## 4 Discussion
Increases in p-PERK, p-eIF2, and ATF4 reduce protein translation activity [37]. The expression levels of p-PERK, p-eIF2, and ATF4 were increased by 4 HR administration (Figure 2a). TUDCA is an inhibitor of ER stress [13], the administration of which decreased the expression of ER stress markers such as ATF6\(\alpha\), p-PERK, p-eIF2, and ATF4, which were increased by 4 HR administration (Figure 2b). TGF-\(\beta\)1 increases VEGF expression in HUVECs and helps differentiate HUVECs [9]. The administration of TUDCA decreased 4 HR-induced TGF-\(\beta\)1 expression (Figure 2c). The expression levels of SIRT1 and SIRT3 were not dramatically changed by 4 HR administration (Figure 5a). Interestingly, the expression level of SIRT6 was increased by 4 HR (Figure 5b). SIRTs gene expression was increased by 4 HR administration (Figure 5c). A discrepancy between the quantity of SIRT1 and -3 proteins and their mRNA molecules was observed. This type of discrepancy has been frequently observed [38]. Though the detailed mechanism remains elusive, interaction with microRNA has been suggested as a reason for this discrepancy [39]. Interestingly, many types of microRNAs interact with SIRT1 [40]. Pan-SIRTs activity was increased by 4 HR administration (Figure 5d). To increase SIRT activity, NAD+ (which is the substrate of SIRT) should be increased [41]. NADH and the NAD+/NADH ratio were significantly increased by 4 HR administration (\(p<0.05\), Figure 5e,f). The NAD+/NADH ratio has been reported as 2-10, and it is different to the cellular type and metabolic status [42; 43]. In this study, the NAD+/NADH ratio was 3-5 and increased following 4 HR administration (Figure 5f). In addition, NAD+ and NADH distribute differently to cellular compartments [44]. In the case of hepatocytes, the NAD+/NADH ratio in cytoplasm is 725, and that in mitochondria is 8 [45]. When cells undergo stress, maintaining the mitochondrial NAD+ level is important for cell survival, despite its drop in the nucleus and cytoplasm [46]. Thus, the change in NAD+ level following 4 HR administration in each cellular compartment could be an interesting topic for future study. The increased apoptotic stress by ER and mitochondria seemed to be ameliorated by SIRT activation. 4 HR and resveratrol (10-100 \(\upmu\)M) show antioxidant activity after hydrogen peroxide treatment in lymphocytes [18]. Resveratrol activates SIRT and ameliorates mitochondrial stress [47]. The increased apoptotic stress induced by 4 HR seemed to be ameliorated by SIRT activation as with resveratrol, which was also induced by 4 HR as a delayed response. In this study, the administration of 4 HR on HUVECs induced ER stress (Figure 2a). As 4 HR has a long alkyl group, it can bind to any hydrophobic domain of proteins with different ranges of affinity [1]. Some proteins show high affinity to 4 HR, which has been confirmed by chromatography with 4 HR-bound beads [16]. The protein conformation would be changed after binding to 4 HR and could be considered as misfolded protein. GRP78 binds to misfolded proteins and is transported to the ER [47]. This protein complex phosphorylates PERK and subsequently activates ATF4 (Figure 6). As a consequence, apoptotic stress increases. In this study, ATP synthesis in mitochondria was decreased (Figure 1a), and NAD+ level was increased by 4 HR administration (Figure 5e). Reduced ATP synthesis and increased NAD+ level increase SIRT transcription [48]. The activation of several kinases by mitochondrial and ER stress phosphorylates Sp1 [27]. By increasing phosphorylation, Sp1 is dissociated from HDACs, and gene promoter activity is increased [49]. 4 HR could bind to class I HDACs and inhibits their enzyme activity [22]. Accordingly, Ac-H3 was increased by 4 HR administration (Figure 3d). When 4 HR binds to HDACs, they dissociate from p-Sp1 via conformation change. In fact, HDACi binding to HDACs induces HDAC conformation change and dissociation from Sp1 [20]. HDACis such as trichostatin A and suberoylanilide hydroxamic acid increase TGF-81 expression through this mechanism [20].