EFFETTO PROTETTIVO DI GHRELINA SUL DANNO DA STRESS OSSIDATIVO NEGLI OSTEOBLASTI

Increasing evidence suggests a role for oxidative stress in age-related decrease in osteoblast number and function, leading to the development of osteoporosis. In this study, we investigated the effects of ghrelin on tert-butyl-hydroperoxide (t-BHP)-induced oxidative damage in MC3T3-E1 osteoblastic cells, as well as the role of the ghrelin receptor (GHS-R) involved in such activity. MTT assay showed that t-BHP treatment resulted in decreased cell viability in a concentration and time dependent manner. Under 250 μM t-BHP treatment, MC3T3-E1 cells displayed significant loss of viability and dramatic morphological changes characterized by cell shrinkage and typical apoptotic alterations such as chromatin condensation. MC3T3-E1 cells pretreated with ghrelin (10-9 M) and cultured with t-BHP (250 μM) for 3h showed increased viability and reduced apoptosis as shown by MTT assay and Hoechst-33258 staining. Furthermore, ghrelin prevented t-BHP-induced osteoblastic dysfunction and changes in the cytoskeleton organization evidenced by the staining of the actin fibers with Phalloidin-FITC by reducing reactive oxygen species generation. Cell treatment with the GHS-R1a agonist, EP1572 (10-7-10-11 M), had no effect against t-BHP-induced cytotoxicity and pretreatment with the selective GHS-R1a antagonist, D-Lys3-GHRP-6 (10-7 M), failed to remove ghrelin (10-9 M)-protective effects against oxidative injury, indicating that GHS-R1a is not involved in such ghrelin activity. Accordingly, unacylated ghrelin (DAG), not binding GHS-R1a, displays the same protective actions of ghrelin against t-BHP-induced cytotoxicity. Consistently with these observations, Ghrelin has been previously shown to activate different intracellular pathways including PI3K/Akt via a specific ghrelin/DAG binding site, that is not GHS-R1a. Furthermore, cell treatment with a specific inhibitor of PI3K/Akt pathway, (Ly294002, 10μM) reduced the protective effect of ghrelin against t-BHP-induced oxidative damage. This observation indicates that ghrelin confers protection against t-BHP-induced oxidative damage via a PI3K/Akt dependent pathway. Akt activation controls the cell survival through phosphorylation of downstream targets such as protein kinase GSK-3b. Inactivation of GSK-3b results in traslocation of b-catenin to the nucleus and nuclear b-catenin is known to inhibit apoptosis. In this study we have shown that ghrelin increased GSK-3b phosphorylation and b-catenin accumulation measured by Western Blot primary in osteoblasts obtained from rat calvary cells (rOB). These findings suggest that ghrelin can function as an antioxidant compound on osteoblasts and may have therapeutic potential to promote bone formation and reduce bone loss associated with aging.