CN115684608B - Metabolic marker for treating myocardial ischemia reperfusion injury by targeting myocardial peptide and application thereof - Google Patents
Metabolic marker for treating myocardial ischemia reperfusion injury by targeting myocardial peptide and application thereof Download PDFInfo
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- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 claims abstract description 28
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Abstract
The invention discloses a metabolic marker for treating myocardial ischemia reperfusion injury by targeting myocardial peptide and application thereof, belonging to the field of biomarkers. The invention discloses a metabolic marker for treating myocardial ischemia reperfusion injury by targeting myocardial peptide, which comprises aspartic acid and/or lysine. Experiments find that the myocardial peptide has therapeutic effect on myocardial ischemia reperfusion injury, and the indication effect of lysine and aspartic acid on myocardial peptide for treating myocardial ischemia reperfusion injury is found by carrying out non-targeted metabonomics index screening on myocardial tissue and targeted metabonomics verification on the screened lysine and aspartic acid. The invention provides a new thought and direction for the treatment of myocardial ischemia reperfusion.
Description
Technical Field
The invention relates to the field of biomarkers, in particular to a metabolic marker for treating myocardial ischemia reperfusion injury by targeting cardiac carnosine and application thereof.
Background
Myocardial ischemia refers to a pathological condition in which blood perfusion of the heart is reduced, oxygen supply to the heart is reduced, myocardial energy metabolism is abnormal, and normal operation of the heart cannot be promoted. Restoring blood perfusion after a period of myocardial ischemia exacerbates the damage to myocardial structure and function, resulting in decreased cardiac function and malignant arrhythmia, a process known as myocardial ischemia reperfusion.
Myocardial peptide is a small molecule polypeptide with biological activity. Studies have shown that the myocardial peptide has significant preventive and therapeutic effects on myocardial ischemia reperfusion injury and can reduce release of myocardial enzymes and severe arrhythmia after ischemia reperfusion. The myocardial peptide can relieve the release of myocardial enzyme after myocardial ischemia reperfusion to relieve myocardial injury. During ischemia reperfusion injury, myocardial cells are denatured and necrotized, so that cell membrane work is performedThe myocardial enzyme activity is increased due to the escape of CK in cells, and clinical researches show that the serum myocardial enzyme activity of the myocardial peptide is obviously reduced after administration compared with that before administration, so that the myocardial injury is protected. One of the causes of arrhythmia is dysfunction of ion channels of myocardial cell membranes, intracellular acidosis, elevated H concentration upon myocardial ischemia reperfusion injury, and through H/Na exchange and Na/Ca 2+ Exchange, resulting in intracellular Ca 2+ Overload, abnormal activity of these ions is prone to induce arrhythmia. And the myocardial peptide can reduce the incidence rate of severe ventricular arrhythmia of the ischemia-reperfusion injury rat by inhibiting ventricular intramuscular flow to calcium.
Metabonomics originated in the 90s of the 20 th century and was used to quantify and characterize small molecule metabolites. Among them, the advantage of non-targeted metabonomics to quantitatively analyze large-scale data simultaneously is widely applied in the pathophysiological mechanism aspect of diseases, and the metabonomics analysis mainly carries out dynamic tracking analysis on body fluid analyzed by cells and organisms, and because metabolic products exist in the body fluid relatively stably, the metabonomics analysis can be used for diagnosis and research of diseases, and new metabolic products are found to be used for research of myocardial ischemia reperfusion, and have important significance for preventing and treating myocardial ischemia reperfusion.
Disclosure of Invention
The invention aims to provide a metabolic marker for treating myocardial ischemia reperfusion injury by targeting myocardial peptide and application thereof, so as to solve the problems in the prior art, and the experiment verifies the indication effect of lysine and aspartic acid on myocardial peptide for treating myocardial ischemia reperfusion injury, thereby providing a new direction and thought for treating myocardial ischemia reperfusion injury.
In order to achieve the above object, the present invention provides the following solutions:
the invention provides a metabolic marker for treating myocardial ischemia reperfusion injury by targeting myocardial peptide, wherein the metabolic marker comprises aspartic acid and/or lysine.
The invention also provides application of the metabolic marker in preparing a product for monitoring the effect of the myocardial peptide in treating myocardial ischemia reperfusion injury.
The invention also provides application of the detection reagent of the metabolic marker in preparation of products for monitoring the effect of the myocardial peptide in treating myocardial ischemia reperfusion injury.
Preferably, the product comprises a reagent or kit.
Preferably, a decrease in the amount of aspartic acid and lysine in myocardial tissue as compared to before the myocardial ischemia reperfusion injury is treated with the myocardial peptide is indicative of the myocardial peptide functioning to treat myocardial ischemia reperfusion injury.
The invention discloses the following technical effects:
according to the invention, experiments show that the myocardial peptide has a treatment effect on myocardial ischemia reperfusion injury, through non-targeted metabonomics index screening is carried out on myocardial tissues, and targeted metabonomics verification is carried out on screened lysine and aspartic acid, after 7 days of myocardial ischemia reperfusion injury is treated by the myocardial peptide, the contents of aspartic acid and lysine in the myocardial tissues are reduced, and after 27 days of treatment, the aspartic acid and lysine show a remarkable reduction, so that the indication effect of lysine and aspartic acid on myocardial peptide for treating myocardial ischemia reperfusion is shown, and the myocardial peptide can be used as a metabolic marker for monitoring myocardial ischemia reperfusion for myocardial peptide treatment. The invention provides a new thought and direction for the treatment of myocardial ischemia reperfusion injury.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows HE staining results;
FIG. 2 shows Masson staining results;
FIG. 3 is myocardial ischemia reperfusion injury serum marker content; ast is aspartate aminotransferase, tnT is troponin T, and MYO is myoglobin;
FIG. 4 is a non-targeted metabonomics detection result;
FIG. 5 is a target metabonomics test result;
FIG. 6 shows the amounts of aspartic acid and lysine in myocardial tissue of rats before and after treatment;
FIG. 7 shows the results of aspartic acid and lysine specificity and sensitivity assays.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
In order to detect the content of various amino acids in animal metabolic tissues and indexes such as precision, recovery rate, repeatability and the like, a plurality of indexes of the amino acids in animal metabolic tissue samples are studied on the basis of SD male rat myocardial tissues.
Example 1
1. Standard curve preparation
Weighing 22 kinds of amino acid standard substances with proper amount, preparing into single standard mother solution with methanol or water, weighing each mother solution with proper amount to prepare mixed standard substances, and diluting with 10% methanol-water 1:1 to proper concentration to prepare into working standard solution. The concentrations of the amino acid substances are shown in Table 1, and the mother solution and the working standard solution are stored in a refrigerating manner. An appropriate amount of isotope standard (Trp-d 3) is weighed, and 10% methanol is used for preparing the mixed standard mother solution with the concentration of 1000ng/mL.
TABLE 1
Standard curve:
the standard curves are shown in table 2 below:
TABLE 2
| Substance (B) | Linear equation | Correlation coefficient | Linear range | Quantitative limit |
| Gly | y＝9.06e-005x+0.00104 | 0.9954 | 40-40000 | 40 |
| Ala | y＝0.00429x+0.0188 | 0.9910 | 10-1000 | 10 |
| GABA | y＝0.00709x+0.00275 | 0.9943 | 1-250 | 1 |
| Ser | y＝0.00724x+0.0414 | 0.9911 | 2-400 | 2 |
| Pro | y＝0.0594x+0.0299 | 0.9901 | 1-100 | 1 |
| Val | y＝0.0314x+0.0249 | 0.9934 | 1-500 | 1 |
| Thr | y＝0.00269x+0.0121 | 0.9921 | 10-500 | 10 |
| Ile | y＝0.00435x+0.00478 | 0.9921 | 2-2000 | 2 |
| Leu | y＝0.0351x+0.0509 | 0.9939 | 2-500 | 2 |
| Asn | y＝0.000104x+0.00546 | 0.9921 | 100-4000 | 100 |
| Orn | y＝0.0143x+0.0306 | 0.9908 | 2-500 | 2 |
| Asp | y＝0.00867x+0.00449 | 0.9929 | 4-200 | 4 |
| Hcy | y＝0.000328x+-6.81e-006 | 0.9930 | 10-1000 | 10 |
| Gln | y＝0.00982x+0.0119 | 0.9927 | 1-250 | 1 |
| Lys | y＝0.00951x+0.0108 | 0.9901 | 1-500 | 1 |
| Glu | y＝0.0136x+0.0338 | 0.9929 | 2-500 | 2 |
| Met | y＝0.00869x+0.00635 | 0.9913 | 2.5-1250 | 2.5 |
| His | y＝0.0287x+0.0503 | 0.9900 | 2-500 | 2 |
| Phe | y＝0.0465x+0.0349 | 0.9905 | 1-500 | 1 |
| Arg | y＝0.0281x+0.0246 | 0.9929 | 2-400 | 2 |
| Tyr | y＝0.0116x+0.0188 | 0.9949 | 2.5-1250 | 2.5 |
| Trp | y＝0.0243x+0.0383 | 0.9939 | 0.5-500 | 0.5 |
2. Metabolite extraction
Accurately weighing a proper amount of animal tissue sample in a 2mL EP tube, accurately adding 600 mu L of 10% methanolic formate solution-H 2 O (1:1, V/V) solution, adding 2 steel balls, and vortex oscillating for 30s; placing the mixture into a tissue grinder, and grinding the mixture for 90s at 60 Hz; centrifuging at 12000rpm and 4deg.C for 5min, collecting 20 μl of supernatant, and adding 380 μl 10% formic acid methanol-H 2 O (1:1, V/V) solution, vortex oscillating for 30s, taking 100 mu L of diluted sample, adding 100 mu L of isotope internal standard with the concentration of 100ppb, vortex oscillating for 30s, filtering supernatant by a 0.22 mu m membrane, and adding filtrate into a detection bottle.
Dilution by a 20-fold content (μg/g) =c0.6x2 x 20/sample size, wherein C: ng/mL; sampling amount: mg.
3. On-machine detection
(1) Chromatographic conditions
By ACQUITYBEH C18 column (2.1X100 mm,1.7 μm, waters, USA) with 5. Mu.L sample injection, column temperature 40 ℃, mobile phase A-10% methanol water (0.1% formic acid), B-50% methanol water (0.1% formic acid).
Gradient elution condition is 0-6.5 min, 10-30% B; 6.5-7 min, 30-100% B; 7-14 min,100% B; 14-17.5 min, 100-10% B. The flow rate is 0-8.0 min,0.3mL/min; 8.0-17.5 min,0.4mL/min.
(2) Mass spectrometry conditions
Electrospray ionization (ESI) source, positive ion ionization mode. The ion source temperature was 500 ℃, the ion source voltage was 5500V, the collision gas was 6psi, the curtain gas was 30psi, and the atomizing gas and the assist gas were 50psi. Scanning was performed using Multiplex Reaction Monitoring (MRM). Ion pairs for quantitative analysis are shown in table 3 below.
Table 3 for analysis of quantitative ion pairs
3. Precision of
Working standard solutions with different concentrations are continuously injected for six times, and the daily precision is calculated and expressed as RSD. Six standard samples of different concentrations were treated daily and measured on three different days, and the precision between days was calculated and expressed as RSD. The daily precision is between 1.29 and 14.63 percent, and the daily precision is between 1.70 and 14.96 percent, which indicates that the precision of the instrument is good. The results are shown in Table 4.
4. Repeatability of
Eight samples were repeatedly processed and performed as "metabolite extraction" to obtain concentration calculation repeatability, expressed as RSD. The results are detailed in Table 4.
5. Recovery rate
Ten samples of each concentration were treated in parallel according to "metabolite extraction" and recovery was determined on the same day. The results are detailed in Table 4.
TABLE 4 within day, daytime precision, recovery and repeatability
To further verify the correlation of amino acids and myocardial peptides in metabolic tissues for treatment of myocardial ischemia reperfusion injury, further verification was performed in animal experiments below.
Example 2
1. Experimental grouping
(1) Control group: normal rats;
(2) Model group: myocardial ischemia reperfusion injury model rats;
(3) Positive control group: myocardial ischemia reperfusion injury model rats were perfused with isosorbide dinitrate;
(4) Low dose treatment group: myocardial ischemia reperfusion injury model rat, low dose (18.9 mg/kg) myocardial peptide injection tail vein injection;
(5) Medium dose treatment group: myocardial ischemia reperfusion injury model rat, medium dose (56.7 mg/kg) myocardial peptide injection tail vein injection;
(6) High dose treatment group: myocardial ischemia reperfusion injury model rat, high dose (170.1 mg/kg) myocardial peptide injection tail vein injection.
Each group had 6 rats, 36 rats. The positive control group and the myocardial peptide drug group were continuously administered for 28 days, and blood was taken from the eyeball 40 days after molding, and animals were sacrificed and subjected to myocardial tissue dissection and treatment.
2. Results of high, medium and low dose myocardial peptides for treatment of myocardial ischemia reperfusion Injury (IR)
1.1 Hematoxylin Eosin (HE) staining
1) Fixing: placing myocardial tissues of each experimental group in 10% formaldehyde for fixation for 48 hours;
2) Washing and dehydrating: washing the fixed tissue with running water to remove residual fixing liquid and impurities. Ethanol with different concentrations is dehydrated step by step, 50%,70%,85%,95% until absolute ethanol is obtained, and each stage is dehydrated for 2 hours;
3) And (3) transparency: soaking in 50% alcohol and 50% xylene for 2h, soaking in pure xylene for 2h, and soaking in alcohol xylene for 2h again;
4) Wax dipping: firstly placing the tissue material blocks in an equal amount of mixed liquid of melted paraffin and dimethylbenzene for 1-2 hours, and then placing the tissue material blocks in 2 melted paraffin liquids for about 3 hours respectively;
5) Embedding: placing the tissue blocks immersed with the wax into a container filled with wax liquid, and placing the tissue blocks. Removing redundant paraffin around after the paraffin is solidified, and preparing a slice;
6) Slicing: the wax block was placed in a-20deg.C refrigerator for at least 30min before slicing to increase hardness. Mounting and fixing a slicing knife on a knife rest of a blade machine, fixing a wax block base or a wax block, adjusting the wax block and the knife to a proper position, forming a 5-degree angle between a cutting edge and the surface of the wax block, adjusting the slicing thickness on the slicing machine to be 4-7 mu m, and then slicing;
7) Baking and dewaxing: baking the slide in a constant temperature oven at 65 ℃ for 30min, soaking in xylene I (para position) for 15min, and soaking in xylene II (para position) for 15min;
8) Hydration: soaking the dewaxed slices in 100%,95%,85% and 75% alcohol for 5min respectively, and washing with tap water for 10min;
9) Hematoxylin staining: the slices after distilled water had been added were stained in an aqueous hematoxylin solution for 5min, color separated in aqueous ammonia for several seconds. Washing with running water for 15min, and dehydrating with 70% and 90% ethanol for 10min;
10 Eosin staining: adding 0.5% alcohol eosin staining solution for 1-2 min, washing with running water, and dehydrating the stained slice with pure alcohol;
11 Transparent and encapsulant): the slide was placed in xylene for 3min x 2 times transparency, sealed with neutral resin and placed in an oven at 65℃for 15min.
12 Image acquisition and analysis: and photographing by a microscope, and collecting and analyzing relevant parts of the sample.
1.2 Masson (Masson) staining
1.2.1 pretreatment
Paraffin section: dewaxing and hydrating according to a conventional method, soaking the slices in xylene for 5min, replacing the xylene, and then soaking for 5min; soaking in absolute ethanol for 5min respectively; soaking in 95% ethanol for 5min; soaking in 85% ethanol for 5min; soaking in 70% ethanol for 5min, and soaking in PBS for 3min×3 times;
1.2.2 Experimental procedure
1) Hematoxylin is dyed for 5min, and is washed clean by distilled water;
2) Differentiation of 0.1% HCl, flushing with tap water, and bluing;
3) Dyeing the ponceau acid reddish brown dyeing liquid for 5min, and slightly washing with distilled water;
4) 1% phosphomolybdic acid aqueous solution treatment for 50s;
5) Spin-drying, namely washing without water, dying for 5s, washing with tap water, and drying in an electrothermal wind-supporting drying oven;
6) Adding neutral gum to the slice after airing, and covering with a glass slide;
7) And (5) observing by a microscope, and photographing.
3. Myocardial injury serum marker content detection
Blood was collected from the eyeballs of each group of rats, and the contents of aspartate aminotransferase (ash), troponin T (TnT) and Myoglobin (MYO) were measured using a fully automatic biochemical analyzer (BECKMAN AU 5800).
4. Detection of the content of aspartic acid and lysine in the respective groups
1) Liquid chromatography-mass spectrometry detection of each metabolism index content
Metabolite extraction: accurately weighing a proper amount of sample in a 2mL EP tube, accurately adding 600 mu L of 10% methanolic formate solution-H 2 O (1:1, V/V) solution, adding 2 steel balls, and vortex oscillating for 30s; placing the mixture into a tissue grinder, and grinding the mixture for 90s at 60 Hz; centrifuging at 12000rpm and 4deg.C for 5min, collecting 20 μl of supernatant, adding 380 μl of 10% methanol-H formate 2 O (1:1, V/V) solution, vortex oscillating for 30s, taking 100 mu L of diluted sample, adding 100 mu L of isotope internal standard with the concentration of 100ppb, vortex oscillating for 30s, filtering supernatant by a 0.22 mu m membrane, and adding filtrate into a detection bottle.
2) On-machine detection
Chromatographic conditions: by ACQUITYBEH C18 column (2.1X100 mm,1.7 μm, waters, USA) with 5. Mu.L sample injection, column temperature 40 ℃, mobile phase A-10% methanol water (0.1% formic acid), B-50% methanol water (0.1% formic acid). Gradient elution condition is 0-6.5 min, 10-30% B; 6.5-7 min, 30-100% B; 7-14 min,100% B; 14-17.5 min, 100-10% B. The flow rate is 0-8.0 min,0.3mL/min; 8.0-17.5 min,0.4mL/min.
Mass spectrometry conditions: electrospray ionization (ESI) source, positive ion ionization mode. The ion source temperature was 500 ℃, the ion source voltage was 5500V, the collision gas was 6psi, the curtain gas was 30psi, and the atomizing gas and the assist gas were 50psi. Scanning was performed using Multiplex Reaction Monitoring (MRM).
5. Differences in the levels of aspartic acid and lysine before and after treatment of each group
And detecting myocardial ischemia reperfusion model group by liquid chromatography-mass spectrometry, treating 7-day group and treating 28-day group, and performing myocardial tissue targeted metabonomics detection.
6. Results
1) As shown in FIG. 1, the HE staining results showed that the myocardial fibers of the control group were regularly arranged without gaps of breakage or necrosis. The myocardial fiber structure of the model group is damaged, the myocardial fiber breaks and dissolves, the muscle gap expands, and inflammatory cells infiltrate. After the positive control group and the myocardial peptide injection are injected, myocardial fiber structure damage and inflammatory cell infiltration are obviously improved.
2) The Masson staining results are shown in FIG. 2, and it can be seen from the figure that the control myocardial tissue fibers are aligned and stained uniformly with less collagen fibers. The myocardial fibrosis degree of the model group is obvious, and the collagen fibers are obviously increased. After the positive control group and the myocardial peptide injection are injected, myocardial fibrosis is obviously improved, and collagen fibers are obviously reduced.
3) The serum marker content of myocardial injury is shown in figure 3, and the figure shows that the content of the myocardial injury marker Ast, tnT, MYO is obviously reduced after the myocardial peptide treatment, so that the protective effect of the myocardial peptide treatment on myocardial injury is shown.
4) Fig. 4 shows the results of non-targeted metabonomics testing of myocardial tissue from 6 rats from 3 animals, screening found significant increases in aspartate and lysine levels in the myocardial ischemia reperfusion model group, and significant decreases in both levels after treatment with myocardial peptide compared to the model group.
After non-targeted metabonomics screening, the results of the targeted metabonomics detection verification are shown in fig. 5, and it can be seen that the contents of aspartic acid and lysine in the myocardial ischemia reperfusion model group are significantly increased compared with the normal rat group, and the contents of aspartic acid and lysine are significantly reduced after treatment with myocardial peptide. Suggesting that aspartic acid and lysine content in myocardial tissue is related to myocardial peptide treatment and has a certain identification effect on prognosis.
5) The results of measuring the amounts of aspartic acid and lysine in the myocardial tissue of the rat before and after the myocardial peptide treatment are shown in FIG. 6. As can be seen from the figure, after 7 days of treatment with the myocardial peptide, the contents of aspartic acid and lysine in the myocardial tissue of the myocardial ischemia reperfusion model were reduced, while after 28 days of treatment, the contents of aspartic acid and lysine in the myocardial tissue were significantly reduced. From this, it can be seen that aspartic acid and lysine can be used as metabonomics to monitor myocardial peptide therapy for myocardial ischemia reperfusion.
6) Determination of specificity and sensitivity
ROC curves were made based on comparing the levels of aspartic acid and lysine in rat cardiomyocytes before and after treatment (28 d). As shown in fig. 7, ROC curves showed that Asp AUC value after treatment was 1.000 and lys AUC value was 0.944, indicating higher sensitivity and specificity of aspartic acid and lysine, and that myocardial peptide treatment ischemia reperfusion could be monitored as metabonomic treatment.
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.
Claims (4)
1. Use of a metabolic marker for the manufacture of a product for monitoring the effect of a myocardial peptide in the treatment of myocardial ischemia reperfusion injury, wherein the metabolic marker comprises aspartic acid and/or lysine.
2. Use of a detection reagent for a metabolic marker for the preparation of a product for monitoring the effect of a myocardial peptide in the treatment of myocardial ischemia reperfusion injury, wherein the metabolic marker comprises aspartic acid and/or lysine.
3. Use according to claim 1 or 2, wherein the product comprises a reagent or a kit.
4. The use of claim 1 or 2, wherein a decrease in the amount of aspartic acid and lysine in myocardial tissue as compared to before the myocardial ischemia reperfusion injury is treated with the myocardial peptide is indicative of the myocardial peptide functioning to treat myocardial ischemia reperfusion injury.
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