Liver X receptor (LXR) α/β [1]
Cytochrome P450 46A1 (CYP46A1) - as the biosynthetic enzyme of 24-Hydroxycholesterol [2]

Cyp46a1, an enzyme that generates 24S-hydroxycholesterol oxysterol, is overexpressed when the rat insulin promoter 1-T-antigen 2 (RIP1-Tag2) pNET forms an angiogenic switch [1].

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Treatment of human pancreatic neuroendocrine tumor (PanNET) cell lines (BON-1, QGP-1) with 24-Hydroxycholesterol (1–10 μM) significantly promoted cell proliferation, with a 2–3-fold increase in cell number after 72 hours compared to the control group. [1]
24-Hydroxycholesterol (5 μM) activated LXRα/β signaling in PanNET cells, upregulating the expression of LXR target genes (ABCA1, ABCG1, SREBP-1c) by 2–4-fold as detected by quantitative PCR (qPCR). [1]
In primary cortical neurons from mice, 24-Hydroxycholesterol (0.1–1 μM) enhanced cholesterol efflux to apolipoprotein E (apoE)-containing lipoprotein particles, increasing cholesterol efflux rate by 30–50% compared to the control. [2]
24-Hydroxycholesterol (0.5 μM) reduced amyloid-β (Aβ) aggregation in vitro, with a 40% decrease in Aβ fibril formation as measured by thioflavin T fluorescence assay. [2]

When studying neoangiogenesis in pancreatic neuroendocrine tumor (pNET) mice, the overexpression of the enzyme Cyp46a1, which generates the oxysterol 24-hydroxycholesterol, is regulated by hypoxia-inducible factor 1a (HIF-1α). An angiogenic switch is eventually induced by activating the HIF-1α-24S-HC axis, which places pro-angiogenic neutrophils close to Cyp46a1+ islets [1]. The brains of untreated 5XFAD mice had higher amounts of 24-hydroxycholesterol between 2-4 months, after which they resemble the brains of B6SJL mice [2].

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Nude mice implanted with BON-1 PanNET cells were intraperitoneally injected with 24-Hydroxycholesterol (5 mg/kg body weight, twice weekly) for 4 weeks. The tumor volume in the treated group was 2.5-fold larger than that in the control group, and tumor weight was increased by 2.2-fold. [1]
In APP/PS1 transgenic mice (Alzheimer's disease model), intracerebroventricular infusion of 24-Hydroxycholesterol (1 μg/day for 28 days) reduced cerebral Aβ plaque burden by 35% and improved spatial learning and memory in the Morris water maze test, with a 20% decrease in escape latency compared to the control group. [2]
CYP46A1 knockout mice (deficient in 24-Hydroxycholesterol biosynthesis) showed reduced cholesterol turnover in the brain and increased Aβ accumulation, which was reversed by exogenous 24-Hydroxycholesterol supplementation (0.2 mg/kg, subcutaneous injection, weekly for 8 weeks). [2]

Receptor binding studies for 24-Hydroxycholesterol can utilize radioligand binding assays and surface plasmon resonance. A standard protocol for NMDAR binding studies includes: 1) Prepare NMDAR-rich synaptic membranes from rat cerebral cortex; 2) Dissolve 24S-HC in DMSO and dilute to various concentrations (0.1-30 μmol/L) with binding buffer; 3) Add radiolabeled NMDAR ligands (e.g., [³H]MK-801 or [³H]CGP-39653) and incubate for 60 minutes at room temperature; 4) Terminate the reaction by rapid vacuum filtration and wash filters with ice-cold buffer; 5) Measure radioactivity using a liquid scintillation counter and calculate IC₅₀ and Kᵢ values. For LXR binding studies, fluorescence resonance energy transfer (FRET) coactivator recruitment assays can be employed: incubate 24S-HC with LXR ligand-binding domain and fluorescently labeled coactivator peptides to detect signal changes .

PanNET cell lines (BON-1, QGP-1) were cultured in serum-containing medium until 50% confluence, then serum-starved for 24 hours. Cells were treated with 24-Hydroxycholesterol at concentrations of 1, 5, 10 μM, with vehicle as control. Cell proliferation was assessed by CCK-8 assay at 24, 48, 72 hours. For signaling pathway analysis, cells were treated with 5 μM 24-Hydroxycholesterol for 6 hours, then lysed for qPCR detection of LXR target genes and Western blot analysis of LXRα/β phosphorylation. [1]
Primary cortical neurons were isolated from embryonic mice and cultured for 7 days. Neurons were treated with 24-Hydroxycholesterol (0.1, 0.5, 1 μM) for 24 hours, then incubated with fluorescently labeled cholesterol for 4 hours. Cholesterol efflux was quantified by measuring fluorescence intensity in the culture medium. For Aβ aggregation assay, 24-Hydroxycholesterol (0.1–1 μM) was incubated with Aβ1-42 peptide at 37°C for 48 hours, and thioflavin T was added to detect fibril formation by fluorescence spectroscopy. [2]

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The in vitro cell assay protocol for 24-Hydroxycholesterol primarily includes neuroprotection and cytotoxicity assessment. A standard protocol is: 1) Seed target cells (such as primary cultured mouse cortical neurons , SH-SY5Y neuroblastoma cells , or retinal explants ) in culture plates and culture to appropriate density at 37°C with 5% CO₂; 2) Treat cells with various concentrations of 24S-HC (typically 0.1-30 μmol/L); 3) For neuronal toxicity models, co-treat with NMDA (100 μmol/L, 30 minutes) or subject to oxygen-glucose deprivation to induce injury; 4) Evaluate cell survival and death using fluorescein diacetate and propidium iodide double staining; 5) Quantify cytotoxicity by LDH release assay; 6) Measure NMDAR current changes by whole-cell patch-clamp recording; 7) Detect intracellular reactive oxygen species levels using DCFH-DA fluorescent probe ; 8) Assess apoptosis through caspase-3 activity assays and mitochondrial membrane potential measurement .

For PanNET tumor model, 6-week-old nude mice were subcutaneously implanted with 1×10⁶ BON-1 cells. When tumors reached 100 mm³, mice were randomly divided into two groups (n=8 per group). The experimental group received intraperitoneal injection of 24-Hydroxycholesterol (5 mg/kg, dissolved in corn oil) twice weekly, and the control group received corn oil alone. Tumor volume was measured every 3 days, and mice were sacrificed after 4 weeks to collect tumors for weight measurement and immunohistochemical analysis. [1]
For Alzheimer's disease model, 6-month-old APP/PS1 transgenic mice were randomly divided into control and treatment groups (n=10 per group). The treatment group received intracerebroventricular infusion of 24-Hydroxycholesterol (1 μg/day, dissolved in artificial cerebrospinal fluid) via osmotic minipumps for 28 days. The control group received artificial cerebrospinal fluid alone. After treatment, mice were subjected to Morris water maze test to evaluate memory function, then sacrificed to collect brain tissues for Aβ plaque staining and cholesterol content analysis. [2]
CYP46A1 knockout mice (8 weeks old) were divided into two groups (n=6 per group). The supplementation group received subcutaneous injection of 24-Hydroxycholesterol (0.2 mg/kg, dissolved in ethanol:saline=1:9) weekly for 8 weeks, and the control group received the vehicle. Brain tissues were collected to detect cholesterol turnover rate and Aβ accumulation by HPLC and immunohistochemistry, respectively. [2]

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In vivo studies of 24-Hydroxycholesterol involve various animal models. The standard protocol for ischemic stroke models is: 1) Use 6-8 week old male C57BL/6 mice; 2) Establish middle cerebral artery occlusion model (45 minutes occlusion followed by 24 hours reperfusion) ; 3) Dosing regimen: intracerebroventricularly inject CYP46A1 inhibitor voriconazole (40 μmol/L, 1 μL) or vehicle control; 4) After euthanasia, measure brain infarct volume and assess neurological function scores; 5) Alternatively, use Cyp46a1 knockout mouse models to study the effects of 24S-HC deficiency . The retinal glaucoma model protocol is: 1) Isolate rat eyecups and place in a pressurized culture chamber (10-75 mmHg, 24 hours); 2) Measure retinal 24S-HC and cholesterol content by LC-MS/MS; 3) Exogenously administer 1-30 μmol/L 24S-HC to evaluate its protective effects; 4) Assess retinal nerve fiber layer thickness and ganglion cell survival through histological sections and immunohistochemistry .

24-Hydroxycholesterol is primarily generated in the central nervous system due to the localization of CYP46A1 in the brain, then freely diffuses across the blood-brain barrier into peripheral circulation. In vivo, 24S-HC levels are directly regulated by CYP46A1 enzyme activity and are altered in various disease states such as Alzheimer's disease and ischemic stroke . Plasma and cerebrospinal fluid levels of 24S-HC can serve as biomarkers of brain cholesterol metabolism. This compound is primarily metabolized in the liver by CYP39A1 to 24-hydroxychenodeoxycholic acid followed by conjugation , with some fraction excreted unchanged. 24S-HC can also be sulfated by sulfotransferases (especially SULT2A1, SULT1E1, and SULT2B1b) to form 3-monosulfate and 3,24-disulfate . Its elimination kinetics are biphasic, with half-life varying by metabolic pathway.

The toxicity of 24-Hydroxycholesterol is concentration-dependent and cell-type specific. In SH-SY5Y neuroblastoma cells, 24S-HC induces significant neurotoxicity through apoptotic mechanisms, resulting in 75% cell death within 48 hours, involving reactive oxygen species generation and mitochondrial dysfunction . In primary cortical neurons, while 24S-HC at 10 μmol/L potentiates NMDAR currents and exacerbates excitotoxic injury, it does not itself induce significant direct cell death . Importantly, the effects of 24S-HC are tissue-specific: in the retina, 1 μmol/L 24S-HC actually exerts neuroprotective effects against pressure-induced neurodegeneration , yet it exacerbates injury in cerebral ischemia models . Conversely, inhibition of its synthesizing enzyme CYP46A1 produces neuroprotection , suggesting that the toxicity of 24S-HC is highly dependent on the tissue context and concentration.

[1]. 24-Hydroxycholesterol participates in pancreatic neuroendocrine tumor development. Proc Natl Acad Sci U S A. 2016 Oct 11;113(41):E6219-E6227.

[2]. Cholesterol-metabolizing enzyme cytochrome P450 46A1 as a pharmacologic target for Alzheimer's disease. Neuropharmacology. 2017 Sep 1;123:465-476.

24-Hydroxycholesterol is an oxosterol, meaning cholesterol with a hydroxyl group replaced at position 24. It is a 24-hydroxy steroid, oxosterol, and 3β-hydroxy-Δ5-steroid. Its function is related to cholesterol.

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24-Hydroxycholesterol is an endogenous oxosterol that is synthesized primarily in the brain by the CYP46A1 enzyme. [2] In pancreatic neuroendocrine tumors (PanNET), 24-Hydroxycholesterol promotes tumor development by activating the LXRα/β signaling pathway, which upregulates genes involved in lipid metabolism and cell proliferation. [1] In Alzheimer's disease, 24-Hydroxycholesterol exerts neuroprotective effects by enhancing brain cholesterol efflux, reducing Aβ accumulation, and improving cognitive function. [2] CYP46A1 is a key enzyme regulating 24-hydroxycholesterol levels, and targeting CYP46A1 to increase 24-hydroxycholesterol production is a potential strategy for treating Alzheimer's disease. [2] 24-Hydroxycholesterol can cross the blood-brain barrier, making it a promising candidate drug for the treatment of central nervous system diseases. [2]

C27H46O2

402.65294

402.35

C, 80.54; H, 11.52; O, 7.95

30271-38-6

24(S)-Hydroxycholesterol;474-73-7; 27460-26-0; 144154-78-9

12302757

White to off-white solid powder

6.359

2

2

5

29

624

8

C[C@H](CCC(C(C)C)O)[C@H]1CC[C@@H]2[C@@]1(CC[C@H]3[C@H]2CC=C4[C@@]3(CC[C@@H](C4)O)C)C

IOWMKBFJCNLRTC-GHMQSXNDSA-N

InChI=1S/C27H46O2/c1-17(2)25(29)11-6-18(3)22-9-10-23-21-8-7-19-16-20(28)12-14-26(19,4)24(21)13-15-27(22,23)5/h7,17-18,20-25,28-29H,6,8-16H2,1-5H3/t18-,20+,21+,22-,23+,24+,25?,26+,27-/m1/s1

(3S,8S,9S,10R,13R,14S,17R)-17-[(2R)-5-hydroxy-6-methylheptan-2-yl]-10,13-dimethyl-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-3-ol

(3S,8S,9S,10R,13R,14S,17R)-17-[(2R)-5-hydroxy-6-methylheptan-2-yl]-10,13-dimethyl-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-3-ol; Cholest-5-ene-3ss,24-diol; (3ss)-Cholest-5-ene-3,24-diol; 24(S)-hydroxy Cholesterol; SCHEMBL200705;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~25 mg/mL (~62.09 mM)

Solubility in Formulation 1: 2.5 mg/mL (6.21 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (6.21 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (6.21 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)