Naringenin alleviates myocardial ischemia reperfusion injury by enhancing the myocardial miR-126-PI3K/AKT axis in streptozotocin-induced diabetic rats

Abstract

Ischemic heart disease (IHD) is a leading cause of death in patients with type 1 diabetes. The key to treating IHD is to restore blood supply to the ischemic myocardium, which inevitably causes myocardial ischemia reperfusion (MI/R) injury. Although naringenin (Nar) prevents MI/R injury, the role of Nar in diabetic MI/R (D-MI/R) injury remains to be elucidated. The PI3K/AKT signaling pathway and microRNA (miR)-126 have previously been shown to serve anti-MI/R injury roles. The present study aimed to investigate the protection of Nar against D-MI/R injury and the role of the miR-126-PI3K/AKT axis. Diabetic rats were treated distilled water or Nar (25 or 50 mg/kg, orally) for 30 days and then exposed to MI/R. The present results revealed that Nar alleviated MI/R injury in streptozotocin (STZ)-induced diabetic rats, as shown below: the reduction myocardial enzymes levels was measured using spectrophotometry, the increase of cardiac viability was detected by MTT assay, the inhibition of myocardial oxidative stress was measured using spectrophotometry and the enhancement of cardiac function were recorded using a hemodynamic monitoring system. Furthermore, Nar upregulated the myocardial miR-126-PI3K/AKT axis in D-MI/R rats. These results indicated that Nar alleviated MI/R injury through upregulating the myocardial miR-126-PI3K/AKT axis in STZ-induced diabetic rats. The current findings revealed that Nar, as an effective agent against D-MI/R injury, may provide an effective approach in the management of diabetic IHD.

Keywords: PI3K/AKT signaling; ischemic heart disease; microRNA-126; myocardial ischemia reperfusion injury; naringenin.

Figure 1

Effect of Nar on body weight and blood glucose levels in rats. (A) Blood glucose levels at days 0, 3 and 30 were determined with a blood glucose meter. (B) Body weight in the different groups was measured once a week. Data are presented as the mean ± SEM (number of repeated measurements: n=8-10/group). ^***P<0.001 compared with the control-sham group. Nar, naringenin; STZ, streptozotocin; MI/R, myocardial ischemia reperfusion.

Figure 2

Effect of Nar on myocardial enzyme levels and cardiac viability in diabetic MI/R rats. Levels of (A) LDH and (B) CK-MB in the coronary effluent were detected using spectrophotometry by commercial assay kits. (C) Cardiac formazan content was measured using an MTT assay. Data are presented as the mean ± SEM (number of repeated measurements: n=3/group). ^**P<0.01 and ^***P<0.001 compared with the STZ-sham group; ^#P<0.05 and ^##P<0.01 compared with the STZ-M/IR group. LDH, lactate dehydrogenase; CK-MB, creatine kinase myocardial band; STZ, streptozotocin; MI/R, myocardial ischemia reperfusion; Nar, naringenin.

Figure 3

Effect of Nar on myocardial oxidative stress in diabetic MI/R rats. Activities of (A) SOD and (B) GSH-Px, and the levels of (C) MDA, (D) 8-OHdG as well as (E) H₂O₂ formation in the myocardium were determined by spectrophotometry using commercially available kits. Data are presented as the mean ± SEM (number of repeated measurements: n=3/group). ^**P<0.01 and ^***P<0.001 compared with the STZ-sham group; ^#P<0.05, ^##P<0.01 and ^###P<0.001 compared with the STZ-M/IR group. GSH-Px, glutathione peroxidase; SOD, superoxide dismutase; MDA, malondialdehyde; 8-OHdG, 8-hydroxy-2 deoxyguanosine; STZ, streptozotocin; MI/R, myocardial ischemia reperfusion; Nar, naringenin.

Figure 4

Effect of Nar on cardiac function in diabetic MI/R rats. (A) LVSP, (B) LVEDP, (C) +dP/dt_max and (D) -dP/dt_max were recorded using a hemodynamic monitoring system. Data are presented as the mean ± SEM (number of repeated measurements: n=6-8/group). ^*P<0.05, ^**P<0.01 and ^***P<0.001 compared with the STZ-sham group; ^#P<0.05 and ^##P<0.01 compared with the STZ-M/IR group. STZ, streptozotocin; MI/R, myocardial ischemia reperfusion; Nar, naringenin; LVSP, left ventricular systolic pressure; LVEDP, left ventricular end-diastolic pressure; +dP/dt_max, peak rate of left ventricular maximal systolic/diastolic velocity rise; -dP/dt_max, peak rate of left ventricular maximal systolic/diastolic velocity fall.

Figure 5

Effect of Nar on PI3K/AKT signaling in diabetic MI/R rats. The protein levels of p-PI3K, PI3K, p-AKT, AKT and GAPDH in myocardium were detected by western blotting. Data are presented the mean ± SEM (number of repeated measurements: n=3/group). ^**P<0.01 and ^***P<0.001 compared with the STZ-sham group; ^#P<0.05, ^##P<0.01 and ^###P<0.001 compared with the STZ-M/IR group. STZ, streptozotocin; MI/R, myocardial ischemia reperfusion; Nar, naringenin.

Figure 6

Effect of Nar on miR-126 expression in diabetic MI/R rats. Relative expression levels of myocardial miR-126 were determined using reverse transcription-quantitative PCR. Data are presented as the mean ± SEM (number of repeated measurements: n=3/group). ^**P<0.01 compared with the STZ-sham group; ^##P<0.01 compared with the STZ-M/IR group. miR, microRNA; STZ, streptozotocin; MI/R, myocardial ischemia reperfusion; Nar, naringenin.