SIRT3调控时钟基因BMAL1去乙酰化在恢复自噬节律减轻糖尿病脑缺血再灌注损伤中的作用

Diabetic patients are more prone to ischemic stroke and have more serious damage after reperfusion, which have been found to be related to the disturbance of autophagy rhythm. Our previous study found that the sustained low expression of clock gene BMAL1 and the disturbance of autophagy rhythm during diabetic cerebral ischemia and reperfusion, which aggravated brain injury; by increasing the expression of BMAL1, autophagy rhythm was restored and brain injury was alleviated. It suggests that increasing the expression of BMAL1 to restore the autophagy rhythm may be the key to alleviating the cerebral ischemia reperfusion injury (CIRI) in diabetes, but the specific mechanism remains unclear. SIRT3 is a newly discovered key factor in the deacetylation of clock gene BMAL1. In our preliminary experiments, we found that in diabetic state, SIRT3 expression decreased while cerebral infarction size increased; by increasing the SIRT3 expression, the expression of BMAL1 deacetylation was also increased and brain damage was significantly reduced. In conclusion, we hypothesize that SIRT3 regulates the low expression of BMAL1 deacetylation, which is an important mechanism of autophagy rhythm disturbance, thus aggravating diabetic CIRI. This project aims to establish a model of diabetic CIRI in vivo/in vitro to elucidate the mechanism by which SIRT3 regulates BMAL1 deacetylation in the recovery of autophagy rhythm to reduce diabetic CIRI, thereby providing a new target for the prevention and treatment of diabetic ischemic stroke.

糖尿病患者易发生缺血性脑卒中且再灌注后损伤更为严重，研究发现其与自噬节律紊乱密切相关。我们前期研究发现糖尿病脑缺血再灌注期间核心时钟基因BMAL1持续低表达且自噬节律紊乱，脑损伤加重；通过增加BMAL1的表达恢复自噬节律，脑损伤减轻。提示我们增加BMAL1的表达进而恢复自噬节律是减轻糖尿病脑缺血再灌注损伤(CIRI)的关键，然而具体机制未明。SIRT3是新近发现调控时钟基因BMAL1去乙酰化的关键因子。我们预实验发现糖尿病CIRI中SIRT3表达降低而梗死面积增加；增加SIRT3的表达可明显上调BMAL1去乙酰化水平进而减轻脑损伤。我们推测SIRT3调控BMAL1去乙酰化，进而恢复自噬节律是减轻糖尿病CIRI的重要机制。本项目拟在在体/离体水平建立糖尿病CIRI模型，通过阐明SIRT3调控BMAL1去乙酰化在恢复自噬节律减轻糖尿病CIRI的机制，以期为糖尿病缺血性脑卒中的防治提供新靶点。

缺血性脑卒中是我国卒中患者最常见的类型，近年来我国缺血性脑卒中患者数量持续增加且呈现出年轻化趋势，给家庭和社会带来了沉重的压力。及时恢复脑血流是目前治疗缺血性脑卒中的首选方案，但治疗时间窗却极为严苛且并发症多，尤其是在恢复灌注后脑组织缺血缺氧状态不但未能改善反而会加速诱导细胞死亡即脑缺血再灌注损伤。糖尿病作为一种全身性系统性改变的疾病，其可加剧脑血管的脆弱性和易损性，是缺血性脑卒中的独立危险因素之一。研究表明与非糖尿病患者相比，传统的溶栓治疗应用在糖尿病缺血性脑卒中患者时其脑出血发生率增加，神经功能预后更差，同时一些有益于非糖尿病缺血性脑卒中患者的救治方法未能成功转化应用到糖尿病缺血性脑卒中患者的治疗中。因此深入探讨糖尿病脑缺血再灌注损伤后的有效治疗策略具有重要的临床意义。.本研究结果证实脑缺血再灌注后非糖尿病小鼠自噬升高，糖尿病小鼠自噬降低表明自噬在糖尿病状态下受到抑制，同时糖尿病小鼠CIR后线粒体损伤较非糖尿病小鼠加重，其可能与抑制SIRT1-BMAL1介导的自噬或下调SIRT3-SOD2介导的线粒体保护有关。褪黑激素作为一种内源性激素，在大脑和脑脊液中浓度较高，是一个十分具有吸引力的神经保护候选药物。我们研究发现褪黑素在糖尿病合并CIRI中具有一定的保护作用，其能以浓度依赖的方式增强SIRT1-BMAL1的激活，下调SIRT1-BMAL1或抑制自噬能削弱其保护作用，表明SIRT1和自噬均参与了褪黑素介导的抗氧化和抗凋亡作用；同时糖尿病状态下褪黑素不但减轻了CIR诱导的线粒体氧化应激、Cyt-cyto C释放和线粒体肿胀，还改善了CIR导致的MMP丢失和ATP合成障碍。以上结果从某种角度解释了为何有些对非糖尿病缺血性脑卒中患者有效的治疗方案在糖尿病缺血性脑卒中患者中无效，亦为糖尿病缺血性脑卒中患者的治疗提供理论依据，具有广泛的临床应用前景。