Peptide fraction from sturgeon muscle by pepsin hydrolysis exerts anti-inflammatory effects in LPS-stimulated RAW264.7 macrophages via MAPK and NF-κB pathways

Ruihng Go,Wnghui Shu,Yng Shen,Quni Sun,Wengng Jin,Djing Li,Ying Li,Li Yun,?

aSchool of Food and Biological Engineering,Jiangsu University,Zhenjiang,Jiangsu Province,212013,China

bBio-resources Key Laboratory of Shaanxi Province,School of Bioscience and Engineering,Shaanxi University of Technology,Hanzhong,723001,China

cInstitute of Agro-product Processing,Jiangsu Academy of Agricultural Sciences,Nanjing,210014,Jiangsu,China

Keywords:

Sturgeon

Enzymatic hydrolysis

Antioxidant

Anti-inflammation mechanism

RAW264.7 macrophages

ABSTRACT

Previous studies have suggested that polypeptides extracted from milk,soybean,fish,eggs,and meat possess potential anti-inflammatory effects.To date,few studies have reported the anti-inflammatory function of sturgeon peptides and their underlying mechanisms are unknown.The current study was therefore to determine the anti-inflammatory potential of sturgeon peptides with lipopolysaccharide(LPS)-induced RAW264.7 inflammatory model.Pepsin hydrolysate(PeH)was purified by ultrafiltration and Sephadex G-15 gel filtration chromatography.PeH significantly reduced the inflammatory mediator(NO)and inflammatory cytokines(IL-6,TNF-α and IL-1β)expression in a dose-dependent manner.Moreover,the purified sturgeon peptide(F2)possessed strong antioxidant potential and effectively inhibited DPPH and ABTS free radicals.F2 significantly suppressed the expression of MAPK,IκBα,and NF-κB p65,indicating that F2 exerted anti-inflammatory influence by the inhibition of MAPK and NF-κB pathways.

1.Introduction

inflammation is the body’s defensive response to a variety of inflammatory factors,and it is the most common pathological process in many diseases[1].When biological tissues are stimulated by bacteria,viruses,endogenous toxic substances,etc.,the¨balanced body¨state maintained by the immune system is broken,resulting in a cascading reaction of inflammatory signals.The aggregation of inflammatory cells is induced and the cellular chemotaxis mechanism is initiated.These cells release a large number of cytokines,including interleukin-6(IL-6),interleukin-1β(IL-1β),tumor necrosis factor-α (TNF-α),and other inflammatory mediators,such as nitric oxide(NO),prostaglandin(PG).IL-6 is a multifunctional cytokine which is elevated in inflammatory diseases and would also induce the expression of multiple factors with anti-inflammatory properties,including IL-10,IL-1R antagonist,etc.[2].Excessive release of inflammatory mediators and cytokines causes organ damage as well as chronic diseases.To date,uncontrolled inflammation is increasingly implicated as a risk factor for cardiovascular disease[3,4],depression[5],cancer[6],etc.,In the past several years,inhibiting the synthesis or activity of inflammatory cytokines has been the mainstream in the treatment of inflammation.The most commonly used treatment drugs are steroids,non-steroidal anti-inflammatory drugs,and monoclonal antibodies[7],but many of them are accompanied by adverse side effects,such as reducing the repairability of tissues and damaging gastrointestinal mucosa.Hence,looking for safer and more efficient natural anti-inflammatory compounds is a promising strategy.

Currently,many studies have confirmed that oxidative damage is the key to the accelerated development of chronic inflammation[8–10].Altered inflammatory response together with redox imbalance usually occur in many noncommunicable diseases(NCDs)[11].Several studies suggest that anti-inflammatory peptides are usually accompanied by strong antioxidant potential[12–14].

Sturgeon is an old and endangered commercial aquaculture species,which has considerable economic and ecological value[15].Previous studies have reported on the antioxidant and cryoprotective effects of bioactive peptides originating from sturgeon[16,17].Noman et al.[18]suggested sturgeon protein hydrolysate possessed appropriate contents of amino acids and good physiochemical properties,which had potential applications in food industries.Currently,with the exception of caviar serving as a global business card of sturgeon products,the comprehensive utilization rate and added value of sturgeon are generally very low,and more studies are urgently needed to better utilize the sturgeon byproducts.Especially sturgeon muscle,which possesses optimum dietary protein and essential amino acids,the research on the biological activity of sturgeon muscle peptides however is very limited[15].Many studies have reported that fish is a good source of antiinflammatory peptides,whereas concerning the anti-inflammatory function of sturgeon peptides and the underlying mechanisms were rarely reported.Hence,this study aimed to identify the potential anti-inflammatory sturgeon muscle peptides and the underlying mechanisms in LPS-induced RAW264.7 cells.

2.Materials and methods

2.1.Materials

Sturgeons were obtained from Quzhou Xunlong Aquatic Products Sci-tech Development Co.,Ltd.(Quzhou,China)and stored at-20?C until use.Pepsin and Trypsin were obtained from Sigma-Aldrich(St.Louis,MO,USA);Neutrase and Papain were obtained from Sinopharm Chemical Reagent Co.,Ltd(Shanghai,China);Alcalase was obtained from Macklin Biochemical Co.,Ltd(Shanghai,China);3-(4,5-dimethylthizaol-2-yl)-2,5-diphenylterazolium bromide(MTT),2,2-diphenyl-1-picrylhydrazyl(DPPH),2,2-azinobis(3-ethylbenzthiazoline)-6-sulfonic acid(ABTS)were obtained from Aladdin Industrial Corporation(California,USA);Fetal bovine serum(FBS),Dulbecco’s modified eagle medium(DMEM)were obtained from Biological Industries(Beit Haemek,Israel);Trypsin-EDTA,Penicillin,Streptomycin,LPS,and NO Griess Reagent were from Beyotime Biotechnology(Shanghai,China);IL-6,TNF-α,and IL-1β enzyme-linked immunosorbent assay(ELISA)kits were obtained from Fcmacs(Nanjing,China);antibodies for p44/42 MAPK,p-p44/42 MAPK,SAPK/JNK,p-SAPK/JNK,p38 MAPK,p-p38 MAPK,IKBα,p-IKBα,p-NF-KB p65,NF-KB p65,GAPDH,and Lamin B were supplied by Cell Signaling Technology(Beverly,MA,USA).

2.2.Preparation of sturgeon muscle hydrolysates

Sturgeon were deboned,skinned,rinsed and their back muscles were taken.The crude protein content of the back muscle was 19.92% as determined by the Kjeldahl method[19].Enzymatic hydrolysis was conducted according to the Ahn et al.method with slight adjustment[20].The fish protein was hydrolyzed with five proteases(Neutrase,Alcalase,Pepsin,Papain,Trypsin)under optimal conditions(Table 1).A 10% substrate and enzyme(E/S ratio of 1:100(m/m))were mixed,then hydrolyzed for 6h in a water bath oscillator(120r/min).After hydrolysis,the mixture was heated for 20min at 100?C to inactivate the enzyme.The solutions were centrifuged for 15min at 10,000r/min(4?C)and the supernatant was collected and lyophilized for further experiments.The degree of hydrolysis(DH)was calculated by the OPA method[21].The amino acid composition of sturgeon peptides was analyzed with an amino acid analyzer(Sykam S-433D,Berchtesgaden,Germany).

Table 1The optimum enzymatic hydrolysis conditions of sturgeon muscle by using various enzymes.

2.3.Ultrafiltration and gel filtration chromatography

The partial enzymatic hydrolysate was fractionated through ultrafiltration with membranes molecular weight cut off(MWCO)of 10kDa and 3kDa(Millipore Co.USA).All fractions(MW＞10kDa,3?10kDa,and ＜ 3kDa)were lyophilized for further analysis,and the fraction exhibiting the best anti-inflammatory effects were selected for the subsequent purification.The antiinflammatory fraction was subjected to a Sephadex G-15 gel column(1.2cm×70cm,GE Healthcare)followed by distilled water elution at a flow rate of 1.0mL/min.The elution was monitored at 280nm,each fraction was collected and lyophilized for further analysis.

2.4.Anti-inflammatory activity

2.4.1.Cell culture

RAW264.7 macrophage cells were from the Chinese Academy of Sciences(Shanghai,China).Cells were cultured in DMEM containing 10%FBS,100μg/mL streptomycin and 100 U/mL penicillin.

2.4.2.Cell viability

Cell viability was determined by MTT assay as previously reported[22,23].Cells were treated with different concentrations(0.125,0.25,0.50,1.0,2.0mg/mL)of samples,while the control group was treated with the medium for 24h.Then cells were stained for another 4h at 37?C in the dark after addition of an MTT working solution.The formazans crystals were diluted with 150μL dimethyl sulfoxide(DMSO).Absorbance at 540 nm was determined in a microplate reader(Tecan Infinite PRO TWIN 200,Swiss).Viability of cells was calculated considering controls as 100% viable.

2.4.3.NO production

NO production was measured as reported[24].RAW264.7 cells were treated with samples for 8 h, followed by 2 μg/mL LPS stimulation for 24h.The supernatant was harvested for NO determination using the NO assay kit.Absorbance at 540nm was determined in a microplate reader(Tecan Infinite PRO TWIN 200,Swiss).The sample NO concentrations(μmol/L)were extrapolated from the sodium nitrite standard calibration curve.

2.4.4.The levels of IL-1β,IL-6 and TNF-αin the cell culture supernatant

The levels of IL-6,IL-1β,and TNF-α in cell culture supernatant were quantified with an ELISA kit following the manufacturer’s instructions.The cell sample IL-6,IL-1β,and TNF-α concentrations(pg/mL)were extrapolated from IL-6,IL-1β,and TNF-α standard curves,respectively.

2.5.Western blotting

Fig.1.(A)Effect of sturgeon muscle enzymatic hydrolysates on cell viability.RAW264.7 cells were determined by MTT assay.(B)Effects of enzymatic hydrolysates on the NO production in LPS-stimulated RAW264.7 cells were detected by Griess Reagent.The results were expressed as means± SD(n=3),a–d superscript indicated significantly different at P＜0.05.

Western Blot procedures were performed as previously reported with a slight modification[25].The protein concentrations were determined with Bicinchoninic Acid (BCA) Protein Assay Kit.The proteins were denatured by boiling in loading buffer.Equal amounts of protein samples were loaded per lane,electrophoresis in 12% sodium dodecyl sulfate-polyacrylamide gels.Then transferred onto polyvinylidene fluoride membranes(Millipore,Bedford,MA),and the membranes were blocked with 5% skim milk in tris-buffered saline containing 0.1%Tween-20(TBST).Subsequently,the membrane was washed with TBST and incubated with primary antibodies overnight at 4?C.After the washing using TBST,membranes were incubated with appropriate secondary antibodies for 1 h at room temperature. The blots were visualized using an ECL kit with an enhanced chemiluminescence detection system(Tanon 5200 Multi,Nanjing,China).Images and results were quantified with ImageJsoftware[23,26].

2.6.Antioxidant activity

2.6.1.ABTS radical scavenging activity

The ABTS assay was conducted as previously described with a slight modification[27].50μL different concentration samples were mixed with 150μL ABTS+solution,and followed by incubating room temperature for 10min in the dark.Absorbance was measured at 734nm.While the blank group was replaced samples by distilled water.Samples were tested in triplicate.The ABTS radical scavenging activity was calculated using the following formula:

The IC50value was defined as the antioxidant compound concentration required to scavenge 50% of the ABTS radical.

2.6.2.DPPH radical scavenging activity

The DPPH assay was also performed as previously reported with a slight modification[28].100μL DPPH solution(0.1mmol/L in 95% ethanol)was mixed with 100μL different concentration samples(dissolved in distilled water),and followed by incubating room temperature for 30min in the dark.The absorbance was measured by a microplate reader(Tecan Infinite PRO TWIN 200,Swiss)at 517nm.For the blank,the samples were replaced with distilled water.Samples were tested in triplicate.The DPPH radical scavenging activity was calculated using the following formula:

The IC50value was defined as the antioxidant compound concentration required to scavenge 50% of the DPPH radical.

2.7.Identification of anti-inflammatory peptides

The most active fraction was subjected to liquid chromatography-tandem mass spectrometry (LC–MS/MS),which was conducted by Gene Denovo Biotechnology Company(Guangzhou,China).In brief,LC–MS/MS analysis of the samples was carried out by using AB SCIEX Triple TOF 5600 Plus mass spectrometer paired with AB SCIEX C18(75μm×100mm,3μm)column.Samples were eluted with solvent A(5% acetonitrile,0.1% formic acid)and B(95% acetonitrile,0.1% formic acid),from 5% to 30% B over 65min,30%–50% B over 5min,50%–80% B over 10min and held for 5min to elute all the compounds.Obtained MS/MS data were analyzed by proteomic software MaxQuant(version 1.5)and sequences were searched in the NCBInr database to match with parent proteins.

2.8.Statistical analysis

Statistical analysis was calculated using SPSS(version 22.0;Chicago,IL,USA).All data were expressed as means±standard deviation(SD)of triplicate measurements. Differences between the means of each group were assessed by one-way ANOVA(Tukey’s multiple range test).aP-value of＜0.05 was regarded as statistically significant.

3.Results

3.1.Anti-inflammatory activity of sturgeon enzymatic hydrolysates

Five proteases(Alcalase,Papain,Neutrase,Pepsin,Trypsin)were used for the hydrolysis of the sturgeon muscle protein.Under optimal conditions,the DH values of Alcalase hydrolysis(AH),Papain hydrolysis(PaH),Neutrase hydrolysis(NH),Pepsin hydrolysis(PeH),and Trypsin hydrolysis(TH)were determined as(29.95±0.42)%,(32.58±0.64)%,(33.35±0.22)%,(15.7±0.46)%,and(31.14±0.14)%,respectively.Subsequently,as shown in Fig.1A,when RAW264.7 cells were incubated with five enzymatic hydrolysates at the concentrations of 0.125,0.25,0.5,and 1.0mg/mL, the cell viability treated with all enzymatic hydrolysates was significantly increased,compared to the control.However,the cell viability decreased to 91% at a concentration of 2.0mg/mL with TH treatment.Furthermore,five enzymatic hydrolysates exhibited the highest cell viability at a concentration of 0.5mg/mL.Accordingly,0.5mg/mL was selected for the anti-inflammatory activityin the subsequent studies.Fig.1B shows that NO production in LPS-treated cells was increased by 5.57 times compared with the unstimulated group,whereas NO production decreased significantly under 0.5mg/mL enzymatic hydrolysates pre-incubation.PeH performed the best in this regard.Next was AH,but NH did not show the potential to reduce NO content.

Table 2Amino acid profile of Sturgeon Muscle,Pepsin hydrolysate,＜3kDa fraction and F2 fraction.The results were expressed as means±SD(n=3).

Fig.2.Effect of PeH ultrafiltration fractions on anti-inflammatory activity in LPS-stimulated RAW264.7 cells.(A)NO production.(B)level of IL-1β.The results were expressed as means± SD(n=3),a–e superscript indicated significantly different at P＜ 0.05.

The amino acid composition of sturgeon peptides is summarized in Table 2.The major amino acids in the sturgeon muscle were Glu,Lys,Asn,Leu,and Arg.After pepsin hydrolysis,Glu,Lys,and Val content increased,which might have arisen from the effect of the enzymatic hydrolysis of pepsin.

3.2.Purification of anti-inflammatory peptides

Next,we purified the PeH with ultrafiltration and Sephadex G-15 gel filtration.As shown in Fig.2A-2B,treatment of RAW264.7 with LPS alone for 24h significantly increased the secretion of NO and IL-1β by 7.76 and 9.22-fold,respectively.While a＜3kDa peptide fraction showed the strongest anti-inflammatory activity with 31.77%NO and 38.72%IL-1β inhibited at 0.5mg/mL,the＜3kDa fraction was therefore further separated using Sephadex G-15 gel filtration chromatography.Three absorption peaks(1,2,3)were observed according to the molecular weight(Fig.3A)(MW:F1＞F2＞F3).F2 exhibited much stronger anti-inflammatory activity than other fractions(Fig.3B-3C).Compared with the LPS-alone group,the inhibitory activities of F2 for NO and IL-1β were 42.61% and 50.10%,respectively.Similarly,F2 had the best anti-inflammatory effect,the inhibitory rate was 39.04% for IL-6 and 47.00% for TNF-α(data not shown).Meanwhile,compared with the LPS group alone,pretreatment with F2 decreased the pro-inflammatory cytokines dose-dependently(Fig.4A-4D).As shown in Table 2,after purification,the Phe,Tyr,and Glu content in F2 increased to(19.35±0.07)%,(15.39±0.13)%,and(11.84±0.11)%,respectively.Furthermore,the hydrophobic amino acid content of F2(57.35%)was markedly increased compared to PeH(37.49%).

3.3.Anti-inflammatory mechanism of F2

It is known that the JAK-STAT,MAPK,and NF-κB pathways play critical roles in inflammatory responses[29].LPS alone(2μg/mL)markedly increased the phosphorylation of ERK1/2,JNK,and p38(4.66-fold,5.9-fold,and 8.0-fold,respectively).After pretreatment with F2,the ERK1/2,JNK,and p38 phosphorylation levels were significantly decreased by 25.5%,29.7%,and 46.29%,respectively(P＜0.01)(Fig.5A).Furthermore,pretreatment with 0.5mg/mL F2 significantly inhibited the phosphorylation and degradation of IκBα in the cytoplasm compared with the LPS-alone treatment(inhibition rate:37.11%)(P＜0.01).Simultaneously,LPS increased the expression of p65 in the nucleus by 2.29 times,and this was significantly reduced by F2 pretreatment(Fig.5B).The results suggested that the anti-inflammatory effect of F2 is at least partially attributed to the MAPK signaling pathways inhibition and suppression of IκBα phosphorylation and p65 nuclear translocation.

Fig.3.Purification of anti-inflammatory peptides from PeH.(A)Chromatogram of＜3kDa peptide fraction separated by Sephadex G-15.(B)NO production.(C)level of IL-1β.The results were expressed as means±SD(n=3),where the a–e superscript indicates significantly different at P＜0.05.

Fig.4.Effects of F2 fraction on the production of pro-inflammatory cytokines in LPS-stimulated RAW264.7 cells.(A)NO production.(B)level of IL-1β.(C)level of IL-6.(D)level of TNF-α.The results were expressed as means± SD(n=3),where an a–e superscript indicates significantly different at P＜ 0.05.

Fig.5.Effects of F2 fraction on the LPS-stimulated MAPKs and NF-κB proteins in RAW264.7 cells.(A)The levels of p-ERK,ERK,p-JNK,JNK,p-p38,and p38 determined by Western blotting.(B)Western blot analysis for NF-κB p65,IκBα in the cytoplasmic and nuclear extracts of RAW264.7 cells.The results were expressed as means±SD(n=3),*P＜0.05 and**P＜0.01 were compared with LPS alone group.

Table 3IC50values of the different fractions of Pepsin hydrolysates(PeH),GSH as a positive control.The results were expressed as means± SD(n=3),a–d superscript indicated DPPH free radical scavenging significantly different at P ＜0.05.A–F superscript indicates ABTS free radical scavenging significantly different at P＜0.05.

Table 4Peptide sequences identified by mass spectrometry in the F2 fraction from sturgeon muscle hydrolysate(results were shown based on the relative intensity of mass spectrum,peptide score,and molecular size).

3.4.Determination of antioxidant activity

We further investigated the free radical scavenging capacity of peptides.GSH was used as a positive control.As shown in Table 3,the IC50of DPPH and ABTS of pepsin hydrolysates were(348.7±45.3)μg/mL and(571.3±35.91)μg/mL,respectively.After ultrafiltration,the＜3kDa fraction exhibited a stronger radical scavenging capacity.Among the F1,F2 and F3 fractions,DPPH and ABTS free radical scavenging activity increased with the decrease of molecular weight.F3 exhibited higher scavenging activity on DPPH and ABTS than F2.The IC50value of F3 up to 207.7μg/mL,211.7μg/mL,indicating that low molecular weights had higher antioxidant activity.

3.5.Identification of peptides from sturgeon muscle

F2 was chosen for the MS/MS analysis(Table 4).These peptides derived from a variety of muscle proteins,such as actin,myosin,troponin,titin,etc.respectively,which were varied in molecular size(7–12 amino acids),charges and amino acids composition.The N-terminals of these peptides are mostly hydrophobic amino acids.

4.Discussion

Studies have demonstrated that hydrolysates derived from fish proteins are rich sources of antioxidant and immunomodulatory peptides[30–32].Different cleavage specificity of enzymes affected the release of immunomodulatory peptides from parent proteins[33].In this study,pepsin hydrolysate exhibited stronger anti-inflammatory activity.This might be because of the random cleavages of hydrophobic amino acids by pepsin.Our results found that F2 had a high content of hydrophobic amino acids such as Phe and Tyr,which might contribute to the strong anti-inflammatory effect of F2.Besides,the potent anti-inflammatory activity of F2 results might be attributed to the low molecular weight peptides(＜1.5kDa of F2),which would be consistent with previous studies that food-origin low molecular weight dipeptides and tripeptides decrease the level of inflammatory cytokines in RAW264.7 cells stimulated by LPS[31,34,35].

Previous studies have demonstrated that persistent pathological stimulation of macrophages activates NF-κB and MAPKs,and the activated NF-κB and MAPKs promote the expression of proinflammatory cytokines IL-1β,IL-6,and TNF-α[36].MAPKs,which are a highly conserved family of protein serine/threonine kinases[29].The kinases are comprised of three major subtypes:c-Jun N-terminal kinase(JNK),p38 and extracellular signal-regulated kinase l/2(ERK l/2),and each MAPK plays a significant role during the inflammatory stage.Thus,inhibiting or regulating the expression of the MAPK pathway is thought to be a good therapeutic strategy for treating inflammation[37].In terms of the NF-κB signaling pathway,when the macrophages are in resting states,the binding of NF-κB dimer with IκB(an inhibitory subunit)exists in the cytoplasm in an inactive form.When the cells are stimulated,IκB will be phosphorylated.Subsequently,NF-κB dimers translocate into the nucleus and activate the transcription of target genes[38].One study reported the NF-κB transcription factor is one of the main modulators of pro-inflammatory gene transcription activated by LPS through TLR4[39].In the current study,the F2 fraction inhibited pro-inflammatory cytokine release(IL-1β,IL-6,TNF-α)and the phosphorylation level of MAPKs and IκBα.The current results are consistent with the previous study of salmon pectoral fin by-products hydrolysates andMytilus edulishydrolysates fraction reduced the levels of pro-inflammatory mediators[30,40].

In biological systems,oxidative stress is closely linked with inflammation[41,42].Numerous studies have found that one peptide may carry a variety of biological activities at the same time[20,43–45].Song et al.speculated that amino acids for example aspartic acid and Glu may serve as a link between anti-inflammatory and antioxidant effects[24].The presence of hydrophobic and specific aromatic residues could increase the antioxidant activity of peptides[46],which is in line with our current results that the purified sturgeon peptides possessed strong DPPH and ABTS free radical scavenging ability.One study reported Gln might be a crucial amino acid involved in the anti-inflammatory activity[47],and the other research suggested that the presence of Arg residues at the N-or C-terminals can bind the LPS and block the inflammation response[48].Our results found that the identified sturgeon peptides contained Arg and Lys in the C-terminal.There are also many hydrophobic amino acids at the N-terminal,such as Ala,Val,Leu,etc.,this might promote peptide binding to membranes and contribute to modulating the downstream inflammatory signaling pathways[49].Our results suggested that the anti-inflammatory activity of sturgeon peptides may be related to the specific position of amino acids in the peptide sequence,the composition of amino acid,and the length of peptides.

5.Conclusion

Our present findings suggest that sturgeon peptides exert anti-inflammatory effects via the downregulation of MAPK and NF-κB pathways in LPS-stimulated RAW264.7 macrophages.Stur-geon muscle hydrolysate is promising as a rich source of anti-inflammatory food ingredients.However,the specific active peptide sequences in the hydrolysate still need to be further explored.Meanwhile,Further studies are necessary to evaluate whether this inhibitory property also affects the secretion of other inflammatory factors and chemokines,and furtherin vivostudies of sturgeon peptides are needed in order to clarify the significance of the current research.

Declaration of Competing Interest

Acknowledgements

There is no conflict of interest associated with authors in this manuscript.This project was supported by China Agriculture Research System(CARS-46).