Method for researching specific distribution of multi-component complex system in animal tissue based on mass spectrum imaging and multivariate statistical analysis

Abstract

The invention discloses a method for researching the specific distribution of a multi-component complex system in animal tissues based on mass spectrum imaging and multivariate statistical analysis, which comprises the following steps: treating the multi-component complex system, performing animal administration for a certain time, killing the animal, taking the animal tissues, slicing the animal tissues, and taking out the sliced animal tissues; and performing mass spectrum imaging analysis and multivariate statistical analysis to obtain a specific distribution result of the multi-component complex system in the animal tissue. On the basis of a frozen section technology and a mass spectrum imaging technology, a DESI-MSI analysis method of the whole mouse tissue section is established, and in-situ distribution characteristics of the active ingredients of the Anefortia divaricata on the brain tissue section are disclosed by combining multivariate statistical analysis.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug screening, and in particular, relates to a method for studying the specific distribution of a multi-component complex system in animal tissues based on mass spectrometry imaging and multivariate statistical analysis. Background Art

[0002] Buwangsan showed significant therapeutic effects in the Alzheimer's disease (AD) mouse model, and multiple active ingredients were able to enter the systemic circulation and be detected in the blood. AD is a complex neurodegenerative disease, and its pathogenesis involves multiple aspects, including abnormal deposition of β-amyloid protein in the brain, neuronal damage, inflammatory response, etc. It is well known that the brain is a heterogeneous organ with different functions in different regions. In addition, many different drug targets and receptors are distributed in different brain regions. In situ analysis of drugs in the brain can directly observe the effects of drugs in the lesion area, revealing the interaction between drugs and disease-related molecules, cells or biological processes, helping to understand and explore the possible multiple pharmacodynamic sites of drugs, and providing spatial clues for the elucidation of their pharmacodynamic mechanisms.

[0003] The blood-cerebrospinal fluid barrier (BCSFB) formed by choroid plexus epithelial cells and the blood-brain barrier (BBB) ​​formed by brain capillary endothelial cells are the first barrier to drug uptake by brain tissue; in addition, the transporters and enzymes expressed on neurons and glial cells, which account for 80% of the brain volume, are considered to be secondary barriers to drug uptake [Bagchi, S., et al., In-vitro blood-brain barrier models for drug screening and permeation studies: an overview. Drug Des Devel Ther, 2019. 13: p. 3591-3605.]. Due to the physiological characteristics of the barrier, only small molecules and fat-soluble drugs (such as chloramphenicol, erythromycin and other antibiotics) can pass through the barrier into the brain, and more than 98% of drugs cannot enter the brain to reach therapeutic concentrations [Harilal, S., et al., Revisiting the blood-brain barrier: A hard nut to crack in the transportation of drug molecules. Brain Res Bull, 2020.160: p.121-140.]. Currently, the development of drugs for the treatment of central nervous system diseases faces many obstacles, the most important of which is how drugs can pass through the blood-brain barrier to reach different areas of the brain and the precise target location to exert their effects.

[0004] It is still unclear which of the blood-migrating active ingredients in Buwangsan can pass through the blood-brain barrier and what are the distribution characteristics of these active ingredients in different areas of the brain.

[0005] The in vivo tissue distribution characteristics of drugs are mostly studied using chromatography-mass spectrometry techniques such as GC-MS, LC-MS, and whole-body autoradiography. Mass spectrometry pre-processing requires complex pre-processing work such as separation of different tissues, tissue homogenization, and protein precipitation. The operation is complicated and the in situ distribution characteristics of the drugs are lost. Whole-body autoradiography is mostly used to study the tissue distribution characteristics of drug monomers or a specific class of drugs, and it is difficult to simultaneously study the in vivo tissue characteristics of complex Chinese medicine systems.

[0006] Mass spectrometry imaging (MSI) is a new imaging method based on mass spectrometry technology, which is used to detect and analyze the spatial distribution and signal intensity of biological molecules in tissue sections [Hazra, A., et al., Corticothalamic network dysfunction and behavioral deficits in a mouse model of Alzheimer's disease. Neurobiol Aging, 2016. 44: p. 96-107.]. The main advantage of MSI of biological tissue samples without the need for specific labeling is that it can analyze different tissue section areas for multi-point detection without labeling or staining, and can simultaneously detect and identify multiple substances and perform imaging. Summary of the invention

[0007] The purpose of the present invention is to provide a method for studying the specific distribution of a multi-component complex system in animal tissues based on mass spectrometry imaging and multivariate statistical analysis.

[0008] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0009] The present invention provides a method for studying the specific distribution of a multi-component complex system in animal tissues based on mass spectrometry imaging and multivariate statistical analysis, comprising the following steps:

[0010] After the multi-component complex system is treated, the animal is medicated for a certain period of time, the animal tissue is obtained after the animal is killed, and the animal tissue is sliced ​​and subjected to mass spectrometry imaging analysis and multivariate statistical analysis to obtain the specific distribution results of the multi-component complex system in the animal tissue.

[0011] The multi-component complex system is a mixed system containing two or more components at the same time, preferably a traditional Chinese medicine system.

[0012] The animal tissue is selected from brain tissue of animals such as mice and rats.

[0013] The animal tissue sectioning comprises the following steps:

[0014] The animal tissue was placed on a base with embedding gel, and the slice thickness parameters (15 μm) were set. During the slice process, the box temperature (set to -20°C) and the sample head temperature (set to -15°C) were set.

[0015] The mass spectrometry imaging analysis adopts analytical electrospray ionization mass spectrometry and quadrupole time-of-flight mass spectrometry, and the content of the analysis is selected from optimization of spray solvent, optimization of spray solvent flow rate, optimization of capillary voltage, etc.

[0016] The optimization reference for the mass spectrometry imaging is the lower limit of quantification of the representative components.

[0017] The spray solvent is selected from a methanol and water system, an acetonitrile and water system, and formic acid or ammonia water, ammonium formate, etc. are added to the above system. The spray solvent is selected from 95% MeOH + 0.1% FA (formic acid), 80% ACN + 0.2% FA, 90% MeOH + 0.1% ammonia water, 90% MeOH, 98% MeOH, 80% MeOH.

[0018] The spray solvent flow rate is selected from 0.5 μL·min -1 ~4μL·min -1 (Preferably 1 μL·min -1 , 2μL·min -1 、3μL·min -1 ).

[0019] The capillary voltage is selected from 0.4 kV to 0.8 kV (preferably 0.45 kV, 0.55 kV, 0.65 kV, and 0.75 kV).

[0020] The analytical electrospray ionization mass spectrometry / quadrupole time-of-flight mass spectrometry conditions were set as follows: nebulizing gas was nitrogen, pressure was 13 psi (about 0.09 MPa), ion source temperature was 120°C, nebulizer incident angle was 60°, collection angle was 10°, positive and negative ion mode scan rates were 200 μL·min -1 The mass spectrometry analysis mode was sensitive mode, the scanning range was m / z 50-1200, the pixel size was 100×100 μm, and the scanning speed was 200 μm / s.

[0021] The mass spectrometry imaging analysis includes: using MassLynx and Progenesis Bridge plug-ins to draw a standard curve of a standard substance, and performing a least squares linear regression simulation analysis on the standard curve, calculating a correlation coefficient r value and a linear equation, and obtaining a relative quantitative lower limit of each compound;

[0022] The raw data of animal tissue sections are collected by MassLynx, and the HDI imaging data processing software is used to perform a series of processing on the data files, including peak detection, calibration, deconvolution, data reduction, isotope elimination analysis, extraction of target compound information of traditional Chinese medicine compound and information of 20 to 1000 (preferably 200) compounds with the highest response in the data file, and the total ion current (TIC) normalization method is used to process the imaging of tissue sections. After compensating for the matrix effect, the mass spectrometry imaging data is analyzed and a specific ion distribution image is generated.

[0023] The multivariate statistical analysis was performed on the ROIs of the amygdala, cerebellum, cortex, striatum, hippocampus and thalamus in the animal tissues, and the unit area (mm) of each region of interest (ROI) was calculated. 2 ) mass spectrometry response values, and orthogonal partial least squares discriminant analysis (OPLS-DA) was used to obtain VIP (variable importance in the projection) values ​​and OPLS-DA / S-plot diagrams. The brain migration components that could represent the differences between the groups were found using R 2 X, R 2 Y and Q 2 To evaluate the model.

[0024] The traditional Chinese medicine system is made of calamus, poria, poria, ginseng and polygala in a mass ratio of 2:5:5:5:7.

[0025] The preparation method of the traditional Chinese medicine system comprises the following steps: weighing calamus, poria, poria, ginseng and polygala in a mass ratio of 2:5:5:5:7 and crushing them, adding 5 to 15 times (preferably 10 times) of 30 to 90% (preferably 60%) ethanol, soaking for 1 to 10 hours (preferably 3 hours), refluxing extraction for 1 to 4 hours (preferably 2 hours), filtering, and repeatedly extracting for at least 2 times to obtain a first part of the extract; then adding 5 to 15 times (preferably 10 times) of pure water, continuing to reflux extraction for 1 to 4 hours (preferably 2 hours), filtering to obtain a second part of the extract; mixing the first part of the extract and the second part of the extract, and freeze-drying them into powder to obtain the traditional Chinese medicine system.

[0026] Due to the adoption of the above technical solution, the present invention has the following advantages and beneficial effects:

[0027] In order to clarify the distribution characteristics of various components of Buwangsan in the mouse brain, the present invention established a DESI-MSI analysis method for whole mouse tissue sections based on frozen section technology and mass spectrometry imaging technology, and combined with multivariate statistical analysis to reveal the in situ distribution characteristics of the active ingredients of Buwangsan on brain tissue sections.

[0028] The present invention innovatively combines mass spectrometry imaging with multivariate statistical methods to analyze the regional distribution of various components of Buwang Powder in brain tissue after mice were gavaged with Buwang Powder. The present invention systematically investigated and optimized the preparation method of mouse brain tissue slices, slice thickness, and the effects of key parameters such as spray solvent, spray solvent flow rate, and mass spectrometry capillary voltage on DESI-MSI analysis, and established a mass spectrometry imaging method and multivariate statistical analysis method suitable for the analysis of active components of Buwang Powder in brain tissue slices. This method preliminarily identified 6 active components of Buwang Powder that are specifically distributed in the brain under negative ion mode, and intuitively demonstrated the specific distribution characteristics of each active component in different brain regions of mice, laying the foundation for subsequent research on the material basis of efficacy and quality control, and this strategy and method will also provide a reference for other drug tissue distribution studies for the treatment of neurodegenerative diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a schematic diagram of the development and optimization process of the DESI-MSI imaging method (taking Pachycolic acid as an example).

[0030] Figure 2 This is a schematic diagram of the selection results of ROI areas in various brain regions of mice in DESI-MSI imaging of mouse sections.

[0031] Figure 3 Schematic diagram of the brain regions in sagittal sections of mice.

[0032] Figure 4 This is a schematic diagram of the OPLS-DA scores of each ROI area of ​​mice under negative ion mode.

[0033] Figure 5 This is a 200 permutation test graph of OPLS-DA analysis of the mouse ROI region in negative ion mode.

[0034] Figure 6 This is the S-plot of each ROI area of ​​the mouse under negative ion mode.

[0035] Figure 7 This is a schematic diagram of the results of DESI-MSI negative ion mode imaging of the brain-specific distribution of the active ingredients of Buwangsan. DETAILED DESCRIPTION

[0036] In order to explain the present invention more clearly, the present invention is further described below in conjunction with preferred embodiments. It should be understood by those skilled in the art that the following specific description is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0037] Example 1

[0038] 1. Materials and Methods

[0039] (I) Reagents and materials

[0040] Chinese herbal medicine slices of calamus, poria, poria, ginseng, and polygala were purchased from Shanghai Bailutang Chinese Medicine Store (ginseng slices / Jiangsu; white poria / Huanggang Jingui; poria / Suzhou Boyuan; calamus / Shanghai Qingpu; polygala / Shanxi).

[0041] Standard substances such as pachymic acid, kaempferol, polygalaxanthin III, polygala tenuifolia xanthenone B, 3,6'-dienasinoylsucrose, pachymic acid A, polygala tenuifolia saponin B, polygala tenuifolia A6, and polygala tenuifolia sapogenin were purchased from Shanghai Standard Standard Technology Service Co., Ltd. The concentration of each reference substance was ≥98%.

[0042] Leucine-enkephalin was purchased from Sigma, USA; mass spectrometry grade acetonitrile and formic acid were purchased from Fisher Chemical; Milli-Q water purification system (Millipore, USA); analytical grade anhydrous ethanol: Shanghai Titan Technology Co., Ltd.; pure water: Watsons Shanghai Watsons Daily Necessities Co., Ltd.; Sanfu red ink pen: Newway Daily Necessities (Shanghai) Co., Ltd.; Sanfu black ink pen: Newway Daily Necessities (Shanghai) Co., Ltd.; other reagents were of analytical grade.

[0043] (II) Experimental instruments

[0044] Electrospray ionization quadrupole time-of-flight mass spectrometer DESI XS / Xevo G3 QTOF: Waters, USA. Microtome: Leica CM1950, Germany. Standard sample slide: SLIDE.26MMX76MM.44WELL.MICRO-44. HDI: V1.7 Waters, USA (equipped with Ezinfo multivariate statistical analysis plug-in). MassLynx acquisition software: V4.2 Waters, USA (equipped with Progenesis Bridge plug-in). Micropipette gun: Eppendorf, Germany. 1 / 10,000 electronic balance. Centrifuge: ThermoFisher Scientific, USA. Nitrogen blower, freeze dryer, vortex shaker.

[0045] (III) Experimental animals

[0046] 3 APP / PS1 male mice (provided by Jiangsu Huachuang Xinnuo Pharmaceutical Technology Co., Ltd., SPF grade, weight (26±2) g, 4 months old, license number: SYXK(Su)2020-0009), and 12 C57BL / 6 male mice of the same age. Before the experiment was carried out, the animal experiment protocol of the present invention had been reviewed by the Experimental Animal Ethics Committee of the Naval Medical University, and the experimental animal operations were carried out in compliance with relevant regulations.

[0047] 2. Experimental Methods

[0048] 1. Do not forget to prepare the in vitro sample solution

[0049] The decoction pieces were weighed and crushed according to the mass ratio of 2:5:5:5:7 of calamus, poria, poria, ginseng, and polygala, and 10 times the amount of 60% ethanol was added, soaked for 3 hours, refluxed for 2 hours, filtered, and extracted twice to obtain the first part of the extract; then 10 times the amount of pure water was added, refluxed for 2 hours, and filtered to obtain the second part of the extract; the first part of the extract and the second part of the extract were mixed, freeze-dried into powder in a freeze dryer, and stored in a desiccator. Before use, pure water was used to prepare a uniform suspension of the corresponding concentration.

[0050] 2. Animal medication

[0051] After the mice were adapted to feeding for 7 days, C57BL / 6 male mice were randomly divided into a blank group and a Buwangsan gavage group, with 6 mice in each group. Drugs were administered by gavage.

[0052] Blank group: Each mouse was gavaged with 100 μL of pure water and then killed on the seventh day to obtain tissues.

[0053] Buwangsan gavage group: 100 μL of drugs of corresponding concentration were administered; the dosage of Buwangsan was 0.224 g / 100 g. After 6 consecutive days of administration, the animals were killed on the seventh day and tissues were collected. The drug was administered once 30 min before sacrifice.

[0054] 3. Collection of drug-containing serum and brain tissue

[0055] The blood of mice was quickly drawn by removing their eyeballs, and the whole brain tissue was quickly separated after cardiac perfusion with pre-cooled saline. After being blotted dry with filter paper, the tissue was placed flat in the lid of a 50 mL centrifuge tube. The centrifuge tube was tightened and quickly frozen in liquid nitrogen, and then transferred to a -80°C ultra-low temperature refrigerator for storage.

[0056] 4. Preparation of Mouse Brain Tissue Slices

[0057] The day before, take the tissue sample out of the -80℃ ultra-low temperature freezer and place it in a -20℃ refrigerator to thaw overnight. At the same time, the freezing slicer should also be turned on one day in advance. Place the mouse brain tissue on a base with a small amount of embedding glue, set the slice thickness parameter to 15μm, set the box temperature to -20℃ during the slice process, and set the sample head temperature to -15℃. Start slicing, and stick and fix it on the slide after slicing.

[0058] 5. Preparation of reference samples

[0059] Accurately weigh the reference substances (including: pachymic acid, kaempferol, polygalaxanthin III, polygala tenuifolia xanthenone B, 3,6'-dienasinoylsucrose, pachymic acid A, polygala tenuifolia saponin B, polygala tenuifolia sugar A6, polygala sapogenin, kaempferol), dissolve them in appropriate amount of methanol to prepare 1 mg / ml reference substance stock solution, mix the stock solutions, and use a 1 μL pipette tip to spot on the center of the standard glass plate.

[0060] 6. DESI-MSI analysis

[0061] The conditions of DESI XS / quadrupole time-of-flight mass spectrometry Xevo G3 QTOF were set as follows: nebulizer gas was nitrogen, pressure was 13 psi (about 0.09 MPa), ion source temperature was 120 °C, nebulizer incident angle was 60°, collection angle was 10°, and positive and negative ion mode scan rates were 200 μL·min -1 The mass spectrometry analysis mode was sensitive mode, the scanning range was m / z 50-1200, the pixel size was 100×100 μm, and the scanning speed was 200 μm / s.

[0062] MassLynx and Progenesis Bridge plug-ins were used to draw the standard curve of the standard sample, and the standard curve was subjected to least squares linear regression simulation analysis, the correlation coefficient r value and the linear equation were calculated, and the relative quantitative lower limit of each compound was obtained.

[0063] The raw data of mouse brain slices were collected by MassLynx version 4.2, and HDI imaging (Waters, USA) data processing software was used to perform a series of processing on the data files, including peak detection, calibration, deconvolution, data reduction, isotope elimination and other analyses, to extract the information of target compounds in the traditional Chinese medicine compound and the information of the 200 compounds with the highest response in the data file, and the imaging of tissue slices was processed using the total ion current (TIC) normalization method. After compensating for the matrix effect, the mass spectrometry imaging data was analyzed and a specific ion distribution image was generated.

[0064] 7. Multivariate statistical analysis

[0065] To avoid interference caused by brain tissue heterogeneity, regions of interest (ROI) were circled according to brain partitions. The ROIs of the amygdala, cerebellum, cortex, striatum, hippocampus and thalamus of the control group (i.e., blank group) and the drug administration group (i.e., Buwangsan gavage group) were selected for analysis. The unit area (mm) of each ROI was calculated. 2 ) mass spectrometry response values, and orthogonal partial least squares discriminant analysis (OPLS-DA) was used to obtain VIP (variable importance in the projection) values ​​and OPLS-DA / S-plot diagrams. The brain migration components that can represent the differences between the groups were found using R 2 Y and Q 2 To evaluate the model.

[0066] 3. Experimental Results

[0067] 1. Development and optimization of DESI-MSI imaging method

[0068] The present invention improves the signal sensitivity and selectivity of multiple components of Fufang Buwang Powder by optimizing different conditions.

[0069] After optimization, the optimal physical parameters were set as follows: nebulizer gas was nitrogen, pressure was 13 psi (about 0.09 MPa), ion source temperature was 120 °C, nebulizer incident angle was 60°, collection angle was 10°, and positive and negative ion mode scanning rates were 200 μL·min -1 The mass spectrometry analysis mode was sensitive mode, the scanning range was m / z 50-1200, the pixel size was 100×100 μm, and the scanning speed was 200 μm / s.

[0070] The imaging method was optimized with pachymic acid, kaempferol, polygala xanthones III, polygala xanthones B, 3,6'-dienasinoyl sucrose, pachymic acid A, polygala saponin B, polygala sibirica A6, and polygala sapogenin as representative components in negative ion mode. The effects of different spray solvents (95% MeOH + 0.1% FA, 80% ACN + 0.2% FA, 90% MeOH + 0.1% ammonia water, 90% MeOH, 98% MeOH, 80% MeOH) and flow rates (1 μL·min-1, 2 μL·min-1, 3 μL·min-1) on the mass spectrometry imaging signals of each Chinese medicine component were compared. It was found that in negative ion mode, pachymic acid and kaempferol responded best under 80% MeOH; 3,6'-diesinapoylsucrose and polygala sapogenin responded best under 90% MeOH+0.1% ammonia water, 90% MeOH and 80% MeOH; pachymic acid A responded best under 98% MeOH and 80% MeOH; polygala tenuifolia saponin B and siberian polygala sugar A6 responded best under 90% MeOH+0.1% ammonia water, followed by 80% MeOH; polygala tenuifolia xanthones III and siberian polygala xanthones B had similar responses under the six conditions; after comprehensive consideration, 80% MeOH was finally determined as the final spray solvent.

[0071] With 80% MeOH as the final spray solvent, the effects of capillary voltage (0.45kV, 0.55kV, 0.65kV, 0.75kV) on the mass spectrometry imaging signals of each Chinese herbal medicine component were investigated. In the negative ion mode, the detection limits of pachymic acid, kaempferol, 3,6'-diesinapoylsucrose, pachymic acid A, and polygala sapogenin were similar at the four voltages, but the signal intensity was relatively higher at 0.55kV; the response of polygala tenuifolia saponin B and polygala tenuifolia xanthones III was the best at 0.75kV; the detection limits of polygala tenuifolia xanthones B at the four voltages were similar; therefore, after comprehensive consideration, 0.55kV was finally determined as the final voltage in the negative ion mode.

[0072] 80% MeOH was used as the final spray solvent, 0.55 kV was the capillary voltage, and the flow rate (1 μL min -1 , 2μL·min -1 , 3μL·min -1 ) on the mass spectrometry imaging signals of each Chinese medicine component. In negative ion mode, the compound was -1 The signal intensity was relatively higher at a flow rate of 2 μL·min. -1 As the spray flow rate in negative ion mode.

[0073] Considering the spray solvent, capillary voltage and flow rate, 80% MeOH was finally selected as the final spray solvent, 0.55 kV as the capillary voltage, and 2 μL min -1 Mass spectrometry signals from mouse brain slices were acquired as a function of flow rate.

[0074] Figure 1 The figure is a schematic diagram of the development and optimization process of the DESI-MSI imaging method (taking pachymic acid as an example). A is a schematic diagram of imaging of a series of concentrations of pachymic acid in different spray solvents; B is a schematic diagram of imaging of different concentrations of pachymic acid under different capillary voltages; C is a schematic diagram of imaging of different concentrations of pachymic acid under different flow rates; Figure 1 According to the results, 80% MeOH was finally selected as the final spray solvent, 0.55 kV as the capillary voltage, and 2 μL·min-1 as the flow rate to collect the mass spectrometry signals of mouse brain slices.

[0075] 2. DESI-MSI imaging and multivariate statistical analysis of mouse brain

[0076] After using MassLynx to collect the raw data of mouse brain slices, HDI imaging data processing software was used to perform a series of processing on the data files, extract the information of the target compounds of the Chinese medicine compound and the information of the 200 compounds with the highest response in the data files, and the total ion current (TIC) normalization method was used to perform preliminary processing on the imaging of the tissue slices. After that, the amygdala, cerebellum, cortex, striatum, hippocampus and thalamus of the control group (i.e., blank group) and the drug-administered group (i.e., Buwangsan gavage group) were selected as regions of interest (ROI). The ROI areas are as follows: Figure 2 As shown, Figure 2 The following is a schematic diagram of the selection results of ROI areas in each brain region of mice in DESI-MSI imaging of mouse slices. A is a schematic diagram of the ROI results of the control group under negative ion mode; B is a schematic diagram of the ROI results of the drug-treated group under negative ion mode. Calculate the unit area (mm) of each ROI 2 )'s mass spectrometry response values ​​were analyzed by orthogonal partial least squares discriminant analysis (OPLS-DA), and VIP (variable importance in the projection) values ​​and OPLS-DA / S-plot were obtained to find the brain migration components that could represent the differences between the groups.

[0077] Figure 3This is a schematic diagram of the brain regions in the sagittal section of the mouse. A is the H&E staining image of the sagittal section of the mouse brain, and B is the mass spectrometry imaging image of the sagittal section of the mouse brain. The mouse brain regions can be simply divided according to the picture, and the abbreviations in the figure have the following meanings: OB: Olfactory Bulb; PC: piriform cortex; BS: brain stem; MB: midbrain; Cortex: cerebral cortex; Hippo: hippocampus; Cpu: corpusstriatum; AN: amygdaloid nucleus; TM: thalamus; CB: cerebellum.

[0078] Figure 4 Figure 2 is a schematic diagram of the OPLS-DA scores of each ROI area of ​​mice under negative ion mode, where AF are the OPLS-DA scores of the amygdala, cerebellum, cortex, striatum, hippocampus, and thalamus, respectively. The control group and the drug-treated group were divided into two discrete groups, and the differences between the groups were obvious; Figure 4 As an example, the amygdala of the control group and the drug-treated group was analyzed by OPLS-DA. 2 X is 0.735, indicating that the model can summarize 73.5% of the overall information of PC1 and PC2, R 2 Y is 0.899, indicating that the model can explain 89.9% of the variation information of the dependent variable, Q 2 It is 0.853, indicating that the model can predict with an accuracy of 85.3%.

[0079] Figure 5 This is a 200 permutation test diagram of the OPLS-DA analysis of the mouse ROI area in the negative ion mode. Among them, AF is a schematic diagram of the 200 permutation model test results of the amygdala, cerebellum, cortex, striatum, hippocampus, and thalamus. The results show that all Q 2 The values ​​are all smaller than the original values ​​on the right, and Q 2 The regression line of the point intersects the Y-axis at the negative axis, indicating that the original model is valid and the next step of differential component analysis can be carried out.

[0080] 3. Analysis of the components of Chinese medicine entering the brain in different brain regions

[0081] In the negative ion mode, S-plot analysis was performed according to the OPLS-DA analysis model. The results are as follows: Figure 6 As shown, Figure 6It is the S-plot of each ROI area of ​​the mouse in the negative ion mode. Among them, AF are the S-plots of the amygdala, cerebellum, cortex, striatum, hippocampus, and thalamus, respectively. The two ends of the figure are compounds that are significantly important to the model and have a strong correlation with the response variable. At the same time, according to the VIP value obtained by the OPLS-DA analysis model, a T test was performed between the control group and the drug-treated group to screen for differential compounds with VIP>1.5 and FoldChange>1.2 (drug-treated group / control group) and P<0.05. The results are shown in Tables 1 and Figure 7 As shown, Figure 7 This is a schematic diagram of the results of DESI-MSI negative ion mode imaging of the brain-specific distribution of the active ingredients of Buwang Powder. A total of 6 Chinese medicinal ingredients were screened, among which AF are the imaging images of succinic acid, asarone, L-uridine, artemisinin, 1-hydroxy-3,7-dimethoxyxanthone, and Polygala tenuifolia saponin d-glucoside, respectively.

[0082] Table 1 Active ingredients of Buwangsan in mouse brain detected under negative ion mode

[0083]

[0084] The present invention innovatively combines mass spectrometry imaging with multivariate statistical methods to analyze the regional distribution of various components of Buwang Powder in brain tissue after mice were gavaged with Buwang Powder. The present invention systematically investigated and optimized the preparation method of mouse brain tissue slices, slice thickness, and the effects of key parameters such as spray solvent, spray solvent flow rate, and mass spectrometry capillary voltage on DESI-MSI analysis, and established a mass spectrometry imaging method and multivariate statistical analysis method suitable for the analysis of active components of Buwang Powder in brain tissue slices. This method preliminarily identified 6 active components of Buwang Powder that are specifically distributed in the brain under negative ion mode, and intuitively demonstrated the specific distribution characteristics of each active component in different brain regions of mice, laying the foundation for subsequent research on the material basis of efficacy and quality control, and this strategy and method will also provide a reference for other drug tissue distribution studies for the treatment of neurodegenerative diseases.

[0085] The above is only a preferred embodiment of the present invention, and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment, it is not used to limit the present invention. Any technician familiar with this patent can make some changes or modify the technical contents suggested above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the solution of the present invention.

Claims

1. A method for studying the specific distribution of a multi-component complex system in animal tissues based on mass spectrometry imaging and multivariate statistical analysis, characterized in that: The following steps are involved: After the multi-component complex system is treated, the animal is administered with drugs for a certain period of time, the animal tissue is obtained after the animal is killed, and the animal tissue is sliced ​​and subjected to mass spectrometry imaging analysis and multivariate statistical analysis to obtain the specific distribution results of the multi-component complex system in the animal tissue; The multi-component complex system is a mixed system containing two or more components at the same time; The mass spectrometry imaging analysis adopts analytical electrospray ionization mass spectrometry / quadrupole time-of-flight mass spectrometry, and the content of the analysis is selected from optimization of spray solvent, optimization of spray solvent flow rate, and optimization of capillary voltage; The multivariate statistical analysis was performed on the ROIs of the amygdala, cerebellum, cortex, striatum, hippocampus and thalamus in the animal tissues, and the mass spectrometry response value per unit area of ​​each ROI was calculated. The VIP value and OPLS-DA / S-plot were obtained by orthogonal partial least squares-discriminant analysis. The brain migration components that can represent the differences between the groups were found using R 2 X, R 2 Y and Q 2 To evaluate the model.

2. The method for studying the specific distribution of a multi-component complex system in animal tissues based on mass spectrometry imaging and multivariate statistical analysis according to claim 1, characterized in that: The multi-component complex system is a traditional Chinese medicine system; The animal tissue is selected from animal brain tissue.

3. The method for studying the specific distribution of a multi-component complex system in animal tissues based on mass spectrometry imaging and multivariate statistical analysis according to claim 1, characterized in that: The animal tissue sectioning comprises the following steps: Place the animal tissue on a base with embedding gel for slicing, and set the slice thickness parameters, chamber temperature, and sample head temperature during slicing.

4. The method for studying the specific distribution of a multi-component complex system in animal tissues based on mass spectrometry imaging and multivariate statistical analysis according to claim 1, characterized in that: The optimization reference of the mass spectrometry imaging is the lower limit of quantification of the representative components; The spray solvent is selected from a methanol and water system, an acetonitrile and water system, and formic acid or ammonia water, ammonium formate is added to the above system.

5. The method for studying the specific distribution of a multi-component complex system in animal tissues based on mass spectrometry imaging and multivariate statistical analysis according to claim 4, characterized in that: The spray solvent is selected from 95% MeOH+0.1% FA, 80% ACN+0.2% FA, 90% MeOH+0.1% ammonia water, 90% MeOH, 98% MeOH, and 80% MeOH.

6. The method for studying the specific distribution of a multi-component complex system in animal tissues based on mass spectrometry imaging and multivariate statistical analysis according to claim 1, characterized in that: The spray solvent flow rate is selected from 0.5 μL·min -1 ~4μL·min -1 ; The capillary voltage is selected from 0.4 kV to 0.8 kV.

7. The method for studying the specific distribution of a multi-component complex system in animal tissues based on mass spectrometry imaging and multivariate statistical analysis according to claim 1, characterized in that: The analytical electrospray ionization mass spectrometry / quadrupole time-of-flight mass spectrometry conditions were set as follows: nebulizer gas was nitrogen, pressure was 13 psi, ion source temperature was 120°C, nebulizer incident angle was 60°, collection angle was 10°, positive and negative ion mode scan rates were 200 μL·min -1 The mass spectrometry analysis mode was sensitive mode, the scanning range was m / z 50-1200, the pixel size was 100×100 μm, and the scanning speed was 200 μm / s.

8. The method for studying the specific distribution of a multi-component complex system in animal tissues based on mass spectrometry imaging and multivariate statistical analysis according to claim 1, characterized in that: The mass spectrometry imaging analysis includes: drawing a standard curve of a standard substance, performing a least squares linear regression simulation analysis on the standard curve, calculating a correlation coefficient r value and a linear equation, and obtaining a relative quantitative lower limit of each compound; After the original data of animal tissue sections are collected, the data processing software is used to perform a series of processing on the data files, including peak detection, calibration, deconvolution, data reduction, isotope elimination analysis, extraction of target compound information of traditional Chinese medicine compound and information of 20 to 1000 compounds with the highest response in the data file, and the total ion current normalization method is used to process the imaging of tissue sections. After compensating for the matrix effect, the mass spectrometry imaging data is analyzed and a specific ion distribution image is generated.

9. The method for studying the specific distribution of a multi-component complex system in animal tissues based on mass spectrometry imaging and multivariate statistical analysis according to claim 2, characterized in that: The traditional Chinese medicine system is made of calamus, poria, poria, ginseng and polygala in a mass ratio of 2:5:5:5:

7.

10. The method for studying the specific distribution of a multi-component complex system in animal tissues based on mass spectrometry imaging and multivariate statistical analysis according to claim 9, characterized in that: The preparation method of the traditional Chinese medicine system comprises the following steps: weighing calamus, poria, poria, ginseng and polygala in a mass ratio of 2:5:5:5:7 and then crushing them, adding 5 to 15 times the amount of 30 to 90% ethanol, soaking for 1 to 10 hours, refluxing extraction for 1 to 4 hours, filtering, and repeatedly extracting for at least 2 times to obtain a first part of the extract; then adding 5 to 15 times the amount of pure water, continuing to reflux extraction for 1 to 4 hours, filtering to obtain a second part of the extract; mixing the first part of the extract and the second part of the extract, and freeze-drying them into powder to obtain the traditional Chinese medicine system.

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