## 4 Discussions  
This study provides the first evidence for a novel mechanism by which magnolol, a widely used traditional medicine, inhibits the activity of thrombin, activation of PAR-1, and subsequently downregulates CTGF expression. We also demonstrated that magnolol attenuated thrombin-induced smooth muscle migration in vitro and balloon angioplasty -induced neointimal formation in vivo. Thrombin-mediated activation of PAR-1 has been reported to have numerous biological effects. Therefore, preventing PAR-1 activation (cleavage) and blocking the activation site of PAR-1 is very important for preventing subsequent cellular responses of PAR-1. This study demonstrated that magnolol (50 \(\upmu\)M) significantly reduced the activity of thrombin (Figure 2A) and decreased thrombin-regulated PAR-1 cleavage (Figure 2C) in VSMCs. We also used protein-ligand docking analysis to indicate that magnolol potentially binds to the activation site of PAR-1. These results suggest that magnolol is a thrombin inhibitor and a PAR-1 antagonist. Therefore, treatment with magnolol may be a beneficial strategy for thrombin/PAR-1-related diseases such as thrombosis formation [32], tumor progression [33], and arterial restenosis [34]. In previous studies, JNK-1 and JNK-2 isoforms have been indicated to exhibit different functions in obesity, insulin resistance, axonal guidance, etc. [35; 36]. In this study, we showed that magnolol specifically downregulated the phosphorylation degree of JNK-1 upon thrombin treatment (Figure 3D-F). JNK-1 has been reported to be the major c-Jun/AP-1 interacting kinase after cells are stimulated by thrombin [37; 38]. We demonstrated that thrombin-induced activation of JNK-1 (Figure 3B), c-Jun (Figure 4A) and AP-1 (Figure 4C,D) in VSMCs. In addition, thrombin-stimulated p-JNK-1 (Figure 3D-F), p-cJun (Figure 4B) and AP-1 (Figure 4C,D) were inactivated by magnolol  
Figure 5: Magnolol inhibits the migratory potential of A10 cells and mitigates restenosis. (**A**) The effect of magnolol treatment on the migratory potential of A10 cells was analyzed through the wound healing assay. Representative photomicrographs of initial and final wounds were shown at 40 \(\times\) magnification and representative examples of three similar experiments. (**B**) Cell migration rate data were presented as the means \(\pm\) SD of the means (n = 3). # \(p\) < 0.05, compared with the DMSO alone group; * \(p\) < 0.05, compared with the DMSO plus thrombin treated group. (**C**) Effects of magnolol on balloon angioplasty-induced neointimal formation in rat common carotid artery. Each experiment group (sham operation group, solvent control group (DMSO), and magnolol (13 mg/kg/day) treated group) were taken microscopic images 14 days after balloon angioplasty. The compiled statistics are shown in (**D**). Data are expressed as mean \(\pm\) standard deviation (\(n\) = 3). ## \(p\) < 0.01, compared with the sham group; * \(p\) < 0.05, compared to the solvent (DMSO) group. LA, lumen area; IEL, internal elastic lamina; EEL, external elastic lamina. administration. According to a previous study, balloon angioplasty-induced smooth muscle cell proliferation is mediated by the rapid activation of JNK-1 [39]. Moreover, it has been reported that cell proliferation of hepatocytes, tumor cells, and endothelial cells is related to JNK-1 but not to the activation of JNK-2 [40; 41; 42]. Based on this evidence, magnolol has the potential to ameliorate VSMC proliferation through specific JNK-1 inhibition and to treat diseases such as cancer and atherosclerosis. However, it is worth noting that JNK-1 deficiency in hematopoietic cells may increase the incidence of atherosclerosis [43]. In our previous work, we found that thrombin induces the expression of CTGF in the rat VSMCs through PAR-1/JNK/AP-1 pathway, suggesting that CTGF may involve in the pathogenesis of in-stenten stenosis and atherosclerosis [22].