Ligustilide ameliorates hippocampal neuronal injury after cerebral ischemia reperfusion through activating PINK1/Parkin-dependent mitophagy

Abstract

Background

Mitophagy plays a critical role in cerebral ischemia/reperfusion by timely removal of dysfunctional mitochondria. In mammals, PINK1/Parkin is the most classic pathway mediating mitophagy. And the activation of PINK1/Parkin mediated mitophagy exerts neuroprotective effects during cerebral ischemia reperfusion injury (CIRI). Ligustilide (LIG) is a natural compound extracted from ligusticum chuanxiong hort and angelica sinensis (Oliv.) diels that exerts neuroprotective activity after cerebral ischemia reperfusion injury (CIRI). However, it still remains unclear whether LIG could attenuates cerebral ischemia reperfusion injury (CIRI) through regulating mitophagy mediated by PINK1/Parkin.

Purpose

To explore the underlying mechanism of LIG on PINK1/Parkin mediated mitophagy in the hippocampus induced by ischemia reperfusion.

Methods

This research used the middle cerebral artery occlusion and reperfusion (MCAO/R) animal model and oxygen-glucose deprivation and reperfusion (OGD/R) as in vitro model. Neurological behavior score, 2, 3, 5-triphenyl tetrazolium chloride (TTC) staining and Hematoxylin and Eosin (HE) Staining were used to detect the neuroprotection of LIG in MCAO/R rats. Also, the levels of ROS, mitochondrial membrane potential (MMP) and activities of Na⁺-K⁺-ATPase were detected to reflect mitochondrial function. Moreover, transmission electron microscope (TEM) and fluorescence microscope were used to observe mitophagy and the western blot was performed to explore the changes in protein expression in PINK1/Parkin mediated mitophagy. Finally, exact mechanism between neuroprotection of LIG and mitophagy mediated by PINK1/Parkin was explored by cell transfection.

Results

The results show that LIG improved mitochondrial functions by mitophagy enhancement in vivo and vitro to alleviate CIRI. Whereas, mitophagy enhanced by LIG under CIRI is abolished by PINK1 deficiency and midivi-1, a mitochondrial division inhibitor which has been reported to have the function of mitophagy, which could further aggravate the ischemia-induced brain damage, mitochondrial dysfunction and neuronal injury.

Conclusion

LIG could ameliorate the neuronal injury against ischemia stroke by promoting mitophagy via PINK1/Parkin. Targeting PINK1/Parkin mediated mitophagy with LIG treatment might be a promising therapeutic strategy for ischemia stroke.

Introduction

Ischemia stroke caused by intracerebral arterial embolism, accounting for about 80% of all stroke, has become one of the leading causes of death and permanent disability worldwide (Liao et al., 2019; Wu et al., 2018). Currently, the most common therapy for the patients with acute ischemia stroke is thrombolytic therapy (Gerzanich et al., 2018; Hafez et al., 2015). And the only drug for clinical application approved by Food and Drug Administration (FDA) in USA is tissue plasminogen activator (t-PA). However, the use of t-PA has been limited because of its narrow therapeutic time window and potential neurological injury caused by resupplied blood flow. Therefore, finding novel therapeutic medicines for cerebral ischemia reperfusion is urgently required.

Mitophagy, characterized as a form of selective autophagy, plays an important role in cellular homeostasis because of its function of clearing the damaged and unwanted mitochondria (Lampert et al., 2019; Lichvarova et al., 2018). Recent evidence indicates that the neuroprotection after cerebral ischemic reperfusion is associated with the activation of mitophagy mediated by such as PINK1/Parkin, FUNDC1, BNIP3 signaling pathways (Lampert et al., 2019; Shen et al., 2017; Wen et al., 2021). Among these, PINK1/Parkin pathway is the most classic mitophagy pathway in central nervous system (CNS) disease. The activation of PINK1/Parkin mediated mitophagy could ameliorate neuronal injury induced by cerebral ischemia reperfusion injury through improving mitochondrial function (Wang et al., 2019; Wen et al., 2021). However, some studies have also showed that the inhibition of mitophagy is benefit for the decrease of neurological injury after cerebral ischemia reperfusion (Feng et al., 2018; Yu et al., 2016). Therefore, the role of mitophagy on ischemia stroke is still controversial.

Ligustilide (LIG) is one of the major bioactive chemical composition isolated from the ligusticum chuanxiong hort and angelica sinensis (Oliv.) diels. The potential pharmacological activity of LIG for neurovascular diseases has been discovered for its angiogenesis and antioxidant in ischemia stroke and other diseases (Ren et al., 2020; Zhou et al., 2019). Our previous experiments had found that LIG could protect against ischemic stroke by reducing reactive oxygen species (ROS) aggregation and Ca²⁺overload in neurons, enhancing mitochondrial membrane potential (MMP) (Wu et al., 2015). Further studies have found that LIG could provide neuroprotective effects by promoting mitochondrial division, which is necessary to mitophagy (Wu et al., 2020b). So, we hypothesized that LIG could alleviate the injury against cerebral I/R via promoting PINK1/Parkin mediated mitophagy. And whether LIG plays a protective role on hippocampal neurons injured by ischemia stroke through the PINK1/Parkin mediated mitophagy will be explored in this article.