## 3 Results
### Platonin Supresses AP-1 Activation and JNK1/2 Phosphorylation in Ox-LDL-Treated VSMCs  
As suggests by Figure 4A(a), treatment with ox-LDL (20 ug/mL) for 6 and 12 h did not show any substantial increase in NF-kB luciferase activity in VSMCs (Figure 4A(a)). Some studies had indicated that ox-LDL could activate the DNA binding activity of AP-1 in VSMCs [25]. We therefore determined the effect of platonin on AP-1 binding  
Figure 3: Effects of platonin on the Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) signaling pathway in vascular smooth muscle cells (VSMCs) stimulated by LPS/IFN-\(\gamma\). (**A**) After transient transfection with NF-kB-luc and Renilla-luc plasmids for 24 h, the VSMCs were administered without (**a**) or with (**b**) platonin (2 uM) for 30 min then stimulated by the combination of LPS (50 ug/mL) and IFN-\(\gamma\) (100 units/mL; LPS/IFN-\(\gamma\)) for 6 h or 12 h (black bar: resting group; gray bar: LPS/IFN-\(\gamma\)-treated group; deep-gray bar: platonin + LPS/IFN-\(\gamma\)-treated group). A luciferase assay was conducted as described in “Materials and Methods.” (**B**) (**a**) Cells were treated with LPS/IFN-\(\gamma\) for the indicated period. (**b**) Cells were pretreated with platonin (0.5-2 uM) for 30 min before treatment with LPS/IFN-\(\gamma\) for another 30 min. (**C**) Cells were pretreated without (**a**) or with platonin (1 uM or 2 uM) (**b**) or okadaic acid (10 nM) + platonin (2 uM) (**c**) for 30 min, then treated with LPS/IFN-\(\gamma\) for the indicated time (**a**), 30 min (**b**), and 24 h (**c**). The extent of 1 kB\(\alpha\) phosphorylated p65, and inducible nitric oxide synthetase (iNOS) were determined using Western blotting assay. Data are represented as mean \(\pm\) S.E.M. (\(n=3\)); * \(p<0.05\), ** \(p<0.01\), *** \(p<0.001\) compared with the resting (R) group; # \(p<0.05\), *** \(p<0.01\) compared with the positive control group.  
activity in ox-LDL-treated VSMCs. The administration of ox-LDL (20 ug/mL) for 12 h significantly stimulated AP-1 DNA binding activity compared with the control group (Figure 4A(b)). As is depicted in Figure 4A(c), the ox-LDL-stimulated increase in AP-1 binding activity was notably suppressed in VSMCs pretreated with platonin (2 uM). Several studies have indicated that ox-LDL-induced AP-1 activation is strongly associated with JNK1/2 phosphorylation [26; 27]. We found that the extent of JNK phosphorylation was apparently increased after exposure to ox-LDL (20 ug/mL) for 30 min (Figure 4B(a)). Furthermore, ox-LDL-induced JNK phosphorylation was significantly inhibited in VSMCs pretreated with 2 uM platonin (Figure 4B(b)). These results indicate that the inhibition of AP-1 binding activity may be responsible for the anti-inflammatory mechanism of platonin in ox-LDL-stimulated VSMCs.  
Figure 4: Effects of platonin on AP-1 activity and JNK phosphorylation in ox-LDL-stimulated vascular smooth muscle cells (VSMCs). (**A(a)**) VSMCs were transiently transfected with NF-kB-luc and Renilla-luc plasmids for 24 h and then treated with ox-LDL (20 ug/mL) for 6 h or 12 h. (**A(b,c)**) Transient transfection with AP-1-luc and Renilla-luc plasmids were also performed in VSMCs for 24 h. After transfection, the cells were pretreated without (**b**) or with (**c**) platonin (2 uM) for 30 min then treated with the ox-LDL (20 ug/mL) for 6 h or 12 h (black bar: resting group; gray bar: ox-LDL-treated group; deep-gray bar: platonin+ ox-LDL-treated group). A luciferase assay was performed as described in “Materials and Methods.” (**B**) Cells were pretreated without (**a**) or with platonin (1 or 2 uM) (**b**) for 30 min, then treated with ox-LDL (20 ug/mL) for (**a**) the indicated time or (**b**) 30 min. The extent of JNK phosphorylation was then determined through Western blotting assay. Data are represented as mean \(\pm\) S.E.M. (\(n=3\)); *** \(p<0.01\), *** \(p<0.001\) compared with the resting (R) group; # \(p<0.05\), # \(p<0.01\) compared with the positive control group.