18α-Glycyrrhizin induces apoptosis and suppresses activation of rat hepatic stellate cells

Abstract

Background: To investigate the potential mechanisms underlying the protective effects of 18α Glycyrrhizin (GL) on rat hepatic stellate cells (HSCs) and hepatocytes in vivo and in vitro.

Material/methods: Sprague-Dawley (SD) rats were randomly divided into 5 groups: normal control group, liver fibrosis group, high-dose 18α GL group (25 mg/ kg/d), intermediate-dose 18α GL group (12.5 mg/kg/d) and low-dose 18α GL group (6.25 mg/ kg/d). The rat liver fibrosis model was induced by carbon tetrachloride (CCl4). The expressions of alpha-smooth muscle actin (αSMA) and NF-kappaB were determined by real-time PCR and immunohistochemistry.

Results: 18αGL dose-dependently inhibited the CCl4-induced liver fibrosis. There were significant differences in the mRNA and protein expressions of αSMA between the fibrosis group and 18α-GL treatment groups, suggesting that 18α GL can suppress the proliferation and activation of HSCs. Few HSCs were apoptotic in the portal area and fibrous septum in the liver fibrosis group. However, the double-color staining of a-SMA and TUNEL showed that 18α-GL treatment groups increased HSC apoptosis. NF-kappaB was mainly found in the nucleus in the fibrosis group, while cytoplasmic expression of NF-kappaB was noted in the 18αGL groups. In the in vitro experiments, 18α GL promoted the proliferation of hepatocytes, but inhibited that of HSCs. HSCs were arrested in the G2/M phase following 18α GL treatment and were largely apoptotic.

Conclusions: 18α-GL can suppress the activation of HSCs and induce the apoptosis of HSCs by blocking the translocation of NF-kappaB into the nucleus, which plays an important role in the protective effect of 18α-GL on liver fibrosis.

Figure 1

Representative Light Photomicrographs of liver Tissue Showing Effects of 18αGL on Histological Changes (H&E and Masson Staining, Original Magnification 100). (A–C) represented H&E staining of the control group, liver fibrosis group, and high-dose 18αGL groups, respectively. (D,E) represented Masson staining of control group, liver fibrosis group, and high-dose 18αGL groups, respectively.

Figure 2

Effects of 18αGL on α-SMA Protein Expression in Rats liver Tissues (Positive as Brown, Original Magnification 100 (A–C) 400 (D,E), and mRNA level of a-SMA in five group. (A–C) represented the α-SMA deposition in control group, liver fibrosis group, and high-dose 18αGL groups, respectively. (D and E) are magnified image of (A and B). (F) bargraph showed the ratio of positive expression of a-SMAs. (G) bargraph showed mRNA level of a-SMA in five groups by qPCR quantity. Values are mean ± S.D * p<0.05 vs. liver fibrosis group.

Figure 3

Effects of 18αGL on apoptosis in Rats liver Tissues by TUNEL stained (the nucleus dark blue is positive Original Magnification 100 (A–C) 400 (D–F). Dual staining for a-SMA and TUNEL was undertaken in representative liver sections to localize apoptotic HSCs (Dark brownish stained positive apoptotic bodies with TUNEL reaction. Red stain signified the area of HSC. Co-localization of both stain represented apoptotic HSCs (original magnification 400 (G–I)). (A–C) represented TUNEL staining of control group, liver fibrosis group, and high-dose 18αGL groups, respectively. (D–F) are magnified image of (A–C), respectively (G–I). Dual staining for a-SMA and TUNEL of control group, liver fibrosis group, and high-dose 18αGL groups, respectively. Single arrow indicates apoptosis of HSCs.

Figure 4

Effects of 18αGL on NF-κB protein expression in rats liver tissues by immunohistochemistry staining (Positive as Brown, Original Magnification 400 (A–C), and mRNA level of NF-κB in five groups. (A–C) represented NF-κB staining of control group, liver fibrosis group, and high-dose 18αGL groups, respectively. Red arrows indicate nuclear-positive, and yellow arrows indicate the plasma positive. (D) The bargraph showed the mRNA level of NF-κB in five groups by qPCR quantity. Values are mean ± S.D; * p<0.05 vs. liver fibrosis group.

Figure 5

Effects of 18α GL at different concentrations (0–1 mg/ml) on the growth of hepatocytes and HSCs. (A) hepatocytes; (B) HSCs.