Reduction of liver ischemia reperfusion injury by silencing of TNF-α gene with shRNA.
BACKGROUND
Tumor necrosis factor-alpha (TNF-α) is a central mediator in the hepatic response to ischemia/reperfusion. Short hairpin RNA (shRNA) has been proven to be an effective means of harnessing the RNA interference pathway in mammalian cells. In the current study, we investigated whether silencing TNF-α gene with shRNA can prevent liver ischemic reperfusion injury (IRI).


METHODS
Male BalB/c mice were randomized to TNF-α shRNA, scramble shRNA, or sham operation groups. TNF-α shRNA and scramble shRNA groups were injected 48 h before inducing IRI. IRI was induced via microaneurysm clamps applied to the left hepatic artery and portal vein. Six hours after reperfusion, IRI injury was examined by serum level of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), liver histopathology, MPO, and MDA level, as well as by relative quantities of TNF-α mRNA.


RESULTS
TNF-α expression induced by ischemia reperfusion in the liver was significantly suppressed after treatment with TNF-α shRNA compared with the group treated with scramble shRNA (P < 0.001). Mice treated with TNF-α shRNA showed lower peak values of AST and ALT than scramble shRNA treated mice (P < 0.001). On histopathologic slides, mice treated with TNF-α shRNA had significantly less ischemia/reperfusion injury based on Suzuki score than the scramble shRNA group, 3.57 ± 2.30 and 8.83 ± 0.98 respectively (P < 0.001), while the sham group was not significantly different from the TNF-alpha shRNA group, 0 ± 0 and 3.57 ± 2.30, respectively (P = 0.075). Liver tissue MDA levels were significantly lower in mice treated with TNF-α shRNA as compared with the group treated with scramble shRNA (P < 0.01). Immunohistochemical staining for MPO was significantly lower in mice treated with TNF-α shRNA compared with the group treated with shRNA (compared with treated with scramble shRNA group.)


CONCLUSIONS
Liver IRI can be minimized through gene silencing of TNF-α. This may represent a novel therapy in the setting of transplantation and in other conditions associated with IRI of the liver.
Multi-Organ Transplant Program, London Health Sciences Centre, The University of Western Ontario, London, Canada. roberto.hernandezalejandro@lhsc.on.ca
Roberto
R
Hernandez-Alejandro
Xusheng
X
Zhang
Kris P
KP
Croome
Xiufen
X
Zheng
Jeremy
J
Parfitt
Dong
D
Chen
Anthony
A
Jevnikar
William
W
Wall
Wei-Ping
WP
Min
Douglas
D
Quan
D000410:Alanine Transaminase / Q000097:blood; D000818:Animals; D001219:Aspartate Aminotransferases / Q000097:blood; D015536:Down-Regulation / Q000235:genetics; D015316:Genetic Therapy / Q000379:methods*; D008099:Liver / Q000473:pathology / Q000502:physiology*; D008297:Male; D008315:Malondialdehyde / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D009195:Peroxidase / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000494:pharmacology*; D015427:Reperfusion Injury / Q000235:genetics* / Q000473:pathology / Q000628:therapy*; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics*
D034741:RNA, Small Interfering; D014409:Tumor Necrosis Factor-alpha; D008315:Malondialdehyde; D009195:Peroxidase; D001219:Aspartate Aminotransferases; D000410:Alanine Transaminase