The Regulation Mechanism Of AMPK/FOXO3 Signal Pathway In The Apoptosis And Differentiation Of Duck Myoblast

AMP-activated protein kinase (AMPK) is generally regarded as a key regulator of cellular processes. FOX03 as an important downstream gene of AMPK, participates in a wide variety of physiological processes including cell proliferation, apoptosis, differentiation and metabolism. Whereas, the effect of AMPK/FOX03 on duck myoblast development have not been clearly elucidated. To investigate the effect of AMPK/FOX03 signal pathway on duck myoblasts apoptosis and differentiation the possible mechanisms, we explored the AMPK and FOX03 expression by induced myoblasts apoptosis and differentiation at first. Furthermore, the cell viability and cell cycle be detected after inhibited and activated AMPK or overexpression FOXO3. And the expression change of apoptosis and differentiation marker genes also were detected.1. The cells were treated by using different concentrations inhibitor (0.25M,0.5M. 1M) and activator (0.5mM. 1mM,1.5mM). The results of CCK and qRT-PCR showed that 0.5M and 1mM as the best treatment concentrations of inhibitors and activators. FOX03 mRNA expression was examined at 12,24,36 and 48h following pEGFP-N1-FOX03 transfection with duck myoblasts. FOXO3 mRNA expression at 24h was elevated in the pEGFP-N 1-FOXO3 group compared to the pEGFP-N1 or control groups.2. Dexamethasone (DEX) could inhibite duck myoblasts viability, and AMPKal, FOX03, Caspase 3, LC3 and Caspase 7 in the 1000ng/mL and 10000ng/mL group but AMPKa2 only in 10000ng/mL group. What is more, DEX also could induce G0G1 cell cycle arrest with 1000ng/mL. Furthermore, the protein expression level of p-AMPK and FOXO3 also increased. In addition, we found that the positive effects of DEX on apoptosis of duck myoblasts could be inhibited by DOR. In addition, we found that the positive effects of DEX on apoptosis of duck myoblasts could be inhibited by DOR. Therefore, it may be that high concentration could promote cells apoptosis.3. After the AMPK/FOX03 signal pathway was activated by AICAR and pEGFP-N1-FOXO3, The G0G1 phase cells significant increased in AICAR and FOX03 group. The expression level of Caspase3 and LC3 in AICAR and FOXO3 group were significantly higher than control, DOR and pEGFP group. However, the expression of Caspase7 and Beclinl in AICAR and FOXO3 group were not significant difference, only Fas expression of AICAR group significantly higher than that of control group.4.2% PMSG was used to induce cells differentiation for 24,48 and 72h. Compared the control group, the mRNA level of AMPKal, FOXO3, MyHC for 48,72h and MRF4 for 48h expression was increased in 2% PMSG treatment group. MyHC group at 2% mRNA expression was significantly higher than that of controls and 2%+ DOR group; MyHC expression significantly increased in AICAR group than DOR group.