## 3 Results  
### The Treatment of Magnolol Will Reduce the Thrombin-Mediated CTGF Expression in VSMCs  
We first test the biocompatibility of magnolol in VSMCs to optimize the concentration of magnolol in this study. The VSMCs were treated with various concentrations of magnolol (range from 10 to 50 \(\upmu\)M) alone or co-treated with thrombin (1 U/mL). Following this, 24 h later, cells were applied for a viability test by CCK8 assay (Figure 1A) and LDH release assay (Figure 1B). These results showed that the addition of magnolol alone or with thrombin were harmless to VSMC at the tested concentration. To reveal the effect of magnolol on thrombin-induced CTGF expression, VSMCs were preincubated respectively with 10, 20 or 50 \(\upmu\)M magnolol of magnolol for 90 min prior to the addition of thrombin (1 U/mL) for 1 h. We found that CTGF showed elevated protein expressions after the addition of thrombin. 2 h later, enhanced expression of CTGF reach nearly 2.5 fold than the resting group (Figure 1C) and magnolol significantly reduced the CTGF expression in a dose-dependent manner (Figure 1D). The protein expressions of CTGF were investigated by immunoblotting and normalized with GAPDH. This result demonstrated that magnolol affects to reduce the thrombin-mediated CTGF expression in VSMCs. Magnolol Hindered the VSMCs from Thrombin-Mediated CTGF Expression via Thrombin Activity Interfering, PAR-1 Cleavage Inhibiting and PAR-1 Active Site Blocking  
To understand whether magnolol plays a role in the thrombin-induced CTGF expression, we analyzed the activity of thrombin and the cleavage (activation form) level of PAR-1 after magnolol and thrombin treatment. In Figure 2A, we demonstrated that magnolol (50 \(\upmu\)M) significantly down-regulates  
Figure 1: Influence of magnolol on thrombin-induced CTGF expression. A10 cells were incubated with magnolol (10, 20 or 50 \(\upmu\)M) or vehicle (0.05% DMSO) for 90 min and then treated with or without thrombin (1 U/mL) for 24 h. The cell viability and cytotoxicity were determined by **(A)** CCK-8 assay and **(B)** lactate dehydrogenase (LDH) test, respectively. **(C)** After serum-starved for 6 h, A10 cells were treated with thrombin (1 U/mL) for the indicated times (0.5–3 h), and the protein lysates were collected and subjected to Western blotting for evaluating the time course of CTGF protein expression. **(D)** To assess the influence of magnolol on the thrombin treated cell, the 6-h serum-starved A10 cells were incubated with magnolol (10–50 \(\upmu\)M) or vehicle (0.05% DMSO) for 90 min and followed by thrombin (1 U/mL) treatment for 2 h. Data (**A** and **B**) were presented as the means \(\pm\) SD (n = 3). # \(p<0.05\) and ### \(p<0.0001\), compared with the **(C)** resting group and **(D)** DMSO alone group; *** \(p<0.001\), compared with the **(D)** DMSO alone group. The profiles are all representative examples of three similar experiments. the activity of thrombin. Further, we analyzed the level of the cleaved PAR-1 to evaluate the activity of PAR-1. These results showed that the truncated PAR-1 protein was significantly up-regulated 10 min after thrombin treatment (Figure 2B) and magnolol (50 uM) inhibited the production of cleaved PAR-1 protein after thrombin treatment (Figure 2C). According to the results of thrombin activity measurement and cleaved PAR-1 production test, magnolol may have the potential effect as a thrombin activity inhibitor. Figure 2: The pivotal role of magnolol in thrombin-mediated protease-activated receptor-1 (PAR-1) activation in A10 cells. (**A**) Thrombin activity was measured by a fluorometric assay kit. (**B**) A10 cells were serum-starved for 6 h and then incubated with thrombin (1 U) for the indicated times (0, 3, 5, 10 min) to evaluate the time course of thrombin-induced cleavage of PAR-1. (**C**) Serum-starved A10 cells were incubated with magnolol (10–50 uM) or 0.05% DMSO for 90 min and followed by thrombin (1 U/mL) treatment for 3 min. (**B**,**C**) The cleavage form of PAR-1 was detected through Western blotting under each condition.