Glycyrrhizin Attenuates Portal Hypertension and Collateral Shunting via Inhibition of Extrahepatic Angiogenesis in Cirrhotic Rats

Figure 1. Experimental design. The rats received bile duct ligation (BDL) or sham operation. Treatments were started on the 15th day after operations, when liver fibrosis developed in BDL groups. After 2 weeks of treatments, experiments were performed on the 28th day after operations.

Figure 2. Effects of glycyrrhizin on splanchnic system. In rats with BDL-induced cirrhosis, the superior mesenteric artery (SMA) flow significantly increased compared with the sham-vehicle group. Glycyrrhizin significantly attenuated SMA flow in cirrhotic rats. The SMA resistance decreased in cirrhotic rats and was not affected by glycyrrhizin (n = 6, 6, 6, 6; BDL, bile duct ligation). * p < 0.05.

Figure 3. Effects of glycyrrhizin on mesenteric angiogenesis and portal-systemic collateral shunting. (A) Mesenteric angiogenesis was evaluated by vascular density of mesenteric window. In cirrhotic rats, the mesenteric vascular density was markedly higher than sham rats. Glycyrrhizin significantly reduced mesenteric vascular density (n = 5, 6, 6, 6). (B) The shunting degree was evaluated by color microsphere method. The shunting degree was significantly decreased in glycyrrhizin-treated group (n = 7, 7). Scale bar = 200 μm. * p < 0.05.

Figure 4. The angiogenic protein expressions in the mesentery. Glycyrrhizin downregulated phospho-VEGFR2 expression. VEGF, Phospho-eNOS, iNOS, COX1, and COX2 expressions were unaffected (n = 6, 6; BC, BDL-control; BG, BDL-glycyrrhizin). * p < 0.05.

Figure 5. Effects of glycyrrhizin on hepatic system. (A) The levels of plasma liver injury markers alanine transaminase (ALT), aspartate transaminase (AST), and total bilirubin were determined. The levels of liver injury markers increased markedly in BDL groups. However, glycyrrhizin did not affect ALT, AST, and total bilirubin levels in cirrhotic rats (n = 6, 6, 6, 6). (B) There was a trend toward increased hepatic vascular resistance (HVR) in cirrhotic rats compared with sham-operated control rats. The HVR of the BDL-vehicle and BDL-glycyrrhizin groups was not significantly different (n = 6, 6, 6, 6). * p < 0.05.

Figure 6. Effects of glycyrrhizin on liver fibrosis. (A) The severity of liver fibrosis was evaluated by fibrosis area ratio. The area ratio was significantly increased in BDL-vehicle group compared with sham-vehicle group. Glycyrrhizin treatment tended to attenuate the fibrosis severity (n = 6, 6, 6, 6). (B) The severity of liver fibrosis was further classified blindly by Metavir scoring system by an independent investigator. The results were compatible with the findings of the area ratio analyses that BDL significantly increased the fibrosis severity. Glycyrrhizin did not affect liver fibrosis in sham or BDL groups (n = 6, 6, 6, 6). Scale bar of upper panel = 1000 μm, scale bar of lower panel = 200 μm. * p < 0.05.

Figure 7. Effects of glycyrrhizin on intrahepatic angiogenesis. (A) Intrahepatic angiogenesis over sinusoidal area was evaluated by CD31 staining targeting endothelial cells. (B) Intrahepatic angiogenesis over portal area was investigated by α-SMA staining for vascular smooth muscle cells. The results were consistent. Intrahepatic vessels increased significantly in BDL-vehicle group compared with sham-vehicle group. Glycyrrhizin did not significantly influence the vascular numbers in the liver (n = 6, 6, 6, 6). Scale bar = 200 μm. * p < 0.05.

Figure 8. The fibrogenic protein expressions in the liver. The protein expressions of α–SMA, procollagen Iα1, MMP13, TIMP1, and TGF-β were not significantly different between BDL-vehicle (BV) and BDL-glycirrhizin (BG) groups (n = 6, 6).

Figure 9. Effects of glycyrrhizin on portal hypertension-related derangements. Glycyrrhizin suppressed mesenteric angiogenesis through VEGFR2 downregulation. The splanchnic blood inflow and collateral shunting thus decreased. The hepatic system was unaffected by glycyrrhizin. The net effect of glycyrrhizin is the amelioration of portal hypertension.