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.
Acute myocardial infarction (AMI) is a life-threatening disease characterized by high morbidity and mortality (
Naringin (NRG), a citrus flavonoid, was initially extracted from pomelo peel (
The PI3K/Akt signaling pathway is a conserved family of signal transduction pathway that participates in the modulation of multiple biological processes, including cell proliferation, oxidative response and cardiac apoptosis (
In the present study whether NRG could alleviate myocardial I/R injury
All procedures involving animals were conducted according to the Guide for the Care and Use of Laboratory Animals (NIH Publication no. 85-23, revised 1996) (
An I/R model was established as previously described (
Rats were first randomly assigned to the following groups to determine the optimal dose of NRG administration: i) Sham group; ii) I/R group; iii) NRG (25 mg/kg/day) + I/R group (NRG25 + I/R); iv) NRG (50 mg/kg/day) + I/R group (NRG50 + I/R); and v) NRG (100 mg/kg/day) + I/R group (NRG100 + I/R). The 100 mg/kg dose of NRG was proven to exert the best cardioprotective effects and subsequently the rats were randomly assigned into the following groups to evaluate the functions of NRG in myocardial I/R injury: i) Sham group; ii) I/R group; iii) NRG100 + I/R group; and iv) NRG100 + I/R + LY294002 group (NRG100 + I/R + LY).
After 180 min of reperfusion, blood specimens were obtained and centrifuged 1,000 x g for 15 min at a room temperature in preparation for the measurement of creatine kinase (CK), lactate dehydrogenase (LDH) and cardiac troponin I (cTnI). Creatine kinase isoenzyme Assay kit (cat. no. E006; Nanjing Jiancheng Bioengineering Institute), lactate dehydrogenase assay kit (cat. no. A020-1; Nanjing Jiancheng Bioengineering Institute) and Troponin Assay kit (no. E019-1; Nanjing Jiancheng Bioengineering Institute) were used for the analyses according to the manufacturer's instructions.
2,3,5-triphenyltetrazolium chloride (TTC; Sigma-Aldrich; Merck KGaA) staining was applied to evaluate the infarcted size as previously described (
Blood specimens were obtained for the measurement of malondialdehyde (MDA) and superoxide dismutase (SOD) using Malondialdehyde assay kit (cat. no. A003-1; Nanjing Jiancheng Bioengineering Institute) and Superoxide Dismutase assay kit (cat. no. A001-3; Nanjing Jiancheng Bioengineering Institute) according to the manufacturer's instructions.
The degree of myocardial apoptosis was assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining using the In Situ Cell Death Detection kit (cat. no. 11684817910; Roche Diagnostics GmBH) in accordance with the manufacturer's instructions. Cardiomyocytes with marked nuclear labeling were considered TUNEL-positive using a fluorescence microscope. A total of 3 fields (magnification, x200) were randomly selected per heart to determine apoptosis index (AI) using image-pro 5.0 (Media Cybernetics, Inc.). The formula for calculating AI was as follows, AI=number of apoptotic cells/total number of cells counted.
The levels of interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) released from myocardium were evaluated using Interleukin-1β Assay kit (cat. no. H002; Nanjing Jiancheng Bioengineering Institute), Interleukin-6 Assay kit (cat. no. H007; Nanjing Jiancheng Bioengineering Institute) and Tumor Necrosis Factor-α Assay kit (cat. no. H052; Nanjing Jiancheng Bioengineering Institute) in accordance with the manufacturer's instructions.
Heart samples were fixed in paraffin and cut into transverse sections (5-µm thick) before staining with hematoxylin and eosin (H&E) (
Cardiac performance was analyzed by non-invasive echocardiography after a 180 min period of reperfusion using an ultrasound Doppler imaging system [Vinno Technology (Suzhou) Co., Ltd.]. The heart rate (HR), left ventricular internal dimension systole (LVIDs), left ventricular internal dimension diastole (LVIDd), ejection fraction (EF) and fractional shortening (FS) were obtained as previously described (
Myocardial tissue was immediately harvested and stored in liquid nitrogen 180 min after reperfusion. Western blotting was carried out using a previously described method (
Secondary antibody was horseradish peroxidase (HRP) goat anti-Rabbit (1:10,000; cat. no. AS1107; Wuhan Aspen Biotechnology Co. Ltd.)
Continuous variables are presented as the mean ± standard deviation (SD) and were analyzed using SPSS 18.0 (SPSS, Inc.). One-way analysis of variance (ANOVA) followed by Tukey's post hoc test was used for multiple comparisons among groups. P<0.05 was defined as statistically significant.
The level of myocardial enzymes can reflect the degree of I/R-induced myocardial damage (
The results of the present study suggested that I/R triggered an inflammatory response by increasing the release of inflammatory factors TNF-α, IL-1β and IL-6 (
A significant reduction in SOD activity and MDA concentration was observed in the NRG + I/R group in comparison with I/R (
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The present results showed that the AI (
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Compared with the sham group, I/R significantly increased the expression of Beclin-1 and the LC3BII/LC3BI ratio (
The results of the present study demonstrated that NRG pretreatment could alleviate I/R-induced myocardial injury and cardiac dysfunction by suppressing apoptosis, the inflammatory response and oxidative stress. I/R-promoted myocardial autophagy was also inhibited by NRG pretreatment. Furthermore, the cardioprotective effects of NRG may be closely related to the activation of the PI3K/Akt signaling cascades.
NRG is a pharmacologically active constituent of tomentose pummelo peel, chemically known as 4,5,7-trihydroxy-flavonone-7-rhamnogglucoside (
Myocardial I/R injury occurs with the restoration of blood flow to the occluded coronary artery (
Myocardial I/R injury is also characterized by an excessive level of inflammation response (
Autophagy is a dynamic process in which cellular waste is encapsulated into double membrane vesicles and then transported to lysosomes for degradation (
There are a number of limitations to the present study. Specific PI3K/Akt inhibitors, such as Dactolisib or IC87114, might better have illustrated the cardioprotective role of NRG against I/R injury and an LY treatment alone group could also have aided the interpretation of the results.
In summary, the results of the present study indicated that NRG alleviated myocardial I/R injury via inhibition of cardiac apoptosis, inflammatory reactions and oxidative stress. In addition, NRG pretreatment also attenuated I/R-induced autophagy. The present data also suggested that such cardioprotective effects of NRG could be ascribed to the activation of the PI3K/Akt pathway. Furthermore, short-term NRG pretreatment may be a novel therapeutic strategy for the prevention of I/R injury in AMI patients.
Not applicable.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
FL and ZZ were responsible for conceiving the study, performing the analysis and experiments and drafting the manuscript. JQ analyzed the data. CC and WY also performed the experiments. NW was involved in drafting the manuscript performing and designing the experiments as well as revising it for important intellectual content. FL and NW confirm the authenticity of all the raw data. All authors have read and approved the final manuscript.
Animal experiments were approved by the Institutional Animal Care and Use Committee of Hubei University of Medicine.
Not applicable.
The authors declare that they have no competing interests.
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.
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.
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.
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.
Effects of NRG on cardiac performance.
Group | HR (beats/min) | LVIDs (mm) | LVIDd (mm) | EF (%) | FS (%) |
---|---|---|---|---|---|
Sham | 442.32±16.32 | 2.41±0.31 | 4.98±0.18 | 84.62±3.21 | 59.48±2.69 |
I/R | 429.85±15.75 | 3.24±0.36 |
5.02±0.21 | 55.36±4.23 |
32.25±1.38 |
NRG100 + I/R | 432.48±17.63 | 2.95±0.25 |
5.07±0.23 | 66.31±2.41 |
37.62±1.57 |
NRG100 + I/R +LY | 425.17±19.32 | 3.19±0.32 |
5.04±0.20 | 53.36±4.06 |
31.24±1.45 |
HR, heart rate; LVIDs, left ventricular internal dimension systole; LVIDd, left ventricular internal dimension diastole; EF, ejection fraction; FS, fractional shortening.
aP<0.05 vs. sham group;
bP<0.05 vs. I/R group;
cP<0.05 vs. NRG+I/R group. Data are presentedas the mean ± standard deviation.