基于Bip-GABAA受体α1蛋白稳态（proteostasis）的MCI大鼠麻醉药物筛选、配伍策略

Mild cognitive impairment (MCI) has substantial morbidity, seems to meet more frequently perioperatively. Therefore, choosing the proper anesthetics for these patients to preserve their compromised brain function is a major research endeavor for anesthetists. In the previous studies, we found that there is a positive relationship between neurotoxicity induced by isoflurane and its doses (published in “Br J Anaesth”). We also confirmed that subanesthetic dosage of propofol provided post-conditioning neuroprotection to cerebral ischemia (Nature Science Foundation of China, 81071059，81100984, all finished). Therefore in this study, following accomplishment of a rat model of MCI (bilateral common carotid artery stenosis), 30 days later, MCI rats received an open tibial fracture on the left hind paw with an intramedullary fixation. We use cognitive function test, Real-time PCR, western blotting method and immunofluorescence, to detect the changes of parameters in ER stress, proteostasis of GABAA receptor α1 subunit during different combinations of isoflurane and propofol. We also use antagonist of GABAA receptor α1 subunit to demonstrate its role during these combinations provided. We culture rat primary hippocampal neurons. After chronic hypoxia provided, we use Bip small interfering RNAs (siRNA) to demonstrate the role of Bip in maintaining proteostasis of GABAA receptor α1 subunit and the changes of this pathway during different combinations of isoflurane and propofol. The present study will provide basis and strategy on how to choose proper anesthetics and combination for MCI patients perioperatively.

轻度认知功能损害（MCI）发病率高、围手术期常见，麻醉药物合理选择及配伍亟需解决。前期工作证实异氟烷神经毒性与剂量正相关（发表于Br J Anaesth）；亚麻醉剂量丙泊酚对脑缺血有后处理保护作用（国家自然科学基金81071059，81100984）。本研究制作大鼠MCI模型（双侧颈总动脉缩窄），30d后行胫骨骨折切开复位内固定术，应用异氟烷和丙泊酚不同组合，利用认知行为学测定、尼氏染色、Real-time PCR、western blot、免疫荧光检测MCI大鼠ER stress、GABAA受体α1 Proteostasis变化，并给予GABAA受体α1拮抗剂探讨α1亚基调控机制；培养海马神经元，制作慢性缺氧模型，应用BipSiRNA探讨Bip对GABAA受体α1亚基Proteostasis调控作用及该通路在麻醉药不同组合中的变化，从而为MCI人群麻醉药物合理选择及配伍提供指导和依据。

研究背景：轻度认知功能障碍（mild cognitive impairment, MCI）患者围术期麻醉药选择是临床上亟待解决的问题。项目组前期发现异氟烷通过内质网应激（ER stress）引起神经元损伤，剂量是是决定其毒性作用的关键因素。不同配伍剂量异氟烷与丙泊酚对MCI大鼠ER stress初始启动分子结合免疫球蛋白（binding immunoglobulin protein，Bip）和GABAA受体α1亚基蛋白稳态的影响尚不清楚。.主要研究内容：研究分三部分：（1）通过在体动物实验，研究两种麻醉药不同应用：1 %异氟烷+丙泊酚20 mg·kg−1·h−1、1.4 %异氟烷+丙泊酚10 mg·kg−1·h−1、1.9 %异氟烷、丙泊酚40 mg·kg−1·h−1，对施行胫骨骨折切开复位内固定手术MCI大鼠ER stress、GABAA受体α1亚基Proteostasis影响及GABAA受体α1亚基调控作用。（2）在离体细胞实验中，培养大鼠原代海马神经元，制作慢性低氧模型，应用Bip小干扰RNA（small interfering RNAs, siRNA）转染技术，研究两种麻醉药不同组合对Bip-GABAA受体α1亚基Proteostasis 通路的影响及Bip细胞水平调控机制。（3）完成对MCI大鼠模型大脑超微结构变化的观察，深化MCI大鼠模型建立过程中认知功能减退机制的探讨。.重要结果与关键数据：（1）在体实验中发现，异氟烷复合丙泊酚麻醉（1 %异氟烷+丙泊酚20 mg·kg−1·h−1）对骨折手术MCI大鼠术后认知功能、ER stress影响较两者单独麻醉时减轻，更适宜骨折手术MCI 大鼠麻醉应用。（2）离体细胞实验中发现，1 %异氟烷和6.7 μmol/L（相当于动物实验20 mg·kg−1·h−1的剂量）丙泊酚配伍通过引起Bip蛋白表达上调，维持神经元GABAA受体α1亚基稳态，避免神经元进一步损伤。（3）严重的双侧颈动脉狭窄可诱发老年大鼠轻度认知功能障碍和脑显微结构损伤，且与MCI模型筛选标准相符，进一步确定了该模型的稳定性，并发现该模型下引发MCI的机制与造成线粒体逆行信号系统关键靶点hnRNPA2表达水平降低有关。.科学意义：为临床AD高危转化状态的MCI患者围手术期麻醉药物的合理选择及应用提供思路和策略。