Microglia, essential immune cells in the brain, play crucial roles in neuroinflammation by performing various functions such as neurogenesis, synaptic pruning, and pathogen defense. These cells are activated by inflammatory factors like β-amyloid (Aβ) and oxysterols, leading to morphological and functional changes, including the secretion of inflammatory cytokines and the upregulation of MHC class II molecules. This study focused on identifying specific markers for microglial activation, with a particular emphasis on the roles of oxysterols in this process. We used the HMC3 human microglial cell line to investigate the induction of heat shock protein 60 (HSP60), a chaperonin protein by oxysterols, specifically in the presence of 25-hydroxycholesterol (25OHChol) and 27-hydroxycholesterol (27OHChol). Our findings obtained by the proteomics approach revealed that these oxysterols significantly increased HSP60 expression on microglial cells. This induction was further confirmed using Western blot analysis and immunofluorescence microscopy. Additionally, Aβ1-42 also promoted HSP60 expression, indicating its role as a microglial activator. HSP60 involved in protein folding and immune modulation was identified as a potential marker for microglial activation. This study underscores the importance of HSP60 in the inflammatory response of microglia, suggesting its utility as a target for new therapeutic approaches in neuroinflammatory diseases such as Alzheimer\'s disease (AD).

OBJECTIVE: This study aimed to identify precise biomarkers and develop targeted therapeutic strategies for preventing premature atherosclerotic cardiovascular disease in patients with familial hypercholesterolemia (FH) by investigating the quantitative and qualitative abnormalities in the metabolic network of lipids in these patients using an advanced lipidomics platform.
METHODS: The study population comprised 18 homozygous (HoFH), 18 heterozygous (HeFH) FH patients, and 20 healthy controls. Cholesterol oxidation products (oxysterol, COPs) and main lipid classes were determined using gas chromatography-mass spectrometry. Results were expressed as percentages of total fat matter for lipid classes and percentages of total COPs for oxysterols. The principal component analysis (PCA) was also carried out, to highlight the correlation between studied parameters and groups investigated.
RESULTS: Patients (both HoFH and HeFH) showed lower content of free fatty acids (FFAs) and greater values of triacylglycerols (TAGs) in comparison to controls. HoFH showed lower monoacylglycerols (P<0.01) and higher free cholesterol (FC) (P<0.05) when compared to HeFH and controls. The total content of COPs ranged from 1.96 to 4.25 mg/dL, from 2.27 to 4.05 mg/dL, and from 0.79 to 4.12 mg/dL in healthy controls, HoFH and HeFH groups, respectively, with no significant differences between patients and controls. In general, the 7α-hydroxycholesterol (7α-HC) was greater than other COPs. However, no significant differences were found between the three studied groups. Moreover, an opposite trend was observed between 7α-HC and 7-ketocholesterol (7-KC). Additionally, when PCA was carried out, the first two PCs explained 92.13 % of the total variance, of which the PC1 describes 53.94 % of variance mainly correlated to TAGs, diacylglycerols (DAGs), and 7-KC. On the other hand, the PC2 was correlated primarily for FFAs, FC and esterified sterols (E-STE).
CONCLUSIONS: In conclusion, abnormal levels of TAGs, DAGs and 7-KC were associated with HeFH while HoFH was associated with the abnormal amount of E-STE.

Intracellular cholesterol metabolism is regulated by the SREBP-2 and LXR signaling pathways. The effects of inflammation on these molecular mechanisms remain poorly studied, especially at the blood-brain barrier (BBB) level. Tumor necrosis factor α (TNFα) is a proinflammatory cytokine associated with BBB dysfunction. Therefore, the aim of our study was to investigate the effects of TNFα on BBB cholesterol metabolism, focusing on its underlying signaling pathways. Using a human in vitro BBB model composed of human brain-like endothelial cells (hBLECs) and brain pericytes (HBPs), we observed that TNFα increases BBB permeability by degrading the tight junction protein CLAUDIN-5 and activating stress signaling pathways in both cell types. TNFα also promotes cholesterol release and decreases cholesterol accumulation and APOE secretion. In hBLECs, the expression of SREBP-2 targets (LDLR and HMGCR) is increased, while ABCA1 expression is decreased. In HBPs, only LDLR and ABCA1 expression is increased. TNFα treatment also induces 25-hydroxycholesterol (25-HC) production, a cholesterol metabolite involved in the immune response and intracellular cholesterol metabolism. 25-HC pretreatment attenuates TNFα-induced BBB leakage and partially alleviates the effects of TNFα on ABCA1, LDLR, and HMGCR expression. Overall, our results suggest that TNFα favors cholesterol efflux via an LXR/ABCA1-independent mechanism at the BBB, while it activates the SREBP-2 pathway. Treatment with 25-HC partially reversed the effect of TNFα on the LXR/SREBP-2 pathways. Our study provides novel perspectives for better understanding cerebrovascular signaling events linked to BBB dysfunction and cholesterol metabolism in neuroinflammatory diseases.

BACKGROUND: Si-Ni-San (SNS), a traditional Chinese medicinal formula derived from Treatise on Febrile Diseases, is considered effective in the treatment of inflammatory bowel diseases based upon thousands of years of clinical practice. However, the bioactive ingredients and underlying mechanisms are still unclear and need further investigation.
OBJECTIVE: This study aimed to evaluate the effect, explore the bioactive ingredients and the underlying mechanisms of SNS in ameliorating ulcerative colitis (UC) and associated liver injury in dextran sodium sulphate (DSS)-induced mouse colitis models.
METHODS: The effect of SNS (1.5, 3, 6 g/kg) on 3% DSS-induced acute murine colitis was evaluated by disease activity index (DAI), colon length, inflammatory cytokines, hematoxylin-eosin (H&E) staining, tight junction proteins expression, ALT, AST, and oxidative stress indicators. HPLC-ESI-IT/TOF MS was used to analyze the chemical components of SNS and the main xenobiotics in the colon of UC mice after oral administration of SNS. Network pharmacological study was then conducted based on the main xenobiotics. Flow cytometry and immunohistochemistry techniques were used to demonstrate the inhibitory effect of SNS on Th17 cells differentiation and the amelioration of Th17/Treg cell imbalance. LC-MS/MS, Real-time quantitative polymerase chain reaction (RT-qPCR), and western blotting techniques were performed to investigate the oxysterol-Liver X receptor (LXRs) signaling activity in colon. Targeted bile acids metabolomics was conducted to reveal the change of the two major pathways of bile acid synthesis in the liver, and the expression of key metabolic enzymes of bile acids synthesis was characterized by RT-qPCR and western blotting techniques.
RESULTS: SNS (1.5, 3, 6 g/kg) decreased the DAI scores, protected intestinal mucosa barrier, suppressed the production of pro-inflammatory cytokines, improved hepatic and splenic enlargement and alleviated liver injury in a dose-dependent manner. A total of 22 components were identified in the colon of SNS (6 g/kg) treated colitis mice, and the top 10 components ranked by relative content were regarded as the potential effective chemical components of SNS, and used to conduct network pharmacology research. The efficacy of SNS was mediated by a reduction of Th17 cell differentiation, restoration of Th17/Treg cell homeostasis in the colon and spleen, and the experimental results were consistent with our hypothesis and the biological mechanism predicted by network pharmacology. Mechanistically, SNS regulated the concentration of 25-OHC and 27-OHC by up-regulated CH25H, CYP27A1 protein expression in colon, thus affected the expression and activity of LXR, ultimately impacted Th17 differentiation and Th17/Treg balance. It was also found that SNS repressed the increase of hepatic cholesterol and reversed the shift of BA synthesis to the acidic pathway in UC mice, which decreased the proportion of non-12-OH BAs in total bile acids (TBAs) and further ameliorated colitis and concomitant liver injury.
CONCLUSIONS: This study set the stage for considering SNS as a multi-organ benefited anti-colitis prescription based on the significant effect of ameliorating intestinal and liver damage, and revealed that derivatives of cholesterol, namely oxysterols and bile acids, were closely involved in the mechanism of SNS anti-colitis effect.

UNASSIGNED: Despite endothelial dysfunction being an initial step in the development of hypertension and associated cardiovascular/renal injuries, effective therapeutic strategies to prevent endothelial dysfunction are still lacking. GPR183 (G protein-coupled receptor 183), a recently identified G protein-coupled receptor for oxysterols and hydroxylated metabolites of cholesterol, has pleiotropic roles in lipid metabolism and immune responses. However, the role of GPR183 in the regulation of endothelial function remains unknown.
UNASSIGNED: Endothelial-specific GPR183 knockout mice were generated and used to examine the role of GPR183 in endothelial senescence by establishing 2 independent hypertension models: desoxycorticosterone acetate/salt-induced and Ang II (angiotensin II)-induced hypertensive mice. Echocardiography, transmission electron microscopy, blood pressure measurement, vasorelaxation response experiments, flow cytometry analysis, and chromatin immunoprecipitation analysis were performed in this study.
UNASSIGNED: Endothelial GPR183 was significantly induced in hypertensive mice, which was further confirmed in renal biopsies from subjects with hypertensive nephropathy. Endothelial-specific deficiency of GPR183 markedly alleviated cardiovascular and renal injuries in hypertensive mice. Moreover, we found that GPR183 regulated endothelial senescence in both hypertensive mice and aged mice. Mechanistically, GPR183 disrupted circadian signaling by inhibiting PER1 (period 1) expression, thereby facilitating endothelial senescence and dysfunction through the cAMP/PKA (protein kinase A)/CREB (cAMP-response element binding protein) signaling pathway. Importantly, pharmacological inhibition of the oxysterol-GPR183 axis by NIBR189 or clotrimazole ameliorated endothelial senescence and cardiovascular/renal injuries in hypertensive mice.
UNASSIGNED: This study discovers a previously unrecognized role of GPR183 in promoting endothelial senescence. Pharmacological targeting of GPR183 may be an innovative therapeutic strategy for hypertension and its associated complications.

Polycystin-1 (PC-1) and PC-2 form a heteromeric ion channel complex that is abundantly expressed in primary cilia of renal epithelial cells. This complex functions as a non-selective cation channel, and mutations within the polycystin complex cause autosomal dominant polycystic kidney disease (ADPKD). The spatial and temporal regulation of the polycystin complex within the ciliary membrane remains poorly understood. Using both whole-cell and ciliary patch-clamp recordings, we identify a cilia-enriched oxysterol, 7β,27-dihydroxycholesterol (DHC), that serves as a necessary activator of the polycystin complex. We further identify an oxysterol-binding pocket within PC-2 and showed that mutations within this binding pocket disrupt 7β,27-DHC-dependent polycystin activation. Pharmacologic and genetic inhibition of oxysterol synthesis reduces channel activity in primary cilia. In summary, our findings reveal a regulator of the polycystin complex. This oxysterol-binding pocket in PC-2 may provide a specific target for potential ADPKD therapeutics.

An UPLC-APCI-MS/MS method was developed for the simultaneous determination of cholesterol, 7-dehydrocholesterol (7DHC) and eight oxysterols including 27-hydroxycholesterol (27OHC), 7α-hydroxycholesterol (7αOHC), 7β-hydroxycholesterol (7βOHC), 24S-hydroxycholesterol (24SOHC), 25-hydroxycholesterol (25OHC), 7α,24S-dihydroxycholesterol (7α,24SdiOHC), 7α,25-dihydroxycholesterol (7α,25diOHC), and 7α,27-dihydroxycholesterol (7α,27diOHC). It has been used for quantitative analysis of cholesterol, 7DHC and eight oxysterols in hepatocellular carcinoma (HCC) cells, plasma and tumor tissue samples. And the above compounds were extracted from the biological matrix (plasma and tissue) using liquid-liquid extraction with hexane/isopropanol after saponification to cleave the steroids from their esterified forms without further derivatization. Then cholesterol, 7DHC and oxysterols were separated on a reversed phase column (Agilent Zorbax Eclipse plus, C18) within 8 min using a gradient elution with 0.1 % formic acid in H2O and methanol and detected by an APCI triple quadrupole mass spectrometer. The lower limit of quantification (LLOQ) of the cholesterol, 7DHC and oxysterols ranged from 3.9 ng/mL to 31.25 ng/mL, and the recoveries ranged from 83.0 % to 113.9 %. Cholesterol, 7DHC and several oxysterols including 27OHC, 7αOHC and 7βOHC were successfully quantified in HCC cells, plasma, tissues and urine of HCC mice. Results showed that 27OHC was at high levels in three kind of HCC cells and tumor tissues as well as plasma samples from both HepG2 and Huh7 bearing mice model，and the high levels of 27OHC in tumors were associated with HCC development. Moreover, the levels of cholesterol in HCC cells and tumor issues varied in different HCC cells and mice model. Oxysterols profiling in biological samples might provide complementary information in cancer diagnosis.

This explorative study aimed to assess the mutagenicity and genotoxicity of stored-cooked beef patties formulated with and without phenols (7.00 mg of phenols/80-g patty) extracted from olive vegetation water (OVW), as related to the formation of cholesterol oxidation products (COPs) and heterocyclic amines (HCAs). The patties were packaged in a modified atmosphere, sampled during cold storage (4 °C) for 9 days, and grilled at 200 °C. The genotoxicity was evaluated by the Comet assay. The patty extract was found to be genotoxic on primary peripheral blood mononuclear cells (PBMCs), while no mutagenicity was detected. The addition of OVW phenols significantly decreased the genotoxicity of the patty extract and reduced the total COPs content in stored-cooked patties (4.59 times lower than control); however, it did not affect the content of total HCAs (31.51-36.31 ng/patty) and the revertants\' number. Therefore, these results demonstrate that the OVW phenols were able to counteract the formation of genotoxic compounds in stored-cooked beef patties.

Diabetic kidney disease (DKD) is a leading cause of end stage renal disease with unmet clinical demands for treatment. Lipids are essential for cell survival; however, renal cells have limited capability to metabolize overloaded lipids. Dyslipidaemia is common in DKD patients and renal ectopic lipid accumulation is associated with disease progression. Unveiling the molecular mechanism involved in renal lipid regulation is crucial for exploring potential therapeutic targets. In this review, we focused on the mechanism underlying cholesterol, oxysterol and fatty acid metabolism disorder in the context of DKD. Specific regulators of lipid accumulation in different kidney compartment and TREM2 macrophages, a lipid-related macrophages in DKD, were discussed. The role of sodium-glucose transporter 2 inhibitors in improving renal lipid accumulation was summarized.

Cholesterol oxidation products (COPs) are contaminants of food of animal origin. Increased levels of these compounds in the human body are associated with an increased risk of many non-communicable diseases. Dairy products are mentioned among the main sources of these compounds in the diet. The objective of this study was to evaluate the contents of cholesterol and its oxidized derivatives in eleven groups of dairy products, willingly consumed in European countries. The levels of COPs were determined by applying the GC-TOF/MS method. In the tested products, cholesterol and its oxidation derivatives, such as 7-ketocholesterol, 7α-hydroxycholesterol, 7β-hydroxycholesterol, 5,6β-epoxycholesterol and 5,6α-epoxycholesterol, were determined. The studied dairy products differed in their contents and profiles of oxysterols. The highest contents of COPs were found in cheese with internal mold (13.8 ± 2.5 mg kg-1) and Cheddar (11.7 ± 3.5 mg kg-1), while the lowest levels were detected in yoghurt (0.94 ± 0.30 mg kg-1) and kefir (0.57 ± 0.11 mg kg-1). 7-ketocholesterol and 5,6β-epoxycholesterol were the dominant oxysterols. The ratio of oxidized derivatives to total cholesterol was on average 1.7%. Our results confirmed that dairy products are an important dietary source of COPs. Their levels should be monitored in dairy products to provide the best health quality.