CN111647640A - Method for rapidly and accurately realizing classification of cardiac function and course of chronic heart failure - Google Patents

Method for rapidly and accurately realizing classification of cardiac function and course of chronic heart failure Download PDF

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CN111647640A
CN111647640A CN202010442068.0A CN202010442068A CN111647640A CN 111647640 A CN111647640 A CN 111647640A CN 202010442068 A CN202010442068 A CN 202010442068A CN 111647640 A CN111647640 A CN 111647640A
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heart failure
chronic heart
stage
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余伯阳
李芳�
赖琼
柴程芝
张鹿
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China Pharmaceutical University
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Abstract

The invention discloses a metabolic marker related to diagnosis or monitoring of chronic heart failure course and application thereof. The invention proves that the xanthurenic acid and the phenylacetylglycine can be used as metabolic markers of B and C grades of the heart function of the chronic heart failure respectively so as to realize the accurate grading of the heart function of the chronic heart failure, and the kynurenine 3-monooxygenase (KMO) and the aldehyde dehydrogenase family 1 member A3(ALDH1A3) can be used as targets for screening the medicines for preventing, relieving and/or treating myocardial ischemia injury with different heart function grades.

Description

Method for rapidly and accurately realizing classification of cardiac function and course of chronic heart failure
Technical Field
The invention belongs to the field of heart diseases, and particularly relates to a method for rapidly and accurately realizing the grading of the cardiac function and the course of chronic heart failure.
Background
With the continuing awareness and progression of disease, the american heart Association (ACC) and the american heart Association (AHC) have developed new grades that emphasize the disease progression and evolution. The concrete grading condition is as follows: stage A is a high-risk and susceptible population with no heart failure symptoms, and the left ventricle functions normally; stage B is asymptomatic, but has developed into an organic, structural heart disease, with abnormal left ventricular function; stage C is a basic structural heart disease with symptoms and signs of heart failure such as shortness of breath, decreased exercise tolerance, fluid retention and the like in the past or at present of the patient; stage D is refractory heart failure and may require treatment or end care such as heart transplantation. Wherein stages B and C are two important stages of the disease process in chronic heart failure.
At present, the diagnosis of chronic heart failure is mainly carried out clinically by measuring the content of myocardial injury markers such as hsCRP, BNP and hs-cTn and evaluating the cardiac function. But the determination of biochemical indicators can be influenced by other physiological conditions leading to erroneous diagnostic results, and there are very few biomarkers currently available for staging the heart failure stage. Based on the above practical problems, a better cardiac function evaluation method is urgently needed to achieve accurate diagnosis of chronic heart failure. The high-resolution and high-flux metabonomics technology can provide related metabolic characterization for different chronic heart failure stages, and then specific biomarkers of different chronic heart failure stages are selected, so that the clinical diagnosis and treatment of different stages of heart failure are facilitated.
Disclosure of Invention
In view of the shortcomings of the existing problems, the first object of the present invention is to provide the use of a metabolic marker for preparing a preparation for diagnosing or monitoring the course of chronic heart failure; it is a second object of the present invention to provide a diagnostic or monitoring formulation for the course of heart failure; the third purpose of the invention is to provide the application of the metabolic marker in preparing or screening the medicine for treating chronic heart failure; the fourth purpose is to provide the application of kynurenine-3-monooxygenase (KMO) and/or aldehyde dehydrogenase family 1 member A3(ALDH1A3) as an detection target point in preparing a preparation for diagnosing or monitoring the chronic heart failure course; the fifth purpose is to provide the application of KMO and/or ALDH1A3 as a therapeutic target in preparing or screening medicaments for treating chronic heart failure.
The technical scheme adopted by the invention for solving the technical problems is as follows:
use of a metabolic marker for the preparation of a diagnostic or monitoring formulation for the B and C stage course of chronic heart failure, said metabolic marker being xanthurenic acid and/or phenylacetylglycine.
A kit for diagnosing or monitoring the course of B and C stages of chronic heart failure contains the reagent for detecting xanthurenic acid and/or phenylacetylglycine.
The application of xanthurenic acid and/or phenylacetyl glycine as a metabolic marker in preparing or screening medicines for treating chronic heart failure.
Preferably, the chronic heart failure is in stage B or C
The application of KMO and/or ALDH1A3 as detection targets in preparations for diagnosing or monitoring the disease course of B and C stages of chronic heart failure.
Use of an agent for detecting KMO and/or ALDH1A3 for the preparation of a diagnostic or monitoring agent for the B-and C-stage course of chronic heart failure.
Kit for diagnosing or monitoring the course of stages B and C of chronic heart failure, comprising reagents for detecting KMO and/or ALDH1A 3.
The application of KMO and/or ALDH1A3 as a therapeutic target in preparing or screening medicines for treating chronic heart failure.
Preferably, the chronic heart failure is in stage B or C.
Use of a substance that inhibits the expression of KMO and/or ALDH1A3 in a medicament for the treatment of chronic heart failure.
Preferably, the chronic heart failure is in stage B or C.
Preferably, the substance for inhibiting the expression of KMO is UPF-648.
More preferably, the addition amount of the UPF-648 is 1-2 mg/kg.
Preferably, the substance inhibiting the expression of ALDH1A3 is CM 10.
More preferably, the addition amount of the CM10 is 0.32 to 1.28. mu.M.
The finding proves that the xanthurenic acid and the phenylacetylglycine can be used as metabolic markers of B and C grades of heart functions of chronic heart failure to realize accurate grading of the heart functions of the chronic heart failure, and the KMO and the ALDH1A3 can be used as targets for screening myocardial ischemia injury medicaments for preventing, relieving and/or treating different heart function grades.
The invention also discloses a method for simulating a mouse model of the chronic heart failure course, which comprises the following steps: carrying out intraperitoneal injection and anesthesia on a mouse by adopting 1% sodium pentobarbital, taking a supine position, removing hair from a left chest, coating iodophor for disinfection, and connecting an artificial respirator; after the cortex is longitudinally cut, separating the precordial muscles layer by layer in a blunt way until the ribs are exposed, puncturing the thoracic cavity in the intercostal space by using a pair of bent forceps, tearing off the pericardium, exposing the heart, and extruding the heart out of the thoracic cavity by slightly pressing the thorax; and (3) rapidly ligating a 6-0 silk thread together with the myocardium which passes through the thread at a position 3mm below the left anterior descending origin of the coronary artery, rapidly putting back the thoracic cavity after the ligation is finished, extruding gas and suturing, wherein the mouse model accords with the B stage of cardiac function of clinical chronic heart failure at 2-3 weeks, and the mouse model accords with the C stage of cardiac function of clinical chronic heart failure at 4-5 weeks.
Preferably, the parameters of the connection artificial respirator are as follows: tidal volume 3mL, breathing ratio 2:1, heart rate 110.
The invention also protects the application of the mouse model obtained by the method in a preparation for diagnosing or monitoring the course of chronic heart failure.
The invention also protects the application of the mouse model obtained by the method in preparing or screening the medicine for treating chronic heart failure.
Advantageous effects
(1) According to the invention, a chronic heart failure model mouse induced by coronary artery ligation is firstly utilized to mainly simulate the heart function B stage of clinical chronic heart failure in 2-3 weeks after the model, and mainly simulate the heart function C stage of clinical chronic heart failure in 4-5 weeks;
(2) the invention also discovers that the specificity of the xanthureic acid and the phenylacetylglycine is respectively increased in the urine of a clinical patient with chronic heart failure at the B stage and the C stage, and the KMO and the ALDH1A3 can be respectively used as key regulating enzymes of the xanthureic acid and the phenylacetylglycine;
(3) the invention also discovers that the inhibition of KMO and ALDH1A3 can effectively relieve myocardial ischemia injury.
Drawings
FIG. 1 shows the time course of heart function of a mouse model of chronic heart failure;#p<0.05 and##p<sham group (Sham group) 0.01vs.
FIG. 2 shows the time course change of the heart dimension of a mouse model of chronic heart failure;#p<0.05 and##p<0.01vs.Shamgroup。
FIG. 3 shows the time course change of the heart organ index of the chronic heart failure model mouse;#p<0.05and##p<0.01vs.Shamgroup。
FIG. 4 shows the time course of the serum Brain Natriuretic Peptide (BNP) and hypersensitive C-reactive protein (hs-CRP) content in chronic heart failure model mice; wherein, A) BNP.B) hs-CRP.#p<0.05 and##p<0.01vs.Sham group。
FIG. 5 shows the pathological changes of myocardial tissue and the degree of fibrosis in a mouse model of chronic heart failure.
FIG. 6 shows the change of edema degree of each organ of a chronic heart failure model mouse;#p<0.05 and##p<0.01vs.Shamgroup。
FIG. 7 is a graph showing the change in exercise tolerance of a mouse model of chronic heart failure;#p<0.05 and##p<0.01vs.Sham group。
FIG. 8 measurement of metabolite content in mouse plasma samples;#p<0.05 and##p<0.01vs.Sham group。
FIG. 9 measurement of metabolite content in mouse serum samples; wherein, A) 2-peanut tetraallylglycerophosphorylcholine (2-arachidonylalkylphosphonocholine). B) Nicotinic acid (Nicotinic acid). C) Picolinic acid (Picolinic acid).#p<0.05 and##p<0.01vs.Sham group。
FIG. 10 shows the measurement of the metabolite content in the urine sample of mouse; wherein A) xanthurenic acid (Xanthus nicotinicacid) B) Phenylacetylglycine (Phenylacetylgycene) C) 1-Methylguanosine (1-Methylguanosine) D) N-methyl-4-pyridone-3-carboxamide (4PY) E) fructosyllysine (Fructoserysine) F) 7-Methylguanine (7-Methylguanine).#p<0.05 and##p<0.01vs.Sham group。
FIG. 11 measurement of metabolite content in clinical plasma samples;#p<0.05 and##p<normal human group (normal group) p.0.01 vs<0.05,**p<0.01。
FIG. 12 measurement of metabolite content in clinical serum samples; wherein, A) 2-arachidonoyl glycerophosphocholine, B) nicotinic acid and C) picolinic acid.#p<0.05 and##p<0.01vs.normal group.*p<0.05,**p<0.01。
FIG. 13 measurement of metabolite content in clinical urine samples; wherein, A) xanthurenic acid, B) phenylacetylglycine, C) 1-methylguanosine, D) 7-methylguanine, E)4PY.F) fructose lysine.#p<0.05 and##p<0.01vs.normalgroup. *p<0.05,**p<0.01。
FIG. 14 shows the change of the content of xanthylic acid in the organs and tissues of the chronic heart failure model mouse at the B stage;#p<0.05,##p<0.01, Sham surgery team vs Model team (Sham vs Model).
FIG. 15 selection of differential genes in the metabolic pathway related to xanthurenic acid in B-stage heart tissue of chronic heart failure model mice using transcriptomics.
FIG. 16: key regulation enzyme protein expression conditions in a xanthurenic acid related metabolic pathway in heart tissues of the B and C stage chronic heart failure model mouse;#p<0.05 and##p<0.01vs.Sham group。
FIG. 17: b-stage chronic heart failure model mouseKMO expression in organ tissues;#p<0.05,##p<0.01,Sham vs Model。
FIG. 18: influence of KMO inhibitor on contents of xanthurenic acid and phenylacetyl glycine in urine of B-stage chronic heart failure model mouse;#p<0.05 and##p<0.01vs.Sham group.*p<0.05 and**p<0.01vs.Model group。
FIG. 19: effect of KMO inhibitors on the pathology of heart tissue and the degree of fibrosis in stage B chronic heart failure model mice.
FIG. 20 shows the effect of KMO inhibitor on biochemical markers associated with heart failure in serum of mice model of stage B chronic heart failure, wherein A) BNP.B) hs-CRP.C) tumor necrosis factor α (TNF- α). D) Malondialdehyde (MDA). E) Nitric Oxide (NO).#p<0.05 and##p<0.01vs.Sham group.*p<0.05 and**p<0.01vs.Model group。
FIG. 21: effect of KMO inhibitors on cardiac tissue ultrastructure in stage B chronic heart failure model mice.
FIG. 22: effect of KMO inhibitors on KMO expression in cardiac tissue of stage B chronic heart failure model mice;#p<0.05and##p<0.01vs.Sham group.*p<0.05and**p<0.01vs.Model group。
FIG. 23: effect of KMO inhibitors on viability of oxygen deprivation (OGD) induced damaged H9c2 cells;#p<0.05 and##p<0.01vs.Control group.*p<0.05 and**p<0.01vs.OGD group。
FIG. 24: effect of KMO inhibitors on Lactate Dehydrogenase (LDH) leakage rate from OGD-induced damaged H9c2 cells;#p<0.05 and##p<0.01vs.Control group.*p<0.05,**p<0.01vs.OGD group。
FIG. 25: the change of the phenylacetyl glycine content in each organ tissue of the chronic heart failure model mouse at the C stage.#p<0.05,##p<0.01,Sham vs Model。
FIG. 26: and selecting differential genes in a phenylacetylglycine related metabolic pathway in the C-stage heart tissue of the chronic heart failure model mouse by using transcriptomics.
FIG. 27 is a schematic view showing: chronic heart in stages B and CThe key regulation enzyme protein expression condition in a phenylacetylglycine related metabolic pathway in heart tissues of a mice failure model;#p<0.05 and##p<0.01vs.Sham group。
FIG. 28: the expression condition of ALDH1A3 in each organ tissue of a C-stage chronic heart failure model mouse;#p<0.05,##p<0.01, Sham vs Model。
FIG. 29: influence of ALDH1A3 inhibitor on phenylacetylglycine and xanthurenic acid content in urine of C stage chronic heart failure model mouse;#p<0.05 and##p<0.01vs.Sham group.*p<0.05 and**p<0.01vs.Model group。
FIG. 30: effect of ALDH1a3 inhibitors on heart histopathology and degree of fibrosis in stage C chronic heart failure model mice.
FIG. 31 shows the effect of ALDH1A3 inhibitor on biochemical markers associated with heart failure in serum of C-stage chronic heart failure model mice, wherein A) BNP.B) hs-CRP.C) MDA.D) TNF- α.#p<0.05 and##p<0.01vs.Sham group.*p<0.05and**p<0.01vs.Model group。
FIG. 32: effect of ALDH1a3 inhibitors on cardiac tissue ultrastructure in stage C chronic heart failure model mice.
FIG. 33: effect of ALDH1A3 inhibitors on ALDH1A3 expression in cardiac tissue of stage C chronic heart failure model mice;#p<0.05 and##p<0.01vs.Sham group.*p<0.05 and**p<0.01vs.Model group。
FIG. 34: effect of ALDH1a3 inhibitors on H9c2 cell viability of OGD-induced injury;#p<0.05 and##p<0.01 vs.Control group.*p<0.05 and**p<0.01vs.OGD group。
FIG. 35: effect of ALDH1a3 inhibitors on LDH leakage rate of OGD-induced damaged H9c2 cells;#p<0.05 and##p<0.01vs.Control group.*p<0.05,**p<0.01vs.OGD group。
Detailed Description
The present invention will be described in further detail with reference to examples. The reagents or instruments used are not indicated by manufacturers, and are regarded as conventional products which can be purchased in the market.
Example 1 time course of stage B and C in Chronic Heart failure model mice
Experimental methods
1 laboratory animal
The experimental animal is a male ICR mouse, the weight is 20-22g, the cleaning grade is provided by the comparative medicine center of Yangzhou university, and the quality standard of the experimental animal is met. The license number is SCXK (Su) 2017-. The mice are raised in cages with 5 mice per cage, the room temperature of the raising is 24 ℃, the relative humidity is 40-80%, each mouse can drink water and eat food freely, and the experiment is started 7 days after adaptive raising. 100 mice were randomly divided into 2 groups: 1) sham group (Sham, n ═ 10); 2) model group (Model, n is 90).
2 preparation of mouse Heart failure model
The mice were anesthetized by intraperitoneal injection of 1% pentobarbital sodium, and were supine, left chest depilated, sterilized by iodophor, and connected to a manual ventilator (tidal volume 3mL, breathing ratio 2:1, heart rate 110). After the cortex is longitudinally cut, the precordial muscles are separated layer by layer in a blunt manner until the ribs are exposed, the thoracic cavity is punctured by bending forceps in the intercostal space, the pericardium is torn open, the heart is exposed, and the heart can be extruded out of the thoracic cavity by slightly pressing the thorax. The part 3mm below the left anterior descending branch of the coronary artery and the myocardium through which the thread passes are ligated together by using a No. 6-0 silk thread quickly (the ligation is not performed when the thread is fed), and the ligation is completed and the thoracic cavity is put back quickly, and the air is squeezed out and sutured. And after the molding is finished, carrying out electrocardiogram test to check whether the molding is successful.
3 high frequency color Ultrasound (US) assay for small animals
Sending to animal experiment center of Nanjing medical university for echocardiography at days 1, 7, 14, 21, 28, 35, 42, 49 and 56 after molding. Isoflurane inhalation anesthetized mice were stood upward, and cardiac Ejection Fraction (EF), Fractional Shortening (FS), left ventricular septal thickness (IVS; d), left ventricular internal diameter (LVID; d), left ventricular posterior wall thickness (LVPW; d), corrected left ventricular mass (LV Masscorrect), left ventricular volume (LV Vol; d), pre-ejection period (PEP) and stroke per output (SV ═ LV Vold-LV Vols) of each group of mice were measured using a Visual sonic Vevo2100 special high-frequency color ultrasound apparatus.
4 measurement of BNP and hs-CRP content in serum
Blood is taken by an eyeball-picking method on days 1, 7, 14, 21, 28, 35, 42, 49 and 56 after the model is built, the blood sample is stood for 60min at room temperature and then centrifuged at 3500rpm for 10min, supernatant is taken, the content of BNP and hs-CRP in serum is measured by an ELISA kit (double antibody sandwich enzyme-linked immunosorbent assay), and the specific operation steps refer to the kit use instruction.
Detection of 5 cardiac organ index
The body weight of the experimental mouse was weighed and recorded on days 1, 7, 14, 21, 28, 35, 42, 49, and 56 after molding, the surface blood stain was wiped off with filter paper after the heart tissue was completely removed, and the wet weight of each organ was weighed and recorded on an electronic balance, whereby each organ index was obtained as the organ mass/body weight.
6 HE staining
The HE dyeing method comprises the following operation steps: placing the paraffin sections in an oven to bake for 1-2 h at 60 ℃; paraffin section normal xylene, ethanol dewaxing to water, hematoxylin staining for 10 minutes, washing with running water, removing residual color, 0.7% hydrochloric acid ethanol differentiation for several seconds, washing with running water, section blue for about 15 minutes, 7.95% ethanol for 30 seconds, 8. alcoholic eosin staining for 30 seconds, I95% ethanol for 30 seconds, II 95% ethanol for 30 seconds, I100% ethanol for 30 seconds, II 100% ethanol for 30 seconds, phenol for 30 seconds, (1: 4 phenol 1-xylene 4) I xylene for 30 seconds, II xylene for 30 seconds, neutral gum sealing piece.
7 Masson staining
Paraffin section is dewaxed to water; chromizing or mercury salt removing precipitation; sequentially washing tap water and distilled water; staining the nucleus with Harris hematoxylin staining solution or Weigart hematoxylin staining solution for 1-2 min; slightly washing with running water; differentiating with 0.5% hydrochloric acid alcohol for 15 s; flushing with running water for 3 min; dyeing the ponceau acid fuchsin liquid for 8 min; slightly washing with distilled water; treating with 1% phosphomolybdic acid water solution for about 5 min; directly re-dyeing with aniline blue solution or brilliant green solution for 5min without washing with water; treating with 1% glacial acetic acid for 1 min; dehydrating with 95% ethanol for 5min × 2 times, and drying with absorbent paper; 100% ethanol for 5min × 2 times, and drying the liquid with absorbent paper; transparent in xylene for 5min × 2 times, and sucking the liquid with absorbent paper; and (5) sealing the neutral gum.
Weight ratio of organ to organ
And completely taking out each organ on days 1, 7, 14, 21, 28, 35, 42, 49 and 56 after molding, wiping off surface bloodstains by using filter paper, weighing by using an electronic balance, recording the wet weight of each organ, baking for 72 hours at 90 ℃ in an oven, and weighing the dry weight of each organ, thereby obtaining the wet/dry mass ratio.
9 exhaustive swimming test
After the model was made, exhaustive swimming tests were performed on days 1, 7, 14, 21, 28, 35, 42, 49 and 56, and a lead block of 5% of the body mass was tied to the tail root of the mouse (the mouse was allowed to swim with a load and rapidly come to a state of exhaustion), the mouse with a load was placed in a swimming tank having a water depth of 30cm, a smooth inner wall and a water temperature of 25. + -. 2 ℃ for swimming, and the time of exhaustive swimming of the mouse (the time from the start of swimming until the head of the mouse completely sinks into the water and cannot float out of the water any more for 8 seconds) was recorded. After each swimming exercise, water is wiped off, and fur is dried by using a blower. Each swimming time to exhaustion standard.
Results of the experiment
1 time course change of heart function of chronic heart failure model mouse
The change of the heart function of the chronic heart failure model mice within 56 days is evaluated by using high-frequency color ultrasound, and the representative M-type echocardiogram of each group of mice is shown in figure 1, and the distance between the whole wave crest and the wave trough of the chronic heart failure model mice is shortened compared with that of a control group. Compared with a sham operation group, the ejection fraction of the mice in the model group is remarkably reduced at the 14 th day after coronary artery ligation and modeling; the fractional shortening continued to decline significantly after day 7; the pre-ejection period starts to be remarkably prolonged on the 28 th day, and is in a descending trend compared with a sham operation group in the first 14 days after the model building; the stroke volume decreased after day 1.
2 time course change of heart dimension of chronic heart failure model mouse
Further examination of the change in cardiac dimensions in the chronic heart failure model mice over 56 days with an echocardiograph revealed that the left ventricular septal thickness (fig. 2A), left ventricular internal diameter (fig. 2B), and left ventricular mass after correction (fig. 2C) in the model mice all increased significantly from day 14 to day 56 after coronary artery ligation modeling, while the left ventricular posterior wall thickness began to increase significantly on day 7 (fig. 2D), as compared to the sham group.
3 time course change of heart organ index of chronic heart failure model mouse
By examining the heart organ indexes of each group of mice, the condition of myocardial hypertrophy in 56 days of the chronic heart failure model mice is evaluated. As shown in FIG. 3, the index of the heart organs of the model group mice gradually increased in the whole body after the 1 st day of coronary artery ligation and modeling, and reached the highest point at the 35 th day, as compared with the sham group.
Time-course change of BNP (brain natriuretic peptide) and hs-CRP (human serum albumin-protein kinase) content in serum of 4 chronic heart failure model mouse
The measurement results of the BNP content in the serum of each group of mice are shown in FIG. 4A, compared with the sham operation group, the BNP content of the model group of mice shows a gradual increase trend after 7 days of coronary artery ligation modeling, and hs-CRP is more sensitive than BNP, and is remarkably increased after 1 day of modeling and gradually increased to 56 days (FIG. 4B).
5 change of myocardial tissue pathology and fibrosis degree of chronic heart failure model mouse
Changes in myocardial histopathology and degree of fibrosis within 56 days of chronic heart failure model mice were further assessed using HE and Masson staining, respectively. As shown in FIG. 5, the chronic heart failure model group mice induced by coronary artery ligation gradually increase the degree of myocardial fibrosis, hyperemia and edematous inflammatory cell infiltration with time, the pathological damage degree of heart tissues is lighter on days 14-21, and the damage degree is obviously increased on days 28-35.
6 change of edema degree of each organ of chronic heart failure model mouse
Fluid retention is a typical clinical representation of a chronic heart failure patient, and the edema degree of a chronic heart failure model mouse is evaluated by inspecting the change of the wet-dry mass ratio of 5 main visceral organs (heart, liver, spleen, lung and kidney) within 56 days of the chronic heart failure model mouse. Compared with the sham operation group, the mice in the model group have remarkable edema phenomena of heart, lung and kidney tissues on the 35 th day of coronary artery ligation and modeling, and obvious edema phenomena of liver and spleen tissues on the 28 th and 35 th days.
7 Change in exercise tolerance in Chronic Heart failure model mice
The chronic heart failure patients are easy to have clinical characteristics such as the decrease of exercise tolerance of different degrees, and the change condition of the exercise tolerance of the chronic heart failure model mouse within 56 days is further investigated through exhaustive swimming tests. As shown in FIG. 7, the mice in the model group showed a significant decrease in exhaustive swimming time after 28 days of coronary artery ligation and modeling, as compared with the sham-operated group.
The above experimental results show that: the chronic heart failure model mouse induced by coronary artery ligation develops organic and structural heart disease in 2-3 weeks after the model, and the left ventricle is abnormal in function, so that the model mouse conforms to the clinical B stage of the heart function of chronic heart failure; on the basis of organic and functional lesions generated in the 4 th to 5 th weeks, the chronic heart failure model mouse has obvious heart failure signs and symptoms such as reduced exercise tolerance and fluid retention, and accords with the clinical heart function C stage of chronic heart failure.
Example 2 xanthine acid and phenylacetylglycine can be used as metabolic markers in stage B and C of chronic Heart failure, respectively
Experimental methods
1 laboratory animal
See example 1.
2 clinical samples
The experiment was carried into 100 study subjects by the traditional Chinese medicine institute of Jiangsu province, and blood plasma, blood serum and urine were collected respectively. The three samples were collected and dispensed into 150. mu.L/tube immediately, and stored frozen at-80 ℃. All patients were clinically diagnosed into Normal healthy group (Normal group), chronic heart failure cardiac stage B group (B stage), and cardiac stage C group (C stage).
3 preparation of mouse Heart failure model
See example 1.
4 animal sample Collection
Urine: during collection, fasting is not forbidden, the mouse is placed in a metabolism cage to collect urine for 24 hours, the urine is placed in ice to keep low temperature, 4000r/min, and after low-temperature centrifugation is carried out for 5min at 4 ℃, supernatant is taken and is stored at minus 80 ℃.
Serum: fasting for 24h before collection without water supply, collecting blood by eye ball picking method, standing blood sample at room temperature for 60min, centrifuging at 3500rpm for 10min, collecting supernatant, packaging, and storing at-80 deg.C.
Plasma: fasting for 24h before collection without water supply, collecting blood by eye ball picking method, taking trisodium citrate as anticoagulant, standing the blood sample at room temperature for 10min, centrifuging at 2500rpm for 10min, collecting supernatant, packaging and storing at-80 deg.C.
5 pretreatment of the sample
Thawing frozen samples (serum, plasma and urine) at normal temperature, taking 50 mu L of biological sample, 10 mu L of internal standard working solution and 140 mu L of methanol, and uniformly mixing by vortex for 1 min. Centrifuging at 13000rpm at 4 ℃ for 30min to precipitate protein, taking 160 mu L of supernatant, centrifuging at 13000r/min at 4 ℃ for 30min again, taking 130ul of supernatant, filtering with a 0.22 mu m organic filter membrane, placing in a sample bottle, refrigerating at 4 ℃ for later use, and waiting for detection on a computer.
6 chromatographic conditions
The plasma sample was obtained using a Synergi Fusion-RP C18 column (50X 2mm i.d.,2.5 μm), the serum and urine samples were obtained using a TSK-GEL Amide-80(Part No.0019696) (150X 2.0mm i.d.,5 μm), the column temperature was 25 ℃, the autosampler temperature was 4 ℃ and the sample size was 5 μ L. Mobile phase composition: phase A is an aqueous solution containing 0.1% formic acid, and phase B is acetonitrile containing 0.1% formic acid. Gradient elution conditions for plasma samples: 5-10% B for 0-5min, 10-50% B for 5-10min, and 50-70% B for 10-15min, and then returning to the initial state and balancing for 5 min; the flow rate was 0.4 mL/min. Gradient elution conditions for serum samples: 80-75% B for 0-3min, 75-60% B for 3-8min, 60-50% B for 8-10min, and 50% B for 10-12min, then returning to initial state, and balancing for 5 min; the flow rate was 0.2 mL/min. Gradient elution conditions for urine samples: 70-65% B for 0-3min, 65-60% B for 3-8min, 60-50% B for 8-10min, and 50% B for 10-22min, then returning to initial state, and balancing for 5 min; the flow rate was 0.2 mL/min.
7 Mass Spectrometry Condition
An electrospray ionization source (ESI) is selected, positive ion mode detection is adopted, and a multi-reaction monitoring mode (MRM) is selected by a signal acquisition technology. Capillary and Nozzle voltages (The capillary and Nozzle voltages) were 3500V and 1500V, respectively. The temperature of the auxiliary gas was 350 ℃, the flow rate of the auxiliary gas was 10L/min, the temperature of the sheath gas was 350 ℃, the flow rate of the sheath gas was 12L/min, and the pressure of the atomizer was 50 psi.
Results of the experiment
1 measurement of metabolite content in mouse and clinical plasma samples
Determining the change of C18-Dihydrosphingosine (C18-Dihydrosphingosine) content in plasma of sham operation and chronic heart failure model mice at days 1, 7, 14, 21, 28, 35, 42, 49 and 56 after coronary artery ligation by using LC-MS; the change in C18-dihydrosphingosine content in clinical plasma samples from stage B and C of chronic heart failure was determined using LC-MS in the normal healthy group.
The results showed that the plasma C18-dihydrosphingosine content gradually decreased, and the results are shown in FIG. 8 and FIG. 11, respectively.
2 determination of metabolite content in mouse and clinical serum samples
LC-MS was used to determine the changes in the serum contents of 2-arachidonoyl glycerophosphorylcholine, nicotinic acid and picolinic acid on days 1, 7, 14, 21, 28, 35, 42, 49 and 56 after coronary artery ligation in sham surgery and chronic heart failure model mice. The results showed that 2-arachidonoyl glycerophosphocholine content in serum increased from day 1 to week 3 after coronary artery ligation, but decreased at weeks 4-5 and significantly decreased at weeks 4-5 compared to weeks 2-3, compared to the sham group (fig. 9A); the nicotinic acid content in the serum gradually decreased after coronary artery ligation, and was more decreased in 2-3 weeks than in 4-5 weeks (fig. 9B); the content of picolinic acid in serum gradually decreased after coronary artery ligation (fig. 9C).
LC-MS was used to determine the changes in 2-arachidonoyl glycerophosphocholine, nicotinic acid and picolinic acid content in clinical serum samples from normal healthy groups, chronic heart failure stage B and C. The result shows that compared with the normal healthy group, the content of the 2-arachidonoyl glycerophosphorylcholine in the serum sample in the B stage of the chronic heart failure is obviously increased, and the serum sample tends to be normal in the C stage of the chronic heart failure; the content of nicotinic acid and picolinic acid in clinical serum samples is obviously reduced in the B stage and the C stage of chronic heart failure.
3 determination result of metabolite content in mouse urine sample
Changes in the contents of phenylacetylglycine, N-methyl-4-pyridone-3-carboxamide, fructosyllysine, 1-methylguanosine, 7-methylguanosine and xanthurenic acid in urine at days 1, 7, 14, 21, 28, 35, 42, 49 and 56 after coronary artery ligation in sham operated and chronic heart failure model mice were determined by LC-MS. The results show that the content of the xanthurenic acid (fig. 10A) in urine is obviously increased 2-3 weeks after coronary artery ligation, and gradually approaches to the content level of the pseudo surgery within 4-5 weeks compared with the pseudo surgery group; both phenylacetylglycine (fig. 10B) and 1-methylguanosine (fig. 10C) levels in the urine did not change significantly 2-3 weeks after coronary artery ligation, but began to rise significantly at 4-5 weeks; the content of N-methyl-4-pyridone-3-carboxamide in urine (fig. 10D) was significantly increased before week 1 after coronary artery ligation, but was significantly decreased at weeks 2-5; the content of fructosyllysine in urine (fig. 10E) decreased after coronary artery ligation; the 7-methylguanine content in urine (fig. 10F) increased significantly between 2 and 5 weeks after coronary artery ligation.
4 determination result of metabolite content in clinical urine sample
Changes in phenylacetylglycine, N-methyl-4-pyridone-3-carboxamide, fructosyllysine, 1-methylguanosine, 7-methylguanine and xanthurenic acid content in normal healthy group, chronic heart failure stage B and C clinical urine samples were determined by LC-MS. The result shows that compared with the normal healthy group, the content of the xanthurenic acid in the urine sample is obviously increased only in the stage B of the chronic heart failure, and the urine sample tends to be normal in the stage C of the chronic heart failure; the contents of phenylacetyl glycine, 1-methylguanosine and 7-methylguanosine in the clinical urine sample are not obviously changed in the B stage of chronic heart failure, but are obviously increased in the urine sample in the C stage; the contents of N-methyl-4-pyridone-3-carboxamide and fructose lysine in clinical urine samples were significantly reduced in both stages B and C of chronic heart failure, and there was no significant difference between the two groups.
The above experimental results show that: the xanthurenic acid and phenylacetyl glycine are specifically increased in urine of clinical patients with chronic heart failure at the B stage and the C stage and mice respectively, and can be used for diagnosing the B grade and the C grade of heart functions of chronic heart failure.
Example 3 KMO regulates the increase of xanthurenic acid and can be used as a novel target for the treatment of stage B chronic heart failure
Experimental methods
1 laboratory animal
See example 1 for additional details, except that 60 mice were randomly assigned to 6 groups:
1) sham group (Sham, n ═ 10): carrying out intraperitoneal injection (ip) on sodium chloride injection with equal volume amount after operation, starting from the 1 st day after molding, and continuously carrying out injection for 14 days for 1 time/day;
2) model group (Model-2w, n is 10): carrying out intraperitoneal injection (ip) on sodium chloride injection with equal volume amount after operation, starting from the 1 st day after molding, and continuously carrying out injection for 14 days for 1 time/day;
3) model group (Model-5w, n 10): carrying out intraperitoneal injection (ip) on sodium chloride injection with equal volume amount after operation, starting from the 1 st day after the model is made, and continuously carrying out 35 days for 1 time/day;
4) KMO inhibitor high dose group (UPF-648-H, n ═ 10): injecting 2mg/kg of UPF-648 solution into abdominal cavity after operation, starting from 1d after model building, and continuing for 14 days and 1 time/day;
5) KMO inhibitor low dose group (UPF-648-L, n ═ 10): injecting (ip) UPF-648 solution into abdominal cavity after operation at 1mg/kg, starting at 1d after molding, 1 time/3 days, and taking 14 days;
6) metoprolol group (Met, n ═ 10): intragastric (ig) metoprolol 5.14mg/kg after surgery, starting at 1d after molding and continuing for 14 days.
2 cells
H9c2(2-1) cardiomyocyte cell line, purchased from Shanghai academy of sciences.
3 preparation of mouse Heart failure model
See example 1.
4 LC-MS tissue sample Collection
After the mice are sacrificed, the tissues such as heart, liver, spleen, lung, kidney, brain and the like are quickly dissected and taken out, blood stains on the surfaces of the tissues are rinsed by normal saline, residual water stains are sucked by filter paper, the tissues are weighed, quantitative normal saline (1g of tissue and 2mL of normal saline) is added, and the tissues are prepared into homogenate by a glass homogenizer and are subpackaged and stored at-80 ℃.
Pretreatment of 5 LC-MS samples
Thawing frozen tissue samples (heart, liver, spleen, lung, kidney and brain) at normal temperature, taking 50 μ L of tissue sample, 10 μ L of internal standard working solution and 140 μ L of methanol, and mixing by vortex for 1 min. Centrifuging at 13000rpm at 4 ℃ for 30min to precipitate protein, taking 160 mu L of supernatant, centrifuging at 13000r/min at 4 ℃ for 30min again, taking 130ul of supernatant, filtering with a 0.22 mu m organic filter membrane, placing in a sample bottle, refrigerating at 4 ℃ for later use, and waiting for detection on a computer.
6 LC-MS chromatographic conditions
The tissue sample was subjected to a chromatographic column TSK-GEL Amide-80(Part No.0019696) (150X 2.0 mm. d.,5 μm) at 25 ℃ in each case, 4 ℃ in each case and 5 μ L in each case. Mobile phase composition: phase A is an aqueous solution containing 0.1% formic acid, and phase B is acetonitrile containing 0.1% formic acid. Gradient elution conditions of the sample: 70-65% B for 0-3min, 65-60% B for 3-8min, 60-50% B for 8-10min, and 50% B for 10-22min, then returning to initial state, and balancing for 5 min; the flow rate was 0.2 mL/min.
7 LC-MS Mass Spectrometry conditions
An electrospray ionization source (ESI) is selected, positive ion mode detection is adopted, and a multi-reaction monitoring mode (MRM) is selected by a signal acquisition technology. Capillary and Nozzle voltages (The capillary and Nozzle voltages) were 3500V and 1500V, respectively. The temperature of the auxiliary gas was 350 ℃, the flow rate of the auxiliary gas was 10L/min, the temperature of the sheath gas was 350 ℃, the flow rate of the sheath gas was 12L/min, and the pressure of the atomizer was 50 psi.
8 Western Blotting method for detecting protein expression
The hearts of the Sham (Sham), Model (Model-2w) and Model (Model-5w) mice were perfused with physiological saline and then removed, the remaining parts except the left ventricle of the heart were removed, homogenized with a homogenate containing 1mM PMSF, and quantified with a BCA kit. Each set of samples was loaded on 10% SDS-PAGE gels and the transmembrane bands were incubated with the corresponding primary antibody overnight at 4 ℃. After washing, protein bands on the PVDF membrane are incubated corresponding to 2 antibodies, an ECL kit is used for developing color, the bands are analyzed after being exposed by a gel imager, and the protein content is expressed as a relative value corresponding to an internal reference band. Western blotting is mainly used for investigating the expression condition of the regulatory enzyme A-E protein in heart tissues of various groups of mice.
9 immunohistochemical staining
Taking each organ tissue, and regulating and controlling the expression condition of the enzyme by utilizing immunohistochemical analysis. The hearts were removed and fixed in 4% paraformaldehyde, embedded in paraffin and cut into 4 μm sections. Sections were hydrated with PBS and placed in 3% hydrogen peroxide solution to block peroxidase. The sections were removed and placed in 37 ℃ blocking solution for 1 hour and primary antibody (1: 100) was incubated overnight at 4 ℃. After washing with PBS, the sections were incubated with HRP-Conjugated Secondary antibody (1: 200) at 37 ℃ for 1 hour. After DAB staining, hematoxylin counterstaining and segmental dehydration, the sections were mounted and observed under a 200X microscope.
10 determination of biochemical index content in serum
Blood is taken by an eyeball method, a blood sample is stood at room temperature for 60min and then centrifuged at 3500rpm for 10min, supernatant is taken, the contents of BNP, hs-CRP, TNF-alpha, MDA and NO in the blood serum are measured by an ELISA kit (double antibody sandwich enzyme-linked immunosorbent assay), and the specific operation steps refer to the kit use instruction.
11 HE staining
See example 1.
12 Masson staining
See example 1.
13 Transmission Electron microscopy
1) Fixing: collecting the treated heart tissue of the mouse, respectively adding 1ml of 2.5% glutaraldehyde, fully shaking for about 2min, shaking for 1 time every 15min, and then placing in a refrigerator at 4 ℃ for fixing for more than 2h for later use;
2) rinsing: the cells were washed 3 times for 15min with 0.1M PBS buffer (pH 7.2). To remove residual glutaraldehyde;
3) post-fixing: moving the tissue specimen into a centrifugal tube filled with 1% osmic acid fixing solution, sufficiently shaking for about 5min to allow the osmic acid to sufficiently soak the heart tissue, and fixing for 1 h;
4) rinsing: the cells were washed 3 times for 15min with 0.1M PBS buffer (pH 7.2). Rinsing with double distilled water once, shaking for about 10min, and removing PBS;
5) block dyeing: treating with 1% uranium acetate staining solution for 2h, sucking off the staining solution when precipitate appears, and continuously washing with double distilled water;
6) gradient dehydration: soaking the specimen in 50%, 70%, 80% and 90% acetone respectively, dehydrating for 15min, and soaking in pure acetone for 2 times for 20 min;
7) and (3) infiltration: acetone/embedding medium ═ 1: 1, drying the mixture in an oven at 37 ℃ for 1 h; acetone/embedding medium ═ 1: 4, oven at 37 ℃ overnight; oven drying at 45 deg.C for 1h in pure embedding liquid;
8) embedding polymerization: drying in an oven;
9) ultrathin slicing;
10) and observing and taking pictures by a transmission electron microscope.
14 cell culture and grouping
14.1 culture of H9c2 cardiomyocytes
Rat cardiomyocyte line (H9c2(2-1)) was cultured in 10% FBS-containing DMEM medium at 37 ℃ and 5% CO2Culturing in an incubator, allowing cells to grow adherently, subculturing when the cells grow over 80%, discarding culture solution, adding 2mL of PBS (phosphate buffer solution), gently washing, discarding washing solution, adding 2mL of 0.25% pancreatin solution, digesting at 37 ℃ until the cell morphology shrinks and becomes round, discarding digestion solution, adding culture medium containing 10% FBS (fetal bovine serum) to stop digestion, repeatedly blowing, transferring cell suspension to a centrifuge tube, centrifuging at 1000rpm for 5min, discarding supernatant, resuspending cells in culture medium containing 10% FBS, and performing 1 × 105cells/mL were inoculated in fresh flasks at 37 ℃ with 5% CO2Culturing in an incubator.
14.2 cryopreservation of H9c2 cardiomyocytes
Taking cells in logarithmic phase, digesting the cells with 0.25% trypsin, adding DMEM medium containing 10% FBS to stop digestion, collecting cell suspension, centrifuging at 1000rpm for 5min, removing supernatant, re-suspending the cells in frozen stock solution, wherein the cells in the frozen stock solution are 2 × 106The number of cells/mL, split into frozen tube. Aseptically sealing the freezing tube, immediately placing in a refrigerator at 4 ℃ for 30min, transferring to-20 ℃, placing for 2h, placing in a low-temperature refrigerator at-70 ℃ overnight (16-18 h), and transferring to liquid nitrogen for preservation the next day.
14.3H 9c2 cardiac myocyte resuscitation
The cell cryopreservation tube was taken out of the liquid nitrogen tank and immediately placed in the container 37And (4) rapidly shaking in a water bath at the temperature of 1 ℃ to completely melt the cells in the frozen tube. Centrifuging at 1000rpm for 5min at room temperature, discarding supernatant, adding 1mL DMEM complete culture medium for resuspension, blowing uniformly, transferring cell suspension to culture flask, adding sufficient culture medium, and culturing at 37 deg.C and 5% CO2Culturing in an incubator.
14.4 Experimental groups
Taking rat myocardial cells of logarithmic growth phase H9c2, digesting, re-suspending with 10% FBS-containing DMEM medium, inoculating in 96-well plate with inoculation density of 6 × 104Each well of each cell/mL was filled with 100. mu.L of a medium containing 10% FBS, and the medium was left at 37 ℃ and 5% CO2Normally culturing in an incubator, and establishing N according to the following method when the cells enter logarithmic growth phase2The Oxygen and Glucose Depletion (OGD) model of the hypoxia chamber is grouped as follows:
1) normal culture group (Control): adopting normal culture based on 5% CO at 37 deg.C2Normally culturing in an incubator;
2) normal culture group + UPF-648 high dose group (Control + UPF-648-H): adding UPF-6482 μ M, and culturing at 37 deg.C with 5% CO2Normally culturing in an incubator;
3) normal culture group + UPF-648 Medium dose group (Control + UPF-648-M): adding UPF-6481 μ M, and culturing at 37 deg.C with 5% CO2Normally culturing in an incubator;
4) normal culture group + UPF-648 Low dose group (Control + UPF-648-L): adding UPF-6480.5 μ M, and culturing at 37 deg.C with 5% CO2Normally culturing in an incubator;
5) OGD model set (OGD): cells entering logarithmic growth phase for experiment were used with cells previously subjected to 94% N2+5％CO2+1％O2The sugar-free culture medium with the mixed gas saturated for 1h replaces the normal culture solution, and then 94% N is introduced2+5％ CO2+1％O2And (3) performing anaerobic culture for 6 hours in a constant-temperature incubator at 37 ℃.
6) OGD model group + UPF-648 high dose group (OGD + UPF-648-H): after addition of UPF-6482. mu.M, 94% N2+ 5% CO was added2+1％O2Replacing the normal culture medium with a sugar-free culture medium with the mixed gas saturated for 1h, and carrying out anoxic culture for 6 h.
7) OGD model group + UPF-648 middle dose group (OGD + UPF-648-M): after addition of UPF-6481. mu.M, 94% N was added2+5％CO 2+1％O2Replacing the normal culture medium with a sugar-free culture medium with the mixed gas saturated for 1h, and carrying out anoxic culture for 6 h.
8) OGD model group + UPF-648 low dose group (OGD + UPF-648-L): after addition of UPF-6480.5. mu.M, 94% N was added simultaneously2+5％CO2+1％O2Replacing the normal culture medium with a sugar-free culture medium with the mixed gas saturated for 1h, and carrying out anoxic culture for 6 h.
15 cell viability assay
Taking rat myocardial cells of logarithmic growth phase H9c2, digesting, inoculating in 96-well culture plate, 37 deg.C, 5% CO2Culturing under saturated humidity. After the cells of each experimental group are treated, the prepared MTT is added for incubation. The OD value of each well was measured by an enzyme-linked immunosorbent assay (measurement wavelength 570nm, reference wavelength 650 nm). And calculating the cell activity.
Detection of LDH Release in 16 cell supernatants
Collecting rat myocardial cells of logarithmic growth phase H9c2, digesting, inoculating to 96-well culture plate, inoculating 100 μ L of cells with cell concentration of 1 × 105/mL, inoculating to the culture plate at 37 deg.C and 5% CO2Culturing under saturated humidity for 24 h. After the cells of each experimental group are subjected to administration or oxygen sugar deprivation treatment, culture solution supernatant is taken and measured according to the description of an LDH kit, and the LDH release amount is measured by adopting a 2, 4-dinitrophenylhydrazine chromogenic method.
Results of the experiment
1 change of content of yellow uric acid in B-stage organ tissues of chronic heart failure model mouse
LC-MS is used for quantitatively detecting the content of the xanthurenic acid in heart, liver, spleen, lung, kidney and brain tissues of a model mouse in the B stage of the pseudo-operation and the chronic heart failure. Specific results are shown in fig. 14, compared with the sham operation group, the content of the xanthureic acid in the heart tissue of the model mouse at the B stage of the chronic heart failure is obviously increased, the content of the xanthureic acid in the liver, the kidney and the spleen tissue is obviously lower than that of the sham operation, and the model mouse at the B stage in the lung and the brain tissue has no obvious difference with the sham operation group.
2 selecting differential genes in xanthurenic acid related metabolic pathway in B-stage heart tissue of chronic heart failure model mouse by using transcriptomics
Xanthurenic acid is mainly involved in tryptophan metabolic pathway, as shown in fig. 15A. Based on the proproteomics results, wherein the key regulatory enzyme genes involved in the xanthureic acid related metabolic pathway comprise a member 1 of the HemK methyltransferase family (HEMK1), kynurenine aminotransferase 1(KAT1) and KMO, the RNA levels of the selected differential genes in heart tissues of mice in the sham operation group and the chronic heart failure model in the B stage are shown in FIGS. 15B-D, and compared with the sham operation group, the RNA levels of HEMK1 and KAT1 in the heart tissues of mice in the chronic heart failure model in the B stage are remarkably reduced, and the KMO is remarkably increased.
Expression condition of key regulatory enzyme protein in Xanthurenic acid related metabolic pathway in 3B and C stage chronic heart failure model mouse heart tissue
The expression conditions of key regulatory enzyme protein KAT1, kynurenine aminotransferase 3(KAT3), KMO, Kynurenine (KYNU) and HEMK1 in Xanthus acid related metabolic pathways in heart tissues of mice in a sham-operated group and a B-stage and C-stage chronic heart failure model are respectively detected by using a Western Blotting method. Specific results are shown in fig. 16, compared with the sham operation, the expression of KAT3, KMO and KYNU in the heart tissue of the B-stage chronic heart failure model mouse is obviously increased, and the expression is not obviously different from that of the sham operation group in the C-stage chronic heart failure model mouse. The expression of HEMK1 is significantly reduced, but the expression also tends to be reduced in the C stage. Furthermore, KAT1 did not change significantly in stage B until stage C rose significantly.
KMO expression condition in organ tissues of 4B stage chronic heart failure model mouse
The KMO expression conditions in heart tissues, liver tissues, spleen tissues, lung tissues, kidney tissues and brain tissues of mice in the sham operation group and the B-stage chronic heart failure model group are inspected by using an immunohistochemical technology, and the results are shown in figure 17.
Influence of 5 KMO inhibitor on contents of xanthurenic acid and phenylacetylglycine in urine of B-stage chronic heart failure model mouse
LC-MS is utilized to investigate whether the KMO inhibitor UPF-648 can effectively inhibit the rise of the content of the xanthurenic acid in the urine of the B-stage chronic heart failure model mouse, the result is shown in figure 18A, the UPF-648 with the dose of 2mg/kg and 1mg/kg can effectively inhibit the rise of the content of the xanthurenic acid in the urine of the B-stage chronic heart failure model mouse, and the positive drug metoprolol has no inhibition effect on the rise of the content. The effect of the KMO inhibitor UPF-648 on the phenylacetylglycine content of the urine of each group of mice was also examined, and the results are shown in FIG. 18B, wherein the UPF-648 was used at a dose of 2mg/kg and 1mg/kg, which had no effect on the phenylacetylglycine content of the urine of each group of mice. .
Effect of 6 KMO inhibitor on heart histopathology and fibrosis degree of B-stage chronic heart failure model mouse
Changes in myocardial histopathology and degree of fibrosis in mice model stage B chronic heart failure following administration of the KMO inhibitor UPF-648 were assessed by HE and Masson staining, respectively. The results are shown in FIG. 19, in the B-stage chronic heart failure mice, the phenomena of obvious myocardial fibrosis, congestion, edematous inflammatory cell infiltration and the like appear. Both UPF-648 doses of 2mg/kg and 1mg/kg were effective in improving the pathology and fibrosis levels in model mice, and the highest effect was achieved with high doses of UPF-648.
Influence of 7 KMO inhibitor on biochemical indexes related to heart failure in serum of B-stage chronic heart failure model mouse
The influence and change of the KMO inhibitor UPF-648 on biochemical indexes related to the heart failure in the serum of a B-stage chronic heart failure model mouse are examined by an ELISA kit method, and the biochemical indexes mainly comprise BNP, hs-CRP, TNF-alpha, MDA and NO. The results are shown in fig. 20, and compared with the sham operation group, the biochemical indexes of the serum central failure of the mice with the chronic heart failure in the B stage are obviously increased, which indicates that the model building is successful. Compared with a model group, the UPF-648 with the dose of 2mg/kg has better regulation and control effects on biochemical indexes of the heart failure such as BNP, hs-CRP, TNF-alpha, MDA and NO in the serum of a B-stage chronic heart failure mouse, and the UPF-648 with the dose of 1mg/kg has better improvement effects on the rest biochemical indexes hs-CRP, TNF-alpha, MDA and NO except the BNP.
Influence of 8 KMO inhibitor on ultrastructure of heart tissue of B-stage chronic heart failure model mouse
The result of the transmission electron microscope is shown in fig. 21, the arrangement of the myocardial cells of the mice in the sham operation group is relatively regular, the arrangement of the muscle fibers is relatively regular, the connection among the cells is clear and uninterrupted, and the structure is relatively complete; the cytoplasm has well-aligned myofibrils; the cytoplasm contains a large number of mitochondria and has complete structure; the nucleus is centrally located and structurally intact. Compared with a sham operation group, the mouse myocardial cell myofibril arrangement of the B-stage chronic heart failure model group is disordered, and the phenomena of separation, fracture, dissolution and the like occur, the intercellular connection is fuzzy, the number of mitochondria is increased and accumulated, the shape is swollen and dissolved, and a plurality of vacuoles appear, thereby indicating that the molding is successful. The phenomena of myocardial fibrolysis of mouse cardiomyocytes in high and low doses (1mg/kg and 2mg/kg) of the KMO inhibitor UPF-648 and the metoprolol positive medicine group are reduced compared with a model group, the arrangement of myofibrils of the cardiomyocytes is recovered to be normal, and the form of cell nuclei also tends to be normal.
Effect of 9 KMO inhibitor on KMO expression in cardiac tissue of mice model of stage B chronic heart failure
The influence of the KMO inhibitor UPF-648 on the KMO expression in the heart tissue of the B-stage chronic heart failure model mouse is examined by using an immunohistochemical technology, the result is shown in figure 22, compared with a false operation, the KMO expression level in the heart tissue of the B-stage chronic heart failure model mouse is obviously increased, the result is consistent with the previous research result, and after the KMO inhibitor UPF-648 is administered, the high-low dose group shows a better KMO expression inhibition effect.
Effect of 10 KMO inhibitors on H9c2 cell viability in OGD-induced injury
H9c2 myocardial cells were cultured in an in vivo ischemic environment simulated by a molding method using oxygen deprivation, and the effect of the KMO inhibitor UPF-648 on the viability of H9c2 cells damaged by OGD induction was examined by the MTT method. The results are shown in fig. 23, and compared with the blank group, the OGD group H9c2 with 6H hypoxia caused the cell viability to be reduced significantly, indicating that the modeling was successful. Compared with the OGD group, the KMO inhibitor UPF-648(1 mu M and 2 mu M) is given to remarkably improve the cell viability of the model cells, and has no cell viability influence on the cells of the white blood group.
Influence of 11 KMO inhibitor on LDH leakage rate of OGD-induced damaged H9c2 cells
The influence of the KMO inhibitor UPF-648 on LDH leakage rate of H9c2 cells induced by OGD is examined by using an LDH kit, and the result is shown in figure 24, the LDH leakage rate of H9c2 cells is obviously increased by OGD stimulation, 2 mu M of UPF-648 has obvious inhibition effect on LDH release of H9c2 cells caused by OGD, which indicates that UPF-648 can reduce cell membrane damage caused by OGD and has no obvious influence on LDH leakage rate of normal group cells.
The above experimental results show that: kynurenine 3-monooxygenase (KMO) is expressed specifically in heart tissues of mice in the B stage of chronic heart failure, and is a key regulatory enzyme for causing the specific increase of xanthurenic acid in urine of the mice in the B stage of chronic heart failure. In addition, myocardial ischemia injury can be effectively relieved by inhibiting KMO.
Example 4 ALDH1A3 modulating the increase in phenylacetylglycine as a novel target for the C-stage treatment of chronic heart failure
Experimental methods
1 laboratory animal
See example 1 for additional details, except that 60 mice were randomly assigned to 6 groups:
1) sham group (Sham, n ═ 10): carrying out intraperitoneal injection (ip) on sodium chloride injection with equal volume amount after operation, starting from the 1 st day after the model is made, and continuously carrying out 35 days for 1 time/day;
2) model group (Model-2w, n is 10): carrying out intraperitoneal injection (ip) on sodium chloride injection with equal volume amount after operation, starting from the 1 st day after molding, and continuously carrying out injection for 14 days for 1 time/day;
3) model group (Model-5w, n 10): carrying out intraperitoneal injection (ip) on sodium chloride injection with equal volume amount after operation, starting from the 1 st day after the model is made, and continuously carrying out 35 days for 1 time/day;
4) ALDH1a3 inhibitor high dose group (CM 10-H, n ═ 10): injecting (ip) CM10 solution into abdominal cavity 2mg/kg after operation, beginning at 1d after molding, 1 time/3 days, and 35 days;
5) low dose group of ALDH1a3 inhibitor (CM 10-L, n ═ 10): injecting (ip) CM10 solution into abdominal cavity 1mg/kg after operation, beginning at 1d after molding, 1 time/3 days, and 35 days;
6) metoprolol group (Met, n ═ 10): intragastric (ig) metoprolol 5.14mg/kg after surgery, beginning at 1d after molding, for 35 consecutive days, 1 time/day.
2 preparation of mouse Heart failure model
See example 1.
3 LC-MS tissue sample Collection
See example 3.
Pretreatment of 4 LC-MS samples
See example 3.
5 LC-MS chromatographic conditions
See example 3.
6 LC-MS Mass Spectrometry conditions
See example 3.
7 Western Blotting method for detecting protein expression
And (3) taking heart homogenates of the mice of the Sham operation group (Sham), the Model group (Model-2w) and the Model group (Model-5w) for Western blotting detection, and mainly inspecting the expression conditions of ALDH1A3, ALDH3B1 and ALDH3B2 proteins in heart tissues of the mice of each group. See 3.1.8 for the rest of the steps.
8 immunohistochemical staining
Taking each organ tissue, and analyzing the expression condition of ALDH1A3 by immunohistochemistry. See 3.1.9 for the remaining steps.
9 determination of content of biochemical index in serum
Blood is taken by an eyeball taking method, a blood sample is stood at room temperature for 60min and then centrifuged at 3500rpm for 10min, supernatant is taken, the contents of BNP, hs-CRP, TNF-alpha and MDA in the blood serum are measured by an ELISA kit (double antibody sandwich enzyme-linked immunosorbent assay), and the specific operation steps refer to the kit use instruction.
10 HE staining
See example 1.
11 Masson staining
See example 1.
12 transmission electron microscopy inspection
See example 3.
13 cell culture and grouping
13.1 culture of H9c2 cardiomyocytes
See example 3.
13.2 cryopreservation of H9c2 cardiomyocytes
See example 3.
13.3H 9c2 cardiac myocyte resuscitation
See example 3.
13.4 Experimental groups
Collecting rat myocardial cells of logarithmic growth phase H9c2, digesting, re-suspending with 10% FBS-containing DMEM medium, inoculating in 96-well plate at density of 6 × 104 cells/mL, adding 100 μ L10% FBS-containing medium per well, standing at 37 deg.C and 5% CO2Normally culturing in an incubator, and establishing an N2 anoxic OGD model according to the following method when cells enter a logarithmic growth phase, wherein the specific grouping conditions are as follows:
1) normal culture group (Control): adopting normal culture based on 5% CO at 37 deg.C2Normally culturing in an incubator;
2) normal culture group + CM10 high dose group (Control + UPF-648-H): after addition of CM 101.28. mu.M, the medium was cultured in a normal medium at 37 ℃ under 5% CO2Normally culturing in an incubator;
3) normal culture group + CM10 Medium dose group (Control + UPF-648-M): after adding CM 100.64. mu.M, the normal medium was used at 37 ℃ with 5% CO2Normally culturing in an incubator;
4) normal culture group + CM10 Low dose group (Control + UPF-648-L): after addition of CM 100.32. mu.M, the medium was cultured in a normal medium at 37 ℃ under 5% CO2Normally culturing in an incubator;
5) OGD model set (OGD): cells entering logarithmic growth phase for experiment were used with cells previously subjected to 94% N2+5％CO2+1％O2The sugar-free culture medium with the mixed gas saturated for 1h replaces the normal culture solution, and then 94% N is introduced2+5％CO2+1％O2And (3) performing anaerobic culture for 6 hours in a constant-temperature incubator at 37 ℃.
6) OGD model group + CM10 high dose group (OGD + UPF-648-H): after addition of CM 101.28. mu.M, 94% N was added simultaneously2+5％CO2+1％O2Replacing the normal culture medium with a sugar-free culture medium with the mixed gas saturated for 1h, and carrying out anoxic culture for 6 h.
7) OGD model group + CM10 mid-dose group (OGD + UPF-648-M): after addition of CM 100.64. mu.M, 94% N was added simultaneously2+5％CO2+1％O2Replacing the normal culture medium with a sugar-free culture medium with the mixed gas saturated for 1h, and carrying out anoxic culture for 6 h.
8) OGD model group + CM10 Low dose group (OGD + UPF-648-L): after addition of CM 100.32. mu.M, 94% N was added simultaneously2+5％CO2+1％O2Replacing the normal culture medium with a sugar-free culture medium with the mixed gas saturated for 1h, and carrying out anoxic culture for 6 h.
14 cell viability assay
See example 3.
15 detection of LDH Release in cell supernatants
See example 3.
Results of the experiment
1 change of phenylacetylglycine content in C-stage organ tissues of chronic heart failure model mouse
LC-MS is used for quantitatively detecting the content of phenylacetyl glycine in heart, liver, spleen, lung, kidney and brain tissues of a model mouse in the C stage of the pseudo-operation and the chronic heart failure. Specific results are shown in fig. 25, compared with the sham group, the content of phenylacetyl glycine in the center and lung tissues of the model mouse at the C-stage of chronic heart failure is obviously increased, while the content of phenylacetyl glycine in the liver and spleen tissues is obviously lower than that of the sham operation, and the model mouse at the C-stage in kidney and brain tissues has no obvious difference with the sham operation group.
2 selecting differential genes in phenylacetylglycine related metabolic pathway in heart tissue at C stage of chronic heart failure model mouse by using transcriptomics
Phenylacetylglycine is mainly involved in phenylalanine metabolic pathway, as shown in fig. 26A. Based on proproteomics results, the key regulatory enzyme genes involved in phenylacetylglycine-related metabolic pathway comprise ALDH1A3, acetaldehyde dehydrogenase family 3 member B1(ALDH3B1) and acetaldehyde dehydrogenase family 3 member B2(ALDH3B2), the RNA levels of the selected differential genes in heart tissues of chronic heart failure model mice in the sham operation group and the C stage are shown in figures 26B-D, and compared with the sham operation group, the RNA levels of ALDH1A3, ALDH3B1 and ALDH3B2 in the heart tissues of the chronic heart failure model mice in the C stage are all obviously reduced.
Expression condition of key regulatory enzyme protein in phenylacetylglycine related metabolic pathway in 3B and C stage chronic heart failure model mouse heart tissue
The Western Blotting method is used for respectively detecting the expression conditions of key regulatory enzyme proteins ALDH1A3, ALDH3B1 and ALDH3B2 in phenylacetylglycine related metabolic pathways in heart tissues of a sham operation group, a B stage chronic heart failure model mouse and a C stage chronic heart failure model mouse. Specific results as shown in fig. 27, compared with the sham operation, the expression of only ALDH1a3 in the heart tissue of the C-stage chronic heart failure model mouse was significantly increased, while the B-stage expression was not significantly different from that of the sham operation group. The expression of ALDH3B1 and ALDH3B2 in hearts of B-stage and C-stage chronic heart failure model mice was not different from that of sham surgery.
ALDH1A3 expression in organ tissues of 4C stage chronic heart failure model mouse
The immunohistochemical technology is utilized to investigate the ALDH1A3 expression conditions in heart, liver, spleen, lung, kidney and brain tissues of mice in a sham operation group and a C-stage chronic heart failure model group, the results are shown in figure 28, compared with the sham operation, the ALDH1A3 expression in the heart tissues of the mice in the C-stage chronic heart failure model group is obviously increased, and the ALDH1A3 expression level in other tissues has no obvious difference with that in the sham operation group.
Influence of 5 ALDH1A3 inhibitor on phenylacetylglycine and xanthurenic acid content in urine of C-stage chronic heart failure model mouse
LC-MS is utilized to investigate whether the ALDH1A3 inhibitor CM10 can effectively inhibit the increase of the phenylacetyl glycine content in the urine of the C-stage chronic heart failure model mouse, and the result is shown in figure 29A, the CM10 with the dose of 2mg/kg and 1mg/kg can effectively inhibit the increase of the phenylacetyl glycine content in the urine of the C-stage chronic heart failure model mouse, and the positive drug metoprolol also shows obvious inhibition effect on the increase of the content. In addition, the influence of the ALDH1A3 inhibitor CM10 on the content of the xanthurenic acid in urine of each group of mice is examined, and the result is shown in figure 29B, wherein the content of the xanthurenic acid in urine of the mice among the groups has no obvious difference.
Effect of 6 ALDH1A3 inhibitor on heart histopathology and degree of fibrosis of C-stage chronic heart failure model mouse
Changes in myocardial histopathology and degree of fibrosis following administration of the ALDH1a3 inhibitor CM10 were assessed in mice model stage C chronic heart failure by HE and Masson staining, respectively. The results are shown in FIG. 30, and the mice in the C-stage chronic heart failure model group showed marked phenomena of myocardial fibrosis, hyperemia, edematous inflammatory cell infiltration, and the like. The CM10 with the dose of 2mg/kg and 1mg/kg can effectively improve the pathological condition and the fibrosis degree of a model mouse, and the improvement effect of the CM10 with high dose is optimal.
Influence of 7 ALDH1A3 inhibitor on biochemical indexes related to heart failure in serum of C-stage chronic heart failure model mouse
The influence and change of the ALDH1A3 inhibitor CM10 on biochemical indexes related to the heart failure in serum of a C-stage chronic heart failure model mouse are examined by using an ELISA kit method, wherein the biochemical indexes mainly comprise BNP, hs-CRP, TNF-alpha and MDA. The results are shown in fig. 31, and compared with the sham operation group, the biochemical indexes of the serum central failure of the mice with the chronic heart failure in the C stage are obviously increased, which indicates that the model building is successful. Compared with a model group, the CM10 with the dosage of 2mg/kg and 1mg/kg has better regulation and control effects on biochemical indexes of the heart failure such as BNP, hs-CRP, TNF-alpha, MDA and the like in the serum of the C-stage chronic heart failure mouse.
Effect of 8 ALDH1A3 inhibitor on cardiac tissue ultrastructure of C-stage chronic heart failure model mouse
The result of the transmission electron microscope is shown in fig. 32, the arrangement of the myocardial cells of the mice in the sham operation group is relatively regular, the arrangement of the muscle fibers is relatively regular, the connection among the cells is clear and uninterrupted, and the structure is relatively complete; the cytoplasm has well-aligned myofibrils; the cytoplasm contains a large number of mitochondria and has complete structure; the nucleus is centrally located and structurally intact. Compared with a sham operation group, the mouse myocardial cell myofibril arrangement of the C-stage chronic heart failure model group is disordered, and the phenomena of separation, fracture, dissolution and the like occur, the intercellular connection is fuzzy, the number of mitochondria is increased and accumulated, the shape is swollen and dissolved, and a plurality of vacuoles appear, thereby indicating that the molding is successful. The phenomena of myocardial fibrolysis of mouse myocardial cells of high and low doses (1mg/kg and 2mg/kg) ALDH1A3 inhibitor CM10 and metoprolol positive medicine group are reduced compared with a model group, the arrangement of myofibrils of the myocardial cells is recovered to be normal, and the form of cell nuclei also tends to be normal.
Effect of 9 ALDH1A3 inhibitor on ALDH1A3 expression in cardiac tissue of C stage chronic heart failure model mice
The influence of the administration of the ALDH1A3 inhibitor CM10 on the ALDH1A3 expression in the heart tissue of the C-stage chronic heart failure model mouse is examined by using an immunohistochemical technology, and the result is shown in FIG. 33, and compared with a sham operation, the ALDH1A3 expression level in the heart tissue of the C-stage chronic heart failure model mouse is obviously increased. After CM10 is given, the high-low dose group shows better ALDH1A3 expression inhibition effect.
Effect of 10 ALDH1A3 inhibitors on H9c2 cell viability in OGD-induced injury
H9c2 myocardial cells were cultured in a simulated in vivo ischemic environment by a molding method involving oxygen deprivation, and the effect of CM10, an ALDH1A3 inhibitor, on the viability of H9c2 cells damaged by OGD induction was examined by the MTT method. As shown in fig. 34, the cells of OGD group H9c2 were significantly decreased in viability by 6H due to hypoxia compared with the blank group, indicating successful molding. Compared with the OGD group, the ALDH1A3 inhibitor CM10 (1 and 2 mu M) is given to remarkably improve the cell viability of the model, and has no effect on the cell viability of the white blood group cells.
Effect of 11 ALDH1A3 inhibitors on LDH leakage Rate of H9c2 cells injured by OGD Induction
The LDH kit is utilized to investigate the influence of ALDH1A3 inhibitor CM10 on LDH leakage rate of H9c2 cells with OGD induced damage, the result is shown in figure 35, OGD stimulation enables the LDH leakage rate of H9c2 cells to be obviously increased, CM10 with doses of 0.32 mu M, 0.64 mu M and 1.28 mu M has obvious inhibition effect on LDH release of H9c2 cells caused by OGD, and the UPF-648 can reduce cell membrane damage caused by OGD, and 1.28 mu M of CM10 also has obvious inhibition effect on the LDH leakage rate of normal group cells.
The above experimental results show that: the aldehyde dehydrogenase family 1 member A3(ALDH1A3) specifically increases in the heart tissue of mice in the C stage of chronic heart failure, and is a key regulatory enzyme for causing the specific increase of phenylacetyl glycine in the urine of the mice in the C stage of chronic heart failure. In addition, myocardial ischemic injury can be effectively alleviated by inhibiting ALDH1a 3.
The protection of the present invention is not limited to the above embodiments. Variations and advantages that may occur to those skilled in the art may be incorporated into the invention without departing from the spirit and scope of the inventive concept and the scope of the appended claims is intended to be protected.

Claims (10)

1. Use of a metabolic marker for the preparation of a diagnostic or monitoring preparation for the B and C stage course of chronic heart failure, wherein said metabolic marker is xanthurenic acid and/or phenylacetylglycine.
2. A kit for diagnosing or monitoring the course of stages B and C of chronic heart failure, which comprises a reagent for detecting the metabolic marker according to claim 1.
3. Use of the metabolic marker of claim 1 for the preparation or screening of a medicament for the treatment of stages B and C of chronic heart failure.
Use of KMO and/or ALDH1A3 as detection targets in diagnostic or monitoring preparations for the B-and C-stage course of chronic heart failure.
5. Use of an agent for detecting KMO and/or ALDH1A3 for the preparation of a diagnostic or monitoring agent for the B-and C-stage course of chronic heart failure.
6. A kit for diagnosing or monitoring the course of B-and C-stage chronic heart failure, which comprises a reagent for detecting KMO and/or ALDH1A 3.
Use of KMO and/or ALDH1A3 as therapeutic targets in the preparation or screening of a medicament for the treatment of chronic heart failure.
8. Use of a substance that inhibits the expression of KMO and/or ALDH1A3 in a medicament for the treatment of chronic heart failure.
9. The use according to claim 8, wherein the agent that inhibits the expression of KMO is UPF-648.
10. The use of claim 8, wherein the agent that inhibits the expression of ALDH1A3 is CM 10.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024007778A1 (en) * 2022-07-05 2024-01-11 上海交通大学医学院附属上海儿童医学中心 Use of plasma molecular marker kynurenine in detection of early heart failure

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060019890A1 (en) * 2004-01-15 2006-01-26 Kapoun Ann M Method for treating cardiac remodeling following myocardial injury
CN101443823A (en) * 2004-12-29 2009-05-27 纳图龙公司 Xanthurenic acid derivative pharmaceutical compositoins and methods related thereto
KR20120131368A (en) * 2011-05-25 2012-12-05 연세대학교 산학협력단 Biomarker for heart failure using urinary metabolites
CN103237901A (en) * 2010-03-01 2013-08-07 卡里斯生命科学卢森堡控股有限责任公司 Biomarkers for theranostics
CN106526156A (en) * 2016-10-26 2017-03-22 王喜军 Method for detecting, screening and identifying kidney-yang deficiency metabolism biomarkers
CN109952102A (en) * 2016-09-05 2019-06-28 代谢研究所 Muscular atrophy is treated using tryptophan metabolism object
CN110161242A (en) * 2019-05-23 2019-08-23 华中科技大学同济医学院附属同济医院 Application of EETs, sEH, sEH inhibitor in chronic heart failure
CN110268048A (en) * 2016-12-04 2019-09-20 大学健康网络 Myocardium of atrium cell and ventricular cardiac myocytes pedigree are generated from human pluripotent stem cells
CN110794074A (en) * 2019-11-18 2020-02-14 广西医科大学 Angelica sinensis Sini decoction cold-resistant blood coagulation stasis syndrome differential metabolite metabolic pathway and research method

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060019890A1 (en) * 2004-01-15 2006-01-26 Kapoun Ann M Method for treating cardiac remodeling following myocardial injury
CN101443823A (en) * 2004-12-29 2009-05-27 纳图龙公司 Xanthurenic acid derivative pharmaceutical compositoins and methods related thereto
CN104069123A (en) * 2004-12-29 2014-10-01 纳图龙公司 Xanthurenic acid derivative pharmaceutical compositions and methods related thereto
CN103237901A (en) * 2010-03-01 2013-08-07 卡里斯生命科学卢森堡控股有限责任公司 Biomarkers for theranostics
KR20120131368A (en) * 2011-05-25 2012-12-05 연세대학교 산학협력단 Biomarker for heart failure using urinary metabolites
CN109952102A (en) * 2016-09-05 2019-06-28 代谢研究所 Muscular atrophy is treated using tryptophan metabolism object
CN106526156A (en) * 2016-10-26 2017-03-22 王喜军 Method for detecting, screening and identifying kidney-yang deficiency metabolism biomarkers
CN110268048A (en) * 2016-12-04 2019-09-20 大学健康网络 Myocardium of atrium cell and ventricular cardiac myocytes pedigree are generated from human pluripotent stem cells
CN110161242A (en) * 2019-05-23 2019-08-23 华中科技大学同济医学院附属同济医院 Application of EETs, sEH, sEH inhibitor in chronic heart failure
CN110794074A (en) * 2019-11-18 2020-02-14 广西医科大学 Angelica sinensis Sini decoction cold-resistant blood coagulation stasis syndrome differential metabolite metabolic pathway and research method

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ANDERS LUND: "3rd World Heart Congress-Kynurenine pathway metabolites are associated with mortality in patients with heart failure", 《J CLIN EXP CARDIOLOGY》 *
FRANCESCO FAZIO等: "Vasorelaxing Action of the Kynurenine Metabolite, Xanthuren Acid: The Missing Link in Endotoxin-Induced Hypotension?", 《FRONTIERS IN PHARMACOLOGY》 *
HOWARD A. LEVY等: "Pyridoxine Deficiency in Congestive Heart Failure", 《P.S.E.B.M.》 *
XIUJIE XIE等: "ALDH1A3 Regulations of Matricellular Proteins Promote Vascular Smooth Muscle Cell Proliferation", 《ISCIENCE》 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024007778A1 (en) * 2022-07-05 2024-01-11 上海交通大学医学院附属上海儿童医学中心 Use of plasma molecular marker kynurenine in detection of early heart failure

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