# Anti-Aging Effects of Polyoxometalates on Skin
## 3 Results
### Effects of VBs on the Production of Intracellular ROS and SOD  
To investigate the effects of VBs as anti-aging agents, the impact of VBs on oxidative stress-induced intracellular ROS production was evaluated using fluorescent DCF-DA as an indicator (Figure 5a). The intensity of fluorescence increased in cells exposed to H\({}_{2}\)O\({}_{2}\) by 3.7-fold from normal cells. When cells were treated with VBs after being exposed to H\({}_{2}\)O\({}_{2}\), intracellular ROS production was significantly suppressed by the treatments with VBs in a concentration-dependent manner. Furthermore, H\({}_{2}\)O\({}_{2}\)-induced ROS levels were markedly suppressed by the previous treatment of cells with VBs. Figure 4: Protective effects of VB2 and VB3 on the viability of NHDF treated with H\({}_{2}\)O\({}_{2}\). NHDF were treated with VBs in the presence of H\({}_{2}\)O\({}_{2}\) as described in the Materials and Methods. Cells were cultured for another 18 h and viability was measured. The significance of differences in the effects of VBs on cell viability was analyzed in comparison with that in the H\({}_{2}\)O\({}_{2}\)-treated group. \({}^{**}\)\(p<0.001\). In contrast to ROS production, SOD levels decreased to 56% of the control following an exposure to H\({}_{2}\)O\({}_{2}\) and recovered with the VB treatments in a concentration-dependent manner. Furthermore, a previous treatment with VBs appeared to confer resistance to H\({}_{2}\)O\({}_{2}\)-induced reductions in the production of SOD (Figure 5b). Figure 5: Effects of VBs on the generation of ROS and SOD in NHDF treated with H\({}_{2}\)O\({}_{2}\). To evaluate the therapeutic effects of VBs on oxidative stress, NHDF were treated with 0.2 mM H\({}_{2}\)O\({}_{2}\) for 2 h, washed with PBS, and continued to be cultured with VBs for 4 h. In another experiment, cells were initially treated with VBs for 4 h, washed, and then cultured with 0.2 mM H\({}_{2}\)O\({}_{2}\) for 2 h to monitor prophylactic effects. The level of ROS generated in cells was measured using DCF-DA as described in the Materials and Methods (**a**). In addition, the level of intracellular SOD was quantitated and expressed as a percentage of that in the control (**b**). \({}^{*}p<0.01\), \({}^{**}p<0.001\). Effects of VBs on the mRNA Expression of AQP-1 and AQP-3 and on the Production of Hyaluronan and Elastin under Oxidative Stress Conditions  
Figure 6a,b show the mRNA levels of AQP-1 and AQP-3, which are cell surface molecules that contribute to skin moisturization. The mRNA expression of AQP-1 and AQP-3 markedly decreased when NHDF were exposed to H\({}_{2}\)O\({}_{2}\), but was recovered by the VB treatments in a concentration-dependent manner after oxidative stress. The previous treatment of cells with VBs conferred resistance to H\({}_{2}\)O\({}_{2}\)-induced reductions in mRNA expression levels. The effects of VBs in cells were observed at concentrations of 1 \(\mu\)g/mL and higher, and no significant differences were noted between VB2 and VB3. Figure 7a,b show the amount of hyaluron secreted in the culture supernatant and the amount of intracellular elastin. The level of hyaluron decreased to approximately 50% that of the control after the H\({}_{2}\)O\({}_{2}\) treatment and this reduction was not recovered by the VB treatments. On the other hand, H\({}_{2}\)O\({}_{2}\)-induced reductions in hyaluron production appeared to be minimized by the previous treatment of cells with VBs at higher concentrations (Figure 7a). Figure 6: Effects of VBs on the mRNA expression of aquaporins in NHDF treated with H\({}_{2}\)O\({}_{2}\). To evaluate the therapeutic effects of VBs on oxidative stress, NHDF were treated with 0.2 mM H\({}_{2}\)O\({}_{2}\) for 2 h, washed with PBS, and continued to be cultured with 10 \(\mu\)g/mL VBs for 4 h. In another experiment, cells were initially treated with 10 \(\mu\)g/mL VBs for 4 h, washed, and then cultured with 0.2 mM H\({}_{2}\)O\({}_{2}\) for 2 h to monitor prophylactic effects.