Pituitary Adenylate Cyclase-Activating Polypeptide Ameliorates Radiation-Induced Cardiac Injury

Abstract

Radiation-induced heart disease (RIHD) is a common sequelae of thoracic irradiation. Oxidative stress is associated with the development of RIHD. Studies have shown that pituitary adenylate cyclase-activating polypeptide 38 (PACAP38) represents the multipotent properties of cytoprotective effect on its anti-inflammatory, anti-apoptotic and antioxidant activities. If PACAP38 plays a role in attenuating RIHD remains investigation. In the present study, the culture of rat H9C2 cardiomyocytes was subjected to irradiation (IR) in vitro. Cardiac injury was performed to expose murine heart for IR. H9C2 cells and C57 mice were pre-treated with PACAP38 at different doses prior to radiation exposure. The effects of PACAP38 was evaluated by cell viability assay (CCK8), plate clone formation assay, cell cycles and apoptosis of flow cytometry. Apoptosis-related proteins, cell cycle-associated proteins and NRF2/HO-1 were determined by qRT-PCR and Western blot. The transient knockdown of NRF2 signaling by siRNA was also examined to explore the possible abortion of cytoprotection of PACAP38. H-E, Masson and TUNEL staining were performed in vivo in mouse model of IR. Cell viability was enhanced for 15-20% higher in H9C2 cardiomyocytes treated with PACAP38 (10-7M)+IR (12Gy) than in cardiomyocytes exposed to radiation (12Gy) alone (PACAP38+IR vs. IR, P<0.01). 10-7M PACAP38 increased colony-forming efficiency by 23%, 41% and 3 fold at IR doses of 2Gy, 4Gy and 8Gy, respectively. PACAP38 suppressed myocardial apoptosis and G2/M arrest through blunting the radiation-induced down-regulation of Bcl-2, CyclinB1 and CDK1, and inhibiting the up-regulation of Bax. IR resulted in activating of NRF2 and HO-1 expressions were further enhanced by PACAP38 at 10-7M concentration in H9C2 cells. The protective effect of PACAP38 was partially blocked by Nrf2 siRNA silencing (PACAP38+IR vs. PACAP38+IR+SiNrf2, P<0.05). Moreover, treatment with PACAP38 (10μg, i.p.) protected mice from histological damage by significantly inhibition of myocardial apoptosis. Our results indicate that PACAP38 has the potential to effectively protect against acute radiation-induced cardiac injury and its cardioprotective effect involves upregulation of Nrf2/HO-1-dependent signaling activation.