Chlorogenic Acid Inhibits Liver Fibrosis by Blocking the miR-21-Regulated TGF-β1/Smad7 Signaling Pathway in Vitro and in Vivo

Abstract

Aims: Chlorogenic acid (CGA) is a phenolic acid that has a wide range of pharmacological effects. However, the protective effects and mechanisms of CGA on liver fibrosis are not clear. This study explored the effects of CGA on miR-21-regulated TGF-β1/Smad7 liver fibrosis in the hepatic stellate LX2 cell line and in CCl4-induced liver fibrosis in Sprague-Dawley rats. Methods: The mRNA expression of miR-21, Smad7, connective tissue growth factor (CTGF), α-smooth muscle actin (α-SMA), tissue inhibitor of metalloproteinase 1 (TIMP-1), matrix metalloproteinase-9 (MMP-9), and transforming growth factor-β1 (TGF-β1) and the protein levels of Smad2, p-Smad2, Smad3, p-Smad3, Smad2/3, p-Smad2/3, Smad7, CTGF, α-SMA, TIMP-1, MMP-9 and TGF-β1 were assayed in LX2 cells and liver tissue. The effects of CGA after miR-21 knockdown or overexpression were analyzed in LX2 cells. The liver tissue and serum were collected for histopathological examination, immunohistochemistry (IHC) and ELISA. Results: The mRNA expression of miR-21, CTGF, α-SMA, TIMP-1, and TGF-β1 and the protein expression of p-Smad2, p-Smad3, p-Smad2/3, CTGF, α-SMA, TIMP-1, and TGF-β1 were inhibited by CGA both in vitro and in vivo. Meanwhile, CGA elevated the mRNA and protein expression of Smad7 and MMP-9. After miR-21 knockdown and overexpression, the downstream molecules also changed accordingly. CGA also lessened the degree of liver fibrosis in the pathological manifestation and reduced α-SMA and collagen I expression in liver tissue and TGF-β1 in serum. Conclusion: CGA might relieve liver fibrosis through the miR-21-regulated TGF-β1/Smad7 signaling pathway, which suggests that CGA might be a new anti-fibrosis agent that improves liver fibrosis.

Keywords: Smad7; TGF-β1; chlorogenic acid; liver fibrosis; miR-21.

FIGURE 1

Cytotoxicity of CGA on LX2 cells was determined by CCK8 assay. (A) LX2 cells plated in 96-well plates were treated with CGA for 24 h, and cell viability was determined. (B) Cell morphologies of LX2 after CGA treatment for 24 h. (C) The mRNA level of miR-21 was measured by quantitative real-time PCR.

FIGURE 2

TGF-β1 increases miR-21 and CTGF expression in a dose-dependent manner and decreases it in a time-dependent manner. (A,B) The protein levels of CTGF were measured by western blot. (C,D) The ratio of CTGF to GAPDH was obtained by densitometric analysis. (E,F) The mRNA levels of miR-21 and CTGF were detected by real-time quantitative PCR. Data are shown as the means ± SD. ^∗P < 0.05, ^∗∗P < 0.01. compared with either time point 0 or the untreated group.

FIGURE 3

Effects of CGA on the TGF-β1/miR-21/Smad7 signaling pathway in LX2 cells after TGF-β1 stimulation. (A–C) The mRNA levels were detected by real-time quantitative PCR. (D) The protein levels were assayed by western blotting. The data from three independent experiments are expressed as the means ± SD. ^∗P < 0.05 compared with the experimental group; ^∗∗P < 0.01 compared with experimental group; ^##P < 0.01 compared with the normal group.

FIGURE 4

Verification of downstream signaling molecules in LX2 cells after miR-21 overexpression. (A) LX2 cells were transfected with lentivirus, and the expression of GFP was observed with a fluorescence microscope after 48 and 72 h. (B) The expression of miR-21, Smad7 and CTGF were measured by quantitative real-time PCR. (C) The protein expression was detected by western blotting. Data are shown means ± SD and significant differences were determined by one-way ANOVA. ^##P < 0.01 for lentivirus-up group vs. normal group.

FIGURE 5

Effects of CGA on the TGF-β1/miR-21/Smad7 signaling pathway in LX2 cells after TGF-β1 overexpression. (A–C) The mRNA levels were detected by real-time quantitative PCR. (D) The protein levels were assayed by western blotting. The data from three independent experiments are expressed as the means ± SD. ^∗P < 0.05 for lentivirus-up/TGF-β1/CGA vs. lentivirus-up/TGF-β1; ^∗∗P < 0.01 for lentivirus-up/TGF-β1/CGA vs. lentivirus-up/TGF-β1; ^#P < 0.05 compared with the normal group; ^##P < 0.01 compared with the normal group.

FIGURE 6

Verification of downstream signaling molecules in LX2 cells after miR-21 knockdown. (A) LX2 cells were transfected with lentivirus, and the expression of GFP was observed with a fluorescence microscope after 48 and 72 h. (B) The expression of miR-21, Smad7 and CTGF was measured by quantitative real-time PCR. (C) The protein expression was detected by western blotting. Data are shown as the means ± SD, and significant differences were determined by one-way ANOVA. ^##P < 0.01 for lentivirus-down group vs. normal group.

FIGURE 7

Effects of CGA on the TGF-β1/miR-21/Smad7 signaling pathway in LX2 cells after TGF-β1 knockdown. (A–C) The mRNA levels were detected by real-time quantitative PCR. (D) The protein levels were assayed by western blotting. The data from three independent experiments are expressed as the means ± SD. ^∗P < 0.05 for lentivirus-down/TGF-β1/CGA vs. lentivirus-down/TGF-β1; ^∗∗P < 0.01 for lentivirus-down/TGF-β1/CGA vs. lentivirus-down/TGF-β1; ^##P < 0.01 lentivirus-down/TGF-β1 vs. lentivirus-down.

FIGURE 8

Effect of CGA on the expression of TGF-β1 in serum, and the level of TGF-β1 was determined by ELISA. Data are shown as the means ± standard deviation. n = 10, ^∗∗P < 0.01 compared with the experimental group; ^##P < 0.01 compared with the normal group, as determined by Student’s t-test.

FIGURE 9

Evaluation of the effect of CGA on liver histopathological and immunohistochemistry (IHC) in liver tissue. (A) Histological images of rat livers stained with H&E (original magnification, ×200). (B) The histopathologic detection of collagen in the liver by Masson’s trichrome stain (original magnification, ×100). (C,D) Effects of CGA on α-SMA and collagen I expression were examined with immunohistochemistry in liver tissue (original magnification, ×100).

FIGURE 10

Effect of CGA on the TGF-β1/miR-21/Smad7 signaling pathway in CCl4-induced rats. (A–C) The mRNA levels were measured by real-time quantitative PCR. (D) The protein levels were assayed by western blotting. The data from three independent experiments are expressed as the means ± SD. ^∗P < 0.05 compared with the experimental group; ^∗∗P < 0.01 compared with experimental group; ^#P < 0.05 compared with the normal group; ^##P < 0.01 compared with the normal group.

FIGURE 11

The illustration of CGA protecting against liver fibrosis in vitro and in vivo by regulating miR-21-regulated TGF-β1/Smad7 signaling pathway.