活化Drp1介导谷胱甘肽代谢调节失血性休克后线粒体功能的研究

Role of activated dynamin-related protein 1-mediated glutathione metabolism in regulation of mitochondrial dysfunction after hemorrhagic shock

目的探究失血性休克后活化Drp1介导线粒体功能改变与能量代谢之间的关系及其作用机制。方法 60只C57雌鼠(8周龄,体质量约20 g)分为休克WT组与常态WT组(n=30)。60只Drp1 KO雌鼠(C57品系,8周龄,体质量约20 g)分为休克Drp1 KO组(作缺血休克处理)与常态Drp1 KO组(n=30)。观察失血性休克后血管组织能量代谢、线粒体代谢、线粒体功能、Drp1活性变化,以及干预Drp1对休克后能量代谢、线粒体代谢及其差异代谢物的影响。对血管平滑肌细胞进行Drp1过表达、外源性谷胱甘肽(10 mmol/L)处理后观察干预Drp1及谷胱甘肽对缺氧后ROS、线粒体膜电位等线粒体功能变化。结果休克后血管组织能量代谢减低、谷胱甘肽等线粒体代谢异常、线粒体ROS产生增高2.3倍、线粒体ATP含量减少67.8%、线粒体呼吸强度OCR减弱50%(P<0.05),缺氧后ROS水平增高4倍、线粒体膜电位减少76.7%(P<0.05),并且休克和缺氧后均发现Drp1发生活化及线粒体转位(P<0.05)。常态WT组血管组织谷胱甘肽水平为(1.35±0.42)nmol/μg,休克WT组谷胱甘肽水平为(0.55±0.20)nmol/μg,休克Drp1 KO组血管组织谷胱甘肽水平(0.94±0.30)nmol/μg明显高于休克WT组(P<0.05)。Drp1过表达后血管平滑肌细胞ROS水平增加3.3倍、线粒体膜电位减少70%(P<0.05),进一步外源性补充谷胱甘肽后ROS水平和线粒体膜电位明显改善(P<0.05)。结论失血性休克后活化Drp1可通过抑制谷胱甘肽线粒体代谢引起线粒体功能障碍和机体能量代谢紊乱。

Objective To explore the relationship between activated dynamin-related protein 1(Drp1)-mediated mitochondrial function and energy metabolism after hemorrhagic shock and clarify its potential mechanisms. Methods Sixty C57 female mice(8-weeks old, weighing about 20 g) were randomly divided into shock+WT group(n=30, treated with hemorrhagic shock) and Normal+WT group(n=30). Thirty Drp1 KO C57 female mice(8-weeks old, weighing about 20 g) were treated with hemorrhagic shock as shock+Drp1 KO group, and the other 30 KO mice were regarded as Normal+Drp1 KO group. The changes of energy metabolism, mitochondrial metabolism, mitochondrial functions as well as Drp1 activity in vascular tissues after hemorrhagic shock were observed in WT and KO mice respectively. Vascular smooth muscle cells(VSMCs) were treated with Drp1 over-expression and exogenous glutathione(10 mmol/L) to confirm the effects of Drp1 and glutathione on mitochondrial functions after shock. Results After hemorrhagic shock, the energy metabolism and mitochondrial metabolism, such as glutathione, were decreased significantly(P<0.05), mitochondrial reactive oxygen species(ROS) production were increased by 2.3 times(P<0.05), ATP content was decreased by 67.8%(P<0.05), and mitochondrial respiratory rates were decreased by 50%(P<0.05). However, hypoxia resulted in the ROS production of VSMCs enhanced by 4 times and the mitochondrial membrane potential(ΔΨm) decreased by 76.7%(P<0.05). What’s more, both Drp1 activation and mitochondrial translocation were observed in shock-treated mice and hypoxia-induced VSMCs. The level of glutathione was 1.35±0.0.42 nmol/μg in normal+WT group, 0.55±0.20 nmol/μg in shock+WT group, while the level in shock+Drp1 KO group(0.94±0.30 nmol/μg) was much higher than that in shock+WT group(P<0.05). Drp1 over-expression induced ROS level in VSMCs increased by 3.3 times and ΔΨm decreased by 70%(P<0.05). After further exogenous glutathione supplementation, both ROS level and ΔΨm were improved significantly(P<0.05). Conclusion Activated Drp1 after hemorrhagic shock induces mitochondrial dysfunctions and abnormal energy metabolism by inhibiting glutathione metabolism.