ABSTRACT

Serine protease Omi/HtrA2, a member of the HtrA family, is closely related to the maintenance of mitochondrial integrity and participates in apoptosis but its role in cerebral ischemia/reperfusion (I/R) injury and cellular oxidative stress response remains unclear. In this study, we found that I/R injury resulted in a time-dependent increase in Omi/HtrA2 expression in rat brain tissue. Inhibition of Omi/HtrA2 significantly inhibited XIAP cleavage in H₂O₂-induced PC12 cells. In addition, inhibition of Omi/HtrA2 significantly inhibited the up-regulation of mitochondrial stress proteins CHOP and ClpP, significantly reduced mitochondrial aggregation, and attenuated the decline of mitochondrial ΔΨm in PC12 cells. Studies show that there is a physical interaction between Omi/HtrA2 and OPA1. We found that Omi/HtrA2 and OPA1 are closely related to the oxidative stress mitochondrial response in PC12 cells. The current study has demonstrated that Omi/HtrA2 is upregulated in brain I/R injury in vivo and is implicated in mitochondrial response to oxidative stress in vitro by regulating mitochondrial stress proteins CHOP and CLpP and by interacting with mitochondrial cristae remodeling protein OPA1. These findings suggest that Omi/HtrA2 could be a candidate molecular target in diseases that involve oxidative stress such as in I/R injury.

Abbreviation: ATP: Adenosine tripHospHate; Bax: BCL2-Associated X; Bcl-2: B-cell lympHoma-2; BSA: Albumin from bovine serum; DMEM: Dulbecco’s Minimum Essential Medium; DMSO: Dimethyl sulfoxide; HSP60: Heat shock protein60, 70; L-OPA1: Long forms of OPA1; Omi/HtrA2: high-temperature-regulated A2; MCAO: Middle cerebral artery occlusion; OPA1: Optic AtropHy; PBS: PHospHate buffered saline; PMSF: pHenylmethyl sulfonylfluoride; ROS: reactive oxygen species; SDS: Sodium dodecyl sulfate; S-OPA1: Short forms of OPA1; TTC: TripHenyltetrazalium chloride; XIAP: X-linked inhibitor apoptosis protein

摘要

丝氨酸蛋白酶 Omi/HtrA2 是 HtrA 家族的一员，与维持线粒体完整性密切相关并参与细胞凋亡，但其在脑缺血/再灌注 (I/R) 损伤和细胞氧化应激反应中的作用尚不清楚。在这项研究中，我们发现 I/R 损伤导致大鼠脑组织中 Omi/HtrA2 表达的时间依赖性增加。Omi/HtrA2 的抑制显着抑制了 H ₂ O _{2 中的}XIAP 裂解-诱导的 PC12 细胞。此外，Omi/HtrA2的抑制显着抑制了线粒体应激蛋白CHOP和ClpP的上调，显着减少了线粒体聚集，并减弱了PC12细胞中线粒体ΔΨm的下降。研究表明 Omi/HtrA2 和 OPA1 之间存在物理相互作用。我们发现 Omi/HtrA2 和 OPA1 与 PC12 细胞中的氧化应激线粒体反应密切相关。目前的研究表明，Omi/HtrA2在体内脑 I/R 损伤中上调，并在体外参与线粒体对氧化应激的反应通过调节线粒体应激蛋白 CHOP 和 CLpP 以及与线粒体嵴重塑蛋白 OPA1 相互作用。这些发现表明 Omi/HtrA2 可能是涉及氧化应激的疾病（如 I/R 损伤）的候选分子靶标。

缩写：ATP：Adenosine tripHospHate；Bax：BCL2-相关X；Bcl-2：B 细胞淋巴瘤-2；BSA：牛血清白蛋白；DMEM：Dulbecco 最低必需培养基；DMSO：二甲基亚砜；HSP60：热休克蛋白60、70；L-OPA1：OPA1 的长形式；Omi/HtrA2：高温调节A2；MCAO：大脑中动脉闭塞；OPA1：视神经萎缩；PBS：磷酸缓冲盐水；PMSF：苯甲基磺酰氟；ROS：活性氧；SDS：十二烷基硫酸钠；S-OPA1：OPA1 的缩写；TTC：三苯基四氮杂氯化物；XIAP：X-连锁抑制剂凋亡蛋白