Adenosine and inosine exert cytoprotective effects in an in vitro model of liver ischemia-reperfusion injury

Abstract

Liver ischemia represents a common clinical problem. In the present study, using an in vitro model of hepatic ischemia-reperfusion injury, we evaluated the potential cytoprotective effect of the purine metabolites, such as adenosine and inosine, and studied the mode of their pharmacological actions. The human hepatocellular carcinoma-derived cell line HepG2 was subjected to combined oxygen-glucose deprivation (COGD; 0-14-24 h), followed by re-oxygenation (0-4-24 h). Adenosine or inosine (300–1,000 μM) were applied in pretreatment. Cell viability and cytotoxicity were measured by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide and lactate dehydrogenase methods, respectively. The results showed that both adenosine and inosine exerted cytoprotective effects, and these effects were not related to receptor-mediated actions, since they were not prevented by selective adenosine receptor antagonists. On the other hand, the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride (EHNA, 10 μM) markedly and almost fully reversed the protective effect of adenosine during COGD, while it did not influence the cytoprotective effect of inosine in the same assay conditions. These results suggest that the cytoprotective effects are related to intracellular actions, and, in the case of adenosine also involve intracellular conversion to inosine. The likely interpretation of these findings is that inosine serves as an alternative source of energy to produce ATP during hypoxic conditions. The protective effects are also partially dependent on adenosine kinase, as the inhibitor 4-amino-5-(3-bromophenyl)-7-(6-morpholino-pyridin-3-yl)pyrido[2,3-d]pyrimidine, 2HCl (ABT 702, 30 μM) significantly reversed the protective effect of both adenosine and inosine during hypoxia and re-oxygenation. Collectively, the current results support the view that during hypoxia, adenosine and inosine exert cytoprotective effects via receptor-independent, intracellular modes of action, which, in part, depend on the restoration of cellular bioenergetics. The present study supports the view that testing of inosine for protection against various forms of warm and cold liver ischemia is relevant.

The Hypoxia-Reoxygenation Injury Model

“…Drug treatments may be administered before the hypoxia induction to test preventive effects or following the hypoxic period to test the therapeutic potential in ischemic diseases [1][2][3]. For gene silencing small interfering RNAs may be added 48 hours prior to the hypoxia exposure to effectively reduce RNA and protein levels of the gene of interest at the time of the hypoxia experiment [4,5].…”

“…Most cells can survive in culture if the cellular ATP concentration will be reduced by less than 75-80% the normal ATP level [1][2][3]5]. Following an OGD injury that does not reduce the cellular ATP concentration below 20% of the initial baseline value full recovery is expectable if optimal culture conditions are provided.…”

“…In vivo ischemia-reperfusion models are technically simple and reproduce many aspects of ischemic diseases, but in vitro models are equally important, because they allow detailed study of the mechanism of cellular damage and make it possible to test large chemical libraries or sets of human small interfering RNAs (siRNAs) that are essential for early phase drug discovery [1][2][3][4][5]. Chemical hypoxia models that use mitochondrial uncoupling agents or respiration blockers reproduce the rapid onset of ischemia but there is no way to study the recovery processes that occur during reperfusion [6][7][8][9].…”

Szabad gyökök és a máj ischaemiás-reperfúziós károsodása

“…kinázokat (RISK) aktiváló hatása mellett [33] újszerű, eddigiekben kevésbé ismert hatásmecha-nizmusaira derült fény laborunk kollaboráló kísérle-tében. A primer májrákból származó in vitro modellen történő kísérlet I-R szimulálását követően azonosította az adenozin védőhatásának hatásos koncentrációját [34]. Továbbá igazolást nyert, hogy az adenozin védőhatását legalábbis részben exogén energiaszubsztrát-funkciót betöltve fejti ki, mivel ic.…”

“…Továbbá igazolást nyert, hogy az adenozin védőhatását legalábbis részben exogén energiaszubsztrát-funkciót betöltve fejti ki, mivel ic. bejutását követően, inozin intermedier metaboliton keresztül foszforilálódik AMP-vé, majd ADP-vé [34]. Szoros értelemben véve nem kondicionálószer a glutamin, azonban számos jótékony hatá-sánál fogva régóta ismeretes pozitív hatása számos katabolikus állapot kapcsán [35].…”