Evaluating toxicity and decoding the underlying mechanisms of active compounds are crucial for drug development.In this study,we present an innovative,integrated approach that combines air flow-assisted desorption electrospray ionization mass spectrometry imaging(AFADESI-MSI),time-of-flight secondary ion mass spectrometry(ToF-SIMS),and spatial metabolomics to comprehensively investi-gate the nephrotoxicity and underlying mechanisms of nitidine chloride(NC),a promising anti-tumor drug candidate.Our quantitive AFADESI-MSI analysis unveiled the region specific of accumulation of NC in the kidney,particularly within the inner cortex(IC)region,following single and repeated dose of NC.High spatial resolution ToF-SIMS analysis further allowed us to precisely map the localization of NC within the renal tubule.Employing spatial metabolomics based on AFADESI-MSI,we identified over 70 discriminating endogenous metabolites associated with chronic NC exposure.These findings suggest the renal tubule as the primary target of NC toxicity and implicate renal transporters(organic cation transporters,multidrug and toxin extrusion,and organic cation transporter 2(OCT2)),metabolic en-zymes(protein arginine N-methyltransferase(PRMT)and nitric oxide synthase),mitochondria,oxidative stress,and inflammation in NC-induced nephrotoxicity.This study offers novel insights into NC-induced renal damage,representing a crucial step towards devising strategies to mitigate renal damage caused by this compound.

Polygala tenuifolia,commonly known as Yuanzhi(YZ)in Chinese,has been shown to possess anti-insomnia properties.However,the material basis and the mechanism underlying its sedative-hypnotic effects remain unclear.Herein,we investigated the active components and neurochemical mechanism of YZ extracts using liquid chromatography tandem mass spectrometry(LC-MS/MS)-based pharmaco-metabolomics and mass spectrometry imaging(MSI)-based spatial resolved metabolomics.According to the results,17 prototypes out of 101 ingredients in the YZ extract were detected in both the plasma and brain,which might be the major components contributing to the sedative-hypnotic effects.Network pharmacology analysis revealed that these prototypes may exert their effects through neuroactive ligand-receptor interaction,serotonergic synapse,dopaminergic synapse,and dopaminergic synapse,among other pathways.LC-MS/MS-based targeted metabolomics and Western blot(WB)revealed that tryptophan-serotonin-melatonin(Trp-5-HT-Mel)and tyrosine-norepinephrine-adrenaline(Tyr-Ne-Ad)are the key regulated pathways.Dopa decarboxylase(DDC)upregulation and phenylethanolamine N-methyltransferase(PNMT)downregulation further confirmed these pathways.Furthermore,MSI-based spatially resolved metabolomics revealed notable alterations in 5-HT in the pineal gland(PG),and Ad in the brainstem,including the middle brain(MB),pons(PN),and hypothalamus(HY).In summary,this study illustrates the efficacy of an integrated multidimensional metabolomics approach in unraveling the sedative-hypnotic effects and neurochemical mechanisms of a Chinese herbal medicine,YZ.

Diabetic cardiomyopathy(DCM)is a metabolic disease and a leading cause of heart failure among people with diabetes.Mass spectrometry imaging(MSI)is a versatile technique capable of combining the molecular specificity of mass spectrometry(MS)with the spatial information of imaging.In this study,we used MSI to visualize metabolites in the rat heart with high spatial resolution and sensitivity.We optimized the air flow-assisted desorption electrospray ionization(AFADESI)-MSI platform to detect a wide range of metabolites,and then used matrix-assisted laser desorption ionization(MALDI)-MSI for increasing metabolic coverage and improving localization resolution.AFADESI-MSI detected 214 and 149 metabolites in positive and negative analyses of rat heart sections,respectively,while MALDI-MSI detected 61 metabolites in negative analysis.Our study revealed the heterogenous metabolic profile of the heart in a DCM model,with over 105 region-specific changes in the levels of a wide range of metabolite classes,including carbohydrates,amino acids,nucleotides,and their derivatives,fatty acids,glycerol phospholipids,carnitines,and metal ions.The repeated oral administration of ferulic acid during 20 weeks significantly improved most of the metabolic disorders in the DCM model.Our findings provide novel insights into the molecular mechanisms underlying DCM and the potential of ferulic acid as a therapeutic agent for treating this condition.

Three-dimensional(3D)cell spheroid models combined with mass spectrometry imaging(MSI)enables innovative investigation of in vivo-like biological processes under different physiological and patho-logical conditions.Herein,airflow-assisted desorption electrospray ionization-MSI(AFADESI-MSI)was coupled with 3D HepG2 spheroids to assess the metabolism and hepatotoxicity of amiodarone(AMI).High-coverage imaging of＞1100 endogenous metabolites in hepatocyte spheroids was achieved using AFADESI-MSI.Following AMI treatment at different times,15 metabolites of AMI involved in N-desethylation,hydroxylation,deiodination,and desaturation metabolic reactions were identified,and according to their spatiotemporal dynamics features,the metabolic pathways of AMI were proposed.Subsequently,the temporal and spatial changes in metabolic disturbance within spheroids caused by drug exposure were obtained via metabolomic analysis.The main dysregulated metabolic pathways included arachidonic acid and glycerophospholipid metabolism,providing considerable evidence for the mechanism of AMI hepatotoxicity.In addition,a biomarker group of eight fatty acids was selected that provided improved indication of cell viability and could characterize the hepatotoxicity of AMI.The combination of AFADESI-MSI and HepG2 spheroids can simultaneously obtain spatiotemporal infor-mation for drugs,drug metabolites,and endogenous metabolites after AMI treatment,providing an effective tool for in vitro drug hepatotoxicity evaluation.

Chiari malformation (CM) is a group of congenital cerebellar tonsillar hernia malformations involving the craniocervical junction. Chiari malformation type I (CMI) is the most common in clinic, however its pathogenesis is still unclear, and there is no consensus on the surgical treatment standard of CMI. At present, the most widely accepted is the theory of posterior fossa incompatibility, so doctors at home and abroad use posterior fossa decompression (PFD) and posterior fossa compression with duraplasty (PFDD) as the gold standard for surgical treatment, and have their own experience and technical improvement. However, the volume of the posterior cranial fossa in some patients is no different from that in healthy people, and about 30% of the patients with CMI have poor results after posterior cranial fossa decompression. As a result, this operation cannot treat all patients with CMI. In recent years, with the development of imaging, the progress of diagnostic technology and the deepening of understanding of CM, some studies have shown that CMI may be related to atlantoaxial instability, and proposed that CMI is the secondary factor of atlantoaxial instability, and atlantoaxial fusion is the standard of surgical treatment, which has caused great controversy in academic circles. Different clinical research results of scholars support or oppose this theory: some studies have shown that the clinical symptom relief rate of patients with CMI treated with atlantoaxial fusion is 96.9%; another study showed that 70% of patients with CMI underwent atlantoaxial fusion had improved neurological function, but the overall postoperative effect was not satisfactory. In short, CMI is related to many diseases and its clinical manifestations are complex. Therefore, individualized management and treatment should be carried out in combination with the clinical manifestations and auxiliary examination results of patients.

Developing analytical methods for the chemical components of natural medicines remains a challenge due to its diversity and complexity. Miao-Fu-Zhi-Tong (MFZT) granules, an ethnic Yi herbal prescription, comprises 10 herbs and has been clinically applied for gouty arthritis (GA) therapy. Herein, a series of chemical profiling strategies including in-house library matching, molecular networking and MS/MS fragmentation behavior validation based on ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) were developed for qualitative analysis of MFZT granules. A total of 207 compounds were identified or characterized in which several rare guanidines were discovered and profiled into alkyl substituted or cyclic subtypes. Moreover, network pharmacology analysis indicated that MFZT's anti-gout mechanism was mostly associated with the nuclear factor kappa-B (NF-κB) signaling, nucleotide oligomerization domain (NOD)-like signaling and rheumatoid arthritis pathways, along with the synergistic effect of 84 potential active compounds. In addition, a quantitative analytical method was developed to simultaneously determine the 29 potential effective components. Among them, berberine, pellodendrine, 3-feruloylquinic acid, neoastilbin, isoacteoside and chlorogenic acid derivatives at higher concentrations were considered as the chemical markers for quality control. These findings provide a holistic chemical basis for MFZT granules and will support the development of effective analytical methods for the herbal formulas of natural medicines.
Humans ; Chromatography, High Pressure Liquid/methods* ; Tandem Mass Spectrometry/methods* ; Drugs, Chinese Herbal/chemistry* ; Quality Control ; Arthritis, Gouty

Deconvolution of potential drug targets of the central nervous system (CNS) is particularly challenging because of the complicated structure and function of the brain. Here, a spatiotemporally resolved metabolomics and isotope tracing strategy was proposed and demonstrated to be powerful for deconvoluting and localizing potential targets of CNS drugs by using ambient mass spectrometry imaging. This strategy can map various substances including exogenous drugs, isotopically labeled metabolites, and various types of endogenous metabolites in the brain tissue sections to illustrate their microregional distribution pattern in the brain and locate drug action-related metabolic nodes and pathways. The strategy revealed that the sedative-hypnotic drug candidate YZG-331 was prominently distributed in the pineal gland and entered the thalamus and hypothalamus in relatively small amounts, and can increase glutamate decarboxylase activity to elevate γ-aminobutyric acid (GABA) levels in the hypothalamus, agonize organic cation transporter 3 to release extracellular histamine into peripheral circulation. These findings emphasize the promising capability of spatiotemporally resolved metabolomics and isotope tracing to help elucidate the multiple targets and the mechanisms of action of CNS drugs.

Postzygotic mutations are acquired in normal tissues throughout an individual's lifetime and hold clues for identifying mutagenic factors.Here,we investigated postzygotic mutation spectra of healthy individuals using optimized ultra-deep exome sequencing of the time-series samples from the same volunteer as well as the samples from different individuals.In blood,sperm,and muscle cells,we resolved three common types of mutational signatures.Signatures A and B represent clock-like mutational processes,and the polymorphisms of epigenetic regulation genes influence the pro-portion of signature B in mutation profiles.Notably,signature C,characterized by C＞T transitions at GpCpN sites,tends to be a feature of diverse normal tissues.Mutations of this type are likely to occur early during embryonic development,supported by their relatively high allelic frequencies,presence in multiple tissues,and decrease in occurrence with age.Almost none of the public datasets for tumors feature this signature,except for 19.6％of samples of clear cell renal cell carcinoma with increased activation of the hypoxia-inducible factor 1(HIF-1)signaling pathway.Moreover,the accumulation of signature C in the mutation profile was accelerated in a human embryonic stem cell line with drug-induced activation of HIF-1α.Thus,embryonic hypoxia may explain this novel signature across multiple normal tissues.Our study suggests that hypoxic condition in an early stage of embryonic development is a crucial factor inducing C＞T transitions at GpCpN sites;and indi-viduals'genetic background may also influence their postzygotic mutation profiles.

Multicellular tumor spheroids (MCTS) can simulate the structure and metabolic characteristics of tumors in vivo, which is of great significance to study the metabolic phenotype of tumor cells and the mechanism of drug intervention. In this study, esophageal cancer MCTS were constructed, and MCTS frozen sections were prepared after treated with different formulations of paclitaxel (PTX) including common PTX injection, PTX liposome and albumin bound PTX. MCTS mass spectrometry imaging analysis method was established by using air flow assisted desorption electrospray ionization mass spectrometry imaging (AFADESI-MSI). The visualization of the permeation and enrichment process of PTX in MCTs after PTX treatment was realized, and the spatially resolved metabolomics of PTX injection group was studied. The results showed that the permeation and enrichment behavior of PTX in MCTs model were related to the formulations. The changes of endogenous metabolites in MCTs of esophageal cancer after treated with PTX injection had temporal and spatial characteristics. The metabolic changes of MCTS during the initial 0-4 hours were dominated by the down-regulation of middle-high polarity metabolites and some lipids in the central region of MCTS, while the metabolic changes of MCTS during 8-72 hours were mainly up-regulated by lipid metabolites in the peripheral region of MCTS. The combination of in vivo tumor-associated MCTs model with label free, highly sensitive and high coverage mass spectrometry imaging technology provided a new method and strategy for the study of pharmacometabolomics.

Objective:To investigate the relationship between simple Chiari malformation type I (CMI) and atlantoaxial instability from the imaging point of view.Methods:A retrospective analysis were performed on 46 patients diagnosed with simple CMI from January 2014 to December 2020. Forty-six normal people matched for age and sex were selected as the normal control group, while 30 patients with atlantoaxial dislocation were selected as the dislocation group. The degree of atlantoaxial joint degeneration in each group was assessed according to Weishaupt degeneration grading; the atlantoaxial joint angulation angle was measured in the control group of patients with simple CMI; and the sagittal imaging parameters of cervical spine X-ray were measured, including C 0-C 1 Cobb angle, C 0-C 2 Cobb angle, C 1-C 2 Cobb angle, C 1-C 7 Cobb angle, C 2-C 7 Cobb angle, C 7 Slope, C 2 Tilt, spino cranial angle (SCA), and C 2-C 7 sagittal vertebral axis (SVA). All radiographic parameters were measured twice independently by two spine surgeons, and intraclass correlation coefficient (ICC) were determined to demonstrate intra- and inter-observer reliability. Results:ICC ranged between 0.842 and 0.974 in the current study, demonstrating "excellent" reliability of radiographic measurements. No significant difference was noted regarding age and the distribution of genders among the three groups. There were significant differences in the distribution of Weishaupt degeneration grading of atlantoaxial joints between simple CMI, normal and dislocation group ( H=53.68, P<0.001 on the left side; H=43.39, P<0.001 on the right side). There were significant differences in the degree of atlantoaxial joint degeneration between the normal group and dislocation group (left, Z=6.60, P<0.001; right, Z=6.29, P<0.001); There were significant differences in the degree of atlantoaxial joint degeneration between the normal group and simple CMI patients (left, Z=5.31, P<0.001; right, Z=4.13, P<0.001); There were significant differences in the degree of atlantoaxial joint degeneration between simple CMI and dislocation group (left, Z=3.20, P=0.001; right, Z=3.15, P=0.002). There were significant difference in the angulation angle of the atlantoaxial articular surface between the normal group and simple CMI patients (left, Z=3.32, P<0.001; right, Z=5.74, P<0.001). There were significant differences in C 0-C 1 Cobb angle ( t=2.41, P=0.018), C 1-C 7 Cobb angle ( t=2.88, P=0.005), C 2-C 7 Cobb angle ( t=3.29, P=0.001), and C 2-C 7 SVA ( t=2.87, P=0.005) between the normal group and simple CMI patients, but there was no significant difference in other parameters. Conclusion:The degree of atlantoaxial joint degeneration in patients with simple CMI is higher than that in normal people, the angulation angle is larger, and the cervical lordosis is larger, suggesting that there may be atlantoaxial joint instability. This study provides further evidence that Chiari malformation type I is associated with atlantoaxial instability.