摘要
目的探究c-Jun氨基末端激酶(JNK)在促进氧糖剥夺(OGD)神经元存活中的作用及机制。方法体外培养大鼠胎鼠大脑皮层原代神经元建立OGD神经元模型,通过CCK-8法筛选自噬特征时间点,进行JNK激活剂茴香霉素处理。将细胞随机分为空白组、茴香霉素处理组(神经元在OGD后,用茴香霉素处理5 h)、溶剂对照组(OGD神经元培养基中加入等量的溶剂二甲基亚砜)。Western blot法和免疫荧光细胞化学染色分别检测OGD神经元beclin 1、微管相关蛋白1轻链3(LC3)和B细胞淋巴瘤因子2(Bcl2),胱天蛋白酶3(caspase-3)、P62、泛素(ubiquitin)、组织蛋白酶B(cathepsin B)和溶酶体相关膜蛋白1(LAMP1)的蛋白表达量;CCK-8法检测神经细胞活性,利用IPP软件测量神经元轴突长度。结果激活JNK可明显提高LC3、beclin 1、Bcl2的表达,并显著降低beclin 1-Bcl2复合物的含量及caspase-3、P62、ubiquitin的表达,而cathepsin B、LAMP1的表达无明显变化。由此,通过激活JNK活性可显著提高OGD神经元存活。结论激活JNK促进beclin 1-Bcl2解离,增强自噬,诱导大鼠OGD神经元凋亡向自噬转换,发挥神经元保护作用。
Objective To investigate the efficacy and mechanism of c-Jun N-terminal kinase(JNK)in boosting survival of oxygen glucose deprivation(OGD)rat neurons.Methods The cortex neurons frcxn fetal rats were primarily cultured to prepare a model of OGD neurons in vitro,and the characteristic endpoints were filtered to intervene with JNK inducer anisomycin(AN),respectively.The cells were randomly divided into control group,solvent control group(a same volume of solvent DMSO was added into the culture medium of the OGD neuron),AN group(OGD neurons were treated with JNK inducer AN for 5 hours at the end of OGD).After that,Western blotting and immunofluorescence cytometry were respectively performed to detect the protein expressions in OGD neurons,including beclin 1,microtubule-associated protein 1 light chain 3(LC3),B cell lymphoma 2(Bcl2),caspase-3,P62,ubiquitin,cathepsin B and lysosomal associated membrane protein 1(LAMP1).The cell activity was evaluated by CCK-8 assay,and the axon length was measured by IPP software.Results Activation of JNK significantly promoted the expressions of beclin 1,LC3,and Bcl2,and markedly reduced the content of beclin 1-Bcl2 complex and attenuated the expressions of P62 and ubiquitin.Meanwhile,the expressions of cathepsin B and LAMP1 were not obviously altered.In this way,the survival rate of OGD neurons was improved.Conclusion Activation of JNK exerts a neuroprotective effect by facilitating dissociation of beclin 1-Bcl2 and inducing a switch from apoptosis to autophagy in OGD neurons.
作者
黄志文
任璐
邓仪昊
何红云
HUANG Zhiwen;REN Lu;DENG Yihao;HE Hongyun(Department of Human Anatomy,Medical College,Kunming University of Science and Technology,Kunming 650500,China)
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2022年第5期412-418,共7页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金(81960418,81860411)
云南省万人计划青年拔尖人才专项(YNWR-QNBJ-2018*034)
云南省应用基础研究计划项目(2019FB098,202001AT070049)
云南省教育厅科研基金项目(2018JS016,2020J0066)
省级人培项目(KKSY201960010)。
