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The novel peptides ICRD and LCGEC screened from tuna roe show antioxi‐dative activity via Keap1/Nrf2-ARE pathway regulation and gut microbiotamodulation

Jiaojiao Han, Zhongbai Huang, Shasha Tang, Chenyang Lu, Haitao Wan, JunZhou, Ye Li, Tinghong Ming, Zaijie Jim Wang, Xiurong Su

PII: S0308-8146(20)30956-0DOI: https://doi.org/10.1016/j.foodchem.2020.127094Reference: FOCH 127094

To appear in: Food Chemistry

Received Date: 10 October 2019Revised Date: 13 May 2020Accepted Date: 16 May 2020

Please cite this article as: Han, J., Huang, Z., Tang, S., Lu, C., Wan, H., Zhou, J., Li, Y., Ming, T., Jim Wang, Z.,Su, X., The novel peptides ICRD and LCGEC screened from tuna roe show antioxidative activity via Keap1/Nrf2-ARE pathway regulation and gut microbiota modulation, Food Chemistry (2020), doi: https://doi.org/10.1016/j.foodchem.2020.127094

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1 The novel peptides ICRD and LCGEC screened from tuna roe show

2 antioxidative activity via Keap1/Nrf2-ARE pathway regulation and gut

3 microbiota modulation

4 Jiaojiao Hana,b, Zhongbai Huangb, Shasha Tanga,b, Chenyang Lua,b, Haitao

5 Wana,b, Jun Zhoua,b, Ye Lia,b, Tinghong Minga,b, Zaijie Jim Wangc, Xiurong Sua,b*

6 a State Key Laboratory for Managing Biotic and Chemical Threats to the Quality

7 and Safety of Agro-products, Ningbo University, Ningbo, China

8 b School of Marine Science, Ningbo University, Ningbo, China

9 c Department of Biopharmaceutical Sciences, University of Illinois, Chicago,

10 USA

11

12 * Corresponding author

13 Dr. Xiurong Su

14 E-mail address: suxiurong_public@163.com (X.R.S.)

15 Postal address: Ningbo University, 169 Qixing South Road, Ningbo, China

16

17 Running title: Peptides with antioxidative activity derived from tuna roe

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26 Abstract

27 In this study, a high-throughput strategy combined with MALDI TOF/TOF-

28 MS and Discovery Studio 2017 was developed to screen peptides with certain

29 functions from hydrolysate. Two dominant peptides, Ile-Cys-Arg-Asp (ICRD)

30 and Leu-Cys-Gly-Glu-Cys (LCGEC), were predicted to have antioxidant activity

31 by Discovery Studio 2017. Then the activity in vitro of peptides had been

32 confirmed via DPPH assay. Both two peptides decreased apoptosis induced by

33 UVB treatment in HaCaT cells and altered Keap1/Nrf2-ARE pathway

34 transcription. Furthermore, the antioxidant activity of LCGEC was achieved

35 after 6-week treatment in mice via regulating the Keap1/Nrf2-ARE pathway,

36 inhibiting the release of proinflammatory cytokines, increasing the abundance

37 of 3-indolepropionic acid and short-chain fatty acids production in feces and

38 modulating gut microbiota composition. This study provided two tuna roe

39 peptides with in vitro and in vivo antioxidant activity.

40

41 Key words: Peptide; high throughput strategty; tuna roe; antioxidant; gut

42 microbiota; metabolism

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51 Abbreviations

52 DPPH: 1,1-diphenyl-2-picrylhydrazyl

53 IPA: 3-indolepropionic acid

54 SCFA: Short-chain fatty acid

55 ROS: Reactive oxygen species

56 GSH-Px: Glutathione peroxidase

57 SOD: Superoxide dismutase

58 BHT: 2,6-ditert-butyl-4-methyl phenol

59 BHA: Butyl hydroxy anisole

60 MTT: [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolinum bromide]

61 LEfSe: Discriminant analysis effect size

62 RDA: Redundancy analysis

63 OTU: Operational taxonomic unit

64 PCoA: Principal coordinate analysis

65 qRT-PCR: Quantitative real time- polymerase chain reaction

66 TNF-α: Tumor necrosis factor-α

67 IL-6: Interleukin 6

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76 1. Introduction

77 Reactive oxygen species (ROS) are products of normal cell metabolism

78 that stimulate and regulate important biochemical pathways within a certain

79 range (Turrens, 2003). However, accumulation of ROS promotes damage to

80 biomacromolecules and results in pathological changes in tissues (Li et al.,

81 2016). Furthermore, ROS-damaged mitochondria in turn produce more ROS,

82 activating necrotic or apoptotic pathways (Zorov, Juhaszova, & Sollott, 2014).

83 At present, the known antioxidant mechanisms include scavenging of free

84 radicals to block the chain reactions of free radicals, chelation of metal ions to

85 inhibit the production of free radicals catalyzed by metals, and regulation of

86 antioxidant enzymes (Seth, Yan, Polk, & Rao, 2008). The Keap1/Nrf2-ARE

87 pathway is an important antioxidant pathway that regulates antioxidant

88 enzymes such as glutathione peroxidase (GSH-Px) and superoxide dismutase

89 (SOD), which play important roles in antioxidation by converting peroxides to

90 less toxic or harmless substances (Rubiolo, Mithieux, & Vega, 2008). Although

91 2,6-di-tert-butyl-4-methyl phenol (BHT) and butyl hydroxy anisole (BHA) can

92 retard lipid oxidation and citric acid can chelate metal ions, and although these

93 compounds are used as antioxidants, their long-term consumption may lead to

94 a series of side effects (Ito, Hirose, Fukushima, Tsuda, Shirai, & Tatematsu,

95 1986). Therefore, development of other effective and safe antioxidants is

96 necessary.

97 The antioxidant activity of peptides is not only reflected in radicals

98 scavenging in vitro, but also in regulating antioxidant pathway in vivo. Peptides

99 have been the focus of research on natural antioxidants in recent decades.

100 Previous studies have reported that peptides purified from vegetables, plants

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101 and fungi show strong 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical-

102 scavenging and lipid peroxidation-inhibiting activity in vitro and participate in

103 MAPK/NF-κB and PI3K/Akt signaling pathways to regulate oxidation in vivo

104 (Chen et al., 2018; Sun, He, & Xie, 2004; M. Zhang et al., 2018). Although

105 various peptides with antioxidant activity have been obtained, there are certain

106 drawbacks to common peptide screening methods. The traditional purify-and-

107 identify methods are labor-intensive and time-consuming due to the complexity

108 of enzymatic hydrolysates, and iterative rounds of purification and identification

109 are normally needed for a peptide screen. At the same time, the mechanisms

110 of antioxidation have not been clearly explained. Therefore, it is necessary to

111 develop a high-throughput strategy for screening of peptides with certain

112 functions and to subsequently clearly analyze the antioxidant mechanisms of

113 identified peptides.

114 In past decades, it has been found that the gut microbiota is closely related

115 to antioxidation, and many antioxidation-related diseases have been proven to

116 be associated with composition and metabolism changes in the gut microbiota,

117 including non-alcoholic fatty liver disease, obesity and aging (Borrelli et al.,

118 2018; Qiao, Sun, Ding, Le, & Shi, 2013; J. Zhang et al., 2017). The mechanisms

119 by which the gut microbiota enhances antioxidant capacity have been partially

120 explained. On the one hand, gut microbes such as Clostridium and

121 Lactobacillus could regulate Keap1/Nrf2-ARE, PKC and MAPK antioxidant

122 pathways to inhibit oxidative stress (Wang et al., 2012). On the other hand,

123 Lactobacillus shows the ability to capture metal ions and inhibit metal ion

124 oxidation (J. Lee, Hwang, Chung, Cho, & Park, 2005). Therefore, it is necessary

125 and feasible to clarify antioxidant mechanisms in terms of the gut microbiota

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126 and to identify key antioxidation-related bacteria.

127 A number of studies have reported that fish roe has abundant antioxidant

128 functions (Intarasirisawat, Benjakul, & Visessanguan, 2011). However, the

129 structures of the specific peptides with antioxidation activity and the antioxidant

130 mechanism remain unclear. In this study, the sequences and abundances of

131 peptides from tuna roe hydrolysate were identified by MALDI TOF/TOF-MS.

132 The antioxidant activity of the dominant peptides ICRD and LCGEC was

133 predicted with Discovery Studio 2017 and subsequently confirmed by DPPH

134 assay, UVB-irradiated HaCaT cell and mouse experiments. The underlying

135 mechanism of antioxidant activity was clarified with regard to Keap1/Nrf2-ARE

136 pathway regulation and gut microbiota composition and metabolism modulation.

137

138 2. Materials and methods

139 2.1 Prediction of peptide functions by reverse molecular docking

140 Tuna roe was purchased from Ningbo Today Food Co., Ltd (Ningbo,

141 Zhejiang, China). Based on previous studies (J. Han et al., 2018), the optimized

142 enzymolysis parameters of tuna roe were trypsin and alkaline protease

143 combination at the ratio of 1:2, 4 h hydrolysis under 55°C, the enzyme

144 concentration was 3% and the solid-liquid ratio was 1:9. The low molecular

145 weight (≤ 2KDa) peptides were obtained from enzymatic hydrolysate by

146 centrifugation, ultrafiltration and freeze-drying. Then the precise molecular

147 mass and amino acid sequence of the peptides were determined using MALDI-

148 TOF/TOF-MS (AB SCIEX, Applied Biosystems, CA, USA) (J. Han et al., 2018),

149 and the peptides Ile-Cys-Arg-Asp (ICRD) and Leu-Cys-Gly-Glu-Cys (LCGEC)

150 were identified as the major peptides. Then, these two peptides were used as

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151 ligands, and their functions were screened by reverse docking in Discovery

152 Studio 2017 software (Beijing Chong Teng Technology Co., Ltd., Beijing, China)

153 based on the fit values. Pharmacophore with fit-value >3 was considered to

154 interact with peptides. Then, pharmacophore crystal structures with fit-value >3

155 were obtained from the protein data bank (PDB) for docking simulations. The

156 ligand-receptor complex energy scores were measured by CDOCKER docking

157 to determine whether the ligands and receptor proteins bonded.

158 2.2 DPPH radical scavenging assay

159 The peptides ICRD and LCGEC were synthesized by MuJin Bio Tech Co.,

160 Ltd. (Shanghai, China) (Fig. S1). The anti-oxidant effect of ICRD and LCGEC

161 in vitro was estimated according to the method described previously(Babini,

162 Tagliazucchi, Martini, Dei Più, & Gianotti). 0.5 mL of peptides solution with

163 different concentration mixed with 1.5 mL of 100 µM DPPH (Sigma-Aldrich, MO,

164 USA) in ethanol. The mixture solutions stayed in the dark environment for 30

165 min at room temperature. Then the solutions measured by Spectrophotometer

166 (Thermo Fisher Scientific, Waltham, MA, USA) at 517 nm. The inhibitory ratio

167 of DPPH corrected for the blank and was calculated by the Eq(1). Equivalent

168 concentration ascorbic acid (Sigma-Aldrich, MO, USA) was used as the positive

169 control.

170 (1)𝐃𝐏𝐏𝐇 𝐬𝐜𝐚𝐯𝐞𝐧𝐠𝐢𝐧𝐠 𝐞𝐟𝐟𝐞𝐜𝐭(%) = (𝟏 ―𝐀𝐛𝐬𝟏𝐀𝐛𝐬𝟐) × 𝟏𝟎𝟎%

171 where Abs1 is the absorbance value of the peptide, Abs2 is the

172 absorbance value of the blank.

173 2.3 Cell experiment design

174 2.3.1 Cell culture

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175 HaCaT cells were purchased from Beijing Beina Chuanglian Biotechnology

176 Co., Ltd. (Beijing, China) and cultured in DMEM (Sigma, St. Louis, MO, USA)

177 supplemented with 10% fetal bovine serum (HyClone Co., Ltd., Logan, UT,

178 USA) and antibiotics (100 U/ml penicillin, 0.25 µg/ml streptomycin) (Thermo

179 Fisher Scientific, Waltham, MA, USA). The cells were grown at 37 °C in a

180 humidified atmosphere with 5% CO2. When the cells reached over 90%

181 confluence, they were passaged, digested with a 0.05% trypsin–EDTA solution

182 (Shanghai Yuanye Biotechnology Co., Ltd., Shanghai, China) and counted for

183 further analysis.

184 2.3.2 Cellular growth inhibition measurement

185 HaCaT cells were plated into 96-well microplates at a density of 104

186 cells/well and allowed to grow for 24 h at 37 °C. After 24 h of culture with the

187 peptides, the cellular growth of HaCaT cells under different concentrations of

188 peptides (10 μg/ml-100 μg/ml) was measured with the 3-(4,5-dimethylthiazol-2-

189 yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich, MO, USA) method.

190 According to previous study(Kobiela et al., 2017), MTT was dissolved in DMEM

191 (Sigma-Aldrich, MO, USA) at the concentration of 0.5 mg/ml. After cell

192 treatment with the investigated peptides, medium was replaced by 100 µl/well

193 of the MTT solution followed by incubation for 2 h at 37 °C, 5%CO2.

194 Solubilization of formazan product was performed by addition of 100 µl/well of

195 dimethyl sulfoxide (DMSO, Sigma-Aldrich, MO, USA) and shaking for 5 min at

196 room temperature. The sample absorbance was determined at 490 nm

197 wavelengths in a microplate reader (iMark, Bio-Rad, USA). The results are

198 expressed as a percent of unteated control and are an average of six

199 experiments.

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200 2.3.3 Peptide treatment of UVB-irradiated HaCaT cells

201 The HaCaT cells were divided into control, model, ICRD and LCGEC

202 groups, with six parallel samples in each group. The cells in the control and

203 model groups were cultured with DMEM for 4 h, while those in the ICRD and

204 LCGEC peptide treatment groups were cultured with 50 μg/ml ICRD and 50

205 μg/ml LCGEC, respectively, for 4 h according to the results of the cellular

206 growth inhibition experiment. Then, for all groups, the culture medium was

207 removed, PBS was added to cover the cells, and the cells were irradiated with

208 40 mJ/cm2 UVB; however, the control group wells were covered with aluminum

209 foil. After UVB irradiation, the original medium was returned to the cells, which

210 were cultured for further 20 h. Laser scanning confocal microscopy and flow

211 cytometry were used to analyze the effects of the peptides on apoptosis with

212 the Annexin V-FITC/PI (Beyotime Biotechnology Co., Ltd, Shanghai, China)

213 double labeling method. Based on previous study(Ghosh, Gelman, & Maxfield,

214 1994a), after 24 h of culture, cells were washed twice with chilled PBS and 106

215 cells were collected. 500 μL buffer were added to suspend cells, followed by

216 the addition of 5 μL Annexin V-FITC and PI respectively. Then, cells were

217 allowed to react under room temperature in dark condition for 15 min. Afterward,

218 105 cells were taken and smeared with a fluorescent glycerol free patch. They

219 were observed immediately by using laser scanning confocal microscopy (Bio-

220 Rad Microscience, Cambridge, MA) and fluorescence images were obtained.

221 (Ghosh, Gelman, & Maxfield, 1994b). Another 105 cells were selected out via

222 flow cytometry (Villamón, González-Fernández, Such, Cervera, Gozalbo, & Gil,

223 2018) with specific voltage and fluorescence compensation. Cytometric data

224 were analyzed through Kaluza software (Beckman Coulter).

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225 2.3.4 Determination of intracellular oxidation indicators

226 After 24 h of culture with the peptides, the medium was discarded, and the

227 cells were collected and washed twice with PBS. The supernatants were

228 discarded after centrifugation, and the pellets were homogenized with

229 physiological saline at a 1:9 ratio (w/v) to obtain whole homogenates. After

230 centrifugation at 12,000 × g for 20 min at 4 °C, the supernatants were collected.

231 The levels of SOD, GSH-Px and MDA in the supernatants were measured with

232 commercial kits (Suzhou Comin Biotechnology Co., Ltd., Suzhou, Jiangsu,

233 China) according to the manufacturer’s instructions.

234 2.3.5 Total RNA extraction and quantification of gene expression

235 As previously described (D. Zhang et al., 2015), total RNA from cell was

236 extracted using commercial TransZol Up Plus RNA Kit (TransGen Biotech,

237 Beijing, China). Subsequently, reverse transcription was achieved using total

238 RNA as the starting material and the TransScript All-in-One First-Stand cDNA

239 kit (TransGen Biotech, Beijing, China). The transcriptional levels of genes were

240 determined by quantitative real-time RT-PCR (qRT-PCR) performed on a

241 Rotor-Gene 6000 realtime PCR machine (Corbett, Australia) with SYBR®

242 Premix Ex TaqTM II according to the manufacturer’s protocols. The primers

243 were designed using software Primer 5 and listed in Supplementary Table S1.

244 The relative expression levels of the target genes were normalized internally to

245 the 16S rDNA level and quantified using the 2-△△CT method with β-actin as the

246 housekeeping gene.

247 2.4 Animal trial

248 2.4.1 Animal experiment design

249 Sixteen male ICR mice (25 ± 2 g) were purchased from the Laboratory

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250 Animal Center of Zhejiang Province (Hangzhou, Zhejiang, China). After two

251 weeks of acclimatization, they were randomly designated into control and

252 experimental groups. Fecal samples were collected immediately upon

253 defecation from each mouse at baseline (0 weeks) and stored at −80 °C. The

254 mice in the experimental group received 10 mg/kg/d of LCGEC by gavage, and

255 the mice in the control group received the same amount of normal saline by

256 gavage. The mice had free access to water and feed in a temperature-

257 controlled room (23-25°C) under a 12-hour dark-light cycle. The body weight of

258 each mouse was recorded every four days. After 6 weeks of feeding, fecal

259 samples were immersed in liquid nitrogen immediately and stored at −80°C,

260 and the mice were anaesthetized with ether. Then, blood samples were

261 collected from the retro-orbital plexus. The serum was separated via

262 centrifugation and stored at −80 °C. The mice were sacrificed by cervical

263 dislocation. The liver and brain were removed from each mouse, weighed and

264 immersed in liquid nitrogen immediately for further analysis.

265 2.4.2 Dosage information

266 Based on our previous study and on other studies focused on antioxidant

267 activity, the dose of hydrolysate suitable for mice is 200-400 mg/kg/d (J. Han et

268 al., 2018; E. J. Lee et al., 2017). In this study, the content of LCGEC in the

269 hydrolysate was 2.6%. Therefore, in the experimental group, the mice were

270 given 10 mg/kg/d of LCGEC, which is equivalent to 49.8 mg/d in humans

271 (Reagan-Shaw, Nihal, & Ahmad, 2008).

272 2.4.3 Analysis of biochemical parameters

273 The activity of MDA, SOD and GSH-Px in the serum and liver was

274 determined according to the instructions for each kit (Nanjing Jiancheng

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275 Bioengineering Institute, Nanjing, Jiangsu, China). The 3-indolepropionic acid

276 (IPA) content in feces was determined according to the instructions of the kit

277 (Shanghai Enzyme Linked Biotechnology Co., Ltd., Shanghai, China).

278 2.4.4 Determination of short-chain fatty acids (SCFAs) in feces

279 As in previous studies, 150±3 mg fecal samples were extracted with 10 mL

280 of a mixture containing water, 50% of H2SO4 and ether (8:2:10). Subsequently,

281 the mixtures were shaken and centrifuged, CaCl2 was added to the supernatant

282 to absorb the water, and a 0.22 μm filter was used before injecting the

283 supernatant into a GC-MS system (Agilent 7890 gas chromatography coupled

284 to an M7-80E mass spectrometric detector) (X. Han et al., 2018). Acetic acid,

285 propionic acid, butyric acid and valeric acid were purchased from Sigma, and

286 standard solutions with known concentrations were prepared and used to

287 construct a GC standard curve (Supplementary Table S2). The content of each

288 SCFAs was calculated by the standard curve of SCFAs and mass of fecal

289 sample.

290 2.4.5 Total DNA extraction, PCR and sequencing of fecal samples

291 Reference to previous studies, a QIAamp DNA Stool Mini Kit was used to

292 extract total DNA from fecal samples(Wu, Lin, Chang, Lin, & Lai, 2018). The

293 PCR primers were designed based on the sequence of the V3-V4 regions of

294 the bacterial 16S rRNA gene and the amplification were conducted using a

295 universal forward primer (5′- (10-base barcode)- CCTACGGGAGGCAGCAG-

296 3′) and a reverse primer (5′- GACTACHVGGGTATCTAATCC -3′). Sequencing

297 was performed using the MiSeq system (Illumina, San Diego, CA, USA) at LC

298 Sciences Co., Ltd. (Hangzhou, China).

299 Raw FASTQ files were multiplexed and filtered using QIIME (version 1.8.0)

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300 according to the following steps: (1) reads with more than 10 bp of overlap were

301 merged via software FLASH (version1.2.11) and reads that could not be

302 merged were removed; (2) data belonging to each sample were identified

303 through the barcode sequence; (3) reads were truncated at any position when

304 the average quality score on the 10-bp sliding window was below 20; and (4)

305 Reads that contained undetected nucleotides (N) or were shorter than 200 bp

306 were removed. Chimera sequences were identified and removed using Uchime

307 (version 4.2.40). The operational taxonomic units (OTUs) were clustered using

308 Usearch (version 7.1). The α-diversity analysis was calculated using mothur

309 (version 1.36.0). The most abundant sequences in each OTU were used for

310 taxonomic classification by Ribosomal Database Project (RDP) Classifier. A

311 principal coordinate analysis (PCoA) was completed via Muscle (version

312 3.8.31). Biomarker identification was performed using the linear discriminant

313 analysis (LDA) effect size (LEfSe) method to characterize the gut microbiota

314 characteristics specific to the different treatments. Redundancy analysis (RDA)

315 was used to construct models with log-converted OTU relative abundances,

316 and key OTUs that were different between the two groups were identified with

317 Canoco 5.0 (Microcomputer Power, Ithaca, NY, USA). The groups were used

318 as the environmental variables. Correlations between key OTU abundances

319 and phenotypes of antioxidation were calculated via Spearman’s correlation

320 analysis (SPSS, version 19.0, Chicago, IL, USA). Correlations were defined as

321 significant for a P<0.05 and a false discovery rate <0.25.

322 2.5 Statistical analysis

323 The data are shown as the mean ± standard deviation (SD). Normally

324 distributed data were assessed by ANOVA followed by Tukey’s post hoc test

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325 (SPSS, version 19.0, Chicago, IL, USA). P<0.05 was considered to be the

326 standard criterion of statistical significance.

327 2.6 Accession Numbers

328 The sequence had been deposited at NCBI Sequence Read Archive

329 Database under the accession number SRP199102.

330 3. Results

331 3.1 ICRD and LCGEC were the major peptides in tuna roe hydrolysate

332 The sequences of amino acids in the tuna roe hydrolysate were determined

333 via MALDI-TOF/TOF-MS, and the peptides with the greatest representation are

334 listed in Supplementary Table S3. In this study, most of the parent ions were

335 less than m/z 1000; the parent ions at m/z 506.1045 and m/z 524.1029 had the

336 highest signal intensities, which were determined to be the peptides Ile-Cys-

337 Arg-Asp (ICRD) and Leu-Cys-Gly-Glu-Cys (LCGEC), respectively (Fig. 1A).

338 3.2 ICRD and LCGEC were predicted to interact with Keap1

339 The peptide functions were predicted via reverse docking in Discovery

340 Studio 2017 and were ordered by fit value (Supplementary Table S4 and S5).

341 Both ICRD and LCGEC showed interactions with Keap1 (PDB ID: 5dad) with

342 high fit values; Keap1 plays an important role in regulating oxidative stress and

343 is involved in the Keap1/Nrf2-ARE pathway to regulate antioxidant enzymes.

344 Therefore, we further verified the interaction via molecular docking in Discovery

345 Studio 2017. Both ICRD and LCGEC shared similar docking sites with TX6

346 (Fig.1B), a confirmed Keap1 inhibitor, and they had a better binding ability than

347 TX6, indicated by a lower CDOCKER interaction energy (CIE) (Supplementary

348 Table S6). In addition, ICRD had three amino acid residues involved in four

349 hydrogen bonds (HBs) with Keap1 residues, while LCGEC had six amino acid

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350 residues that established eight HBs (Fig.1B). Moreover, there were 10 and 9

351 amino acid residues of Keap1 that interacted with LCGEC and ICRD,

352 respectively (Fig.1C). Therefore, the peptides ICRD and LCGEC were

353 predicted to have antioxidant activity.

354 3.3 DPPH radical scavenging capacity

355 Based on the prediction results of DS, DPPH method was used to verify

356 the antioxidant activity of peptides in vitro, and the ascorbic acid was used as

357 a standard (Fig. 2A and Supplementary Table S7). Results showed that the

358 antioxidant capacity of ICRD and LCGEC was increased with the increase of

359 concentration. The DPPH inhibitory ratio of LCGEC was higher than ICRD,

360 however, it was significantly lower than that of ascorbic acid at the same

361 concentration.

362 3.4 Growth inhibitory effects of ICRD and LCGEC on HaCaT cells

363 The viability of HaCaT cells was analyzed by MTT assay after 24 h of

364 treatment with different peptide concentrations (Fig. 2B). The results showed

365 that neither peptide inhibited the cells in the investigated concentration range

366 (10 μg/ml-100 μg/ml), and the beneficial effect on cell proliferation was

367 maximized when the concentration was 50 μg/mL for both peptides.

368 3.5 ICRD and LCGEC treatment attenuated apoptosis induced by UVB

369 Laser scanning confocal microscopy was used to qualitatively analyze the

370 effects of the peptides on apoptosis in UVB-irradiated HaCaT cells. Compared

371 with those in the model group, the numbers of early apoptotic (green) and

372 middle/late apoptotic (red) cells were decreased in the ICRD and LCGEC

373 groups (Fig. 2C).

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374 Next, flow cytometry was used to quantitatively analyze apoptosis rates.

375 Compared with those in the control group (0%), the percentages of necrotic

376 cells (Q1 quadrant, 0.07±0.01%), late apoptotic cells (Q2 quadrant, 51.0±1.90%)

377 and early apoptotic cells (Q3 quadrant, 15.80±1.93%) in the model group

378 significantly increased after UVB radiation, whereas the percentages of live

379 cells (Q4 quadrant, 33.13±1.27%) decreased significantly after UVB radiation,

380 and the total apoptosis rate reached 66.8±1.27%. Compared with those in the

381 model group, the percentages of necrotic cells, late apoptotic cells and early

382 apoptotic cells were significantly decreased in the ICRD group (to 0%,

383 6.62±0.51%, and 4.13±0.95%, respectively) and the LCGEC group (to 0%,

384 6.42±0.60%, and 3.68±0.74%, respectively), whereas the percentages of live

385 cells were significantly increased in the ICRD group (to 89.25±0.46%) and the

386 LCGEC group (to 89.90±0.79%); the total apoptosis rate was reduced to

387 10.75±0.46% and 10.1±0.79% in the ICRD group and the LCGEC group,

388 respectively (Fig. 2D).

389 3.6 ICRD and LCGEC improved antioxidant activity and regulated the

390 Keap1/Nrf2-ARE pathway in cells

391 Compared with the control group, the model group showed significantly

392 decreased levels of SOD (62.77±4.18 U/ml, P<0.01) and GSH-Px (45.17±1.11

393 U/ml, P<0.01) but increased levels of MDA (11.83±2.32 nmol/ml, P<0.05).

394 However, both ICRD and LCGEC treatment restored these three indices, and

395 LCGEC had a better antioxidant effect than ICRD (Fig. 3A).

396 After UVB radiation, the transcription of Keap1 and Nrf2 was upregulated,

397 but the transcription of Maf and antioxidant enzymes was downregulated. After

398 peptide treatment, the transcription of Keap1, Maf, CAT, GST and Mn-SOD was

17

399 restored to the level in the control group, while the transcription of Nrf2, Cu-

400 SOD and GSH-Px was further increased compared to that in the model group

401 (Fig. 3B and Fig. S2). In addition, compared with ICRD, LCGEC treatment

402 caused relatively higher transcription of antioxidation-related genes.

403 3.7 LCGEC improved antioxidant activity and regulated the Keap1/Nrf2-

404 ARE pathway in healthy mice

405 Considering that LCGEC had better antioxidant activity than ICRD in the

406 cell experiments, LCGEC was chosen for follow-up animal experiments.

407 Compared with the values in the control group, the body weight in the LCGEC

408 group increased gradually, whereas the liver and brain indices decreased (Fig.

409 4A and 4B). The levels of serum GSH-Px (888.42±209.40 U/ml) and SOD

410 (86.57±6.45 U/ml) in the LCGEC group were significantly increased, while the

411 levels of MDA (6.60±1.58 nmol/ml) were significantly decreased. The condition

412 in the liver was similar to that in the serum (Fig. 4C), with increased GSH-Px

413 and SOD levels and decreased MDA levels in the LCGEC group compared to

414 the control group. In addition, the transcription of Keap1 and Maf was

415 significantly downregulated in the liver and brain, whereas that of the

416 antioxidant enzymes was upregulated (Fig. 4D).

417 3.8 Effects of LCGEC on the IPA and SCFA levels in feces

418 LCGEC treatment significantly increased the content of 3-indolepropionic

419 acid (IPA) in feces from 3.47±0.11 μg/g to 3.93±0.10 μg/g (Fig. 5A), and the

420 levels of acetic acid, propionic acid, butyric acid and valeric acid were increased

421 by 8%, 15.3%, 22.7% and 1.75%, respectively (Fig. 5B).

422 3.9 LCGEC altered gut microbiota structure in mice

423 After 6 weeks of feeding, fecal samples were sequenced to elucidate the

18

424 effects of LCGEC treatment on gut microbiota structure. The richness

425 (observed species) and diversity (Shannon) were increased in the LCGEC

426 treatment group compared to the control group (Fig. 5C).

427 The overall structure of the gut microbiota in the two groups was analyzed

428 via weighted UniFrac PCoA (Fig. 5D). There were no obvious differences in gut

429 microbiota composition at 0 weeks between the control group and the LCGEC

430 group (red circle vs. blue circle). However, after 6 weeks of treatment, the gut

431 microbiotas of the groups were different and had shifted far from each other.

432 RDP Classifier was used to analyze variations in gut microbiota

433 composition in the mice given LCGEC. The most abundant phyla in the control

434 group included Bacteroidetes (69.79±0.99%), Firmicutes (25.94±0.72%) and

435 Proteobacteria (1.84±0.22%), and the ratio of Firmicutes to Bacteroidetes was

436 0.37±0.01. Compared to the control treatment, LCGEC treatment decreased

437 the abundance of Bacteroidetes (to 68.59±1.82%) and Proteobacteria (to

438 1.03±0.17%) and increased the abundance of Firmicutes (to 27.58±1.90%) as

439 well as the ratio of Firmicutes to Bacteroidetes (to 0.40±0.04). In addition,

440 Bacteroidia, Bacilli and Clostridia were the major classes. There was no

441 significant difference in the abundance of Bacteroidia between control group

442 (69.79±0.99%) and LCGEC group (67.59±1.39%, P>0.05), and the abundance

443 of Bacilli and Clostridia increased from 10.06±1.58% and 14.38±3.16% in the

444 control group to 11.35±3.15% and 14.91±3.02% in the LCGEC group,

445 respectively (P>0.05) (Fig. 5E and Supplementary Table S8).

446 LEfSe was used to identify biomarkers and revealed that the phylum

447 Proteobacteria, the class Epsilonproteobacteria and the order

448 Campylobacterales were prevalent in the control group, while the class

19

449 Clostridia and the order Clostridiales were prevalent in the LCGEC group (Fig.

450 5F).

451 In addition, 71 key OTUs responding to LCGEC treatment were identified

452 via RDA (Fig. S3 and Supplementary Table S9). Among the 71 OTUs, 14 OTUs

453 were upregulated (7 with a fold change>4), and 57 OTUs were downregulated

454 (46 with a fold change>4) in the LCGEC group compared with the control group

455 (Fig. S4). Spearman’s correlation analysis was carried out to correlate the 71

456 OTUs with SOD, GSH-Px, and MDA levels in the serum and liver. Among the

457 71 OTUs, 25 OTUs were positively correlated with antioxidant indices, and 46

458 OTUs were negatively correlated with antioxidant indices. Twenty-three key

459 OTUs were significantly associated with at least three physiological indices;

460 these OTUs belonged to Clostridia (n=10), Firmicutes (n=4), Bacteroidia (n=4),

461 Epsilonproteobacteria (n=2) and Bacilli (n=2) at the class level. Among them, 4

462 key OTUs were significantly associated with five indices; these OTUs belonged

463 to the genera Helicobacter, Ruminococcus, Clostridium and Lactobacillus (Fig.

464 6).

465 4. Discussion

466 ROS cause oxidative damage and dysregulation of normal metabolism and

467 physiology. Generally, synthetic antioxidants have been used to reduce

468 oxidation, but there are safety concerns over the use of synthetic antioxidants.

469 Developing new safe and effective antioxidation alternatives is necessary.

470 Peptides have been reported to be important antioxidants, but it is hard to

471 directionally screen peptides with certain functions with high throughput from

472 enzymatic hydrolysates and to clarify the antioxidant mechanisms. In this study,

473 the functions of ICRD and LCGEC were predicted in Discovery Studio 2017

20

474 with high throughput. Then, the antioxidant activity of these peptides was

475 verified via DPPH assay, cell and animal experiments, and the underlying

476 mechanisms were elucidated by gene transcription quantification, metabolite

477 measurement and gut microbiota composition analysis.

478 Previous studies have indicated that the activity of peptides depends on

479 their amino acid sequences. Due to the high oxygen radical-scavenging activity

480 of Met (M), Trp (W), Tyr (Y) and Cys (C), peptides rich in these amino acids,

481 such as Trp-Tyr-Ser-Leu-Ala-Met-Ala-Ala-Ser-Asp-Ile (WYSLAMAASDI)

482 isolated from whey protein, show strong antioxidant activity (Hernández-

483 Ledesma, Dávalos, Bartolomé, & Amigo, 2005). Both ICRD and LCGEC have

484 Cys, which may be the reason for peptides have antioxidant activity. In addition,

485 peptide chain length is also considered to be an important factor in antioxidant

486 activity. Peptides with molecular weights in the range of 0.5-3 kDa (with a length

487 of approximately 4-24 amino acids) have been suggested to have better

488 antioxidant activity than peptides with molecular weights >3 kDa (Nalinanon,

489 Benjakul, Kishimura, & Shahidi, 2011). This better activity may be due to the

490 high activity, easy absorption and low toxicity of peptides with small molecular

491 weights (Dei Piu et al., 2014).

492 Peptides can not only scavenge free radical activity in vitro, but also play

493 an antioxidant role in vivo. In this study, ICRD and LCGEC were predicted to

494 bind with Keap1. Keap1 is not only a multidomain repressor protein but also an

495 inhibitor of Nrf2, which is the main regulatory factor of cellular redox reactions

496 and is widely distributed in various organs of the body. When cells are

497 stimulated by ROS, Nrf2 is uncoupled from Keap1 and activated. From the

498 results of qRT-PCR in the cell and animal experiments, the levels of Nrf2 were

21

499 increased significantly after peptide treatment. After uncoupling, Nrf2

500 translocates into the cell nucleus and binds to antioxidant response elements

501 (AREs), which are specific DNA binding sequences located in the 5' terminal

502 promoter sequence of the protective gene SOD; Nrf2 thus regulates the

503 transcriptional activity of antioxidant enzymes, playing a role in resistance to

504 oxidative damage (J.-M. Lee, Calkins, Chan, Kan, & Johnson, 2003) and

505 inducing increased transcription levels of HO-1, NQO1, GCLC, GCLM, GST,

506 GSH-Px, Cu-SOD and Mn-SOD in both cells and mice after LCGEC treatment.

507 In addition, the relatively higher mRNA levels of antioxidation-related genes

508 after LCGEC treatment than after ICRD treatment indicated that LCGEC had

509 better antioxidant effects. This study confirmed the antioxidant activity of

510 peptides, and found the regulation effects of them on Keap1/Nrf2-ARE pathway,

511 but current data cannot make the conclusion whether these peptides directly

512 bind to Keap1, which is needed to be confirmed in the future research.

513 Gut microbiota metabolites, such as IPA and SCFAs, have effects on

514 antioxidant activity. IPA is a strong antioxidant that eliminates free radicals and

515 acts synergistically with glutathione, which is synthesized by Clostridium in the

516 intestine (Hwang et al., 2009). In this study, the content of IPA in fecal samples

517 was increased after LCGEC treatment, as was the abundance of Clostridium.

518 In addition, SCFAs produced by SCFA-producing bacteria (e.g., Ruminococcus

519 and Clostridium) have been reported to promote antioxidant activity by reducing

520 the expression of inducible nitric oxide synthase in the liver and promoting the

521 synthesis of melatonin in the duodenum (Jin, Sellmann, Engstler, Ziegenhardt,

522 & Bergheim, 2015). Moreover, one SCFA, butyric acid, which is specifically

523 produced by butyric acid producers (Bacteroides, Bacilli and Clostridium, etc.),

22

524 exerts additional antioxidant properties by increasing the activity of antioxidant

525 enzymes. In this study, the content of SCFAs increased after LCGEC treatment,

526 and the abundance of SCFA producers and butyric acid producers increased.

527 On the other hand, SCFAs show anti-inflammatory activity. There is a

528 complementary relationship between inflammation and oxidation. Previous

529 studies have indicated that the NLRP3 inflammasome regulates the expression

530 of proinflammatory factors (such as TNF-α and IL-6), and ROS are the basic

531 activators of NLRP3. Effectively inhibiting the expression of proinflammatory

532 factors can also reduce the accumulation of ROS (Zhou, Yazdi, Menu, &

533 Tschopp, 2011). In this study the proinflammatory factors TNF-α and IL-6 were

534 decreased after LCGEC treatment (Fig. S5). In addition, some bacteria, such

535 as Helicobacter and Lactobacillus, also regulate inflammatory factors (Axling et

536 al., 2012; Davì et al., 2005). The abundance of Helicobacter was decreased

537 after LCGEC treatment, while the abundance of Lactobacillus was increased.

538 These findings were consistent with the transcription levels of the inflammatory

539 factors.

540 In addition to indirectly exerting antioxidant activity through anti-

541 inflammatory activity, Lactobacillus, which was significantly increased after

542 LCGEC treatment, also plays an antioxidant role directly. It has been well

543 recognized that probiotic Lactobacillus possesses strong antioxidant activity

544 and is able to reduce the risk of accumulation of ROS (Achuthan et al., 2012).

545 Studies have shown that Lactobacillus plantarum YW11 isolated from Tibetan

546 kefir can modulate the gut microbiota and improve antioxidant status in aging

547 mice (J. Zhang et al., 2017). It has been reported that two strains of

548 Lactobacillus with significant antioxidant activity serve as defensive agents in

23

549 the gut microbiota ecosystem and overcome exogenous and endogenous

550 oxidative stress (Kullisaar et al., 2002).

551 In conclusion, the peptides ICRD and LCGEC obtained in this study

552 regulated the Keap1/Nrf2-ARE pathway and exhibited antioxidant activity in

553 DPPH assay, cells and mice experiment. In addition, LCGEC treatment

554 modulated gut microbiota composition and increased the production of gut-

555 derived metabolites (IPA and SCFAs).

556

557 Ethics statement

558 All of the experimental procedures and animal care were performed in

559 accordance with the Guide for the Care and Use of Laboratory Animals

560 prepared by the Ningbo University Laboratory Animal Center (affiliated with the

561 Zhejiang Laboratory Animal Common Service Platform), and all of the animal

562 protocols were approved by the Ningbo University Laboratory Animal Center

563 under permit number No. SYXK (ZHE 2008-0110).

564

565 Acknowledgments

566 This work was sponsored by the National Key R&D Program of China

567 (grant number 2018YFD0901102), the Natural Science Foundation of Zhejiang

568 Province (grant number LY18C010001 and LY19C010003), and K.C. Wong

569 Magna Fund of Ningbo University. We thank Nature Research Editing Service

570 for English language editing.

571

572 Conflict of interest

573 All authors declare that they have no conflict of interest.

24

574

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695 Figure Legends

696 Fig. 1 Structural identification and functional prediction of major peptides.

697 (A) MALDI-TOF/TOF tandem mass spectra of the peptides. (B)The interactions

698 of the peptides with Keap1. (C) The interactions of the amino acid residues of

699 TX6, ICRD and LCGEC with Keap1. Red indicates amino acid residues shared

700 by all three peptides. Blue indicates amino acid residues shared by TX6 and

701 ICRD. Gray indicates amino acid residues shared by TX6 and LCGEC.

702 Fig. 2 The DPPH radical scavenging assay and inhibitory effects of the

703 peptides on apoptosis in UVB-irradiated HaCaT cells. (A) The DPPH radical

704 scavenging ability of LCGEC and ICRD, with ascorbic acid as a control. The

705 data are expressed as the mean ± SD, n=3. *, P<0.05; **, P<0.01, compared to

706 the ascorbic acid. (B) The viability of HaCaT cells treated with LCGEC and

707 ICRD at different concentrations. The data were normalized to the mean control

708 value. (C) Qualitative analysis of apoptosis in UVB-irradiated HaCaT cells

709 treated with peptides via laser scanning confocal scanning microscopy. The

710 first column shows PI fluorescence, the second column shows Annexin V-FITC

711 fluorescence, and the third column shows double labeling. The early cell

712 apoptotic cells are green, and the middle and late apoptotic cells are red. (D)

713 Quantitative analysis of apoptosis in UVB-irradiated HaCaT cells treated with

714 peptides via flow cytometry. Four kinds of cells can be distinguished in the

715 scatter plot determined by flow cytometry: necrotic cells, in the Q1 quadrant,

716 are Annexin V-FITC negative and PI positive (FITC-PI+); late apoptotic cells, in

717 the Q2 quadrant, are Annexin V-FITC and PI positive (FITC+PI+); early

718 apoptotic cells, in the Q3 quadrant, are Annexin V-FITC positive and PI

719 negative (FITC+PI-); and live cells, in the Q4 quadrant, are Annexin V-FITC and

28

720 PI negative (FITC-PI-). The graph in C shows the representative results for

721 each group. The total apoptotic rate of each group is shown on the right side.

722 The results are shown as the mean ± SD, n=6.

723 Fig. 3 Effects of the peptides on antioxidant enzyme activity and related

724 gene transcription in UVB-irradiated HaCaT cells. (A) Effects of the peptides

725 on antioxidant enzyme activity in HaCaT cells irradiated with UVB. (B) Analysis

726 of the antioxidant effects of the peptides on cells at the mRNA level. The results

727 are expressed as the mean ± SD, n=6. a-d represent significant differences

728 among groups according to the one-way ANOVA and Tukey's post hoc test (P

729 < .05).

730 Fig. 4 Effects of the peptides on antioxidant enzyme activity and related

731 gene transcription in mice. (A) The body weights of the mice in the two groups.

732 (B) Effects of the peptides on the liver and brain indexes in mice. (C) Effects of

733 LCGEC on antioxidant enzyme activity in mice. (D) Effects on the transcription

734 of related proteins in the Keap1/Nrf2-ARE antioxidant pathway in the mouse

735 liver and brain. The data are expressed as the mean ± SD, n=8. *, P<0.05; **,

736 P<0.01, compared to the control group.

737 Fig. 5 Effect of LCGEC treatment on gut microbiota metabolism and

738 microbiota structure. (A) The content of IPA in feces. (B) The content of

739 SCFAs in feces. (C) The alpha diversity of the gut microbiota in the two groups.

740 (D) Variations in the gut microbiota structures of mice treated with LCGEC via

741 weighted UniFrac PCoA. (E) RDP classifications of microbial composition at the

742 phylum level and class level. (F) The biomarkers identified via LEfSe in the fecal

743 microbiota. Red, control group; green, LCGEC group. The LDA score is

744 displayed for taxa meeting an LDA significance threshold of > 3. The data are

29

745 expressed as the mean ± SD, n=8. *, P<0.05; **, P<0.01, compared to the

746 control group.

747 Fig. 6 Heatmap of key OTUs responding to LCGEC treatment identified via

748 RDA. (A) Heatmap of the abundance 71 key OTUs. (B) The correlation of 71

749 OTUs with SOD, GSH-Px and MDA levels in the liver and serum. (C) The

750 bacterial taxon information on the OTUs (genera and phyla). The colors of the

751 spots in panel A represent the OTU relative abundance values of the groups.

752 The OTUs are sorted according to their phylogenetic positions. The OTUs

753 labeled in red are OTUs with enhanced abundance, and the OTUs labeled in

754 gray are OTUs with reduced abundance (compared with the control group). The

755 spot colors in panel B represent the Spearman’s correlation R-values between

756 the OTUs and SOD, MDA, and GSH-Px levels in the liver and serum. *, P<

757 0.05; **, P< 0.01; ★, key OTUs that were significantly associated with at least

758 four physiological indexes. The taxonomy (phyla and genera) of the OTUs are

759 shown on the right.

30

760

761 Fig.1

31

762

763 Fig.2

764

765

32

766

767 Fig.3

33

768

769 Fig.4

34

770

771 Fig.5

35

772

773 Fig.6

774

775

776 Credit Author Statement：777

778 Jiaojiao Han: Conceptualization, Methodology, Writing - Original Draft, Writing

779 - Review and Editing. Zhongbai Huang: Data Curation, Formal analysis,

780 Writing - Original Draft. Shasha Tang: Data Curation, Validation. Chenyang

781 Lu: Project administration, Supervision. Haitao Wan: Investigation. Jun Zhou:

782 Visualization, Software. Ye Li: Formal analysis. Tinghong Ming: Resources.

783 Zaijie Jim Wang: Supervision. Xiurong Su: Project administration,

784 Supervision.

36

785

786

787 Declaration of interests

788

789 ☒ The authors declare that they have no known competing financial interests or personal

790 relationships that could have appeared to influence the work reported in this paper.

791

792 ☐The authors declare the following financial interests/personal relationships which

793 may be considered as potential competing interests:

794

795796

797

798

799

800 HIGHLIGHTS

801 1. Two antioxidant peptides ICRD and LCGEC were screened and identified from tuna roe.

802 2. ICRD and LCGEC regulated Keap1/Nrf2-ARE pathway transcription in cell and mice.

803 3. LCGEC regulated gut microbiota structure and metabolism in mice.

37

804

805

806