Naringin reduces mitochondrial fragmentation and excessive mitophagy in cerebral ischemia-reperfusion injury via the S-nitrosylated Dynamin-related protein 1 (SNO-DRP1) / Leucine-rich repeat kinase 2 (LRRK2) / Mitochondrial Calcium Uniporter (MCU) pathway.

BACKGROUND: Mitophagy is vital for preventing cerebral ischemia-reperfusion (CI/R) injury. Naringin, a flavanone glycoside, reduces CI/R injury by blocking harmful mitophagy induced by peroxynitrite (ONOO⁻). Our earlier studies identified that mitochondrial calcium (mtCa2+) efflux stabilizes mitochondrial endosymbiosis and initiates protective mitophagy. However, it is unclear whether and how naringin inhibits harmful mitophagy in association with mtCa2+.

PURPOSE: This study aimed to examine the influence of the S-nitrosylated dynamin-related protein 1 (SNO-DRP1)/leucine-rich repeat kinase 2 (LRRK2)/mitochondrial calcium uniporter (MCU) pathway on harmful mitophagy, which is inhibited by naringin in CI/R injury.

METHODS: To validate the hypotheses, a transient middle cerebral artery occlusion/reperfusion (tMCAO/R) model in rats and oxygen-glucose deprivation/reoxygenation (OGD/R)-induced PC12 cells were used to investigate the effects of naringin. Specifically, linsidomine hydrochloride (SIN-1) was used to enhance SNO-DRP1, whereas spermine (Spm) was used to activate MCU. Mitochondrial fragmentation was assessed by measuring the average mitochondrial diameter via transmission electron microscopy (TEM) and evaluating the protein levels of mitofusin 1 (MFN1) and dynamin-related protein 1 (DRP1) through western blotting (WB). The SNO-DRP1 formation was determined by a biotin switch assay. The extent of SNO-DRP1-mediated excessive mitophagy was examined by quantifying the number of autophagosomes using TEM and assessing the colocalization of translocase of the outer mitochondrial membrane complex subunit 20 (TOMM20)-DRP1-3-NT and 3-NT-DRP1-LC3 via immunofluorescence (IF). The interaction between SNO-DRP1 and LRRK2 was confirmed using co-immunoprecipitation and IF colocalization. MtCa2+ overload induced by MCU was evaluated by analyzing MCU protein levels, measuring mtCa2+ fluorescence intensity following Rhod-2 AM treatment, and assessing the opening of the mitochondrial permeability transition pore. The mitochondrial reactive oxygen species and mitochondrial DNA release were used to evaluate the mitochondrial endosymbiosis failure.

RESULTS: Naringin exhibited a dose-dependent protective effect against CI/R injury in rats subjected to the tMCAO/R model. It effectively reduced mitochondrial fragmentation and SNO-DRP1-mediated mitophagy, attenuated the interaction between SNO-DRP1 and LRRK2, and decreased the protein level of MCU in the penumbra region of the cortex. SIN-1 and Spm counteracted the neuronal protection of naringin in OGD/R-induced PC12 cells. They also mitigated the impact of naringin on mitochondrial fragmentation, harmful mitophagy, and MCU-mediated mtCa2+ overload and mitochondrial endosymbiosis disruption. Notably, only SIN-1 reversed naringin's suppression of SNO-DRP1 formation and its interaction with LRRK2 in vitro.

CONCLUSIONS: This study demonstrates that naringin reduces ONOO⁻-caused harmful mitophagy in CI/R injury by blocking the SNO-DRP1/LRRK2/MCU pathway, which helps prevent mitochondrial fragmentation and preserve mitochondrial endosymbiosis. This pathway may be among the mechanisms by which naringin exerts its protective effects.