Biomimetic peptides protect cells from oxidative stress

Abstract

Most degenerative diseases are caused by free radicals. Antioxidin-RL peptide is a free radical scavenger found in the skin of plateau frog Odorrana livida, which is more stable than vitamin C as it resists light-induced degradation. However, whether and how antioxidin-RL protects cells from oxidative stress was not clear. Here we addressed this issue, and in addition, we designed a series of antioxidin cognates by adding tyrosine residues to enhance free radical-binding capability. We performed free radical-clearing assays in solution to screen the mutants, and found a mutant antioxidin-2 that was as stable as antioxidin-RL and cleared free radical faster. By using PC-12 cells as a model, we demonstrated that both antioxidin-2 and antioxidin-RL inhibited the accumulation of intracellular free radicals triggered by H₂O₂, reduced mitochondria membrane potential dissipation, maintained mitochondrial morphology, and decreased the expression of dynamin-related protein-1 in mitochondria, with antioxidin-RL more effective. Antioxidin-RL also attenuated the changes in SOD1 and GPx1 expression induced by H₂O₂. These findings provide insight into the anti-oxidative mechanisms of antioxidin-RL and its derivatives, which will provide rational basis for the development of more effective antioxidants to cure diseases.

Keywords: Antioxidin-RL; PC12 cell; metabolic activity; mitochondria; oxidative injury.

Figure 1

Amino acid sequence alignment among antioxidin peptides and ABTS+ free radical scavenging assay. (A) Amino acid sequence alignment between AR and its homologs antioxidin-RP1 and antioxidin-RP2. The consensus was highlighted in frame. (B) ABTS+ free radical scavenging was tested at 340 nm. The in vitro reaction was started by addition of AR, Vc or the mutants to ABTS solution. Final concentrations of antioxidants and ABTS+ free radical in the reaction mixture were 3 and 40 μM respectively. (C) The same as (B), except that samples was kept at room temperature and exposed to light and air for 1 week before the testing.

Figure 2

PC12 Cell metabolic activity examined with CCK-8 kit and intracellular ROS level examined with DCFH-DA probe. A. Metabolic activity of cells treated with antioxidants in the absence of H₂O₂. B. Metabolic activity of H₂O₂-insulted cells with antioxidants. C. intracellular ROS accumulation of H₂O₂-insulted cells with antioxidants. All experiments were carried out in triplicates. Data were shown as the percentage of the values corresponding to the uninjured cells and represented as mean ± SD. *P<0.05 compared to H₂O₂-treated cells. ※P<0.05 compared to untreated cells. #P<0.05 compared to cells treated with H₂O₂ and Vc at respective concentration.

Figure 3

MMP evaluation with JC-10 probe and mitochondrial morphology with MitoTracker Green. A. Relative JC-10 ratio representing MMP. JC-10 ratio equals the intensity at 590 nm divided by that at 525 nm, and relative JC-10 ratio is represented as the percentage of values corresponding to the uninjured cells. All experiments were carried out in triplicates and the data were shown as mean ± SD. ※P<0.05, *P<0.05 and #P<0.05 as indicated in Figure 2. B. Confocal microscopy image of mitochondrial morphology.

Figure 4

Drp1 expression by Western blotting analysis. (A) Images of the Western blot. β-actin was used as the internal reference. (B) Bar graph showing quantified densitometry from (A). Relative density was represented as the percentage of values corresponding to the uninjured cells. All experiments were carried out in triplicates. Data were shown as mean ± SD. ※P<0.05, *P<0.05 and #P<0.05 as indicated in Figure 2.

Figure 5

Oxidative stress responsive gene expression detected by qPCR. A. SOD1 mRNA expression level. B. GPx1 mRNA expression level. ※P<0.05, *P<0.05 and #P<0.05 as indicated in Figure 2.