Thalidomide Improves the Intestinal Mucosal Injury and Suppresses Mesenteric Angiogenesis and Vasodilatation by Down-Regulating Inflammasomes-Related Cascades in Cirrhotic Rats

Abstract

Background and aims: By blocking TNFα-related effects, thalidomide not only inhibits hepatic fibrogenesis but improves peripheral vasodilatation and portal hypertension in cirrhotic rats. Nonetheless, the investigation of thalidomide's effects on splanchnic and collateral microcirculation has been limited. Our study explored the roles of intestinal and mesenteric TNFα along with inflammasome-related pathway in relation to cirrhosis and the splanchnic/collateral microcirculation.

Methods: Using in vivo and in vitro approaches, mechanisms of the effects of thalidomide on intestinal and mesenteric inflammatory, vasodilatory and angiogenic cascades-related abnormalities were explored in cirrhotic rats that had received 1-month thalidomide (C-T) treatment.

Results: In cirrhotic rats, high tumor necrosis factor (TNF)α, vascular endothelial growth factor (VEGF) and nitric oxide (NO)x levels were associated with the NOD-like receptors protein 3 (NLRP3), IL-1β and caspase-1 inflammasome over-expression in splenorenal shunt and mesenteric tissues. The thalidomide-related inhibition of mesenteric and splenorenal shunt inflammasome expression was accompanied by a significantly decreased intestinal mucosal injury and inflammasome immunohistochemical staining expression. Suppression of various angiogenic cascades, namely VEGF-NOS-NO, was paralleled by a decrease in mesenteric angiogenesis as detected by CD31 immunofluorescence staining and by reduced portosystemic shunting (PSS) in C-T rats. The down-regulation of the mesenteric and collateral vasodilatory VEGF-NOS-NO cascades resulted in a correction of vasoconstrictive hypo-responsiveness and in an attenuation of vasodilatory hyper-responsiveness when analyzed by in situ perfusion of the superior mesenteric arterial (SMA) and portosystemic collaterals. There was also a decrease in SMA blood flow and an increase in SMA resistance in the C-T rats. Additionally, acute incubation with thalidomide abolished TNFα-augmented VEGF-mediated migration of and tube formation of human umbilical vein endothelial cells, which was accompanied by corresponding changes in inflammatory and angiogenic substances release.

Conclusions: The suppression of inflammasome over-expression by chronic thalidomide treatment ameliorates inflammatory, angiogenic and vasodilatory cascades-related pathogenic changes in the splanchnic and collateral microcirculation of cirrhotic rats. Thalidomide seems to be a promising agent that might bring about beneficial changes to the disarrangements of peripheral, hepatic, splanchnic and collateral systems in cirrhosis.

Fig 1. Dose-response curves and area under curves (AUC) of in situ perfusion of cirrhotic microvessels.

Chronic thalidomide treatment improves vasoconstrictive hypo-responsiveness to arginine vasopressin (AVP) (A,B) and attenuates vasodilatory hyper-responsiveness to acetycholine (ACh) (C,D) in superior mesenteric arteries (SMA) and portosystemic collateral vessels (PS collaterals); ^#: P < 0.05 vs. C-V group.

Fig 2. In vivo and in vitro effects of thalidomide treatment on mesenteric and human umbilical vein endothelial cells (HUVECs)-induced angiogenesis.

Representative CD31 immunofluoresence (IF) staining angiogenesis images (A) and bar graphs of vascular length (B) and area (C) in mesenteric window. Representative images and bar graph of HUVECs tube formation (D,E) and migration (F,G) assays after 36hr of treatment; ^†: P<0.05 vs. S-V; ^δ: p< 0.05 vs. C-T rats;^#: p < 0.01 vs. VEGF+TNFα; *: p < 0.05 vs. buffer group.

Fig 3. Effects of thalidomide treatment on various cirrhotic vascular/mesenteric/intestinal inflammatory, angiogenic and vasodilatory markers.

Protein and mRNA expressions in splenorenal shunts (BDL) and left renal vein (sham). (A,B), and mesenteric tissues (C,D); (E).H-E staining for severity of mucosal injury (suzuki score) of small intestine; (F). IHC staining for inflammasome expression (NLRP3/caspase-1/IL-1β) of ileum. †p<0.05 vs. S-V rats; δ p< 0.05 vs. C-T rats. Genes were normalized to 18S RNA as an internal control.

Fig 4. Acute effects of thalidomide on various cirrhotic vascular/mesenteric/intestinal inflammatory and angiogenic markers in human umbilical vein endothelial cells (HUVECs) system.

Various protein (A-B), mRNA (C) and cytokines (D) levels in supernatant of HUVECs with different treatments. #: p < 0.01 vs. VEGF+TNFα; *: p < 0.05 vs. buffer group.

Fig 5. Schematic representative hypothesis of chronic thalidomide treatment effects on cirrhotic rats of our study.

TNFα: tumor necrosis factor-α; IVC: inferior vena cava; SMV: superior mesenteric vein; eNOS: endothelial nitric oxide synthase; NO: nitric oxide.