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Mar Drugs
2024 Jul 21;227:. doi: 10.3390/md22070325.
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Proteomics Analysis of the Protective Effect of Polydeoxyribonucleotide Extracted from Sea Cucumber (Apostichopus japonicus) Sperm in a Hydrogen Peroxide-Induced RAW264.7 Cell Injury Model.
Shu Z
,
Ji Y
,
Liu F
,
Jing Y
,
Jiao C
,
Li Y
,
Zhao Y
,
Wang G
,
Zhang J
.
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Sea cucumber viscera contain various naturally occurring active substances, but they are often underutilized during sea cucumber processing. Polydeoxyribonucleotide (PDRN) is an adenosine A2A receptor agonist that activates the A2A receptor to produce various biological effects. Currently, most studies on the activity of PDRN have focused on its anti-inflammatory, anti-apoptotic, and tissue repair properties, yet relatively few studies have investigated its antioxidant activity. In this study, we reported for the first time that PDRN was extracted from the sperm of Apostichopus japonicus (AJS-PDRN), and we evaluated its antioxidant activity using 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS), and hydroxyl radical scavenging assays. An in vitro injury model was established using H2O2-induced oxidative damage in RAW264.7 cells, and we investigated the protective effect of AJS-PDRN on these cells. Additionally, we explored the potential mechanism by which AJS-PDRN protects RAW264.7 cells from damage using iTRAQ proteomics analysis. The results showed that AJS-PDRN possessed excellent antioxidant activity and could significantly scavenge DPPH, ABTS, and hydroxyl radicals. In vitro antioxidant assays demonstrated that AJS-PDRN was cytoprotective and significantly enhanced the antioxidant capacity of RAW264.7 cells. The results of GO enrichment and KEGG pathway analysis indicate that the protective effects of AJS-PDRN pretreatment on RAW264.7 cells are primarily achieved through the regulation of immune and inflammatory responses, modulation of the extracellular matrix and signal transduction pathways, promotion of membrane repair, and enhancement of cellular antioxidant capacity. The results of a protein-protein interaction (PPI) network analysis indicate that AJS-PDRN reduces cellular oxidative damage by upregulating the expression of intracellular selenoprotein family members. In summary, our findings reveal that AJS-PDRN mitigates H2O2-induced oxidative damage through multiple pathways, underscoring its significant potential in the prevention and treatment of diseases caused by oxidative stress.
Figure 1. AJS-PDRN scavenging assays for DPPH, ABTS, and hydroxyl radicals. (A) AJS-PDRN scavenging assay of DPPH radicals; (B) AJS-PDRN scavenging assay of ABTS radicals; and (C) AJS-PDRN scavenging assay of hydroxyl radicals. a,b IC50 data with different alphabets show significantly different values (p < 0.05).
Figure 2. Effects of AJS-PDRN on RAW264.7 cell viability with/without H2O2 induction. (A) Cells treated with the indicated concentrations of H2O2 for 4 h; (B) cells treated with the indicated concentrations of AJS-PDRN for 6 h; and (C) cells pretreated with the indicated concentrations of AJS-PDRN for 6 h and then stimulated with 400 μM H2O2 for 4 h. The results are presented as the mean ± SD of three replicates. Different letters at the top of the columns indicate statistically significant differences.
Figure 3. Effects of AJS-PDRN on the levels of oxidative stress biomarkers in H2O2-induced RAW264.7 cells. (A) SOD activity; (B) CAT activity; (C) GSH content; and (D) MDA content. The results are presented as the mean ± SD of three replicates. Different letters at the top of the columns indicate statistically significant differences.
Figure 4. Effect of AJS-PDRN on H2O2-induced protein expression profile in RAW264.7 cells. (A) PCA based on quantitative data of the selected proteins in each group (n = 3 for each group); (B) Venn diagram of DEPs in the H2O2 group and the AJS-PDRN group; and (C) Venn diagram demonstrating the number of DEPs identified in each comparison.
Figure 5. GO enrichment analysis of DEPs. (A) The H2O2 group vs. the control group; (B) the AJS-PDRN group vs. the H2O2 group. The number after each term represents the number of differential proteins annotated to that term.
Figure 6. The bubble diagram of KEGG pathway analysis of DEPs. (A) The H2O2 group vs. the control group; (B) the AJS-PDRN group vs. the H2O2 group. The larger the bubble the greater the number of differential proteins contained in the entry. The color of the bubbles changes from blue to red, indicating that the smaller the enrichment p-value, the greater the degree of significance.
Figure 7. PPI network analysis of DEPs. (A) PPI network diagram of all DEPs with interactions in the H2O2 group vs. the control group. (B) The highest-scoring sub-network in the H2O2 group was selected using the MCODE algorithm. (C) The major GO BP terms were significantly enriched by 11 proteins in the sub-network, as well as the related DEPs. (D) PPI network diagram of all DEPs with interactions in the AJS-PDRN group vs. the H2O2 group. (E) The highest-scoring sub-network in the AJS-PDRN group was selected using the MCODE algorithm. (F) The major GO BP terms and KEGG pathway were significantly enriched by 9 proteins in the sub-network, as well as the related DEPs.
