Cholesterol neutralized vemurafenib treatment by promoting melanoma stem-like cells via its metabolite 27-hydroxycholesterol.

Vemurafenib has been used as first-line therapy for unresectable or metastatic melanoma with BRAFV600E mutation. However, overall survival is still limited due to treatment resistance after about one year. Therefore, identifying new therapeutic targets for melanoma is crucial for improving clinical outcomes. In the present study, we found that lowering intracellular cholesterol by knocking down DHCR24, the limiting synthetase, impaired tumor cell proliferation and migration and abrogated the ability to xenotransplant tumors. More importantly, administration of DHCR24 or cholesterol mediated resistance to vemurafenib and promoted the growth of melanoma spheroids. Mechanistically, we identified that 27-hydroxycholesterol (27HC), a primary metabolite of cholesterol synthesized by the enzyme cytochrome P450 27A1 (CYP27A1), reproduces the phenotypes induced by DHCR24 or cholesterol administration and activates Rap1-PI3K/AKT signaling. Accordingly, CYP27A1 is highly expressed in melanoma patients and upregulated by DHCR24 induction. Dafadine-A, a CYP27A1 inhibitor, attenuates cholesterol-induced growth of melanoma spheroids and abrogates the resistance property of vemurafenib-resistant melanoma cells. Finally, we confirmed that the effects of cholesterol on melanoma resistance require its metabolite 27HC through CYP27A1 catalysis, and that 27HC further upregulates Rap1A/Rap1B expression and increases AKT phosphorylation. Thus, our results suggest that targeting 27HC may be a useful strategy to overcome treatment resistance in metastatic melanoma.

Cholesterol Metabolite 27-Hydroxycholesterol Enhances the Secretion of Cancer Promoting Extracellular Vesicles by a Mitochondrial ROS-Induced Impairment of Lysosomal Function.

Extracellular vesicles (EVs) serve as crucial mediators of cell-to-cell communication in normal physiology as well as in diseased states, and have been largely studied in regard to their role in cancer progression. However, the mechanisms by which their biogenesis and secretion are regulated by metabolic or endocrine factors remain unknown. Here, we delineate a mechanism by which EV secretion is regulated by a cholesterol metabolite, 27-Hydroxycholesterol (27HC), where treatment of myeloid immune cells (RAW 264.7 and J774A.1) with 27HC impairs lysosomal homeostasis, leading to shunting of multivesicular bodies (MVBs) away from lysosomal degradation, towards secretion as EVs. This impairment of lysosomal function is caused by mitochondrial dysfunction and subsequent increase in reactive oxygen species (ROS). Interestingly, cotreatment with a mitochondria-targeted antioxidant rescued the lysosomal impairment and attenuated the 27HC-mediated increase in EV secretion. Overall, our findings establish how a cholesterol metabolite regulates EV secretion and paves the way for the development of strategies to regulate cancer progression by controlling EV secretion.

Roseburia intestinalis Supplementation Could Reverse the Learning and Memory Impairment and m6A Methylation Modification Decrease Caused by 27-Hydroxycholesterol in Mice.

The abnormality in N6-methyladenosine (m6A) methylation is involved in the course of Alzheimer's disease (AD), while the intervention of 27-Hydroxycholesterol (27-OHC) can affect the m6A methylation modification in the brain cortex. Disordered gut microbiota is a key link in 27-OHC leading to cognitive impairment, and further studies have found that the abundance of Roseburia intestinalis in the gut is significantly reduced under the intervention of 27-OHC. This study aims to investigate the association of 27-OHC, Roseburia intestinalis in the gut, and brain m6A modification in the learning and memory ability injury. In this study, 9-month-old male C57BL/6J mice were treated with antibiotic cocktails for 6 weeks to sweep the intestinal flora, followed by 27-OHC or normal saline subcutaneous injection, and then Roseburia intestinalis or normal saline gavage were applied to the mouse. The 27-OHC level in the brain, the gut barrier function, the m6A modification in the brain, and the memory ability were measured. From the results, we observed that 27-OHC impairs the gut barrier function, causing a disturbance in the expression of m6A methylation-related enzymes and reducing the m6A methylation modification level in the brain cortex, and finally leads to learning and memory impairment. However, Roseburia intestinalis supplementation could reverse the negative effects mentioned above. This study suggests that 27-OHC-induced learning and memory impairment might be linked to brain m6A methylation modification disturbance, while Roseburia intestinalis, as a probiotic with great potential, could reverse the damage caused by 27-OHC. This research could help reveal the mechanism of 27-OHC-induced neural damage and provide important scientific evidence for the future use of Roseburia intestinalis in neuroprotection.

Plasma oxysterols are associated with serum lipids and dementia risk in older women.

INTRODUCTION: Apolipoprotein E4 (APOE4) carriers' tendency toward hypercholesterolemia may contribute to Alzheimer's disease (AD) risk through oxysterols, which traverse the blood-brain barrier. METHODS: Relationships between baseline plasma oxysterols, APOE status, serum lipids, and cognitive impairment risk were examined in 328 postmenopausal women from the Women's Health Initiative Memory Study. Women were followed for 25 years or until incident dementia or cognitive impairment. RESULTS: Levels of 24(S)-hydroxycholesterol (24-OHC), 27-hydroxycholesterol (27-OHC), and 24-OHC/27-OHC ratio did not differ by APOE status (p's > 0.05). Higher 24-OHC and 27-OHC were associated with higher total, low density lipoprotein (LDL), non-high density lipoprotein (HDL), remnant, LDL/HDL, and total/HDL cholesterol and triglycerides (p's < 0.05). Higher 24-OHC/27-OHC was associated with greater dementia risk (hazard ratio = 1.51, 95% confidence interval:1.02-2.22), which interaction analyses revealed as significant for APOE3 and APOE4+, but not APOE2+ carriers. DISCUSSION: Less favorable lipid profiles were associated with higher oxysterol levels. A higher ratio of 24-OHC/27-OHC may contribute to dementia risk in APOE3 and APOE4+ carriers.

Impaired astrocytic synaptic function by peripheral cholesterol metabolite 27-hydroxycholesterol.

Astrocytes represent the most abundant cell type in the brain, where they play critical roles in synaptic transmission, cognition, and behavior. Recent discoveries show astrocytes are involved in synaptic dysfunction during Alzheimer's disease (AD). AD patients have imbalanced cholesterol metabolism, demonstrated by high levels of side-chain oxidized cholesterol known as 27-hydroxycholesterol (27-OH). Evidence from our laboratory has shown that elevated 27-OH can abolish synaptic connectivity during neuromaturation, but its effect on astrocyte function is currently unclear. Our results suggest that elevated 27-OH decreases the astrocyte function in vivo in Cyp27Tg, a mouse model of brain oxysterol imbalance. Here, we report a downregulation of glutamate transporters in the hippocampus of CYP27Tg mice together with increased GFAP. GLT-1 downregulation was also observed when WT mice were fed with high-cholesterol diets. To study the relationship between astrocytes and neurons, we have developed a 3D co-culture system that allows all the cell types from mice embryos to differentiate in vitro. We report that our 3D co-cultures reproduce the effects of 27-OH observed in 2D neurons and in vivo. Moreover, we found novel degenerative effects in astrocytes that do not appear in 2D cultures, together with the downregulation of glutamate transporters GLT-1 and GLAST. We propose that this transporter dysregulation leads to neuronal hyperexcitability and synaptic dysfunction based on the effects of 27-OH on astrocytes. Taken together, these results report a new mechanism linking oxysterol imbalance in the brain and synaptic dysfunction through effects on astrocyte function.

High level 27-HC impairs trophoblast cell invasion and migration via LXR in pre-eclampsia.

INTRODUCTION: To explore the potential impact of 27-hydroxycholesterol (27-HC) on trophoblast cell function in pre-eclampsia. RESULTS: The levels of 27-HC and the expression of CYP27A1 are upregulated in clinical samples of PE. Furthermore, high concentrations of 27-HC can inhibit the invasion and migration ability of trophoblast cells in vitro, and this inhibitory effect is weakened after LXR silencing. In HTR8/SVneo cells treated with 27-HC, the expression of ABCA1/ABCG1 are increased. Finally, we established a mouse model of PE using l-NAME (N-Nitro-l-Arginine Methyl Ester). We found an increase in the levels of 27-HC in the peripheral blood serum of the PE mouse model, and an upregulation of CYP27A1 and LXR expressions in the placenta of the PE mouse model. CONCLUSION: 27-HC inhibits the invasion and migration ability of trophoblast cells by activating the LXR signaling pathway, which is involved in the pathogenesis of Pre-eclampsia(PE).

The role of 27-hydroxycholesterol in meta-inflammation.

27-Hydroxycholesterol (27OHChol), a prominent cholesterol metabolite present in the bloodstream and peripheral tissues, is a kind of immune oxysterol that elicits immune response. Recent research indicates the involvement of 27OHChol in metabolic inflammation (meta-inflammation) characterized by chronic responses associated with metabolic irregularities. 27OHChol activates monocytic cells such that they secrete pro-inflammatory cytokines and chemokines, and increase the expression of cell surface molecules such as pattern-recognition receptors that play key roles in immune cell-cell communication and sensing metabolism-associated danger signals. Levels of 27OHChol increase when cholesterol metabolism is disrupted, and the resulting inflammatory responses can contribute to the development and complications of metabolic syndrome, including obesity, insulin resistance, and cardiovascular diseases. Since 27OHChol can induce chronic immune response by activating monocyte-macrophage lineage cells that play a crucial role in meta-inflammation, it is essential to understand the 27OHChol-induced inflammatory responses to unravel the roles and mechanisms of action of this cholesterol metabolite in chronic metabolic disorders.

27-Hydroxycholesterol inhibits trophoblast fusion during placenta development by activating PI3K/AKT/mTOR signaling pathway.

Trophoblast cell syncytialization is essential for placental and fetal development. Abnormal trophoblast cell fusion leads to pregnancy pathologies, such as preeclampsia (PE), intrauterine growth restriction (IUGR), and miscarriage. 27-hydroxycholesterol (27-OHC) is the most abundant oxysterol in human peripheral blood synthesized by sterol 27-hydroxylase (CYP27A1) and is considered a critical mediator between hypercholesterolemia and a variety of related disorders. Gestational hypercholesterolemia was associated with spontaneous preterm delivery and low birth weight (LBW) in term infants, yet the mechanism is unclear. In this study, two trophoblast cell models and CD-1 mice were used to evaluate the effects of 27-OHC on trophoblast fusion during placenta development. Two different kinds of trophoblast cells received a dosage of 2.5, 5, or 10 uM 27-OHC. Three groups of pregnant mice were randomly assigned: control, full treatment (E0.5-E17.5), or late treatment (E13.5-E17.5). All mice received daily intraperitoneal injections of saline (control group) and 27-OHC (treatment group; 5.5 mg/kg). In vitro experiments, we found that 27-OHC inhibited trophoblast cell fusion in primary human trophoblasts (PHT) and forskolin (FSK)-induced BeWo cells. 27-OHC up-regulated the expression of the PI3K/AKT/mTOR signaling pathway-related proteins. Moreover, the PI3K inhibitor LY294002 rescued the inhibitory effect of 27-OHC. Inhibition of trophoblast cell fusion by 27-OHC was also observed in CD-1 mice. Furthermore, fetal weight and placental efficiency decreased and fetal blood vessel development was inhibited in pregnant mice treated with 27-OHC. This study was the first to prove that 27-OHC inhibits trophoblast cell fusion by Activating PI3K/AKT/mTOR signaling pathway. This study reveals a novel mechanism by which dyslipidemia during pregnancy results in adverse pregnancy outcomes.

27-Hydroxycholesterol activates the GSK-3ß/ß-catenin signaling pathway resulting in intestinal fibrosis by inducing oxidative stress: effect of dietary interventions.

OBJECTIVE: Intestinal fibrosis, a common and serious complication of inflammatory bowel disease (IBD), results from chronic inflammation. A high-cholesterol diet may be a risk factor for IBD and 27-hydroxylcholesterol (27HC) is the main human cholesterol metabolite. This study investigated whether 27HC can induce intestinal fibrosis. METHODS: The effects of cholesterol and 27HC on intestinal fibrosis were assessed in zebrafish and human intestinal epithelial Caco-2 cells. RESULTS: Cholesterol and 27HC induced intestinal inflammation and collagen deposition, inhibited E-cadherin (E-ca) expression in the intestinal epithelium, and promoted nuclear translocation of ß-catenin in zebrafish. Cholesterol and 27HC up-regulated expression of COL-1, α-SMA, CTGF, TIMP1, N-cadherin, vimentin, glycogen synthesis kinase-3ß (GSK-3ß) and ß-catenin, but inhibited E-ca, in Caco-2 cells. The expression of these proteins was inhibited by CYP27A1 knockdown and ß-catenin knockdown. 27HC-induced nuclear translocation of ß-catenin occurs in Caco-2 cells. p38, ERK, and AKT activate ß-catenin and thereby participate in 27HC-induced epithelia-mesenchymal transition (EMT) and fibrosis. 27HC-increased oxidative stress and the fibrosis and EMT markers, the nuclear translocation of ß-catenin, and the up-regulation of p-cell kinase proteins promoted by 27HC were inhibited by N-acetyl-L-cysteine (NAC). Folic acid (FA), resveratrol (RES), and NAC all ameliorated the 27HC-induced effects in Caco-2 cells and zebrafish. CONCLUSION: A high-cholesterol diet caused intestinal fibrosis in zebrafish, mediated by a major cholesterol metabolite, 27HC. 27HC increased oxidative stress and activated p38, ERK, AKT, and ß-catenin, leading to EMT of epithelial cells and intestinal fibrosis. FA and RES both ameliorated intestinal fibrosis by restraining 27HC-induced ß-catenin activation.

27-Hydroxycholesterol induces liver fibrosis via down-regulation of trimethylation of histone H3 at lysine 27 by activating oxidative stress; effect of nutrient interventions.

BACKGROUND: Chronic liver injury caused by activation of hepatic stellate cells (HSCs) is a key event in the development of liver fibrosis (LF). A high-cholesterol diet can prompt accumulation of free cholesterol in HSCs, which promotes HSC activation and progression of LF. OBJECTIVE: 27-Hydroxycholesterol (27HC) is the most abundant cholesterol metabolite. Here, we investigated whether the HSC activation and LF induced by high cholesterol is caused by its metabolite 27HC, and whether TGFß classical signaling were involved in these processes. METHODS: In vitro, LX2 and HSC-T6 cells were used to explore the effects of 27HC on activation of HSCs, while LSECs were used to observe the effects of 27HC on capillarization. In vivo, zebrafish were used to assess the effect of 27HC on LF. RESULTS: The cholesterol metabolite 27HC promoted the proliferation of HSCs and up-regulated expression of COL-1 and α-SMA as well as CTGF and TIMP1. Also, 27HC up-regulated expression of Smad2/3 and phosphorylated Smad2/3 in HSCs. Furthermore, 27HC-induced up-regulation of COL-1, α-SMA, CTGF, and TIMP1 protein levels was inhibited by Smad2/3 knockout. In addition, 27HC down-regulated H3K27me3 by inhibition of EZH2 and promotion of UTX and JMJD3 expression via the TGFß signaling, thereby inducing activation of HSCs. Notably, 27HC significantly aggravated the pathological damage induced by DEN, and induced deposition of collagen fibers in zebrafish liver. Folic acid (FA) and resveratrol (RES) both reduced 27HC-induced production of reactive oxygen species (ROS) and inhibited the effects of TGFß signaling on EZH2, UTX, and JMJD3, thereby increasing H3K27me3, and finally jointly inhibiting LF. CONCLUSION: Cholesterol is metabolized to 27HC, which mediates activation of HSCs and onset of LF. Reduced expression of H3k27me3 by TGFß signaling is crucial to 27HC-induced LF. FA and RES ameliorated activation of HSCs and LF by reducing 27HC-induced production of ROS and regulating of H3K27me3.

CYP27A1-27-hydroxycholesterol axis in the respiratory system contributes to house dust mite-induced allergic airway inflammation.

BACKGROUND: 27-Hydroxycholesterol (27-HC) derived from sterol 27-hydroxylase (CYP27A1) has pro-inflammatory biological activity and is associated with oxidative stress and chronic inflammation in COPD. However, the role of regulation of CYP27A1- 27-HC axis in asthma is unclear. This study aimed to elucidate the contribution of the axis to the pathophysiology of asthma. METHODS: House dust mite (HDM) extract was intranasally administered to C57BL/6 mice and the expression of CYP27A1 in the airways was analyzed by immunostaining. The effect of pre-treatment with PBS or CYP27A1 inhibitors on the cell fraction in the bronchoalveolar lavage fluid (BALF) was analyzed in the murine model. In vitro, BEAS-2B cells were treated with HDM and the levels of CYP27A1 expression were examined. Furthermore, the effect of 27-HC on the expressions of E-cadherin and ZO-1 in the cells was analyzed. The amounts of RANTES and eotaxin from the 27-HC-treated cells were analyzed by ELISA. RESULTS: The administration of HDM increased the expression of CYP27A1 in the airways of mice as well as the number of eosinophils in the BALF. CYP27A1 inhibitors ameliorated the HDM-induced increase in the number of eosinophils in the BALF. Treatment with HDM increased the expression of CYP27A1 in BEAS-2B cells. The administration of 27-HC to BEAS-2B cells suppressed the expression of E-cadherin and ZO-1, and augmented the production of RANTES and eotaxin. CONCLUSIONS: The results of this study suggest that aeroallergen could enhance the induction of CYP27A1, leading to allergic airway inflammation and disruption of the airway epithelial tight junction through 27-HC production.

Enzymatically Formed Oxysterols and Cell Death.

The side-chain hydroxylation of cholesterol by specific enzymes produces 24(S)-hydroxycholesterol, 25-hydroxycholesterol, 27-hydroxycholesterol, and other products. These enzymatically formed side-chain oxysterols act as intermediates in the biosynthesis of bile acids and serve as signaling molecules that regulate cholesterol homeostasis. Besides these intracellular functions, an imbalance in oxysterol homeostasis is implicated in pathophysiology. Furthermore, growing evidence reveals that oxysterols affect cell proliferation and cause cell death. This chapter provides an overview of the pathophysiological role of side-chain oxysterols in developing human diseases. We also summarize our understanding of the molecular mechanisms underlying the induction of various forms of cell death by side-chain oxysterols.

Histone deacetylase inhibitor attenuates the effects of 27-hydroxycholesterol on the rat brain.

Hypercholesterolemia is a risk factor for Alzheimer's disease (AD). Plasma cholesterol does not pass the blood-brain barrier whereas its metabolite 27-hydroxycholesterol (27-OHC) can enter the brain. High 27-OHC in the brain has been suggested to mediate hypercholesterolemia-induced impairments of learning and memory through promoting amyloid-ß accumulation and facilitating synaptic disruption. In AD brains, the activity of histone deacetylase (HDAC) is elevated. Treating AD animals with HDAC inhibitors decreases amyloid-ß levels and synaptic damages, which leads to memory improvement. Whether HDAC activity is involved in the actions of 27-OHC is still uncertain. In this study, 4 weekly injections of 27-OHC/vehicle were given to rats followed by 3 daily injections of HDAC inhibitor trichostatin (TSA)/vehicle. The results of Morris water maze test reveal that all rats have intact spatial learning ability during the 5-d training phase. However, the behavioral performance during the probe trial was impaired by 27-OHC treatment, which was improved by adding TSA treatments. Furthermore, 27-OHC treatments reduced the hippocampal levels of acetylated histone H3, acetylated α tubulin, insulin-degrading enzyme and postsynaptic protein PSD-95, indicating that 27-OHC treatments may induce enhanced HDAC activity, decreased amyloid-ß clearance and synaptic disruption. All reduced levels returned to the basal levels by adding TSA treatments. These findings support our hypothesis that HDAC activity is enhanced following long-term exposure to excess 27-OHC.

The Cytotoxicity of 27-Hydroxycholesterol in MCF-7 and MDA-MB-231.

Background: Although several roles of 27-hydroxycholesterol (27-HC), the most abundant oxysterol in blood circulation, in cancers have been elucidated, its impact on breast cancer proliferation and its pathway remain unknown. Materials and Methods: The effect of 27-HC on breast cancer cell proliferation and its pathway was evaluated using Michigan Cancer Foundation - 7 (MCF-7) and M.D. Anderson - Metastatic Breast 231 (MDA-MB-231) cell lines. The MTT assay was applied after 24- and 48-hour incubation to distinguish cell proliferation. To determine the cause of different viability results from the MTT assay, the Annexin-FITC/PI test was used at concentrations of 0.1, 1, and 10 µM after 24- and 48-hour incubation. Results: 27-HC in concentrations of 5, 10, and 20 µM induced cell cytotoxicity compared with control. Also, the annexin V conjugated with fluorescein isothiocyanate/propidium iodide (Annexin-FITC/PI) test revealed an increase in total apoptotic cells treated with 0.1, 1, and 10 µM of 27-HC after 48 hours (P value < 0.05). Besides, the cytotoxic effect of 27-HC was observed at 10 µM concentration in both cell lines, MCF-7 and MDA-MB-231 (P value < 0.05). Conclusion: The identification of 27-HC's cytotoxic effects on both estrogen receptor (ER)-negative and ER-positive breast cancer cell lines is a novel discovery that may be linked to LXRß.

Adipose Tissue-Derived Mesenchymal Stem Cells Alter Metabolites of Brain Cholesterol Homeostasis in An Alzheimer's Model.

OBJECTIVE: Disruption of cholesterol homeostasis in Alzheimer's disease (AD) plays a crucial role in disease pathogenesis, making it a potential therapeutic target. Mesenchymal stem cells (MSCs) show promise in treating cognitive impairment and provide a novel therapeutic approach. This study aims to investigate the effects of MSCs on specific metabolites associated with brain cholesterol homeostasis in an AD rat model. MATERIALS AND METHODS: In this experimental study, animals were divided into three groups: control, AD, and AD+MSCs. AD was induced using amyloid beta (Aß) and confirmed through the Morris water maze (MWM) behavioural test and Congo red staining. MSCs were extracted, characterised via flow cytometry, subjected to osteoblast and adipose differentiation, and injected intraventricularly. The cholesterol metabolite levels were measured using gas chromatography-mass spectrometry (GC)-MS and compared among the groups. RESULTS: Treatment with MSCs significantly improved memory function in the AD+MSCs group compared to the AD group and the number of beta-amyloid plaques decreased according to histological assessment. Disturbances in the brain cholesterol metabolites that included desmosterol, 7-dehydrocholesterol, 24S-hydroxycholesterol, 27-hydroxycholesterol and cholesterol were observed in the AD group compared to the control group. Treatment with MSCs resulted in significant alterations in the levels of these metabolites. CONCLUSION: The findings indicate that MSC therapy has the potential to improve AD by modulating brain cholesterol homeostasis and promoting the differentiation of stem cells into nerve cells. The results emphasize the importance of investigating the role of cholesterol metabolites in the context of MSC therapy to gain deeper insights into underlying mechanisms of the therapeutic efficacy of MSCs in AD.

27-hydroxycholesterol causes cognitive deficits by disturbing Th17/Treg balance and the related immune responses in mild cognitive impairment patients and C57BL/6J mice.

BACKGROUND: Cognitive impairment is associated with dysregulated immune responses. Emerging evidence indicates that Th17 cells and their characteristic cytokine-IL-17 are receiving growing interest in the pathogenesis of cognitive decline. Here, we focus on the involvement of Th17 cells in mild cognitive impairment (MCI) and the possible mechanism of cholesterol metabolite-27-hydroxycholesterol (27-OHC). METHODS: 100 individuals were recruited into the nested case-control study who completed cognition assessment and the detection of oxysterols and Th17-related cytokines in serum. In addition, mice were treated with 27-OHC and inhibitors of RORγt and Foxp3 (Th17 and Treg transcription factors), and the factors involved in Th17/Treg balance and amyloidosis were detected. RESULTS: Our results showed there was enhanced 27-OHC level in serum of MCI individuals. The Th17-related cytokines homeostasis was altered, manifested as increased IL-17A, IL-12p70, IL-23, GM-CSF, MIP-3α and TNF-α but decreased IL-13, IL-28A and TGF-ß1. Further, in vivo experiments showed that 27-OHC induced higher immunogenicity, which increased Th17 proportion but decreased Treg cells in peripheral blood mononuclear cells (PBMCs); Th17 proportions in hippocampus, and IL-17A level in serum and brain were also higher than control mice. The fluorescence intensity of amyloid-ß (Aß) and the precursor of amyloid A amyloidosis-serum amyloid A (SAA) was increased in the brain of 27-OHC-treated mice, and worse learning and memory performance was supported by water maze test results. While by inhibiting RORγt in 27-OHC-loaded mice, Th17 proportions in both PBMCs and hippocampus were reduced, and expressions of IL-17A and TGF-ß1 were down- and up-regulated, respectively, along with a decreased amyloidosis in brain and improved learning and memory decline. CONCLUSIONS: Altogether, our results demonstrate that excessive 27-OHC aggravates the amyloidosis and leads to cognitive deficits by regulating RORγt and disturbing Th17/Treg balance.

Tissue-Specific Oxysterols as Predictors of Antidepressant (Escitalopram) Treatment Response in Patients With Major Depressive Disorder.

Background: There is growing evidence that disturbances in cholesterol metabolism may be involved in major depressive disorder (MDD). However, it is not known if cholesterol metabolites present in the brain and periphery can be used to diagnose and predict an MDD patient's response to antidepressant treatment. Methods: A total of 176 subjects (85 patients with MDD and 91 healthy control subjects) were included in this study. The expression of peripheral and brain-specific oxysterols and related gene polymorphisms were investigated in all subjects. The severity of depression was measured using the 17-item Hamilton Depression Rating Scale, 16-item Quick Inventory of Depressive Symptoms-Self-Report, and Patient Health Questionnaire-9 for all patients with MDD before and after 12 weeks of antidepressant treatment. Results: Patients with MDD expressed higher plasma levels of 24(S)-hydroxycholesterol (24OHC) (mainly secreted from the brain) compared with healthy control subjects, and the higher levels of 24OHC were associated with 24OHC synthetase (CYP46A1) gene polymorphisms. In patients with MDD, an improved response to the 12-week antidepressant treatment was associated with a reduction of both 24OHC and 27OHC (mainly secreted from the peripheral system) levels relative to baseline levels. Nonresponders exhibited increased levels of oxysterols at the end of treatment compared with baseline. The superior reduction in oxysterol levels correlated with better outcomes from the antidepressant treatment. Conclusions: These data suggest a potential role for oxysterols as diagnostic and treatment response-related indicators for MDD.

27-Hydroxycholesterol impairs learning and memory ability via decreasing brain glucose uptake mediated by the gut microbiota.

Brain glucose hypometabolism is a significant manifestation of Alzheimer's disease (AD). 27-hydroxycholesterol (27-OHC) and the gut microbiota have been recognized as factors possibly influencing the pathogenesis of AD. This study aimed to investigate the link between 27-OHC, the gut microbiota, and brain glucose uptake in AD. Here, 6-month-old male C57BL/6 J mice were treated with sterile water or antibiotic cocktails, with or without 27-OHC and/or 27-OHC synthetic enzyme CYP27A1 inhibitor anastrozole (ANS). The gut microbiota, brain glucose uptake levels, and memory ability were measured. We observed that 27-OHC altered microbiota composition, damaged brain tissue structures, decreased the 2-deoxy-2-[18 F] fluorodeoxyglucose (18F-FDG) uptake value, downregulated the gene expression of glucose transporter type 4 (GLUT4), reduced the colocalization of GLUT1/glial fibrillary acidic protein (GFAP) in the hippocampus, and impaired spatial memory. ANS reversed the effects of 27-OHC. The antibiotic-treated mice did not exhibit similar results after 27-OHC treatment. This study reveals a potential molecular mechanism wherein 27-OHC-induced memory impairment might be linked to reduced brain glucose uptake, mediated by the gut microbiota.

Development and validation of a liquid chromatography-tandem mass spectrometry assay to quantify plasma 24(S)-hydroxycholesterol and 27-hydroxycholesterol: A new approach integrating the concept of ion ratio.

Accurate quantification of 24(S)-hydroxycholesterol and 27-hydroxycholesterol holds substantial biological significance due to their involvement in pivotal cellular processes, encompassing cholesterol homeostasis, inflammatory responses, neuronal signaling, and their potential as disease biomarkers. The plasma determination of these oxysterols is challenging considering their low concentrations and similarities in terms of empirical formulae, molecular structure, and physicochemical properties across all human endogenous plasma oxysterols. To overcome these sensitivity and specificity issues, we developed and validated a quantification method using liquid chromatography coupled to a tandem mass spectrometry instrument. Validation studies were designed inspired by Clinical and Laboratory Standards Institute (CLSI) C62-A Guidelines. The linearity ranged between 20 and 300 nM for both oxysterols with limits of quantification at 20 nM and 30 nM for 24(S)-OHC and 27-OHC, respectively. Inter-day precision coefficient variations (CV) were lower than 10% for both oxysterols. An optimal separation of 25-OHC was obtained from 24(S)-OHC and 27-OHC with a resolution (Rs) > 1.25. The determination and validation of ion ratios for 24(S)-OHC and 27-OHC enabled another quality check in identifying interferents that could impact the quantification. Our developed and validated LC-MS/MS method allows consistent and reliable quantification of human plasmatic 24(S)-OHC and 27-OHC that is warranted in fundamental and clinical research projects.

Inhibition of Marek's Disease Virus Replication and Spread by 25-hydroxycholesterol and 27-hydroxycholesterol In Vitro.

Marek's disease virus (MDV) causes a deadly lymphoproliferative disease in chickens, resulting in huge economic losses in the poultry industry. It has been suggested that MDV suppresses the induction of type I interferons and thus escapes immune control. Cholesterol 25-hydroxylase (CH25H), a gene that encodes an enzyme that catalyses cholesterol to 25-hydroxycholesterol (25-HC), is an interferon-stimulating gene (ISG) known to exert antiviral activities. Other oxysterols, such as 27-hydroxycholesterols (27-HC), have also been shown to exert antiviral activities, and 27-HC is synthesised by the catalysis of cholesterol via the cytochrome P450 enzyme oxidase sterol 27-hydroxylase A1 (CYP27A1). At 24 h post infection (hpi), MDV stimulated a type I interferon (IFN-α) response, which was significantly reduced at 48 and 72 hpi, as detected using the luciferase assay for chicken type I IFNs. Then, using RT-PCR, we demonstrated that chicken type I IFN (IFN-α) upregulates chicken CH25H and CYP27A1 genes in chicken embryo fibroblast (CEF) cells. In parallel, our results demonstrate a moderate and transient upregulation of CH25H at 48 hpi and CYP27A1 at 72hpi in MDV-infected CEF cells. A significant reduction in MDV titer and plaque sizes was observed in CEFs treated with 25-HC or 27-HC in vitro, as demonstrated using a standard plaque assay for MDV. Taken together, our results suggest that 25-HC and 27-HC may be useful antiviral agents to control MDV replication and spread.