Naringin attenuates rat myocardial ischemia/reperfusion injury via PI3K/Akt pathway-mediated inhibition of apoptosis, oxidative stress and autophagy

Abstract

Naringin (NRG) has been reported to exert cardioprotective effects against multiple cardiovascular diseases, including lipopolysaccharide-induced and hyperglycemia-induced myocardial injury. However, the role of NRG in myocardial ischemia/reperfusion (I/R) injury remains unclear. In the present study, the PI3K/Akt pathway was investigated to evaluate the possible mechanisms underlying the roles of NRG in myocardial ischemia/reperfusion (I/R) injury. The levels of cardiac enzymes were measured by ELISA to evaluate the optimal dosage of NRG that could protect against myocardial I/R injury. Rats were administered 100 mg/kg of NRG and activities of myocardial enzymes, the level of cardiac apoptosis and inflammation, oxidant response, autophagy indicators and echocardiography were evaluated. The level of corresponding proteins was measured using western blotting. The results indicated that NRG elicited the best cardioprotective effects at a dose of 100 mg/kg by significantly reducing the levels of myocardial enzymes, apoptosis, inflammation, oxidative response and infarct size. Furthermore, NRG alleviated contractile dysfunction by increasing the left ventricular ejection fraction and fractional shortening. In addition, NRG markedly promoted the phosphorylation of Akt, while decreasing the level of autophagy indicator beclin-1 and the microtubule-associated protein 1B-light chain 3 (LC3B) II/ LC3BI ratio. However, PI3K/Akt inhibitor (LY294002) partially reduced the NRG induced phosphorylation of Akt and the reduction in beclin-1, along with the LC3BII/LC3BI ratio. The results of the present study demonstrated that NRG could attenuate myocardial I/R injury.

Keywords: PI3K/Akt; apoptosis; autophagy; myocardial ischemia/reperfusion injury; naringin; oxidative stress.

Figure 1

NRG pretreatment attenuates cardiac I/R injury. Serum levels of (A) CK-MB, (B) cTnI and (C) LDH (n=5) after I/R and following pretreatment with different NRG doses. The expression levels of (D) CK-MB, (E) cTnI and (F) LDH (n=5) after I/R with use of a PI3K/Akt inhibitor. (G) Representative images of heart tissues following hematoxylin and eosin staining. (H) Damage score (n=5). NRG, naringin; I/R, ischemia reperfusion; CK-MB, creatine kinase myocardial band; cTn1, cardiac troponin 1; LDH, lactate dehydrogenase; LY, PI3K/Akt inhibitor LY294002; NRG25, 25 mg/kg naringin; NRG50, 50 mg/kg naringin; NRG100, 100 mg/kg naringin. ^aP<0.05 vs. sham group; ^bP<0.05 vs. I/R group; ^cP<0.05 vs. I/R group; ^dP<0.05 vs. NRG50 + I/R group.

Figure 2

NRG pretreatment alleviates I/R-accelerated inflammatory reactions, oxidative stress and infarct size. The expression levels of (A) TNF-α, (B) IL-1β and (C) IL-6 (n=5); (D) The level of MDA (n=5). (E) The level of SOD (n=5). (F) Representative images of 2,3,5-triphenyltetrazolium chloride stain. (G) Quantitative analysis of infarct size (n=3). NRG, naringin; I/R, ischemia reperfusion; TNF-α, tumor necrosis factor-α; IL, interleukin; MDA, malondialdehyde; SOD, superoxide dismutase; NRG100, 100 mg/kg naringin; LY, PI3K/Akt inhibitor LY294002. ^aP<0.05 vs. sham group; ^bP<0.05 vs. I/R group; ^cP<0.05 vs. NRG100 + I/R group.

Figure 3

NRG pretreatment suppresses I/R-induced myocardial apoptosis. (A) Representative images of TUNEL stain; (B) Apoptosis index (n=5). NRG, naringin; I/R, ischemia reperfusion; LY, PI3K/Akt inhibitor LY294002; NRG100, 100 mg/kg naringin. ^aP<0.05 vs. sham group; ^bP<0.05 vs. I/R group; ^cP<0.05 vs. NRG100 + I/R group.

Figure 4

NRG pretreatment represses myocardial autophagy through PI3K/Akt signaling cascades. (A) Representative images of western blotting. Quantitative analysis of the (B) Bax, and (C) Bcl levels and the (D) cleaved caspase: caspase 3 and (E) p-Akt: Akt ratio and the (F) beclin-1 and (G) LC3BII: LC3BI ratio (n=3). NRG, naringin; I/R, ischemia reperfusion; LY, PI3K/Akt inhibitor LY294002; NRG100, 100 mg/kg naringin; p-Akt, phosphorylated Akt; LC3B, microtubule-associated protein 1B-light chain 3. ^aP<0.05 vs. sham group; ^bP<0.05 vs. I/R group; ^cP<0.05 vs. NRG100 + I/R group.