Crocetin protects cardiomyocytes against hypoxia/reoxygenation injury by attenuating Drp1-mediated mitochondrial fission via PGC-1α

BACKGROUND Saffron(Crocus sativus L.)has been traditionally used as food,spice,and medicine.Crocetin(CRT),as main bioactive component of saffron,has accumulated pieces of beneficial evidence on myocardial ischemia/reperfusion(I/R)injury.However,the mechanisms are poorly explored.This study aims to investigate the effects of CRT on H9c2 cells under hypoxia/reoxygenation(H/R)and elucidated the possible underlying mechanism.METHODS H/R attack was performed on H9c2 cells.Cell counting kit-8 was used to detect the cell viability.Cell samples and culture supernatants were evaluated via commercial kits to measure the superoxide dismutase(SOD)activity,malondialdehyde(MDA)content,and cellular adenosine triphosphate(ATP)content.Various fluorescent probes were used to detect cell apoptosis,intracellular and mitochondrial reactive oxygen species(ROS)content,mitochondrial morphology,mitochondrial membrane potential(MMP),and mitochondrial permeability transition pore(mPTP)opening.Proteins were evaluated via Western Blot.RESULTS H/R exposure severely reduced cell viability and increased LDH leakage.Peroxisome proliferator-activated receptorγcoactivator-1α(PGC-1α)suppression and dynamin-related protein 1(Drp1)activation were coincided with excessive mitochondrial fission,mitochondrial permeability transition pore(mPTP)opening and mitochondrial membrane potential(MMP)collapse in H9c2 cells treated with H/R.Mitochondria fragmentation under H/R injury induced ROS over-production,oxidative stress,and cell apoptosis.Notably,CRT treatment significantly prevented mitochondrial fission,mPTP opening,MMP loss,and cell apoptosis.Moreover,CRT sufficiently activated PGC-1α and inactivated Drp1.Interestingly,mitochondrial fission inhibition with mdivi-1 similarly suppressed mitochondrial dysfunction,oxidative stress and cell apoptosis.However,silencing PGC-1α with small interfering RNA(siRNA)abolished the beneficial effects of CRT on H9c2 cells under H/R injury,accompanied with increased Drp1 and p-Drp1ser616 levels.Furthermore,over-expression of PGC-1αwith adenovirus transfection replicated the beneficial effects of CRT on H9c2 cells.CONCLUSIONS Our study identified PGC-1α as a master regulator in H/R-injured H9c2 cells via Drp1-mediated mitochondrial fission.We also presented the evidence that PGC-1α might be a novel target against cardiomyocyte H/R injury.Our data revealed the role of CRT in regulating PGC-1α/Drp1/mitochondrial fission process in H9c2 cells under the burden of H/R attack,and we suggested that modulation of PGC-1α level may provide a therapeutic target for treating cardiac I/R injury.