Hydrolyzed fish proteins reduced activation of caspase-3 in H2O2 induced oxidative stressed liver cells isolated from Atlantic salmon (Salmo salar)

Abstract

Hydrolyzed fish proteins (H-pro) contains high concentrations of free amino acids and low molecular peptides that potentially benefit health. The following study aimed to test whether the water soluble phase of H-pro could reduce apoptosis and inflammation in primary liver cells isolated from Atlantic salmon following H2O2 provoked oxidative stress. Cells were grown as monocultures or co-cultured with head kidney cells to assess possible cross talk in inflammation and metabolism during treatments. Cells were grown in media with or without H-pro for 2 days before being stressed with 200 µM H2O2 then harvested 24 h post exposure. Both treatments were compared to the respective treatments without H2O2 supplementation. Oxidative stressed cells had increased activation of caspase-3, but supplementation with H-pro in the media prior to the oxidative stress reduced caspase-3 activation. In conclusion, free amino acids and low molecular weight peptides from H-pro attenuated oxidative stress, and made cells able to withstand apoptosis after H2O2 provoked oxidative stress.

Keywords: Atlantic salmon; Caspase-3; Co-cultures; Head kidney cells; Hydrolyzed proteins; Oxidative stress; Primary liver cells; Viability.

Fig. 1

The relative normalized gene expression in liver cells grown as mono and co-cultures. The normalized gene expression of mono-cultured cells grown in the cL-15 media without H₂O₂ supplementation (i.e. the positive control) was set equal to 100 and the other treatments calculated relative to these. The white bars represent the mono cultured cells, while the filled bars represent the co-cultured cells. Differences were assessed by two tailed Mann–Whitney U test and p < 0.05 was accepted as statistically different. Values are mean of 5 fish ± SE. The overall outcome in treatments * conditions (asterisk). Also the within each culture condition treatments effects (letters) are indicated

Fig. 2

From Western blots it was clear that activated cleaved caspase-3 was more profound in the co-cultured liver cells than in the corresponding mono-cultured liver cells (a). Lanes 1–4 are mono-cultured liver cells while lanes 5–8 is liver cells co-cultured with head kidney cells. Lanes 1 and 5 are controls, lanes 2 and 6 are cells grown in 20 % H-pro, lanes 3 and 7 are the control cells stressed with 200 µM H₂O₂ and lanes 4 and 8 are the cells grown in 20 % H-pro stressed with 200 µM H₂O_2. (b) The abundance means of activated cleaved caspase-3 relative to the abundance in the control that are set equal to 100 after the abundance was normalized. Cells grown with H-pro had less active cleaved caspase 3 as compared to controls (p = 0.024, Mann–Whitney U-test n = 5 ± SE)

Fig. 3

Immunostaining for active cleaved caspase-3 (AbCam, ab77973) verified that cleaved active caspase-3 was higher in the cells cytosol when grown in control media and stressed with 200 µM  H₂O₂, while the cells grown in media supplemented with H-pro before being stressed with 200 µM H₂O₂ had almost no active cleaved caspase-3. Providing evidence that the free amino acids and low molecular weight peptides of H-pro had the ability to attenuate activation of caspase-3

Fig. 4

The design chosen for the cell study. The primary liver cells and head kidney cells were grown in complete Leibowitz-15 media (cL-15) that either contained 20 % H-pro (H-pro solved in cL-15 media) or without H-pro supplementation (control). On day two 200 µM H₂O₂ was added or not to the cells with the aim to study the ability of H-pro to attenuate H₂O₂ induced oxidative stress and inflammation responses in primary liver and head kidney cells cultured as the respective monocultures and together in co-culture