35. Tissue protection mediated by diallyl disulfide in rat liver hypothermic preservation

Abstract

The beneficial effects of dietary garlic (Allium sativum) have been recognized for centuries. In particular, garlic consumption has been frequently associated with the reduction of multiple risk factors such as increased reactive oxygen species, high blood pressure and high cholesterol. Hydrogen sulfide (H 2 S) could be implicated in those garlic cytoprotective effects. This gas is the newest member of the gaseous transmitter family, known to promote vasodilatation, antioxidant defenses, attenuate inflammation and inhibit apoptosis. These properties make this gas ideally suited to protect tissues from ischemia–reperfusion injuries. We evaluated the cytoprotective potential of a garlic derived compound (Diallyl disulfide, DADS) during cold storage of rat livers with an isolated normothermic reperfusion experimental approach. Male adult Wistar rats (250–300 g) were hepatectomized by a widespread surgical technique. Livers were preserved in HTK solution (Custodiol®) + (1, 5, 50 and 500 μM) or without DADS (control) up to 48 h at 0–4°C, after that, they were evaluated in an ex vivo perfusion system. Fresh control livers were immediately reperfused. The following parameters were assessed: intrahepatic resistance (IR), bile production (BP), oxygen consumption (OC) and lactate dehydrogenase release (LDH). Also, 5 mg of bromosulfophtalein (BSP) was added to the perfusion media at t = 0, to evaluate depuration capacity through BSP spectrophotometric determination and thin layer chromatography (TLC) of bile samples. Hypothermic preservation injuries were manifested during reperfusion through the parameters evaluated (versus control fresh livers). Intrahepatic resistance and LDH release after 60 min of reperfusion, were lessened when DADS in a 5 μM concentration was added to the preservation solution. IR (mmHg min gliv/mL): 3.16 ± 0.60 (HTK−5 μM), 3.39 ± 0.57 (HTK); LDH activity in perfusate (U/L.gliv): 48.74 ± 2.80 (HTK−5 μM), 58.95 ± 5.50 (HTK). Also, bile production was augmented to some extent by this dose when compared to the HTK without DADS group: 0.50 ± 0.18 μL/min gliv (HTK−5 μM); 0.38 ± 0.13 μL/min gliv (HTK). Oxygen consumption constitutes a marker for active metabolism, 5 μM doses of DADS showed a 21.5% increase in O 2 uptake ( p < 0.05, vs. HTK alone). Interestingly, the other assayed doses not only did not improve protection but, in some cases, seemed to deteriorate grafts. Percentages of BSP excreted in bile at the end of ex vivo perfusion (90 min) indicated an increased depuration competence for livers preserved in HTK+5 and 50 μM of DADS: 67.17 ± 8.10% (HTK−5 μM), 60.90 ± 7.02%(HTK−50 μM) and 41.93 ± 9.86% (HTK) ( p < 0.05). TLCs revealed four spots with different R f corresponding to free BSP, BSP-GSH conjugates and BSP- γ Glu-Cys/ γ Glu species (100%). This study reflected that cold storage in HTK barely affected BSP conjugation to glutathione (GSH) since % BSP-GSH of fresh control livers did not differ from preserved ones. No statistical differences were found between preserved groups when comparing BSP-GSH proportions. In conclusion, addition of diallyl disulfide in a 5 μM concentration to preservation solution may improve static cold storage outcomes for rat livers.