Estrogen Deficiency Potentiates Thioacetamide-Induced Hepatic Fibrosis in Sprague-Dawley Rats

Abstract

Hepatic fibrosis is characterized by persistent deposition of extracellular matrix proteins and occurs in chronic liver diseases. The aim of the present study is to investigate whether estrogen deficiency (ED) potentiates hepatic fibrosis in a thioacetamide (TAA)-treated rat model. Fibrosis was induced via intraperitoneal injection (i.p.) of TAA (150 mg/kg/day) for four weeks in ovariectomized (OVX) female, sham-operated female, or male rats. In TAA-treated OVX rats, the activities of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and γ-glutamyl transferase (GGT) were significantly increased compared to those in TAA-treated sham-operated OVX rats or TAA-treated male rats. Furthermore, α-smooth muscle actin (α-SMA) expression was significantly increased compared to that in TAA-treated sham-operated rats. This was accompanied by the appearance of fibrosis biomarkers including vimentin, collagen-I, and hydroxyproline, in the liver of TAA-treated OVX rats. In addition, ED markedly reduced total glutathione (GSH) levels, as well as catalase (CAT) and superoxide dismutase (SOD) activity in TAA-treated OVX rats. In contrast, hepatic malondialdehyde (MDA) levels were elevated in TAA-treated OVX rats. Apoptosis significantly increased in TAA-treated OVX rats, as reflected by elevated p53, Bcl-2, and cleaved caspase 3 levels. Significant increases in interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) concentrations were exhibited in TAA-treated OVX rats, and this further aggravated fibrosis through the transforming growth factor-β (TGF-β)/Smad pathway. Our data suggest that ED potentiates TAA-induced oxidative damage in the liver, suggesting that ED may enhance the severity of hepatic fibrosis in menopausal women.

Keywords: collagen I; estrogen deficiency; liver fibrosis; thioacetamide; α-SMA.

Figure 1

Experimental design for the liver fibrosis model. Liver fibrosis was induced by repeated injections of TAA (150 mg/kg/day) for 4 weeks in ovariectomized female or male rats. TAA = thioacetamide; i.p. = intraperitoneal injection; OVX = ovariectomized.

Figure 2

Effects of estrogen deficiency on hepatic damage in TAA-treated rats for 2 weeks. (a) Changes in serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and r-glutamyl transferase (r-GTT) activities in the serum of TAA-treated rats. Data are expressed as the mean ± S.D (6 animals/group). Statistical analysis was performed by one-way ANOVA followed by a Tukey’s HSD post hoc test for multiple comparisons. * p < 0.05 and ** p < 0.01 compared to control group; ^# p < 0.05 and ^## p < 0.01 compared to sham/TAA group. (b) Alterations in the pro-inflammatory cytokines interleukin 6 (IL-6) and tumor necrosis factor-alpha (TNF-α) levels in the serum of TAA-treated rats. Data are expressed as the mean ± S.D (6 animals/group). Statistical analysis was performed by one-way ANOVA followed by a Tukey’s HSD post hoc test for multiple comparisons. ** p < 0.01 compared to control group; ^# p < 0.05 compared to sham/TAA group. (c) Representative histology of hematoxylin and eosin (H&E) stained liver sections from experimental groups. TAA-induction for 10 days showed small nodules with degenerative hepatocytes, absence of sinusoid, an increase of fibrous tissue thickness (black arrows), and expansion of the portal tract with hepatic central vein and hepatic nodule increases (cirrhosis). CV, the central vein; PV, the portal vein. Original magnification: ×100.

Figure 3

Effects of estrogen deficiency on estrogen receptor α (ERα) expression in livers of TAA-treated rats for 4 weeks. (a) Representative western blots of ERα expression in liver, using an experimental model of TAA-induced hepatic fibrosis. β-Actin expression was used as the loading control. (b) Protein bands were quantified by densitometric analysis and normalized to β-actin. Values are expressed as the mean ± S.D. ** p < 0.01 compared to control group; ^# p < 0.05 compared to sham/TAA group. (c) Representative immunohistochemical staining of ERα expression from experimental groups. Original magnification: ×100, scale bar: 100 μm. Arrow indicated the ERα positive cells.

Figure 4

Effect of estrogen deficiency on antioxidant enzyme activity and apoptosis in the liver of TAA-treated rats. (a) Malondialdehyde (MDA), catalase (CAT), glutathione (GSH), and total superoxide dismutase (SOD) activities were measured in the liver of TAA-treated rats. Data are expressed as the mean ± S.D of duplicate experiments (6 animals/group). Statistical analysis was performed by one-way ANOVA followed by a Tukey’s HSD post hoc test for multiple comparisons. * p < 0.05 and ** p < 0.01 compared to control group; ^# p < 0.05 compared to OVX/TAA group. (b) Expression of p53, Bax, Bcl-2, and cleaved caspase-3 were measured by western blot analysis, using an experimental model of TAA-induced hepatic fibrosis. β-Actin expression was used as the loading control. The western blot results are representative of three separate experiments. (c) Apoptosis was determined by terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling (TUNEL) staining of liver sections from TAA-treated rats. The arrowheads indicate TUNEL-positive hepatocytes and Stars indicate TUNEL-positive NPC. Arrows indicate hepatocytes. NPC, non-parenchymal cells. Magnification ×200.

Figure 5

Effect of estrogen deficiency on extracellular matrix protein formation in the liver of TAA-treated rats. (A) Expression of α-SMA, collagen I, and vimentin were measured by western blot analysis using an experimental model of TAA-induced hepatic fibrosis. β-Actin expression was used as the loading control. The western blot results representative three separate experiments. (B) Protein bands were quantified by densitometric analysis and normalized to β-actin. Values are expressed as the mean ± S.D. * p < 0.05 and ** p < 0.01 compared to control group; ^## p < 0.01 compared to sham/TAA group. (C) Representative immunohistochemical staining of α-SMA and Masson’s trichrome-stained liver sections. Black arrows represent α-SMA and collagen accumulation. Original magnification: 40×, scale bar: 50 μm. (D) 4-Hydroxyproline contents in the serum and liver of TAA-treated rats. Data are expressed as the means ± S.D. of duplicate experiments (6 animals/group). Statistical analysis was performed by one-way ANOVA followed by a Tukey’s HSD post hoc test for multiple comparisons. ** p < 0.01 compared to control group; ^# p < 0.05 compared to sham/TAA group.

Figure 6

Effect of estrogen deficiency on hepatic fibrosis pathways in TAA-treated rats. (a) Expression of p-Smad2/3, Smad2/3, and Smad4 was measured by western blot analysis using an experimental model of TAA-induced hepatic fibrosis. β-Actin expression was used as the loading control. The western blot results representative three separate experiments. (b) Protein bands were quantified by densitometric analysis and normalized to β-actin. Values are expressed as the mean ± S.D. * p < 0.05 and ** p < 0.01 compared to control group; ^## p < 0.01 compared to sham/TAA group. (c) Representative immunohistochemical staining of TGF-β1 (Black arrows) in the liver. Original magnification: ×100 (d) Expression of phosphoinositol-3′-kinase (PI3K), p-PI3K, protein kinase B (Akt), p-Akt, phosphatase, and tensin homolog (PTEN) were measured by western blot analysis using an experimental model of TAA-induced hepatic fibrosis. β-Actin expression was used as the loading control. The western blot results representative three separate experiments.