生物钟基因Bmal1调控自噬在高糖诱导心肌损伤中的作用

High glucose-induced cardiomyocyte injury is the major cause of diabetic cardiomyopathy (DCM), the regulation mechanisms of which are not full clear. It has been known that suppression of autophagy was protective in high glucose-induced cardiomyocyte injury, there was an innate circadian rhythm to the regulation of autophagy, there were alterations of circadian clock in the heart by streptozotocin-induced diabetes. Based on our previous study, we hypothesized that the clock gene, brain and muscle Arnt-like 1(bmal1), regulates autophagy in high glucose-induced cardiomyocyte injury. We are going to make cardiomyocyte injury model in response to high glucose with cardiomyocytes derived from both neonatal rats and adult mice. we will use the molecular biology and cell biology techniques to address three questions: (1) if the alteration expression of circadian clock gene, bmal1, has an impact upon cardiomyocyte death and survival in response to high glucose; (2) how altered autophagy was involved in the impact of altered bmal1 expression upon cardiomyocyte death and survival in response to high glucose. (3) what is the molecular mechanism underlying the observed autophagic activity change in response to altered bmal1 expression? in the present study, we will uncover the molecular mechanism for high glucose-induced cardiomyocyte injury from new point of view and provide a potential therapeutic target site for DCM prevention and treatment.

高糖诱导心肌细胞损伤是糖尿病性心肌病的主要原因，但此过程的调控机制尚未完全阐明。现有证据表明：抑制自噬在高糖诱导的心肌损伤中发挥保护作用，自噬受到生物钟节律的调控，糖尿病大鼠心脏节律基因表达异常。我们在前期研究的基础上提出生物钟基因bmal1表达异常通过调控自噬参与高糖诱导心肌损伤的假说。我们拟制备高糖新生大鼠和成鼠心肌细胞损伤模型，应用分子生物学、细胞生物学等技术，阐明三个问题：(1)bmal1表达异常是否影响高糖诱导的心肌损伤；(2) 自噬改变是如何在bmal1表达异常影响高糖诱导的心肌损伤中发挥作用的；(3) 高糖环境中bmal1表达异常影响自噬改变的信号转导机制是什么；本课题将从新的视觉阐明高糖心肌损伤发生的分子机制，为糖尿病心肌病的防治提供新思路和新靶点。

高糖诱导心肌细胞损伤是糖尿病性心肌病的主要原因，但此过程的调控机制尚未完全阐明。本项目应用细胞生物学、分子生物学等技术，以心肌细胞为研究对象，对生物钟基因bmal1在DCM发生发展中的作用进行了深入探讨。我们腺病毒载体转染Bmal1 shRNA到新出生的SD大鼠的心室肌细胞，以沉默bmal1基因造成该基因的低表达，转染SC shRNA作为对照组。转染Bmal1 cDNA到心肌细胞，以产生Bmal1基因过表达，转染PcDNA作为对照组。然后分别用含正常葡萄糖浓度（5.5mM）的培养液和含高浓度葡萄糖浓度（25mM）的培养液体外培养，结果显示：高糖环境下Bmal1基因过表达可减轻心肌细胞损伤，并对抑制的自噬有恢复作用，进一步用3-MA抑制自噬，可观察到Bmal1过表达对心肌细胞损伤的抑制作用有所减轻；相反Bmal1基因低表达可进一步加重高糖诱导的心肌细胞损伤，并对高糖抑制的自噬有进一步下调作用，进一步用雷帕霉素诱导自噬，可观察到Bmal1低表达对高糖诱导的心肌细胞损伤的加重作用有所下降。研究还发现，高糖环境下，mTORC1通路中的p-p70S6K、p-S6和p-4EBP表达明显升高，Bmal1基因过表达对mTORC1通路中的p-p70S6K、p-S6和p-4EBP有明显上调作用，在使用mTORC1的抑制剂雷帕霉素后，原本被上调的自噬受到了一定程度的抑制，并且原本减轻的心肌损伤又有所加重，而过表达mTOR后可抑制高糖条件下的自噬，并可显著减少高糖导致的心肌细胞损伤。综上所述，生物钟基因Bmal1表达异常通过调控自噬参与高糖诱导的心肌损伤， mTORC1信号通路介导高糖环境中Bmal1表达异常对自噬的调控。