Melatonin alleviates oxidative stress induced damage in human iPSCs harboring mtDNA 3243A>G mutation via MAPK pathway

Mitochondrial diseases are a group of serious inherited multisystem disorders caused by mutations in mitochondrial DNA (mtDNA) or nuclear DNA and still have faced a significant challenge to therapy due to their complicated genotype-phenotype relationships and diverse clinical manifestations. Human induced pluripotent stem cell (hiPSC) offered novel opportunities for cell-based modeling mitochondrial diseases in a patient-specific level. This study aims to explore possibility to potential strategy against mutation-associated oxidative damage through hiPSCs derived from mitochondrial diseases patients.

A human induced pluripotent stem cell line (mt-hiPSCs) from a patient harboring 70.70% heteroplasmic m.3243A>G mutation was established and exposed to hydrogen peroxide (H₂O₂). The cell viability, apoptosis level and mitochondrial function were measured through CCK-8, western blot, flow cytometry, RT-qPCR, fluorescence staining and compared to wild-type hiPSCs. Thereafter, the participation of mitogen-activated protein kinases (MAPK) pathway in the melatonin-mediated protection against H₂O₂-induced oxidative injury was also evaluated.

Under prolonged low-dose hydrogen peroxide (H₂O₂) exposure, mt-hiPSCs showed significantly reduced viability, elevated apoptosis (52.13 vs. 25.62% in wild-type hiPSCs, P < 0.001), and exacerbated mitochondrial dysfunction, including superoxide accumulation and membrane potential depolarization. Melatonin pretreatment effectively mitigated H₂O₂-induced damage, restoring cell viability, reducing lactate dehydrogenase release, and suppressing apoptosis by normalizing BAX/BCL2 ratios and CASPASE-3 activation. Moreover, melatonin preserved mitochondrial fusion dynamics (MFN1) and respiratory chain integrity (COX IV), counteracting H₂O₂-induced abnormalities. Mechanistically, mt-hiPSCs displayed hyperactivation of MAPK signaling (p-p38, p-ERK, p-JNK) under oxidative stress, which was attenuated by melatonin. Consistently, administration of the MAPK inhibitor SB203580 further confirmed that melatonin's protective effects are closely associated with modulation of MAPK pathway activity in mt-hiPSCs exposed to oxidative stress.

These findings highlight the vulnerability of m.3243A>G mutant cells to oxidative stress and demonstrate melatonin's therapeutic potential in alleviating mitochondrial dysfunction via MAPK pathway modulation. This study provides a patient-derived model for exploring mitochondrial disorders and identifies melatonin as a promising cytoprotective agent against mutation-associated oxidative damage.