## 3 Results
### The Treatment of Magnolol Will Reduce the Thrombin-Mediated CTGF Expression in VSMCs
Magnolol Performed Different Effect on Thrombin Induced JNK-1 and JNK-2 in Vascular Smooth Muscle Cells  
Next, we explored the thrombin-induced expression of JNK-1/2, the upstream molecules of CTGF, under magnolol treatment. After thrombin (1 U/mL) treated for 3, 5, and 10 min, the VSMCs were harvested to analyze the phosphorylation of JNK-1/2. We found that 3 min after 1 U/mL thrombin addition, both P-JNK-1 and P-JNK-2 expression increased (Figure 3A). In Figure 3B,C, these results showed that the protein levels of \(p\)-JNK-1 and \(p\)-JNK-2 are 3 and 2.5 times higher than the resting group, respectively. The expressions were digitalized and the \(p\)-JNK1/2 expressions were normalized with total JNK1/2 and plotted. Afterward, we pretreated VSMCs with 10, 20 and 50 \(\upmu\)M magnolol, and all the cells were harvested 3 min after treatment with 1 U/mL thrombin (Figure 3D). The results showed that compare with vehicle control (DMSO), the expression of \(p\)-JNK-1 was significantly reduced after pretreatment with magnolol in a dose-dependent manner (Figure 3E). In contrast, magnolol has no significant effect on the expression of \(p\)-JNK-2 after thrombin treatment (Figure 3F). The expression level of \(p\)-JNK1 and \(p\)-JNK2 were digitalized and normalized with the expression of total JNK1/2. Magnolol Down-Regulate Thrombin-Induced Transcription Factor Activation in Vascular Smooth Muscle Cells  
Thrombin is an indicator of c-Jun through the JNK pathway [26,27]. To determine whether magnolol affects the activation of the transcription factor in VSMCs under thrombin treatment, VSMCs were pre-treated with magnolol and harvested after 1 U/mL thrombin treatment. The results indicated that phosphorylated c-Jun showed an elevated level in a time-dependent manner after thrombin treatment (Figure 4A). In Figure 4B, magnolol (10, 20 and 50 \(\upmu\)M) inhibit the p-c-Jun level in a concentration-dependent manner. The c-Jun expressions and the phosphorylated level were normalized with GAPDH. As shown in Figure 4C, after 10 min of thrombin (1 U/mL) treatment, phosphorylated c-Jun (red) increased and accumulated significantly in the nucleus (blue). In addition, the red fluorescence intensities were markedly disrupted when the cells were exposed to 50 \(\upmu\)M magnolol (Figure 4C, the bottom of the panels). Next, the VSMCs, that transfected with AP-1-responsive firefly luciferase construct and constitutively expressing Renilla luciferase construct, were used to examine the activating protein-1 (AP-1) activity by AP-1-luciferase reporter gene assay. Transcription factor AP-1, which plays an important role in the regulation of gene expression, cell proliferation, and cell transformation, is mainly composed of Jun (c-Jun, JunB, and JunD) and Fos (cFos, fosB, Fra1, and Fra2) protein families [28,29]. In addition, phosphorylation of AP-1 proteins such as c-Jun probably enhances the transcriptional activity of AP-1 [30]. We found that the AP-1 transactivation activity increased significantly compared with the solvent control group after incubation with thrombin (Figure 4D, lane 3), and the up-regulated AP-1 transactivation activity was reversed by magnolol treatment (Figure 4D, lane 4). These results suggest that magnolol plays an essential role in thrombin-induced c-Jun activity in smooth muscle cells.