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. 2024 Feb 22;19(2):e0290526.
doi: 10.1371/journal.pone.0290526. eCollection 2024.

17-Oxime ethers of oxidized ecdysteroid derivatives modulate oxidative stress in human brain endothelial cells and dose-dependently might protect or damage the blood-brain barrier

Máté Vágvölgyi  1 , Dávid Laczkó  1 , Ana Raquel Santa-Maria  2   3 , Judit P Vigh  2   4 , Fruzsina R Walter  2 , Róbert Berkecz  5 , Mária A Deli  2 , Gábor Tóth  6 , Attila Hunyadi  1   7   8
Affiliations

17-Oxime ethers of oxidized ecdysteroid derivatives modulate oxidative stress in human brain endothelial cells and dose-dependently might protect or damage the blood-brain barrier

Máté Vágvölgyi et al. PLoS One. .

Abstract

20-Hydroxyecdysone and several of its oxidized derivatives exert cytoprotective effect in mammals including humans. Inspired by this bioactivity of ecdysteroids, in the current study it was our aim to prepare a set of sidechain-modified derivatives and to evaluate their potential to protect the blood-brain barrier (BBB) from oxidative stress. Six novel ecdysteroids, including an oxime and five oxime ethers, were obtained through regioselective synthesis from a sidechain-cleaved calonysterone derivative 2 and fully characterized by comprehensive NMR techniques revealing their complete 1H and 13C signal assignments. Surprisingly, several compounds sensitized hCMEC/D3 brain microvascular endothelial cells to tert-butyl hydroperoxide (tBHP)-induced oxidative damage as recorded by impedance measurements. Compound 8, containing a benzyloxime ether moiety in its sidechain, was the only one that exerted a protective effect at a higher, 10 μM concentration, while at lower (10 nM- 1 μM) concentrations it promoted tBHP-induced cellular damage. Brain endothelial cells were protected from tBHP-induced barrier integrity decrease by treatment with 10 μM of compound 8, which also mitigated the intracellular reactive oxygen species production elevated by tBHP. Based on our results, 17-oxime ethers of oxidized ecdysteroids modulate oxidative stress of the BBB in a way that may point towards unexpected toxicity. Further studies are needed to evaluate any possible risk connected to dietary ecdysteroid consumption and CNS pathologies in which BBB damage plays an important role.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Oxidative cleavage of the sterol sidechain of calonysterone (1).
Fig 2
Fig 2. Synthesis of oxime (3) and oxime ether (4–8) derivatives of compound 2.
Fig 3
Fig 3. The effects of compound 8 at concentrations of 0.01, 0.1, 1, and 10 μM treatment on human brain microvascular endothelial cells (hCMEC/D3) were evaluated using impedance-based assays to assess cell viability and barrier integrity in the absence and presence of oxidative stress promoted by tert-butyl hydroperoxide (tBHP).
A: Time-dependent impact of 8 on cell viability following co-treatment with tBHP (350 μM). B: Impact of 8 on cell viability at 4 hours co-treatment with tBHP (350 μM). C: Impact of 8 on cell viability at 24 hours co-treatment with tBHP (350 μM). The data are presented as the mean ± standard deviation (SD) and were obtained from a minimum of two independent experiments (n = 2−3) with 3−9 technical replicates. Data analysis was performed using one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparisons test. The results were statistically significant with *p < 0.05, ****p < 0.0001, compared to the control group, and ####p < 0.0001, compared to the tBHP group.
Fig 4
Fig 4. Reactive oxygen species (ROS) measurement in human brain microvascular endothelial cells (hCMEC/D3) after 4-hour treatment with 0.01–10 μM compound 8 in the absence and presence of oxidative stress promoted by tert-butyl hydroperoxide (tBHP, 350 μM).
Data is given in fluorescent intensity corresponding to ROS amount produced intracellularly measured by DCFDA assay. Data are presented as mean ± standard deviation (SD; n = 7–8). Data analysis was performed using one-way analysis of variance (ANOVA) followed by Bonferroni’s multiple comparisons test. Statistical significance: ***p < 0.001, compared to the control group, ##p < 0.01; ###p < 0.001, compared to the tBHP group.
Fig 5
Fig 5. Effects of compound 8 (10 μM) on the barrier integrity of human brain microvascular endothelial cells (hCMEC/D3) in the absence and presence of oxidative stress promoted by tert-butyl hydroperoxide (tBHP, 350 μM).
All treatments were performed for 4 hours. A: Transendothelial electrical resistance (TEER) measurement. B: Permeability measurement for the paracellular marker molecule FITC-dextran 4 kDa (FD4). C: Permeability measurement for the transcellular marker molecule Evans blue labeled-albumin (EBA, 67 kDa). Data are presented as mean ± standard deviation (SD; n = 4). Data analysis was performed using one-way analysis of variance (ANOVA) followed by Bonferroni’s multiple comparisons test. Statistical significance: ***p < 0.001, compared to the control group, and ###p < 0.001, compared to the tBHP group. D: Immunocytochemistry for β-catenin and zonula occludens-1 (ZO-1) junctional associated molecules. Green: junctional staining. Blue: cell nuclei. Bar: 20 μm.
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Grants and funding

This study was funded by National Research, Development and Innovation Office, Hungary (NKFIH; K134704) and NKFIH and the Ministry of Innovation and Technology, Hungary (TKP2021-EGA-32) awarded to AH; by the Gedeon Richter Plc. Centenarial Foundation Research Grant awarded to FRW; by the European Training Network H2020-MSCA-ITN-2015 (Grant no. 675619) awarded to MD and financing ARSM; by the New National Excellence Program of the Ministry for Culture and Innovation (ÚNKP-22-4-SZTE-169) awarded to MV; by NKFIH, 2022-1.2.6-TÉT-IPARI-TR-2022-00024 awarded to MD and AH; and by NKFIH, cooperative doctoral program (KDP2020-1005968) awarded to AH and DL.