Antifibrotic Effect of Marine Ovothiol in an In Vivo Model of Liver Fibrosis

Abstract

Liver fibrosis is a complex process caused by chronic hepatic injury, which leads to an excessive increase in extracellular matrix protein accumulation and fibrogenesis. Several natural products, including sulfur-containing compounds, have been investigated for their antifibrotic effects; however, the molecular mechanisms underpinning their action are partially still obscure. In this study, we have investigated for the first time the effect of ovothiol A, π-methyl-5-thiohistidine, isolated from sea urchin eggs on an in vivo murine model of liver fibrosis. Mice were intraperitoneally injected with carbon tetrachloride (CCl₄) to induce liver fibrosis and treated with ovothiol A at the dose of 50 mg/kg 3 times a week for 2 months. Treatment with ovothiol A caused a significant reduction of collagen fibers as observed by histopathological changes and serum parameters compared to mice treated with control solution. This antifibrotic effect was associated to the decrease of fibrogenic markers involved in liver fibrosis progression, such as the transforming growth factor (TGF-β), the α-smooth muscle actin (α-SMA), and the tissue metalloproteinases inhibitor (TIMP-1). Finally, we provided evidence that the attenuation of liver fibrosis by ovothiol A treatment can be regulated by the expression and activity of the membrane-bound γ-glutamyl-transpeptidase (GGT), which is a key player in maintaining intracellular redox homoeostasis. Overall, these findings indicate that ovothiol A has significant antifibrotic properties and can be considered as a new marine drug or dietary supplement in potential therapeutic strategies for the treatment of liver fibrosis.

Figure 1

(a) Histological analysis. H&E staining and sirius red dye to highlight the collagen fibers of liver section: (A, B) healthy hepatic tissue; (C, D) fibrotic liver tissue induced by CCl₄; (E, F) liver tissue with hepatic fibrosis treated with ovothiol A. (b) Evaluation of serum levels of liver enzymes. The levels of AST, ALT, and ALP were determined in the serum from mice affected by liver fibrosis and treated with ovothiol A or control solution. Data are expressed as mean ± SD, n = 7. The significance was determined by the ANOVA and post hoc analysis: (^∗p < 0.05) and (^∗∗p < 0.01) represent significance compared to vehicle + CCl₄; (^#p < 0.05) and (^##p < 0.01) compared to nontreated (NT) healthy mice.

Figure 2

Gene expression analysis of markers of liver fibrosis by real-time qPCR. The levels of gene expression of the fibrotic markers in tissues after treatment with ovothiol A or control solution were compared to tissues from healthy mice (reference baseline). Data were analyzed through the REST software, which considers fold differences ≥ +/−2 to be significant. (^∗∗∗p < 0.001) represents the significance compared to the treated mice with vehicle + CCl₄.

Figure 3

Protein expression of liver fibrosis markers. (a) A representative experiment of Western blot analysis of cytosolic extracts obtained from hepatic tissues of mice treated with ovothiol A or vehicle, after induction of liver fibrosis, compared to samples of healthy mice (NT), using antibodies specific for TGF-β, α-SMA, and TIMP1. Histograms of the densitometry analysis of protein bands obtained by Western blot for liver markers: (b) TGF-β; (c) α-SMA; and (d) TIMP1. Data were normalized for GAPDH. Data are expressed as mean ± SD, n = 7. The significance was determined by ANOVA test. (#p < 0.05) and (##p < 0.01) represent significance compared to NT; (^∗∗p < 0.01) represents significance compared to the treated with vehicle + CCl₄.

Figure 4

Protein expression of GGT. (a) Representative Western blot performed with GGT-specific antibody using microsomal extracts of hepatic tissues of mice with liver fibrosis treated with ovothiol or control solution compared to nontreated healthy mice (NT). (b) Histogram of the densitometric analysis of the GGT bands at 64 and 50 kDa. Data were normalized for GAPDH. (c) Representative Western blot performed on mice serum using GGT-specific antibody. Data are expressed as mean ± SD, n = 6. Statistical significance was determined by the one-way ANOVA test; (#p < 0.05) and (###p < 0.001) represent the significance compared to the corresponding NT band; (^∗p < 0.05) represents the significance compared to fibrotic mice treated with control solution.

Figure 5

GGT activity and glutathione content. (a) The enzymatic activity of GGT was evaluated on liver tissue microsomal extracts containing membrane-bound GGT. The activity of GGT was normalized compared to the mature protein band (50 kDa) detected by Western blot. (b) The levels of glutathione were determined in hepatic tissue of mice treated with ovothiol A or vehicle, after induction of hepatic fibrosis, compared to samples of healthy tissue mice (NT). Data are expressed as mean ± SD, n = 6. The bars indicated the mean of 7 measures +/− SD (standard deviation). The significance was determined by the ANOVA and post hoc analysis: (#p < 0.05) represents significance compared to healthy control; (^∗∗p < 0.01) represents significance compared to mice treated with vehicle + CCl₄.

Figure 6

Proposed mechanism of action for ovothiol. During the development of liver fibrosis, membrane-bound GGT activity increases, leading to ROS overproduction. ROS can activate TGF-β, which in turn upregulates α-SMA and TIMP1, favoring ECM deposition. Ovothiol acts as a GGT inhibitor and in turn reduces TGF-β activation, thus inducing a cascade of events leading to downregulation of profibrogenic molecules and induction of fibrolytic enzymes.