硒在减缓糖尿病加重脑缺血损伤中的作用及与线粒体分裂/融合关系研究

To clear the prevention and treatment function of selenium and selenoprotein H (SelH) in hyperglycemia exacerbated ischemic brain injury and the relations with mitochondrial fission/fusion. The cultured hippocampal pyramidal cells (HT22), the SelH transfected HT22 cells and the corresponding empty vector transfected cells were injured by high lactate and high glucose+ hypoxia, which are similar with the state of hyperglycemic ischemic cerebral injury simulation. Selenite treatment will be given at different time points. Cell activity, mitochondrial membrane potential, mitochondrial respiratory function and ROS generation will be observed to detect whether selenium/selH can release the damage of the nerve cell and of the mitochondrial function; Electro microscopy will be applied to observe the mitochondria morphology; Double labeling of mitochondrial specific labeled probe (Mito Tracker Green) and lysosomal specific labeled probe (Lyso Tracker red) evaluate mitochondrial autophagy status; immunofluorescence and western blotting assay of mitochondrial fission/fusion relating factors, including Drp1, Fis1, OPA1, Mfn2 and mitochondrial autophagy-related factors LC3 expression. With which to explore whether selenium/SelH slow the nerve cell damage by stabilizing fission/fusion balance. Animal experiments was applied to further verify the results. The aim of all this study was testing whether high-selenium drinking water can reduce hyperglycemia aggravated cerebral ischemic damage and the relationship with mitochondrial fission/fusion.or further validate the test results, the test will be applied in diabetic rat model with focal cerebral ischemic injury. Testing whether high-selenium drinking water can reduce hyperglycemia aggravated cerebral ischemic damage and the relationship with mitochondrial fission/fusion.

为明确硒和硒蛋白H(SelH)在预防及治疗高血糖加重脑缺血损伤中的作用及与线粒体分裂/融合的关系，本课题将培养海马椎体细胞（HT22），SelH转染HT22细胞和相应空载体转染细胞，高乳酸及高糖+缺氧培养，模拟高血糖脑缺血损伤状态，损伤不同时间点给予亚硒酸钠保护。检测细胞活性，线粒体膜电位, 线粒体呼吸功能和ROS的生成，验证硒/SelH能否减缓该状态对神经细胞的损伤和线粒体功能的破坏；透射电镜观察线粒体，线粒体特异性标记探针（Mito Tracker Green）与溶酶体特异性标记探针（Lyso Tracker red）双重标记评价线粒体自噬状态；免疫荧光和western blotting 法检测线粒体裂解/融合相关因子Drp1，Fis1，OPA1，Mfn2 和线粒体自噬相关因子LC3等的表达。糖尿病大鼠脑缺血模型进一步验证实验结果。探讨硒/SelH减缓神经损伤的可能机制。

糖尿病/高血糖加重脑缺血再灌注损伤，糖尿病/高血糖可进一步加重脑缺血后线粒体相关活性氧簇（ROS）的产生。硒可以稳定线粒体内膜电位和减少 ROS 的形成。但硒在高血糖加重脑缺血损伤中的作用及与线粒体分裂/融合的关系目前尚不清楚。本课题将培养海马椎体细胞（HT22），SelH转染HT22细胞和相应空载体转染细胞，高乳酸及高糖+缺氧培养，模拟高血糖脑缺血损伤状态，损伤不同时间点给予亚硒酸钠保护。检测细胞活性，线粒体膜电位，线粒体呼吸功能和ROS的生成，验证硒/SelH能否减缓该状态对神经细胞的损伤和线粒体功能的破坏；透射电镜观察线粒体，线粒体特异性标记探针（Mito Tracker Green）与溶酶体特异性标记探针（Lyso Tracker red）双重标记评价线粒体自噬状态；免疫荧光和 western blotting 法检测线粒体裂解/融合相关因子Drp1，Fis1，OPA1，Mfn2和线粒体自噬相关因子LC3等的表达。糖尿病大鼠脑缺血模型进一步验证实验结果。探讨硒/SelH减缓神经损伤的可能机制。结果发现：1）硒可以缓解高乳酸，高糖+缺氧对海马椎体细胞的损伤；2）SelH 可以缓解高乳酸，高糖+缺氧对海马椎体细胞的损伤；3）线粒体分裂增加，融合减少参与了高乳酸，高糖+缺氧对海马椎体细胞的损伤；4）硒和 SelH 通过减少线粒体分裂，促进线粒体融合减缓了高乳酸，高糖+缺氧对海马椎体细胞的损伤。本项目的研究结果有可能为脑中风伴随糖尿病/高血糖患者提供新的治疗方向（低剂量的硒补充可能成为该类疾病的治疗途径）。. 该项目已发表 SCI 收录论文6篇，中文核心期刊1篇。获2019年宁夏科技进步奖三等奖1项。培养博士研究生2名，硕士研究生3名。