Propofol protects against hydrogen peroxide-induced injury in cardiac H9c2 cells via Akt activation and Bcl-2 up-regulation

Abstract

Propofol is a widely used intravenous anesthetic agent with antioxidant properties secondary to its phenol based chemical structure. Treatment with propofol has been found to attenuate oxidative stress and prevent ischemia/reperfusion injury in rat heart. Here, we report that propofol protects cardiac H9c2 cells from hydrogen peroxide (H(2)O(2))-induced injury by triggering the activation of Akt and a parallel up-regulation of Bcl-2. We show that pretreatment with propofol significantly protects against H(2)O(2)-induced injury. We further demonstrate that propofol activates the PI3K-Akt signaling pathway. The protective effect of propofol on H(2)O(2)-induced injury is reversed by PI3K inhibitor wortmannin, which effectively suppresses propofol-induced activation of Akt, up-regulation of Bcl-2, and protection from apoptosis. Collectively, our results reveal a new mechanism by which propofol inhibits H(2)O(2)-induced injury in cardiac H9c2 cells, supporting a potential application of propofol as a preemptive cardioprotectant in clinical settings such as coronary bypass surgery.

Figure 1. Effects of H₂O₂ and/or propofol on H9c2 cells viability and apoptosis

(A, B) Cells were incubated with increasing concentrations of H₂O₂ (0 to 500μM) or propofol (0 to 100μM) for 24 hours. Cell viability was measured by MTS assay. (A) H₂O₂ decreased H9c2 cells viability in a concentration-dependent manner. (B) Cell viability was not significantly affected by any tested propofol concentrations. (C, D, E) Cells were treated with propofol (5μM) or vehicle (DMSO) in the presence or absence of H₂O₂ (400μM) for 24 hours. (C) Cell viability was measured by MTS assay. (D) Cell apoptosis was evaluated by Hoechst33258 staining. Representative immunocytochemistry photomicrographs and corresponding histogram were displayed. (E) Caspase-3 activity was measured by the Fluorometric CaspACE™ Assay. *p<0.05 vs control, **p<0.01 vs control. # p<0.01 vs H₂O₂ only group.

Figure 2. Effects of propofol on Akt phosphorylation and PI3K inhibition on the protection of propofol against H₂O₂-stimulated cell death and apoptosis

(A) H9c2 cells were treated with vehicle (DMSO) or propofol (50μM) for various times as indicated. Cell lystates were harvested and western blot was performed to examine Akt phosphorylation. Propofol increased Akt phosphorylation at both serine 473 and threonine 308 sites while DMSO has no effects on Akt phosphorylation. (B, C, D) H9c2 cells were pre-incubated with 500nM wortmannin for 30 min and then treated with H₂O₂ (400μM) and propofol (50μM) for 24 hours. Wortmannin reversed the increases in cell viability (B) and the decreases in caspase-3 activity (C) induced by propofol in H₂O₂-stimulated cells. Western blotting results further showed that wortmannin treatment decreased Akt phosphorylation induced by propofol (D). *p<0.01 vs control, #p<0.01 vs H₂O₂ only group, $ p<0.01 vs H₂O₂+propofol+DMSO group.

Figure 3. Modulation of propofol on BCl-2

(A, B) H9c2 cells were treated with propofol (50μM) or H₂O₂ (400μM) for various times as indicated. (A) Cell lystates were harvested and western blot was performed to examine Bcl-2 protein expression. Expression of β-actin was also examined for protein loading control. A decrease in Bcl-2 expression was observed in response to H₂O₂, while propofol increased Bcl-2 expression time-dependently. (B) Total cellular RNA were extracted and real-time RT-PCR was performed to examine Bcl-2 mRNA expression. Expression of GAPDH was also examined for internal control. Propofol increased Bcl-2 mRNA expression beginning at 1 hour after treatment and was still elevated after 6 hour treatment. (C, D) H9c2 cells were pre-incubated with 500nM wortmannin for 30 min and then treated with H₂O₂ (400μM) and propofol (50μM) for 24 hours. Wortmannin treatment decreased the Bcl-2 protein expression induced by propofol (C). Of interest, wortmannin treatment had no effects on propofol-induced Bcl-2 gene expression (D). *p<0.01 vs control, #p<0.01 vs H₂O₂ only group, $ p<0.01 vs H₂O₂+propofol+DMSO group.

Figure 4. Postulated Akt -dependent and -independent modulation on Bcl-2 by propofol

Akt phosphorylation induces the phosphorylation of Bad, which leads to the release and activation of Bcl-2. Moreover, Akt interact with and activate inhibitor of kappa B kinases (IKKs) and lead to the activation of nuclear factor (NF)-κB, which then translocates to the nucleus and transcribes anti-apoptotic Bcl-2 gene. We postulated that propofol might modulate Bcl-2 activation and expression through both Akt -dependent and -independent (STAT-3, signal transducer and activator of transcription 3) pathways.