Oral Treatment with Plant-Derived Exosomes Restores Redox Balance in H2O2-Treated Mice

Abstract

Plant-derived exosomes (PDEs) are receiving much attention as a natural source of antioxidants. Previous research has shown that PDEs contain a series of bioactives and that their content varies depending on the fruit or vegetable source. It has also been shown that fruits and vegetables derived from organic agriculture produce more exosomes, are safer, free of toxic substances, and contain more bioactives. The aim of this study was to investigate the ability of orally administered mixes of PDE (Exocomplex^®) to restore the physiological conditions of mice treated for two weeks with hydrogen peroxide (H₂O₂), compared with mice left untreated after the period of H₂O₂ administration and mice that received only water during the experimental period. The results showed that Exocomplex^® had a high antioxidant capacity and contained a series of bioactives, including Catalase, Glutathione (GSH), Superoxide Dismutase (SOD), Ascorbic Acid, Melatonin, Phenolic compounds, and ATP. The oral administration of Exocomplex^® to the H₂O₂-treated mice re-established redox balance with reduced serum levels of both reactive oxygen species (ROS) and malondialdehyde (MDA), but also a general recovery of the homeostatic condition at the organ level, supporting the future use of PDE for health care.

Keywords: anti-aging; anti-stress; antioxidants; health; natural bioactives; organic agriculture; plant-derived exosomes; plants.

Figure 1

Analysis of concentration and size distribution of Exocomplex^® by Nanoparticle tracking analysis (NTA). (a) NTA profile of Exocomplex^® in the liquid state. (b) NTA profile of Exocomplex^® in the freeze-dried state.

Figure 2

Transmission electron microscopy (TEM) of Exocomplex^®. (a) Typical rounded structure and nanometer size of Exocomplex^®. (b) Structural integrity of Exocomplex^®.

Figure 3

Serum ROS levels in C57BL mice after treatment with Exocomplex^®. Analysis of the total ROS levels (Mean Fluorescent Intensity, M.F.I.) was performed on the serum of blood samples collected just before the sacrifice of the mice. Analysis was performed with a fluorimetric assay, and the signals emitted were measured on a microplate reader at 488 nm (blue laser) in the FITC channel. Data are expressed as means ± SE. *** p < 0.001.

Figure 4

Serum lipid peroxidation measurement in C57BL mice after treatment with Exocomplex^®. Quantification of the lipid peroxidation was performed on the serum of blood samples collected just before the sacrifice of the mice. Lipid peroxidation was evaluated through the concentration of malondialdehyde (MDA) (nmol/mL), resulting from oxidative damage. Analysis was performed with a fluorimetric assay, and the relative fluorescence units (RFU) were measured at Ex = 532 nm/Em = 553 nm on a microplate reader (green laser). Data are expressed as means ± SE. **** p < 0.0001.

Figure 5

Number of bone marrow cells and splenocytes after treatment with Exocomplex^®. Effect of Exocomplex^® treatment on the number of bone marrow cells and splenocytes obtained just after the sacrifice of the mice. The cells were counted by trypan blue exclusion under an optical microscope. (a) Number of bone marrow cells. (b) Number of Splenocytes. Data are expressed as means ± SE. **** p < 0.0001.

Figure 6

Proliferation of bone marrow cells and splenocytes after treatment with Exocomplex^®. Effect of Exocomplex^® treatment on the proliferation of bone marrow cells and splenocytes obtained just after the sacrifice of the mice. The optical density values (a.u.) were read at 405 nm in a microplate reader after the reaction with alkaline phosphatase. (a) Proliferation of bone marrow cells. (b) Proliferation of Splenocytes. Data are expressed as means ± SE. **** p < 0.0001.

Figure 7

Effect of Exocomplex^® treatment on oxidative stress in bone marrow cells and splenocytes. (a) ROS levels (M.F.I., a.u.) in bone marrow cells measured in a fluorescence microplate reader at 488 nm (blue laser). (b) ROS levels (M.F.I., a.u.) in splenocytes measured in a fluorescence microplate reader at 488 nm (blue laser). (c) Mitochondrial membrane potential (M.F.I., a.u.) measurement in bone marrow cells. Green fluorescence values were read at Ex/Em = 490/516 nm in a fluorescence microplate reader. (d) Mitochondrial membrane potential (M.F.I., a.u.) measurement in splenocytes. Green fluorescence values were read at Ex/Em = 490/516 nm in a fluorescence microplate reader. (e) Comparison between the mitochondrial superoxide (M.F.I., a.u.) levels and ROS levels (M.F.I., a.u.) measured in bone marrow cells. Red fluorescence values related to mitochondrial superoxide were read at Ex/Em = 540/590 nm in a fluorescence microplate reader. Green fluorescence values related to ROS were read at 488 nm in a fluorescence microplate reader. (f) Comparison between the mitochondrial superoxide (M.F.I., a.u.) levels and ROS levels (M.F.I., a.u.) measured in splenocytes. Red fluorescence values related to mitochondrial superoxide were read at Ex/Em = 540/590 nm in a fluorescence microplate reader. Green fluorescence values related to ROS were read at 488 nm in a fluorescence microplate reader. Data are expressed as means ± SE. ** p < 0.01, **** p < 0.0001.

Figure 8

Effect of Exocomplex^® treatment on Ig tot expression. Protein levels of total Ig were detected and quantified on the serum of blood samples collected just before the sacrifice of the mice. The optical density values were read at 450 nm in a microplate reader, and the concentration of total Ig (µg/mL) was calculated from the standard curve. Data are expressed as means ± SE. **** p < 0.0001.

Figure 9

Effect of Exocomplex^® treatment on the oxidative stress in ovarian germ cells and in blood. Ovarian germ cells and serum of blood samples were obtained right after and before the sacrifice of the mice, respectively. (a) Number of ovarian germ cells. The cells were counted by trypan blue exclusion under an optical microscope. (b) Comparison between the DNA damage in serum and telomere length in ovarian germ cells. DNA damage was measured by the formation of 8-hydroxy-2′-deoxyguanosine (8-OHdG), a ubiquitous marker of oxidative stress. The absorbance was read at 450 nm in a microplate reader, and the concentration of 8-OHdG (ng/mL) was calculated from the standard curve. Telomere length was measured as fluorescence emission by flow cytometry after excitation at 488 nm. Data are expressed as means ± SE. **** p < 0.0001.

Figure 10

Effect of Exocomplex^® treatment on levels of serotonin and melatonin in murine body fluids. (a) Comparison between the ROS (M.F.I., a.u.) and serotonin levels (ng/mL) measured in serum of blood samples obtained right before the sacrifice of the mice. Green fluorescence values related to ROS (M.F.I., a.u.) were read at 488 nm in a fluorescence microplate reader. The optical density values related to serotonin were read at 405 nm in a microplate reader, and the serotonin concentration (ng/mL) was calculated from the standard curve. (b) Serotonin concentration (ng/mL) in urine samples collected just before the sacrifice of mice. The optical density values related to serotonin were read at 405 nm in a microplate reader, and the serotonin concentration (ng/mL) was calculated from the standard curve. (c) Comparison between the ROS (M.F.I., a.u.) and melatonin levels (ng/mL) measured in serum of blood samples obtained right before the sacrifice of the mice. Green fluorescence values related to ROS (M.F.I., a.u.) were read at 488 nm in a fluorescence microplate reader. The optical density values related to serotonin were read at 450 nm in a microplate reader, and the melatonin concentration (ng/mL) was calculated from the standard curve. Data are expressed as means ± SE. * p < 0.1, **** p < 0.0001.