微量胰岛素对七氟醚吸入麻醉诱导新生大鼠认知功能障碍的预防作用及其可能的作用机制

doi:10.16098/j.issn.0529-1356.2021.06.007

摘要/Abstract

摘要：

目的探讨微量胰岛素对七氟醚吸入麻醉诱导大鼠认知功能障碍的预防作用，及其可能的作用机制。方法将60只新生大鼠随机分为对照组（CON）、低剂量胰岛素预防组（LIP）、高剂量胰岛素预防组（HIP）和七氟醚模型组（MOD），其中预防组和模型组均采用七氟醚诱导构建大鼠认知功能障碍模型。采用Morris水迷宫定向航行实验和空间探索实验评价大鼠的学习和记忆功能； HE染色观察大鼠海马组织病理学变化；流式细胞术检测大鼠海马组织细胞的凋亡情况；RT-PCR检测海马组织雷帕霉素靶蛋白（mTOR）、真核细胞肽链延伸因子2（eEF-2） mRNA表达水平； Western blotting检测脑源性神经营养因子（BDNF）、突触后致密蛋白-95（PSD-95）、突触素-Ⅰ（synapsin-Ⅰ）、钙调蛋白激酶Ⅱα（CaMKⅡα）、mTOR及eEF-2蛋白表达水平。结果 Morris水迷宫实验结果显示，胰岛素能够显著缩短大鼠逃避潜伏期时间及游泳距离，提高穿越平台次数；流式细胞术结果表明，胰岛素预防组能够显著抑制大鼠脑神经细胞的凋亡，且高剂量胰岛素预防组抑制效果更为明显；RT-PCR 及Western blotting检测发现，模型组大鼠海马组织中mTOR、eEF-2 mRNA和蛋白表达水平显著升高，而BDNF、PSD-95、synapsin-Ⅰ、CaMKⅡα蛋白表达水平显著降低；与模型组相比，胰岛素预防给药组大鼠海马组织中mTOR、eEF2 mRNA和蛋白表达水平显著下调，而BDNF、PSD-95、synapsin-Ⅰ、CaMKⅡα蛋白表达水平明显上调，差异具有统计学意义（P<0.05）。结论微量胰岛素可增加认知功能障碍大鼠海马组织中突触相关蛋白的表达，降低其mTOR、eEF-2 mRNA表达水平，预防七氟醚诱导的大鼠认知功能的障碍，其机制可能与调节mTOR-eEF2途径有关。

关键词: 胰岛素, 雷帕霉素靶蛋白, 真核细胞肽链延伸因子2, 认知功能障碍, 免疫印迹法, 大鼠

Abstract:

Objective To study the preventive effect of microinsulin on cognitive dysfunction induced by sevoflurane inhalation anesthesia in rats and its possible mechanism. Methods Sixty newborn rats were randomly divided into control group(CON), insulin prevention low dose group (LIP), insulin prevention hihg dose group (HIP), and sevoflurane model group(MOD). The prevention group and the model group were induced by sevoflurane to construct rat cognitive dysfunction model. Morris water maze directional sailing test and space exploration test were used to evaluate the learning and memory function of rats; HE staining was used to observe the pathological morphological changes of hippocampus in rats; Flow cytometry was used to detect the hippocampus of rats cell apoptosis; Detection of rapamycin target protein (mTOR) and eukaryotic peptide chain elongation factor 2 (eEF-2) mRNA levels in hippocampus by RT-PCR; The expression levels of brain-derived neurotrophic factor (BDNF), post-synaptic dense protein-95 (PSD-95), synapsin-Ⅰ, and calmodulin kinase Ⅱα(CaMKⅡα)， mTOR and eEF-2 protein were detected by Western blotting. Results The result of the Morris water maze experiment showed that insulin significantly reduced the escape latency and swimming distance of rats, and increased the number of crossing platforms; Flow cytometry result showed that the insulin prevention group significantly inhibited the apoptosis of rat brain neurons, and the inhibition effect of high-dose insulin prevention group was more obvious; RT-PCR and Western blotting analysis found that the expression levels of mTOR and eEF-2 mRNA and proteins in the hippocampus of the model group increased significantly, while the expression levels of BDNF, PSD-95, synapsin-Ⅰ, and CaMKⅡα proteins reduced significantly. The expression levels of mTOR and eEF-2 mRNA and proteins in the hippocampus of rats in the insulin prevention group decreased significantly, while the expression levels of BDNF, PSD-95, synapsin-Ⅰ, and CaMKⅡα protein increased significantly. The difference was statistically significant (P<0.05). Conclusion Trace insulin can increase the expression of synapse-related proteins in the hippocampus of cognitive dysfunction rats, reduce their mTOR and eEF-2 mRNA expression levels, and prevent sevoflurane-induced cognitive impairment in rats. The mechanism may be related to the regulation of mTOR-eEF-2 approach.

Key words: Insulin, Rapamycin target protein, Eukaryotic peptide chain elongation factor 2, Cognitive dysfunction, Western blotting, Rat

中图分类号:

R575.1

吴勇陈健陈爱鸾李成洁沈伯雄 . 微量胰岛素对七氟醚吸入麻醉诱导新生大鼠认知功能障碍的预防作用及其可能的作用机制[J]. 解剖学报, 2021, 52(6): 882-888.

WU Yong CHEN Jian CHEN Ai-luan LI Cheng-jie SHEN Bo-xiong. Preventive effect of trace insulin on cognitive dysfunction induced by sevoflurane inhalation anesthesia in newborn rats and its possible mechanism[J]. Acta Anatomica Sinica, 2021, 52(6): 882-888.

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