Mechanism of Hepatoprotective Effect of Boesenbergia rotunda in Thioacetamide-Induced Liver Damage in Rats

Abstract

Background. Researchers focused on developing traditional therapies as pharmacological medicines to treat liver cirrhosis. Objectives. Evaluating the hepatoprotective activity of Boesenbergia rotunda (BR) rhizome ethanolic extract on thioacetamide-induced liver cirrhosis in rats. Methods. Male Sprague-Dawley rats were intraperitoneally injected with 200 mg/kg TAA 3 times/week and daily oral administration of 250 mg/kg, 500 mg/kg of BR extract, and 50 mg/kg of the reference drug Silymarin for 8 weeks. At the end of the experiment, Masson's trichrome staining was used to measure the degree of liver fibrosis. Hepatic antioxidant enzymes (CAT and GPx), nitrotyrosine, cytochrome (P450 2E1), matrix metalloproteinase (MMP-2 and MMP-9), tissue inhibitor of metalloproteinase (TIMP-1), and urinary 8-hydroxyguanosine were measured. Serum levels of transforming growth factor TGF- β 1, nuclear transcription factor NF- κ B, proinflammatory cytokine IL-6, and caspase-3 were evaluated. Serum protein expression and immunohistochemistry of proapoptotic Bax and antiapoptotic Bcl-2 proteins were measured and confirmed by immunohistochemistry of Bax, Bcl-2, and proliferating cell nuclear antigen (PCNA). Results. BR treatment improved liver histopathology, immunohistochemistry, and biochemistry, triggered apoptosis, and inhibited cytokines, extracellular matrix proteins, and hepatocytes proliferation. Conclusion. Liver cirrhosis progression can be inhibited by the antioxidant and anti-inflammatory activities of BR ethanolic extract while preserving the normal liver status.

Figure 1

Effect of B. rotunda (BR) extract on hepatic levels of cytochrome enzyme CYP2E1 in rats at the end of the experiment. Data were expressed as mean ± SEM. **P < 0.01 compared with the normal group. *P < 0.01 compared with cirrhosis group.

Figure 2

Effect of BR extract on the serum level of TGF-β1, NF-κB, and IL-6 in rats at the end of the experiment. (a) TGF-β1, (b) NF-κB, and (c) IL-6. Data were expressed as mean ± SEM. Means among groups (n = 6 rats/group) show significant difference. **P < 0.001 compared to the normal group. *P < 0.001 compared to cirrhosis group. ^# P < 0.01 compared to cirrhosis group.

Figure 3

Effect of BR extract on the serum level of (a) Bax and Bcl-2, (b) Bax/Bcl-2 ratio, (c) caspase-3, and (d) relation between Bax, Bcl-2, and caspase-3 at the end of the experiment. Data were expressed as mean ± SEM. *P < 0.05 compared with the cirrhosis group. **P < 0.05 compared to the normal group.

Figure 4

Masson's trichrome staining of representative livers sampled from rats in different experimental groups. (a) Normal liver did not show signs of collagen deposition in livers from a normal rat. (b) Severe collagen deposition (arrow) and severe fibrosis were seen in the livers from a cirrhosis rat. (c) Minor collagen deposition in the liver of a hepatoprotected rat treated with Silymarin. (d) Moderated collagen deposition and moderate congestion around the central vein in the liver of rats treated with low dose BR extract. (e) Mild collagen deposition was observed in the livers of rats treated with high dose BR extract (original magnification ×20).

Figure 5

Immunohistochemistry staining of (a) Bax and (b) Bcl-2 of representative livers sampled from rats in different experimental groups. Less apoptosis indicated by (ia) few Bax-positive hepatocytes (white arrow) and (ib) more Bcl-2-positive hepatocytes (black arrow) in liver tissues from a cirrhosis rat group. (iia) High numbers of Bax-positive hepatocytes (White arrow) and (iib) very less Bcl-2-positive hepatocytes (black arrow) in the liver from a hepatoprotected rat treated with Silymarin. Moderate apoptosis as indicated by (iiia) moderate Bax staining (white arrow) and (iiib) moderate Bcl-2 staining (black arrow) indicating moderate apoptosis in the liver from the rats treated with low dose BR extract. (iva) High numbers of Bax-positive cells (white arrow) with severe apoptosis and (ivb) very less Bcl-2-positive hepatocytes (black arrow) were observed in the liver of the rats treated with high dose BR extract (original magnification ×40).

Figure 6

Immunohistochemistry staining of PCNA of livers sampled from rats in different experimental groups. (a) Normal livers did not show signs of PCNA expression in hepatocytes from control rats. (b) Cirrhosis control liver showed severe fibrosis with greater PCNA expression in the necrotized hepatocytes. (c) Silymarin-treated liver showed less PCNA-stained hepatocytes (arrow) indicating less hepatocyte proliferation. (d) The low dose BR-treated liver showed moderate hepatocyte proliferation as indicated by moderate PCNA staining (arrow) in the hepatocytes. (e) High dose BR-treated livers showed minor PCNA expression (arrow) with few proliferated necrotized hepatocytes which were observed in the liver (original magnification ×40).

Figure 7

Possible mechanism of hepatoprotective effect of Boesenbergia rotunda on TAA-induced liver damage in rats.