## 3 Results
### The Effects of Genes and Biological Functions Caused by 4-MSP-Treated HSCs  
To examine the alterations of gene expression profile after TGF-\(\beta\)1 and 4-MSP treatment, the microarray experiments were performed. In microarray analysis, the signal intensity with a 1.5-fold increase or decrease was defined as significantly altered in mRNA expression (Figure 4a). To narrow down genes that were affected after 4-MSP treatment, a Venn diagram was performed. The result showed that 74 genes that were downregulated and 92 genes that were upregulated after TGF-\(\beta\)1 treatment were reversed after being treated with 4-MSP (Figure 4b,c). These genes (166 genes) were further used to investigate the pathways changed by 4-MSP treatment. In KEGG pathway enrichment analysis, we found that 4-MSP affected the pathways related to NOD-like receptor signaling, transcriptional misregulation in cancer, TGF-beta signaling, and NF-kappa B signaling pathways (Figure 4d). GO term enrichment results showed that inflammatory response, negative regulation of cell proliferation, MAPK cascade, and signal transduction were affected with the 4-MSP treatment (Figure 4e). These results imply the possibility that 4-MSP treatment influences HSCs activation and proliferation. Figure 3: Compound 4-MSP treatment did not induce apoptosis in HSCs. Treatment of LX2 and HSC-T6 cells with PDGF-BB (10 ng/mL) and 4-MSP (5 uM) for 24 h. (**a**,**c**) Protein levels of Caspase-9, PARP cleavage, and \(\alpha\)-tubulin were analyzed by western blotting. PC: positive control. (**b**,**d**) mRNA levels of Bcl-xl and Bax, relative to that of GAPDH, were measured by real-time RT-PCR. Data shown are at least three independent experiments with similar results. Compound 4-MSP Ameliorates Fibrosis Process in Mice With CCl\({}_{4}\)-Induced Liver Fibrosis Without Inducing Adverse Effects  
To further examine the antifibrotic efficacy of 4-MSP, we used the intraperitoneal injection of CCl\({}_{4}\), a widely-used experimental mouse model of liver fibrosis, in the experiments. In Figure 5a, 4-MSP treatment inhibited the CCl\({}_{4}\)-induced upregulation of hepatic \(\alpha\)-SMA expression. Besides this, the protein expression levels of phosphor-PDGFR and TGF-\(\beta\)R remained unchanged in four groups. Surprisingly, we found that CCl\({}_{4}\) administration induced upregulation of PDGF, and treatment of 4-MSP significantly reversed the effects (Figure 5a). The QPCR results showed that treatment of 4-MSP inhibited the expression of \(\alpha\)-SMA, col1A2, TGF-\(\beta\), and MMP2 in the liver (Figure 5b). Besides this, the PDGF-BB mRNA levels were also measured. Figure 5b shows that 4-MSP treatment did not change the PDGF-BB expression. H&E staining was used for hepatic pathological examination, and the results demonstrated that CCl\({}_{4}\) induced significantly irregular infiltration borders, perinuclear vacuoles, changes in liver fat, and regional inflammation in the liver, whereas treatment with the 4-MSP demonstrated a normal hepatic morphology (Figure 5c). Collagen deposition was determined by [PERSON]'s Trichrome staining. Results showed that the deposition of collagen was significantly increased in CCl\({}_{4}\)-treated mice, whereas this deposition event was reduced in 4-MSP-treated mice (Figure 5c). The results of hydroxyproline assay showed that 4-MSP treatment slightly reduced the CCl\({}_{4}\)-induced hydroxyproline accumulation (Figure 1d). To further investigate the effect of 4-MSP in affecting liver inflammation, the TNF-\(\alpha\) and F4/80 mRNA expression levels were analyzed. The QPCR results found that treatment of 4-MSP significantly inhibited TNF-\(\alpha\) and F4/80 expression (Figure 5b). Figure 4: Gene expression profile of TGF-\(\beta\)1 and 4-MSP-treated LX2 cells. (**a**) The volcano plot shows the distribution of differentially expressed genes according to 1.5-fold change (red axis). (**b**) The Venn diagrams show the correlations for up- and downregulated genes between LX2 \(\pm\) TGF-\(\beta\)1 and LX2+ TGF-\(\beta\)1 \(\pm\) 4-MSP.