Britanin relieves ferroptosis-mediated myocardial ischaemia/reperfusion damage by upregulating GPX4 through activation of AMPK/GSK3β/Nrf2 signalling

Abstract

Context: Ferroptosis was described as an important contributor to the myocardial ischaemia/reperfusion (MIR) injury, and britanin (Bri) was reported to exert antitumor and anti-inflammatory activities.

Objective: Our study explores the effect and mechanism of Bri on MIR damage.

Materials and methods: The rat model of MIR was established by ligation of the left anterior descending coronary artery. Male Sprague-Dawley (SD) rats were divided into three groups: sham group (n = 6), MIR group (n = 6) and MIR + Bri group (n = 6; 50 mg/kg). Rats were intragastrically pre-treated with Bri or normal saline once daily for 3 days. To further verify the role and mechanism of Bri, H9C2 cells were subjected to hypoxia plus reoxygenation (H/R) to induce the in vitro model of MIR.

Results: Compared with MIR rats, Bri significantly decreased infarct area (22.50% vs. 38.67%), myocardial apoptosis (23.00% vs. 41.5%), creatine phosphokinase (0.57 U/mL vs. 0.76 U/mL), and lactate dehydrogenase levels (3.18 U/mL vs. 5.17 U/mL), concomitant with alleviation of ferroptosis. Mechanistically, Bri treatment induced the activation of the adenosine monophosphate activated protein kinase (AMPK)/glycogen synthase kinase 3β (GSK3β)/nuclear factor erythroid 2-related factor 2 (Nrf2) pathway in vivo. In addition, the AMPK/GSK3β/Nrf2 pathway participated in the regulation of glutathione peroxidase 4 (GPX4) expression, and silencing of Nrf2 attenuated the effect of Bri on H/R-induced cell injury.

Discussion and conclusions: Bri protected against ferroptosis-mediated MIR damage by upregulating GPX4 through activation of the AMPK/GSK3β/Nrf2 signalling, suggesting that Bri might become a novel therapeutic agent for MIR.

Keywords: Myocardial infarction; apoptosis; hypoxia-reoxygenation injury; iron; oxidative stress.

Figure 1.

Bri protected against MIR injury. SD rats were intragastrically administered with Bri, followed by undergoing MIR surgery. (A) Representative images and quantification of myocadiac infarction area in the sham, MIR, and MIR + Bri group. (B) Representative images and quantification of myocardial apoptosis in the sham, MIR, and MIR + Bri group. (C and D) Commercial kits were used to analyse the levels of serum CPK and LDH in the sham, MIR, and MIR + Bri group. **p < 0.01 and ***p < 0.001.

Figure 2.

Bri alleviated MIR-induced ferroptosis. SD rats were intragastrically administered with Bri, followed by undergoing MIR surgery. The levels of ROS (A), MDA (B), GSH (C), and iron (D) in the myocardium of rats were determined using commercial kits. (E and F) qRT-PCR and western blot analysis of GPX4 expression in the myocardium of rats. **p < 0.01 and ***p < 0.001.

Figure 3.

Bri inhibited ferroptosis-dependent H/R injury by facilitating GPX4 expression in vitro. H9C2 cells were transfected with si-GPX4 or si-NC and then treated with Bri. After 6 h of treatment, H9C2 cells were exposed to H/R condition. (A and B) qRT-PCR and western blot analysis of GPX4 expression in H9C2 cells transfected with si-GPX4 or si-NC. (C and D) GPX4 expression was detected in H9C2 cells with different treament. (E) The viability of H9C2 cells was determined using CCK-8 assay. (F) LDH release assay was conducted to determine the level of LDH in H9C2 cells. The levels of ROS (G), MDA (H), GSH (I), and iron (J) in H9C2 cells were determined using commercial kits. *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 4.

Bri exerted regulatory effects on the AMPK/GSK3β/Nrf2 pathway in MIR rats. SD rats were intragastrically administered with Bri, followed by undergoing MIR surgery. (A) Representative images and (B–D) quantification of western blot results of AMPK, p-AMPK, GSK3β, p-GSK3β, and Nuc-Nrf2 expression in the myocardium of MIR rats. *p < 0.05 and ***p < 0.001.

Figure 5.

The AMPK/GSK3β/Nrf2 pathway was involved in the regulation of GPX4 expression. H9C2 cells were transfected with si-AMPK, si-Nrf2, GSK3β-OE or matched controls. Western blot analysis of Nuc-Nrf2 (A) and GPX4 (B) expression in H9C2 cells. ***p < 0.001.

Figure 6.

Nrf2 attenuated the effect of Bri on H/R-induced cell injury in H9C2 cells. H9C2 cells were transfected with si-Nrf2 or si-NC. At 18 h after transfection, H9C2 cells were treated with Bri, followed by induction of H/R injury. (A) The viability of H9C2 cells was determined using CCK-8 assay. (B) LDH release assay was conducted to determine the level of LDH in H9C2 cells. The levels of ROS (C), MDA (D), GSH (E), and iron (F) in H9C2 cells were determined using commercial kits. *p < 0.05, **p < 0.01, and ***p < 0.001.