## 3 Results
### Suppressive Effect of HEBI in UPM-Induced NF-kB Activation  
The phosphorylation levels of 1 kB-\(\alpha\) (Figure 6a), p50, and p65 were compared using Western blot analysis. According to the Western blot analysis, UPM increased the phosphorylation levels of 1 kB-\(\alpha\) compared to the unstimulated cells (Figure 6b). In parallel to _P_-1 kB-\(\alpha\), the cytosolic _P_-p50 and _P_-p65 were significantly increased in the UPM-exposed groups (Figure 6c); however, the elevated phosphorylation levels of the aforementioned proteins were dose-dependently decreased with the treatment of HEBI (Figure 6d). These results suggest that the isolated compound has an inhibitory effect on the phosphorylation of 1 kB-\(\alpha\), inhibiting the phosphorylation of p50 and p65 in the cytosol. Besides the cytosolic NF-kB inhibition, we attempted to evaluate NF-kB translocation levels in the nucleus using Western blot analysis (Figure 6e). In response to UPM, levels of p50 and p65 were dramatically increased in UPM treated MH-S cells (Figure 6f); however, co-treatment of HEBI before UPM stimulation decreased the elevated p50 and p65 in the nucleus observed in the UPM-stimulated MH-S macrophages.  
Figure 3: 3-Hydroxy-5,6-epoxy-\(\beta\)-ionone (HEBI) inhibits urban fine dust (UPM)-induced iNOS and COX2 in MH-S cells. MH-S murine lung macrophages were exposed to different concentrations of HEBI for 1 h and then stimulated with UPM (31.3 ug/mL) for 24 h. The total lysates of proteins were analyzed by Western blots (**a**) and quantified using ImageJ software (**b**). The relative mRNA expressions of _iNOS_ (**c**) and _COX2_ (**d**) were determined by RT-qPCR. Data are expressed as mean \(\pm\) S.D. (\(n=3\)). * _p_\(<\) 0.05 or **_p_\(<\) 0.01 compared with UPM-stimulated group and *_p_\(<\) 0.01 compared to control group.  
Figure 4: Effect of 3-hydroxy-5_6_-epoxy-\(\beta\)-ionone (HEBI) against urban fine dust (UPM)-induced pro-inflammatory cytokine production in MH-S macrophages. MH-S macrophages were exposed to HEBI (15.6–62.5 \(\upmu\)g/mL) for 1 h. The protein levels of IL-1\(\beta\) (**a**), IL-6 (**b**), and TNF-\(\alpha\) (**c**) in the culture media were detected by ELISA after UPM (31.3 \(\upmu\)g/mL) stimulated for 24 h. The relative mRNA expression levels of IL-1\(\beta\) (**d**), IL-6 (**e**), and TNF-\(\alpha\) (**f**) were detected with RT-q PCR followed by UPM treatment for 24 h. Data are expressed as mean \(\pm\) S.D. (\(n\) = 3). * \(p\) < 0.05 or ** \(p\) < 0.01 compared with UPM-stimulated group and ** \(p\) < 0.01 compared to control group.  
Figure 5: Effect of 3-Hydroxy-5,6-epoxy-\(\beta\)-ionone (HEBI) against urban fine dust (UPM)-induced _TLR2_ (**a**) and _TRL4_ (**b**) expressions in MH-S macrophages. MH-S macrophages were exposed to different concentrations of HEBI for 1 h. The mRNA transcriptional activities of TLR2 and TLR4 were detected with RT-q PCR followed by UPM induced for 24 h. Data are expressed as mean \(\pm\) S.D. (\(n\) = 3). **_p_ \(<\) 0.01 compared with UPM-stimulated group and **_p_ \(<\) 0.01 compared to control group.  
Figure 6: Effect of 3-hydroxy-5,6-epoxy-\(\beta\)-ionone (HEBI) against urban fine dust (UPM)-induced NF-kB phosphorylation in MH-S macrophages. MH-S macrophages were exposed to different concentrations of HEBI for 60 min and then stimulated with UPM (31.3 ug/mL) for 30 min. The cytosolic phosphorylation levels of cytosolic IxB-\(\alpha\) (**a,b**), p50 (**c**), and p65 were analyzed by Western blots and quantified using ImageJ software (**d**). The translocation levels of cytosolic p50 and p65 in the nucleus were analyzed by Western blots (**e**) and quantified using the ImageJ image processing program (**f**). Nucleus p50 and p65 levels were normalized against the corresponding Nucleoin. Data are expressed as mean \(\pm\) S.D. (\(n\) = 3). *_p_ \(<\) 0.05 or **_p_ \(<\) 0.01 compared with the UPM-stimulated group and **_p_ \(<\) 0.01 compared to control group.