±0.08、0.94±0.08和1.09±0.16。上述指标,模型组与对照组比较,差异均有统计学意义(均P<0.01);中、高剂量实验组与模型组比较,差异均有统计学意义(P<0.01,P<0.05)。结论淫羊藿苷可能通过激活Keap1/Nrf2/抗氧化反应元件(ARE)信号通路,调节相关蛋白表达,降低ROS水平,从而有效缓解神经元细胞的氧化应激损伤。

Objective To explore the protective mechanism of icariin on neuronal oxidative damage,providing a basic pharmacological basis for the treatment of cognitive impairment.Methods Glutamate was used to induce oxidative stress injury in HT22 cells.HT22 cells were divided into control group(normal cultured cells),model group(glutamate injury model)and experimental-L,-M,-H groups(5,10 and20μmol·L^(-1)icariin pretreatment for modeling,respectively).Cell proliferation was detected by cell counting kit-8(CCK-8)method;cytotoxicity was detected by lactate dehydrogenase(LDH)method;reactive oxygen species(ROS)levels were detected by flow cytometry;superoxide dismutase(SOD)levels were detected by biochemical kits;the expression levels of Kelch-like epichlorohydrinrelated protein-1(Keap1),nuclear factor E2-related factor 2(Nrf2)were detected by Western blotting;the corresponding mRNA expression was detected by real-time fluorescence quantification polymerose chain reaction.Results The cell viability of control group,model group and experimental-L,-M,-H groups were(100.00±1.31)%,(66.38±2.44)%,(72.07±4.95)%,(82.41±3.57)%and(87.97±4.98)%;LDH release were(0.48±0.52)%,(18.82±2.09)%,(15.32±1.17)%,(10.37±1.39)%and(6.51±0.87)%;ROS level were(14.23±1.13)%,(41.74±1.60)%,(35.69±1.08)%,(33.28±1.69)%and(30.32±2.03)%;SOD levels were(54.84±1.17),(37.95±1.13),(48.02±1.28),(50.56±1.34)and(52.55±1.04)U·mg^(-1);Keapl protein levels were 0.36±0.01,0.52±0.03,0.46±0.04,0.39±0.09 and 0.35±0.12;Nrf2 protein levels were 0.29±0.02,0.13±0.08,0.18±0.03,0.21±0.11 and 0.26±0.04;catalase(CAT)mRNA levels were1.01±0.08,0.81±0.06,0.90±0.04,1.05±0.15 and 1.33±0.26;SOD mRNA levels were 1.09±0.12,0.83±0.03,0.86±0.08,0.94±0.08 and 1.09±0.16.Among the above indicators,the differences between the model group and the control group were statistically significant(all P<0.01);the differences between the experimental-M,-H groups and the model group were statistically significant(P<0.01,P<0.05).Conclusion Icariin may activate the Keap1/Nrf2/antioxidant response element(ARE)signaling pathway,regulate the expression of related proteins,and reduce the level of ROS to effectively alleviate oxidative stress injury in neuronal cells.