The Citrus Flavanone Naringenin Protects Myocardial Cells against Age-Associated Damage

Abstract

In recent years, the health-promoting effects of the citrus flavanone naringenin have been examined. The results have provided evidence for the modulation of some key mechanisms involved in cellular damage by this compound. In particular, naringenin has been revealed to have protective properties such as an antioxidant effect in cardiometabolic disorders. Very recently, beneficial effects of naringenin have been demonstrated in old rats. Because aging has been demonstrated to be directly related to the occurrence of cardiac disorders, in the present study, the ability of naringenin to prevent cardiac cell senescence was investigated. For this purpose, a cellular model of senescent myocardial cells was set up and evaluated using colorimetric, fluorimetric, and immunometric techniques. Relevant cellular senescence markers, such as X-gal staining, cell cycle regulator levels, and the percentage of cell cycle-arrested cells, were found to be reduced in the presence of naringenin. In addition, cardiac markers of aging-induced damage, including radical oxidative species levels, mitochondrial metabolic activity, mitochondrial calcium buffer capacity, and estrogenic signaling functions, were also modulated by the compound. These results suggested that naringenin has antiaging effects on myocardial cells.

Figure 1

(a) The structure of Nar. (b) Schematic depiction of the cell culture treatment protocol.

Figure 2

Nar effects on H9c2 cell senescence hallmarks. (a) Senescence-associated b-galactosidase staining. The data are shown as the percentages of β-galactosidase-positive cells. Each bar represents the mean ± SEM of three replicates from three independent experiments. ^∗∗∗p < 0.01 versus the control group; (b) the data are shown as percentage of β-galactosidase-positive cells. Each bar represents the mean ± SEM of three replicates from three independent experiments. ^∗∗p < 0.01 versus the H₂O₂-treated cells. (c) Representative phase contrast photomicrographs of treated cells; (A) H₂O₂-treated cells; (B) 40 μM Nar-cotreated cells. (d, e) Cell cycle arrest machinery. (d) p16 and p21 mRNA fold induction. Each bar represents the mean ± SEM of three replicates from three independent experiments. ^∗∗∗p < 0.001 versus the control group; ^###p < 0.001 versus the H₂O₂-treated group. (e) Cell cycle phases. Each bar represents the mean ± SEM of three replicates from three independent experiments. °°°p < 0.001 versus the control group; ^∗∗p < 0.01 versus the H₂O₂-treated group.

Figure 3

ROS production and DNA damage. (a) ROS generation in H9c2 cells. Each bar represents the mean ± SEM of three replicates from three independent experiments. ^∗∗∗p < 0.001 versus the control cells, and ^###p < 0.001 versus the H₂O₂-treated cells. (b) Representative photomicrographs of cells assessed using the Comet assay. (c) DNA damage was calculated as classes of DNA damage, as described in Section 2.8. ^∗∗∗p < 0.001 and ^∗∗p < 0.01 versus control; ^###p < 0.001, ^##p < 0.01, and ^#p < 0.05 versus the H₂O₂-treated cells.

Figure 4

Effects of H₂O₂ and Nar on the mitochondrial oxidative metabolism activity and CRC. (a) H9c2 cells were treated with H₂O₂ and DMSO or Nar, and the mitochondrial oxidative metabolism activity was examined. (b–e) The cells were suspended in CRC medium and permeabilized with digitonin. To these cells, 0.25 M Calcium Green-5N and 5 mM succinate were added. This addition was followed by a series of Ca²⁺ pulses (10 nmoles) at 3-minute intervals until onset of the permeability transition (plateau). The Relative Fluorescence Unit (RFU) was recorded by spectrophotometer technique. ^∗p < 0.05 versus DMSO-treated cells. (b) Representative traces of the H9c2 cell treatments with H₂O₂ and vehicle are shown in dashed black and gray, respectively. (c) The results from the H₂O₂-treated cells are shown as the CRC normalized to the CRC of control (CRC₀). The data shown are the means ± SEM of three independent experiments. ^∗p < 0.05 versus vehicle. (d) Representative traces of senescent H9c2 cells cotreated with DMSO (dashed black), Nar (dark gray), and Nar + Pax (light gray). (e) The results from senescent cells treated with Nar and Nar + Pax are shown as the CRC normalized to the CRC of control (CRC₀). The data shown are the means ± SEM of three independent experiments. ^∗∗p < 0.01 versus DMSO-treated cells.

Figure 5

Effect of Nar on the levels of released estradiol and estrogen-regulated gene mRNA expression. (a) Each bar represents the mean ± SEM of three replicates from three independent experiments. The measured estradiol levels were within the reference range [24]. Specific calibration curves and appropriate positive/negative controls were carried out during the assay. ^∗p < 0.05 and ^∗∗∗p < 0.001 versus the control; ^###p < 0.001 versus H₂O₂-treated cells. (b) ER β and (c) VDR mRNA expression as indicators of estrogenic activity in the senescent H9c2 cells. Each bar represents the mean ± SEM of three replicates from three independent experiments. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001 versus the control; ^#p < 0.05 and ^##p < 0.01 versus H₂O₂-treated cells.