Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2023 Jan 9;24(2):1290.
doi: 10.3390/ijms24021290.

Identification of Side Chain Oxidized Sterols as Novel Liver X Receptor Agonists with Therapeutic Potential in the Treatment of Cardiovascular and Neurodegenerative Diseases

Na Zhan  1   2 , Boyang Wang  1 , Nikita Martens  2   3 , Yankai Liu  1 , Shangge Zhao  1 , Gardi Voortman  2 , Jeroen van Rooij  2 , Frank Leijten  2 , Tim Vanmierlo  3   4 , Folkert Kuipers  5   6 , Johan W Jonker  5 , Vincent W Bloks  5 , Dieter Lütjohann  7 , Marcella Palumbo  8 , Francesca Zimetti  8 , Maria Pia Adorni  9 , Hongbing Liu  1 , Monique T Mulder  2
Affiliations

Identification of Side Chain Oxidized Sterols as Novel Liver X Receptor Agonists with Therapeutic Potential in the Treatment of Cardiovascular and Neurodegenerative Diseases

Na Zhan et al. Int J Mol Sci. .

Abstract

The nuclear receptors-liver X receptors (LXR α and β) are potential therapeutic targets in cardiovascular and neurodegenerative diseases because of their key role in the regulation of lipid homeostasis and inflammatory processes. Specific oxy(phyto)sterols differentially modulate the transcriptional activity of LXRs providing opportunities to develop compounds with improved therapeutic characteristics. We isolated oxyphytosterols from Sargassum fusiforme and synthesized sidechain oxidized sterol derivatives. Five 24-oxidized sterols demonstrated a high potency for LXRα/β activation in luciferase reporter assays and induction of LXR-target genes APOE, ABCA1 and ABCG1 involved in cellular cholesterol turnover in cultured cells: methyl 3β-hydroxychol-5-en-24-oate (S1), methyl (3β)-3-aldehydeoxychol-5-en-24-oate (S2), 24-ketocholesterol (S6), (3β,22E)-3-hydroxycholesta-5,22-dien-24-one (N10) and fucosterol-24,28 epoxide (N12). These compounds induced SREBF1 but not SREBP1c-mediated lipogenic genes such as SCD1, ACACA and FASN in HepG2 cells or astrocytoma cells. Moreover, S2 and S6 enhanced cholesterol efflux from HepG2 cells. All five oxysterols induced production of the endogenous LXR agonists 24(S)-hydroxycholesterol by upregulating the CYP46A1, encoding the enzyme converting cholesterol into 24(S)-hydroxycholesterol; S1 and S6 may also act via the upregulation of desmosterol production. Thus, we identified five novel LXR-activating 24-oxidized sterols with a potential for therapeutic applications in neurodegenerative and cardiovascular diseases.

Keywords: Alzheimer’s disease; LXR agonists; cardiovascular disease; cholesterol efflux; oxidized sterols.

PubMed Disclaimer

Conflict of interest statement

All authors declare that they have no conflict of interest that might be perceived as influencing the impartiality of the reported research.

Figures

Figure 1
Figure 1
Chemical structures of S1-S9 and N10-N1.
Figure 2
Figure 2
Effects of sidechain oxidized sterols on transcriptional activity of LXRα and β in HEK 293 cells. Using the luciferase reporter assay the capacity of oxidized sterols on LXR-mediated transcription was determined. Transfected cells were incubated with different concentrations of oxysterols (2.5, 5.0 and 7.5 µM) for 24 h. T0901317 (T09, 1 µM) and GW3965 (GW, 5 µM) were used as positive controls. Data represent the mean ± SD of three separate experiments, each performed in triplicate (n = 9). Significance is compared to the control (DMEM/F-12 medium with EtOH or DMSO) value: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.
Figure 3
Figure 3
Differential effects of five oxidized sterols on cholesterol turnover genes and CYP46A1 mRNA levels. (A) The expression of APOE, ABCA1 and ABCG1 in CCF-STTG1, CHME3 and SH-SY-5Y cells. (B) CYP46A1 expression in CCF-STTG1 and SH-SY-5Y cells. Cells were incubated with 2.5 or 5.0 μM of oxidized sterols and 1 μM T0901317 (T09) and 5 μM GW3965 (GW) compound for 24 h. Total RNA was isolated from the cells and analyzed by qPCR as described in Materials and Methods. Gene expression was normalized to mean of the most stable housekeeping genes (SDHA, B2M, ACTB and HPRT1) and expressed as Relative Expression compared to the vehicle control. The values are presented as the means of three experiments ± SD (n = 3).
Figure 4
Figure 4
Effects of LXR-activating oxidized sterols on the gene’s expression of lipogenesis. (A) SREBF1, SCD1, FASN and ACACA expression in CCG-STTG1 or HepG2 cells. (B) ABCA1 and ABCG1 expression in HepG2 cells. Cells were incubated with EtOH (0.1%, v/v), DMSO (0.1%, v/v), 1 μM T0901317 (T09), 5 μM GW3965 (GW), 2.5 or 5.0 μM of oxysterols for 24 h and gene expression was assessed by qPCR. Gene expression was normalized to the most stable housekeeping genes (SDHA, B2M, ACTB, and HPRT1) and expressed as Relative. Expression compared to the vehicle control (EtOH or DMSO) given as means of three experiments ± SD (n = 3).
Figure 5
Figure 5
Effect of LXR ligands on cholesterol efflux from HepG2 cells. HepG2 cells were loaded for 24 h with 2 μCi/mL [3H] cholesterol in the presence of 1% FCS and then incubated for 20 h with vehicle (ethanol), 2.5 μM or 5 μM LXR ligands. Cholesterol efflux was promoted to (A) human APOA1 (10 µg/mL), (B) human HDL (12.5 µg/mL), and (C) a pool of serum from normolipidemic individuals at 2% (v/v) for 4 h and assayed as described under Methods. Each cell treatment was performed in triplicate and data are expressed in percentage as mean ± SD (n = 3). Significance is compared to the control (EtOH) value: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.
Figure 6
Figure 6
S1 and S6 regulate cholesterol biosynthesis in a cell type-specific manner. Lanosterol (Lano), lathosterol (Lath), desmosterol (Desmo), and cholesterol (Chol) were quantified in S1 or S6 loaded cells (HepG2, CCF-STTG1 and SHSH-5Y) using GC-MS method. Fold change compared to the vehicle control. Each bar represents the mean ± SD of three separate experiments, each performed in triplicate (n = 9). Significance is compared to the control (EtOH) value: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.
Figure 7
Figure 7
Effects of oxysterols on DHCR7 and DHCR24 mRNA levels. Cells were incubated with 2.5 or 5.0 μM of 24-oxidized sterols and 1 μM T0901317 (T09) and 5 μM GW3965 (GW) for 24 h. Gene expression was determined by qPCR and normalized to the most stable housekeeping genes (SDHA, B2M, ACTB and HPRT1) and expressed as Relative Expression compared to the vehicle control. The values are presented as the means of three experiments ± SD (n = 3).
Figure 8
Figure 8
Synthesis of oxidized sterols, reagents and conditions: (a) CH3OH, H2SO4, 8 h, 95 °C, 89.0%; (b) TsCl, pyridine (Py), 24 h, room temperature (r.t)., 77.9%; (c) AcOK, DMF, H2O, 7 h, 105 °C, S1 50.9%, S2 4.2%, S3 4.5%; (d) 2% KOH-CH3OH, 3 h, r.t., 96.7%. (e) Isopropylmagnesium chloride solution, N,O-dimethylhydroxylamine hydrochloride, tetrahydrofuran(THF), 12 h, 0 °C-r.t., S4 85.5%; (f) Isopropylmagnesium bromide solution, triethylamine (Et3N), THF, 18 h, 0 °C-r.t., S6 26.8%, S7 2.8%, S8 8.2%; (g) Isopropylmagnesium chloride solution, THF, 18 h, 0 °C-r.t., S6 52.0%; (h) Vinylmagnesium chloride solutionchlorovinylmagnesium, THF, 18 h, 0 °C-r.t., S5; (i) Isopropylmagnesium chloride solution, THF, 12 h, 0 °C-r.t., S9 90.8%; (j) Semi-PHPLC equivalent elution, MeOH-H2O, 90:10 (v/v), S8a and S8b in a 1:1 ratio; (k) Semi-PHPLC equivalent elution, MeOH- CH3CN-H2O, 85:1:14 (v/v/v), S9a and S9b in a 1:1 ratio.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Fessler M.B. The challenges and promise of targeting the Liver X Receptors for treatment of inflammatory disease. Pharmacol. Ther. 2018;181:1–12. doi: 10.1016/j.pharmthera.2017.07.010. - DOI - PMC - PubMed
    1. Xu X., Xiao X., Yan Y., Zhang T. Activation of liver X receptors prevents emotional and cognitive dysfunction by suppressing microglial M1-polarization and restoring synaptic plasticity in the hippocampus of mice. Brain Behav. Immun. 2021;94:111–124. doi: 10.1016/j.bbi.2021.02.026. - DOI - PubMed
    1. Ramírez C.M., Torrecilla-Parra M., Pardo-Marqués V., de-Frutos M.F., Pérez-García A., Tabraue C., de la Rosa J.V., Martín-Rodriguez P., Díaz-Sarmiento M., Nuñez U., et al. Crosstalk Between LXR and Caveolin-1 Signaling Supports Cholesterol Efflux and Anti-Inflammatory Pathways in Macrophages. Front. Endocrinol. 2021;12:635923. doi: 10.3389/fendo.2021.635923. - DOI - PMC - PubMed
    1. Zelcer N., Tontonoz P. Liver X receptors as integrators of metabolic and inflammatory signaling. J. Clin. Investig. 2006;116:607–614. doi: 10.1172/JCI27883. - DOI - PMC - PubMed
    1. Wang X., Lu K., Luo H., Liang D., Long X., Yuan Y., Wu C., Bao J. In silico identification of small molecules as novel LXR agonists for the treatment of cardiovascular disease and cancer. J. Mol. Model. 2018;24:57. doi: 10.1007/s00894-018-3578-y. - DOI - PubMed