摘要
目的探讨MK801(dizocilpine,地佐环平)对全脑缺血/再灌注后神经型一氧化氮合成酶(nNOS)介导的凋亡信号调节激酶1(apoptosis signal-regulating kinase 1,ASK1)信号通路及海马CA1区细胞凋亡的影响。方法采用四动脉结扎法构建大鼠全脑缺血模型。SD大鼠腹腔注射MK801(30mg·kg-1)。通过"生物素转化法"(Biotin-Swich method)检测蛋白质的S-亚硝基化。然后运用免疫印迹、免疫共沉淀和免疫组织化学等方法对蛋白质的磷酸化以及蛋白质之间的相互作用进行研究;应用焦油紫染色的方法检测脑缺血/再灌注后海马CA1区的细胞凋亡情况。结果脑缺血/再灌注引起了ASK1的S-亚硝基化;MK801明显地抑制了脑缺血/再灌注诱导的ASK1的S-亚硝基化的增加;MK801明显降低了Thr845的磷酸化水平、增加了Ser83位的磷酸化水平,从而降低了ASK1的活性;MK801引起ASK1下游MKK4/7-JNK信号通路及下游凋亡的核通路也产生了相应的变化。结论 MK801能够抑制SD大鼠全脑缺血/再灌注引起ASK1的S-亚硝基化,进而影响ASK1凋亡信号通路,对神经元损伤起到保护作用。
Aim To investigate the effects of MKS01 on the activation of apoptosis signal kinase 1 （ ASK1 ） -mediated c-Jun N-terminal kinase （JNK） signaling pathway and protection of neuron. Methods Transi- ent cerebral ischemia was induced by a four-vessel oc-cluded （4-VO） method. Sprague-Dawley rats were treated intraperitoneally with （ ＋ ）-5-methyl-10, l l- dibenzol a, d-cyclohepten-5, 10-imine meleate （MK801, 30 mg . kg-1）. The study was performed mainly by SDS-PAGE, Western blot, immunoprecipi- tation and immunohistochemistry to exam phosphoryla- tion and the interaction of proteins. Cresyl violet stai-ning was also performed to examine the survival and apoptosis of neurons in the pyramidal cell layer of the hippocamal CA1 subfield. S-nitrosylation was exam- ined mainly by the biotin switch assay. Results S-ni-trosylation of ASK1 could be induced by cerebral is-chemia-reperfusion, and that administration of MK801, an antagonist of N-methyl-D-aspartate （ NMDAR）, di- minished the S-nitrosylation of ASK1. The results also showed that ASK1 S-nitrosylation decreased the level of p-ASK1 （Thr845） phosphorylation, on the contrary, it made the level of p-ASK1 （ Ser83 ） phosphorylation ele- vate. Furthermore, our results showed that ASK1 downstream MKK4/7-JNK signaling routes and nuclear or non-nuclear apoptosis pathways were involved in the above signaling routes. Conclusion MK801 can af-fect ASK1 signaling pathways by inhibiting ASK1 S-ni-trosylation during ischemia/reperfusion in SD rat, and then reduce neuron damage.
出处
《中国药理学通报》
CAS
CSCD
北大核心
2014年第2期179-185,共7页
Chinese Pharmacological Bulletin
基金
国家自然科学基金资助项目(No 31172171)
