Nutr Res Pract.  2015 Jun;9(3):219-226. 10.4162/nrp.2015.9.3.219.

Anti-inflammatory effect of enzymatic hydrolysates from Styela clava flesh tissue in lipopolysaccharide-stimulated RAW 264.7 macrophages and in vivo zebrafish model

• Ko SC
• Jeon YJ

• KMID: 2313833
• DOI: http://doi.org/10.4162/nrp.2015.9.3.219

Abstract

BACKGROUND/OBJECTIVES
In this study, potential anti-inflammatory effect of enzymatic hydrolysates from Styela clava flesh tissue was assessed via nitric oxide (NO) production in lipopolysaccahride (LPS) induced RAW 264.7 macrophages and in vivo zebrafish model.
MATERIALS/METHODS
We investigated the ability of enzymatic hydrolysates from Styela clava flesh tissue to inhibit LPS-induced expression of pro-inflammatory mediators in RAW 264.7 macrophages, and the molecular mechanism through which this inhibition occurred. In addition, we evaluated anti-inflammatory effect of enzymatic hydrolysates against a LPS-exposed in in vivo zebrafish model.
RESULTS
Among the enzymatic hydrolysates, Protamex-proteolytic hydrolysate exhibited the highest NO inhibitory effect and was fractionated into three ranges of molecular weight by using ultrafiltration (UF) membranes (MWCO 5 kDa and 10 kDa). The above 10 kDa fraction down-regulated LPS-induced expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), thereby reducing production of NO and prostaglandin E2 (PGE2) in LPS-activated RAW 264.7 macrophages. The above 10 kDa fraction suppressed LPS-induced production of pro-inflammatory cytokines, including interleukin (IL)-1beta, IL-6, and tumor necrosis factor (TNF)-alpha. In addition, the above 10 kDa fraction inhibited LPS-induced phosphorylation of extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinase (JNK), and p38. Furthermore, NO production in live zebrafish induced by LPS was reduced by addition of the above 10 kDa fraction from S. clava enzymatic hydrolysate.
CONCLUSION
The results of this study suggested that hydrolysates derived from S. clava flesh tissue would be new anti-inflammation materials in functional resources.

Keyword

Anti-inflammatory; Styela calva; enzymatic hydrolysates; macrophages; zebrafish model

MeSH Terms

Cyclooxygenase 2
Cytokines
Dinoprostone
Extracellular Signal-Regulated MAP Kinases
Interleukin-6
Interleukins
JNK Mitogen-Activated Protein Kinases
Macrophages*
Membranes
Molecular Weight
Nitric Oxide
Nitric Oxide Synthase Type II
Phosphorylation
Tumor Necrosis Factor-alpha
Ultrafiltration
Zebrafish*
Cyclooxygenase 2
Cytokines
Dinoprostone
Extracellular Signal-Regulated MAP Kinases
Interleukin-6
Interleukins
JNK Mitogen-Activated Protein Kinases
Nitric Oxide
Nitric Oxide Synthase Type II
Tumor Necrosis Factor-alpha

Figure

• 

  Fig. 1 Inhibitory effect of enzymatic hydrolysates from S. clava protein on LPS-induced NO production in RAW 264.7 macrophages (▪) and cell viability (-♦-). Production of nitric oxide was assayed in the culture medium of macrophages stimulated with LPS (1 µg/ml) for 24 h in the presence of enzymatic hydrolysates (200 µg/ml). Each value indicates the mean ± SD from three independent experiments. *P < 0.05. A, Alcalase; B, Protamex; C, Neutrase; D, Flavourzyme; E, Kojizyme; F, Pepsin; G, Trypsin; H, Papain.

• 

  Fig. 2 Inhibitory effects of molecular weight fractions from Protamex hydrolysate on LPS-induced NO production in RAW 264.7 macrophages (▪) and cell viability (-♦-). Production of nitric oxide was assayed in the culture medium of macrophages stimulated with LPS (1 µg/ml) for 24 h in the presence of molecular weight fractions (50, 100 and 200 µg/ml). Each value indicates the mean ± SD from three independent experiments. *P < 0.05.

• 

  Fig. 3 Effect of SFTPH-I on LPS-induced expression of iNOS and COX-2 and production of PGE2 in RAW 264.7 macrophages. Cells were stimulated with LPS (1 µg/ml) in the presence of SFTPH-I (50, 100, and 200 µg/ml) for 24 h at 37℃. (A) Culture media were collected for measurement of (A) PGE2 production by ELISA. Values are expressed as mean ± SD of triplicate experiments. *P < 0.05 for comparison with the LPS-stimulated group. (B) Cells were stimulated with LPS (1 µg/ml) in the presence of SFTPH-III (50, 100, and 200 µg/ml) for 24 h at 37℃. The levels of iNOS and COX-2 proteins in cell lysates were analyzed by Western blot.

• 

  Fig. 4 Inhibitory effect of SFTPH-I on LPS-stimulated IL-6, IL-1β and TNF-α production in RAW 264.7 macrophages. Production of IL-6, IL-1β and TNF-α was assayed in the culture medium of macrophages stimulated with LPS (1 µg/ml) for 24 h in the presence of SFTPH-I (50, 100 and 200 µg/ml). Levels of IL-6, IL-1β and TNF-α were measured in culture media using an ELISA kit. Each value indicates the mean ± SD from three independent experiments. *P < 0.05, **P < 0.01.

• 

  Fig. 5 Inhibitory effect of SFTPH-I on the protein level of p38, ERK and JNK in RAW 264.7 macrophages. RAW 264.7 macrophages were pre-incubated for 18 h, followed by stimulation with LPS (1 µg/ml) for 20 min in the presence of SFTPH-I (50, 100 and 200 µg/ml). The levels of p-ERK, ERK, p-JNK, JNK, p-p38 and p-38 were determined via Western blotting.

• 

  Fig. 6 Survival rate after treatment with LPS or co-treatment with SFTPH-I. The embryos were treated with LPS (3 µg/ml) and SFTPH-I (50, 100 and 200 µg/ml). Each value indicates the mean ± SD from three independent experiments.

• 

  Fig. 7 Inhibitory effect of SFTPH-I on LPS-induced NO production in zebrafish embryos. The embryos were treated with LPS (3 µg/ml) and SFTPH-I (50, 100 and 200 µg/ml). After incubation, NO was detected by fluorescence after DAF-FM DA staining. The NO level was measured using an image analysis fluorescence microscope. Each value indicates the mean ± SD from three independent experiments. *P < 0.05.

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