自噬抑制剂3-MA对机械性神经元损伤后神经元凋亡的影响  

The effect of autophagy inhibitor 3-MA on apoptosis induced by mechanical neuronal injury

作　　者：赵明明[1] 罗鹏[2] 赵永博[3] 尹丰[1] 费舟[2] 

机构地区：[1]海军总医院神经外科,北京100048 [2]第四军医大学西京医院神经外科,陕西西安710032 [3]解放军第323医院神经外科,陕西西安710054

出　　处：《中华神经外科疾病研究杂志》2016年第3期196-200,共5页Chinese Journal of Neurosurgical Disease Research

基　　金：国家自然科学基金资助项目(30930093);全军医学科技青年培育基金资助项目(13QPN026)

摘　　要：目的研究自噬抑制剂6-氨基-3-甲基嘌呤(3-MA)对机械性损伤后神经元凋亡的影响。方法小鼠皮层神经元原代培养2 w后,采用机械性神经元损伤模型,通过蛋白印迹法(Western blot)定量分析损伤后不同时间点自噬相关分子微管相关蛋白轻链3(LC3)Ⅰ/Ⅱ的表达情况;通过免疫荧光染色分析机械性损伤后24 h神经元自噬相关分子LC3的表达情况;小鼠皮层神经元原代培养2 w后,用自噬抑制剂3-MA预处理1 h,采用机械性神经元损伤模型,通过乳酸脱氢酶(LDH)活性测定及碘化丙啶(PI)/Hoechst 33342双染测定神经元损伤程度以及3-MA的保护作用,并通过Western blot研究凋亡相关指标caspase-3和自噬相关指标微管相关蛋白轻链3(LC3)和Beclin-1的表达变化。结果Western blot方法和免疫荧光组化同时证明机械性神经元损伤后24 h LC3Ⅱ表达明显增加;LDH活性测定表明3-MA能抑制机械性损伤造成的LDH活性的增高;PI/Hoechst 33342双染表明3-MA可以明显减少机械性损伤后神经元的凋亡;通过Western blot法证明3-MA抑制LC3Ⅱ,并导致cleaved caspase-3表达明显降低。结论自噬抑制剂3-MA可能通过抑制自噬而减少机械性神经元损伤后神经元凋亡,进而对机械性损伤后神经元起保护性作用。Objective The effect of autophagy inhibitor 3-methyladenine （3-MA） on apoptosis induced by mechanical neuronal injury is investigated. Methods Mouse cortical neurons were cultured for 2 w in vitro. After establishment of mechanical neuronal injury model, the expression of microtubule-associated protein light chain 3 （Ⅰ/23） Ⅰ/Ⅱ was measured by Western blot. LC3, as an autophagy related molecular, was detected by immunofluoreseence staining in the neurons at 24 h after mechanical neuronal injury. Neurons were pre-treated with autophagy inhibitor 3-MA for 1 h before mechanical neuronal injury model. Then, cell viability was evaluated by lactate dehydrogenase （LDH） activity assay and immtmofluoreseence double staining of propidium iodide （P I） and Hoechst 33342. The expressions of caspase-3, LC3, and Beclin-1 were rneasured by Western blot. Results Western blot and staining indicated that the significant increase of LC3 expression at 24 h after mechanical neuronal injury. LDH assay showed that pre-treatment of 3-MA suppressed mechanical injury-induced elevation of LDH activity. PI/Hoechst double staining suggested that pre-treatment of 3-MA also decreased the neuronal apoptosis after mechanical injury. Furthermore, Western blot indicated that 3-MA inhibited the expression of LC3 II and then resulted in the decrease of cleaved caspase-3. Conclusion Autophagy inhibitor 3-MA might attenuate mechanical neuronal injmy-induced neuronal apoptosis via inhibiting autophagy, thereby leading to the neuroprotective effects after mechanical neuronal injury.

关 键 词：机械性神经元损伤 凋亡 自噬 

分 类 号：R322[医药卫生—人体解剖和组织胚胎学]