CN105440103B - Anti-inflammatory peptide separated from haliotis discus hannai abalone viscera and application thereof - Google Patents

Abstract

The invention is used to evaluate the beneficial effects of haliotis discus hannai, using a multi-stage HPLC purification system to purify anti-inflammatory peptides (AAIP, abalone anti-inflammatory peptides) from abalone. In tandem MS analysis, fragmentation results demonstrate that there is Nitric Oxide (NO) inhibitory activity (IC)₅₀55.8 μ M) of AAIP is Pro-Phe-Asn-Glu-Gly-Thr-Phe-Ala-Ser (1175.2Da), while the anti-inflammatory effect of AAIP on Lipopolysaccharide (LPS) -stimulated RAW264.7 macrophages is also studied and the molecular mechanisms are elucidated the results indicate that AAIP peptides inhibit LPS-induced Nitric Oxide (NO) production via Inducible Nitric Oxide Synthase (iNOS) expression in a dose-dependent manner, and also significantly reduce gene transcription of proinflammatory cytokines such as interleukin (IL-1 β), tumor necrosis factor (TNF- α) and IL-6.

Description

Anti-inflammatory peptide separated from haliotis discus hannai abalone viscera and application thereof

Technical Field

The invention belongs to the field of biotechnology, in particular to the field of bioactive peptides. In particular, the invention relates to anti-inflammatory peptides isolated from the gut of abalone using a continuous HPLC purification system and uses thereof.

Background

Inflammation represents a highly coordinated set of events that allow tissues to respond to injury or infection, requiring the involvement of expression of various cell types and responses to a variety of mediators in a sequential manner (Sebban & Courtois, 2006.) macrophages are the primary immune cells in the innate immune system when infected by pathogens, activation of macrophages plays a critical role in inflammatory responses.

Lipopolysaccharide (LPS), a major component of gram-negative bacteria, elicits many major cellular responses that play a key role in the pathogenesis of inflammatory responses, and has been used to induce macrophage activation. Mitogen-activated protein kinase (MAPK) also regulates key proinflammatory pathways after stimulation with LPS. MAPK (ERK 1/2, p38 and JNK) are a group of serine/threonine kinases that are activated in response to various arrays of extracellular stimuli and mediate signal transduction from the cell surface to the nucleus (Cobb & Goldsmith, 1995). Furthermore, MAPK has previously been implicated in signaling pathways associated with LPS-induced inflammation. Phosphorylation and activation of three major MAPKs have been shown to cause inflammatory gene expression in LPS-induced macrophages (Hommes, peppellenbosch, & van Deventer, 2004). LPS treatment resulted in the regulation of iNOS expression via the MAPK signaling pathway by phosphorylating p38, ERK and JNK, the main three distinct groups of the MAPK subfamily (caroo et al, 2000), thought to play distinct roles with distinct properties in inflammatory diseases. Thus, the involvement of MAPK is often involved in the development of disease and the expression of immune or inflammatory responses (Inoue & Kubo, 2004).

Bioactive peptides are specific protein fragments that have a positive impact on a body function or condition and may ultimately affect human health; it is intended to be provided by a safe, reliable and consistent oral delivery system. Gastrointestinal proteolytic enzymes help to release the peptides after digestion, and the fraction of these absorbed components is sufficient to be physiologically active to humans. In addition, bioactive peptides derived from gastric digestive juices have also been reported to have beneficial effects on anti-inflammatory, anti-hypertensive, anti-oxidant, anti-cancer, anti-bacterial and anti-arthritic (Jung, Rajapakse, & Kim, 2005; Rajapakse, Mendis, Jung, Je, & Kim, 2005; Ryu, Qian, & Kim, 2010). In particular, gastric digestive peptides can be potentially beneficial ingredients in nutritional formulations due to their rapid and reproducible process for producing relatively large amounts of bioactive peptides (Kapsokefalou & Miller, 1991).

Abalone is a marine gastropod which is one of the important fisheries and food industry resources for the massive marine culture in asia, africa, australia and america. Pacific abalone, Haliotis discus hannai, has been cultivated in great quantities offshore in east Asia regions since the nineties of the twentieth century.

Marine organisms possess a variety of bioactive natural components with many physiological functions related to nutritional and pharmaceutical activity, such as antioxidant, anti-inflammatory, anticancer, antibacterial, antihypertensive, anticoagulant, anti-cardiovascular diseases, etc. (Mayer, rodri guez, Berlinck, & Fusetani, 2011). However, although much research has been conducted on the biological and physiological properties of abalone with respect to pathology, genetics and aquaculture techniques, the beneficial effects of abalone on health have been rarely reported (Sun et al, 2010; Ekanayake et al, 2008; Zoysa et al, 2009; Wan, Whang, & Lee, 2005).

In the present invention, applicants disclosed the purification and characterization of peptides from abalone visceral proteins and examined the effect of the peptides on their inhibition of Nitric Oxide (NO) production and reduction of pro-inflammatory cytokine gene transcription in macrophages by Inducible Nitric Oxide Synthase (iNOS) expression. These events are consistent with the activation of mitogen-activated protein kinase (MAPK). Inhibition of this signaling pathway by peptides can modulate LPS-induced inflammatory responses.

Disclosure of Invention

Recently, bioactive peptides obtained by proteolysis have received a lot of attention due to their extended structure, composition and sequence properties as well as their biological activity. They are useful as universal raw materials for the production of human nutraceuticals and pharmaceuticals. Bioactive peptides have been obtained from a variety of raw materials containing high protein concentrations (Vo, Ryu, & Kim, 2013; Harnedy & FitzGerald, 2012). Therefore, abalone (haliotis discus hannai) is considered a major source for the production of biologically active peptides, such as anti-inflammatory peptides. In vitro simulated gastrointestinal digestion-gastrointestinal endopeptidase hydrolysis including pepsin, trypsin and a-chymotrypsin was used. It is believed that prior hydrolysis with endopeptidase can increase the bioavailability of the active peptide and avoid further digestion in the gastrointestinal tract. Previous studies have shown that gastrointestinal enzymatic digestion results in more potent peptides than single enzymatic digestion (Vo, Ryu, & Kim, 2013). Notably, bioactive peptides with low molecular weight may cross the intestinal barrier. Numerous studies have demonstrated that low molecular weight peptides exhibit potent anti-inflammatory biological activity. Lee et al (2009) prepared protein peptides with a size distribution below 1.3kDa and these peptides attenuated the symptoms of inflammatory bowel disease. Anti-inflammatory LDAVNR (683Da) and MMLDF (655Da) from Spirulina maxima (spirulinamaxima), QCQCAVEGGL (1007.28Da) from Crassostrea gigas (Crassostrea gigas), and QCQQAVQSAV204(1061.32Da) from Ruditapes philippinarum (Ruditapes), exhibited anti-inflammatory effects (Vo, Ryu, & Kim, 2013; Hwang et al, 2012; Lee et al, 2012). According to the literature, anti-inflammatory peptides have a wide range of molecular weights. The present inventors have conducted extensive studies to isolate the anti-inflammatory peptide AAIP (Pro-Phe-Asn-Glu-Gly-Thr-Phe-Ala-Ser, 1175.2Da) from the intestine of abalone (Haliotis discus hannai) using a continuous HPLC purification system and found that it has a potent anti-inflammatory effect. In another aspect, the invention relates to the use of the anti-inflammatory peptide AAIP in anti-inflammatory therapy, and as a functional health food. In particular, the invention also relates to application of the anti-inflammatory peptide AAIP in preparing medicines and health-care foods for treating inflammation.

Specifically, the present invention uses the gastric digestion process (i.e., the process described in Kapsokefalou & Miller (1991)) by sequentially adding amounts of pepsin, trypsin, and α -chymotrypsin for hydrolysis at a temperature and pH, then, fractionating by a UF membrane bioreactor system, followed by purification by ion exchange chromatography and HPLC.

Drawings

FIG. 1 purification of NO production inhibiting peptides from AIGID III. (A) Flash Protein Liquid Chromatography (FPLC) of AIGID III by Hiprep 16/10DEAE FF anion exchange chromatography and elution was performed at a flow rate of 2.4mL/min using a linear gradient solution (0-100%) of 2.0M NaCl in 20mM sodium acetate buffer (pH 4.0). (B) Activity to suppress NO production at each stage on Hiprep 16/10DEAE FF column using FPLC. Error bars represent the average and s.d from triplicate experiments after subtracting the background value from the original value.

FIG. 2(A) Fr IV active ingredient at Primesphere10C₁₈Reversed phase HPLC plot on column and HPLC operation was performed using 15% acetonitrile as mobile phase at flow rate of 1mL/min using UV detector at 215 nm. (B) The activity of a fraction having a reversed phase HPLC profile on a Primesphere10C18 column chromatography to inhibit NO production. Error bars represent the average and s.d from triplicate experiments after subtracting the background value from the original value. (C) The active fraction peak with the highest production activity was further isolated by final purification on a Synchropak RPP-100 analytical column. HPLC operation was carried out using 10% acetonitrile as mobile phase at a flow rate of 1mL/min at 215nm using a UV detector. (D) Molecular mass and amino acid sequence recognition of AAIP. MS/MS experiments were performed on a Q-TOF tandem mass spectrometer (Micromass co., Manchester, UK) equipped with a nano-ESI source. Sequencing of active peptides was obtained in the m/z range 50-2500 and calculated by using PepSeq de node sequencingSequencing by the method.

Fig. 3(a) cytocompatible effect of AAIP on RAW264.7 cell viability. Cells were treated with AAIP at the indicated concentration (0-500. mu.M). After 24h of AAIP treatment, cell viability was assessed by MTT assay as described in the text. Results are mean ± standard error of three independent experiments. (B) Effect of AAIP on LPS-induced NO production in RAW264.7 cells. Cells cultured in serum-free medium were pretreated with AAIP at various concentrations (10-500. mu.M) for 1h, followed by stimulation with LPS (100 ng/mL). Conditioned media was collected after 48h and NO concentration was measured using grits reaction.

FIG. 4. Effect of AAIP on LPS-induced mRNA and iNOS protein expression in RAW264.7 cells. Cells cultured in serum-free medium were pretreated with AAIP at various concentrations (10-500. mu.M) for 1h, and then with LPS (100ng mL)^-1) GAPDH and β -actin expression were used as internal controls for genes and proteins in RT-PCR and western blot, respectively, (a) cell lysates were extracted and genes (IL-1 β, TNF- α and IL-6) were analyzed by RT-PCR, (B) cell lysates were extracted and protein levels of iNOS were analyzed by western blot, respectively.

FIG. 5 Effect of AAIP on LPS-induced phosphorylation of ERK 1/2, JNK 1/2, and p38MAP kinases in RAW264.7 cells. Cells were treated with vehicle or AAIP (10-250 μ M) at the indicated concentration for 1h, followed by incubation with LPS (100 ng/mL). Cell lysates were then prepared and subjected to western blotting with antibodies specific for phosphorylated forms of ERK 1/2, JNK 1/2, and p 38. Results are representative of three independent experiments.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, embodiments of the present invention will be described in detail below. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.

Materials and sources

Macrophages (RAW264.7) were obtained from the American Type of Culture Collection (Manassas, Va., USA). Dulbecco's Modified Eagle's Medium (DMEM), trypsin-EDTA, penicillin/streptomycin/amphotericin (10,000U/mL, 10,000. mu.g/mL and 2,500. mu.g/mL, respectively), Phosphate Buffered Saline (PBS) and Fetal Bovine Serum (FBS) were obtained from Gibco BRL, Life Technologies (USA). 3- (4, 5-dimethyl-2-yl) -2, 5-diphenyltetrazolium bromide (MTT), griiss reagent, a reagent derived from escherichia coli serotype 0111: lipopolysaccharide (LPS) of B4 reagent was purchased from sigma chemical Co, (st. Specific antibodies against iNOS, ERK, JNK and p38 were purchased from Santa Cruz Biotechnology (Santa Cruz, Calif.). Antibodies against phosphorylated (p) -ERK, p-p38, and p-JNK were purchased from CellSignaling Technology (Beverly, MA). Other chemicals and reagents used were of commercial analytical grade.

Preparation of in vitro gastrointestinal digestion and fractionation by UF Membrane bioreactor System (fraction)

The gastric digestion process used was by Kapsokefalou&Pepsin was added at an enzyme to substrate ratio of 1/100(w/w) and then incubated at 37 ℃ on a shaker (stage I) after 2h, pH was set to 6.5 to obtain conditions for intestinal digestion (stage II) likewise, both trypsin and α -chymotrypsin were supplemented at an enzyme to substrate ratio of 1/100 (w/w). solution was further incubated at 37 ℃ for 2.5 h. when samples were taken at the beginning and end of digestion, pH was adjusted to 6.5. samples were centrifuged at 10,000 × g for 15min at 4 ℃ and supernatant was lyophilized to obtain AIGID dry powder>M of 10kDa_WDistributed AIGID-I, AIGID-II with MW distribution of 10-5kDa and M with 5-1kDa_WDistributed AIGID-III. All recovered AIGIDs were lyophilized in a freeze-dryer for 5 days.

Purification of peptides inhibiting NO production

Ion exchange chromatography

Lyophilized AIGID III was dissolved in 20mM sodium acetate buffer (pH4.0) and equilibrated with 20mM sodium acetate buffer (pH4.0) using FPLC (AKTA, Amersham Bioscience co., Uppsala, Swedern) on Hiprep 16/10DEAE FF anion exchange column (16 × 100mM) and eluted with a linear gradient solution of NaCl (0-2.0M) in the same buffer at a flow rate of 2.4 mL/min. Each fraction was monitored at 280nm, collected in a volume of 4mL and concentrated using a rotary evaporator. The active fraction was lyophilized and in the next step chromatography was used.

High Performance Liquid Chromatography (HPLC)

Fractions showing NO generation inhibitory activity were further purified using RP-HPLC on a Primesphere10C18 (20 × 250mm) column using a linear gradient solution of acetonitrile containing 0.1% TFA (0-35%, 30min) at a flow rate of 1.0 mL/min. The elution peak was detected at 215nm and the active peak was concentrated using a rotary evaporator. The valid peaks were collected, assessed for NO production, and then lyophilized. The active fraction from the column was further applied to a Synchropak RPP-100 column and eluted at a flow rate of 1mL/min using a linear gradient of acetonitrile containing 0.1% TFA (20% v/v at 15 min). The final purified peptide was analyzed for amino acid sequence.

Determination of amino acid sequence

The exact molecular mass and amino acid sequence of the purified peptide was determined using a Q-TOF mass spectrometer (Micromass, Altrincham, UK) coupled with an electrospray ionization (ESI) source. Purified peptide (AAIP) was injected separately into the electrospray source after dissolution in methanol/water (1: 1, v/v) and molecular mass was measured by double charge (M +2H) in the mass spectrum⁺²The status is determined.

Cell culture and viability determination

Cell viability is measured by methods such as those described by Hansen, Nielson,&berg (1989) by the MTT reduction assay. Briefly, cells were plated at 1 × 10 in 96-well plates⁴The density of each cell per well was preincubated overnight and then washed with PBS buffer. Cells were treated with different concentrations (10-500. mu.M) of purified peptide for 24h, added 100. mu.L of MTT and incubated at 37 ℃ for 4 h. After removing the culture supernatant, the resulting dark blue crystalsDissolved with DMSO. The absorbance values were read at 540nm on an enzyme linked immunosorbent assay (ELISA) microplate reader (ThermoMax, CA, USA).

Nitric oxide generation assay

Nitric Oxide (NO) levels in culture supernatants were measured by grilis reaction as described by Lee et al (2007). Briefly, RAW264.7 cells were plated in 96-well plates using DMEM at 1 × 10 without phenol red⁴The density of each cell per well was pre-incubated overnight, followed by treatment of samples at different concentrations for 1 h. Thereafter, NO production was stimulated by addition of LPS (100ng/mL final concentration) and incubation for 48 h. Then, 50 μ L of culture supernatant from each sample was mixed with the same volume of grignard reagent, followed by incubation for 15 min. The absorbance values were read at 540nm on an ELISA microplate reader (ThermoMax, CA, USA).

RNA isolation and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis

Total RNA was extracted from RAW264.7 cells after treatment with different concentrations (10-250 μ M) of AAPI (Ren et al, 2005). polymerase chain reaction was performed in an automated Whatman temperature cycler (Biometra, Kent, UK) to amplify IL-6, iNOS, and IL-1 β mRNA. RNA was stored at-70 ℃ prior to use.

PCR was performed using primers selective for IL-6 (5'-ATGAGCACAGAAAGCATGATC-3' and 5'-TACAGGCTTGTCACTCGAATT-3'), iNOS (5'-ATGTCCGAAGCAAACATCAC-3' and 5'-TAATGTCCAGGAAGTAGGTG-3'), IL-1 β (5'-ATGGCAACTGTTCCTGAACTCAACT-3' and 5'-TTTCCTTTCTTAGATATGGACAGGAC-3'), and GAPDH (5'-TTTGTGATGGGTGTGAACCACGAG-3' and 5'-GGAGACAACCTGGTCCTCAGTGTA-3'). the PCR products were subjected to electrophoresis in a 12% agarose gel and stained with ethidium bromide.

Western blot analysis

Cells were allowed to grow at 1 x 10 in 6-well plates⁶The cells were seeded at a density of one cell/well and grown in 2ml growth medium for 24 h. Typically, cells were pretreated with various test materials for 1h, then incubated for 24h, and protein extracts were separated using 10% SDS-PAGE. Proteins were transferred to nitrocellulose membranes using an electrophoretic method. Blocking solution for membranes (in bags)Tween-20 in Tris buffered saline 5% skim milk) was preincubated at room temperature for 2h, then incubated with iNOS, ERK, P-ERK, JNK, P-38, and P-38 (1: 1000) incubate at room temperature for 2 h. After washing, the blot was incubated with horseradish peroxidase conjugated goat anti-mouse IgG secondary antibody (1: 5000; Amersham Pharmacia Biotech, Little Chalfot, UK) for 30 min. Bands were visualized on X-ray membranes using ECL detection reagents (Amersham Biosciences, Piscataway, NJ, USA).

Statistical analysis

Statistical analysis was performed by student t-test. Values of p <0.05 based on at least three or more independent experiments were considered statistically significant.

Biological test results

Purification characteristics of peptides from abalone intestinal digestive juice that inhibit NO production

In previous studies, the formation of abalone gastrointestinal digestive juices (AIGID), gastric digestive juices with different behaviors (stage 1) and intestinal digestive juices (stage 2) were subjected to a hydrolysis process that mimics physiological digestion gastric digestive juices (stage 1) correspond to preparations of pepsin-based hydrolyzed abalone proteins, and intestinal digestive juices (stage 2) correspond to pepsin-hydrolyzed abalone proteins hydrolyzed by two enzymes (trypsin and α -chymotrypsin.) the abalone intestinal juices (stage 2) were further separated into AIGID-I (M gid) in four MW groups using UF membranes (MWCO ═ 10, 5 and 1kDa)_W>10kDa)、AIGID-II(M_W＝10-5kDa)、AIGID-III(M_W5-1kDa) and AIGID-IV (M)_W＝5<1 kDa). Four groups were studied by NO generating activity. The results showed that AIGID-III with MW ═ 1-3kDa showed higher effect in inhibiting NO production than the other groups (data not shown). Therefore, AIGID III was selected for further purification.

Lyophilized AIGID III was dissolved in 20mM sodium acetate buffer (pH4.0) and eluted using a linear gradient of 2.0M NaCl (0-100%) on a Hiprep 16/10DEAE FF anion exchange column using Fast Protein Liquid Chromatography (FPLC). The elution peak was monitored at 280nm and each fraction was collected at 4ml and divided into two non-adsorbed fractions and two adsorbed fractions (FIG. 1A). Merging each stageSeparately, lyophilized, and measured for its activity of inhibiting NO production. Fraction IV showed the highest activity of inhibiting NO production (fig. 1B, in RAW264.7 cells, at 500 μ g/mL, 78.6%). Lyophilized active fraction IV was purified by RP-HPLC at Primesphere10C₁₈On a (20mm × 250mm) column, further separation was performed using a 15% acetonitrile solution containing 0.1% trifluoroacetic acid (TFA). This fraction was divided into three clear fractions FrIV-1, FrIV-2 and FrIV-3 (target peptides) (FIG. 2A). Each fraction was pooled, lyophilized, and measured for its activity in inhibiting NO production. FrIV-3 (the target peptide) showed higher activity of inhibiting NO production than the other fractions (FIG. 2B, in RAW264.7 cell line, at 200. mu.g/mL, 82.5%). FrIV-3 was pooled and further purified on a Synchropak RPP-100 analytical column (10 mm. times.250 mm) using a 10% acetonitrile solution containing 0.1% TFA (FIG. 2C). The amino acid sequence of the purified peptide was determined to be Pro-Phe-Asn-Glu-Gly-Thr-Phe-Ala-Ser (target peptide, AAIP 3; 1175.2Da, FIG. 2D). The masses determined by ESI/MS spectroscopy agree perfectly with the theoretical masses according to the sequence.

From this, it was found that AAIP (Pro-Phe-Asn-Glu-Gly-Thr-Phe-Ala-Ser, 1175.2Da) isolated from abalone intestine according to the present invention showed potent anti-inflammatory effect (FIG. 2).

Cell viability and inhibition of NO production by AAIP

Cytocompatibility effects of AAIP on RAW264.7 cell viability. The results indicate that AAIP showed no cytotoxic effect on the three cultured cell lines tested at different concentrations of 10-500 μ M, as shown in figure 3A. Stimulation of RAW264.7 cells with LPS (100ng/mL) showed significantly increased cellular Nitrite (NO) levels compared to the non-stimulated group. Various concentrations of AAIP were tested for their ability to inhibit NO production stimulated by this LPS. NO is a stable product of the conversion of NO released by LPS stimulated cells. As shown in fig. 3B, AAIP has the highest ability to inhibit NO production in LPS-stimulated RAW264.7 cells, and the inhibition is dose-dependent (50-500 μ M).

Inhibition of NO production by AAIP through inhibition of iNOS expression and inhibition of proinflammatory cytokines

Macrophages are the major cells involved in inflammation and they are responsible for the major functions of inflammation, in particular for immune regulation by the production of various cytokines and growth factors (Fujiwara & Kobayashi, 2004). Inflammation is a complex biological process in which macrophages play a key role in initiation, maintenance and lysis, including the overproduction of pro-inflammatory cytokines and inflammatory mediators such as TNF-a, IL-1b, IL-6, NO and prostaglandins (PGE2) (Kim & Moudgil, 2008; Yoon et al, 2009). TNF-a and IL-1b are pro-inflammatory cytokines that mediate both acute and chronic inflammation by triggering a cascade of inflammatory mediators, such as platelet activating factor and leukotrienes, prostaglandins, NO, IL-6 and IL-8 (Dinarello, 2000). Inhibition of TNF-a and IL-1b production in vitro and in vivo has been widely used in screening for anti-inflammatory agents (Yoon et al, 2009; Dinarello, 2010). IL-6 is an interleukin that acts as both a pro-inflammatory cytokine and an anti-inflammatory cytokine (Petersen & Pedersen, 2006; Opal & DePalo, 2000). Although IL-6 can down-regulate IL-1 and TNF-a synthesis (Opal & DePalo, 2000), overproduction of IL-6 has been shown to underlie many autoimmune and inflammatory diseases, and blockade of IL-6 signaling is considered therapeutic in diseases characterized by pathological overproduction of IL-6 (Nishimoto, 2010). Agents that can reduce IL-6 production are considered anti-inflammatory (Park, Lee, Lee, Kim, & Kim, 2007; Tedelind, Westberg, Kjerrulf, & Vidal, 2007).

To examine the mechanism of inhibition of LPS-stimulated NO production, the mRNA and protein expression levels of iNOS were investigated. As shown in fig. 3A and 3B, LPS treatment substantially increased iNOS mRNA and protein expression compared to non-treated cells. Treatment with AAIP (10, 50, 100 and 250. mu.M) for 24h resulted in a significant decrease in the levels of iNOS mRNA and protein expression. Collective analysis of this data indicates that inhibition of NO production is the result of down-regulation of inossmrna and protein expression by AID treatment in LPS-stimulated RAW264.7 cells. The resulting dose-dependent inhibition of NO has led applicants to interest in studying their corresponding gene and protein expression. RAW264.7 cells were stimulated with LPS for 24h to analyze the effect of AAIP on iNOS transcription and translation, since previous studies have disclosed that maximal iNOS mRNA expression occurs around this time period. As expected, both the translational and transcriptional levels of iNOS were dose-dependent inhibited by AAIP treatment.

To investigate the anti-inflammatory activity of AAIP more deeply, its effect on the production and transcription of the pro-inflammatory cytokines TNF- α, IL-1 β, and IL-6 was analyzed (fig. 4A) we also performed RT-PCR to determine if AAIP inhibits the expression of these cytokines at the translational level as shown in fig. 4A, LPS-induced production at the translational level of IL-6, IL-1 β, and TNF- α mRNA was significantly reduced and concentration dependent due to treatment with AAIP (10, 50, 100, and 250 μ M).

It can be seen that AAIP significantly reduced the release of pro-inflammatory factors including TNF-a, IL-1b, IL-6 and NO in LPS activated RAW264.1 cells (FIG. 4).

Down-regulation of MAPK signals by AAIP

The study of signaling pathways that modulate anti-inflammatory activity plays a crucial role in drug discovery (Lee et al, 2006). Inflammation may be induced by different signaling pathways. We determined which signaling pathway is involved in AAIP-mediated iNOS regulation and pro-inflammatory cytokine production by LPS. The MAPK pathway has been identified as a major signaling pathway that regulates the inflammatory response (Jung, Yoon, Park, Han, & Park, 2009; Himaya, Ryu, Qian, & Kim, 2010). To find pathways that may be involved in the inhibition of iNOS and proinflammatory cytokines by AAIP, three different MAPK molecules, p38, Jun amino terminal kinase (JNK), and extracellular signal-regulated kinase (ERK), were analyzed for non-phosphorylation and phosphorylation. LPS induces rapid phosphorylation of ERK, JNK and p38, and it has been disclosed that this phosphorylation induction is performed within 10-30min of LPS treatment. Therefore, cells for Western blotting were collected after AAIP treatment for 1h followed by LPS (100ng/mL) stimulation for 30 min. As shown in fig. 5, the inhibitory effect of AAIP (10, 50, 100 and 250 μ M) on JNK, ERK and p38 phosphorylation was dose-dependent. Thus, these results indicate that inhibition of iNOS is regulated via inhibition of signal transduction through a MAP kinase-mediated pathway in LPS-stimulated RAW264.7 cells.

Through applicants' studies, it was found that inhibition of all forms of MAPK molecules by AAIP treatment, only the p38 and JNK molecules were dose-dependently inhibited, while ERK was not inhibited. These results indicate that the p38 and JNK pathways are molecular mechanisms mediating inhibitory activity of iNOS expression after AAIP. In addition, previous studies have disclosed that JNK and p38 are important modulators of iNOS and proinflammatory cytokine production, and their down-regulation can lead to inhibition of those inflammatory mediators (joinel, 1995).

It can be concluded from this that AAIP might attenuate LPS-induced gene and protein expression of iNOS and proinflammatory cytokines via the MAPK, ERK and p38MAPK pathways. Therefore, AAIP has potential in anti-inflammatory therapy, and thus can be used for drugs or functional health foods for treating inflammatory diseases.

Claims (3)

1. An anti-inflammatory peptide isolated from abalone viscera, having an amino acid sequence of Pro-Phe-Asn-Glu-Gly-Thr-Phe-Ala-Ser, and a molecular weight of 1175.2 Da.

2. An anti-inflammatory peptide according to claim 1, wherein the abalone is Haliotis discus hannai.

3. Use of an anti-inflammatory peptide according to claim 1 or 2 in the manufacture of a medicament for the treatment of inflammation.

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