Inhibitory effects of armepavine against hepatic fibrosis in rats

Abstract

Activation of hepatic stellate cells (HSCs) plays a crucial role in liver fibrogenesis. armepavine (Arm, C19H23O3N), an active compound from Nelumbo nucifera, has been shown to exert immunosuppressive effects on T lymphocytes and on lupus nephritic mice. The aim of this study was to investigate whether Arm could exert anti-hepatic fibrogenic effects in vitro and in vivo. A cell line of rat HSCs (HSC-T6) was stimulated with tumor necrosis factor-alpha (TNF-alpha) or lipopolysaccharide (LPS) to evaluate the inhibitory effects of Arm. An in vivo therapeutic study was conducted in bile duct-ligated (BDL) rats. BDL rats were given Arm (3 or 10 mg/kg) by gavage twice daily for 3 weeks starting from the onset of BDL. Liver sections were taken for fibrosis scoring, immuno-fluorescence staining and quantitative real-time mRNA measurements. In vitro, Arm (1-10 microM) concentration-dependently attenuated TNF-alpha- and LPS-stimulated alpha-SMA protein expression and AP-1 activation by HSC-T6 cells without adverse cytotoxicity. Arm also suppressed TNF-alpha-induced collagen collagen deposition, NFkappaB activation and MAPK (p38, ERK1/2, and JNK) phosphorylations. In vivo, Arm treatment significantly reduced plasma AST and ALT levels, hepatic alpha-SMA expression and collagen contents, and fibrosis scores of BDL rats as compared with vehicle treatment. Moreover, Arm attenuated the mRNA expression levels of col 1alpha2, TGF-beta1, TIMP-1, ICAM-1, iNOS, and IL-6 genes, but up-regulated metallothionein genes. Our study results showed that Arm exerted both in vitro and in vivo antifibrotic effects in rats, possibly through anti-NF-kappaB activation pathways.

Figure 1

(a) Effects of armepavine on collagen deposition by HSC-T6 cells after TNF-α stimulation for 24 hrs. Collagen deposition by HSC-T6 cells was quantified by Sircol collagen assay (n = 3). *p < 0.05 for TNF-α alone vs. Control; ^#p < 0.05 for TNF-α + armepavine vs. TNF-α alone. (b) Armepavine reduced the protein expression of α-SMA induced by lipopolysaccharide (LPS, 1 μg/ml) in HSC-T6 cells for 24 hrs. Representative results from three independent experiments are shown here, with N-acetylcysteine (NAC) as a positive control. *p < 0.05 for LPS alone vs. Control; ^#p < 0.05 for LPS + armepavine or NAC vs. LPS alone.

Figure 2

(a) Effects of armepavine on TNF-α-induced IκBα phosphorylation in the cytoplasmic extract and translocation of NFκB in the nuclear extract of HSC-T6 cells for 6 hrs, with PCNA and α-tubulin as internal controls. (b) Effects of armepavine on TNF-α-induced NFκB transcriptional activity using luciferase reporter gene assay in HSC-T6 cells for 6 hrs, with N-acetylcysteine (NAC) as a positive control. Representative results from three independent experiments are shown here *p < 0.05 for TNF-α alone vs. Control; ^#p < 0.05 for TNF-α + armepavine or NAC vs. TNF-α alone.

Figure 3

(a) Effects of armepavine on TNF-α-induced ERK1/2, p38 and JNK 1/2 phosphorylation levels in HSC-T6 cells. HSC-T6 cells were treated with TNF-α for 0-120 minutes. Representative results from three independent experiments are shown here. ^#p < 0.05 for phosphorylation of JNK 1/2 induced by TNF-α + armepavine vs. TNF-α alone at the same time point; ^†p < 0.05 for phosphorylation of ERK 1/2 induced by TNF-α + armepavine vs. TNF-α alone at the same time point; *p < 0.05 for phosphorylation of p38 induced by TNF-α + armepavine vs. TNF-α alone at the same time point. (b) Quantitative real-time PCR analysis for the expressions of iNOS, procollagen type I (Col 1α2), α-SMA and TIMP-1 genes in HSC-T6 cells treated with TNF-α and armepavine for 24 hrs. Representative results from three independent experiments are shown here, with N-acetylcysteine (NAC) as a positive control. *p < 0.05 for TNF-α alone vs. Control; ^#p < 0.05 for TNF-α + (S)-armepavine or NAC vs. TNF-α alone.

Figure 4

Histological examination of liver sections in control and bile-duct-ligated (BDL) rats. Representative liver sections were obtained from sham-operated rats (a), BDL rats receiving vehicle (b), BDL rats receiving 50 mg/kg silymarin (c), BDL rats receiving 3-mg/kg armepavine (d), and BDL rats receiving 10-mg/kg armepavine (e). Sections were stained with Sirius red. Scale bar = 200 m.