# Anti-Aging Effects of Polyoxometalates on Skin
## 4 Discussion  
We previously reported that VBs are very stable molecules that exhibit strong antiviral and anti-bacterial activities [19]. In addition to those findings, VBs are characterized by their extremely low cytotoxicity. In fact, it has been found that they do not exert any cytotoxicity even when cells are treated with concentrations 3000 times higher than those used in this study. To investigate the versatility of 2 PMs (VB2 and VB3), we herein focused on their anti-aging effects on skin as well as their ability to induce cytoprotective responses in NHDF exposed to AGE and H2O2. The results obtained clearly demonstrated that the addition of VBs to normal cutaneous fibroblasts alone did not induce marked changes in cells, but up-regulated the expression of cytoprotective molecules and ameliorated adverse responses in cells exposed to stress. Specifically, neither VB2 nor VB3 affected the mRNA expression of AGE receptors or Hsps in NHDF when used alone (Figures 2 and 3). In NHDF exposed to AGE, VB2 and VB3 markedly increased the transcription of AGE receptors, particularly FEEL-1, FEEL-2, and RAGE. A previous study reported that FEEL-1 and FEEL-2 played a role in cytoprotection by participating in the endocytosis of free AGEs in cells and their degradation by lysosomes [29]. On the other hand, RAGE is known to contribute to the generation of ROS by activating NADPH oxidase. The enhanced transcription of RAGE may be linked to increased ROS production. In the case of RAGE, splice mutations occur during transcription, resulting in two variants; one is RAGE that lacks intracellular domains (C-terminally truncated RAGE: C-RAGE), which is a soluble RAGE protein released from cells, and the other is RAGE that lacks an extracellular V domain (N-terminally truncated RAGE: N-RAGE) [30; 31]. Because these two truncated RAGE did not exert any function as AGE receptors, the increased transcription of RAGE may not necessarily promote the production of ROS. This issue warrants further study. We then examined the effects of VBs on the mRNA expression of Hsps. The expression of Hsps is generally up-regulated by environmental stress, such as heat, ultraviolet light, and ROS. Hsps perform chaperone functions by stabilizing newly synthesized proteins to ensure correct folding or by helping to refold proteins damaged by stress. Hsp104 has been shown to mediate the disaggregation of proteins [32] and gp96 is involved in intracellular antigen presentation [33]. Hsp90 assists proteins to fold properly and remain stable when exposed to stress and facilitates the maturation of strictly selected proteins [34]. Hsp70 has been shown to regulate the quality of proteins by controlling their folding, transport, and degradation [35; 36]. Hsp60 maintains the folding of proteins in mitochondria and is involved in the membrane penetration of proteins in mitochondria [37]. Hsp32 is involved in heme degradation, the products of which exhibit antioxidant activities [38]. Because VB2 and VB3 are both capable of similarly inducing the expression of Hsps in cells subjected to AGE stress, VBs appear to function as potent cytoprotective agents under stress conditions. In an experiment to investigate the response of cells exposed to H2O2, we observed an approximately 40% reduction in cell viability. In contrast, the VB treatments markedly increased cell viability in a concentration-dependent manner. Furthermore, the response of cells treated with VBs and then incubated with H2O2 (the prophylactic effects of VBs) was stronger than that of cells incubated with H2O2 prior to the VB treatments (the therapeutic effects of VBs; Figure 4). To elucidate the underlying mechanisms, we assessed intracellular ROS and SOD production levels. Intracellular ROS levels were markedly increased by the H2O2 treatment, but suppressed by VB2 and VB3 in a concentration-dependent manner. This prophylactic effect of VBs on ROS production was also stronger than its therapeutic effect (Figure 5a).