Annexin V assay-proven anti-apoptotic effect of ascorbic acid 2-glucoside after cold ischemia/reperfusion injury in rat liver transplantation.
Controversy exists over whether the predominant cell death of hepatocytes is due to apoptosis or necrosis after ischemia/reperfusion injury. In this study we investigated the predominant cell death of hepatocytes after cold ischemia/reperfusion injury using the Annexin V-based assay, and evaluated the anti-apoptotic effect of ascorbic acid 2-glucoside (AA-2G) added to the University of Wisconsin solution (UW solution) in rat liver transplantation. The retrieved liver was preserved in 4 UW solution for 24 h, and then transplanted orthotopically to the syngeneic Wistar recipient. The animals were divided into 2 groups, a control group (n=10), in which liver grafts were preserved in UW solution (4), and an AA-2G group (n=10), in which liver grafts were preserved in UW solution (4) with AA-2G (100 ug/ml). The serum AST level 4 h after reperfusion in the control group was significantly suppressed in the AA-2G group, and the bile production of the liver graft in the AA-2G group was well recovered. The mean survival time in the AA-2G group was significantly improved compared with that in the control group. Annexin-V and Propidium iodide staining 4 h after reperfusion showed a significantly higher percentage of viable hepatocytes in the AA-2G group compared with the control group (93.4 +/- 2.0 vs. 80.3 +- 2.1%, P<0.05). In the control group, the main cell death of hepatocytes was apoptosis (early apoptosis: 10.0 +- 4.7%, late apoptosis: 6.4 +/- 1.7%). The addition of AA-2G to the UW solution significantly inhibited both early and late apoptotic cell death 4 h after reperfusion (early apoptosis: 0.98 +/- 0.88%, late apoptosis: 2.2 +/- 1.1%). The expression of caspase 9 in the immunostaining of the liver graft was suppressed in the AA-2G group compared with in the control group. Our study using the Annexin V-based assay provided evidence that the predominant cell death of hepatocytes was apoptosis after 24 h cold ischemia/reperfusion injury in rat liver transplantation. The addition of AA-2G to the UW solution attenuated 24 h cold ischemia/reperfusion injury by inhibiting the apoptosis of hepatocytes.
Liu
Jie
J
Department of Gastroenterological Surgery, Transplant, and Surgical Oncology, Okayama University Graduate School of Medicine and Dentistry, Okayama 700-8558, Japan.
Yagi
Takahito
T
Sadamori
Hiroshi
H
Matsukawa
Hiroyoshi
H
Sun
Dong-Sheng
DS
Mitsuoka
Naoshi
N
Yamamura
Masao
M
Matsuoka
Junji
J
Jin
Zaishun
Z
Yamamoto
Itaru
I
Tanaka
Noriaki
N
D000818:Animals; D017304:Annexin A5 / Q000032:analysis; D017209:Apoptosis / Q000187:drug effects*; D001205:Ascorbic Acid / Q000031:analogs & derivatives* / Q000627:therapeutic use*; D001219:Aspartate Aminotransferases / Q000378:metabolism; D001646:Bile / Q000378:metabolism; D053453:Caspase 9; D020169:Caspases / Q000032:analysis; D015925:Cryopreservation; D005434:Flow Cytometry; D022781:Hepatocytes / Q000201:enzymology; D007150:Immunohistochemistry / Q000379:methods; D008099:Liver / Q000737:chemistry / Q000201:enzymology / Q000473:pathology; D008102:Liver Circulation; D016031:Liver Transplantation; D008297:Male; D051381:Rats; D017208:Rats, Wistar; D015427:Reperfusion Injury / Q000209:etiology / Q000503:physiopathology*; D013194:Staining and Labeling; D016019:Survival Analysis
D017304:Annexin A5; C065378:ascorbic acid 2-O-glucoside; D001219:Aspartate Aminotransferases; C506435:Casp9 protein, rat; D053453:Caspase 9; D020169:Caspases; D001205:Ascorbic Acid