Effects of Ad-myr-HA-Akt on cirrhotic portal hypertension in rats

Abstract

Objective To probe the effects of recombinant adenovirus containing Akt on carbon tetrachioride-induced rat liver cirrhosis and portal hypertension. Methods Cirrhosis was induced in rats by a complex method of carbon tetrachloride. Recombinant adenovirus Ad-myr-HA-Akt was produced by homologous recombination in 293 cells. Rats received Ad-myr-HA-Akt via the tail vein at the second and the sixth week respectively. The pathological changes in liver tissues were observed after Van Gieson (VG) staining. Fas antigen in rat livers were determined by immunohistochemical method. The levels of alanine minotransferase( ALT), aspartate aminotransferase ( AST), albumin( ALB ) and hydroxyproline (Hyp) were measured. Fas antigen in rat livers were determined with immunohistochemical method. Expression of Akt, p-Akt, Fas and DR5 were evaluated by Western blotting. Frozen sections of the liver, heart,lung,kidney, brain,spleen and testis were made to examine the expression of enhance green flourescent protein (EGFP) by fluorescence microscopy in EGFP group. After 8-week CCl4 treatment, portal hypertensive rats in the saline group and Ad-Akt group received saline and Ad-myr-HA-Akt via the tail vein respectively. Portal vein pressure, mean arterial pressure and heart rate were measured in all rats on Day 3. Results In comparison with other cirrhosis rats, the pathological changes in the Akt group was markedly attenuated, and the levels of ALT, AST and Hyp were significantly lowered. Western blotting showed that the protein expression of p-Akt in the Akt group was higher significantly as compared with those in the negtive control group, saline group and EGFP group. Western blot also showed that the protein expression of Fas and DR5 in the Akt group was lower significantly. EGFP expression was mainly demonstrated by fluorescence microscopy on the frozen section of liver, very little fluorescene were detected in lung and kidney and there was no detectable EGFP in the other organs. Conclusions Ad-myr-HA-Ak inhibits CCl4-induced liver cirrhosis and is a potential pharmacological target for gene therapy in liver cirrhosis. Key words: Liver cirrhosis; Hypertension, portal; Recombinant adenovirus vector; Gene therapy