## 3 Results
### DNJ Promotes Oxidative DNA Damage Repair via Stimulating NRF2/Ogg1  
NRF2 is a master regulator of cellular oxidative equilibrium. As mentioned above, DNJ ameliorates high-glucose-induced oxidative damage. We therefore examined whether DNJ stimulated the NRF2 anti-oxidative response in HUVEC cultured in the medium containing 11 mmol/L glucose. As shown in Figure 3A, the induction of NRF2 expression in HUVEC was observed at different time intervals post the 5 umol/L DNJ treatment. At 24 h post DNJ treatment, the NRF2 expression increased by around 26%. We next detected the expression of NRF2 in cells cultured in the medium containing 11 mmol/L glucose and 50 mmol/L glucose after DNJ treatment, respectively. As shown in Figure 3B, the addition  
Figure 2: (**A**) Immuno-staining of 8-OHdG. HUVEC were cultured in 11 mmol/L glucose (1), 11 mmol/L glucose and DNJ (2), 50 mmol/L glucose (3), and 50 mmol/L glucose and DNJ. Scale bar is 50 μm. (**B**) Western blotting detection of the expression levels of OGG1 in cells cultured in 11 mmol/L and 50 mmol/L glucose with or without DNJ treatment. (**C**) SA-\(\beta\)-gal staining of senescent cells. HUVEC were cultured in 11 mmol/L glucose (1), 11 mmol/L glucose and DNJ (2), 50 mmol/L glucose (3), and 50 mmol/L glucose and DNJ. Scale bar is 50 μm. (**D**) Western blotting detection of the expression levels of p21 in cells cultured in 11 mmol/L and 50 mmol/L glucose with or without DNJ treatment. Three replicates were set for each sample, and each experiment was performed three times. # means the \(p\) value < 0.05, ## means the \(p\) value < 0.01. One-way ANOVA with Tukey test was used to analyze the significance.  
of DNJ increased the expression of NRF2 by around 23% in cells cultured in the medium containing 11 mmol/L, and by around 50% in cells cultured in the medium containing 50 mmol/L glucose. Heme oxygenase 1 (HO-1) is a downstream target of NRF2 [18]. To confirm the activation of NRF2 by DNJ, the expression of HO-1 was detected. As shown in Figure 3B, the addition of DNJ enhanced the expression of HO-1 in cells cultured in media containing 11 mmol/L and 50 mmol/L glucose. These results indicate that DNJ stimulates the NRF2 anti-oxidative response.  
OGG1 was reported as another downstream target of NRF2 [24]. Since we have shown that DNJ increases the expression of OGG1, we next used ML385, a specific chemical inhibitor  
Figure 3: (**A**) Western blotting detection of NRF2 expression levels in HUVEC 0, 6, 12, 24 h post the treatment using DNJ. (**B**) Western blotting detection of the expression levels of NRF2 and HO-1 in HUVEC cultured in media containing 11 mmol/L and 50 mmol/L glucose, with or without DNJ treatment. (**C**) Western blotting detection of the expression levels of HO-1 and OGG1 in HUVEC. The cells were cultured in media containing 11 mmol/L and 50 mmol/L glucose. DNJ and ML385 treatments are shown in the figure. (**D**) Immuno-fluorescence staining of 8-OHdG in HUVEC. The cells were cultured in media containing 11 mmol/L and 50 mmol/L glucose. DNJ and ML385 treatments are indicated in the figure. Three replicates were set for each sample, and each experiment was performed three times. # means the \(p\) value < 0.05, # means the \(p\) value < 0.01. One-way ANOVA with Tukey test was used to analyze the significance.  
of NRF2, to study whether DNJ treatment enhances OGG1 expression via NRF2. As shown in Figure 3C, 10 umol/L ML385 inhibited the induced expression of HO-1 and OGG1 by DNJ. In addition, oxidative DNA damage levels under the application of 10 umol/L ML385 were detected via immuno-staining of 8-OHdG. As shown in Figure 3D, the effect of DNJ in ameliorating 50 mmol/L glucose-induced 8-OHdG formation was blocked by the use of ML385 treatment. These results indicate that DNJ treatment decreases high-glucose-induced oxidative DNA damage through activating NRF2 and its downstream target, OGG1.