The peptide glycyl-ʟ-histidyl-ʟ-lysine is an endogenous antioxidant in living organisms, possibly by diminishing hydroxyl and peroxyl radicals

Abstract

Despite evidence that tripeptide glycyl-ʟ-histidyl-ʟ-lysine (GHK) is an endogenous antioxidant, its mechanism and importance are not fully understood. In the present study, the ability of GHK to reduce levels of reactive oxygen species (ROS) in Caco-2 cells was evaluated by flow cytometry with the oxidation-sensitive fluorescent dye 2',7'-dichlorodihydrofluorescein diacetate. Further, types of ROS diminished by GHK were assessed by utilizing an electron spin resonance (ESR) spin-trapping technique. GHK reduced the tert-butyl hydroperoxide-induced increase in ROS levels in Caco-2 cells at concentrations of 10 µM or less. Experiments utilizing an ESR spin-trapping technique revealed that, among hydroxyl (·OH), superoxide (O₂^-·), and peroxyl (ROO·) radicals generated by respective chemical reaction systems, GHK diminished signals of both ·OH and ROO·, but not O₂^-·. Additionally, the GHK effect on the signal of ·OH was much stronger than those of other well-known antioxidative, endogenous peptides, carnosine and reduced glutathione. These results suggest that GHK can function as an endogenous antioxidant in living organisms, possibly by diminishing ·OH and ROO·.

Keywords: Glycyl-ʟ-histidyl-ʟ-lysine; endogenous antioxidant; hydroxyl radical; oxidative stress; peroxyl radical.

Figure 1

Alterations in ROS levels in Caco-2 cells treated with t-BOOH in combination with GHK. (A) Representative univariate histograms of flow cytometry data. (B) Quantitative assessment of the MFI flow cytometry data. Results are presented as means ± standard errors of the mean (n = 3-12). ^α P < 0.01 vs. None. ^β P < 0.01, ^γ P < 0.01 vs. t-BOOH. ^δ P < 0.01 vs. NAC. MFI, mean fluorescence intensity (shown as dashed lines in part A); GHK, glycyl-ʟ-histidyl-ʟ-lysine; t-BOOH, tert-butylhydroperoxide; NAC, N-acetyl-ʟ-cysteine.

Figure 2

Effect of GHK on the amounts of spin signal adducts of ·OH, O₂ ^-·, and t-BOO· generated by their respective chemical reaction systems. A. Representative ESR signal spectra of (a) DMPO-OH reflecting ·OH, (b) DMPO-OOH reflecting O₂ ^-·, and (c) POBN adduct signal reflecting t-BOO·. (a) Fe²⁺, 0.25 mM; H₂O₂, 0.5 mM. (b) xanthine, 200 mM; XO, 0.1 U/mL. (c) Ce⁴⁺, 0.2 mM; t-BOOH, 400 mM. B. The effect of GHK on the amounts of spin signal adducts of ·OH, O₂ ^-·, and t-BOO·. The radical intensity was defined as the ratio of the peak height of respective signal [indicated as arrows in part Aa-Ac] to that of Manganese (Mn). Results are presented as means ± standard errors of the mean (n = 3-8). ^α P < 0.01 vs. Control. GHK, 250 μM. ·OH, hydroxyl radicals; O₂ ^-·, superoxide radicals; t-BOO·, tert-butyl peroxyl radicals; GHK, glycyl-ʟ-histidyl-ʟ-lysine; XO, xanthine oxidase; SOD, superoxide dismutase; ESR, electron spin resonance; t-BOOH, tert-butyl hydroperoxide.

Figure 3

Effects of carnosine and GSH as well as GHK on the amounts of spin signal adduct of ·OH generated by the Fenton-type reaction. The radical intensity was defined as the ratio of the peak height of the signal [indicated as arrows in Figure 2Aa] to that of Manganese (Mn). Results are presented as means ± standard errors of the mean (n = 3-8). ^α P < 0.05, ^β P < 0.01 vs. None., ^γ P < 0.01 vs. GSH. ^δ P < 0.05, εP < 0.01 vs. carnosine. ·OH, hydroxyl radicals; GHK, glycyl-ʟ-histidyl-ʟ-lysine; GSH, reduced glutathione.