## 3 Results
### The Treatment of Magnolol Will Reduce the Thrombin-Mediated CTGF Expression in VSMCs
(**D**) The docking pose of magnolol (top left, blue) in the PAR-1 active site. The docking pose in 2D (top right) highlights both hydrogen and hydrophobic interactions as dotted lines and green lines, respectively. The co-crystal ligand, VPX (bottom, yellow), is superimposed onto magnolol (blue). They contain similar aromatic ring structures that occupy a similar space within the PAR-1 active site (green circle). Green dashed lines denote hydrogen bonds in 3D renderings. Data (**A**–C) were presented as the means \(\pm\) SD (n = 3). ### \(p\) \(<\)0.001, ### \(p\) \(<\) 0.01 and # \(p\) \(<\) 0.05, compared with the resting group; **_p_\(<\)0.01, compared with the DMSO plus thrombin treated group. In addition, we performed a docking analysis to predict the potential of magnolol as a PAR-1 antagonist. Here, we docked magnolol into the PAR-1 crystal structure (PDB ID: 3 VW7) and compared its interactions to the co-crystal ligand VPX. Our docking result suggests that magnolol forms interactions within the PAR-1 active site (Figure 3D). Magnolol consists of two chavicol moieties linked together by a carbon-carbon bond between their benzene rings. Two hydrogen bonds, one for each chavicol moiety, are formed between residues H255 and Y353 (Figure 2D). The chavicol moiety consists of a benzene ring and an aliphatic side chain. This facilitates hydrophobic interactions that sandwich the compound in the PAR-1 active site. For example, one chavicol moiety not only forms a hydrogen bond to Y353, but also hydrophobic interactions with residues L237, L263, F271, and L333. The benzene ring of the chavicol moiety also facilitates stable stacking interaction with the phenol ring of Y183. Similar hydrophobic interactions with, residues such as L258, L262, and Y337, sandwich the other chavicol moiety. Then, we compared the binding conformation of magnolol with that of the co-crystal ligand, VPX (Figure 2D). The VPX structure contains two aromatic ring structures linked together (Figure 2D). This is reminiscent of the benzene rings found on magnolol. We found that the aromatic ring structures of VPX occupy a similar space compared to magnolol. Figure 3: Regulatory roles of magnolol in thrombin-induced c-Jun N-terminal kinase (JNK) expression in A10 cells. (**A–C**) After 6 h of serum starvation, A10 cells were treated with thrombin (1 U/mL) for 3, 5 or 10 min. The protein lysates were collected and subjected to Western blotting for evaluating the time course of (**A**) JNK1/2, (**B**) JNK-1 and (**C**) JNK-2 phosphorylation that inducted by thrombin. (**D–F**) Starved (6 hr) cells pre-treated by magnolol (10–50 uM) or 0.05% DMSO for 90 min and then thrombin (1 U/mL) incubated for 5 min. The (**D**) \(p\)-JNK1/2 (**E**) \(p\)-JNK-1 and (**F**) \(p\)-JNK-2 were detected through Western blotting. (**A–C**) \(\#\)\(p\)\(<\) 0.05, \(\#\)\(\#\)\(p\)\(<\) 0.001, \(\#\)\(p\)\(<\) 0.01 and \(\#\)\(p\)\(<\) 0.05, compared with the resting group. (**D–F**) \(\#\)\(p\)\(<\) 0.001, compared with the DMSO alone group; \({}^{***}\)\(p\)\(<\) 0.001, \({}^{**}\)\(p\)\(<\) 0.01 and \({}^{*}\)\(p\)\(<\) 0.05, compared with the DMSO plus thrombin treated group. All data were presented as the means \(\pm\) standard error of the means (n = 3). The presence of the hydrophobic interactions sandwiching the aromatic rings of both magnolol and VPX suggests the importance of this feature for potential PAR-1 inhibitors. Further analysis found similar hydrophobic interactions between magnolol, VPX, and PAR-1 residues L237, H255, L262, F271, L333, Y337, and Y353 (Figure 2D). Residue Y183 forms a stable stacking interaction between one of the benzene rings of both compounds. Magnolol forms additional hydrophobic interactions with residues L258 and L263. This is due to the presence of the aliphatic side chains of magnolol. Together, the magnolol can form similar interactions as the ring structures of the co-crystal ligand VPX. This result suggests that magnolol can potentially bind to the PAR1 active site.