[Mitophagy mediated by ligustilide relieves OGD/R-induced injury in HT22 cells].

Mitochondrion, as the main energy-supply organelle, is the key target region that determines neuronal survival and death during ischemia. When an ischemic stroke occurs, timely removal of damaged mitochondria is very important for improving mitochondrial function and repairing nerve damage. This study investigated the effect of ligustilide(LIG), an active ingredient of Chinese medicine, on mitochondrial function and mitophagy based on the oxygen and glucose deprivation/reperfusion(OGD/R)-induced injury model in HT22 cells. By OGD/R-induced injury model was induced in vitro, HT22 cells were pre-treated with LIG for 3 h, and the cell viability was detected by the CCK-8 assay. Immunofluorescence and flow cytometry were used to detect indicators related to mitochondrial function, such as mitochondrial membrane potential, calcium overload, and reactive oxygen species(ROS). Western blot was used to detect the expression of dynamin-related protein 1(Drp1, mitochondrial fission protein) and cleaved caspase-3(apoptotic protein). Immunofluorescence was used to observe the co-localization of the translocase of outer mitochondrial membrane 20(TOMM20, mitochondrial marker) and lysosome-associated membrane protein 2(LAMP2, autophagy marker). The results showed that LIG increased the cell viability of HT22 cells as compared with the conditions in the model group. Furthermore, LIG also inhibited the ROS release, calcium overload, and the decrease in mitochondrial membrane potential in HT22 cells after OGD/R-induced injury, facilitated Drp1 expression, and promoted the co-localization of TOMM20 and LAMP2. The findings indicate that LIG can improve the mitochondrial function after OGD/R-induced injury and promote mitophagy. When mitophagy inhibitor mdivi-1 was administered, the expression of apoptotic protein increased, suggesting that the neuroprotective effect of LIG may be related to the promotion of mitophagy.

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

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.

Ligustilide attenuates ischemic stroke injury by promoting Drp1-mediated mitochondrial fission via activation of AMPK.

BACKGROUND: Ischemic stroke is a major global cause of death and permanent disability. Studies have suggested that mitochondria play a critical role in maintaining cellular energy homeostasis and inevitably involved in neuronal damage during cerebral ischemic. Ligustilide is the main active ingredient of Angelica sinensis and Ligusticum chuanxiongs with neuroprotective activity. PURPOSE: These study sought to exlopre the role of LIG in improving mitochondrial function and the relationship between LIG induced mitochondrial fission and mitophagy in ischemic stroke. METHODS: Cerebral I/R injury was established by the model of Oxygen-glucose deprivation/reperfusion (OGD/R) in HT22 cells and middle cerebral artery occlusion (MCAO) in rats. Mitochondrial functions of were detected by flow cytometry and immunofluorescence, and mitochondrial fission were detected by western blots. Furthermore, we studied the role of AMPK pathway in the neuroprotective effect of LIG. RESULTS: LIG treatment significantly increased the MMP and ATP production, decreased the reactive oxygen species (ROS) generation and Ca2+ overload, and further induced mitochondrial fission and mitophagy. Moreover, we found that blocking mitochondrial fission by mdivi-1 resulted in accumulation of damaged mitochondria mainly through selectively blocking mitophagy, thereby inhibiting viability of HT-22 cells after OGD/R. Also, Drp-1 inhibitor mdivi-1 increased the infarct volume and aggravated the neurological deficits after MCAO operation in vivo. Additionally, LIG triggered AMP-activated protein kinase (AMPK) pathway. AMPKα2 knockdown attenuated LIG-induced mitochondrial fission through inhibiting the expression of Drp1 and Fis1, and led to nerve cell apoptosis. CONCLUSION: Our study indicate that LIG attenuated the injury of ischemic stroke by improving mitochondrial function and highlight the critical role of LIG in the regulation of LIG-induced mitochondrial fission and mitophagy via an AMPK-dependent manner. These findings indicate that LIG protects nerve damage against ischemic stroke by inducing Drp1-mediated mitochondrial fission via activation of AMPK signaling pathway in vivo and in vitro.