解偶联蛋白-2促进高血糖脑缺血时星形胶质细胞活化的研究

Mitochondrial abnormalities and the generation of ROS involved in hyperglycemia aggravate cerebral ischemic injury. We confirmed that astroyctes activation was inhibitied by hyperglycemia, which was involved in hyperglcyemia enhanced brain ischemia. However, the underlying mechemism was unclear. Uncoupling proteins (UCPs) are inner mitochondrial membrane protein family members. UCP2 can against oxidative stress, play a neuroprotective effect. For investigation of UCP2 role in diabetes model with cerebral ischemia, to make clear whether stabilizing mitochondril and inhibition of mitochondrial the o O2 were involved in the cerebral protect and in astrocytes protection, and further to detect the involved molecular mechanisms. The Astrocytes would be primary cultured from Tg-UCP2 (high expression of UCP2 genotype) KO-the UCP2 (not express UCP2 gene knockout type) and the C57BL/6j (wild-type genotype) mouse. And the corresponding astrocytes cell lines will be cultured. High oxygen deprivation experiments (OD) with high blood sugar and/or high lactate conditions will be applied to mimic hyperglycemia with stroke state. Cerebral ischemia model (MCAO) will be built on the diabetic mouse to explore whether increased expression of UCP2 can slow hyperglycemia aggravated cerebral ischemic injury and protect astrocytes from injury. And further to investigate the involved possible molecular mechanisms. The results will provide a new direction to treatment of stroke patients with diabetes / hyperglycemia.

线粒体功能障碍ROS过多参与了高血糖加重缺血性脑损伤，我们前期研究提示，高血糖可抑制星形胶质细胞活化和增殖，从而加重缺血性脑损伤，其机制尚不清楚。本项目拟通过体外和体内实验，对解偶联蛋白-2（UCP2）促进高血糖脑缺血时星形胶质细胞活化进行较深入的研究。对比观察高表达UCP2（Tg-UCP2）, 不表达UCP2 （KO-UCP2）和野生型（C57BL/6j）小鼠原代培养的星形胶质细胞，以及MA-h星形胶质细胞株，在高血糖和高乳酸条件下缺氧时细胞活化和增殖能力；采用大脑中动脉缺血模型，对比观察Tg-UCP2，KO-UCP2和C57BL/6j小鼠在高血糖脑缺血再灌注时，过氧化损伤和星形胶质细胞凋亡、活化和增殖能力；采用免疫组化、Western blot、定量PCR等方法测定线粒体通路相关基因表达。探讨增加UCP2表达是否可促进星形胶质细胞活化和增殖、减缓高血糖加重脑缺血损伤，及可能的分子机制。

本研究按照申请书计划进行了研究工作，基本达到了预期的研究目标。研究结果提示，糖尿病高血糖加重缺血再灌注脑组织损伤，解偶联蛋白-2（UCP2）基因敲除（KO-UCP2）可以明显加重糖尿病/高血糖加重的缺血再灌注脑损伤，增加脑梗死的面积及神经元损伤。高血糖通过破坏线粒体膜电位 ，增加•O2.的生成，最终下调UCP2表达，相反，UCP2稳定星形胶质细胞线粒体膜电位 ，调节•O2的生成。KO-UCP2可能促进神经细胞凋亡。糖尿病高血糖脑缺血再灌注损伤中，KO-UCP2上调Cyt C、Caspase-3、Cleaved Caspase-9的表达,KO-UCP2可能通过线粒体介导的凋亡通路，参与高血糖脑缺血再灌注的神经元损伤。在缺血再灌注损伤激发的氧化应激反应下，星形胶质细胞细胞受到氧化损伤，在高血糖条件下，损伤进一步加重，激活受到抑制，从而失去对神经元的支持和营养。糖尿病/高血糖可以明显加重缺血性脑损伤，脑缺血性损伤的加重可能与eNOS，VEGF和VEGFR-1的表达降低有关。枸杞多糖是本地中药枸杞的有效成分，可通过促进线粒体融合及降低线粒体分裂，减轻高血糖条件下脑缺血再灌注损伤。