Immunostimulatory Effect of Ovomucin Hydrolysates by Pancreatin in RAW 264.7 Macrophages via Mitogen-Activated Protein Kinase (MAPK) Signaling Pathway

Abstract

Ovomucin (OM), which has insoluble fractions is a viscous glycoprotein, found in egg albumin. Enzymatic hydrolysates of OM have water solubility and bioactive properties. This study investigated that the immunostimulatory effects of OM hydrolysates (OMHs) obtained by using various proteolytic enzymes (Alcalase^®, bromelain, α-chymotrypsin, Neutrase^®, pancreatin, papain, Protamax^®, and trypsin) in RAW 264.7 cells. The results showed that OMH prepared with pancreatin (OMPA) produced the highest levels of nitrite oxide in RAW 264.7 cells, through upregulation of inducible nitric oxide synthase mRNA expression. The production of pro-inflammatory cytokines such as tumor necrosis factor-α and interleukin-6 were increased with the cytokines mRNA expression. The effect of OMPA on mitogen-activated protein kinase signaling pathway was increased the phosphorylation of p38, c-Jun NH2-terminal kinase, and extracellular signal-regulated kinase in a concentration-dependent manner. Therefore, OMPA could be used as a potential immune-stimulating agent in the functional food industry.

Keywords: enzymatic hydrolysis; immunostimulating; mitogen-activated protein kinase (MAPK) signaling pathway; ovomucin; pancreatin.

Fig. 1.. SDS-PAGE analysis of ovomucin hydrolysates (OMH).

M, marker; lane 1, ovomucin hydrolysate prepared with Alcalase (OMAC); lane 2, ovomucin hydrolysate prepared with bromelain (OMBM); lane 3, ovomucin hydrolysate prepared with α-chymotrypsin (OMCT); lane 4, ovomucin hydrolysate prepared with Neutrase (OMNT); lane 5, ovomucin hydrolysate prepared with pancreatin (OMPA); lane 6, ovomucin hydrolysate prepared with Papain (OMPP); lane 7, ovomucin hydrolysate prepared with Protamax (OMPT); lane 8, ovomucin hydrolysate prepared with trypsin (OMTP). SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Fig. 2.. Effects of ovomucin hydrolysates (OMHs) on RAW 264.7 cell viability.

All values are mean±SD. ^a–m Different letters among samples indicate significant differences (p<0.05). OMAC, ovomucin hydrolysate prepared with Alcalase; OMBM, ovomucin hydrolysate prepared with bromelanin; OMCT, ovomucin hydrolysate prepared with α-chymotrypsin; OMNT, ovomucin hydrolysate prepared with Neutrase; OMPA, ovomucin hydrolysates prepared with pancreatin; OMPP, ovomucin hydrolysate prepared with papain; OMPT, ovomucin hydrolysate prepared with Protamax; OMTP, ovomucin hydrolysate prepared with trypsin.

Fig. 3.. Effects of ovomucin hydrolysates (OMH) on the production of (A) nitric oxide and effects of OMPA on the expression of (B) iNOS mRNA in RAW 264.7 cells.

Control: non-treated group. ^a–m Different letters among samples indicate significant differences (p<0.05). NO, nitrite oxide; LPS, lipopolysaccharide; OMAC, ovomucin hydrolysate prepared with Alcalase; OMBM, ovomucin hydrolysate prepared with bromelanin; OMCT, ovomucin hydrolysate prepared with α-chymotrypsin; OMNT, ovomucin hydrolysate prepared with Neutrase; OMPA, ovomucin hydrolysates prepared with pancreatin; OMPP, ovomucin hydrolysate prepared with papain; OMPT, ovomucin hydrolysate prepared with Protamax; OMTP, ovomucin hydrolysate prepared with trypsin; iNOS, inducible nitric oxide synthase.

Fig. 4.. Effects of ovomucin hydrolysate prepared with pancreatin (OMPA) on the (A) morphological change and (B) cell number of RAW 264.7 cells.

The group without OMPA was used as the control group, and LPS (10 ng/mL) was used as the positive control. LPS, lipopolysaccharide.

Fig. 5.. Effects of ovomucin hydrolysate prepared with pancreatin (OMPA) on the (A) TNF-α and (B) IL-6 production in RAW 264.7 cells. Effects of OMPA on the (C) TNF-α and (D) IL-6 mRNA expression in RAW 264.7 cells.

The group without OMPA was used as the control group, and LPS (10 ng/mL) was used as the positive control. The results are expressed as the mean±SD of independent experiments. ^a–e Different letters on each bar indicate a statistically significant difference between values (p<0.05). TNF-α, tumor necrosis factor-α; LPS, lipopolysaccharide; IL-6, interleukin-6.

Fig. 6.. Effects of ovomucin hydrolysate prepared with pancreatin (OMPA) on the MAPK phosphorylation in RAW 264.7 cells.

(A) Protein expression levels of MAPKs. Relative protein expression levels of (B) p-p38/p38, (C) p-JNK/JNK, (D) p-ERK/ERK. The group without OMPA was used as the control group, and LPS (10 ng/mL) was used as the positive control. GAPDH was used as the loading control. ^a–e Different letters indicate statistical differences in the group of samples (p<0.05). LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase.