CN110346466B - Construction method for enhancing model of seleno-aminopolysaccharide on immune mechanism of black porgy based on liver metabonomics research - Google Patents
Construction method for enhancing model of seleno-aminopolysaccharide on immune mechanism of black porgy based on liver metabonomics research Download PDFInfo
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Abstract
A method for constructing an enhancement model of selenized aminopolysaccharide on an immune mechanism of black porgy based on liver metabonomics research relates to the field of biomedical analysis and comprises the following steps: 1) dividing the juvenile sea bream into an experimental group and a blank group; 2) adding seleno-amino polysaccharide into the feed of the experimental group; 3) carrying out starvation treatment on the juvenile sea breams in the experimental group and the blank group, after anesthesia, taking out liver tissues and storing for later use; 4) preparing a liver tissue sample, preparing a quality control sample at the same time, and processing by using an ultra-high performance liquid chromatography-time-of-flight mass spectrometry combined technology; 5) and analyzing and processing the collected liver metabonomics data of the black porgy, identifying and screening liver metabonomics biomarkers for enhancing the immune mechanism of the black porgy by the selenoamino-polysaccharide, and constructing and analyzing metabolic pathways pointed by the liver metabonomics biomarkers. The invention has the advantages of good systematicness, high accuracy, low cost and simple operation.
Description
Technical Field
The invention relates to the field of biomedical analysis, in particular to a method for constructing an enhancement model of a seleno-aminopolysaccharide on an immune mechanism of black porgy based on liver metabonomics research.
Background
Selenium is used as an essential trace element for organisms, and plays an important role in the aspects of growth and health maintenance of the organisms in the forms of selenoprotein, selenium-dependent enzyme accessory factors and the like. Much research is currently done on organic selenium, such as amino-selenized polysaccharide, which is relatively less toxic and easily absorbed and utilized. The amino polysaccharide selenide is obtained by combining polysaccharide with inorganic selenium through a chemical modification method, the physiological activity and the pharmacological function of the selenium and the polysaccharide are optimized, the biological activity is generally higher than that of the polysaccharide and the selenium, and the amino polysaccharide selenide is easier to absorb and utilize by organisms. The selenized aminopolysaccharides can be used as a selenium-containing dietary supplement with the potential of enhancing adaptive immunity.
The black porgy (Acathopagrus schlegelii) is an economic fish cultured in seawater in the southeast coastal region and the western pacific coast of China, but the research on the immunoregulation effect of the seleno-glycosaminoglycan on the black porgy is less. The liver is the central organ of the metabolism of body substances, and many metabolic processes such as synthesis, decomposition and transformation of substances are performed in the liver. The biochemical change of the liver is in the important crossing position of basic research and clinical research, so that the metabonomics research on the liver has high basic and clinical research value, preliminary research is already carried out on the utilization of the black porgy to the selenium at present, but the research on the immunoregulation function of the selenoamino polysaccharide to the black porgy is less, and the existing evaluation method for researching the immune mechanism of the selenoamino polysaccharide to the black porgy has the technical problems of poor systematicness, low accuracy, high cost, complex operation and the like.
For example, the literature "influence of different types of selenium on the growth and serum immunity index of young black sea breams [ J ] Aquaculture, 2018(5): 577) 583", which studies the influence of different types of exogenous selenium on the growth performance, body composition and serum immunity index of young black sea breams by feeding black sea breams with a test feed (control group) containing nitrogen such as protein 41.57%, fat 13.63%, and the like, and adding 2.35mg/kg of selenylated polysaccharide and 0.88mg/kg of sodium selenite (so that the addition amount of exogenous selenium is 0.4mg/kg) for 8 weeks. The growth performance and serum immunocompetence of the juvenile black porgy can be obviously improved by adding a proper amount of exogenous selenium, the bioavailability of the organic selenium is obviously higher than that of an inorganic selenium test result, but the evaluation method has the technical problems of poor systematicness, low accuracy, high cost, complex operation and the like.
Disclosure of Invention
The invention provides a construction method for researching an enhancement model of an immune mechanism of black porgy by using amino polysaccharide selenide based on liver metabonomics, aiming at overcoming the technical problems of poor systematicness, low accuracy, high cost, complex operation and the like of the existing evaluation method for researching the immune mechanism of black porgy by using amino polysaccharide selenide.
In order to achieve the purpose, the invention adopts the following technical scheme:
a construction method for researching a black porgy immune mechanism enhancement model by using seleno-aminopolysaccharide based on liver metabonomics comprises the following steps:
1) dividing the young black porgy fish used for animal experiment into experiment group and blank group;
2) feeding the experimental group and the blank group with the same feed, and adding the selenized aminopolysaccharide into the feed of the experimental group;
3) after feeding is finished, carrying out starvation treatment on the juvenile sea breams in the experimental group and the blank group, carrying out anesthesia by using an anesthetic, taking out liver tissues, and storing for later use;
4) preparing liver tissue samples and quality control samples, and then processing by using an ultra-high performance liquid chromatography-time-of-flight mass spectrometry combined technology, wherein the quality control samples are inserted among the liver tissue samples during sample introduction;
5) analyzing and processing liver metabonomics data of the black porgy collected by the ultra-performance liquid chromatography-time-of-flight mass spectrometry combined technology, identifying and screening liver metabonomics biomarkers for enhancing the immune mechanism of the black porgy by the amino-selenide polysaccharide, and constructing and analyzing metabolic pathways pointed by the liver metabonomics biomarkers.
Metabonomics can directly reflect the changes of biochemical processes and states in vivo by carrying out a series of dimensionality reduction on original complex data reflecting sample information, and has unique advantages in the aspect of systematically researching the overall and dynamic change rules of biological endogenous micromolecular metabolites. The invention combines and utilizes a high-throughput, high-sensitivity and high-resolution mass spectrum detection technology and a non-targeted metabonomics method to explain the potential mechanism and target approach of the selenoamino-polysaccharide to the immunoregulation of the black porgy, and provides reference for the development of the black porgy immunopotentiator.
Preferably, the specific method for treating the juvenile black porgy in the step 1) comprises the following steps: and (4) stopping feeding the black porgy juvenile fish, and then putting the black porgy juvenile fish into a glass fiber cylinder for microflow feeding.
The specific feeding method of the experimental group and the blank group in the step 2) comprises the following steps: feeding the experimental group and the blank group by adopting common feed, adding 0.5-0.7 mg Se/kg of seleno-amino polysaccharide into the common feed of the experimental group, and continuously feeding for 8-10 weeks.
The specific method for treating the juvenile Sparus macrocephalus after feeding in step 3) comprises the following steps: after starving the juvenile sea breams of the experimental group and the blank group for 20-30h, anesthetizing by using anesthetic, taking out liver tissues, weighing parallel samples with the same quantity in the experimental group and the blank group, quickly freezing by using liquid nitrogen, and finally storing in an ultralow-temperature refrigerator at the temperature of-80 ℃ for later use.
The preparation method of the liver tissue sample in the step 4) comprises the following steps: taking the standby liver tissue in a centrifuge tube, adding 5-15 times volume of methanol frozen at minus 10-minus 30 ℃, homogenizing at 10000-14000rpm for 0.5-1.5min, centrifuging at 4 ℃ and 12000-14000rpm for 3-5min, taking the supernatant, drying by nitrogen at 30-50 ℃, re-dissolving by methanol water solution, and finishing the preparation of the liver tissue sample within 12 min; the quality control sample preparation method comprises the following steps: mixing the above supernatants, drying with nitrogen at 30-50 deg.C, and re-dissolving with methanol water solution.
The specific method for processing by using the ultra-high performance liquid chromatography-time-of-flight mass spectrometry combined technology in the step 4) comprises the following steps: after being filtered by a microporous filter membrane, the liver tissue sample redissolved by the methanol water solution enters an ultra-high performance liquid chromatography-mass spectrometer for analysis under fixed chromatographic conditions and mass spectrum conditions, and in the detection process, a quality control sample is inserted into every other liver tissue sample with the same quantity for detection and analysis so as to monitor the detection stability.
The specific method for analyzing and processing the liver metabonomics data of the black porgy in the step 5) comprises the following steps: using XCMSplusComprehensively performing non-targeted metabonomic analysis on the collected black porgy liver metabonomics data, adopting a Centwave feature detection algorithm (the peak width is from 5 to 20s, and the mass tolerance is 5ppm) to discover and match peaks, finding different biomarkers, automatically linking XCMS Plus software to a METLIN data spectrum library (more than 24 ten thousand metabolite information, wherein 12127 metabolites have high-resolution secondary maps), and combining endogenous generationMetabolite secondary spectrum library (comprising 550 common endogenous metabolic compounds including molecular formula, molecular weight, CAS number, chemical name and structure diagram), identifying different biomarkers according to the first-level m/z and isotope abundance ratio of the compounds in the spectrum library and the information of second-level mass spectrum, and finding out metabolic pathway by clustering analysis of the biomarkers.
In the step 4), because the liver of the black porgy contains abundant metabolic enzymes, the whole sample pretreatment process adopts low-temperature operation, the activity of the metabolic enzymes in the liver is greatly reduced at the environmental temperature of 4 ℃, and the contents of protein, fat and the like in the liver are kept relatively stable; secondly, a high-speed rapid homogenization method is adopted, so that the liver sample and the frozen methanol extracting solution are rapidly and fully mixed, and the speed of extracting potential markers in the liver by using methanol is increased; thirdly, adopting the steps of extracting liquid nitrogen, blowing, re-dissolving residues and detecting, aiming at concentrating trace potential markers in the extracting solution and then detecting by an ultra-high performance liquid chromatography-time of flight mass spectrometer, so that the detection sensitivity of the potential markers is increased, and the potential markers in the liver are objectively reflected; finally, because the liver contains abundant metabolic enzymes, the whole appearance of the potential markers in the liver can be completely and faithfully embodied only by accelerating extraction and shortening extraction time as much as possible, so that the preparation of the whole liver tissue sample is basically completed within 12min, the extraction process is fast and efficient, errors caused by manual operation are reduced, the potential markers in the liver are extracted to the maximum extent, and the accuracy and precision of mass spectrum data acquisition and analysis are guaranteed.
Preferably, there are 32 liver metabonomics biomarkers for enhancing immune mechanism of black porgy by using the selenized aminopolysaccharide, and the biomarkers are respectively: l-histidine, dimethylglycine, 4-guanidinobutyric acid, L-valine, L-leucine, L-isoleucine, L-phenylalanine, L-tryptophan, succinic acid, adenine, adenosine, deoxyinosine, inosine, deoxyguanosine, guanosine, betaine, L-methionine, glyceric acid, L-proline, L-threonine, L-glutamine, L-serine, adenosine monophosphate, deoxyadenosine monophosphate, gamma-aminobutyric acid, L-glutamic acid, D-ribulose 5-phosphate, guanosine monophosphate, ornithine, L-arginine, creatine, arginosuccinic acid.
Preferably, the biomarkers are directed predominantly to 7 metabolic pathways, respectively: aminoacyl-tRNA biosynthesis, valine, leucine and isoleucine biosynthesis, arginine and proline metabolism, purine metabolism, nitrogen metabolism, glycine, serine and threonine metabolism, alanine, aspartate and glutamate metabolism.
Preferably, C is18The chromatographic conditions of the chromatographic column are as follows: flow rate: 0.3ml/min, sample tray temperature: 4 ℃, column temperature: at 40 ℃, the mobile phase A is 2mmol/L ammonium formate and 0.05% formic acid aqueous solution, the mobile phase B is a mixed solution of acetonitrile and isophthalic acid, the volume ratio of the acetonitrile to the isophthalic acid is 1:1, and the sample injection amount is as follows: 2 μ L, mobile phase gradient elution is shown in the following table.
The chromatography conditions of the HILLIC chromatographic column are as follows: flow rate: 0.3ml/min, sample tray temperature: 4 ℃, column temperature: at the temperature of 45 ℃, the mobile phase C is 10mmol/L ammonium formate and 0.1% formic acid aqueous solution, the mobile phase D is acetonitrile aqueous solution, wherein the volume ratio of acetonitrile to water is 95:5, and the water contains 10mmol/L ammonium formate and 0.1% formic acid, the sample amount is as follows: 1 μ L, mobile phase gradient elution is shown in the following table.
Preferably, the mass spectrometry conditions are: electrospray ionization (ESI) source positive and negative ion scan mode was used, spray voltage (IS): a positive ion of 5500V and a negative ion of-4500V; ionization Temperature (TEM): 550 ℃; atomizing gas (GS 1): 60 psi; auxiliary heating gas (GS 2): 60 psi; air curtain gas (CUR): 35 psi. The primary mass spectrum acquisition range is m/z 100-1250, and the accumulation time is 0.10 s; DP: 80V. And (2) acquiring a secondary mass spectrum by adopting an IDA mode, wherein the acquisition range is m/z 50-1250, the accumulation time is 0.05000s, and the DP: 80V; CE is 40 +/-20 eV. The IDA conversion criteria are: the signal intensity is more than 100cps, the molecular weight error is 50mDa, and isotopes are excluded within 4 Da.
In processing this complex matrix of liver, the use of different chromatographic techniques is a key issue to achieve maximum detection characteristics. The liquid phase conditions used for metabolomics experiments, including the solvents and elution gradients employed, should not be used to enhance or target the separation of any particular metabolite, but rather should have general applicability. The invention adopts two different mobile phases and combines two different chromatographic columns to analyze liver tissue samples, wherein C is18In the chromatographic column, the mobile phase A is 2mmol/L ammonium formate and 0.05% formic acid aqueous solution, the mobile phase B is a mixed solution of acetonitrile and isophthalic acid, the volume ratio of the acetonitrile to the isophthalic acid is 1:1, and the mobile phase is mainly used for some compounds with smaller polarity, and can be used for preparing the compounds with smaller polarity at C18Eluting on the chromatographic column, and simultaneously ensuring the chromatographic peak shape of the chromatographic column, thereby better separating small molecular metabolites with small polarity; in HILIC chromatographic column, mobile phase C is 10mmol/L ammonium formate and 0.1% formic acid aqueous solution, mobile phase D is acetonitrile aqueous solution, wherein the volume ratio of acetonitrile to water is 95:5, and the water contains 10mmol/L ammonium formate and 0.1% formic acid, which are mainly aimed at some C18The system reserves compounds with poor polarity and large polarity, so that the separation effect on small molecule metabolites with large polarity is the best, and the separation and data acquisition are respectively carried out on liver samples in a positive ion mode and a negative ion mode by combining a time-of-flight mass spectrometry technology, so that the small molecule metabolites can be detected as much as possible, and the accuracy and the correctness of metabonomics analysis are improved.
However, time of flight (TOF) mass spectrometers are sensitive to temperature fluctuations and require periodic calibration to ensure accuracy of metabonomic experimental data. Metabolites are determined according to the mass number measured, and therefore, accurate mass measurement is crucial for the success of metabolomics experiments. The invention uses the TripleTOFTM 5600+ system and adopts an external calibration method, the measured quality error is controlled within 5ppm, and the accuracy and effectiveness of the data obtained in the data acquisition process are ensured. In IDA mode, when TOF MS/MS is triggered, the maximum number of TOF MS/MS needs to be monitored, which affects the information amount of data acquisition. While it is desirable to maximize the number of TOF MS/MS events to increase the number of product ions collected, it is desirable to ensure that there are sufficient data points collected for chromatographic peaks. The IDA method adopted by the invention comprises the following steps: the scanning time is 0.15 seconds and 10 MS/MS events, the ion accumulation time of each event is 0.05 seconds, the cycle time is 0.7 seconds, 10-12 collected data points of each chromatographic peak can be realized, and high-quality TOF MS and TOF MS/MS data are provided for the statistical evaluation of mass spectrum and the simultaneous identification of metabolites.
Preferably, the juvenile black porgy used for the animal experiment in the step 1) has healthy constitution and uniform size, and the initial weight is 12.8-13.2 g.
Preferably, the selenium content of the selenized aminopolysaccharide is 27.3 mg/g.
Preferably, the anesthetic is MS-222 at a concentration of 60 mg/L.
Preferably, the volume ratio of methanol to water in the aqueous methanol solution is 4: 1.
Therefore, the invention has the following beneficial effects: the invention adopts the ultra-high performance liquid chromatography-time-of-flight mass spectrometry combined technology to carry out metabonomics research on the liver of the black porgy fed with selenized aminopolysaccharide, and adopts two chromatographic columns of C18 and HILLIC in combination with XCMSplusThe software is used for analyzing, 32 different biomarkers are screened out, the immune action mechanism is identified and high-throughput analysis is realized by utilizing the callback change of the content of the biomarkers, then MetabioAnalyst 3.0 is used for analyzing the metabolic pathway of the different biomarkers, and the immune enhancement effect of the selenoamino polysaccharide is comprehensively evaluated from the whole level, so that the establishment of the immune mechanism enhancement model of the selenoamino polysaccharide on the black porgy based on liver metabonomics research lays a certain experimental foundation for deeply discussing the immune enhancement mechanism of the selenoamino polysaccharide, and compared with other methods for establishing or evaluating the immune mechanism model of the black porgy by the selenoamino polysaccharide, the method disclosed by the invention is good in systematicness, high in accuracy, low in cost and simple to operate.
Drawings
FIG. 1 shows a total ion current chromatogram of a quality control sample of the present invention, wherein (a) is C18Column, positive ion scan mode, b) is C18A chromatographic column and a negative ion scanning mode, c) is a HILIC chromatographic column and a positive ion scanning mode, d) is a HILIC chromatographic column and a negative ion scanning mode).
FIG. 2 shows the total ion current chromatogram of samples of experimental group and blank group under different ion scanning modes (a) is C18Column, positive ion scan mode, b) is C18A chromatographic column and a negative ion scanning mode, c) is a HILIC chromatographic column and a positive ion scanning mode, d) is a HILIC chromatographic column and a negative ion scanning mode).
FIG. 3 shows the PCA of the liver metabolism profile analysis under different ion scanning modes of the present invention (a) is C18Chromatographic column, negative ion scanning mode, b) is C18Chromatographic column, positive ion scanning mode, c) HILIC chromatographic column, negative ion scanning mode, d) HILIC chromatographic column, positive ion scanning mode, black experimental group, grey blank group).
Detailed Description
The invention is further described with reference to specific embodiments.
Example 1:
a construction method for researching a black porgy immune mechanism enhancement model by using seleno-aminopolysaccharide based on liver metabonomics comprises the following steps:
1) stopping feeding the black porgy juvenile fish for 1 day, and then randomly dividing the black porgy juvenile fish with healthy physique, uniform size and 12.8-13.2 g of initial weight into an experimental group and a blank group, wherein the blank group is used for feeding common feed, the experimental group is added with 0.6mg Se/kg of selenoamino polysaccharide in the common feed and is used for feeding for 8 weeks, after the feeding is finished, the black porgy juvenile fish is starved for 24 hours and is anesthetized by 60mg/L of MS-222, liver tissues are taken out, wherein 10 parallel samples are respectively selected from the experimental group and the blank group, are weighed, are quickly frozen by liquid nitrogen, and are placed in an ultra-low temperature refrigerator at minus 80 ℃ for storage for later use;
2) taking the 20 spare liver tissues in a centrifuge tube, adding methanol with 10 times volume of frozen at-2 ℃, homogenizing at 12000rpm for 1min, centrifuging at 13000rpm for 4min at 4 ℃, taking supernate, drying the supernate with nitrogen at 40 ℃, redissolving with methanol water solution (the volume ratio of methanol to water is 4:1) to prepare a liver tissue sample, and finishing the preparation of the liver tissue sample within 12 min; mixing the supernatants, drying with nitrogen at 40 deg.C, and redissolving with methanol water solution to obtain quality control sample;
3) filtering the liver tissue samples and the quality control samples through a microporous filter membrane, then, entering an ultra-high performance liquid chromatography-mass spectrometer for analysis, during detection, firstly, 2 quality control samples are put in, then, 20 liver tissue samples are put in, one quality control sample is inserted between every 5 liver tissue samples, and after the liver tissue samples are put in, 2 quality control samples are put in. Wherein C in liquid chromatography18Combined with HILLIC column, C18The chromatographic conditions of the chromatographic column are as follows: flow rate: 0.3ml/min, sample tray temperature: 4 ℃, column temperature: at 40 ℃, mobile phase a was 2mmol/L ammonium formate and 0.05% formic acid in water, mobile phase B was acetonitrile, and mobile phase gradient elution is shown in the following table:
the chromatography conditions of the HILLIC chromatographic column are as follows: flow rate: 0.3ml/min, sample tray temperature: 4 ℃, column temperature: at the temperature of 45 ℃, the mobile phase C is 10mmol/L ammonium formate and 0.1% formic acid aqueous solution, the mobile phase D is acetonitrile aqueous solution, wherein the volume ratio of acetonitrile to water is 95:5, and the water contains 10mmol/L ammonium formate and 0.1% formic acid, the sample amount is as follows: 1 μ L, mobile phase gradient elution is shown in the following table:
the mass spectrum conditions are as follows: electrospray ionization (ESI) source positive and negative ion scan mode was used, spray voltage (IS): a positive ion of 5500V and a negative ion of-4500V; ionization Temperature (TEM): 550 ℃; atomizing gas (GS 1): 60 psi; auxiliary heating gas (GS 2): 60 psi; air curtain gas (CUR): 35 psi. The primary mass spectrum acquisition range is m/z 100-1250, and the accumulation time is 0.10 s; DP: 80V. And (2) acquiring a secondary mass spectrum by adopting an IDA mode, wherein the acquisition range is m/z 50-1250, the accumulation time is 0.05000s, and the DP: 80V; CE is 40 +/-20 eV. The IDA conversion criteria are: the signal intensity is more than 100cps, the molecular weight error is 50mDa, and isotopes are removed within 4 Da;
4) analyzing and processing black porgy liver metabonomics data collected by an ultra-high performance liquid chromatography-time-of-flight mass spectrometry combined technology, carrying out comprehensive non-targeted metabonomics analysis on the collected black porgy liver metabonomics data by using XCMSplus, finding and matching peaks by using a Centwave characteristic detection algorithm (the peak width is 5-20 s, and the mass tolerance is 5ppm) to find different biomarkers, automatically linking XCMS Plus software to a METLIN database (more than 24 ten thousand metabolite information, wherein 12127 metabolites have high-resolution secondary spectrums), combining an endogenous metabolite secondary spectrum library (comprising 550 common endogenous metabolic compounds comprising molecular formulas, molecular weights, CAS numbers, chemical names and structural diagrams), carrying out primary and secondary mass spectrometry information according to the accurate primary m/z, isotope abundance ratio and the secondary mass spectrometry information of the compounds in the spectrum library, and identifying the biomarkers with the differences, and finding out metabolic pathways by clustering analysis of the biomarkers.
Example 2:
the difference from the example 1 is that in the step 1), 0.5mg Se/kg of selenoamino polysaccharide is added into the common feed in the experimental group, and the feed is fed for 10 weeks; step 2) taking the 20 spare liver tissues into a centrifuge tube, adding methanol with 15 times volume frozen at minus 10 ℃, homogenizing for 0.5min at 10000rpm, centrifuging for 5min at 12000rpm at 4 ℃, taking supernatant, drying by nitrogen at 30 ℃, redissolving by using methanol water solution (the volume ratio of methanol to water is 4:1) to prepare a liver tissue sample, and finishing the preparation of the liver tissue sample within 12 min; and (3) uniformly mixing the supernatants in the preparation of the liver tissue samples, drying the mixed supernatants at 50 ℃ by using nitrogen, and redissolving the mixed supernatants by using a methanol aqueous solution to prepare a quality control sample.
Example 3:
the difference from the example 1 is that in the step 1), 0.7mg Se/kg of seleno-amino polysaccharide is added into the common feed in the experimental group, and the feed is fed for 9 weeks; step 2) taking the 20 spare liver tissues into a centrifuge tube, adding methanol with 5 times volume frozen at minus 30 ℃, homogenizing for 0.5min at 14000rpm, centrifuging for 3min at 14000rpm at 4 ℃, taking supernate, drying the supernate with nitrogen at 50 ℃, redissolving with methanol water solution (the volume ratio of methanol to water is 4:1) to prepare a liver tissue sample, and finishing the preparation of the liver tissue sample within 12 min; and (3) uniformly mixing the supernatants in the preparation of the liver tissue samples, drying the mixed supernatants at the temperature of 30 ℃ by using nitrogen, and redissolving the mixed supernatants by using a methanol aqueous solution to prepare a quality control sample.
Before preprocessing the analysis data, the quality and the effectiveness of the obtained analysis data are firstly verified. The total ion current chromatogram of the quality control sample in different ion scanning modes is shown in figure 1, and data validity verification results show that RSD of representative ion chromatogram peak intensities distributed in different retention times in the quality control sample is less than 5%, tR drift is less than 0.1min, m/z fluctuation range is not more than 5ppm, and system stability is good.
The total ion current chromatograms of the blank group and the experimental group are shown in fig. 2, and it can be seen from the graphs that the small molecule metabolites of the samples of the blank group and the experimental group have certain differences.
The metabonomics data of the black porgy livers of the experimental group and the blank group under the positive and negative ion scanning mode are processed by XCMSplusSW software analyzes the mass spectrum collected data, the given outline analysis result of the major components of the liver metabolites of the black porgy is shown in figure 3, and the HILIC column and the C column of the experimental group are shown to be compared with the blank group18The liver metabolites of the black porgy are obviously changed in the positive and negative ion scanning mode.
The identification method of the potential biomarker comprises the steps of determining relative molecular weight through primary mass spectrum information and obtaining structural fragment information of the potential biomarker by utilizing secondary mass spectrum information. Will be described in example C18The mass spectrum detection results of the chromatographic column and the HILIC chromatographic column in the positive and negative ion modes are obtained by searching Metabolite HR MS2 library database (SCIEX), 32 different biomarkers are accurately identified, and compared with a blank group, the levels of the metabolic biomarkers of the seleno-glycosaminoglycan addition group are increased by 26, and the levels of the metabolic biomarkers of the seleno-glycosaminoglycan addition group are reduced by 6. The specific results are shown in table 1 below.
Table 1: differential biomarkers for non-targeted metabolomic analysis.
According to the accurately identified potential biomarkers, selecting fish as a model by using a MetabioAnalyst 3.0 website, and carrying out related metabolic pathway analysis; the analysis result shows that 32 potential biomarkers with significant differences mainly point to 7 metabolic pathways (P <0.05) covering aminoacyl-tRNA biosynthesis, amino acid metabolism, nucleotide metabolism, nitrogen metabolism and the like, and the specific characteristics are shown in the following table 2.
Table 2: and analyzing the metabolic pathway.
Amino acids are basic structural substances constituting the immune system of the body, and have close relation with the formation of the immune system, the development of organs and the exertion of functions. According to metabolic pathway analysis, the experimental group has more significant differences in amino acid synthesis or metabolism, aminoacyl-tRNA biosynthesis, purine metabolism and nitrogen metabolism compared with the blank group, and the results of the potential biomarkers (Table 1) show that 18 potential biomarkers are involved in 11 amino acid biosynthesis or metabolism pathways, wherein the concentration level of 13 of the potential biomarkers is significantly up-regulated after the seleno-glycosaminoglycan is administered. In the arginine and proline metabolic pathways, the content of arginine in the experimental group was in a downward trend compared to the blank group. Arginine is one of essential amino acids of fish, and arginine and its metabolite Nitric Oxide (NO), ornithine and citrulline can play a role in enhancing phagocytosis and sterilization of cells and promoting immune regulation such as synthesis of immunoglobulin through endocrine hormones such as growth hormone. Arginine is involved in immune regulation in the body mainly through 2 metabolic pathways, the first metabolic pathway is the arginase pathway, i.e. arginine is converted into ornithine and further polyamine is generated, and ornithine and polyamine play an important role in immune regulation of animals; the other way is the way of metabolizing arginine into NO, the NO not only plays an important role in exerting phagocytic effect on macrophages, but also has an influence on connection and activation of the macrophages and lymphocytes, and is beneficial to improving respiratory burst activity, phagocytic effect and lysozyme activity of immune cells.
Glutamine can promote mitosis and differentiation and proliferation of macrophage and lymphocyte, increase production of inflammatory factor, and increase organism immunity; branched chain amino acids such as valine, leucine, and isoleucine can stimulate proliferation of monocytes, regulate secretion of cytokines, and enhance immune response. The contents of glutamine, threonine, valine, leucine, isoleucine and the like in the experimental group are higher than those in the blank group, and the result shows that the contents of the glutamine, the threonine, the valine, the leucine, the isoleucine and the like in the experimental group have a certain enhancement effect on the influence of amino acid on the immunoregulation.
Claims (7)
1. A construction method of a black porgy immune mechanism enhancement model by selenylation aminopolysaccharide based on liver metabonomics research is characterized by comprising the following steps:
1) dividing the young black porgy fish used for animal experiment into experiment group and blank group;
2) feeding the experimental group and the blank group with the same feed, and adding the selenized aminopolysaccharide into the feed of the experimental group;
3) after feeding is finished, carrying out starvation treatment on the juvenile sea breams in the experimental group and the blank group, carrying out anesthesia by using an anesthetic, taking out liver tissues, and storing for later use;
4) preparation of liver tissue samples and quality control samples, followed by use of ultra performance liquid chromatography-TripleTOFTM5600+ combined technology, and inserting quality control sample between liver tissue samples during sample introduction; using ultra performance liquid chromatography-TripleTOFTM5600+ combination technique is as follows: passing the liver tissue sample through a microwellAfter the filtration of the filter membrane, the mixture enters an ultra-high performance liquid chromatography-mass spectrometer for analysis under the fixed chromatographic condition and the mass spectrum condition, wherein C in the liquid chromatography18The chromatographic column is combined with the HILIC chromatographic column, and in the detection process, a quality control sample is inserted into every other liver tissue sample with the same quantity for detection and analysis so as to monitor the detection stability;
C18the chromatographic conditions of the chromatographic column are as follows: flow rate: 0.3ml/min, sample tray temperature: 4 ℃, column temperature: at 40 ℃, the mobile phase A is 2mmol/L ammonium formate and 0.05% formic acid aqueous solution, the mobile phase B is a mixed solution of acetonitrile and isophthalic acid, the volume ratio of the acetonitrile to the isophthalic acid is 1:1, and the sample injection amount is as follows: 2 μ L, mobile phase gradient elution is shown in the following table:
The chromatographic conditions of the HILIC chromatographic column are as follows: flow rate: 0.3ml/min, sample tray temperature: 4 ℃, column temperature: at the temperature of 45 ℃, the mobile phase C is 10mmol/L ammonium formate and 0.1% formic acid aqueous solution, the mobile phase D is acetonitrile aqueous solution, wherein the volume ratio of acetonitrile to water is 95:5, and the water contains 10mmol/L ammonium formate and 0.1% formic acid, the sample amount is as follows: 1 μ L, mobile phase gradient elution is shown in the following table:
5) For ultra-high performance liquid chromatography-tripleTOFTM5600+ liver metabonomics data of black porgy collected by combined technology is analyzed and processed, liver metabonomics biomarkers related to enhancement of black porgy immune mechanism by selenoamino-polysaccharide are identified and screened, and metabolic pathways pointed by the liver metabonomics biomarkers are constructed and analyzed;
the selenized aminopolysaccharide has 32 liver metabonomics biomarkers for enhancing the immune mechanism of the black porgy, and the biomarkers are respectively as follows: l-histidine, dimethylglycine, 4-guanidinobutyric acid, L-valine, L-leucine, L-isoleucine, L-phenylalanine, L-tryptophan, succinic acid, adenine, adenosine, deoxyinosine, inosine, deoxyguanosine, guanosine, betaine, L-methionine, glyceric acid, L-proline, L-threonine, L-glutamine, L-serine, adenosine monophosphate, deoxyadenosine monophosphate, gamma-aminobutyric acid, L-glutamic acid, D-ribulose 5-phosphate, guanosine monophosphate, ornithine, L-arginine, creatine, arginosuccinic acid;
the biomarkers mainly point to 7 metabolic pathways, which are: aminoacyl-tRNA biosynthesis, valine, leucine and isoleucine biosynthesis, arginine and proline metabolism, purine metabolism, nitrogen metabolism, glycine, serine and threonine metabolism, alanine, aspartate and glutamate metabolism.
2. The method for constructing the black porgy immune mechanism enhancing model based on the liver metabonomics research selenoamidopolysaccharide of claim 1, wherein the method for processing the black porgy juvenile fish in the step 1) comprises the following steps: stopping feeding the black porgy juvenile fish, and then placing the black porgy juvenile fish into a glass fiber cylinder for microflow feeding;
the specific feeding method of the experimental group and the blank group in the step 2) comprises the following steps: feeding the experimental group and the blank group by adopting common feed, adding 0.5-0.7 mg Se/kg of seleno-amino polysaccharide into the common feed of the experimental group, and continuously feeding for 8-10 weeks;
the specific method for treating the juvenile Sparus macrocephalus after feeding in step 3) comprises the following steps: after starving the juvenile sea breams of the experimental group and the blank group for 20-30h, anesthetizing by using anesthetic, taking out liver tissues, weighing parallel samples with the same quantity in the experimental group and the blank group, quickly freezing by using liquid nitrogen, and finally storing in an ultralow-temperature refrigerator at the temperature of-80 ℃ for later use;
the preparation method of the liver tissue sample in the step 4) comprises the following steps: placing the prepared liver tissue in a centrifuge tube, adding 5-15 times volume of methanol frozen at-10 to-30 ℃, homogenizing at 10000-; the quality control sample preparation method comprises the following steps: mixing the supernatants, drying with nitrogen at 30-50 deg.C, and re-dissolving with methanol water solution;
the specific method for analyzing and processing the liver metabonomics data of the black porgy in the step 5) comprises the following steps: using XCMSplusThe method comprises the steps of carrying out comprehensive non-targeted metabonomic analysis on collected liver metabonomics data of the black porgy, finding and matching peaks by adopting a Centwave characteristic detection algorithm to find different biomarkers, automatically linking XCMS Plus software to a METLIN data spectral library, combining an endogenous metabolite secondary spectral library, identifying the different biomarkers according to the accurate primary m/z and the primary and secondary mass spectrum information of the isotope abundance ratio of compounds in the spectral library, and finding a metabolic pathway by clustering analysis on the biomarkers.
3. The method for constructing the black porgy immune mechanism enhancing model based on liver metabonomics research selenoamidopolysaccharide according to claim 2, wherein the mass spectrum conditions are as follows: adopting an electrospray ionization source positive and negative ion scanning mode, spraying voltage: a positive ion of 5500V and a negative ion of-4500V; ionization temperature: 550 ℃; atomizing: 60 psi; auxiliary heating gas: 60 psi; air curtain air: 35 psi; the primary mass spectrum acquisition range is m/z 100-1250, and the accumulation time is 0.10 s; DP: 80V; and (2) acquiring a secondary mass spectrum by adopting an IDA mode, wherein the acquisition range is m/z 50-1250, the accumulation time is 0.05000s, and the DP: 80V; CE is 40 +/-20 eV; the IDA conversion criteria are: the signal intensity is more than 100cps, the molecular weight error is 50mDa, and isotopes are excluded within 4 Da.
4. The method for constructing the black porgy immune mechanism enhancing model based on the liver metabonomics research selenoamido polysaccharide of claim 1 or 2, wherein the black porgy juvenile fish used for the animal experiment in the step 1) has healthy body and uniform size, and the initial weight is 12.8-13.2 g.
5. The method for constructing the model for enhancing the immune mechanism of black porgy by using the amino polysaccharide selenide based on liver metabonomics research according to claim 1 or 2, wherein the selenium content of the amino polysaccharide selenide is 27.3 mg/g.
6. The method for constructing the model for enhancing the immune mechanism of black porgy by using the selenized aminopolysaccharide based on liver metabonomics research according to claim 1 or 2, wherein the anesthetic is MS-222 and the concentration is 60 mg/L.
7. The method for constructing the model for researching immune mechanism enhancement of the sparus macrocephalus by the amino selenide polysaccharides based on liver metabonomics according to claim 2, wherein the volume ratio of methanol to water in the methanol aqueous solution is 4: 1.
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