CN109813814A - Application of the phenylalanine metabolic alterations in cerebral ischemia/reperfusion injury degree evaluation - Google Patents
Application of the phenylalanine metabolic alterations in cerebral ischemia/reperfusion injury degree evaluation Download PDFInfo
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Abstract
The invention discloses phenylalanine metabolic alterations in the application in cerebral ischemia/reperfusion injury degree evaluation: being detected using content of the LC-MS technology to phenylalanine in organism or cell, according to the changes of contents of the phenylalanine before and after giving drug or other intervening measures, to evaluate cerebral ischemia/reperfusion injury degree variation under intervening measure.The present invention carries out intuitive judgment to the variation of cerebral ischemia/reperfusion injury degree by the situation of change of phenylalanine content in sample, it is convenient and efficient, effective supplement can be played to the evaluation of existing cerebral ischemia/reperfusion injury degree and supported, provide new reference for the evaluating drug effect in drug or the effect assessment of other intervening measures, new drug development.
Description
Technical field
The invention belongs to neuromedicine fields, relate to the use of Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS method) detection
Phenylalanine content changes in peripheral blood equal samples, for evaluating the change of cerebral ischemia/reperfusion injury degree in drug test
Change.
Background technique
Cerebral apoplexy medically refers to the brain cell death as caused by thrombosis.Cerebral apoplexy is broadly divided into ischemic
(since blood supply deficiency causes) and hemorrhagic (since bleeding causes) two classes.The occlusion of internal carotid and vertebral artery, narrow etc.,
Easily cause cerebral arterial thrombosis, leads to some lost of brain function, and male is multiple.According to statistics, there are about 42,400,000 brains in the whole world
The patient of stroke medical history is survived.Between 1990 to 2010 years, the quantity of developed country's patients with cerebral apoplexy is with every year about 10%
Speed decline, and be then with every year about 10% speed increase in developing country.Stroke Death patient in 2016 is up to 630
Ten thousand, account for the 11% of overall death toll.Approximately half of patients with cerebral apoplexy life cycle was less than 1 year.China every year there are about 150~
200 Wan Xinfa cerebral apoplexy cases have become China and are only second to malignant tumour and painstaking effort wherein about 70% is cerebral arterial thrombosis
The third-largest cause of death of pipe disease and primary Pathogenic factors.The main reason for cerebral arterial thrombosis is angiemphraxis or narrow,
Cause local brain tissue blood supply insufficient；And hemorrhagic apoplexy is then to cause blood straight due to ruptured cerebral aneurysm etc.
It taps into caused by brain tissue or spatium interdurale.Country defend that planning commission 2017 announces " Chinese findings in acute ischemic cerebral stroke is examined
Treat guidance specification " it points out, the processing of acute ischemic cerebral apoplexy is it should be emphasized that early diagnosis, early treatment, early rehabilitation and early stage
Prevention is sent out again.Thromboembolism treatment be currently the most important ones restore blood flow measure, rt-PA (rtPA) and
Urokinase is China's main Thrombolytic Drugs used at present.However, a large number of studies show that, blood flow Reperfu- sion after quick cerebral ischemia,
The a large amount of activation of inflammatory cell in ischemic focus part and the release of inflammatory factor are easily induced, reactivity keto in nerve cell is promoted
(ROS) horizontal increase and Apoptosis eventually lead to nerve cell and ischemia/reperfusion (I/R) damage occur.The machine of I/R damage
Complex processed is related to the toxic reaction of excitatory amino acids such as mitochondria and ROS damage, glutamic acid, intracellular Ca2+It is super
The number of mechanisms such as load, inflammatory reaction, Apoptosis and autophagy.
Metabolism group (Metabonomics) is carried out to a certain individual, all metabolites organized or contained into the cell
One new branch of science of qualitative and quantitative analysis.Currently, metabolism group with genomics, transcription group, proteomics
Etc. the group investigative technique platform that a variety of omics technologies collectively form systems biology.In recent years, with mass spectrum, nuclear magnetic resonance etc.
The maturation of detection technique platform, metabolism group gradually penetrate into every field, and application during cerebral ischemia is also day
Benefit is deeply.In recent years, the laboratory Duo Jia is reported in sharply changing along with cell metabolism state during cerebral ischemia in succession
Become, and it was found that the metabolic marker objects of significant changes.These endogenous metabolism objects mainly with energetic supersession, amino acid metabolism and
Lipid metabolism is related: (1) amino acids: including excitatory amino acid (EAAs), branched-chain amino acid, taurine, glycine, paddy ammonia
Amide, homocysteine, γ-aminobutyric acid etc.；(2) energetic supersession object: including glucose, lactic acid, pyruvic acid, ketoboidies,
α-ketoglutaric acid etc.；(3) other: arachidonic acid and its metabolite, 5-HT, NO, catecholamine, glycerol, inositol etc..Its
In, energetic supersession object and excitatory amino acid are at present using more endogenous metabolism analyte detection index.
When evaluating coincident with severity degree of condition, in addition to using marking scales, " Chinese acute ischemic cerebral apoplexy diagnosis and treatment guide
2018 " recommend, it includes plain CT (first choice), multi-mode CT, routine MRI, multi-mode MRI etc. that cerebral lesion inspection, which uses,.Angiosis
Become and checks common carotid ultrasound, transcranial Doppler (TCD), magnetic resonance cerebral angiography (MRA), High-resolution MRI
(HRMRI), CT angiography (CTA) and digital subtraction angiography (DSA) etc..However, these check there is also: need to infuse
The influence of the limitations, operating technology level such as radioiodine contrast agent, costly, review time slightly length, the contraindication of patient itself.
AHA/ASA does not recommend routinely to carry out MRI inspection before intravenous thrombolysis therapy to check the micro- bleeding of encephalic, does not recommend in morbidity 6h
Ischemic cerebral stroke patients checked with perfusion to select to be suitable for machinery and take the patient of bolt.In addition to the above evaluation measures, at present
The ancillary measure that the drug test of evaluation in to(for) cerebral injury generally uses generally requires to sample from tissue.It will be clear that it is not
Only step is complicated, time-consuming, and can not be widely used for human medicine clinical test.
Safflower (Carthamus tinctorius) belongs to the tubular flower of composite family class plant, and property pungent-warm has promoting blood circulation
The effect of, its related preparations is widely used in clinical treatment at present, and development prospect is very considerable.Carthamin yellow (SY) be containing
The crude extract of hydroxyl radical carthamin yellow carthamus A (Hydroxysafflor yellow A, HSYA), HSYA be in safflower pharmacological effect most
Effective water soluble ingredient.Existing a large amount of research confirms that HSYA has specific protective effect to brain and cardiac muscle I/R damage, can
To inhibit, Apoptosis, reduction ROS is horizontal, help to repair injured neurons, promotes cell survival.Inhibited in addition, also having
The functions such as lipid oxide and Calcium overload help to protect cell mitochondrial.Studies have found that HSYA can significantly inhibit
The infiltration of the cell nuclear translocation and neutrophil leucocyte of NF- κ B key molecule p65 reduces the content of plasma angiotensinogen II, and
Inhibit the inflammatory reaction etc. of blood clotting enzyme induction.At the same time, HSYA can also by inhibit nerve cell take in too much calcium from
Son, and increase rat brain natriuretic peptide BNP level, it is related that this may reduce the function of brain edema with HSYA.HSYA can also be significantly inhibited
The aggtegation of blood platelet increases vascular endothelial cell quantity, the activity of Adhering capacity and cell mitochondrial, inhibits vascular thrombosis
It generates.The neuroprotection and mechanism of HSYA is substantially clear.
Summary of the invention
The purpose of the present invention is to provide phenylalanine metabolic alterations answering in cerebral ischemia/reperfusion injury degree evaluation
With.
In order to achieve the above objectives, the invention adopts the following technical scheme:
A method of phenylalanine metabolism in detection organism and cell, comprising the following steps:
1) organism or biological cell culture are sampled using LC-MS technology and sample is (by sampling
To) detection, obtain the content of phenylalanine in the sample of the organism or biological cell culture；
2) after step 1), drug-treated or other intervening measures are given to same organism or same organism；
Alternatively, giving drug-treated to same biological cell culture or same biological cell culture or other interventions are arranged
It applies；
3) using LC-MS technology to after giving intervening measure organism or biological cell culture be sampled
And pattern detection, obtain the content of phenylalanine in the sample of the organism or biological cell culture；
4) content of phenylalanine in sample that step 1) and step 3) obtain is compared, is obtained according to comparison result
The situation of change of organism or biological cell culture phenylalanine in the metabolic process under intervening measure, to be arranged to intervention
It applies the effect of cerebral ischemia/reperfusion injury degree is evaluated and (if the content of phenylalanine reduces, show cerebral ischemia/again
Perfusion injury degree reduces).
Preferably, the LC-MS technology is selected from Liquid Chromatography-Tandem Mass Spectrometry.
Preferably, the sample is selected from the relevant cells such as brain tissue, peripheral blood or neuronal cell, PC12 cell.
Preferably, the sample passes through before detection extracts metabolin, phenylalanine internal standard compound and derivatization is added, then
It is scattered in mobile phase, the sample detection sample of liquid chromatography-tandem mass spectrometry analysis is made.
Preferably, extractant is added after the extraction is specifically includes the following steps: brain tissue or cell are ground, it is ground
After being mixed and being centrifuged, supernatant is taken；Or mix peripheral blood (blood filter paper) with extractant, by standing, take supernatant.
Preferably, the extractant is selected from mixed liquor, anhydrous methanol, methanol aqueous solution, the dichloro of ethyl acetate and ethyl alcohol
One of mixed liquor of methane and methanol is a variety of, for brain tissue or cell, by successively using above-mentioned four after grinding
Kind extractant utmostly extracts the metabolins such as phenylalanine in sample, spirit accurate in conjunction with Liquid Chromatography-Tandem Mass Spectrometry
Quick feature obtains the accurate content analysis result of phenylalanine in sample.
Preferably, the intervening measure is selected from drug-treated.
Preferably, the drug is selected from carthamin yellow or hydroxyl radical carthamin yellow carthamus A.
Preferably, in the step 1), organism is selected from animal brain ischemia reperfusion model, biological cell training
It supports object and deprives reoxygenation model selected from cell oxygen sugar.
A kind of kit detecting phenylalanine metabolism in organism and cell, including above-mentioned extractant and liquid chromatogram-
Tandem Mass Spectrometry Analysis phenylalanine internal standard compound.
The method that phenylalanine is metabolized in above-mentioned detection organism and cell is based on cerebral ischemia/reperfusion injury degree
Application (for example, in test cell line, animal experiment or clinical test of drug) in intervening measure effect assessment.
The beneficial effects of the present invention are embodied in:
The present invention detects phenylalanine content in peripheral blood or its hetero-organization, cell equal samples by LC-MS technology,
Convenient and efficient, object under inspection physiological status does not influence, and is not required to the exogenous materials such as injection diodone, passes through tissue or cell
The situation of change of middle phenylalanine content carries out intuitive judgment to the variation of cerebral ischemia/reperfusion injury degree, lacks to existing brain
The evaluation of blood/reperfusion injury degree can play effective supplement and support, be cerebral ischemia/reperfusion injury clinical treatment medicine
Evaluating drug effect in object or the effect assessment of other intervening measures, new drug development provides new reference.
Further, before the present invention is to the different biological samples such as brain tissue, peripheral blood, neuronal cell and PC12 cell
Handle it is simple and effective, by Liquid Chromatography-tandem Mass, phenylalanine content can be carried out quickly, it is accurate, reliable
Detection is analyzed in conjunction with phenylalanine metabolic alterations, provides convenient approach for evaluation cerebral ischemia/reperfusion injury degree variation.
Detailed description of the invention
Fig. 1 is phenylalanine generation in control group (Sham) and I/R damage group (cerebral ischemia re-pouring group) Mice brain tissues
Thank to variation statistical chart.
Fig. 2 is phenylalanine generation in control group (Sham) and I/R damage group (cerebral ischemia re-pouring group) mouse peripheral blood
Thank to variation statistical chart.
Fig. 3 is phenylpropyl alcohol in control group (untreated fish group) and OGD/R group (oxygen sugar deprives reoxygenation group) mice neuronal cells
Propylhomoserin metabolic alterations statistical chart.
Fig. 4 is phenylpropyl alcohol ammonia in control group (untreated fish group) and OGD/R group (oxygen sugar deprives reoxygenation group) P of Rats C12 cell
Acid metabolic changes statistical chart.
Fig. 5 is phenylalanine metabolic pathway schematic diagram.
Fig. 6 is that RT-qPCR detects phenylalanine metabolism pass in control group (Sham) and I/R damage group Mice brain tissues
The differential expression of key enzyme Pah (phenylalanine hydroxylase), Got1 (aspartate transaminase) and Tat (tyrosine transaminase) count
Figure.
Fig. 7 is that RT-qPCR detects control group (untreated fish group) and OGD/R group mice neuronal cells phenylalanine generation
Thank to the differential expression statistical chart of key enzyme Pah, Got1 and Tat.
Fig. 8 is that RT-qPCR detects control group (untreated fish group) and OGD/R group P of Rats C12 cell phenylalanine is metabolized
The differential expression statistical chart of key enzyme Pah, Got1 and Tat.
Specific embodiment
The present invention is described in further detail with reference to the accompanying drawings and examples, and the embodiment is to of the invention
It explains, rather than limiting the scope of the invention.
(1) metabolite content in LC-MS/MS method detection brain tissue, peripheral blood and cell
1. reagent and instrument
1.1 main agents
Amino acid Isotopic Internal Standard (Cambridge Isotope Labs)；
Ethyl acetate, ethyl alcohol, anhydrous methanol, methylene chloride, formic acid, methanol (chromatography), n-butanol, acetonitrile (chromatography), second
Acyl chlorides (is purchased from Shanghai Sinopharm Chemical Reagent Co., Ltd.).
1.2 key instrument
2. amino acid Isotopic Internal Standard is prepared
12 kinds of amino acid Isotopic Internal Standards are shared, respectively15N,2-13C- glycine (N1,C1-Gly)、2H4Alanine
(D4-Ala)、2H8Valine (D8-Val)、2H3Leucine (D3-Leu)、2H3Methionine (D3-Met)、2H5Phenylalanine
(D5-Phe)、13C6Tyrosine (C6-Tyr)、2H3Aspartic acid (D3-Asp)、2H3Glutamic acid (D3-Glu)、2H2Ornithine
(D2-Orn)、2H2Citrulling (D2-Cit),2H4,13C- arginine (D4,Cl-Arg).All internal standards are work with methanol dilution
Liquid, for concentration in addition to Gly is 12.5 μm of ol/L, remaining is 2.5 μm of ol/L.
3. sample collection
Brain tissue sample is dissected to obtain by C57BL/6 mouse；Peripheral blood sample is taken a blood sample to obtain by C57BL/6 mouse rat-tail；
PC12 cell is purchased from Chinese Academy of Sciences Shanghai American Type Culture Collection committee cell bank.
4.LC-MS/MS operating condition
4.1 liquid chromatogram operating conditions
Mobile phase uses 80% acetonitrile, and speed change: the 140 μ μ L/min of L/min, 0.3min → 30 is arranged in quaternary flow rate pump,
The μ of 1.2min → 300 L/min, 0.4min.Autosampler is set as every 20 μ L of sub-sampling.
4.2 mass spectrums (MS/MS) operating condition
Ion source temperature (Probe temperature): 350 DEG C；Transformation residence time (Dwell time per every time
Transition): 200ms；Sprayer (Nebulizer, GAS 1): 30psi；Heater (Heater gas, GAS 2):
40psi；Gas curtain gas (Curtain gas): 25psi；Ion source voltage (Ion spray voltage): 5500V.
Mass spectrograph is scanned using cation, and amino acid detects 2 kinds of different scan methods, and more reaction detections are used for sweet ammonia
Sour (Gly), ornithine (Orn), arginine (Arg) and citrulling (Cit), other amino acid detection neutral loss scan sides
Formula.
5. phenylalanine content detecting step
The detection method of phenylalanine content in 5.1C57BL/6 Mice brain tissues
A. tissue grinder: taking tissue about 50mg, and 200 μ L ultrapure waters are added and are ground in grinder with the frequency of 40Hz
5min。
B. fractional extraction: the 1. mixed liquor (1:1, v/v) of 400 μ L ethyl acetate and ethyl alcohol, 2. 200 μ L anhydrous methanol, 3.
The mixed liquor (3:1, v/v) of 200 μ L anhydrous methanols and water, the 4. mixed liquor (3:1, v/v) of the methylene chloride of 200 μ L and methanol；
It is ground 2min in grinder after often adding a kind of solution, the sample after grinding is put into refrigerated centrifuge, 4 DEG C,
12000rpm is centrifuged 5min, then Aspirate supernatant, and whole process operates on ice, and remainder continues to extract.
C. nitrogen is blown: after the supernatant of sample is mixed, 1mL is sucked out in EP pipe, dries up under nitrogen evaporator room temperature.
D. internal standard solution prepares: the Isotopic Internal Standard working solution being stored in 4 DEG C of refrigerators being shaken 15min at room temperature, is taken
100 μ L are added in the EP pipe, are stored at room temperature 1h；65 DEG C are dried with nitrogen, about 15min.
E. derive: taking 50 μ L derivating agents (the chloro- hydrochloric acid n-butanol of acetyl, 9:1, V/V) that the EP pipe is added, be placed in 65 DEG C of perseverances
Derivative 20min in incubator；It is dried with nitrogen in 65 DEG C, about 20min.
F. it dissolves: taking 150 μ L mobile phase solution (acetonitrile-water mixed liquor, 8:2, V/V) that the EP pipe is added, be placed at room temperature for
10min obtains sample detection sample.
G. sample detection inspection product are analyzed using Liquid Chromatography-Tandem Mass Spectrometry, obtains phenylpropyl alcohol ammonia in tissue samples
The content of acid.
The detection method of phenylalanine content in 5.2C57BL/6 mouse peripheral blood sample (blood filter paper)
A. dry blood spot: filter paper model 903#, mouse tail vein drop of blood is on filter paper, diameter about 10mm, room
It is dried under temperature, -20 DEG C of refrigerators save to be measured.
B. it samples: dry blood spot being broken into 3mm circle Blood piece (being equivalent to 3.2 μ L plasma samples) with punch, is placed in
In 96 orifice plates；
C. it extracts: the Isotopic Internal Standard working solution being stored in 4 DEG C of refrigerators being shaken into 15min at room temperature, 100 μ L is taken to add
Enter to place in 96 orifice plates of Blood piece, be stored at room temperature 1h, supernatant is taken to be placed in 96 new orifice plates；
D. nitrogen is blown: 65 DEG C are dried with nitrogen, about 15min；
E. derive: being added 50 μ L derivating agents (the chloro- hydrochloric acid n-butanol of acetyl, 9:1, V/V), be placed in 65 DEG C of insulating boxs derivative
20min；
F. nitrogen is blown: 65 DEG C are dried with nitrogen, about 20min；
G. it dissolves: 150 μ L mobile phase solution (acetonitrile-water mixed liquor, 8:2, V/V) is added, is placed at room temperature for 10min, obtains
Sample detection sample.
H. sample detection inspection product are analyzed using Liquid Chromatography-Tandem Mass Spectrometry, obtains phenylpropyl alcohol ammonia in tissue samples
The content of acid.
The detection of phenylalanine content in 5.3 mouse primary developing approach cells and P of Rats C12 cell
Method (with 5.1).
6. data processing
Analytical calculation is carried out to data by SPSS15.0 software, using Internal standard curve method, according to metabolin and accordingly
Kurtosis ratio is substituted into standard curve and is calculated, and obtains the concentration of phenylalanine in sample.
(2) the metabolic marker object of brain I/R damage
(1) metabonomic analysis, screening are carried out to C57BL/6 mouse sham-operation group and I/R damage group brain tissue, peripheral blood
Metabolic marker object.(2) C57BL/6 mice neuronal cells and P of Rats C12 cell control group, OGD/R group are metabolized
Group credit analysis, verifies the variation tendency of phenylalanine.(3) Mice brain tissues, neuronal cell and PC12 cell are extracted respectively
RNA carries out reverse transcription and PCR analysis, clear under the conditions of I/R damage and OGD/R, the expression hair of phenylalanine key enzyme
The expression of raw change, the metabolic marker object relevant enzyme further obtained to screening carries out laboratory proofing.Specific steps and knot
Fruit is as follows.
1. screening the metabolin of content significant changes in brain I/R damage.
1.1 experimental design
6 pairs of C57BL/6 mouse I/R damage brain tissue samples and sham-operation group (control group) tissue samples are collected ,-
80 DEG C freeze.Tissue samples inspection carries out metabolism group detection (including above-mentioned LC-MS/MS method) to sample.
The preliminary treatment of 1.2 data
A total of 79 metabolins detected in brain tissue, go out 6 kinds of amino acid generations by data processing preliminary screening
Thank to object.
1.3 paired t-test methods
It is handled, metabolin is analyzed by t inspection, wherein 5 kinds of amino acid metabolisms using SPSS18.0 statistical software
Object content dramatically increases, and a kind of amino acid metabolite content significantly reduces, and wherein phenylalanine variation tendency is more obvious, knot
Fruit is as shown in Figure 1, phenylalanine content increases i.e. in focal cerebral ischemia/reperfusion injury tissues following MCAO in rats.Meanwhile referring to fig. 2, small
Phenylalanine levels increase in blood plasma after mouse cerebral ischemia/reperfusion injury.Therefore, phenylalanine is in I/R damage group mouse brain group
Knitting has raising with content in peripheral blood.
2. confirmatory experiment
OGD/R experiment is carried out using neuronal cell and PC12 cell, in-vitro simulated cerebral ischemia/reperfusion injury is collected
Inspection after cell sample carries out metabolism group detection (referring to above-mentioned LC-MS/MS method) to metabolite content variation in sample,
Verify previous experiments result.As shown in figure 3, phenylalanine content increases after mouse primary developing approach oxygen sugar deprives reoxygenation.
As shown in figure 4, phenylalanine content increases after P of Rats C12 cell oxygen sugar deprives reoxygenation.
3. the variation that phenylalanine content increases regulation key gene expression in association metabolic pathway.
3.1 phenylalanine metabolic pathways
Key metabolic enzymes are Pah, Got1, Tat in phenylalanine metabolic pathway as can be seen from Figure 5.
3.2Pah, Got1, Tat design of primers
1. mouse cell key enzyme PCR of table reacts primer
Note: the design of primers deadline is in May, 2017, and GAPDH is internal reference
2. rat cell key enzyme PCR of table reacts primer
Note: the design of primers deadline is in September, 2017, and GAPDH is internal reference
3.3RNA is extracted and content analysis
A. it takes 50mg Mice brain tissues to be put into homogenizer, 1mL TRIzol reagent is added, is placed in and is homogenized repeatedly on ice；
B. the TRIzol lysate of tissue or cell is transferred in 1.5ml EP pipe, 0.2mL chloroform is added, mixes well,
5min is placed at room temperature；
C. sample is placed in 4 DEG C of 12000rpm centrifugation 10min；
D. it takes upper strata aqueous phase to be placed in new 1.5mL EP pipe, 0.5mL isopropanol is added and mixes, is stored at room temperature 10min, 4
DEG C 12000rpm is centrifuged 10min；
E. it inhales and abandons supernatant, the visible RNA precipitate of naked eyes；
F. 75% ethanol washing of 1mL is added, 4 DEG C of 12000rpm are centrifuged 5min, abandon supernatant；It is repeated once；
G. it spontaneously dries；According to RNA amount, RNA precipitate is dissolved with 30~50 μ L Rnase-free DEPC water；
H. it is quantitative RNA to be carried out using NanoDrop-2000 nucleic acids instrument, and according to the ratio of OD260nm/280nm point
RNA purity is analysed, remaining sample freezes in -80 DEG C.
3.4cDNA reverse transcription
It is carried out according to cDNA reverse transcription reagent box operational manual, 1 μ g RNA is taken to carry out reverse transcription.
3.5RT-qPCR
PCR reaction condition:
(1) template denaturation: 95 DEG C of 5min；
(2) product amplification: 95 DEG C of 15s, 56 DEG C of 30s, 72 DEG C of 1min, 40 circulations；
(3) product extends: 72 DEG C of 10min.
Using the RNA of each group Mice brain tissues, reverse transcription and PCR reaction, the key of comparative analysis amino acid metabolism are carried out
As a result the content difference of metabolic enzyme mRNA is shown in Fig. 6, Pah, Got1 expression in I/R damage group brain tissue is prompted to lower, and Tat table
Up to up-regulation.
Group of cells RNA extraction method is same as above, and carries out reverse transcription and PCR reaction, the key of comparative analysis amino acid metabolism
The content difference of metabolic enzyme mRNA, is as a result shown in Fig. 7, Fig. 8, consistent with brain tissue testing result height.
Experimental result is shown: Pah, Got1 of I/R damage group and OGD/R group expression are lowered, Tat expression up-regulation, phenylpropyl alcohol ammonia
Acid content increases.On this basis, peripheral blood phenylalanine content can be changed to the new finger as evaluation I/R damage variation
Mark provides convenience for evaluation brain I/R degree of injury.
(3) the neuroprotection pharmacodynamics mechanism analysis of carthamin yellow or hydroxyl radical carthamin yellow carthamus A
(1) to C57BL/6 mouse sham-operation group, I/R damage group and 5mg/kg, 20mg/kg SY administration group brain tissue and
Peripheral blood carries out metabonomic analysis (referring to above-mentioned LC-MS/MS method), finds phenylalanine in I/R damage group mouse brain group
Knitting has raising with content in peripheral blood, but the phenylalanine content of SY intervention group is decreased obviously.(2) to C57BL/6 mouse mind
Metabonomic analysis (ginseng is carried out through first cell and PC12 cell control group, OGD/R group and 1 μM, 10 μM of HSYA administration groups
See above-mentioned LC-MS/MS method), verify the variation tendency of phenylalanine.As a result, it has been found that the variation tendency of phenylalanine with body reality
It is consistent to test group.(3) brain tissue, neuronal cell and PC12 cell RNA are extracted respectively, are carried out reverse transcription and PCR analysis, are defined
I/R damage is under the conditions of OGD/R, and the expression of phenylalanine key enzyme changes, further to metabolic markers (phenylpropyl alcohol
Propylhomoserin) relevant enzyme expression carry out laboratory proofing.Experimental result is shown: Pah, Got1 of I/R damage group and OGD/R group expression
It lowers, Tat expression up-regulation, phenylalanine content increases.And under drug (SY or HSYA) intervention, the expression of Pah, Got1, Tat
It is reversed, phenylalanine content decline.Thus prove that SY and HSYA can be by intervening phenylalanine key enzyme
Expression plays neuroprotection to reduce phenylalanine content.
In short, the present invention carries out metabolism group detection to C57BL/6 Mice brain tissues and peripheral blood first, discovery I/R
Phenylalanine significantly increases under faulted condition.It is detected by the metabolism group of mice neuronal cells and P of Rats C12 cell, into
One step confirms that OGD/R damage can cause phenylalanine significantly to increase.Therefore it is close to speculate that phenylalanine metabolic pathway is damaged with I/R
Cut phase is closed.Expression analysis further is carried out to the key metabolic enzymes of phenylalanine metabolic pathway, in conjunction with clearly with neuroprotection
As a result the safflower active constituent intervention of effect illustrates that the access of phenylalanine metabolism is obstructed the phenylpropyl alcohol ammonia caused in tissue and cell
Acid content increases, and prompting phenylalanine variation is to evaluate an important references of I/R damage variation.
<110>the Fourth Military Medical University of P.L.A
<120>application of the phenylalanine metabolic alterations in cerebral ischemia/reperfusion injury degree evaluation
<160> 16
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Claims (10)
1. a kind of method of phenylalanine metabolism in detection organism and cell, it is characterised in that: the following steps are included:
1) using LC-MS technology is to organism or biological cell culture is sampled and pattern detection, the biology is obtained
The content of phenylalanine in the sample of body or biological cell culture；
2) after step 1), drug-treated or other intervening measures are given to same organism or same organism；Alternatively,
Drug-treated or other intervening measures are given to same biological cell culture or same biological cell culture；
3) using LC-MS technology to after giving intervening measure organism or biological cell culture is sampled and sample
This detection obtains the content of phenylalanine in the sample of the organism or biological cell culture；
4) content of phenylalanine in sample that step 1) and step 3) obtain is compared, is intervened according to comparison result
The variation of organism or biological cell culture phenylalanine in the metabolic process under measure.
2. a kind of method for detecting phenylalanine metabolism in organism and cell according to claim 1, it is characterised in that: institute
It states LC-MS technology and is selected from Liquid Chromatography-Tandem Mass Spectrometry.
3. a kind of method for detecting phenylalanine metabolism in organism and cell according to claim 1, it is characterised in that: institute
It states sample and is selected from brain tissue, peripheral blood or cell.
4. a kind of method for detecting phenylalanine metabolism in organism and cell according to claim 1, it is characterised in that: institute
It states intervening measure and is selected from drug-treated.
5. according to claim 1 or a kind of 4 methods for detecting phenylalanine metabolism in organism and cell, feature exist
In: the drug is selected from carthamin yellow or hydroxyl radical carthamin yellow carthamus A.
6. a kind of method for detecting phenylalanine metabolism in organism and cell according to claim 1, it is characterised in that: institute
It states organism and is selected from animal brain ischemia reperfusion model, biological cell culture is selected from cell oxygen sugar and deprives reoxygenation damage
Wound model.
7. it is a kind of detection organism and cell in phenylalanine metabolism kit, including metabolin extractant and be used for liquid phase color
Spectrum-Tandem Mass Spectrometry Analysis phenylalanine internal standard compound.
8. in a kind of as described in claim 1 detection organism and cell the method for phenylalanine metabolism based on cerebral ischemia/
Application in the intervening measure effect assessment of reperfusion injury degree.
9. application according to claim 8, it is characterised in that: the intervening measure is selected from drug-treated.
10. application according to claim 9, it is characterised in that: the drug is selected from carthamin yellow or hydroxyl safflower yellow
Pigment A.
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| CN115684608A (en) * | 2022-11-03 | 2023-02-03 | 大连珍奥药业股份有限公司 | Metabolic marker for treating myocardial ischemia reperfusion injury by targeting myocardial peptide and application thereof |
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