作为新型分子成像技术，质谱成像无需特异性标记和复杂样品前处理，可对生物组织切片中内源性或外源性等多种分子进行同时成像检测，获得分子定性定量和定位信息，具有高分辨率，高灵敏度，高特异性，质量范围宽，实验周期短等特点，近年来该技术应用于生命科学领域在药物及其代谢产物组织的分布、疾病诊断标志物的发现、疾病发展进程机制探索等应用引起了高度关注。目前，代谢组学通过定性定量分析研究生物体受外在刺激或者扰动后整体代谢物的变化及变化规律已成为代谢生物标志物筛选及内源性分子调控代谢机制探索的强大工具。基于质谱成像技术衍生的空间分辨代谢组学通过表征代谢物分子的原位空间信息尤其适合可视化器官或组织在不同组织微区的代谢轮廓改变，在药物损伤的发生、发展、消失进程中生物标志物的筛选具有独特优势，可更深层次和准确了解代谢物在疾病发生发展过程中的作用与机制。

基于药物所质谱课题组前期自主研发的空气动力辅助解吸电喷雾电离质谱成像技术（Air-flow-assisted desorption electrospray ionization mass spectrometry imaging，AFADESI-MSI）技术，本课题通过合作引入该技术到药物毒理学领域，开展了以下研究内容突出该技术应用至毒理学研究中的独特优势及广阔前景，其主要内容包括以下2个部分：

1. 基于质谱成像技术探究5-羟甲基糠醛的肾毒性作用机制

5-羟甲基糠醛（5-Hydroxymethylfurfural，5-HMF）是美拉德反应（Maillard reaction，MR）的副产物。葡萄糖注射液高温灭菌过程中可产生5-HMF，该化合物被报道具有肾毒性，课题组前期考察了5-HMF静脉给药ICR小鼠的急性毒性，半数致死剂量（median lethal dose，LD 50）为871.12 mg/kg，14天后病理学检查结果显示存活小鼠肾脏呈现慢性进行性肾病改变，其肾脏相关的毒性机制及潜在早期毒性预测指标有待进一步研究。

基于AFADESI-MSI分析5-HMF在不同时间点对小鼠肾脏微区的内源性代谢物点的改变情况，筛选5-HMF肾毒性早期相关的生物标志，探究可能具有的肾毒性机制。ICR小鼠以300 mg/kg剂量静脉给药5-HMF，对照组给予生理盐水,于1 h、4 h、24 h时间点分别取肾组织，通过AFADESI-MSI质谱成像技术可视化肾脏组织微区的空间轮廓，检测到代谢物在组织微区中的特异性分布，进一步分别从肾髓质和肾皮质中提取质谱数据进行峰对齐和归一化，采用多变量统计分析筛选出各时间点下肾髓质和肾盂中差异代谢物，进行初步鉴定后发现检测到的代谢物包括脂肪酸类、磷脂类、氨基酸类、有机酸类、核苷类、嘌呤类，其中代表性代谢物有苯丙氨酸，腺苷，腺嘌呤，次黄嘌呤，鸟苷单磷酸，FA-22:6等，进一步将差异代谢物富集分析发现5-HMF给药后发生显著变化的代谢通路主要涉及：嘌呤代谢，丙氨酸天冬氨酸和谷氨酸代谢，精氨酸合成，三羧酸循环和嘧啶代谢5个通路。将综合各类代谢物在不同时间点的变化趋势，筛选出次黄嘌呤，FA-22:6，LPG-22:6可能作为5-HMF早期肾毒性的预测指标，推荐5-HMF给药后4 h是检测药物毒性标志物的最佳时间。基于质谱成像技术可视化5-HMF引起的肾脏结构微区的代谢变化，揭示了5-HMF可能的肾毒性机制，为5-HMF肾毒性靶点研究提供前期理论基础，也为药物肾脏毒理学机制研究提供了新的技术支持。

2. 整合网络毒理学和质谱成像技术研究何首乌D组分的肝毒性机制

何首乌作为传统中药，生何首乌具有解毒、除疟、润肠、助便等功效，制何首乌可调理肝肾、补益精血、乌黑须发、强健筋骨，但是由何首乌引发肝毒性的不良反应频频报道，而目前毒性机制尚不完全明确，本实验室前期采用斑马鱼胚胎模型对何首乌提取成分进行毒性评价，发现70% 乙醇提取物中95% EtOH洗脱的D组分（何首乌D组分，PM-D）肝毒性最强，进一步鉴定PM-D出的25种主要成分并基于斑马鱼胚胎模型评价各成分的肝毒性，结果显示8种化合物具有明细的肝毒性。本研究拟采用网络毒理学和空间分辨代谢组学阐明何首乌D组分的肝毒性机制。

本研究首先对PM-D进行了急性毒性实验（剂量分别为500, 1000, 2000, 5000 mg/kg），结果显示所有剂量的小鼠均未出现死亡和异常毒性症状，大体解剖未见明显病理改变。然而，前期PM-D在斑马鱼模型急性毒性实验中显示出非常明显的肝毒性。考虑物种差异性，进一步重复给药PM-D 2 g/kg连续7天后，通过病理和生化检查评估其对小鼠肝脏的损伤作用。生化指标显示给药组肝功能相关指标出现明显上调，病理结果表现给药组肝细胞肥大，肝窦扩张，微肉芽肿，轻度变性坏死。

针对8种关键毒性化合物，包括大黄素（Emodin），大黄酸（Chrysophanol），大黄素-6-β-D-葡萄糖苷（Emodin-6-O-β-D-glucopyranoside），大黄素-8-β-D-葡萄糖苷（Emodin-8-O-β-D-glucopyranoside），1,3,8-Trihydroxy-6-methyl-10H-anthracen-9-one， 顺式-大黄素-大黄素二蒽酮（Cis-emodin-emodin dianthrones），反式-大黄素-大黄素二蒽酮（Trans-emodin-emodin dianthrones），Polygonumnolide C4，进行网络毒理学分析发现PM-D的30个潜在肝毒性靶点。GO和KEGG富集分析显示，共同靶点涉及多种生物活性和信号通路，包括有机氮化合物代谢过程、细胞对内源性刺激的反应、激酶活性的正向调节、凋亡过程的调控、活性氧代谢过程的正调控、PI3K-Akt信号通路、AMPK信号通路、mTOR信号通路、HIF-1信号通路、Ras信号通路及MAPK信号通路等。分子对接结果证实，8种关键毒性成分与10个核心靶点(mTOR、PIK3CA、AKT1、EGFR等)具有较高的结合活性。质谱成像检测PM-D代谢物在小鼠肝脏中高度富集。空间分辨代谢组学结果表明PM-D给药后代谢谱发生了显著变化，代谢产物如牛磺酸、牛磺胆酸、腺苷、酰基肉碱等与PM-D诱导的肝损伤有关。代谢通路富集分析显示，亚麻酸和亚油酸代谢、肉碱合成、支链脂肪酸氧化等6种代谢途径发生显著变化。综合分析揭示PM-D引起的肝毒性与胆汁淤积、线粒体损伤、氧化应激导致能量代谢、脂质代谢紊乱等密切相关。

本研究通过网络毒理学和空间分辨代谢组学相结合的方法，全面阐述了PM-D多靶点肝毒性作用机制，为进一步研究PM的毒性机制和临床安全使用提供了基础理论。

综上所述，质谱成像技术将整体动物或各器官为研究对象，可快速确定药物毒性作用靶器官，进一步开展相关毒性标志物及毒性代谢机制的研究可对药物尤其中草药复杂组分毒性进行科学的、综合的阐释，为药物毒性预测，安全性评价以及现代中药的药理毒理评价体系提供新的技术支持，由于代谢物生物学过程的复杂性，仍需分子生物学实验进一步确证。

As a powerful molecular imaging technology, mass spectrometry imaging (MSI) is that can obtain qualitative, quantitative, and location information by simultaneously detecting and mapping endogenous or exogenous molecules in biological tissue slices without specific chemical labeling or complex sample pretreatment. With the characteristics of high resolution, high sensitivity, high specificity, wide quality range, and short experimental period, MSI has attracted great attention in the field of life science such as distribution of drugs and their metabolites, discovery of disease diagnostic markers, and exploration of mechanism of disease development. At present, metabonomics is an emerging science to study the changes and rules of whole metabolites after external stimulation or disturbance by qualitative and quantitative analysis and has become a powerful tool for exploring metabolic process, biomarker identification, and metabolism regulation mechanism of endogenous molecular. Spatial-resolved metabolomics based on MSI can characterize the in-situ spatial information of metabolite molecules, which is especially suitable for displaying metabolic profile changes of different macro-areas in organs or tissues, screening biomarkers in the process of drug damage, and understanding the metabolites mechanism in the process of disease development at a deeper and accurate level.

 In this study, the air flow-assisted desorption electrospray ionization mass spectrometry imaging (AFADESI-MSI) technology independently developed by our research group was used to highlight the unique advantages and broad prospects of the MSI application in toxicological studies through the following research contents. The main content of this study mainly includes the following two parts:

1. Exploring the mechanism of 5-hydroxymethylfurfural induced nephrotoxicity based on mass spectrometry imaging

5-HMF is a by-product of Maillard reaction (MR), which can be produced in the process of high-temperature sterilization of glucose injection, and has been reported to have renal toxicity. The acute toxicity of ICR mice administrated intravenously with 5-HMF was investigated in the early stage of our research group. The median lethal dose (LD50) was 871.12 mg/kg, and pathological examination 14 days later revealed chronic progressive nephropathy in the kidney of the surviving mice. The nephrotoxicity-related mechanism and early predictive biomarkers need to be further studied.

ICR mice were intravenously administered with 300mg/kg 5-HMF, and the control group was given normal saline. The renal tissues were collected at 1h, 4h, and 24h, respectively. AFADESI-MSI was used to visualize the spatial profile of renal tissue micro-areas, detect the specific distribution of metabolite, and further extract mass spectrometry data from the renal medulla and renal cortex respectively for peak alignment and normalization. Multivariate statistical analysis was used to screen out the differential metabolites in the renal medulla and renal pelvis at each time point. After preliminary identification, it was found that the types of detected metabolites included fatty acids, phospholipids, amino acids, organic acids, nucleosides, and purines. Representative metabolites include phenylalanine, adenosine, adenine, hypoxanthine, guanosine monophosphate, FA-22:6, etc. Further pathway enrichment analysis showed that the metabolic pathways with significant changes mainly involved purine metabolism, alanine aspartic acid, and glutamate metabolism, arginine synthesis, tricarboxylic acid cycle, and pyrimidine metabolism. In addition, based on the variation trend of metabolites at different time points, the study screened out hypoxanthine, FA-22:6, and LPG-22:6 as possible biomarkers for predicting early nephrotoxicity, and recommended that 4 hours after 5-HMF administration was the best time to detect toxicity biomarkers.

In this study part, the metabolic changes of renal structure caused by 5-HMF were visualized based on MSI, revealing the possible nephrotoxicity mechanism of 5-HMF, providing a preliminary theoretical basis and new technical support for the drug renal toxicological mechanism study.

2. Integrated network toxicology and spatially resolved metabolomics to investigate the hepatotoxicity mechanisms of component D of Polygonum multiflorum Thunb

Reynoutria multiflora (Thunb.) Moldenke. (Polygonaceae) (Polygonum multiflorum Thunb, PM) is a traditional Chinese medicine (TCM). Raw PM has the effects of detoxifying, eliminating carbuncle, eliminating malaria, moistening the intestine, and assisting defecation. Processed PM nourishes the liver, kidney, and blood, strengthens muscles and bones, and blackens hair. However, adverse liver toxicity reactions of PM have been reported frequently, and the toxic mechanism is not completely clear at present. Our previous studies on the toxicity of different extracts and components of PM were therefore assessed in zebrafish embryos, with the results showing that the acute toxicity and hepatotoxicity of component D [95% ethanol (EtOH) extracted elution] in a 70% EtOH extract of PM (PM-D) were significantly higher than that of other extracts. Furthermore, the 25 components in PM-D were identified and hepatotoxicity of each component was evaluated based on zebrafish embryo model. The results showed that 8 compounds had significant hepatotoxicity. Whereas the hepatotoxicity mechanism of PM-D is unknown. This work aimed to investigate the hepatotoxicity mechanisms of PM-D by integrating network toxicology and spatially resolved metabolomics strategy.

In this study, acute toxicity experiment of PM-D was performed first and the dosage was set at 500, 1000, 2000, 5000 mg/kg. No death and no abnormal toxicity symptoms in mice were observed at all doses within 14 days. Furthermore, no obvious pathological change was found in gross anatomy. However, Acute toxicity exploration of PM-D in zebrafish model showed very obvious hepatotoxicity. Considering the difference of species, the liver injury effect of mice induced by PM-D was evaluated by pathological and biochemical examination after repeated administration of 2 g/kg dose for 7 days. Pathological results showed that hepatocyte hypertrophy, hepatic sinus dilatation, microgranuloma, and mild degeneration necrosis were observed in the model group.

Based on eight key toxic compounds, including emodin, chrysophanol, emodin-6-O-β-D-glucopyranoside，emodin-8-O-β-D-glucopyranoside, 1,3,8-trihydroxy-6-methyl-10H-anthracen-9-one, cis-emodin-emodin dianthrones, trans-emodin-emodin dianthrones and polygonumnolide C4, network toxicology identified 30 potential targets of liver toxicity of PM-D. The GO and KEGG enrichment analyses showed that the common targets involved multiple biological activities and signaling pathways, including organonitrogen compound metabolic process, cellular response to endogenous stimulus, positive regulation of kinase, regulation of apoptotic process, positive regulation of reactive oxygen species metabolic process, PI3K-Akt signaling pathway, AMPK signaling pathway, mTOR signaling pathway, HIF-1 signaling pathway, Ras signaling pathway and MAPK signaling pathway, etc. The Molecular docking results confirmed that 8 key toxic components had high binding activity with 10 core targets (mTOR, PIK3CA, AKT1, EGFR, etc.). The high distribution of metabolites of PM-D in the liver of mice in the administration group was detected by mass spectrometry imaging.

Furthermore, spatially resolved metabolomics results revealed significant changes in metabolic profile after PM-D administration and metabolites such as taurine, taurocholic acid, adenosine, acyl-carnitines, etc. were associated with PM-D-induced liver injury. Enrichment analysis of metabolic pathways revealed linolenic acid and linoleic acid metabolism, carnitine synthesis, and oxidation of branched-chain fatty acids, and other 6 metabolic pathways were significantly changed. Comprehensive analysis showed that the hepatotoxicity caused by PM-D was closely related to cholestasis, mitochondrial damage, oxidative stress and energy metabolism, and lipid metabolism disorders.

This work comprehensively demonstrates the multi-target hepatotoxicity mechanisms of PM-D via combined network toxicology and spatially resolved metabolomics strategy, which provided a basic theory for further study on the toxicity mechanism of PM and clinical safety use.

What has been discussed above, mass spectrometry imaging takes the whole animal or organ as the research object to quickly determine the drug toxic target organs, and further explore toxicity biomarkers and toxicity metabolic mechanism, scientifically and comprehensively explain the toxicity of drugs, especially for complex components of Chinese herbal medicine, providing new technical support for the toxicity prediction, safety evaluation, pharmacology and toxicological evaluation system of modern Chinese herbal medicine. Due to the abundance of metabolite biological processes, molecular biological experiments are still required for further confirmation.