Fig. 6

Hesperetin activates FOXO3a and its downstream target genes, and promoting mitochondrial function. A Significant activation of FOXO3a transcriptional signaling based on activation z-score (z-score > + 2.0 and p-value < 0.01) and IPA upstream regulator analysis of Hes-modulated DEGs in HEK001 keratinocytes. The mRNA levels of the DEGs are associated with FOXO3a-related transcriptional changes. The DEGs can be classified according to the different functions of the downstream target genes of FOXO3a. B The mRNA levels of TFAM and TFB1M in HEK001 keratinocytes. C A heatmap illustrating that hesperetin modulates the expression of OXPHOS-related genes in HEK001 keratinocytes. The criteria for the gene list in the heatmap is absolute fold change > 1.1 and p < 0.05 (Hes vs Veh). D A graphic summary of the DEGs and pathways associated with FOXO3a and modulated by hesperetin. Several upstream inhibitory pathways of FOXO3a are down-regulated by hesperetin. These include PI3K-AKT-mTOR, ERK-MAPK and other regulators (SET9, CBP and USP7) and these are known to suppress FOXO3a transcriptional activity by modulating its subcellular localization and DNA binding affinity. Conversely, ATM-LKB1-AMPK signaling and p38α are able to enhance FOXO3a activity via promoting its nuclear localization; both expression of LKB1 and p38α are increased by hesperetin. Additionally, AMPKα promotes an accumulation of FOXO3a in mitochondria in order to promote mitochondrial functions. Furthermore, ARF-p53-PTEN pathway indirectly activates FOXO3a via suppressing PI3K-AKT-mTOR signaling; ARF and p53 are found to be increased by hesperetin. Red boxes indicate upregulation and blue boxes indicate downregulation of mRNA levels. Red lines indicate activation and blue lines indicate suppression of FOXO3a activity. Data are presented as mean ± SD. *p < 0.05; **p < 0.005 by Student’s t test; not significant (n.s.). DBS DNA-binding sequence, RNS reactive nitrogen species, SASP senescence-associated secretory phenotype