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Saponin-Based Nanoemulsification Improves the Antioxidant Properties of Vitamin A and E in AML-12 Cells

View Article: PubMed Central - PubMed

ABSTRACT

Our work aimed to investigate the protective effects of saponin-based nanoemulsions of vitamin A and E against oxidative stress-induced cellular damage in AML-12 cells. Saponin nanoemulsions of vitamin A (SAN) and vitamin E (SEN) were prepared by high-pressure homogenization and characterized in terms of size, zeta potential, and polydispersity index. SEN and SAN protect AML-12 cells against oxidative stress-induced cellular damage more efficiently via scavenging reactive oxygen species (ROS), and reducing DNA damage, protein carbonylation, and lipid peroxidation. These results provide valuable information for the development of nanoemulsion-based delivery systems that would improve the antioxidant properties of vitamin A and E.

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Effect of nanoemulsion on the cellular redox status of AML-12 cells exposed to H2O2. (a) Cells were co-treated with H2O2 and stained with DCF-DA; (b) The CMFDA fluorescence probe revealed GSH levels in AML-12 cells. Images were obtained under fluorescence microscopy from three separate experiments; (c) Quantification of DCF-DA fluorescence intensity using ImageJ software; (d) The mean fluorescence intensity of GSH was measured using ImageJ software; (e) GSSG expression in different groups was analyzed by immunocytochemistry. Images were obtained by light microscopy; (f) Effect of nanoemulsion on NADPH level was tested by immunocytochemistry. Images were obtained by light microscopy. C, control; SN, empty saponin nanoemulsion; VA, vitamin A; SAN, saponin nanoemulsion of vitamin A; VE, vitamin E; SEN, saponin nanoemulsion of vitamin E. All values are represented as mean ± SEM from three or more independent studies. *** p < 0.001.
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ijms-17-01406-f003: Effect of nanoemulsion on the cellular redox status of AML-12 cells exposed to H2O2. (a) Cells were co-treated with H2O2 and stained with DCF-DA; (b) The CMFDA fluorescence probe revealed GSH levels in AML-12 cells. Images were obtained under fluorescence microscopy from three separate experiments; (c) Quantification of DCF-DA fluorescence intensity using ImageJ software; (d) The mean fluorescence intensity of GSH was measured using ImageJ software; (e) GSSG expression in different groups was analyzed by immunocytochemistry. Images were obtained by light microscopy; (f) Effect of nanoemulsion on NADPH level was tested by immunocytochemistry. Images were obtained by light microscopy. C, control; SN, empty saponin nanoemulsion; VA, vitamin A; SAN, saponin nanoemulsion of vitamin A; VE, vitamin E; SEN, saponin nanoemulsion of vitamin E. All values are represented as mean ± SEM from three or more independent studies. *** p < 0.001.

Mentions: To investigate whether the difference in hepatocyte viability after H2O2 and nanoemulsion exposure was correlated with ROS formation, we analyzed intracellular peroxide levels in AML-12 cells using the oxidant-sensitive probe DCF-DA with fluorescence microscopy. As shown in Figure 3a, H2O2 exposure increased DCF-DA fluorescence, which was reduced in cells treated with vitamin A and E. However, treatment with SEN and SAN resulted in comparatively lower levels of intracellular ROS (p < 0.001; Figure 3a,c). Cellular oxidative stress can also be measured by an alternative method, i.e., determining the intracellular level of reduced glutathione (GSH) concentration [33]. Cellular GSH levels, determined with the GSH–sensitive fluorescent dye CMFDA in AML-12 cells exposed to H2O2, were considerably depleted. However, GSH depletion in SAN and SEN cells was significantly mitigated compared with that in vitamin A, vitamin E, and SN cells (p < 0.001; Figure 3b,d). Under oxidative stress, GSH is oxidized to GSSG. We evaluated GSSG levels under same conditions to further confirm our results. When cells were exposed to H2O2, cellular GSSG was significantly higher in control cells, and it decreased more in SAN and SEN cells than in vitamin A and E cells (Figure 3e).


Saponin-Based Nanoemulsification Improves the Antioxidant Properties of Vitamin A and E in AML-12 Cells
Effect of nanoemulsion on the cellular redox status of AML-12 cells exposed to H2O2. (a) Cells were co-treated with H2O2 and stained with DCF-DA; (b) The CMFDA fluorescence probe revealed GSH levels in AML-12 cells. Images were obtained under fluorescence microscopy from three separate experiments; (c) Quantification of DCF-DA fluorescence intensity using ImageJ software; (d) The mean fluorescence intensity of GSH was measured using ImageJ software; (e) GSSG expression in different groups was analyzed by immunocytochemistry. Images were obtained by light microscopy; (f) Effect of nanoemulsion on NADPH level was tested by immunocytochemistry. Images were obtained by light microscopy. C, control; SN, empty saponin nanoemulsion; VA, vitamin A; SAN, saponin nanoemulsion of vitamin A; VE, vitamin E; SEN, saponin nanoemulsion of vitamin E. All values are represented as mean ± SEM from three or more independent studies. *** p < 0.001.
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ijms-17-01406-f003: Effect of nanoemulsion on the cellular redox status of AML-12 cells exposed to H2O2. (a) Cells were co-treated with H2O2 and stained with DCF-DA; (b) The CMFDA fluorescence probe revealed GSH levels in AML-12 cells. Images were obtained under fluorescence microscopy from three separate experiments; (c) Quantification of DCF-DA fluorescence intensity using ImageJ software; (d) The mean fluorescence intensity of GSH was measured using ImageJ software; (e) GSSG expression in different groups was analyzed by immunocytochemistry. Images were obtained by light microscopy; (f) Effect of nanoemulsion on NADPH level was tested by immunocytochemistry. Images were obtained by light microscopy. C, control; SN, empty saponin nanoemulsion; VA, vitamin A; SAN, saponin nanoemulsion of vitamin A; VE, vitamin E; SEN, saponin nanoemulsion of vitamin E. All values are represented as mean ± SEM from three or more independent studies. *** p < 0.001.
Mentions: To investigate whether the difference in hepatocyte viability after H2O2 and nanoemulsion exposure was correlated with ROS formation, we analyzed intracellular peroxide levels in AML-12 cells using the oxidant-sensitive probe DCF-DA with fluorescence microscopy. As shown in Figure 3a, H2O2 exposure increased DCF-DA fluorescence, which was reduced in cells treated with vitamin A and E. However, treatment with SEN and SAN resulted in comparatively lower levels of intracellular ROS (p < 0.001; Figure 3a,c). Cellular oxidative stress can also be measured by an alternative method, i.e., determining the intracellular level of reduced glutathione (GSH) concentration [33]. Cellular GSH levels, determined with the GSH–sensitive fluorescent dye CMFDA in AML-12 cells exposed to H2O2, were considerably depleted. However, GSH depletion in SAN and SEN cells was significantly mitigated compared with that in vitamin A, vitamin E, and SN cells (p < 0.001; Figure 3b,d). Under oxidative stress, GSH is oxidized to GSSG. We evaluated GSSG levels under same conditions to further confirm our results. When cells were exposed to H2O2, cellular GSSG was significantly higher in control cells, and it decreased more in SAN and SEN cells than in vitamin A and E cells (Figure 3e).

View Article: PubMed Central - PubMed

ABSTRACT

Our work aimed to investigate the protective effects of saponin-based nanoemulsions of vitamin A and E against oxidative stress-induced cellular damage in AML-12 cells. Saponin nanoemulsions of vitamin A (SAN) and vitamin E (SEN) were prepared by high-pressure homogenization and characterized in terms of size, zeta potential, and polydispersity index. SEN and SAN protect AML-12 cells against oxidative stress-induced cellular damage more efficiently via scavenging reactive oxygen species (ROS), and reducing DNA damage, protein carbonylation, and lipid peroxidation. These results provide valuable information for the development of nanoemulsion-based delivery systems that would improve the antioxidant properties of vitamin A and E.

No MeSH data available.


Related in: MedlinePlus