Cytoprotection by bcl-2 gene transfer against ischemic liver injuries together with repressed lipid peroxidation and increased ascorbic acid in livers and serum.

Abstract

The maximum gene exhibition was shown to be achieved at 48 h after transfection with human bcl-2 (hbcl-2) genes built in an SV40 early promoter-based plasmid vector and HVJ-liposome for cultured rat hepatocytes. The similar procedure of hbcl-2 transfection was therefore conducted for livers in rats via the portal vein, and after 48 h followed by post-ischemic reperfusion (I/R) operation for some hepatic lobes. The I/R-induced hepatic injuries were in situ observed as both cell morphological degeneration and cellular DNA strand cleavages around capillary vessels of the ischemic liver lobes as detected by HE stain and TUNEL assay, and were biochemically observed as release of two hepatic marker enzymes AST and ALT into serum. All the I/R-induced injuries examined were appreciably repressed for rats transfected with hbcl-2; hbcl-2 was expressed in hepatocytes around the capillaries of ischemic regions such as the median lobe and the left lobe, but scarcely around those of non-ischemic regions. Thus cytoprotection against I/R-induced injuries may be attributed to the I/R-promoted expression of transferred hbcl-2 genes. The possibility was examined firstly by methylphenylindole method, which showed that I/R-enhanced lipid peroxidation in the reference vector-transfected livers were markedly repressed in the hbcl-2-transfected livers. Contents of ascorbic acid (Asc) in serum and livers of hbcl-2-transfected rats were enriched, unexpectedly, versus those of non-transfected rats, and were as abundant as 1.90-fold and 1.95- to 2.60-fold versus those in the pre-ischemic state, respectively. After I/R, an immediate decline in serum Asc occurred in hbcl-2-transfectants, and was followed by prompt restoration up to the pre-ischemic Asc levels in contrast to the unaltered lower Asc levels in non-transfectants except a transient delayed increase. Hepatic Asc contents were also diminished appreciably at the initial stage after I/R in the ischemic lobes of hbcl-2-transfectants, which however retained more abundant Asc versus non-transfectants especially at the initial I/R stage when scavenging of the oxidative stress should be most necessary for cytoprotection. The results showed a close correlation between cytoprotection by exogenously transferred hbcl-2 and repressive effects on the lipid peroxidation associated with Asc consumption or redistribution.