Intermittent hypoxia preconditioning protects WRL68 cells against oxidative injury: Involvement of the PINK1/Parkin-mediated mitophagy regulated by nuclear respiratory factor 1

Highlights

• IH preconditioning induced the PINK1/Parkin-mediated mitophagy.

• IH preconditioning enhanced the capacity of mitophagy.

• The IH preconditioning-induced mitophagy is regulated by NRF-1.

• IH-induced mitochondrial ROS generation increased the expression of NRF-1.

Abstract

The protective effect of intermittent hypoxia (IH) preconditioning against oxidative injury in hepatic cells was investigated and the involvement of the PINK1/Parkin-mediated mitophagy regulated by nuclear respiratory factor 1 (NRF-1) was evaluated. The results showed that IH preconditioning protected HepG2 cells against oxygen and glucose deprivation/reperfusion (OGD/Rep)-induced injury and protected WRL68 cells against H₂O₂ or AMA-induced oxidative injury. IH preconditioning up-regulated the protein level of NRF-1, PINK1, Parkin, and LC3 II, promoted the recruitment of the cytosolic Parkin, indicating the initiation of the PINK1/Parkin-mediated mitophagy in WRL68 cells. When NRF-1 was down-regulated by NRF-1 specific shRNA, the protein level of PINK1 and Parkin as well as the mitophagy level were significantly decreased. After IH preconditioning, the protein level of PINK1 and the recruitment of Parkin in CCCP-treated group were significantly higher than that of the control group, indicating the increased mitophagy capacity. And the increased mitophagy capacity induced by IH preconditioning was also reduced by down-regulation of NRF-1. Furthermore, the protective effect of IH preconditioning against H₂O₂-induced oxidative injury in WRL68 cells was inhibited when NRF-1 or PINK1 was down-regulated by specific shRNA. Mitochondrial ROS generation may be responsible for the increased expression of NRF-1 induced by IH preconditioning. In conclusion, the PINK1/Parkin-mediated mitophagy regulated by NRF-1 was involved in IH preconditioning-induced protective effect against oxidative cellular injury in hepatic cells.

Introduction

Intermittent hypoxia (IH) refers to the process of repeated alternation of hypoxia and reoxygenation which can trigger a bewildering array of both detrimental and beneficial effects in multiple physiological systems (Navarrete-Opazo and Mitchell, 2014, Serebrovska et al., 2016). Accumulating evidence suggests that modest/acute IH exposures often lead to beneficial effects without detectable pathology (Navarrete-Opazo and Mitchell, 2014, Qiao et al., 2019, Serebrovska and Shatilo, 2015). It has been reported that IH preconditioning reduced myocardial infarct size after global ischemia–reperfusion in rats (Beguin et al., 2005). And IH preconditioning-induced ischemic tolerance persists longer than ischemic preconditioning (Qiao et al., 2019). IH preconditioning also alleviated ischemia–reperfusion-induced injury in liver and brain in rodents (Chouker et al., 2012, Wang et al., 2017). Although more and more studies have focused on the protective effect of IH preconditioning in recent years, the exact mechanism underlying how IH preconditioning exerts its protective effect is still unclear (Li et al., 2019, Serebrovska et al., 2016).

Mitochondria are highly dynamic organelles that are essential for cellular physiology (Ma et al., 2020, Palmer et al., 2011). Mitophagy, known as the mitochondria specific autophagy, is a major player in the stability of the mitochondrial network by removing damaged or abnormal mitochondria (Garza-Lombo et al., 2020, Ma et al., 2020). It has been reported that PINK1/Parkin-mediated mitophagy is the most important pathway that contributes to protecting mitochondria against oxidative stress-induced injury (Eiyama and Okamoto, 2015, Wei et al., 2015, Zhao et al., 2019). When mitochondria are damaged, PINK1 acts as a mitochondrial serine/threonine kinase driving Parkin recruitment to mitochondria, starting the PINK1/Parkin-mediated mitophagy (Kane et al., 2014, Sekine and Youle, 2018). Therefore, mitophagy mediated by PINK1/Parkin plays an essential role in mitochondrial quality control under stress conditions (Georgakopoulos et al., 2017, Zhu et al., 2013).

Nuclear respiratory factor 1 (NRF-1) has always been considered as the important transcription factor in regulating genes that essential for mitochondrial biogenesis (Dhar et al., 2008, Wang et al., 2016). In recent years, it has been found that NRF-1 can function as the transcription factor in regulating other genes that are not closely related to mitochondrial biogenesis suggesting the more potential functions of NRF-1 (Satoh et al., 2013). In our earlier studies, we found that NRF-1 has a positive regulatory effect on the expression of PINK1 and Parkin genes, and involves in mitochondrial quality control through regulating the PINK1/Parkin-mediated mitophagy (Lu et al., 2020).

Although mitochondrial protection has been involved in the IH preconditioning-mediated protection (Chang et al., 2019, Jung and Mallet, 2018, Wang et al., 2012). However, it is not clear whether the PINK1/Parkin-mediated mitophagy plays an important role in it. Therefore, the involvement of the PINK1/Parkin-mediated mitophagy in IH preconditioning-mediated protection against oxidative stress-induced injury in human hepatic cell was studied in this study, and the regulatory role of NRF-1 was also evaluated.