CN109813814B - Application of phenylalanine metabolism change in evaluation of cerebral ischemia/reperfusion injury degree - Google Patents
Application of phenylalanine metabolism change in evaluation of cerebral ischemia/reperfusion injury degree Download PDFInfo
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Abstract
The invention discloses application of phenylalanine metabolism change in evaluation of cerebral ischemia/reperfusion injury degree, which comprises the following steps: and detecting the content of phenylalanine in the organism or cell by adopting a liquid chromatography-mass spectrometry technology, and evaluating the change of the cerebral ischemia/reperfusion injury degree under the intervention measure according to the content change of the phenylalanine before and after the administration of the medicine or other intervention measures. The method provided by the invention can be used for visually judging the change of the cerebral ischemia/reperfusion injury degree through the change condition of the phenylalanine content in the sample, is convenient and quick, can be used for effectively supplementing and supporting the evaluation of the existing cerebral ischemia/reperfusion injury degree, and provides a new reference for the effect evaluation of medicines or other intervention measures and the efficacy evaluation in new medicine development.
Description
Technical Field
The invention belongs to the field of neurology medicine, and relates to a method for detecting content change of phenylalanine in samples such as peripheral blood and the like by using a liquid chromatography-tandem mass spectrometry (LC-MS/MS method) for evaluating change of cerebral ischemia/reperfusion injury degree in a drug test.
Background
Stroke medically refers to the death of brain cells due to poor blood flow. Cerebral apoplexy is mainly divided into ischemic (due to insufficient blood supply) andbleeding (due to hemorrhage) is of two types. Occlusion and stenosis of the internal carotid and vertebral arteries are both likely to cause ischemic stroke, leading to partial loss of brain function, and many men suffer. Statistically, about 4240 million patients with a history of stroke globally survive. The number of stroke patients in developed countries has declined at a rate of approximately 10% per year between 1990 and 2010, while in developing countries it has increased at a rate of approximately 10% per year. In 2016, the number of dead patients with stroke reaches 630 ten thousand, which accounts for 11 percent of the total death. About half of stroke patients have a survival time of less than 1 year. About 150-200 ten thousand new cases of cerebral apoplexy occur in China every year, wherein about 70 percent of cases are ischemic cerebral apoplexy and become the third leading cause of death and the first disabling cause of malignant tumors and cardiovascular diseases in China. The main reason of cerebral ischemic stroke is blood vessel blockage or stenosis, resulting in local cerebral tissue blood supply deficiency; hemorrhagic stroke is caused by direct blood entering into brain tissue or meningeal space due to rupture of cerebral aneurysm. The Chinese medical guidance for treating ischemic stroke in the acute stage published by Wei Ji Wei of China in 2017 indicates that the treatment of acute ischemic stroke emphasizes early diagnosis, early treatment, early rehabilitation and early prevention of recurrence. Thrombolytic therapy is the most important blood flow restoration measure at present, and recombinant tissue plasminogen activator (rtPA) and urokinase are the main thrombolytic drugs used in China at present. However, a great deal of research shows that rapid cerebral ischemia-followed blood flow reperfusion easily induces activation of a large number of inflammatory cells and release of inflammatory factors in local ischemic foci, promotes increase of Reactive Oxygen Species (ROS) level in nerve cells and apoptosis, and finally leads to ischemia/reperfusion (I/R) damage of nerve cells. The mechanism of I/R injury is abnormally complex, and toxic reaction and intracellular Ca of excitable amino acids such as mitochondria, ROS injury, glutamic acid and the like are involved2+Overload, inflammatory responses, apoptosis, and autophagy.
Metabolomics (metabolomics) is an emerging discipline for the qualitative and quantitative analysis of all metabolites contained within an individual, tissue or cell. At present, metabonomics and various omics technologies such as genomics, transcriptomics and proteomics form an omics research technology platform of system biology. In recent years, with the maturity of detection technology platforms such as mass spectrometry and nuclear magnetic resonance, metabonomics gradually permeates into various fields, and the application in the process of cerebral ischemia injury is increasingly deep. In recent years, multiple laboratories have successively reported that there is a dramatic change in the metabolic state of cells during cerebral ischemic injury, and that metabolic markers have been found that vary significantly. These endogenous metabolites are mainly associated with energy metabolism, amino acid metabolism and lipid metabolism: (1) amino acids: including Excitatory Amino Acids (EAAs), branched chain amino acids, taurine, glycine, glutamine, homocysteine, gamma-aminobutyric acid, and the like; (2) energy metabolites: including glucose, lactic acid, pyruvic acid, ketone bodies, alpha-ketoglutaric acid, and the like; (3) and others: arachidonic acid and its metabolites, 5-HT, NO, catecholamines, glycerol, inositol, etc. Among them, energy metabolites and excitatory amino acids are currently the most widely used indicators for detection of endogenous metabolites.
In the evaluation of the severity of the disease, except for using a rating scale, the Chinese guideline 2018 for acute ischemic stroke treatment recommends that the examination of the brain lesion uses flat scan CT (first choice), multi-mode CT, conventional MRI, multi-mode MRI and the like. Carotid ultrasound, transcranial doppler (TCD), magnetic resonance cerebrovascular angiography (MRA), High Resolution Magnetic Resonance Imaging (HRMRI), CT angiography (CTA), Digital Subtraction Angiography (DSA), and the like are commonly used for vascular disease examination. However, these checks also exist: the iodine contrast agent needs to be injected, the cost is high, the examination time is long, the patient has limitations such as contraindications, and the like, and the operation technical level is influenced. AHA/ASA does not recommend that MRI examination is routinely performed to investigate intracranial microhemorrhage before intravenous thrombolysis treatment, and does not recommend that perfusion examination is applied to ischemic stroke patients within 6h of onset to select patients suitable for mechanical thrombus removal. In addition to the above evaluation measures, the current auxiliary measures generally adopted in the drug test for the evaluation of brain injury often require sampling from tissues. It is not only, obviously, complicated and time consuming, but also not widely applicable for clinical trials of drugs for human use.
Safflower (Carthamus tinctorius) belongs to tubular flowers of Compositae plants, is pungent and warm in nature, has the effects of activating blood circulation and removing blood stasis, is widely applied to clinical treatment at present, and has a very considerable development prospect. Safflower Yellow (SY) is a crude extract containing Hydroxysafflor yellow a (HSYA), which is the most effective water-soluble component in the pharmacological efficacy of safflower. A large number of studies have proved that HSYA has definite protective effect on brain and myocardial I/R injury, and can inhibit apoptosis, reduce ROS level, help repair damaged neurons, and promote cell survival. In addition, the preparation also has the functions of inhibiting lipid peroxidation, calcium ion overload and the like, and is beneficial to protecting cell mitochondria. Research shows that HSYA can obviously inhibit nuclear translocation of NF-kB key molecule p65 and infiltration of neutrophils, reduce the content of angiotensin II in blood plasma, and inhibit inflammatory reaction induced by hemagglutinase. At the same time, HSYA may also increase rat brain natriuretic peptide BNP levels by inhibiting excessive uptake of calcium ions by nerve cells, which may be associated with HSYA's function in reducing brain edema. HSYA can also significantly inhibit platelet aggregation, increase the number of vascular endothelial cells, adhesion ability and activity of cell mitochondria, and inhibit the generation of vascular thrombosis. The neuroprotective effects and mechanisms of HSYA are essentially clear.
Disclosure of Invention
The invention aims to provide application of phenylalanine metabolism change in evaluation of cerebral ischemia/reperfusion injury degree.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for detecting phenylalanine metabolism in organisms and cells, comprising the steps of:
1) sampling the organism or organism cell culture by adopting a liquid chromatography-mass spectrometry technology and detecting a sample (obtained by sampling) to obtain the content of phenylalanine in the sample of the organism or organism cell culture;
2) after step 1), administering a pharmaceutical treatment or other intervention to the same organism or to the same organism; alternatively, a drug treatment or other intervention is administered to a cell culture of the same organism or a cell culture of the same organism;
3) sampling and detecting a sample of the organism or the organism cell culture subjected to the intervention measure by adopting a liquid chromatography-mass spectrometry technology to obtain the content of phenylalanine in the sample of the organism or the organism cell culture;
4) comparing the content of phenylalanine in the samples obtained in the step 1) and the step 3), and obtaining the change condition of phenylalanine in the organism or organism cell culture in the metabolic process under the intervention measure according to the comparison result, thereby evaluating the effect of the intervention measure on the degree of cerebral ischemia/reperfusion injury (if the content of phenylalanine is reduced, the degree of cerebral ischemia/reperfusion injury is reduced).
Preferably, the LC-MS technique is selected from LC-tandem mass spectrometry.
Preferably, the sample is selected from brain tissue, peripheral blood or neuronal cells, PC12 cells and other related cells.
Preferably, the sample is subjected to metabolite extraction, phenylalanine internal standard substance addition and derivatization before detection, and then is dispersed in a mobile phase to prepare a sample loading detection sample for liquid chromatography-tandem mass spectrometry.
Preferably, the extraction specifically comprises the following steps: grinding brain tissue or cells, adding an extracting agent, grinding, uniformly mixing and centrifuging, and taking supernate; or mixing peripheral blood (blood filter paper sheet) with the extractant, standing, and collecting supernatant.
Preferably, the extractant is selected from one or more of a mixed solution of ethyl acetate and ethanol, anhydrous methanol, a methanol aqueous solution, and a mixed solution of dichloromethane and methanol, and for brain tissues or cells, the four extractants are used in sequence after grinding, so that metabolites such as phenylalanine in a sample are extracted to the maximum extent, and an accurate content analysis result of phenylalanine in the sample is obtained by combining the accurate and sensitive characteristics of a liquid chromatography-tandem mass spectrometry.
Preferably, the intervention is selected from drug treatment.
Preferably, the drug is selected from safflower yellow or hydroxysafflower yellow A.
Preferably, in the step 1), the organism is selected from an animal brain ischemia/reperfusion injury model, and the organism cell culture is selected from a cell oxygen sugar deprivation reoxygenation injury model.
A kit for detecting phenylalanine metabolism in organisms and cells comprises the extracting agent and a phenylalanine internal standard substance for liquid chromatography-tandem mass spectrometry.
The method for detecting phenylalanine metabolism in organisms and cells is applied to the evaluation of the effect of intervention measures based on the degree of cerebral ischemia/reperfusion injury (for example, in cell tests, animal tests or clinical tests of medicines).
The invention has the beneficial effects that:
the method detects the phenylalanine content in samples such as peripheral blood or other tissues and cells by a liquid chromatography-mass spectrometry technology, is convenient and quick, is not influenced by the physiological state of a detected object, does not need to inject exogenous substances such as an iodophor and the like, visually judges the change of the cerebral ischemia/reperfusion injury degree by the change condition of the phenylalanine content in the tissues or the cells, can effectively supplement and support the evaluation of the existing cerebral ischemia/reperfusion injury degree, and provides a new reference for the effect evaluation of clinical treatment medicines or other intervention measures for the cerebral ischemia/reperfusion injury and the efficacy evaluation in the development of new medicines.
Furthermore, the method is simple and effective in pretreatment of different biological samples such as brain tissues, peripheral blood, neuron cells, PC12 cells and the like, can be used for quickly, accurately and reliably detecting the content of phenylalanine by liquid chromatography-tandem mass spectrometry, and provides a convenient way for evaluating the change of the degree of cerebral ischemia/reperfusion injury by combining with the analysis of phenylalanine metabolic change.
Drawings
FIG. 1 is a statistical chart of the phenylalanine metabolism changes in the brain tissues of mice in the control group (Sham) and the I/R injury group (cerebral ischemia-reperfusion group).
FIG. 2 is a statistical graph showing the metabolic changes of phenylalanine in peripheral blood of mice in the control group (Sham) and the I/R injury group (cerebral ischemia-reperfusion group).
FIG. 3 is a statistical graph showing the change in phenylalanine metabolism in neuronal cells of mice in the control group (untreated group) and the OGD/R group (oxygen sugar deprived reoxygenation group).
FIG. 4 is a statistical graph showing the metabolic changes of phenylalanine in PC12 cells of rats in the control group (untreated group) and the OGD/R group (oxygen sugar deprived reoxygenation group).
FIG. 5 is a schematic diagram of the phenylalanine metabolic pathway.
FIG. 6 is a statistical chart showing RT-qPCR detection of the expression differences of the key enzymes Pah (phenylalanine hydroxylase), Got1 (aspartate aminotransferase) and Tat (tyrosine aminotransferase) for phenylalanine metabolism in brain tissues of mice in the control group (Sham) and I/R injury groups.
FIG. 7 is a statistical graph of RT-qPCR detection of expression differences of phenylalanine metabolism key enzymes Pah, Got1 and Tat in control group (untreated group) and OGD/R group mouse neuronal cells.
FIG. 8 is a statistical graph of RT-qPCR detection of expression differences of phenylalanine metabolism key enzymes Pah, Got1 and Tat in control group (untreated group) and OGD/R group rat PC12 cells.
Detailed Description
The present invention will be described in further detail with reference to the following drawings and examples, which are illustrative of the present invention and are not intended to limit the scope of the present invention.
LC-MS/MS method for detecting metabolite content in brain tissue, peripheral blood and cells
1. Reagent and apparatus
1.1 Primary reagents
Amino acid Isotope internal standards (Cambridge Isotope Labs);
ethyl acetate, ethanol, anhydrous methanol, dichloromethane, formic acid, methanol (chromatography), n-butanol, acetonitrile (chromatography), acetyl chloride (all available from Shanghai pharmaceutical group chemical Co., Ltd.).
1.2 Main instruments
2. Preparation of amino acid isotope internal standard
There are 12 kinds of amino acid isotope internal labels, respectively15N,2-13C-Glycine (N)1,C1-Gly)、2H4-alanine (D)4-Ala)、2H8-valine (D)8-Val)、2H3-leucine (D)3-Leu)、2H3Methionine (D)3-Met)、2H5-phenylalanine (D)5-Phe)、13C6-tyrosine (C)6-Tyr)、2H3-aspartic acid (D)3-Asp)、2H3-glutamic acid (D)3-Glu)、2H2-ornithine (D)2-Orn)、2H2Citrulline (D2-Cit),2H4,13C-arginine (D)4Cl-Arg). All internal standards were diluted with methanol as working solution at a concentration of 2.5. mu. mol/L, except Gly at 12.5. mu. mol/L.
3. Sample collection
Brain tissue samples were dissected from C57BL/6 mice; peripheral blood samples were collected from rat tails of C57BL/6 mice; PC12 cells were purchased from the Shanghai culture Collection cell Bank of China academy of sciences.
LC-MS/MS working conditions
4.1 working conditions of liquid chromatography
The mobile phase adopts 80% acetonitrile, and the speed of the quaternary pump is changed: 140 μ L/min, 0.3min → 30 μ L/min, 1.2min → 300 μ L/min, 0.4 min. The autosampler is set to sample 20 μ L each time.
4.2 Mass Spectrometry (MS/MS) working conditions
Ion source temperature (Probe temperature): 350 ℃; dwell time per transition (Dwell time): 200 ms; nebulizer (Nebulizer, GAS 1): 30 psi; heater GAS (GAS 2): 40 psi; air Curtain gas (curtaingas): 25 psi; ion source voltage (Ion spray voltage): 5500V.
The mass spectrometer adopts positive ion scanning, 2 different scanning methods are used for amino acid detection, multiple reaction detection is used for glycine (Gly), ornithine (Orn), arginine (Arg) and citrulline (Cit), and a neutral loss scanning mode is used for other amino acid detection.
5. Phenylalanine content detection step
5.1 detection method of phenylalanine content in brain tissue of C57BL/6 mouse
A. Grinding the tissues: about 50mg of the tissue was taken, and 200. mu.L of ultrapure water was added thereto and ground in a grinder at a frequency of 40Hz for 5 min.
B. Step-by-step extraction: 400 mu L of mixed solution of ethyl acetate and ethanol (1:1, v/v), 200 mu L of anhydrous methanol, 200 mu L of mixed solution of anhydrous methanol and water (3:1, v/v), and 200 mu L of mixed solution of dichloromethane and methanol (3:1, v/v); grinding and mixing in a grinder for 2min after adding one solution, placing the ground sample in a low-temperature centrifuge, centrifuging at 12000rpm for 5min at 4 deg.C, sucking supernatant, operating on ice, and extracting the rest.
C. Nitrogen blowing: after the supernatant of the sample is mixed evenly, 1mL of the mixture is sucked out of an EP tube, and the mixture is dried at normal temperature by a nitrogen blowing instrument.
D. Preparing an internal standard solution: oscillating the isotope internal standard working solution stored in a refrigerator at 4 ℃ for 15min at room temperature, adding 100 mu L of isotope internal standard working solution into the EP tube, and standing for 1h at room temperature; blowing at 65 ℃ for about 15min with nitrogen.
E. Derivatization: adding 50 μ L derivatization agent (acetyl chloride-n-butanol hydrochloride, 9:1, V/V) into the EP tube, and placing in a thermostat at 65 deg.C for derivatization for 20 min; blowing the mixture at 65 ℃ for about 20min by using nitrogen.
F. Dissolving: and adding 150 mu L of mobile phase solution (acetonitrile-water mixed solution, 8:2, V/V) into the EP tube, and standing at room temperature for 10min to obtain a sample for loading detection.
G. Analyzing the sample by liquid chromatography-tandem mass spectrometry to obtain the content of phenylalanine in the tissue sample.
Method for detecting content of phenylalanine in 5.2C57BL/6 mouse peripheral blood sample (blood filter paper sheet)
A. Dry blood filter paper sheet: the model of the filter paper sheet is 903#, the tail vein blood of the mouse is dripped on the filter paper sheet, the diameter of the filter paper sheet is about 10mm, and the filter paper sheet is dried in the air at room temperature and stored in a refrigerator at the temperature of-20 ℃ to be tested.
B. Sampling: a piece of dry blood filter paper was punched into a 3mm circular blood piece (equivalent to 3.2. mu.L of a plasma sample) with a punch and placed in a 96-well plate;
C. and (3) extraction: oscillating isotope internal standard working solution stored in a refrigerator at 4 ℃ for 15min at room temperature, adding 100 mu L of isotope internal standard working solution into a 96-well plate for placing blood slices, standing for 1h at room temperature, and placing supernatant into a new 96-well plate;
D. nitrogen blowing: drying with nitrogen at 65 ℃ for about 15 min;
E. derivatization: adding 50 μ L derivatization agent (acetyl chloride-n-butanol hydrochloride, 9:1, V/V), and placing in a thermostat at 65 deg.C for derivatization for 20 min;
F. nitrogen blowing: drying with nitrogen at 65 ℃ for about 20 min;
G. dissolving: adding 150 μ L mobile phase solution (acetonitrile-water mixture, 8:2, V/V), standing at room temperature for 10min to obtain sample.
H. Analyzing the sample by liquid chromatography-tandem mass spectrometry to obtain the content of phenylalanine in the tissue sample.
5.3 detection of phenylalanine content in mouse Primary culture neuronal cells and rat PC12 cells
Method (same as 5.1).
6. Data processing
And analyzing and calculating the data by SPSS15.0 software, and substituting the data into a standard curve according to the ratio of the metabolite to the corresponding kurtosis by adopting an internal standard curve method to calculate so as to obtain the concentration of phenylalanine in the sample.
Metabolic markers for (di) brain I/R injury
(1) And carrying out metabonomic analysis on brain tissues and peripheral blood of a C57BL/6 mouse sham operation group and an I/R injury group, and screening metabolic markers. (2) And carrying out metabonomic analysis on C57BL/6 mouse neuron cells, a control group of rat PC12 cells and an OGD/R group, and verifying the change trend of phenylalanine. (3) Respectively extracting mouse brain tissue, neuron cells and PC12 cell RNA, carrying out reverse transcription and PCR analysis, determining the change of expression of phenylalanine metabolism key enzyme under the conditions of I/R injury and OGD/R, and further carrying out laboratory verification on the expression of metabolic marker related enzyme obtained by screening. The specific steps and results are as follows.
1. Metabolites with significantly varying levels in brain I/R injury were screened.
1.1 design of the experiment
6 pairs of I/R injured brain tissue samples from C57BL/6 mice and tissue samples from the sham operated group (control group) were collected and frozen at-80 ℃. Tissue samples were submitted for metabolomics testing of the samples (including the LC-MS/MS method described above).
1.2 preliminary processing of data
A total of 79 metabolites were detected in brain tissue, and 6 amino acid metabolites were preliminarily selected by data processing.
1.3 paired t test method
The metabolites were analyzed by t-test using SPSS18.0 statistical software, wherein the content of 5 amino acid metabolites was significantly increased and the content of 1 amino acid metabolite was significantly decreased, wherein the phenylalanine change trend was more pronounced, and the results are shown in fig. 1, i.e., the phenylalanine content in brain tissue was increased after the mouse cerebral ischemia/reperfusion injury. Also, with reference to figure 2, phenylalanine levels in plasma increased following cerebral ischemia/reperfusion injury in mice. Therefore, the content of phenylalanine in the brain tissue and the peripheral blood of the mice in the I/R injury group is increased.
2. Verification experiment
The OGD/R experiment is carried out by using neuron cells and PC12 cells, cerebral ischemia/reperfusion injury is simulated in vitro, cell samples are collected and then sent for detection, metabonomics detection is carried out on the content change of metabolites in the samples (see the LC-MS/MS method), and the early-stage experiment result is verified. As shown in fig. 3, phenylalanine content increased following oxygen sugar deprivation reoxygenation of primary cultured neurons in mice. As shown in FIG. 4, the phenylalanine content increased after oxygen deprivation reoxygenation of rat PC12 cells.
3. The increase of the content of phenylalanine is related to the change of regulating the expression of key enzyme genes in metabolic pathways.
3.1 phenylalanine Metabolic pathway
From FIG. 5, it is understood that the key metabolic enzymes in the phenylalanine metabolic pathway are Pah, Got1 and Tat.
3.2Pah, Got1, Tat primer design
TABLE 1 PCR reaction primers for key enzymes of mouse cell metabolism
Note: the primer design completion time is 2017, 5 months, and GAPDH is an internal reference
TABLE 2 PCR reaction primers for key enzymes of rat cell metabolism
Note: the primer design completion time is 2017, 9 months, and GAPDH is an internal reference
3.3RNA extraction and content analysis
A. Placing 50mg of mouse brain tissue into a homogenizer, adding 1mL of TRIzol reagent, and repeatedly homogenizing on ice;
B. transferring the TRIzol lysate of the tissue or the cell into a 1.5mL EP tube, adding 0.2mL of chloroform, fully and uniformly mixing, and standing for 5min at room temperature;
C. centrifuging the sample at 12000rpm at 4 deg.C for 10 min;
D. placing the upper water phase in a new 1.5mL EP tube, adding 0.5mL isopropanol, mixing, standing at room temperature for 10min, and centrifuging at 4 deg.C at 12000rpm for 10 min;
E. the supernatant was aspirated and RNA precipitation was visualized;
F. adding 1mL of 75% ethanol for washing, centrifuging at 4 ℃ and 12000rpm for 5min, and removing supernatant; repeating the steps once;
G. naturally drying; according to the RNA amount, 30-50 mu L of RNase-free DEPC water is used for dissolving the RNA precipitate;
H. RNA quantification was performed using a NanoDrop-2000 nucleic acid analyzer and RNA purity was analyzed according to OD260nm/280nm ratio, with the remaining samples frozen at-80 ℃.
3.4cDNA reverse transcription
1. mu.g of RNA was used for reverse transcription according to the instructions of the cDNA reverse transcription kit.
3.5RT-qPCR
And (3) PCR reaction conditions:
(1) template denaturation: 5min at 95 ℃;
(2) and (3) amplification of a product: at 95 ℃ for 15s, at 56 ℃ for 30s, at 72 ℃ for 1min, for 40 cycles;
(3) product extension: 10min at 72 ℃.
The RNA of each group of mouse brain tissues is utilized to carry out reverse transcription and PCR reaction, the content difference of mRNA of key metabolic enzyme of amino acid metabolism is analyzed in a comparison mode, and the result is shown in figure 6, which indicates that the expression of Pah and Got1 is reduced and the expression of Tat is increased in the brain tissues of the I/R injury group.
The RNA extraction method of each group of cells is the same as the above, reverse transcription and PCR reaction are carried out, the content difference of key metabolic enzyme mRNA of amino acid metabolism is analyzed in a contrast way, and the result is shown in figure 7 and figure 8 and is highly consistent with the detection result of brain tissue.
The experimental results show that: the Pah and Got1 expression of the I/R injury group and the OGD/R group is down-regulated, the Tat expression is up-regulated, and the phenylalanine content is increased. Based on the change of the content of the phenylalanine in the peripheral blood, the change of the content of the phenylalanine in the peripheral blood can be used as a new index for evaluating the change of the I/R injury, and convenience is provided for evaluating the I/R injury degree of the brain.
Pharmacodynamic mechanism analysis of neuroprotective effect of (III) safflower yellow or hydroxysafflor yellow A
(1) Metabonomics analysis (see the LC-MS/MS method) is carried out on brain tissues and peripheral blood of a C57BL/6 mouse sham operation group, an I/R injury group, a 5mg/kg SY administration group and a 20mg/kg SY administration group, and the content of phenylalanine in the brain tissues and the peripheral blood of mice in the I/R injury group is increased, but the content of phenylalanine in the SY intervention group is obviously reduced. (2) Metabonomic analysis (see the LC-MS/MS method) is carried out on C57BL/6 mouse neuron cells, a PC12 cell control group, an OGD/R group and a 1 mu M and 10 mu M HSYA administration group, and the change trend of phenylalanine is verified. As a result, the trend of phenylalanine was found to be consistent with that of the in vivo experimental group. (3) Respectively extracting RNA of brain tissues, neuron cells and PC12 cells, carrying out reverse transcription and PCR analysis, definitely changing the expression of phenylalanine metabolism key enzyme under the conditions of I/R injury and OGD/R, and further carrying out laboratory verification on the expression of metabolic marker (phenylalanine) related enzyme. The experimental results show that: the Pah and Got1 expression of the I/R injury group and the OGD/R group is down-regulated, the Tat expression is up-regulated, and the phenylalanine content is increased. Under the intervention of drugs (SY or HSYA), the expression of Pah, Got1 and Tat is reversed, and the content of phenylalanine is reduced. Therefore, the SY and the HSYA can interfere the expression of key enzyme of phenylalanine metabolism, so that the phenylalanine content is reduced, and the neuroprotection effect is exerted.
In conclusion, the invention firstly carries out metabonomic detection on the brain tissue and the peripheral blood of the C57BL/6 mouse, and the phenylalanine is obviously increased under the I/R injury state. The significant increase of phenylalanine caused by OGD/R injury is further proved by metabonomics detection of mouse neuron cells and rat PC12 cells. Therefore, it is presumed that the phenylalanine metabolic pathway is closely related to I/R impairment. And further carrying out expression analysis on key metabolic enzymes of phenylalanine metabolic pathways, and combining with the intervention of the safflower active ingredients with definite neuroprotective effect, the result shows that the pathway of phenylalanine metabolism is blocked to cause the increase of the phenylalanine content in tissues and cells, and the fact that the phenylalanine change is an important reference for evaluating the I/R damage change is suggested.
<110> the fourth military medical university of the Chinese people liberation army
<120> application of phenylalanine metabolic change in evaluation of degree of cerebral ischemia/reperfusion injury
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Claims (5)
1. A method for detecting phenylalanine metabolism in organisms and cells, comprising: the method comprises the following steps:
1) sampling and detecting a sample of the organism or the organism cell culture by adopting a liquid chromatography-mass spectrometry technology to obtain the content of phenylalanine in the sample of the organism or the organism cell culture; wherein, the metabolite is extracted from the sample of the organism before detection by the following steps;
A. grinding the tissues: taking 50mg of tissues, adding 200 mu L of ultrapure water, and grinding for 5min in a grinder at the frequency of 40 Hz;
B. step-by-step extraction: 400 muL of mixed liquid of ethyl acetate and ethanol is mixed with the anhydrous methanol in a ratio of 1:1, v/v, 200 muL of mixed liquid of the anhydrous methanol and water in a ratio of 3:1, v/v, and 200 muL of mixed liquid of dichloromethane and methanol in a ratio of 3:1, v/v; grinding and uniformly mixing the solution in a grinder for 2min after each addition of one solution, placing the ground sample in a low-temperature centrifuge for centrifugation, and then sucking the supernatant;
C. nitrogen blowing: uniformly mixing the supernatant of the sample, sucking 1mL of the mixture out of an EP tube, and drying the mixture at normal temperature by using a nitrogen blowing instrument;
D. preparing an internal standard solution: oscillating isotope internal standard working solution stored in a refrigerator at 4 ℃ for 15min at room temperature, adding 100 mu L of isotope internal standard working solution into the EP pipe, and standing for 1h at room temperature; drying with nitrogen at 65 ℃;
E. derivatization: adding a 50 mu L derivatization agent into the EP pipe, and placing the EP pipe in a thermostat at 65 ℃ for derivatization for 20 min; drying with nitrogen at 65 ℃; the derivatization agent is acetyl chloride-n-butyl alcohol hydrochloride with the proportion of 9:1 and V/V;
F. dissolving: adding 150 mu L of mobile phase solution into the EP tube, and standing at room temperature for 10min to obtain a sample loading detection sample;
analyzing the sample to be detected by using a liquid chromatography-tandem mass spectrometry method to obtain the content of phenylalanine in the tissue sample;
the sample of the organism is selected from brain tissue;
the method of extracting metabolites of the cell culture of the organism is the same as the method of extracting metabolites of the organism;
2) after step 1), administering a drug treatment to the same organism or to the same organism; alternatively, the same organism cell culture or a cell culture of the same organism is administered a drug treatment;
3) sampling and detecting a sample of the organism or the organism cell culture treated by the administration of the medicament by adopting a liquid chromatography-mass spectrometry technology to obtain the content of phenylalanine in the sample of the organism or the organism cell culture;
4) comparing the content of phenylalanine in the samples obtained in the steps 1) and 3), and obtaining the change of phenylalanine in the metabolic process of the organism or organism cell culture under the treatment of the medicament according to the comparison result;
the medicine is selected from safflower yellow or hydroxy safflower yellow A;
the organism is selected from animal cerebral ischemia/reperfusion injury model, and organism cell culture is selected from cell oxygen deprivation reoxygenation injury model.
2. The method of claim 1, wherein the method comprises: the sample also includes peripheral blood.
3. Use of the method of claim 1 for the evaluation of the effect of the intervention based on the degree of cerebral ischemia/reperfusion injury.
4. Use according to claim 3, characterized in that: the intervention is selected from drug treatment.
5. Use according to claim 4, characterized in that: the medicine is selected from safflower yellow or hydroxy safflower yellow A.
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