Baicalein pre-treatment alleviates hepatic ischemia/reperfusion injury in mice by regulating the Nrf2/ARE pathway

Abstract

Hepatic ischemia-reperfusion injury (HIRI) is caused by blood flow recovery following ischemia. Baicalein (BAI), a natural antioxidant used in traditional Chinese medicine, eliminates excessive free radicals and protects the structure of the cell membrane. However, its protective mechanism against HIRI is still unclear. The present study investigated underlying mechanism using a mouse HIRI model. Liver injury was evaluated using serum levels of alanine aminotransferase and aspartate aminotransferase, and hematoxylin-eosin staining was performed to evaluate the pathological changes in liver tissue. Apoptosis of hepatocytes was detected by TUNEL staining. The expression levels of reactive oxygen species (ROS), malondialdehyde (MDA) and superoxide dismutase (SOD) in the liver were detected to evaluate oxidative stress. Western blotting was performed to assess expression levels of nuclear factor E2-related factor 2 (Nrf2)/antioxidant response elements (ARE) pathway proteins in liver tissue. BAI pre-treatment significantly decreased elevation of serum aminotransferase levels induced by IR and alleviated histological damage to the liver. BAI decreased production of ROS and MDA in liver tissue induced by IR and increased the activity of SOD. At the same time, BAI inhibited apoptosis of liver cells induced by oxidative stress. Furthermore, BAI promoted the translocation of Nrf2 into the nucleus and increased the expression of total heme oxygenase-1 and NAD(P)H dehydrogenase quinone-1. The Nrf2 inhibitor ML385 reversed the protective effect of BAI on HIRI. These results indicated that BAI served a protective effect in HIRI by regulating the Nrf2/ARE pathway.

Keywords: baicalein; hepatic ischemia/reperfusion injury; nuclear factor E2-related factor 2 signaling; reactive oxygen species.

Figure 1

Structure of BAI and schematic diagram of the experimental protocol. (A) Chemical structure of BAI. (B) Experimental model design. BAI, baicalein; AST, aspartate aminotransferase; ALT, alanine aminotransferase; ROS, reactive oxygen species; Nrf2, nuclear factor E2-related factor 2; ARE, antioxidant response elements; IR, ischemia/reperfusion.

Figure 2

BAI ameliorates liver damage induced by IR. Mice were pre-treated with either vehicle (DMSO) or BAI at 10, 50 or 100 mg/kg. Serum levels of (A) ALT and (B) AST were assessed and (C) Hematoxylin and eosin staining was performed 6 h after hepatic IR. Scale bar=50 µm. (D) Suzuki score. ^*P<0.05 vs. Sham; ^#P<0.05 vs. IR. The effect of BAI on serum (E) ALT and (F) AST levels in mice subjected to Sham or ischemia treatment followed by reperfusion. ^*P<0.05 vs. Sham; ^#P<0.05 vs. DMSO at the same time point. BAI, baicalein; AST, aspartate aminotransferase; ALT, alanine aminotransferase; IR, ischemia/reperfusion.

Figure 3

BAI decreases oxidative stress and hepatic apoptosis following I/R. Samples were collected 6 h after liver reperfusion in the I/R and BAI (100 mg/kg) + IR groups. (A) ROS (red) in liver tissue. (B) Representative images of TUNEL staining. Arrows indicate positive cells. Scale bar=50 µm. (C) Percentage of TUNEL-positive hepatocytes. (D) MDA content and (E) SOD activity were measured in liver tissue. ^*P<0.05 vs. Sham; ^#P<0.05 vs. IR. BAI, baicalein; IR, ischemia/reperfusion; ROS, reactive oxygen species; MDA, malonaldehyde; SOD, superoxide dismutase.

Figure 4

BAI upregulates Nrf2/ARE pathway protein expression in the liver. Samples were collected 6 h after liver reperfusion in the IR and BAI (100 mg/kg) + IR groups. (A) Levels of Nrf2 nucleoprotein, total HO-1 and NQO-1 protein were analyzed by western blotting. Histone H3 and GAPDH were used as the internal controls. (B) Nrf2 nucleoprotein relative to histone H3 and total HO-1 and NQO-1 protein relative to GAPDH. ^*P<0.05 vs. Sham. ^#P<0.05 vs. IR. BAI, baicalein; IR, ischemia/reperfusion; Nrf2, nuclear factor E2-related factor 2; ARE, antioxidant response elements; HO01, heme oxygenase-1; NQO-1, NAD(P)H dehydrogenase quinone-1.

Figure 5

Nrf2/ARE pathway inhibitor reverse the protective effect of BAI on hepatic IR injury. Samples were collected 6 h after liver perfusion in all groups. (A) Levels of Nrf2 nucleoprotein, total HO-1, and NQO-1 protein were analyzed by western blotting. GAPDH and histone H3 were used as the internal controls. (B) Nrf2 nucleoprotein expression relative to histone H3 and HO-1 and NQO-1 protein expression relative to GAPDH. (C) Serum ALT and AST levels. (D) MDA content and (E) SOD activity were measured in liver tissue. (F) Pathological damage in the liver was measured by HE and TUNEL staining and ROS activity was measured by fluorescent-labeled DHE staining. Apoptotic cells are indicated by red arrows. Scale bar=50 µm. (G) Suzuki score. (H) Percentage of TUNEL-positive hepatocytes. ^*P<0.05 vs. IR; ^#P<0.05 vs. IR + BAI 100 mg/kg. BAI, baicalein; IR, ischemia/reperfusion; Nrf2, nuclear factor E2-related factor 2; ARE, antioxidant response elements; HO01, heme oxygenase-1; NQO-1, NAD(P)H dehydrogenase quinone-1; MDA, malonaldehyde; SOD, superoxide dismutase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; HE, hematoxylin and eosin; ROS, reactive oxygen species.