The Role of the Sirt1/Foxo3a Pathway in Mitigating Myocardial Ischemia-Reperfusion Injury by Dexmedetomidine

Abstract

Myocardial ischemia-reperfusion injury (MIRI) significantly affects the prognosis of cardiac surgery patients. The anesthetic dexmedetomidine (Dex) has shown protective effects against ischemia-reperfusion injury in cardiomyocytes; however, its exact mechanism remains unclear. In this study, hypoxia/reoxygenation (H/R) and ischemia/reperfusion (I/R) models were used to investigate the effects of Dex on H9c2 cells and MIRI in mice. The roles of the Sirtuin 1/Forkhead box O3a (Sirt1/FoxO3a) pathway in the protective effects of Dex were explored using the Sirt1 inhibitor EX527 and FoxO3a gene silencing. Results showed that H/R significantly reduced H9c2 cell viability, increased Lactate Dehydrogenase (LDH) leakage, and elevated reactive oxygen species (ROS) production. Dex pretreatment reversed these effects. Additionally, Dex significantly reduced the expression of Bcl-2-associated X protein/B-cell lymphoma 2 (Bax/Bcl-2), cleaved caspase-3, Beclin-1, and microtubule-associated protein 1A/1B-light chain 3B (LC3B), inhibiting apoptosis and autophagy while increasing the expression of p62, Sirt1, and FoxO3a. The protective effects of Dex against H/R injury were abolished by EX527 or FoxO3a silencing. In the mouse MIRI model, Dex pretreatment decreased serum LDH and Creatine Kinase-MB (CK-MB) levels, reduced myocardial infarct size and cardiac injury, and significantly improved left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS). These protective effects were markedly reversed by EX527. These findings indicate that Dex alleviates MIRI by restoring Sirt1 expression and activating FoxO3a.

Keywords: FoxO3a; Sirt1; apoptosis; autophagy; dexmedetomidine; myocardial ischemia–reperfusion injury.

FIGURE 1

Construction of the H/R injury model in H9c2 cells under Dex treatment. (A) Effect of Dex concentrations on the cell viability. (B) Effect of Dex concentrations on the cell viability following H/R injury. (C) Effect of Dex concentrations on the LDH release following H/R injury. The cells were subjected to hypoxia treatment for 6 h and reoxygenation for 12 h. Then, LDH release was measured. CN: Control, H/R: Hypoxia/reoxygenation, Dex: Dexmedetomidine. ^ns p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. One‐way ANOVA was applied, followed by Tukey's post hoc test, a multiple comparison test.

FIGURE 2

Effect of treatment with Dex at 10 μM on apoptosis, autophagy, and oxidative stress of H/R‐induced H9c2 cells. (A) Western blot analysis of Sirt1, FoxO3a, and densitometry analysis of proteins. (B) Western blot analysis of Bax, Bcl‐2, Cleaved caspase‐3, and densitometry analysis of proteins. (C) Western blot analysis of p62, Beclin‐1, LC3B, and densitometry analysis of proteins. (D) ROS degrees measured by DHE‐DAPI staining (20×) and statistical graph of DHE. (E) The content of MDA. (F) Representative TUNEL‐staining images (20×) and statistical graph of TUNEL‐positive cells. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. One‐way ANOVA was applied, followed by Tukey's post hoc test, a multiple comparison test.

FIGURE 3

Effect of Dex treatment on apoptosis, autophagy, and oxidative stress of H/R‐induced H9c2 cells through the inhibition of Sirt1 expression. (A) Cell viability. (B) LDH leakage. (C) Western blot analysis of Sirt1, FoxO3a, and densitometry analysis of proteins. (D) Western blot analysis of Bax, Bcl‐2, Cleaved caspase‐3, and densitometry analysis of proteins. (E) Western blot analysis of p62, Beclin‐1, LC3B, and densitometry analysis of proteins. (F) ROS degrees measured by DHE‐DAPI staining (20×) and statistical graph of DHE. (G) The content of MDA. (H) Representative TUNEL‐staining images 20×) and statistical graph of TUNEL‐positive cells. (I) qPCR shows the mRNA expression levels of genes related to apoptosis, oxidative stress, and autophagy. ^ns p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. One‐way ANOVA or two‐way ANOVA was applied, followed by Tukey's post hoc test, a multiple comparison test.

FIGURE 4

Effect of Dex treatment on apoptosis, autophagy, and oxidative stress of H/R‐induced H9c2 cells through the silencing of FoxO3a expression. (A) Western blot analysis of FoxO3a silence vs. CN or CN + NC siRNA. NC: Negative control of FoxO3a. (B) Cell viability. (C) LDH Leakage. (D) Western blot analysis of Sirt1, FoxO3a, and densitometry analysis of proteins. (E) Western blot analysis of Bax, Bcl‐2, Cleaved caspase‐3, and densitometry analysis of proteins. (F) Western blot analysis of p62, Beclin‐1, LC3B, and densitometry analysis of proteins. (G) ROS degrees measured by DHE‐DAPI staining (20×) and statistical graph of DHE. (H) The content of MDA. (I) Representative TUNEL‐staining images (20×) and statistical graph of TUNEL‐positive cells.*p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. One‐way ANOVA was applied, followed by Tukey's post hoc test, a multiple comparison test.

FIGURE 5

Dex treatment attenuates H/R‐induced injury via Sirt1/FoxO3a signal pathway. (A) Cell viability. (B) LDH leakage. (C) The content of MDA. (D) Representative TUNEL‐staining images (20×) and statistical graph of TUNEL‐positive cells. (E) Western blot analysis of Sirt1, FoxO3a, and densitometry analysis of proteins. ^ns p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. One‐way ANOVA was applied, followed by Tukey's post hoc test, a multiple comparison test.

FIGURE 6

Dex treatment alleviates myocardial ischemia–reperfusion injury in vivo. (A) Evans blue and TTC double staining were used to evaluate the myocardial infarction area. The Evans blue‐staining area represented in blue, the TTC‐staining area in red (indicating the area at risk), and the TTC‐negative staining area appearing pale (indicating infarcted myocardium). (B) HE staining was used to evaluate myocardial injury (40×). (C) Echocardiography of mouse and statistical graph of LVEF and LVFS. (D) Serum LDH leakage and Serum CK‐MB levels were measured. (E) Western blot analysis of Sirt1, FoxO3a, and densitometry analysis of proteins. (F) Western blot analysis of Bax, Bcl‐2, Cleaved caspase‐3, and densitometry analysis of proteins. (G) Western blot analysis of p62, Beclin‐1, LC3B, and densitometry analysis of proteins. I/R: Ischemia/reperfusion. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. One‐way ANOVA was applied, followed by Tukey's post hoc test, a multiple comparison test.

FIGURE 7

Schematic diagram displays the role of Dex treatment alleviate myocardial ischemia–reperfusion injury. The upward red arrow represents upregulation of gene expression, and the downward blue arrow represents downregulation of gene expression.