Hepatic preconditioning of doxorubicin in stop-flow chemotherapy: NF-κB/IκB-α pathway and expression of HSP72

Abstract

To provide hepatic protection through administration of doxorubicin before stop-flow chemotherapy (SFC) and to investigate the expression of heat shock protein 72 (HSP72) and role of nuclear factor kappa B (NF-kappaB) in this effect. The hepatic preconditioning of doxorubicin was established in a porcine model by injection of doxorubicin (1 mg/kg) before SFC. The experimental animals were randomized into two groups: groups receiving doxorubicin (DOX) and normal saline (NS). Serial serum and tissue samples were taken from both groups to evaluate the protection of doxorubicin. Western blot and immuno-precipitation were applied to detect the expression of HSP72, NF-kappaB p65 protein, inhibitor kappaB-alpha (IkappaB-alpha) and phosphorylated IkappaB-alpha as well. The expression of tumor necrosis factor alpha (TNF-alpha) was estimated by semiquantitative RT-PCR. And the extent of the hepatic injury was estimated with the level of serum aminotransferases. An abundance production of HSP72 in porcine liver was observed after 24 h of intravenous administration of doxorubicin, but without any change in the expression of NF-kappaB p65 subunit in cytoplasm. NF-kappaB p65 subunit accumulated in nuclei at the end of SFC and reached its highest level at 30 min after the restoration of the abdominal circulation and decreased gradually during the 6 h after SFC in NS group, while there was little change in DOX group. There was also a slight decrease of IkappaB-alpha at 30 min after the restoration of the abdominal circulation in NS group accompanying with the appearance of phosphorylated IkappaB-alpha. The expression of TNF-alpha was significantly higher in NS group than that in DOX group (average 1.40+/-0.17 vs 0.62+/-0.22, P<0.01) at serial time points after SFC. Serum ALT and AST levels of NS group were higher after 24 h than those of DOX group (93.2+/-7.8 IU/L vs 53.3+/-13.9 IU/L, 217.0+/-29.4 IU/L vs 155.0+/-15.6 IU/L for ALT and AST respectively, P<0.05) and after 48 h than those of DOX group (66.6+/-18.1 IU/L vs 43.3+/-16.7 IU/L, 174.4+/-21.3 IU/L vs 125.7+/-10.5 IU/L for ALT and AST respectively, P<0.05). Doxorubicin renders the liver to be tolerant to the hepatic influence in SFC in a porcine model through the NF-kappaB/IkappaB-alpha pathway with the expression of HSP72.