Role of Keap1-Nrf2/ARE signal transduction pathway in protection of dexmedetomidine preconditioning against myocardial ischemia/reperfusion injury

Abstract

Objective: To explore the role and mechanism of the Kelch sample related protein-1-nuclear factor erythroid-2 related factor 2/antioxidant response element (Keap1-Nrf2/ARE) signaling pathway in protection of dexmedetomidine (DEX) preconditioning against myocardial ischemia/reperfusion injury (MIRI).

Methods: A total of 70 male SD rats were randomly divided into seven equal groups (n=10): blank control (S group), ischemia/reperfusion injury (C group), DEX preconditioning (DEX group), tertiary butylhydroquinone (tBHQ) control (tBHQ group), combined tBHQ and DEX preconditioning (tBHQ+DEX group), all-trans retinoic acid (ATRA) control (ATRA group), and combined ATRA and DEX preconditioning (ATRA+DEX group). Serum creatine kinase-MB (CK-MB) and cardiac troponin I (cTnI) concentrations were measured by ELISA kits, and the infarct size (IS) was assessed by Evan's blue and 2,3,5-triphenyltetrazolium chloride (TTC) staining. Oxidative stress was assessed through Western blotting for expression of Keap1-Nrf2/ARE pathway members and oxidative stress markers.

Results: Cardioprotection of DEX, tBHQ, and tBHQ+DEX preconditioning treatments were shown as lower concentrations of serum CK-MB and cTnI and a smaller IS following MIRI in rats compared with those of MIRI rats without pre-treatment. In addition, tBHQ+DEX preconditioning exhibited stronger myocardial protection compared with DEX preconditioning. Mechanistically, the cardioprotection offered by DEX, tBHQ, and tBHQ+DEX preconditioning treatments was mediated via exerting antioxidant stress through activation of the Keap1-Nrf2/ARE signal transduction pathway. Conversely, the protective effects of DEX were diminished by blocking the Keap1-Nrf2/ARE pathway with inhibitor ATRA.

Conclusion: DEX preconditioning protects against MIRI by exerting antioxidant stress through activation of the Keap1-Nrf2/ARE signal transduction pathway, while inhibition of the Keap1-Nrf2/ARE signal transduction pathway reverses the protective effect of DEX preconditioning on MIRI.

Keywords: Dexmedetomidine; Keap1-Nrf2/ARE signal transduction pathway; Myocardial ischemia/reperfusion injury; Oxidative stress.

Figure 1. Experimental protocol of the groups

Group S: Blank control group; Group C: IRI group; Group DEX: DEX preconditioning group; Group tBHQ: tBHQ control group; Group tBHQ+DEX: combined tBHQ and DEX preconditioning group; Group ATRA: ATRA control group; Group ATRA+DEX: combined ATRA and DEX preconditioning group.

Figure 2. Typical arrhythmia changes

The electrocardiogram of VPC (A), ventricular bigeminy (B), VT (C), and ventricular fibrillation (D).

Figure 3. IS, serum CK-MB, and cTnI concentrations in the seven groups

Typical stained myocardial slices (A), infarct size (B), and serum cTnI (C) and CK-MB (D) concentrations in the blank control (S), IRI (C), DEX preconditioning (DEX), tBHQ control (tBHQ), combined tBHQ and DEX preconditioning (tBHQ+DEX), ATRA control (ATRA), and combined ATRA and DEX preconditioning (ATRA+DEX) groups. All data were expressed as mean ± standard deviation , n=5.

Figure 4. Western blotting for myocardial expression of Nrf2, HO-1, and NQO1

Western blots for myocardial expression of Nrf2, HO-1, and NQO1 (A), quantitative analysis for myocardial expression of Nrf2 (B), HO-1 (C), and NQO1 (D) in the blank control (S), IRI (C), DEX preconditioning (DEX), tBHQ control (tBHQ), combined tBHQ and DEX preconditioning (tBHQ+DEX), ATRA control (ATRA), and combined ATRA and DEX preconditioning (ATRA+DEX) groups. All data were expressed as mean ± standard deviation , n=5.

Figure 5. Levels of oxidative stress markers

SOD (A), MDA (B), and GSH-Px (C) levels in the blank control (S), IRI (C), DEX preconditioning (DEX), tBHQ control (tBHQ), combined tBHQ and DEX preconditioning (tBHQ+DEX), ATRA control (ATRA), and combined ATRA and DEX preconditioning (ATRA+DEX) groups. All data were expressed as mean ± standard deviation , n=5.