Melatonin attenuated OGD/R-induced H9c2 cell injury
The protective effect of melatonin on OGD/R-induced H9c2 cell injury was firstly investigated. The results of CCK-8 and LDH assays showed that OGD/R exposure resulted in significant decrease in cell viability and increase in LDH release, which was attenuated by pretreatment with different concentrations of melatonin (Fig. 1A and 1B). The results indicated that the use of 0.1 mM and 0.5 mM melatonin were less effective, and no comparable attenuation effects on cell viability and LDH release were observed at the concentrations of 1 mM and 5 mM. Thus, 1 mM melatonin was chosen in the subsequence experiments. As shown in Fig. 1C, the cell apoptosis rates under OGD/R were significantly higher than those under OGD/R with melatonin treatment. Western blot analysis revealed that melatonin pretreatment significanltly down-regulated pro-apoptotic proteins (Bad and Bax) and up-regulated the anti-apoptotic Bcl-2 in OGD/R-treated H9c2 cells (Fig. 1D). These data suggest that melatonin attenuated OGD/R-induced H9c2 cell injury.
Melatonin protected H9c2 cells against OGD/R injury via inhibiting Foxo3a/Bim signaling pathway
As Foxo3a has been demonstrated to play a critical role in I/R-induced cardiomyocyte apoptosis [14, 15, 21], we then determined whether melatonin exerts any influence on Foxo3a in H9c2 cells upon OGD/R exposure. As shown in Fig. 2A, OGD/R exposure induced up-regulation of Foxo3a, whereas melatonin treatment reversed OGD/R-induced up-regulation of Foxo3a and Bim, the well-known downstream of Foxo3a. OGD/R exposure promoted the nuclear translocation of Foxo3a, which could be inhibited by melatonin treatment (Fig. 2B). Moreover, under OGD/R condition, Foxo3a knockdown by siRNA significantly up-regulated cell viability (Fig. 2C), down-regulated LDH release (Fig. 2D) and Bim expression (Fig. 2E). Together, these data indicate that melatonin protected H9c2 cells against OGD/R injury via inhibiting Foxo3a/Bim signaling pathway.
Inhibition of Rac1 activation contributed to the protective effect of melatonin against OGD/R injury in H9c2 cells
Rac1 plays a crucial role in the modulation of OGD/R-induced cell injury [22, 23]. The regulatory effect of melatonin on Rac1 was then investigated. As shown in Fig. 3A, OGD/R induced an increase in Rac1-GTP, while the level of total Rac1 protein in H9c2 cells remained unmodified. The increased Rac1 activation by OGD/R exposure was suppressed by melatonin. To determine the involvement of Rac1 activation in OGD/R-induced cell injury, H9c2 cells were pretreated with Rac1-GTP inhibitor, NSC23766. The results showed that when Rac1 activity was inhibited by NSC23766 (Fig. 3B), cell viability was increased (Fig. 3C) and LDH release was decreased (Fig. 3D) in OGD/R-treated H9c2 cells. These data indicated that Rac1 activation is required for OGD/R-induced H9c2 cell injury. To further determine whether the inactivation of Rac1 was contributed to melatonin mediated protection against OGD/R, H9c2 cells were transfected with Rac1-V12, an active mutant of Rac1. The results showed that Rac1-V12 overexpression (Fig. 3E) significantly reversed the effect of melatonin on cell viability (Fig. 3F) and LDH release (Fig. 3G) in OGD/R-treated H9c2 cells. Collectively, these results indicate that inhibition of Rac1 activation contributed to the protective effect of melatonin against OGD/R-induced injury in H9c2 cells.
Melatonin inhibited OGD/R-activated Foxo3a/Bim signaling pathway through inactivation of Rac1
The above results indicated that Rac1 and Foxo3a signaling mainly contribute to the protective effect of melatonin on OGD/R-induced H9c2 cell injury. Previous studies have shown that activation of Foxo3a/Bim signaling pathway is primarily regulated by Rac1 [19, 20]. Thus, we investigated whether Rac1 is involved in melatonin-induced activation of Foxo3a/Bim signaling pathway in our system. The results showed that inactivation of Rac1 by NSC23766 significantly inhibited expression of Foxo3a and Bim in OGD/R-treated H9c2 cells (Fig. 4A). Furthermore, the inhibitory effects of melatonin on OGD/R-induced up-regulation of Foxo3a and Bim were reversed by overexpression of Rac1-V12 (Fig. 4B). Together, these results indicated that melatonin inhibited OGD/R-activated Foxo3a/Bim signaling pathway through inactivation of Rac1.
JNK inactivation was responsible for Rac1 inhibition-mediated inactivation of Foxo3a/Bim signaling pathway and decreased cell injury in melatonin-treated H9c2 cells
To explore the mechanism underlying Rac1-mediated inactivation of Foxo3a/Bim signaling pathway in our system, we first examined the activation of JNK pathway in the melatonin-treated H9c2 cells. The western blotting results demonstrated that the level of phosphorylated JNK was increased by OGD/R exposure (Fig. 5A). However, the promotion to the phosphorylated JNK was inhibited by melatonin treatment (Fig. 5A). The inhibitory effect of melatonin on OGD/R-induced phosphorylation of JNK was reversed by overexpression of Rac1-V12 (Fig. 5B). Moreover, Rac1-V12 overexpression-induced up-regulation of Foxo3a and Bim in melatonin-treated H9c2 cells were reversed by pretreatment with SP600125, a JNK inhibitor (Fig. 5C). These results indicated that JNK acts as a downstream effector of Rac1 in mediating inactivation of Foxo3a/Bim signaling pathway in melatonin-treated H9c2 cells.
To investigate the role of JNK de-phosphorylation in the protective effect of melatonin, we then measured the cell viability and LDH release of melatonin-treated H9c2 cells, in the presence of anisomycin, a potent JNK agonist. The results showed that anisomycin treatment markedly deteriorated melatonin–mediated viability increase and LDH release decrease of OGD/R-treated H9c2 cells (Fig. 5D and 5E). Together, these experiments demonstrated that JNK inactivation was responsible for Rac1 inhibition-mediated inactivation of Foxo3a/Bim signaling pathway and decreased cell injury in melatonin-treated H9c2 cells.