Study On The Mechanism Of FoxO3a/PGC-1α Involved In Zinc Oxide Nanoparticles-induced Mitochondrial Biogenesis Dysfunction In Cardiomyocytes

Objective To investigate the specific mechanism of FoxO3a/PGC-1α in regulating mitochondrial biosynthesis to protect cardiomyocytes from the toxic effects of ZnO NPs.Background Zinc oxide nanoparticles(ZnO NPs)is a new type of multifunctional inorganic compound,which has been widely used in biological,industrial and medical fields.It has been proved that ZnO NPs can be absorbed into the blood while crossing most biological barriers in organisms,causing toxicity to the cardiovascular system.Previous studies have shown that ZnO NPs-induced myocardial injury is closely related to oxidative stress of cardiomyocytes and disruption of mitochondrial biogenesis.In view of the important roles of FoxO3a and PGC-1α in regulating mitochondria biogenesis and REDOX,this study aims to achieve the results of in vitro experiments.To observe the myocardial toxic damage effects of reactive oxygen species(ROS)and PGC-1α at different levels,and to explore the mechanism of FoxO3a/PGC-1α involvement in mitochondria biogenesis dysfunction induced by ZnO NPs in cardiomyocytes.Methods In this study,human cardiomyocyte AC16 was used as an in vitro model.Dynamic light scattering(DLS)method was used to characterize ZnO NPs.The morphology of AC16 cells was observed under an inverted microscope.After exposing AC16 cells to ZnO NPs at different doses and for different times,the cell survival rate was determined by CCK-8 method,and the cell damage was observed by lactate dehydrogenase(LDH)leakage rate.The changes of ROS and mitochondrial membrane potential(MMP)were observed by fluorescence quantification.TRIzol was used to extract total RNA from cells for transcriptome sequencing,and gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis were performed for differentially expressed genes.The expressions of FoxO3a/PGC-1α and its downstream proteins were detected by western blotting.To observe the protective effects of reactive oxygen scavher Mito Tempo and Resveratrol(RSV)on the cytotoxicity induced by ZnO NPs.The nuclear translocation of FoxO3a was observed by immunofluorescence and western blotting.Finally,the co-localization of FoxO3a/PGC-1α was observed by immunofluorescence.Results1.Dynamic light scattering(DLS)results show that ZnO NPs have a certain aggregation property.After exposing AC16 cells to different doses of ZnO NPs for 24 h,cell morphology was changed,cell survival rate decreased and LDH leakage rate increased.After short-term exposure(6 h),ROS and malondialdehyde(MDA)levels were increased and MMP levels decreased,leading to oxidative stress and mitochondrial damage.2.Transcriptome analysis showed that 1 071 genes were enriched in the 50 μM ZnO NPs group compared with the control group,including 561 up-regulated genes and 510 down-regulated genes.Compared with the control group,7 164 genes were enriched in 200 μM ZnO NPs group,including 4 098 up-regulated genes and 3 066 down-regulated genes.GO and KEGG analysis showed that the differential genes were mainly concentrated in reactive oxygen species,antioxidant activity,mitochondrial cytochrome C release,apoptosis and other signaling pathways.Pretreatment of myocardial cells with reactive oxygen scavher Mito Tempo improved the cytotoxicity,ROS accumulation and MMP reduction induced by ZnO NPs.3.Immunoimographic results suggest that ZnO NPs can significantly up-regulate the expression of FoxO3a antioxidant pathway and downstream protein SOD2,and downregulate the expression of PGC-1α mitochondria biogenesis pathway and down-stream proteins NRF1,TFAM and COX IV.After preconditioning cardiomyocytes with RSV,mitochondria biogenesis disorders were further improved in addition to the same protective effect as Mito Tempo.4.Immunofluorescence and western blotting showed that FoxO3a gradually transferred into the nucleus with the increase of ZnO NPs dose.Immunofluorescence showed that FoxO3a and PGC-1α were co-expressed with the increase of ZnO NPs dose.Conclusion1.ZnO NPs can produce cytotoxicity to AC16 cells in a dose-and time-dependent manner,mainly manifested as ROS accumulation and mitochondrial membrane potential decline.2.After cardiomyocytes were exposed to ZnO NPs,transcriptomic sequencing revealed that the differential genes were mainly reflected in the related pathways of antioxidant and respiratory chain,and the results of western blotting suggested that the mitochondria biogenesis was impaired.Active oxygen scavenge agent Mito Tempo and mitochondrial generation agonist RSV can alleviate myocardial cell damage induced by ZnO NPs by regulating oxidative stress state and promoting mitochondrial generation.3.FoxO3a/PGC-1α pathway is an important regulator of myocardial toxicity and mitochondria biogenesis disorders induced by ZnO NPs.