Glucobrassicin Metabolites Ameliorate the Development of Portal Hypertension and Cirrhosis in Bile Duct-Ligated Rats

Abstract

Patients suffering from liver cirrhosis are often complicated with the formation of portosystemic collateral vessels, which is associated with the progression of a splanchnic hyperdynamic circulatory state. Alleviating pathological angiogenesis has thus been proposed to be a feasible treatment strategy. Indole-3-carbinol (C9H9NO, I3C) and 3,3'-diindolymethane (DIM), formed by the breakdown of glucosinolate glucobrassicin, are prevalent in cruciferous vegetables and have anti-angiogenesis properties. We aimed to evaluate their influences on portal hypertension, the severity of mesenteric angiogenesis, and portosystemic collaterals in cirrhosis. Sprague-Dawley rats with common bile duct ligation (CBDL)-induced liver cirrhosis or sham operation (surgical control) were randomly allocated to receive I3C (20 mg/kg/3 day), DIM (5 mg/kg/day) or vehicle for 28 days. The systemic and portal hemodynamics, severity of portosystemic shunting, mesenteric angiogenesis, and mesenteric proangiogenic factors protein expressions were evaluated. Compared to vehicle, both DIM and I3C significantly reduced portal pressure, ameliorated liver fibrosis, and down-regulated mesenteric protein expressions of vascular endothelial growth factor and phosphorylated Akt. DIM significantly down-regulated pErk, and I3C down-regulated NFκB, pIκBα protein expressions, and reduced portosystemic shunting degree. The cruciferous vegetable byproducts I3C and DIM not only exerted a portal hypotensive effect but also ameliorated abnormal angiogenesis and portosystemic collaterals in cirrhotic rats.

Keywords: 3,3′-diindolymethane (DIM); angiogenesis; indole-3-carbinol (I3C); liver cirrhosis; portosystemic collaterals.

Figure 1

(A) The degree of portosystemic shunting of CBDL rats treated with vehicle, DIM (3,3′-diindolymethane), or I3C (Indole-3-carbinol) analyzed with ANOVA. I3C significantly decreased the degree of shunting (* p < 0.05, I3C vs. corresponding vehicle group). (B) The plasma VEGF (vascular endothelial growth factor) concentrations analyzed with ANOVA. Compared with vehicle, I3C significantly decreased the plasma VEGF concentration (* p < 0.05, I3C vs. vehicle).

Figure 1

(A) The degree of portosystemic shunting of CBDL rats treated with vehicle, DIM (3,3′-diindolymethane), or I3C (Indole-3-carbinol) analyzed with ANOVA. I3C significantly decreased the degree of shunting (* p < 0.05, I3C vs. corresponding vehicle group). (B) The plasma VEGF (vascular endothelial growth factor) concentrations analyzed with ANOVA. Compared with vehicle, I3C significantly decreased the plasma VEGF concentration (* p < 0.05, I3C vs. vehicle).

Figure 2

Representative images of CD31-labelled mesenteric window microvascular networks in CBDL rats treated with vehicle, DIM, or I3C: (A) The magnification 40×, (B) the magnification 100×. (C) The vascular density of different experimental groups analyzed with ANOVA under the magnification 100×. DIM markedly decreased the vascular length (* p < 0.05, DIM vs. vehicle) and vascular area of mesenteric window (* p < 0.05, DIM vs. vehicle).

Figure 3

Mesenteric protein expressions of piNOS/iNOS, peNOS/eNOS, MMP-9, NFκB p65, pIκBα/IκBα, VEGF in CBDL rats treated with vehicle, DIM, or I3C analyzed with ANOVA (* p < 0.05, DIM- or I3C-treated group vs. vehicle group). The representative images are shown below.

Figure 4

Mesenteric protein expressions of pVEGFR2/VEGFR2, pAkt/Akt, HIF1-α, pErk44/42/Erk44/42, pRaf-1/Raf-1 in CBDL rats treated with vehicle, DIM, or I3C analyzed with ANOVA (* p < 0.05, DIM or I3C vs. vehicle). The representative images are shown below.

Figure 5

(A) The Sirius red-stained area of liver in CBDL rats treated with vehicle, I3C, or DIM analyzed with ANOVA. I3C and DIM significantly decreased the Sirius red-stained area (* p < 0.05, DIM or I3C vs. vehicle). The representative images of (B) CBDL-vehicle, (C) CBDL-DIM, and (D) CBDL-I3C groups are shown (in magnification 40×).

Figure 5

(A) The Sirius red-stained area of liver in CBDL rats treated with vehicle, I3C, or DIM analyzed with ANOVA. I3C and DIM significantly decreased the Sirius red-stained area (* p < 0.05, DIM or I3C vs. vehicle). The representative images of (B) CBDL-vehicle, (C) CBDL-DIM, and (D) CBDL-I3C groups are shown (in magnification 40×).

Figure 6

The angiogenesis-related signaling pathways influenced by I3C and DIM in mesentery of rats with CBDL-induced liver cirrhosis. The dashed line denotes the down-regulated signaling transduction, the solid line indicates the blocked angiogenesis, and the t-bar represents the direct inhibitory actions of I3C and DIM on the targets.