[Akt gene therapy for cirrhotic rats with portal hypertension].

Abstract

To determine whether there is an impaired Akt and eNOS activation in cirrhotic livers, and to investigate the feasibility of transferring adenovirus-mediated Akt gene to the liver for portal hypertension.
Recombinant adenovirus Ad-myr-HA-Akt and Ad-EGFP were produced by homologoas recombination in 293 cells . The Methods of compound factor, carbon tetrachloride (CCl4), corn flour, and cholesterol plus alcohol were used to construct the hepatic cirrhosis rat models. Ten normal rats were served as a normal control group, and 40 cirrhotic rats were divided into 4 groups randomly: an untreated group, an Ad-myr-HA-Akt treated group, an Ad-EGFP group, and a saline group. Ad-myr-HA-Akt, Ad-EGFP, and saline were transduced into the Ad-myr-HA-Akt treated group, Ad-EGFP group, and saline group via the tail vein respectively. Portal vein pressure, mean arterial pressure, and heart rate were measured in all rats. Protein abundance and phosphorylation status of Akt and eNOS were examined by Western blot. Spectrophotometry was used to measure the NO level. Frozen sections of the liver, heart, lung, kidney, brain, spleen, and testis were made to examine the expression of enhanced green fluorescent protein (EGFP) by fluorescence microscopy on Day 3 in the Ad-EGFP group.
The concentration of recombinant adenovirus Ad-myr-HA-Akt after the purification was 5.5 x 10(11)vp/mL and that of Ad-EGFP was 6.0 x 10(11)vp/mL. Akt and eNOS phosphorylations in the liver of cirrhotic rats were obviously impaired. Adenoviral delivery of myr-Akt restored eNOS phosphorylation, increased the NO level and decreased the portal pressure after 3 days of adenoviral infection. In contrast, the livers infected with Ad-EGFP and saline were not changed. The EGFP expression was mainly found under the fluorescence microscopy on the frozen section of liver. Very little fluorescence was detected in the lung and kidney; and there was no detectable EGFP in other organs.
There is an impaired Akt and eNOS activation in the cirrhotic livers; myr-Akt gene therapy can restore the Akt activation and NO production in the cirrhotic liver, suggesting that this therapy may be helpful in treating portal hypertension.