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| 亚低温对OGD/R模型新生大鼠海马神经元损伤的保护分子机制研究 |
| Molecular mechanism of protective effects of mild hypothermia on hippocampal neuron damage in OGD/R model newborn rats |
| 投稿时间：2022-12-01 |
| DOI：10.3969/j.issn.1000-0399.2023.06.002 |
| 中文关键词: 海马神经元 新生大鼠 氧糖剥夺/复糖复氧 亚低温 自噬通量 |
| 英文关键词: Hippocampal neuron Neonatal rats Oxygen-glucose deprivation/reoxygenation Mild hypothermia Autophagic flux |
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| 中文摘要: |
| 目的 观察亚低温(MH)对新生大鼠海马神经元氧糖剥夺/复糖复氧(OGD/R)损伤的保护作用,并探讨其作用机制。方法 体外培养新生大鼠海马神经元,将实验细胞随机分为对照组、OGD/R组、OGD/R+MH组和OGD/R+MH+Chl组;OGD/R组神经元给予OGD处理6 h,再于37℃复糖复氧处理24 h建立OGD/R模型。OGD/R+MH组和OGD/R+MH+Chl组神经元均给予OGD处理6 h,再分别于32℃不加或加氯喹(Chl)条件下复糖复氧处理24 h。收集各组复糖复氧处理24 h后的细胞及细胞培养上清液;采用电镜观察神经元超微结构变化,MTT法检测神经元活力,ELISA法检测乳酸脱氢酶(LDH)漏出率,TUENL染色法检测神经元凋亡情况,West-ern blot检测半胱氨酸天冬氨酸蛋白酶3剪切体(Cleaved caspase-3)、聚ADP核糖聚合酶剪切体(Cleaved PARP)、溶酶体相关膜蛋白2(LAMP2)、泛素结合蛋白P62、微管相关蛋白1轻链3-Ⅱ(LC3Ⅱ)表达水平。结果 与对照组比较,OGD/R组出现明显的细胞器损伤、降解、线粒体肿胀等神经元损伤表现,神经元活力下降、LDH漏出率增加、凋亡神经元增多(P<0.05),凋亡标志物Cleaved caspase-3、Cleaved PARP表达升高(P<0.05),LAMP2、P62蛋白表达降低,LC3Ⅱ蛋白表达升高(P<0.05)。MH干预后,OGD/R+MH组较OGD/R组细胞器损伤减轻,神经元活力升高、LDH漏出率减少、凋亡神经元减少(P<0.05),凋亡标志物Cleaved caspase-3、Cleaved PARP表达降低(P<0.05),LAMP2蛋白表达升高,P62、LC3Ⅱ蛋白表达降低(P<0.05)。而OGD/R+MH+Chl组同时给予MH和自噬通量抑制剂Chl干预后,MH的上述作用被逆转(P<0.05)结论 MH可能通过改善自噬通量、增加海马神经元自噬活性,从而减轻OGD/R所致的神经损伤。 |
| 英文摘要: |
| Objective To observe the protective effect of mild hypothermia (MH) against oxygen-glucose deprivation/reoxygenation (OGD/R) injury of hippocampal neurons in neonatal rats, and to explore its action mechanism. Methods The hippocampal neurons of neonatal rats were cultured in vitro, and then they were randomly divided into control group, OGD/R group, OGD/R+MH group and OGD/R+MH+Chl group. The neurons in OGD/R group were treated with OGD for 6 h, and then treated with reoxygenation for 24 h under 37℃ to construct OGD/R models. The neurons in the OGD/R+MH group and OGD/R+MH+Chl group were treated with OGD for 6 h, and then treated with reoxygenation without or with chloroquine (Chl) for 24 h under 32℃,respectively.The cells and cell supernatants after 24 h of reoxygenation in each group were collected. The ultrastructural changes of neurons were observed by electron microscope. The activity of neurons was detected by MTT. The leakage rate of lactate dehydrogenase(LDH) was detected by ELISA. Neuronal apoptosis was detected by TUENL staining. The expression levels of cleaved cysteine proteinase 3 (Cleaved caspase-3), cleaved poly ADP-ribose polymerase (Cleaved PARP), lysosomeassociated membranes protein 2 (LAMP2),ubiquitin-binding protein P62 and microtubule-associated protein 1 light chain 3-II (LC3II) were detected by Western blot. Results Compared with control group, there were obvious neuron injuries (organelle damage, degradation, mitochondrial swelling), declined neurons activity, increased leakage rate of LDH and apoptotic neurons in OGD/R group (P<0.05), the expressions of Cleaved caspase-3 and Cleaved PARP increased (P<0.05), the expressions of LAMP2 and P62 decreased, and LC3II increased (P<0.05). Compared with the OGD/R group, organelle damage was reduced,neuron activity increased, the leakage rate of LDH and apoptotic neurons decreased in the OGD/R+MH group (P<0.05), the expressions of Cleaved caspase-3 and Cleaved PARP decreased (P<0.05), LAMP2 increased, and P62 and LC3II decreased (P<0.05). However, in the OGD/R+MH+Chl group, the above-mentioned effects of MH were significantly reversed(P<0.05). Conclusions MH may alleviate OGD/R-induced nerve injury by improving autophagic flux and increasing autophagic activity of hippocampal neurons. |
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