Ferulic acid attenuates liver fibrosis and hepatic stellate cell activation via inhibition of TGF-β/Smad signaling pathway

Abstract

Purpose: Liver fibrosis is a worldwide health issue. Development of effective new drugs for treatment of this disease is of great importance. This study investigated the therapeutic effects of ferulic acid on liver fibrosis in vitro and in vivo.

Materials and methods: Human hepatic stellate cell line (HSC) LX-2 was used for in vitro assays. Transforming growth factor β1 (TGF-β1) was used to induce hepatic fibrosis in LX-2 cells. Western blot was used to detect protein levels of collagen I, fibronectin, α-smooth muscle actin (SMA), p-Smad2, p-Smad3, p-p38, and p-JNK. Gene expression was measured by RT-qPCR. Fluorescence staining was used to determine localization of Smad4. CCl4-induced hepatic fibrosis in SD rats was used as an in vivo model. Histological features were detected by hematoxylin and eosin staining. Levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), hexadecenoic acid (HA), and hydroxyproline (Hyp) were measured by ELISA.

Results: TGF-β1 treatment significantly increased levels of collagen I, fibronectin, α-SMA, p-Smad2, p-Smad3, and Smad4 in LX-2 cells. Ferulic acid improved TGF-β1-induced hepatic fibrosis via regulation of the TGF-β1/Smad pathway. Consistent with in vitro data, CCl4 caused severe hepatic fibrosis in SD rats, as determined by ALT, AST, HA, and Hyp upregulation. Protein levels of p-Smad2 and p-Smad3 in liver tissues were significantly increased following treatment with CCl4. All CCL4-induced changes were markedly attenuated by ferulic acid treatment.

Conclusion: Ferulic acid potently improved hepatic fibrosis via inhibition of the TGF-β1/Smad pathway in vitro and in vivo. These findings provided evidence for potential use of ferulic acid to treat or prevent liver fibrosis.

Keywords: CCl4; Smad signaling pathway; TGF-β1; ferulic acid; hepatic fibrosis.

Figure 1

The expression of collagen I, fibronectin, and α-SMA were significantly upregulated by TGF-β1 in LX-2 cells. Exponentially growing LX-2 cells were treated with 5 ng/mL TGF-β1 for 24 hours. (A) The chemical structure of ferulic acid. (B) Collagen I, fibronectin, and α-SMA gene levels in the control or TGF-β1 group were detected by RT-qPCR. (C) The representative Western blot image of collagen I, fibronectin, and α-SMA in the control group or TGF-β1 group. (D–F) The quantification of collagen I, fibronectin, and α-SMA proteins expressions in LX-2 cells. Notes: n=3 in each group. ^**P<0.01 vs control group. Abbreviations: α-SMA, α-smooth muscle actin; TGF-β1, transforming growth factor-β1.

Figure 2

Ferulic acid improved TGF-β1-induced in vitro liver fibrosis. (A) LX-2 cells were treated with indicated concentrations of ferulic acid for 24 hours and cell viability was detected with CCK-8 kit. (B) A density of 5×103 cells/well was planted in 96-well plate and exposed to ferulic acid for 12 hours before TGF-β1 treatment. Collagen I, fibronectin, and α-SMA gene levels in control, TGF-β1, ferulic acid group, TGF-β1 plus ferulic acid groups were detected with RT-qPCR. (C) The representative Western blot image of collagen I, fibronectin, and α-SMA in control, TGF-β1, ferulic acid, or TGF-β1 plus ferulic acid group. (D–F) The quantification of collagen I, fibronectin, and α-SMA proteins expressions in LX-2 cells. The relative gene expression of GAPDH was used as the internal standard in RT-qPCR assay. The quantification results of the Western blot was normalized to the expression of β-actin by Image Pro Plus. Notes: n=3 in each group. ^*P<0.05 vs control group; ^**P<0.01 vs control group, ^##P<0.01 vs TGF-β1 group. Abbreviations: α-SMA, α-smooth muscle actin; TGF-β1, transforming growth factor-β1.

Figure 3

Ferulic acid attenuated TGF-β1-induced liver fibrosis in LX-2 cells via inhibition of the TGF-β1/Smad pathway. (A) The representative Western blot image of p-Smad2, p-Smad3, p-p38, and p-JNK in LX-2 cells treated with TGF-β1 and/or ferulic acid. (B–E) The quantification of proteins p-Smad2, p-Smad3, p-p38, and p-JNK in LX-2 cells by normalizing to the expression of β-actin with Image Pro Plus. (F) The representative image of cell location of Smad4 in LX-2 by immunocytochemistry and DAPI staining (magnification ×200). Notes: Nuclei were stained with DAPI for 15 minutes at 37°C; n=3 in each group. ^**P<0.01 vs control group, ^##P<0.01 vs TGF-β1 group. Abbreviations: DAPI, 4′, 6-diamidino-2-phenylindole; TGF-β1, transforming growth factor-β1.

Figure 4

Ferulic acid improved CCl4-induced rat liver fibrosis. (A) The representative H&E images of liver in control, CCl4, ferulic acid, or CCl4 plus ferulic acid groups. The images were captured under a light microscope with 200 times amplification. (B–E) Rat serums and liver tissues in each group were collected at the end of the animal experiment. Notes: The levels of ALT, AST in serums and HA, Hyp in liver tissues were determined by using commercial available kits; n=6 in each group. ^**P<0.01 vs control group, #P<0.05, ^##P<0.01 vs CCl4 group. Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; HA, hexadecenoic acid; H&E, hematoxylin and eosin; Hyp, hydroxyproline.

Figure 5

Ferulic acid regulated Smad pathway in CCl4-treated rat. (A) The representative Western blot image of p-Smad2 and p-Smad3 in rats treated with CCl4 and/or ferulic acid group. (B, C) The quantification of proteins p-Smad2 and p-Smad3 in liver tissues by normalizing to the expression of β-actin with Image Pro Plus; (D) A flow diagram to represent the molecular pathway regulated by ferulic acid in CCl4-treated rat. Notes: n=6 in each group. ^**P<0.01 vs control group, ^##P<0.01 vs CCl4 group.