Apigenin Alleviates Myocardial Reperfusion Injury in Rats by Downregulating miR-15b

Abstract

BACKGROUND We investigated whether apigenin could mitigate myocardial reperfusion injury in rats, and a possible mechanism was proposed. MATERIAL AND METHODS The I-R injury model was established in rats along with a sham group as control, and the expressions of microRNA-15b (miR-15b), JAK2, and p-JAK2 in the myocardia of the 2 groups were detected. Apoptosis and reactive oxygen species (ROS) were also detected. Rats in the I-R injury model were divided into 3 groups in vivo: the 1I-R group, the 2I-R+solvent group, and the 3I-R+apigenin group. Expression of miR-15b, JAK2, p-JAK2, STAT3, and p-STAT3 in the myocardia of the 3 groups were detected. ROS content, apoptosis, MDA content, SOD, and CAT activities were detected. Rat myocardial H9C2 cells were cultured in vitro and divided into 5 treatment groups in vitro; expressions of miR-15b, JAK2, p-JAK2, STAT3, and p-STAT3 in H9C2 cells were detected, and the apoptosis and ROS content were detected by flow cytometry. RESULTS We found that the increased miR-15b expression during myocardial I-R injury in rats downregulated the expression of JAK2 and activity of the JAK2-STAT3 pathway, promoted myocardial apoptosis and ROS production, and aggravated myocardial I-R injury. Apigenin treatment can downregulate miR-15b expression, increase the expression of JAK2 and the activity of JAK2-STAT3 pathway, reduce myocardial apoptosis and ROS production, and alleviate myocardial I-R injury. CONCLUSIONS Api treatment downregulated the expression of miR-15b and upregulated the expression of JAK2 and the activity of the JAK2-STAT3 pathway, thereby alleviating myocardial I-R injury, cardiomyocyte apoptosis, and ROS production in vitro.

Figure 1

Relationship between miR-15b and JAK2. (A) A schematic diagram of the interaction site between miR-15b and JAK2 mRNA’s 3′-UTR. (B) Double-luciferase reporter gene assay. * P<0.05 compared with miR-NC.

Figure 2

The oxidative stress and apoptosis of myocardium in I-R rats increased significantly. (A) Detection of MDA content in rat myocardium. (B) Caspase-3 activity in rat myocardium was detected by spectrophotometry. (C) SOD activity in rat myocardium was detected by kits. (D) CAT activity in rat myocardium was detected by kits. (E) Flow cytometry was used to detect ROS content in rat myocardium. * P<0.05 compared with the sham group; ^# P<0.05 at 24 h compared with 12 h.

Figure 3

The expressions of miR-15b and JAK2 in the myocardium of I-R rats were increased significantly. (A) qRT-PCR detection of the expression of miR-15b in rat myocardium. (B) qRT-PCR detection of the expression of JAK2 mRNA in rat myocardium. (C) Western blot detection of protein expressions in rat myocardium. * P<0.05 versus sham; ^# P<0.05 at 24 h versus 12 h.

Figure 4

Api treatment reduced oxidative stress and apoptosis in the myocardium of I-R rats. (A) MDA content in rat myocardium. (B) Caspase-3 activity in rat myocardium. (C) SOD activity in rat myocardium. (D) CAT enzyme activity in rat myocardium. (E) Flow cytometry detection of ROS content in rat myocardium. * P<0.05 versus I-R; ^# P<0.05 versus I-R+solvent.

Figure 5

Api reduced the expressions of miR-15b in the myocardium of I-R rats and increased the expression of JAK2. (A) qRT-PCR detection of miR-15b expression in rat myocardium. (B) qRT-PCR detection of the expression of JAK2 mRNA in rat myocardium. (C) Western blot detection of protein expressions in rat myocardium. * P<0.05 versus I-R; ^# P<0.05 versus I-R+Solvent.

Figure 6

Api reduced the expression of miR-15b in H9C2 cells and increased the expression of JAK2. (A) qRT-PCR was used to detect the expression of miR-15b in H9C2 cells. (B) qRT-PCR was used to detect the expression of JAK2 mRNA in H9C2 cells. (C) Western blot detection of protein expressions in H9C2 cells. * P<0.05 versus control; ^# P<0.05 versus I-R+solvent+miR-NC.

Figure 7

Api significantly decreased H9C2 cell apoptosis and ROS production under I-R condition. (A) ROS content in H9C2 cells of each treatment group. (B) Apoptosis rate of H9C2 cells in each treatment group. (C) Flow cytometry was used to detect ROS content in H9C2 cells. (D) Flow cytometry indicated apoptosis of the H9C2 cells. * P<0.05 versus control; ^# P<0.05 versus I-R+solvent+miR-NC.