ABSTRACT
1. The aim of the present study was to investigate the role of proteasome in the pathogenesis of liver injury induced by intestinal ischaemia-reperfusion (I/R) and the effect of the proteasome inhibitor lactacystin on neutrophil infiltration, intracellular adhesion molecule (ICAM)-1 and nuclear factor (NF)-kappaB expression in the liver tissues of rats. 2. Thirty-two Wistar rats were randomly divided into four groups (n = 8 in each group) as follows: (i) a control, sham-operated group; (ii) an I/R group subjected to 1 h intestinal ischaemia and 4 h reperfusion; (iii) a group pretreated with 0.2 mg/kg lactacystin 1 h before intestinal I/R; and (iv) a group pretreated with 0.6 mg/kg lactacystin 1 h before intestinal I/R. Liver and intestine histology were observed. Serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH), as well as 20S proteasome activity in circulating white blood cells, were measured. Myeloperoxidase (MPO) activity in liver tissues and the immunohistochemical expression of liver NF-kappaB and ICAM-1 were assayed. In addition, a western blot of liver NF-kappaB was performed. 3. Compared with the sham-operated control group, liver and intestine injury was induced by intestinal I/R, characterized as histological damage including oedema, haemorrhage and infiltration by inflammatory cells, as well as a significant increase in serum AST (365 +/- 121 vs 546 +/- 297 IU/L, respectively; P < 0.05), ALT (65 +/- 23 vs 175 +/- 54 IU/L, respectively; P < 0.01) and LDH levels (733 +/- 383 vs 1434 +/- 890 IU/L, respectively; P < 0.05). Compared with the control group, MPO activity in the liver tissues increased significantly in the I/R group (2.05 +/- 0.69 vs 3.42 +/- 1.11 U/g, respectively; P < 0.05). Strong positive expression of liver ICAM-1 and NF-kappaB p65 was observed. 4. Compared with the intestinal I/R group, administration of 0.6 mg/kg lactacystin markedly reduced 20S proteasome activity in circulating white blood cells (15.47 +/- 4.00 vs 2.07 +/- 2.00 pmol 7-amino-4-methylcoumarin (AMC)/s per mg, respectively; P < 0.01) and ameliorated liver injury, which was demonstrated by decreased levels of serum AST (546 +/- 297 vs 367 +/- 86 IU/L, respectively; P < 0.05), ALT (175 +/- 54 vs 135 +/- 26 IU/L, respectively; P < 0.05) and LDH (1434 +/- 890 vs 742 +/- 218 IU/L, respectively; P < 0.05) and a reduced liver pathological score (2.13 +/- 0.64 vs 1.25 +/- 0.46, respectively; P < 0.01). Compared with the intestinal I/R group, MPO activity in liver tissues decreased significantly following lactacystin pretreatment (3.42 +/- 1.11 vs 2.58 +/- 0.61 U/g, respectively; P < 0.05) and the expression of liver NF-kappaB and ICAM-1 was markedly ameliorated. 5. The present study reveals that the proteasome inhibitor lactacystin ablates liver injury induced by intestinal I/R. One possible mechanism responsible for this effect is the inhibition of enhanced ICAM-1 and neutrophil infiltration by inhibition of NF-kappaB activity. The results suggest the feasibility of using proteasome inhibitor clinically in the treatment of intestinal I/R.
Subject(s)
Acetylcysteine/analogs & derivatives , Cysteine Proteinase Inhibitors/pharmacology , Intestines/blood supply , Liver Diseases/prevention & control , Liver/drug effects , Proteasome Endopeptidase Complex/drug effects , Reperfusion Injury/complications , Acetylcysteine/pharmacology , Acetylcysteine/therapeutic use , Alanine Transaminase/metabolism , Animals , Aspartate Aminotransferases/metabolism , Cysteine Proteinase Inhibitors/therapeutic use , Disease Models, Animal , Dose-Response Relationship, Drug , Feasibility Studies , Intercellular Adhesion Molecule-1/metabolism , L-Lactate Dehydrogenase/metabolism , Liver/enzymology , Liver/metabolism , Liver/pathology , Liver Diseases/etiology , Liver Diseases/metabolism , Liver Diseases/pathology , Male , Neutrophil Infiltration/drug effects , Peroxidase/metabolism , Proteasome Endopeptidase Complex/blood , Rats , Rats, Wistar , Reperfusion Injury/drug therapy , Reperfusion Injury/metabolism , Reperfusion Injury/pathology , Transcription Factor RelA/metabolismABSTRACT
AIM: To investigate the protective effects and possible mechanisms of Veratrum nigrum L.var. ussuriense Nakai alkaloids (VnA) on hepatic ischemia/reperfusion (I/R) injury in rats. METHODS: Forty male Wistar rats were randomly divided into four experimental groups (n = 10 in each): (A) Control group (the sham operation group); (B) I/R group (pretreated with normal saline); (C) Small-dose (10 microg/kg) VnA pretreatment group; (D) Large-dose (20 microg/kg) VnA pretreatment group. Hepatic ischemia/reperfusion (Hepatic I/R) was induced by occlusion of the portal vein and the hepatic artery for 90 min, followed by reperfusion for 240 min. The pretreatment groups were administered with VnA intraperitoneally, 30 min before surgery, while the control group and I/R group were given equal volumes of normal saline. Superoxide dismutase (SOD) activity, myeloperoxidase (MPO) activity and nitric oxide (NO) content in the liver tissue at the end of reperfusion were determined and liver function was measured. The expression of intercellular adhesion molecule-1 (ICAM-1) and E-selectin (ES) were detected by immunohistochemical examinations and Western blot analyses. RESULTS: The results showed that hepatic I/R elicited a significant increase in the plasma levels of alanine aminotransferase (ALT: 74.53 +/- 2.58 IU/L vs 1512.54 +/- 200.76 IU/L, P < 0.01) and lactic dehydrogenase (LDH: 473.48 +/- 52.17 IU/L vs 5821.53 +/- 163.69 IU/L, P < 0.01), as well as the levels of MPO (1.97 +/- 0.11 U/g vs 2.57 +/- 0.13 U/g, P < 0.01) and NO (69.37 +/- 1.52 micromol/g protein vs 78.39 +/- 2.28 micromol/g protein, P < 0.01) in the liver tissue, all of which were reduced by pretreatment with VnA, respectively (ALT: 1512.54 +/- 200.76 IU/L vs 977.93 +/- 89.62 IU/L, 909.81 +/- 132.76 IU/L, P < 0.01, P < 0.01; LDH: 5821.53 +/- 163.69 IU/L vs 3015.44 +/- 253.01 IU/L, 2448.75 +/- 169.4 IU/L, P < 0.01, P < 0.01; MPO: 2.57 +/- 0.13 U/g vs 2.13 +/- 0.13 U/g, 2.07 +/- 0.05 U/g, P < 0.01, P < 0.01; NO: 78.39 +/- 2.28 micromol/g protein vs 71.11 +/- 1.73 micromol/g protein, 68.58 +/- 1.95 micromol/g protein, P < 0.05, P < 0.01). The activity of SOD (361.75 +/- 16.22 U/mg protein vs 263.19 +/- 12.10 U/mg protein, P < 0.01) in the liver tissue was decreased after I/R, which was enhanced by VnA pretreatment (263.19 +/- 12.10 U/mg protein vs 299.40 +/- 10.80 U/mg protein, 302.09 +/- 14.80 U/mg protein, P < 0.05, P < 0.05). Simultaneously, the histological evidence of liver hemorrhage, polymorphonuclear neutrophil infiltration and the overexpression of ICAM-1 and E-selectin in the liver tissue were observed, all of which were attenuated in the VnA pretreated groups. CONCLUSION: The results demonstrate that VnA pretreatment exerts significant protection against hepatic I/R injury in rats. The protective effects are possibly associated with enhancement of antioxidant capacity, reduction of inflammatory responses and suppressed expression of ICAM-1 and E-selectin.
