Andrographolide Ameliorates Liver Fibrosis in Mice: Involvement of TLR4/NF- κ B and TGF- β 1/Smad2 Signaling Pathways

Abstract

Liver fibrosis is characterized by activated hepatic stellate cells (HSC) and extracellular matrix accumulation. Blocking the activation of HSC and the inflammation response are two major effective therapeutic strategies for liver fibrosis. In addition to the long history of using andrographolide (Andro) for inflammatory disorders, we aimed at elucidating the pharmacological effects and potential mechanism of Andro on liver fibrosis. In this study, liver fibrosis was induced by carbon tetrachloride (CCl₄) and the mice were intraperitoneally injected with Andro for 6 weeks. HSC cell line (LX-2) and primary HSC were also treated with Andro in vitro. Treatment of CCl₄-induced mice with Andro decreased the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), Sirius red staining as well as the expression of α smooth muscle actin (α-SMA) and transforming growth factor- (TGF-) β1. Furthermore, the expression of Toll-like receptor (TLR)4 and NF-κB p50 was also inhibited by Andro. Additionally, in vitro data confirmed that Andro treatment not only attenuated the expression of profibrotic and proinflammatory factors but also blocked the TGF-β1/Smad2 and TLR4/NF-κB p50 pathways. These results demonstrate that Andro prevents liver inflammation and fibrosis, which is in correlation with the inhibition of the TGF-β1/Smad2 and TLR4/NF-κB p50 pathways, highlighting Andro as a potential therapeutic strategy for liver fibrosis.

Figure 1

Andrographolide (Andro) improved CCl₄-induced liver fibrosis in mice. (a) Representative histology of Sirius Red and immunohistochemical staining of α-SMA and TGF-β1. (b–d) Quantification of positive staining areas was measured by ImageJ software. (e) Hepatic hydroxyproline content. (f) The protein expression of α-SMA and TGF-β1 was examined by Western blot. (g, h) Serum levels of ALT and AST. n = 6. ^### p < 0.001 versus control mice. ^∗ p < 0.05 and ^∗∗ p < 0.01 versus mice induced by CCl₄.

Figure 2

Andro attenuated hepatic inflammation in mice induced by CCl₄. (a) Representative histology of H&E and immunohistochemical staining of CD68. (b) Quantification of CD68-positive staining areas was measured by ImageJ software. (c) The protein expression of CD68 was examined by Western blot. (d–f) The mRNA levels of IL-1β, IL-6, and MCP-1 were measured by q-PCR. n = 6. ^## p < 0.01 and ^### p < 0.001 versus control mice. ^∗ p < 0.05, ^∗∗ p < 0.01, and ^∗∗∗ p < 0.001 versus mice induced by CCl₄.

Figure 3

Andro reduced inflammation in CCl₄-induced mice through inhibition of the TLR4/NF-κB signaling pathway. (a) Representative immunohistochemical staining of TLR4. (b) Representative immunofluorescent staining of NF-κB p-p50. (c, d) Quantification of positive staining areas was measured by ImageJ software. (e) The protein expression of TLR4 was examined by Western blot. n = 6. ^### p < 0.001 versus control mice. ^∗∗ p < 0.01 versus mice induced by CCl₄.

Figure 4

Andro attenuated HSC activation through inhibition of the TGF-β1/Smad2 signaling pathway. (a) Representative immunofluorescent staining of α-SMA. (b) The mRNA levels of α-SMA were measured by q-PCR. (c) The protein expression of α-SMA, TGF-β1, p-Smad2, p-Smad3, and Smad2/3 was examined by Western blot. n = 3. ^∗∗ p < 0.01 and ^∗∗∗ p < 0.001 versus control.

Figure 5

Andro suppressed proinflammatory chemokines mediated by TLR4/NF-κB p50 signaling in HSC. (a–c) The mRNA levels of IL-1β, IL-6, and MCP-1 were measured by q-PCR. (d) The protein expression of TLR4, p-p50, and p50 was examined by Western blot. (e, f) p50 levels in the cytosol and nucleus were assayed. (g) Representative immunofluorescent staining of TLR4. (h) Nuclear translocation of NF-κB p50 in HSC was assayed by immunofluorescence. n = 3. ^∗ p < 0.05, ^∗∗ p < 0.01, and ^∗∗∗ p < 0.001 versus control.