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.

Oxysterols, as metabolites of cholesterol, play a key role in cholesterol homeostasis, autophagosome formation, and regulation of immune responses. Disorders in oxysterol metabolism are closely related to the pathogenesis of neurodegenerative diseases. To systematically investigate the profound molecular regulatory mechanisms of neurodegenerative diseases, it is necessary to quantify oxysterols and their metabolites in central and peripheral biospecimens simultaneously and accurately. However, there are a lot of unsolved problems with the existing methods, such as the hindrance of applying a single method to different biological specimens or the challenge of simultaneous quantification due to differential groups on the ends of the oxysterol side chains. Herein, according to the physicochemical properties and structure of oxysterols, an optimized liquid chromatography-tandem mass spectrometry method for the quantification of oxysterols was established by optimizing the sample preparation process, chromatographic conditions, mobile phase pH, and solvent selection. Seven oxysterols were detected by this method, including 27-hydroxycholesterol, 7α-hydroxycholesterol, 7α,27-dihydroxycholesterol, 7-dehydrocholesterol, 7α-hydroxy-3-oxo-4-cholestenoic acid, 3-hydroxy-5-cholestenoic acid, and 24(S)-hydroxycholesterol. Non-derivatization extraction with methyl tert-butyl ether was used for different biospecimens, followed by simultaneous chromatographic separation of oxysterols on a phenyl hexyl column. By repeated validation, this method exhibited satisfactory linearity, precision, recovery, sensitivity, repeatability, and stability, and it was successfully applied to the detection of oxysterols in the plasma, cerebral cortex, and liver of mouse. In summary, our optimized method enables concurrent analysis and quantification of oxysterols and their metabolites in various biospecimens, presenting a broad range of applicability.

Background/Objectives: Alterations of oxysterols and gut microbiota have been recognized as indicators affecting mild cognitive impairment (MCI) and sarcopenia, respectively, whereas their association with co-dysfunction has not been investigated. Methods: In this study, a total of 1035 individuals were divided into Control (n = 264), MCI (n = 435), and MCI with possible sarcopenia (MPS, n = 336) groups. Cognition and muscle indexes, serum oxysterols, and gut microbiota were measured. Spearman’s rank coefficients were calculated to determine their correlations. Results: Performances of global and multidimensional cognitive tests was successively worse in the Control, MCI, and MPS groups. Longer duration of five-time chair stand test, lower 6-meter walk speed, and handgrip strength were observed in the MPS group, along with increased 27-hydroxycholesterol (27-OHC) and 5α,6α-epoxycholesterol and decreased 5α-Cholest-8(14)-ene-3β,15α-diol (15-HC). Higher concentrations of amyloid precursor protein (APP), neurofilament, and C-terminal agrin fragment (CAF) were discovered in the MCI and MPS groups. The α-diversity of gut microbiota in the MCI and MPS group was remarkably decreased, followed by a shifted abundance of microbial taxa, such as Alistipes and Rikenellaceae. Multiple significant correlations were found between cognition and muscle indexes and with oxysterols. Conclusions: Our study indicates that oxysterols and gut microbiota are prominently involved in the co-dysfunction of cognition and muscle.