BCL-2 and BAX expression and cell proliferation, after partial hepatectomy with and without ischemia, on cholestatic liver in rats: an experimental study.
BACKGROUND
Liver regeneration after partial hepatectomy (PHx) is regulated by several factors that activate or inhibit hepatocyte proliferation. Apoptosis seems to play an important role in cellular proliferation and liver regeneration. This study investigates the expression apoptosis-associated genes bcl-2 and bax, and the presence of apoptosis and cell proliferation after PHx, in normal and jaundiced rats with or without superimposed ischemia.


METHODS
The study included 50 male Wistar rats assigned into; five groups (10 rats each). On day 0, rats of groups II, IV, and V underwent common bile duct ligation (BDL). On day 10, total liver ischemia (TLI) (occlusion of hepatic artery and portal vein-TLI) for 30 min was performed on animals of group V. When TLI was completed, all 30 animals (of groups I, IV, and V) underwent PHx (68%). Animals of group III underwent only TLI for 30 min. Rats of groups I, IV, and V were sacrificed 24 and 48 h after PHx was completed. Rats of group II were sacrificed 10, 11, and 12 days after BDL. Rats of group III were sacrificed immediately, 24 and 48 h after TLI completion. Liver tissue was obtained and pathologic examination included: (a) H&E stain, (b) in situ hybridization (detection of bcl-2 and bax mRNA) in paraffin sections, (c) Western blot analysis for the evaluation of bcl-2 and bax protein levels, (d) in situ hybridization (TUNEL) for the detection of apoptotic bodies, and (e) immunohistochemical stains (streptavidin-biotin method) in paraffin sections to detect cells that (i) express bcl-2 and bax proteins and (ii) undergo proliferation (Ki67+ cells). Results were expressed following morphometric analysis.


RESULTS
Before hepatectomy, bcl-2 (protein or mRNA) levels were higher in jaundiced rats vs controls. Furthermore, bax (protein or mRNA) levels and apoptotic body index (ABI) were higher in cholestatic livers. After hepatectomy, there was an early decrease in the protein and mRNA levels of antiapoptotic gene bcl-2 and a late increase of proapoptotic gene bax and the ABI, compared to controls. Cell proliferation of hepatocytes was lower in group V (BDL + TLI) compared to that of groups II and IV (BDL).


CONCLUSIONS
This study shows that apoptosis takes place in cholestatic livers with or without superimposed ischemia and may contribute in the impaired regenerative response observed in livers of jaundiced rats after partial hepatectomy.
Karavias
Dionissios D
DD
Department of Surgery, University of Patras School of Medicine, Rion University Hospital, Rion, Patras 26500, Greece. tsamanda@otenet.gr
Tsamandas
Athanassios C
AC
Tepetes
Konstantinos
K
Kritikos
Neoklis
N
Kourelis
Theodore
T
Ravazoula
Panagiota
P
Vagenas
Konstantinos
K
Siasos
Nikolaos
N
Mirra
Natale
N
Bonikos
Dionissis S
DS
D000818:Animals; D017209:Apoptosis / Q000235:genetics / Q000502:physiology; D002455:Cell Division / Q000235:genetics / Q000502:physiology; D001651:Cholestasis, Extrahepatic / Q000378:metabolism / Q000601:surgery*; D003250:Constriction; D015870:Gene Expression; D019254:Genes, bcl-2 / Q000235:genetics / Q000502:physiology*; D006498:Hepatectomy / Q000379:methods*; D007511:Ischemia; D008099:Liver / Q000098:blood supply* / Q000502:physiology / Q000601:surgery; D008115:Liver Regeneration / Q000235:genetics / Q000502:physiology*; D008297:Male; D023421:Models, Animal; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000502:physiology*; D019253:Proto-Oncogene Proteins c-bcl-2; D051381:Rats; D017208:Rats, Wistar; D051028:bcl-2-Associated X Protein
C494828:Bax protein, rat; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein