Study on the role of autophagy in heme oxygenase 1 preventing hepatic ischemia/reperfusion injury in rats

Abstract

To identify the role of autopahgy in the protective mechanism of heme oxygenase 1 (HO-1) against hepatic ischemia/reperfusion (I/R) injury. Forty healthy male Sprague-Dawley (SD) rats were randomly (random number table) divided into five groups (n = 8 in each group), namely sham group, model group, cobalt protoporphyrin (CoPP) group, zinc protoporphyrin (ZnPP) group and 6-amino-3-methylpurine (3-MA) group. Partial hepatic I/R model was established by clamping the pedicles of left and median lobes for 1 hour and reopening for 6 hours in rats, and the rats in sham group were only received celiotomp without hepatic I/R. In the CoPP group, CoPP (a HO-1 inducer, 5 mg/kg) was administered i.p 24 hours before I/R. In the ZnPP or 3-MA group, besides pretreatment with CoPP, the rats were given ZnPP (a HO-1 inhibitor, 25 mg/kg) or 3-MA (an autophagy inhibitor, 30 mg/kg) i.p 1 hour before I/R. Serum alanine aminotransferase (ALT) was determined with automatic biochemistry analyzer. The hepatic pathological scores (PS) were determined under light microscope using hematoxylin-eosin (HE) staining. The hepatocyte apoptosis index (AI) was assessed with terminal dexynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. Autophagosomes in liver tissue were counted under electron microscope. The mRNA expressions of HO-1, caspase-3, Beclin-1 and Atg-5 in the liver were determined by reverse transcription-polymerase chain reaction (RT-PCR). The HO-1 activity was also measured by the generation of bilirubin with the method of double-wave spectrophotometry. Compared with the sham group, the level of serum ALT significantly increased in the I/R group (U/L: 560.3±73.6 vs. 49.1±13.8, P < 0.01), HE staining showed a severe hepatic injury (PS: 12.0±2.0 vs. 1.3±0.9, P < 0.01), TUNEL staining showed a higher hepatocytes apoptosis and the expression of caspase-3 significantly increased [AI: (19.38±3.07)% vs. (3.25±1.28)%, caspase-3 mRNA (2-ΔΔCt): 4.62±0.40 vs. 1.05±0.15, both P < 0.01]. However, there was no significant difference in the expression of HO-1 and the genes associated with autophagy between the two groups. In the CoPP group, the hepatic injury was blunted compared with that in the I/R group [ALT (U/L): 223.3±34.4 vs. 560.3±73.6, PS: 5.6±2.3 vs. 12.0±2.0, AI: (11.38±2.39)% vs. (19.38±3.07)%, caspase-3 mRNA (2-ΔΔCt): 2.42±0.33 vs. 4.62±0.40, all P < 0.01]. HO-1 was induced in the CoPP group and autophagy was also increased significantly after I/R when compared with those in the I/R group [HO-1 mRNA (2-ΔΔCt): 3.01±0.71 vs. 1.14±0.20, HO-1 activity (pmol×mg-1×h-1): 259±37 vs. 113±26, the number of autophagosomes: 8.75±0.87 vs. 1.25±0.71, Beclin-1 mRNA (2-ΔΔCt): 2.85±0.28 vs. 1.15±0.11, Atg-5 mRNA (2-ΔΔCt): 2.44±0.25 vs. 1.14±0.12, all P < 0.01]. In the ZnPP group, the activity of HO-1 was much lower than that in the CoPP group, and as a result autophagy was decreased and liver injury was increased. In the 3-MA group, although there was no difference in the activity of HO-1 compared with that in the CoPP group, autophagy was inhibited, and the protective effect of CoPP was eliminated. HO-1 could regulate the level of autophagy during liver I/R, and in turn autophagy might mediate the protective effects of HO-1 against liver I/R injury.