摘要
本研究旨在探讨在H_2O_2诱导氧化应激损伤条件下,雷公藤红素对肌萎缩性侧索硬化症细胞模型SOD1^(G93A)NSC34的保护作用及其相关分子机制。用不同剂量H_2O_2、雷公藤红素处理表达人突变SOD1^(G93A)基因的NSC34细胞24 h后,CCK-8试剂检测细胞存活率;丙二醛试剂盒检测细胞内丙二醛水平;real-time PCR检测细胞谷氨酸半胱氨酸连接酶催化亚基(glutamatecysteine ligase catalytic subunit,GCLC)和谷胱甘肽硫转移酶(glutathione S-transferases,GST)的表达水平;Western blot检测药物处理后细胞内MEK/ERK和PI3K/Akt细胞信号通路的激活。结果显示,50 nmol/L雷公藤红素预处理可提高H_2O_2(10μmol/L)损伤后SOD1^(G93A)NSC34细胞的生存率,并使细胞内MDA生成减少,逆转H_2O_2诱导的SOD1^(G93A)NSC34细胞内谷胱甘肽合成相关酶GCLC和GST mRNA表达下调。雷公藤红素处理0.5 h、1 h分别激活SOD1^(G93A)NSC34细胞内MEK/ERK和PI3K/Akt信号通路达峰值,且MEK抑制剂PD98059和Akt抑制剂MK2206可阻断雷公藤红素对相关信号通路的激活效应。PD98059、MK2206预处理30 min均抑制雷公藤红素引起的SOD1^(G93A)NSC34细胞内GCLC和GST上调。以上研究结果提示,雷公藤红素对肌萎缩性侧索硬化症细胞模型SOD1^(G93A)NSC34细胞有抗氧化应激的保护作用,该神经保护作用与雷公藤红素调节SOD1^(G93A)NSC34细胞内谷胱甘肽合成相关酶类生成有关,细胞内MEK/ERK和PI3K/Akt信号通路参与此调节过程。
To investigate the anti-oxidative effect of celastrol on H2O2-induced oxidative stress in the cell model of amyotrophic lateral sclerosis （ALS） and its molecular mechanism, NSC34 motor neuron-like cells were transfected with EGFP-G93A-SOD1 plasmid and used as in vitro ALS cell model. SOD1G93A transfected NSC34 cells were treated with different doses of H2O2 and celastrol. The survival rate of the cells was detected by CCK-8 assay, and malondialdehyde （MDA） content was detected by corresponding kit. The mRNA expression of glutamate-cysteine ligase catalytic subunit （GCLC） and glutathione S-transferases （GST） were detected by real-time PCR. The activation of intracellular MEK/ERK and PI3K/Akt signal pathways was detected by Western blot. The results showed that pre-incubation of celastrol （50 nmol/L） for 4 h prior to H2O2 （10 μmol/L） co-treatment for another 24 h significantly attenuated H2O2-induced cell death and MDA level in SOD1G93A transfected NSC34 cells. Real-time PCR showed that the mRNA expressions of GCLC and GST were enhanced with pre-incubation of celastrol. Celastrol quickly induced phosphorylation of ERK1/2 and Akt within 30 min and 1 h respectively in SOD1G93A transfected NSC34 cells. Pharmacological inhibitors of MEK （PD98059, 10 μmol/L） or Akt （MK2206, 10 μmol/L） could reverse the phosphorylation of ERK1/2 and Akt, and abolish up-regulation of GCLC and GST induced by celastrol at mRNA levels. Taken together, we conclude that celastrol exerts a beneficial antioxidant effect in SOD1G93ANSC34 cells, which might be dependent on MEK/ERK and PI3K/Akt signaling pathway activation.
作者
李映慧
刘少波
张皓云
周风华
刘永新
卢强
杨苓
LI Ying-Hui;LIU Shao-Bo;ZHANG Hao-Yun;ZHOU Feng-Hua;LIU Yong-Xin;LU Qiang;YANG Ling(College of Biology Science and Technology, Weifang Medical University, Weifang 261053, China;College of Clinic Medicine, Weifang Medical University, Weifang 261053, China;College of Pharmacy, Weifang Medical University, Weifang 261053, China)
出处
《生理学报》
CAS
CSCD
北大核心
2017年第6期751-758,共8页
Acta Physiologica Sinica
基金
supported by the National Natural Science Foundation of China(No.81271413)
the Natural Science Foundation(No.ZR2015HL047)
the College Research Projects(No.J15LK10)of Shandong Province
China
the Students’Science and Technology Innovation Fund of Weifang Medical University
China(No.KX2016042)
