Preventive Effect of YGDEY from Tilapia Fish Skin Gelatin Hydrolysates against Alcohol-Induced Damage in HepG2 Cells through ROS-Mediated Signaling Pathways

Abstract

According to a previous study, YGDEY from tilapia fish skin gelatin hydrolysates has strong free radical scavenging activity. In the present study, the protective effect of YGDEY against oxidative stress induced by ethanol in HepG2 cells was investigated. First, cells were incubated with YGDEY (10, 20, 50, and 100 μM) to assess cytotoxicity, and there was no significant change in cell viability. Next, it was established that YGDEY decreased the production of reactive oxygen species (ROS). Western blot results indicated that YGDEY increased the levels of superoxide dismutase (SOD) and glutathione (GSH) and decreased the expression of gamma-glutamyltransferase (GGT) in HepG2 cells. It was then revealed that YGDEY markedly reduced the expressions of bax and cleaved-caspase-3 (c-caspase-3); inhibited phosphorylation of Akt, IκB-α, p65, and p38; and increased the level of bcl-2. Moreover, the comet assay showed that YGDEY effectively decreased the amount of ethanol-induced DNA damage. Thus, YGDEY protected HepG2 cells from alcohol-induced injury by inhibiting oxidative stress, and this may be associated with the Akt/nuclear factor-κB (NF-κB)/mitogen-activated protein kinase (MAPK) signal transduction pathways. These results demonstrate that YGDEY from tilapia fish skin gelatin hydrolysates protects HepG2 cells from oxidative stress, making it a potential functional food ingredient.

Keywords: Alcohol metabolism; DNA damage; HepG2 cells; Oxidative stress; YGDEY.

Figure 1

(A) The cytotoxic effects of YGDEY on HepG2 cells. Cells were co-cultured with YGDEY (10, 20, 50, and 100 μM) for 24 h, and cell viability was evaluated by MTT assay. Data are shown as means ± SD (n = 3). (B) Cell viability of ethanol-induced HepG2 cells. Cells were treated with ethanol of different concentrations (0, 0.25, 0.5, 0.75, 1, 1.5, 1.75, and 2 M) for 24 h. Cell viability was evaluated by MTT assay. Data are shown as means ± SD (n = 3). *** compared with the no-alcohol blank group, p < 0.001. (C) Protective effects of YGDEY in HepG2 cells. Cells were pretreated with YGDEY for 24 h prior to treatment with 0.75 M ethanol for 2 h. After the treatment, cell viability was evaluated by MTT assay. Data are shown as means ± SD (n = 3). ** compared with the control group, p < 0.01. *** compared with the control group, p < 0.001.

Figure 2

Effect of YGDEY on the intracellular reactive oxygen species (ROS) level. (A) HepG2 cells were pretreated with YGDEY for 24 h before treatment with 0.75 M ethanol for 2 h. Then, the cells were exposed to DCFH-DA for 20 min. DCF fluorescence of the treated cells was measured by using an inverted fluorescence microscope. (a) HepG2 cells without treatment (the blank group); (b) cells exposed to 0.75 M ethanol (the control group); (c, d, e, and f) cells pretreated with YGDEY (10, 20, 50, and 100 μM, respectively) prior to treatment with 0.75 M ethanol. (B) The relative DCF fluorescence intensity. Data are shown as means ± SD (n = 3). ** compared with the control group, p < 0.01. *** compared with the control group, p < 0.001.

Figure 3

(A) Western blot analysis to examine the effect of YGDEY on SOD, GSH, and GGT in ethanol-induced HepG2 cells. YGDEY (10, 20, and 50 μM) was added to cells for 24 h before treatment with 0.75 M ethanol for 2 h. β-Actin was used as an internal control. (B) Protein expression (relative to β-Actin) was evaluated. Data are shown as means ± SD (n = 3). ** compared with the control group, p < 0.01. *** compared with the control group, p < 0.001.

Figure 4

(A) The expressions of bax, bcl-2, procaspase-3, and caspase-3 p20 in HepG2 cells. Cells were pretreated with YGDEY (10, 20, and 50 μM) for 24 h before induction by 0.75 M ethanol for 2 h. β-Actin was used as an internal control. (B) and (C) The ratios of bax/bcl-2 and cleaved-caspase-3/procaspase-3 were calculated. Data are shown as means ± SD (n = 3). ** compared with the control group, p < 0.01. *** compared with the control group, p < 0.001.

Figure 5

(A) Expression changes of phosphor-Akt and phosphor-NF-κB in HepG2 cells. Cells were exposed to YGDEY (10, 20, and 50 μM) for 24 h before treatment with 0.75 M ethanol for 2 h. β-Actin was used as an internal control. (B) Relative protein expression was normalized. Data are shown as means ± SD (n = 3). ** compared with the control group, p < 0.01. *** compared with the control group, p < 0.001.

Figure 6

Effect of YGDEY on the cellular localization of p65 in HepG2 cells. Cells were treated with YGDEY (20 and 50 μM) for 24 h prior to ethanol treatment for 2 h. Images were obtained using an inverted fluorescence microscope.

Figure 7

(A) Effect of YGDEY on the MAPK signaling pathway in HepG2 cells. Cells were pretreated with YGDEY (10, 20, and 50 μM) for 24 h before induction by 0.75 M ethanol for 2 h. β-Actin was used as an internal control. (B) Relative protein expression was normalized. Data are shown as means ± SD (n = 3). *** compared with the control group, p < 0.001.

Figure 8

(A) Comet assay of (a) the blank group; (b) the control group; (c) 10, (d) 20, (e) 50, and (f) 100 μM of YGDEY for 24 h prior to ethanol treatment for 2 h followed by staining with DAPI. Images were obtained using an inverted fluorescence microscope with blue fluorescence (magnification: 10×). (B) Tail lengths of the comets were analyzed. Data are shown as means ± SD (n = 3). *** compared with the control group, p < 0.001.

Figure 9

(A) The optimal docking structure of YGDEY and bcl-2; (B) 2D model of the interaction between YGDEY with the active site of bcl-2. YGDEY is represented by lines, and hydrogen bonds are shown with green dashed lines.