Abstract: BACKGROUND: Recent studies have shown that Chlorella possesses potential value in treating rheumatoid arthritis. The pathological progression of rheumatoid arthritis is closely associated with an imbalance in oxidative stress, abnormal macrophage polarization, aggressive activation of fibroblast-like synoviocytes, and disturbances in the vascular endothelial system. However, the optimization of extraction processes for peptides derived from Chlorella and their regulatory effects and mechanisms on key pathological links in rheumatoid arthritis require systematic validation.
OBJECTIVE: To optimize the extraction process of antioxidant peptides from Chlorella, clarify their antioxidant activity and biosafety, and investigate their regulatory effects on the pathological phenotypes of rheumatoid arthritis-related cells (RAW 264.7 mouse monocyte-macrophage leukemia cells, fibroblast-like synoviocytes, and human umbilical vein endothelial cells), thereby providing experimental evidence for the therapeutic potential of Chlorella peptides in rheumatoid arthritis.
METHODS: (1) Peptide extracts from Chlorella were prepared using bromelain enzymatic hydrolysis combined with the phosphomolybdic acid precipitation method. Using peptide yield as the indicator, extraction parameters including solid-to-liquid ratio, hydrolysis time, and reaction system pH were optimized through single-factor experiments. (2) Peptide content was determined by the BCA method. Antioxidant capacity was detected by the ABTS assay. The biosafety of the peptides on RAW 264.7, fibroblast-like synoviocytes, and human umbilical vein endothelial cells was evaluated using the CCK-8 assay. (3) A lipopolysaccharide-induced RAW 264.7 inflammation model was established. The effects of the peptides on intracellular reactive oxygen species levels and M1/M2 polarization phenotypes were detected via DCFH-DA staining, flow cytometry, and real-time quantitative reverse transcription polymerase chain reaction. (4) A tumor necrosis factor-α-induced fibroblast-like synoviocytes activation model was established. The effects of the peptides on fibroblast-like synoviocytes migration, proliferation, invasion capabilities, and the expression of related genes were assessed using scratch wound assay, EdU proliferation assay, Transwell invasion assay, and real-time quantitative reverse transcription polymerase chain reaction. (5) A vascular endothelial growth factor-A-induced abnormal activation model of human umbilical vein endothelial cells was established. The effects of the peptides on human umbilical vein endothelial cell migration, tube formation ability, and the expression of hypoxia-inducible factor 1-alpha, and vascular endothelial growth factor A genes were evaluated via scratch wound assay, Transwell assay, tube formation assay, and real-time quantitative reverse transcription polymerase chain reaction.  
RESULTS AND CONCLUSION: (1) The optimal extraction conditions for Chlorella peptides were a solid-to-liquid ratio of 2:1 (g:100 mL), an enzymatic hydrolysis time of 60 minutes, and a reaction system pH of 6.5, under which the highest peptide yield was achieved. (2) Chlorella peptides exhibited concentration-dependent antioxidant activity and showed no significant cytotoxicity against RAW 264.7, fibroblast-like synoviocytes, and human umbilical vein endothelial cells within the concentration range of 1–10 μg/mL, indicating good biocompatibility. (3) The Chlorella peptides dose-dependently inhibited lipopolysaccharide-induced reactive oxygen species generation in RAW 264.7 cells, downregulated the expression of M1-type pro-inflammatory genes such as interleukin-1β and tumor necrosis factor-α, and upregulated the expression of M2-type anti-inflammatory genes such as interleukin-10 and arginase-1, thereby promoting macrophage polarization from the M1 phenotype towards the M2 phenotype. (4) The Chlorella peptides significantly inhibited tumor necrosis factor-α-induced migration, proliferation, and invasion capabilities of fibroblast-like synoviocytes and downregulated the expression of related genes including interleukin-6, matrix metalloproteinase-13, tumor necrosis factor receptor superfamily member 11A, and C-X-C motif chemokine ligand 12. (5) The Chlorella peptides effectively inhibited vascular endothelial growth factor A-induced migration and tube formation ability of human umbilical vein endothelial cells and reduced the expression levels of hypoxia-inducible factor-1α and vascular endothelial growth factor A genes. It is concluded that Chlorella peptides exert multi-target regulatory effects on rheumatoid arthritis pathological links through antioxidative stress, modulation of macrophage polarization, inhibition of the aggressive phenotype of fibroblast-like synoviocytes, and amelioration of vascular endothelial disturbances, demonstrating potential therapeutic value for rheumatoid arthritis.

Key words: Chlorella peptide, rheumatoid arthritis, macrophage polarization, fibroblast-like synoviocytes, angiogenesis, antioxidative stress