Melatonin Protects Against Apoptosis of Megakaryocytes Via Its Receptors and Activation of PI3k/Akt Pathway

Melatonin has been shown to be able to lower the frequency of thrombocytopenia in patients with cancer undergoing radiotherapy or chemotherepy in clinical studies (Lissoni et al, J Pineal Res, 1999). Melatonin is an acetylated product of serotonin. Our previous study demonstrated that serotonin stimulates megakaryopoiesis and thrombopoiesis (Yang et al, Stem Cells, 2007; 2014). However, the underlying mechanism of melatonin on platelet formation/survival has not been well investigated. We hypothesized that melatonin has therapeutic effects on thrombocytopenia by reducing apoptosis of megakaryocytes (MKs)/platelets via melatonin receptors and activation of Akt signaling. We investigated the in-vitro effect of melatonin on CFU-MK formation at different doses (0-500 nM). The results showed that melatonin significantly promoted CFU-MK formation at a dose dependant manner. The maximal concentration was at 200 nM (P<0.01, n=6). The size of CFU-MK with melatonin treatment was much larger and each MK cell was more mature looking. Using megakaryocytic cell CHRF-288-11, we identified the expression of melatonin receptors MT2 on these MK cells by RT-PCR and western blotting. We further found that melatonin exerted protective effect on serum-free induced apoptosis of CHRF cells. The cell viability was significantly increased with the treatment of melatonin. Flow cytometric dot-plot analysis demonstrated that the population of apoptotic cells (Annexin V/PI staining) was significantly decreased with melatonin treatment (200nmol/L) for 72 hrs (n=4). It also showed trends of amelioration of caspase-3 expression and the proportion of cells containing JC-1 monomers (indicating a trend in the drop of mitochondrial membrane potential). The population of phospho-Akt were significantly increased after treatment by melatonin and this effect was abolished by melatonin receptor MT2 blocker. The effect of melatonin on in-vivo thrombopoiesis was also investigated in an irradiated mouse model. Eighteen mice were randomly divided into three groups (6 each). Group 1 (normal control) received no irradiation or melatonin; Group 2 (model control) and Group 3 (melatonin treated) both received 4 Gy total body irradiation. After irradiation, melatonin (10mg/kg.d) was injected intraperitoneally into Group 3 consecutively for 21 days. Peripheral blood platelets, white blood cells (WBC), and red blood cells (RBC) were analyzed from the three groups on day 0, 7, 14, and 21.We found that melatonin enhanced the recovery of platelets and WBC production in Group 3. Our results showed that melatonin enhanced platelet formation and survival in radiation induced thrombocytopenia. The underlying mechanisms might involve in direct stimulation of megakaryopoiesis and anti-apoptotic effect on megakaryocytes via it receptors and activation of Akt signaling.