CN113281448A - Metabonomics analysis method based on cardiogenic shock rat model - Google Patents
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Abstract
The invention relates to a metabonomics analysis method based on a cardiogenic shock rat model, which is characterized in that a ligation method of a left anterior descending branch near cardiac apex and a far cardiac apex is adopted to duplicate early and middle cardiogenic shock rat models, and metabolic map information of early and middle plasma samples of the cardiogenic shock of the rats is collected based on an ultra-high performance liquid chromatography-quadrupole flight time tandem mass spectrometer; the Progenisis QI is used for data preprocessing, the EZinfo 2.0 statistical software is used for multivariate statistical analysis, biomarker groups with significant differences are screened out, the metabolic pathways involved in the biomarker groups are analyzed through Metabioanalysis. The invention researches the influence and change rule of external stimulation on endogenous micromolecules of organisms from the integral angle, and provides a new research idea and method for the traditional Chinese medicine syndrome and the traditional Chinese medicine integral action mechanism.
Description
Technical Field
The invention belongs to the field of bioinformatics, and relates to a metabonomics analysis method based on a cardiogenic shock rat model.
Background
Cardiogenic shock is induced by pump failure due to cardiac insufficiency, resulting in peripheral tissues in a hypoperfusion state and hemodynamics abnormality. The development process of cardiogenic shock can be divided into early stage, middle stage and late stage, and the clinical hemodynamic indexes of the early stage are as follows: the blood pressure is normal, even slightly increased or slightly decreased, the heart rate is accelerated, and the pulse pressure difference becomes small; the mid-term hemodynamic parameters are expressed as: the blood pressure is reduced obviously, the heart rate is increased faster than that in the early stage, and the pulse pressure difference is further reduced than that in the early stage; in the late stage, symptoms of disseminated intravascular coagulation and multiple organ failure may occur. Clinical treatment of cardiogenic shock advocates finding high-risk patients as early as possible, implements effective treatment measures as soon as possible, and especially, it is important to discuss intervention action and mechanism of cardiogenic shock in early and middle stages.
The main death cause of patients with cardiogenic shock is heart failure, and the ginseng and aconite injection can remarkably improve the cardiac function of patients with heart failure in acute attack stage. At present, the research on the action mechanism of the ginseng and aconite injection for treating cardiogenic shock is less, and the stage of the cardiogenic shock is not determined. The TCM is well focused on treatment based on syndrome differentiation, and during the development of diseases, the physiological and pathological states of the disease are constantly changed, so that the disease treatment is performed in response to the treatment of the disease.
The ginseng and aconite injection has the functions of strengthening heart, promoting urination, restoring yang, rescuing from collapse and relieving depletion for patients with heart failure, and is widely applied clinically.
Disclosure of Invention
The invention aims to provide a metabonomics analysis method based on a cardiogenic shock rat model, which is used for researching the influence of the ginseng and aconite injection on the plasma metabolites of the cardiogenic shock early and middle model rats, searching possible biomarkers and discussing the action mechanism of the ginseng and aconite injection for treating the cardiogenic shock early and middle.
The invention is realized by the following technical scheme. A metabonomics analysis method based on a cardiogenic shock rat model comprises the following steps:
firstly, duplicating early and middle cardiogenic shock rat models by adopting a left coronary artery anterior descending branch proximal apex and distal apex ligation method;
collecting metabolic map information of early and middle plasma samples of the cardiogenic shock of the rat based on the ultra-high performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometer;
step three, preprocessing data;
step four, performing multivariate statistical analysis to screen out biomarker groups with significant differences;
and step five, analyzing the participating metabolic pathways, and discussing the action mechanism of the ginseng and aconite injection for treating early and middle periods of cardiogenic shock.
In the third step, based on ProgenesisQi software, peak extraction, peak alignment, peak matching, peak intensity correction and the like are carried out on the acquired spectrogram, so that a csv format file containing compound retention time and mass-to-charge ratio information is obtained.
In the fourth step, multivariate statistical analysis is carried out by EZinfo 2.0 statistical software, and biomarker groups with significant differences are screened out.
And step five, analyzing the participating metabolic pathways through a Metabioanalysis. ca website, and discussing the action mechanism of the radix aconiti lateralis preparata injection for treating early and middle cardiogenic shock.
The invention has the beneficial effects that: researching the influence of the ginseng and aconite injection on the plasma metabolites of rats in early and middle models of cardiogenic shock, searching possible biomarkers and discussing the action mechanism of the ginseng and aconite injection for treating the early and middle models of cardiogenic shock; the metabonomics analysis method is identical with the integral view of the traditional Chinese medicine, the influence and change rule of external stimulation on endogenous micromolecules of an organism are integrally researched, and a new research idea and method are provided for the research on the traditional Chinese medicine syndrome and the integral action mechanism of the traditional Chinese medicine.
Detailed Description
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments.
A metabonomics analysis method based on a cardiogenic shock rat model comprises the following steps:
step one, adopting a ligature method of anterior descending branch proximal apex of left coronary artery and distal apex of left coronary artery to duplicate early and middle cardiogenic shock rat models. Rats were fasted for 12h before the experiment and were anesthetized with 2% sodium pentobarbital (50mg/kg) and fixed to rat plates. The right femoral vein was isolated and cannulated for infusion. Separating trachea, and performing trachea intubation. After the chest is opened, a small animal respirator is opened quickly, the pericardium is cut, and the heart is lifted to ligate the anterior descending branch of the left coronary artery to the apex of the heart, so that an early cardiogenic shock model is generated; ligating the anterior descending branch of the left coronary artery and the distal tip to form a cardiogenic shock metaphase model, closing the thoracic cavity and stabilizing for 10 min.
Secondly, collecting metabolic map information of the plasma samples of the rats in early and middle phases of cardiogenic shock by utilizing the characteristics of rapid separation and high sensitivity of mass spectrum of the ultra-high performance liquid chromatography based on the ultra-high performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometer (UPLC/Q-TOF-MS) combined technology;
the specific process of acquiring the map information comprises the following processes:
1. plasma sample preparation method
Thawing frozen rat plasma at room temperature, placing 100ul plasma in 1.5ml EP tube, adding 300ul methanol, mixing uniformly for 1min, standing at constant temperature of 4 deg.C for 3 hr, centrifuging (4 deg.C, 15000RCF, 10min), taking supernatant, placing in 1.5ml EP tube, drying with 30 deg.C water bath nitrogen, and storing to-80 deg.C. Redissolving: adding water-methanol solution (water: methanol: 85:15) to constant volume of 200ul, mixing by vortex for 1min, centrifuging (4 deg.C, 15000RCF, 15min), and collecting supernatant in sample bottle.
2. Plasma sample measurement conditions
(1) The mass spectrum conditions are that the temperature of an ionization source is 100 ℃, the cone hole gas is nitrogen, and the flow rate is 50L/h; the desolvation gas is nitrogen, the temperature is 400 ℃, and the flow rate is 800L/h. Under the positive ion mode, the capillary voltage is 3.0kV, the taper hole voltage is 40V, the extraction taper hole voltage is 80V, the acquisition time is 0-25 min, and the mass number range is 50-1000 Da; the capillary voltage under the negative ion mode is 2.5kV, the taper hole voltage is 40V, and the compensation voltage is 80V. In order to ensure the accuracy and repeatability of the quality, a standard curve of a quality axis is established by adopting sodium formate, and the leucine enkephalin is used for real-time quality correction. The tandem mass spectrometry collision gas is argon, the low collision energy is 4eV, and the high collision energy is 20-40 eV.
To ensure the stability of the entire assay system, the assay was performed using Quality control samples (QC) which were mixed at 5. mu.L per normal sample. During the detection of the samples, the QC samples were run 1 time after every 7 normal samples were detected to measure the stability of the system. To avoid errors, the order of detection of the samples is performed randomly.
(2) Chromatographic conditions are as follows: wherein, the chromatographic conditions of the positive ion mode are that water phase A: ultrapure water (0.1% formic acid), organic phase B: acetonitrile; an aqueous phase A: ultrapure water (0.1% formic acid), organic phase B: acetonitrile; gradient elution: 0-2 min, 0.1% B; 2-6.4 min, 0.1% -22% of B; 6.4-9 min, 22% -35% of B; 9-22 min, 35% -100% B; 22-24 min, 100% B; 24-32 min, 100% -90% B; 32-34 min, 90% -0.1% B; 34-36 min, 0.1% B. The column temperature was 40 ℃, the sample chamber temperature was 10 ℃, the flow rate was 0.4ml/min, and the sample size was 1 ul. Anion mode chromatography conditions: an aqueous phase A: ultrapure water (0.1% formic acid), organic phase B: acetonitrile; gradient elution: 0-20 min, 5% -95% B; 20-23 min, 95% -5% of B. The column temperature is 40 ℃; the temperature of the sample chamber is 10 ℃; the flow rate is 0.3 ml/min; the sample size was 1 ul.
(3) Assay instrument, chromatographic column and profile acquisition software
Waters Acquity UPLC liquid chromatography System, Q-TOF SYNAPT G2 HDMS Mass spectrometer, and Waters ACQUITY UPLC BEH C18The metabolic profiles of early and middle-stage cardiogenic shock plasma samples of rats were collected by a chromatographic column (50 mm. times.2.1 mm, 1.7 μm) through MarkerLynx software.
Thirdly, preprocessing data by using Progenetics QI;
and performing peak extraction, peak alignment, peak matching, peak intensity correction and the like on the acquired spectrogram based on ProgenesisQi software to obtain a csv format file containing compound retention time and mass-to-charge ratio information.
And step four, performing multivariate statistical analysis by using EZinfo 2.0 statistical software to screen out the biomarker groups with significant differences.
Designing an experimental group, importing the processed data matrix into EZinfo 2.0 statistical software for multivariate statistical analysis, performing supervised partial least squares discriminant analysis (PLS-DA), classifying the groups, discriminating the difference between the groups, selecting a variable with a significance difference P of less than 0.05 and a projection Variable (VIP) of more than 1 as a biomarker, and obtaining the retention time and the nucleus-to-nucleus ratio information of the biomarker. Based on a biological database such as HMDB, KEGG and the like, screening out a rat endogenous biomarker and identifying the biomarker.
The rat model in early cardiogenic shock caused 1 pathological biomarker to fluctuate significantly, but due to limited resources, the pathological marker could not be identified. And 4 potential biomarkers with significant differences are found in the early process of treating cardiogenic shock by the ginseng and aconite injection, and the obtained MS is usedEAnd matching MS/MS data information with HMDB database fragment information, and finally identifying 3 biomarkers related to the ginseng and aconite injection for treating early cardiogenic shock, wherein the biomarkers are thymidine, 2-methoxy-4-ethylphenol and cysteine glutathione disulfide respectively. The content of thymidine and 2-methoxy-4-ethylphenol can reach the variation trend of positive regulation in the low dose group, but the medium and high doses are the reverse regulation effect. The content of cysteine glutathione disulfide can reach the change trend of positive regulation by three administration doses.
The cardiogenic mid-shock rat model enables 14 pathological biomarkers to fluctuate significantly, and finally 3 pathological markers are identified, namely, adenosine hexaphosphate, L-gamma-glutamyl-L-isoleucine and dimethyl benzimidazole. Compared with the normal group, the pathologic markers of the cardiogenic mid-shock model group all showed an upward trend.
And step five, analyzing the participating metabolic pathways of the differential metabolic markers screened and confirmed in the step three through a Metabioanalysis.ca website, and discussing the action mechanism of the radix aconiti lateralis preparata injection for treating early and medium cardiogenic shock. The differential metabolic marker group is put into Metabionalyst. ca website to automatically give metabolic pathway, and then the biological function of the differential biomarker is inquired and explained by combining related metabolite databases such as HMBD (human Metabolome database) and KEGG (Kyoto Encyclopedia of Genes and genomes). Early in cardiogenic shock, the disease develops slowly and the fluctuation of endogenous metabolome is small. (2) The cardiogenic mid-shock model causes the differential expression of adenosine hexaphosphate, L-gamma-glutamyl-L-isoleucine and dimethyl benzimidazole in rats, and presumably influences the carbohydrate metabolism and amino acid synthesis in rats according to the biological functions.
According to the invention, the time change rule of each index of hemodynamics and mesentery microcirculation is observed by ligating different parts of the left coronary artery of a rat, and compared with the research and clinical indexes of predecessors, the accuracy of model research is judged according to the data of experimental indexes, preliminarily, the extremely significant difference exists between each index group after respectively ligating the anterior descending proximal apex and the distal apex of the left coronary artery of the rat for 20min, and therefore, the early and middle cardiogenic shock models are successfully established.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (6)
1. The metabonomics analysis method based on the cardiogenic shock rat model is characterized by comprising the following steps:
firstly, duplicating early and middle cardiogenic shock rat models by adopting a left coronary artery anterior descending branch proximal apex and distal apex ligation method;
collecting metabolic map information of early and middle plasma samples of the cardiogenic shock of the rat based on the ultra-high performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometer;
step three, preprocessing data;
step four, performing multivariate statistical analysis to screen out biomarker groups with significant differences;
and step five, analyzing the participating metabolic pathways, and discussing the action mechanism of the ginseng and aconite injection for treating early and middle periods of cardiogenic shock.
2. The cardiogenic shock rat model-based metabonomic analysis method of claim 1, wherein the specific process of step two is as follows:
1. plasma sample preparation: thawing frozen rat plasma at room temperature, placing 100ul of plasma in a 1.5ml EP tube, adding 300ul of methanol, mixing uniformly for 1min by vortex, standing at constant temperature of 4 ℃ for 3h, centrifuging, placing supernate in a 1.5ml EP tube, drying with nitrogen in water bath at 30 ℃, and storing to-80 ℃; redissolving: adding 200ul of water-methanol solution to constant volume, mixing for 1min, centrifuging, and collecting supernatant in a sample bottle to obtain the final product;
2. plasma sample measurement conditions:
(1) the mass spectrum conditions are that the temperature of an ionization source is 100 ℃, the cone hole gas is nitrogen, and the flow rate is 50L/h; the desolventizing gas is nitrogen, the temperature is 400 ℃, and the flow rate is 800L/h; under the positive ion mode, the capillary voltage is 3.0kV, the taper hole voltage is 40V, the extraction taper hole voltage is 80V, the acquisition time is 0-25 min, and the mass number range is 50-1000 Da; under the negative ion mode, the capillary voltage is 2.5kV, the taper hole voltage is 40V, and the compensation voltage is 80V; adopting sodium formate to establish a mass axis standard curve, and carrying out real-time mass correction on leucine enkephalin; the tandem mass spectrometry collision gas is argon, the low collision energy is 4eV, and the high collision energy is 20-40 eV;
performing method verification by using a quality control sample, wherein the quality control sample is obtained by mixing 5 mu L of each normal sample; in the detection process of the samples, running the QC samples for 1 time after detecting 7 normal samples to measure the stability of the system, wherein the detection sequence of the samples is randomly carried out;
(2) chromatographic conditions are as follows: wherein, the chromatographic conditions of the positive ion mode are that water phase A: ultrapure water (0.1% formic acid), organic phase B: acetonitrile; an aqueous phase A: ultrapure water (0.1% formic acid), organic phase B: acetonitrile; gradient elution: 0-2 min, 0.1% B; 2-6.4 min, 0.1% -22% of B; 6.4-9 min, 22% -35% of B; 9-22 min, 35% -100% B; 22-24 min, 100% B; 24-32 min, 100% -90% B; 32-34 min, 90% -0.1% B; 34-36 min, 0.1% B; the column temperature was 40 ℃, the sample chamber temperature was 10 ℃, the flow rate was 0.4ml/min, and the sample size was 1 ul. Anion mode chromatography conditions: an aqueous phase A: ultrapure water, 0.1% formic acid; and (3) organic phase B: acetonitrile; gradient elution: 0-20 min, 5% -95% B; 20-23 min, 95% -5% of B; the column temperature is 40 ℃; the temperature of the sample chamber is 10 ℃; the flow rate is 0.3 ml/min; the sample amount is 1 ul;
(3) assay instrument, chromatographic column and profile acquisition software
Waters Acquity UPLC liquid chromatography System, Q-TOF SYNAPT G2 HDMS Mass spectrometer, and Waters ACQUITY UPLC BEH C18And (3) collecting metabolic maps of the early and middle plasma samples of the cardiogenic shock of the rats by a chromatographic column through MarkerLynx software.
3. The cardiogenic shock rat model-based metabonomics analysis method of claim 1, wherein in step three, peak extraction, peak alignment, peak matching, peak intensity correction and the like are performed on the acquired spectrogram based on ProgenesisQi software, so as to obtain a csv format file containing compound retention time and mass-to-charge ratio information.
4. The cardiogenic shock rat model-based metabonomic analysis method of claim 1, wherein in step four, multivariate statistical analysis is performed by Ezinfo 2.0 statistical software to screen out biomarker groups with significant differences.
5. The cardiogenic shock rat model-based metabonomic analysis method of claim 4, wherein in step four, experimental grouping is designed, the processed data matrix is imported into EZinfo 2.0 statistical software for multivariate statistical analysis, supervised partial least squares discriminant analysis is performed, the groups are classified and the difference between the groups is discriminated, a variable with a significance difference P less than 0.05 and a projection variable greater than 1 is selected as a biomarker, and then the retention time and the plasmid-to-nuclear ratio information are obtained; the endogenous biomarkers of rats were screened and identified based on a biological database.
6. The cardiogenic shock rat model-based metabonomics analysis method of claim 1, wherein in step five, metabolic pathways involved in cardiogenic shock are analyzed through Metabioanalysis.ca website, and the action mechanism of the aconite injection for treating early and middle stages of cardiogenic shock is discussed.
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| CN114487188A (en) * | 2022-01-25 | 2022-05-13 | 上海交通大学 | Metabonomics method for preparing animal model for treating primary dysmenorrhea based on radix aconiti kusnezoffii slices |
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| CN1951432A (en) * | 2005-09-13 | 2007-04-25 | 山东绿叶天然药物研究开发有限公司 | Gingseng and aconite composition, its preparation, preparation process and usage |
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| CN114487188A (en) * | 2022-01-25 | 2022-05-13 | 上海交通大学 | Metabonomics method for preparing animal model for treating primary dysmenorrhea based on radix aconiti kusnezoffii slices |
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