Antioxidants, Vol. 11, Pages 2333: Purification and Identification of Antioxidant Peptides from Rice Fermentation of Lactobacillus plantarum and Their Protective Effects on UVA−Induced Oxidative Stress in Skin

Q.M.: Formal analysis, Investigation, Data curation, Writing—original draft preparation, Writing—review and editing, Visualization. S.Y.: Methodology, Formal analysis, Investigation, Data curation, Writing—original draft preparation. H.F.: Formal analysis, Investigation, Data curation, Writing—original draft preparation, Visualization. D.W.: Methodology, Data curation, Supervision. J.Z.: Methodology, Data curation, Supervision. C.W.: Conceptualization, Validation, Resources. M.L.: Conceptualization, Resources, Project administration, Validation, Writing—review and editing, Visualization. All authors have read and agreed to the published version of the manuscript.

Figure 1. Purification of peptides from fermentation broth: (a) Purification curves of rice fermentation peptides in chromatography column; (b) elution curves of RP−HPLC for standard samples; and (c) elution curves of RP−HPLC for RFP.

Figure 1. Purification of peptides from fermentation broth: (a) Purification curves of rice fermentation peptides in chromatography column; (b) elution curves of RP−HPLC for standard samples; and (c) elution curves of RP−HPLC for RFP.

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Figure 2. Antioxidant capacity of RFP determined by chemical model method: (a) scavenging rate of DPPH•; (b) scavenging rate of O2•−; (c) scavenging rate of •OH; (d) total antioxidant capacity (ABTS assay); and (e) total antioxidant capacity (FRAP assay). $ p < 0.033 as compared to RP group. Values do not have a common mark ($) when p > 0.033, the same below.

Figure 2. Antioxidant capacity of RFP determined by chemical model method: (a) scavenging rate of DPPH•; (b) scavenging rate of O2•−; (c) scavenging rate of •OH; (d) total antioxidant capacity (ABTS assay); and (e) total antioxidant capacity (FRAP assay). $ p < 0.033 as compared to RP group. Values do not have a common mark ($) when p > 0.033, the same below.

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Figure 3. Effects of UVA, RFP, and RP on survival rate and morphology of HSF: (a) effects of RP and RFP on proliferation of HSF cells; (b) effects of different doses of UVA on survival rate of HSF cells; (c) cell survival rate of HSFs protected by samples; (d) control group; (e) model group; (f) positive group; (g) RP group; and (h) RFP group. # p < 0.033, ## p < 0.002, ### p < 0.001 as compared to control group (C); ** p < 0.002, *** p < 0.001 as compared to damage model group (M). Values do not have a common mark (#, *) when p > 0.033.

Figure 3. Effects of UVA, RFP, and RP on survival rate and morphology of HSF: (a) effects of RP and RFP on proliferation of HSF cells; (b) effects of different doses of UVA on survival rate of HSF cells; (c) cell survival rate of HSFs protected by samples; (d) control group; (e) model group; (f) positive group; (g) RP group; and (h) RFP group. # p < 0.033, ## p < 0.002, ### p < 0.001 as compared to control group (C); ** p < 0.002, *** p < 0.001 as compared to damage model group (M). Values do not have a common mark (#, *) when p > 0.033.

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Figure 4. Antioxidant capacity of RFP determined by cell biology model: (a) effects on ROS content; (b) effects on MDA content; (c) effects on total antioxidant capacity (ABTS assay); and (d) effects on total antioxidant capacity (FRAP assay). # p < 0.033, ## p < 0.002, ### p < 0.001 as compared to control group (C); *p < 0.033, *** p < 0.001 as compared to damage model group (M); $ p < 0.033, $$ p < 0.002 as compared to RP group; Values do not have a common mark (#, *, $) when p > 0.033.

Figure 4. Antioxidant capacity of RFP determined by cell biology model: (a) effects on ROS content; (b) effects on MDA content; (c) effects on total antioxidant capacity (ABTS assay); and (d) effects on total antioxidant capacity (FRAP assay). # p < 0.033, ## p < 0.002, ### p < 0.001 as compared to control group (C); *p < 0.033, *** p < 0.001 as compared to damage model group (M); $ p < 0.033, $$ p < 0.002 as compared to RP group; Values do not have a common mark (#, *, $) when p > 0.033.

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Figure 5. Transcriptome sequencing analysis: (a) principal component analysis (PCA); (b) differentially expressed genes between control and model groups; (c) differentially expressed genes between model and RFP groups; (d) results of GO functional enrichment of the differentially expressed genes of three groups; and (e) results of KEGG functional enrichment of the differentially expressed genes of three groups.

Figure 5. Transcriptome sequencing analysis: (a) principal component analysis (PCA); (b) differentially expressed genes between control and model groups; (c) differentially expressed genes between model and RFP groups; (d) results of GO functional enrichment of the differentially expressed genes of three groups; and (e) results of KEGG functional enrichment of the differentially expressed genes of three groups.

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Figure 6. Effects of RFP on nuclear translocation of NRF2: (a) effect on relative expression of KEAP1; (b) effect on relative expression of NRF2; and (c) effect on protein content of cytoplasm and nucleus NRF2. # p < 0.033, ## p < 0.002, ### p < 0.001 as compared to control group; ** p < 0.002, *** p < 0.001 as compared to damage model group; & p < 0.033, && p < 0.002, &&& p < 0.001 as compared to the group with ATRA. Values do not have a common mark (#, *, &) when p > 0.033.

Figure 6. Effects of RFP on nuclear translocation of NRF2: (a) effect on relative expression of KEAP1; (b) effect on relative expression of NRF2; and (c) effect on protein content of cytoplasm and nucleus NRF2. # p < 0.033, ## p < 0.002, ### p < 0.001 as compared to control group; ** p < 0.002, *** p < 0.001 as compared to damage model group; & p < 0.033, && p < 0.002, &&& p < 0.001 as compared to the group with ATRA. Values do not have a common mark (#, *, &) when p > 0.033.

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Figure 7. Effects of RFP on relative expression of GST and GCL genes: (a) effect on GSTM1; (b) effect on GSTT1; (c) effect on GCLC; and (d) effect on GCLM. # p < 0.033, ## p < 0.002, ### p < 0.001 as compared to control group; * p < 0.033, ** p < 0.002, *** p < 0.001 as compared to damage model group; & p < 0.033, &&& p < 0.001 as compared to the group with ATRA. Values do not have a common mark (#, *, &) when p > 0.033.

Figure 7. Effects of RFP on relative expression of GST and GCL genes: (a) effect on GSTM1; (b) effect on GSTT1; (c) effect on GCLC; and (d) effect on GCLM. # p < 0.033, ## p < 0.002, ### p < 0.001 as compared to control group; * p < 0.033, ** p < 0.002, *** p < 0.001 as compared to damage model group; & p < 0.033, &&& p < 0.001 as compared to the group with ATRA. Values do not have a common mark (#, *, &) when p > 0.033.

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Figure 8. Effects of RFP on transcription expression and content of antioxidant enzymes: (a) effect on transcription expression of HO−1; (b) effect on content of HO−1; (c) effect on transcription expression of NQO1; (d) effect on NQO1content; (e) effect on transcription expression of SOD; (f) effect on SOD content; (g) effect on transcription expression of CAT; (h) effect on CAT content; (i) effect on transcription expression of GSH−Px; and (j) effect on GSH−Px content. # p < 0.033, ## p < 0.002, ### p < 0.001 as compared to control group; * p < 0.033, ** p < 0.002, *** p < 0.001 as compared to damage model group; & p < 0.033, && p < 0.002, &&& p < 0.001 as compared to the group with ATRA. Values do not have a common mark (#, *, &) when p > 0.033.

Figure 8. Effects of RFP on transcription expression and content of antioxidant enzymes: (a) effect on transcription expression of HO−1; (b) effect on content of HO−1; (c) effect on transcription expression of NQO1; (d) effect on NQO1content; (e) effect on transcription expression of SOD; (f) effect on SOD content; (g) effect on transcription expression of CAT; (h) effect on CAT content; (i) effect on transcription expression of GSH−Px; and (j) effect on GSH−Px content. # p < 0.033, ## p < 0.002, ### p < 0.001 as compared to control group; * p < 0.033, ** p < 0.002, *** p < 0.001 as compared to damage model group; & p < 0.033, && p < 0.002, &&& p < 0.001 as compared to the group with ATRA. Values do not have a common mark (#, *, &) when p > 0.033.

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Figure 9. Effects of RFP on H&E staining and antioxidant capacity of mice skin tissues: (a) control group (double steam water); (b) model group (double steam water + UVA); (c) positive control group (1 g/L GSH + UVA); (d) sample group (1 g/L RFP + UVA); (e) effect on antioxidant capacity; and (f) effect on CAT activity. Single arrow indicates irregular epidermal hyperplasia; the double arrow points to the corner plug of the hair follicle. ## p < 0.002, ### p < 0.001 as compared to control group; * p < 0.033, *** p < 0.001 as compared to damage model group. Values do not have a common mark (#, *) when p > 0.033.

Figure 9. Effects of RFP on H&E staining and antioxidant capacity of mice skin tissues: (a) control group (double steam water); (b) model group (double steam water + UVA); (c) positive control group (1 g/L GSH + UVA); (d) sample group (1 g/L RFP + UVA); (e) effect on antioxidant capacity; and (f) effect on CAT activity. Single arrow indicates irregular epidermal hyperplasia; the double arrow points to the corner plug of the hair follicle. ## p < 0.002, ### p < 0.001 as compared to control group; * p < 0.033, *** p < 0.001 as compared to damage model group. Values do not have a common mark (#, *) when p > 0.033.

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Table 1. Primer sequences for real−time PCR.

Table 1. Primer sequences for real−time PCR.

GeneDirectionPrimer Pair Sequence (5′→3′)β−actinFTGGCACCCAGCACAATGAARCTAAGTCATAGTCCGCCTAGAAGCANRF2FCAACTCAGCACCTTGTATCRTTCTTAGTATCTGGCTTCTTKEAP1FGGAGGCGGAGCCCGARGATGCCCTCAATGGACACCAHO−1FCAAGCGCTATGTTCAGCGACRGCTTGAACTTGGTGGCACTGNQO1FCAGCCAATCAGCGTTCGGTARCTTCATGGCGTAGTTGAATGATGTCGCLCFCAGTCAAGGACCGGCACAAGRCAAGAACATCGCCTCCATTCAGGCLMFTAAATCCCGATGAAAGAGRAACAGGAGGTGAAGCAATGSTM1FGAACTCCCTGAAAAGCTAAAGCRGTTGGGCTCAAATATACGGTGGGSTT1FTTCCTTACTGGTCCTCACATCTCRTCACCGGATCATGGCCAGGCGCASODFTGGAGATAATACAGCAGGCTRAGTCACATTGCCCAAGTCTCCATFCCTTCGACCCAAGCAARCGATGGCGGTGAGTGTGSH−PxFAGAAGTGCGAGGTGAACGGTRCCCACCAGGAACTTCTCAAA

Table 2. Peptide sequencing of RFP.

Table 2. Peptide sequencing of RFP.

SequenceLengthMass (Da)PLL3341.23146YNDGDAPIVA101033.4716LLLP4454.31552YNDGDAPIV9962.43453VRVF5575.3319LLSPF4519.31692FYNDGDAPIV101109.5029YNEGDAPVVA101033.4716

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