MCU/ROS在布比卡因加重糖尿病小鼠神经损伤中的作用

Diabetic patients are susceptible to local anesthetics-induced neurotoxicity, which is closely related to hyperglycemia-mediated enhancement of local anesthetics-induced reactive oxygen species (ROS) overproduction. Mitochondrial calcium uniporter (MCU) is one of major pathway for the regulation of mitochondrial Ca2+ level. We found that hyperglycemia upregulated MCU expression, and enhanced bupivacaine-induced ROS overproduction and neurotoxicity. Inhibition of MCU activity could restrain the above phenomenon. Ca2+/Calcium/calmodulin-dependent protein kinase (CaMKII)/cyclic AMP response element binding protein (CREB) pathway was involved in hyperglycemia- and bupivacaine-mediated the expression of MCU activity. It is envisaged that hyperglycemia can elevate the expression of MCU activity through Ca2+/CaMKII/CREB pathway, and enhance bupivacaine-mediated the mitochondrial calcium overload and ROS overproduction, which aggravate the nerve damage of diabetic mice. Lentiviral technology will be used to construct the high expression and low expression of MCU in SH-SY5Y cell line. Constructing mice model of diabetic mellitus and using molecular biological technique will be involved in our study. This study will prove the target that hyperglycemia acts on the enhancement of local anesthetics neurotoxicity, and lay a theretical foundation on how to avoid and control nerve damage in diabetes after local anesthetics treatment.

研究表明：糖尿病患者对局麻药神经毒性损伤敏感性增加。我们证实该现象与高血糖增强局麻药致神经元活性氧簇（ROS）爆发密切相关，但其调控机制未明。预实验发现：①线粒体钙单向转运体（MCU）与高血糖增强布比卡因致神经元线粒体钙超载及ROS爆发相关；②Ca2+/钙调蛋白依赖性激酶Ⅱ（CaMKⅡ）/环磷腺苷反应元件结合蛋白（CREB）通路与高血糖及布比卡因调控MCU表达活性相关；由此设想：高血糖可通过Ca2+/CaMKⅡ/CREB通路上调MCU表达活性，增强布比卡因致神经元线粒体钙超载与ROS爆发，加重糖尿病小鼠神经毒性损伤。运用慢病毒感染技术构建MCU高表达及低表达SH-SY5Y细胞株，同时建立糖尿病小鼠模型，运用钙离子成像、免疫荧光、PCR、Western Blot等多种分子生物学技术证实上述设想，为糖尿病患者对局麻药神经毒性损伤敏感性增加的机制与防治研究提供理论依据。

项目背景：.中国糖尿病患病人数已居世界第一位。糖尿病神经病变是糖尿病慢性并发症中最常见类型之一，其发生、发展与高血糖持续状态及其相关病理生理改变有关，临床麻醉中越来越多需接受神经阻滞的患者伴发糖尿病，已有临床研究表明: 糖尿病患者接受神经阻滞麻醉或镇痛后，发生神经损伤的风险明显增加，或原有多发性神经病变加重，其机制未明。本课题组前期研究证实：高血糖增强活性氧簇（reactive oxygen species，ROS）爆发及DNA氧化损伤，加重局麻药致神经细胞线粒体氧化应激与毒性损伤，但高糖环境下局麻药致ROS爆发增强的调控机制未明。.主要研究内容：.1.研究MCU对高血糖调控布比卡因致神经细胞ROS生成和毒性损伤的影响。.2.研究Ca2+/CaMKⅡ/CREB在高血糖与布比卡因调控神经细胞MCU表达活性中的作用。.3.研究MCU对糖尿病大鼠鞘内注射布比卡因致脊髓神经线粒体钙超载和氧化损伤的影响。.4.研究Ca2+/CaMKⅡ/CREB在糖尿病与鞘内注射布比卡因调控大鼠脊髓神经MCU表达中作用。.重要结果与关键数据：.1. 高血糖提高MCU活性表达加重布比卡因致神经细胞线粒体ROS爆发增强其神经毒性损伤。.2.布比卡因引发神经细胞（SH-SY5Y与DRG细胞）胞浆钙离子超载，激活CaMKⅡ/CREB通路，高血糖可增强上述通路激活，提高MCU表达，加重布比卡因神经毒性损伤。.3. 布比卡因上调MCU表达活性，增强氧化应激，加重糖尿病大鼠脊髓神经损伤与提高其机械痛与热痛阈值。.4. Ca2+/CaMKⅡ/CREB在鞘内注射布比卡因提高大鼠脊髓神经MCU表达，进而加重其脊髓神经损伤中发挥重要作用。.科学意义：.本研究提示可通过调控MCU的活性防治围手术期糖尿病患者实施神经阻滞后神经损伤发生，减少围手术期神经并发症，利于术后快速康复。