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. 2016 Jun;37(6):1501-10.
doi: 10.3892/ijmm.2016.2571. Epub 2016 Apr 20.

Antioxidant Effects of Hydroxysafflor Yellow A and acetyl-11-keto-β-boswellic Acid in Combination on Isoproterenol-Induced Myocardial Injury in Rats

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Antioxidant Effects of Hydroxysafflor Yellow A and acetyl-11-keto-β-boswellic Acid in Combination on Isoproterenol-Induced Myocardial Injury in Rats

Minchun Chen et al. Int J Mol Med. .
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Abstract

Oxidative stress plays an important role in the initiation and development of myocardial injury (MI). The peroxisome proliferator-activated receptor gamma coactivator-1α (PGC‑1α)/nuclear factor erythroid 2-related factor 2 (Nrf2) pathway is considered to be a potential target for cardioprotection in MI. Acetyl-11-keto-β-boswellic acid (AKBA) is the major organic acid component extracted from Boswellia serrata Roxb. ex Colebr. Hydroxysafflor yellow A (HSYA) is the principal active constituent of Carthamus tinctorius L. In the present study, we aimed to investigate the cardioprotective effects of HSYA and AKBA in combination in vivo and in vitro, as well as the underlying mechanisms responsible for these effects. For this purpose, MI was produced in Sprague-Dawley rats by subcutaneous injection with isoproterenol. To model ischemic-like conditions in vitro, H9C2 cells were subjected to oxygen-glucose deprivation (OGD). The levels of creatine kinase-MB (CK‑MB), lactate dehydrogenase (LDH), malondialdehyde (MDA) as well as superoxide dismutase (SOD) activity were examined as well as apoptotic cell death. Mitochondrial reactive oxygen species (ROS) production and mitochondrial membrane potential (ΔΨm or MMP) were measured using MitoSOX Red and 5,5',6,6'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) dye. The expression of PGC-1α and Nrf2 was quantified by western blot analysis and immunohistochemistry. HSYA and AKBA prevented myocardial pathological changes, significantly reduced the blood levels of CK-MB and LDH, and decreased apoptotic cell death. They significantly increased the expression of PGC-1α and Nrf2, and the activity of the antioxidant enzyme SOD and also decreased the levels of MDA and ROS. Moreover, the reduction in MMP was partly prevented by HSYA and AKBA. Taken together, these findings elucidate the underlying mechanisms through which HSYA and AKBA protect against MI. Additionally, HSYA and AKBA appear to act synergistically in order to exert cardioprotective effects.

Figures

Figure 1
Figure 1
Chemical structure of (A) hydroxysafflor yellow A (HSYA; molecular weight, 612] and (B) acetyl-11-keto-β-boswellic acid (AKBA; molecular weight, 512).
Figure 2
Figure 2
Hydroxysafflor yellow A (HSYA) and acetyl-11-keto-β-boswellic acid (AKBA) prevent isoproterenol hydrochloride (ISO)-induced pathological changes in rat heart tissue (heart tissues were stained with H&E and visualized under a light microscope at ×400 magnification).
Figure 3
Figure 3
Hydroxysafflor yellow A (HSYA) and acetyl-11-keto-β-boswellic acid (AKBA) protect against myocardial injury (MI). The induction of MI by isoproterenol hydrochloride (ISO; 100 mg/kg) was performed after treating the rats with either HSYA (100 mg/kg) or AKBA (100 mg/kg) alone, or a combination of HSYA (50 mg/kg) and AKBA (50 mg/kg) for 14 days. We measured (A) serum creatine kinase-MB (CK-MB) levels and (B) serum lactate dehydrogenase (LDH) levels. (C and D) HSYA and/or AKBA protected H9C2 cells against oxygen-glucose deprivation (OGD)-induced cell damage. The H9C2 cells were subjected to OGD following treatment with either HSYA (10 µM) or AKBA (10 µM), or a combination of HSYA (5 µM) and AKBA (5 µM) for 24 h. We measured (C) CK-MB activity and (D) LDH activity in the culture supernatant. The results are presented as the means ± SD; *P<0.05 vs. ISO or OGD group; #P<0.05 vs. HSYA or AKBA group; **P<0.01 vs. Sham group.
Figure 4
Figure 4
Pre-treatment with hydroxysafflor yellow A (HSYA) and acetyl-11-keto-β-boswellic acid (AKBA) inhibits apoptosis. (A) The induction of myocardial injury by isoproterenol hydrochloride (ISO; 100 mg/kg) was performed after treating the rats with either HSYA (100 mg/kg) or AKBA (100 mg/kg) alone, or a combination of HSYA (50 mg/kg) and AKBA (50 mg/kg) for 14 days. The inhibitory effects of HSYA and AKBA on apoptosis in the rats exposed to ISO were evaluated using the TUNEL assay. (B) The quantitative results of TUNEL staining in rat hearts following different treatments are shown. (C) Effects of HSYA and AKBA on nuclear morphological changes in cardiomyocytes subjected to oxygen-glucose deprivation (OGD). The H9C2 cells were stained with Hoechst 33258 (magnification, ×400). Results are presented as the means ± SD; *P<0.05 vs. ISO group; #P<0.05 vs. HSYA or AKBA group.
Figure 5
Figure 5
Hydroxysafflor yellow A (HSYA) and acetyl-11-keto-β-boswellic acid (AKBA) attenuate oxidative stress. H9C2 cells were treated with HSYA (10 µM) and AKBA (10 µM) for 24 h prior to oxygen-glucose deprivation (OGD) for 4 h, followed by incubation under normal conditions. Data are presented as the means ± SD of five independent experiments. (A) Intracellular reactive oxygen species (ROS) level. (B) The cells were incubated with JC-1 (10 ng/ml) for 30 min, and mitochondrial membrane potential (ΔΨm or MMP) was analyzed by flow cytometry. The bar diagram shows the percentage of red fluorescence to green fluorescence. (C and D) The rats were treated with either HSYA (100 mg/kg) or AKBA (100 mg/kg) alone, or a combination of HSYA (50 mg/kg) and AKBA (50 mg/kg) for 14 days prior to the administration of isoproterenol hydrochloride (ISO;100 mg/kg) for 2 days, to induce myocardial injury. Determination of (C) malondialdehyde (MDA) levels and (D) superoxide dismutase (SOD) activity in the rat myocardium. Determination of (E) MDA levels and (F) SOD activity under OGD conditions. *P<0.05 vs. ISO or OGD group; #P<0.05 vs. HSYA or AKBA group.
Figure 6
Figure 6
Expression of peroxisome proliferator activated receptor gamma coactivator-1α (PGC-1α) and nuclear factor erythroid 2-related factor 2 (Nrf2). (A) PGC-1α and (B) Nrf2 expression was induced by hydroxysafflor yellow A (HSYA) and/or acetyl-11-keto-β-boswellic acid (AKBA) in the rats with isoproter-AKBA) in the rats with isoproterenol hydrochloride (ISO)-induced MI (magnification, ×400). Immunohistochemical analysis was performed on the tissue samples in order to determine Nrf2 and PGC-1α expression. (C) PGC-1α and Nrf2 expression in cultured H9C2 cells was evaluated by western blot analysis. The intensity of each band was quantified by densitometry. Data represent the means ± SD; *P<0.05 compared with oxygen-glucose deprivation (OGD) group; #P<0.05 vs. HSYA or AKBA group.

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