CN118831080B - Application of 5-oxyproline in preparing medicine for preventing or treating cardiomyopathy - Google Patents

Abstract

The present invention discloses a new use of 5-oxyproline in preparing a drug for preventing or treating cardiomyopathy, especially a new use in preventing or treating cardiomyopathy induced by chemotherapy in tumor patients. Through further research on chemotherapy-induced cardiomyopathy in tumor patients, the present invention finds that 5-oxyproline can partially restore decreased cardiac function, improve myocardial cell atrophy and myocardial tissue fibrosis, and reduce myocardial tissue oxidative stress and myocardial cell apoptosis. It can be used to prevent or treat chemotherapy-induced cardiomyopathy in tumor patients, solves the problem that there is currently no small molecule metabolite treatment method for chemotherapy-induced cardiomyopathy in tumor patients, is expected to significantly reduce the mortality rate of tumor patients due to cardiovascular disease, and has important clinical prevention and treatment significance.

Description

Application of 5-oxyproline in preparation of medicines for preventing or treating cardiomyopathy

Technical Field

The invention relates to a novel application of 5-oxyproline, in particular to an application of 5-oxyproline in preparing a medicament for preventing or treating myocardial diseases induced by chemotherapy of tumor patients.

Background

Heart failure is the final stage of various cardiovascular diseases, has high mortality disability rate, and is the last fort to be overcome in the cardiovascular field. Heart failure, tumor heart disease, has become an emerging leading edge area. While various types of chemotherapeutics bring survival benefits, various adverse reactions of the drugs exist, particularly cardiac injury caused by chemotherapy, and 1/3 tumor patients die from cardiovascular diseases rather than tumors themselves, so that the adverse reactions of the drugs are the primary causes of non-cancer related death of the tumor patients at present.

The most classical chemotherapeutic anthracyclines for tumor treatment are widely used in clinical therapy due to their powerful therapeutic effects, but their clinical application is largely affected by cumulative and dose-dependent cardiotoxicity, which may ultimately lead to irreversible structural changes of the myocardium and progressive heart failure. Right-hand raschig is currently the only drug approved by the FDA for preventing myocardial toxicity caused by anthracyclines, but is banned from pediatric hematological tumors due to its serious myelosuppressive side effects. In addition, studies have been made on the prevention of breast cancer treatment-related cardiac insufficiency (anthracyclines, trastuzumab) by anti-heart failure drugs such as angiotensin converting enzyme inhibitors, angiotensin receptor blockers and beta receptor blockers, but such drugs are not targeted, and the efficacy, the choice of use and the applicable population are not defined.

Therefore, the exacerbation of myocardial injury induced by chemotherapy of a tumor patient can be specifically and effectively prevented or reversed, the non-cancer cardiovascular death rate can be effectively reduced, and the overall survival prognosis of the tumor chemotherapy patient can be improved.

Disclosure of Invention

The invention aims to provide an application of 5-oxyproline (5-oxoproline, 5-OXO) in preparing a medicament for preventing or treating myocardial diseases induced by chemotherapy of tumor patients.

In order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows: application of 5-oxyproline in preparing medicines for preventing or treating cardiomyopathy.

The inventor finds a new application of 5-oxyproline in preventing or treating cardiomyopathy in research, can combine the 5-oxyproline with other active ingredients or auxiliary materials and the like to prepare a medicament for preventing or treating cardiomyopathy, and provides a new treatment choice for treating cardiomyopathy.

As a preferred embodiment of the application of the 5-oxyproline in preparing medicines for preventing or treating cardiomyopathy, the cardiomyopathy is cardiomyopathy induced after chemotherapy of tumor patients. The application discloses a novel application of 5-oxyproline in preventing or treating cardiomyopathy, and the cardiomyopathy is especially cardiomyopathy induced by tumor patients after chemotherapy, especially cardiomyopathy induced by myocardial irreversible structural change, progressive heart failure and the like caused by the influence of accumulation of chemotherapeutic drugs, dose dependence and the like on cardiotoxicity in the chemotherapy process.

As a preferred embodiment of the application of the 5-oxyproline in preparing the medicine for preventing or treating cardiomyopathy, the medicine for chemotherapy is an anthracycline. More preferably, the anthracycline is doxorubicin. The chemotherapeutic agent is preferably but not limited to anthracyclines, such as doxorubicin and the like, and the 5-oxoproline has good effect on the prevention or treatment effect of cardiomyopathy, especially on the cardiomyopathy induced by the classical drug anthracycline in the current treatment of tumor patients.

In addition, the application also provides the application of the 5-oxyproline in preparing medicaments for recovering the reduced cardiac function, recovering the reduced cardiac weight, improving the myocardial cell atrophy, improving the myocardial tissue fibrosis, reducing the oxidative stress of the myocardial tissue and reducing the myocardial cell apoptosis. The inventor finds that the 5-oxyproline has a certain recovery effect on the reduced cardiac function and cardiac weight, can improve myocardial cell atrophy and myocardial tissue fibrosis, can reduce myocardial tissue oxidative stress and myocardial apoptosis, and can be used for preparing medicines with any one of cardiac function and cardiac weight recovery, myocardial cell atrophy improvement, myocardial tissue fibrosis reduction, myocardial tissue oxidative stress reduction and myocardial apoptosis reduction.

Preferably, the above-mentioned decrease in cardiac function, decrease in cardiac weight, cardiomyocyte atrophy, myocardial tissue fibrosis, myocardial tissue oxidative stress, and myocardial apoptosis are induced after chemotherapy in a tumor patient. In the new application of the 5-oxyproline, the 5-oxyproline has good recovery and improvement effects on symptoms caused by chemotherapy of tumor patients, especially on heart function decline, heart weight decline, myocardial cell atrophy, myocardial tissue fibrosis, myocardial tissue oxidative stress and myocardial cell apoptosis caused by chemotherapy drugs.

Preferably, the chemotherapy is performed with anthracyclines. More preferably, the anthracyclines include those commonly available in chemotherapy currently in clinical use, including, but not limited to, doxorubicin, for example. The symptoms are generally caused by the accumulation and dosage of chemotherapeutics, in particular the accumulation and dosage of anthracyclines, on irreversible structural changes and damages caused by cardiac functions and the like, including, but not limited to, doxorubicin and the like, and the 5-oxyproline has good preventive and therapeutic effects on the irreversible structural changes and damages of cardiac muscles induced by the chemotherapeutics.

Finally, the invention also provides a medicament for preventing or treating cardiomyopathy induced by chemotherapy of a tumor patient, wherein the medicament comprises 5-oxyproline and a pharmaceutically acceptable carrier. The 5-oxyproline can be prepared into a medicament for preventing or treating cardiomyopathy induced by chemotherapy of a tumor patient together with a pharmaceutically acceptable carrier.

Preferably, the medicament may be prepared in various dosage forms in the art, including, for example, but not limited to, injections.

According to the invention, through further research on cardiomyopathy induced by chemotherapy of a tumor patient, the 5-oxyproline can partially recover the reduced cardiac function and heart weight, improve myocardial cell atrophy and myocardial tissue fibrosis, relieve myocardial tissue oxidative stress and myocardial apoptosis, can be used for preventing or treating the cardiomyopathy induced by the chemotherapy of the tumor patient, solves the problem that no treatment means for small molecular metabolites for the cardiomyopathy induced by the chemotherapy of the tumor patient exists at present, is expected to obviously reduce the death rate of the tumor patient due to cardiovascular diseases, and has important clinical prevention and treatment significance.

Drawings

FIG. 1 is a graph showing the results of changes in 5-hydroxyproline in serum and myocardial tissue of mice and its correlation with early heart function decline.

FIG. 2 is a graph of the cells of the control, DOX, DOX+5-OXO groups.

Figure 3 is a graph of cardiac weight/body weight of mice in the control, 5-OXO, DOX, dox+5-OXO groups after dosing.

Fig. 4 is an ultrasound examination of the left ventricle of the control, 5-OXO, DOX, dox+5-OXO group.

Fig. 5 is a graph comparing changes in left ventricular end-systole volume (LVEDV), ejection Fraction (EF), short axis contraction (FS%) and stroke volume (CO) for the control group, 5-OXO group, DOX group, dox+5-OXO group.

FIG. 6 is a map of Masson staining and myocardial collagen area quantification for the control, 5-OXO, DOX, DOX+5-OXO groups.

FIG. 7 is a schematic representation of hearts and HE staining of the control, 5-OXO, DOX, DOX+5-OXO groups.

FIG. 8 is a statistical plot of WGA staining of control, 5-OXO, DOX, DOX+5-OXO, and cardiomyocyte cross-sectional area.

FIG. 9 shows the results of immunohistochemical staining of myocardial tissue 4-HNE in the control group, 5-OXO group, DOX group, DOX+5-OXO group.

FIG. 10 shows the cardiomyocyte apoptosis and apoptosis statistics of the control, 5-OXO, DOX, DOX+5-OXO groups.

Detailed Description

For a better description of the objects, technical solutions and advantages of the present invention, the present invention will be further described with reference to the accompanying drawings and specific examples.

Example 1

Anthracycline-induced changes in 5-oxyproline content in early blood of cardiomyopathy

1. Laboratory animals and groups

A total of 16C 57 mice were selected from 6-8 weeks, and randomly divided into experimental and control groups of 8 mice each.

The mice of the experimental group are subjected to modeling of tumor cardiomyopathy (doxorubicin, 5mg/kg,1 time), and cardiac performance is evaluated by cardiac color Doppler ultrasound after 1 week.

2. Extraction of metabolites from blood

(1) Anesthetizing experimental mice and control mice after modeling of tumor heart diseases, taking down hearts, cleaning the hearts with pre-cooled normal saline at 4 ℃, discharging residual blood, and taking part of cardiac apex to be rapidly frozen by liquid nitrogen for metabolome analysis of myocardial tissues;

(2) Slowly thawing myocardial tissue frozen by liquid nitrogen at 4 ℃, taking a proper amount of sample, adding precooled methanol/acetonitrile/water solution (2:2:1, v/v), vortex mixing, performing low-temperature ultrasonic treatment for 30min, standing at-20 ℃ for 10min, centrifuging at 14000g at 4 ℃ for 20min, taking supernatant, vacuum drying, adding 100 mu L acetonitrile water solution (acetonitrile: water=1:1, v/v) for redissolution during mass spectrometry, vortex, centrifuging at 14000g at 4 ℃ for 15min, and taking supernatant to obtain metabolic extract.

3. Detection of 5-oxyproline content

The metabolic extract is used as a sample to detect the content of 5-oxyproline, and the detection method comprises the following steps:

(1) Sample pretreatment

Synthesis of 5-oxoproline internal standard: preparing 1mg/mL glutamic acid-d 5 solution by using 0.1N hydrochloric acid, heating at 80 ℃ for 15 hours to synthesize 5-oxyproline-d 5 internal standard solution, and regulating the pH value back to neutrality by using ammonia water to prepare 5-oxyproline internal standard;

preparing an internal standard working solution: diluting the internal standard 5-oxyproline by 200 times by using a 75% acetonitrile aqueous solution containing 0.2% formic acid to prepare an internal standard 5-oxyproline working solution;

Sample processing: and respectively taking 50 mu L of standard curve, quality control product and sample to be tested, adding the standard curve, quality control product and sample to be tested into a 1.5 mL centrifuge tube, adding 400 mu L of internal standard working solution, carrying out vortex oscillation for 60 seconds, centrifuging at 13000rpm and 4 ℃ for 10 minutes, absorbing 200 mu L of supernatant, transferring into a 2.2mL 96-well plate, and waiting for loading.

(2) Liquid phase process

The metabolites in the sample were separated by column Phenomenex Kinetex F (2.6 um, 2.1X100 mm) under the following specific chromatographic conditions:

mobile phase a was 0.1% formic acid water;

Mobile phase B was 0.1% acetonitrile formate;

the flow rate is 0.25ml/min;

The column temperature is 25 ℃;

Gradient elution conditions are shown in table 1 below.

TABLE 1 gradient elution conditions

(3) Mass spectrometry method

AB SCIEX Triple Quad ™ 4500MD liquid chromatography tandem mass spectrometry detection system adopts ESI+ and Multiple Reaction Monitoring (MRM) mode to carry out mass spectrometry scanning; the Curtain Gas (Curtain Gas, CUR) is 35 psi; collision Gas (CAD) 9 psi; the voltage is +5500V; the desolventizing gas temperature is 500 ℃; heating gas (GS 1) was 50psi; the assist gas (GS 2) was 50psi. The mass spectral channel parameters are shown in table 2 below.

Table 2 mass spectral channel parameters

(4) Calibrator and quality control product

The standard 5-oxyproline is prepared into a primary mother solution with the concentration of 50mM by water, diluted into a secondary mother solution with the concentration of 4mM by water, diluted by water and uniformly mixed to obtain standard yeast and quality control products with different concentrations, and the specific details are shown in the following table 3.

TABLE 3 standard curves and quality control products with different concentrations

The detection result is shown in figure 1. As can be seen from FIG. 1, the content of 5-oxyproline in the blood of mice with doxorubicin-induced cardiomyopathy is significantly reduced and is inversely related to the early cardiac function reduction index, i.e., myocardial global longitudinal strain (global longitudinal strain, GLS).

The inventor discovers that the content of 5-oxyproline is obviously reduced in the chemotherapy-induced cardiomyopathy for the first time, and further researches on the chemotherapy-induced cardiomyopathy of tumor patients through the discovery, and discovers that the 5-oxyproline can partially recover the reduced cardiac function and heart weight, improve myocardial cell atrophy and myocardial tissue fibrosis and relieve myocardial tissue oxidative stress and myocardial cell apoptosis.

Example 2

Experiment of the Effect of oxyproline on Adriamycin-induced Primary myocardial cytotoxicity in rats

1. Extraction and culture of rat primary myocardial cells (NRCMs)

1-3 Days of newborn Sprague-Dawley rats (purchased from the animal experiment center of Renzhijie hospital) were used, and after sterilization by first immersing in 70% alcohol for several seconds, the chest was opened from the two armpits of the rats vertically by using an instrument, and the heart was grasped with curved forceps. After the heart was subsequently washed clean in PBS, it was placed in an Erlenmeyer flask and approximately 8ml of digest (25 ml 2mg/ml collagenase II, 23ml 0.25% pancreatin without EDTA, 2ml PBS) was injected each time. The conical flask was placed on a constant temperature magnetic stirrer at 37℃and 200rmp, and subjected to a first stirring digestion for 20 minutes, the supernatant was collected, the residue was removed, and then the digestion solution was added again for a second 15 minutes of digestion, the supernatant was collected again, and the residue was removed, and then each mechanical digestion was performed on the stirrer for about 10 minutes. On the third digestion, the washed hearts were aspirated with supernatant and placed in DMEM medium with 5% serum and subjected to intense blow and neutralization of the digestive fluids. This process is repeated until the heart has completely digested or only fiber remains.

The treated heart supernatant was then centrifuged at 1200 revolutions per minute and the supernatant discarded. 2ml of erythrocyte lysate was added to each tube of cells, and the cells were blown off, allowed to stand in the lysate for 2 minutes, and then added to the medium to polymerize the cells.

Finally, centrifugation was performed again, and the centrifugation conditions were adjusted to 1000 revolutions per minute and the centrifugation time was 5 minutes. After discarding the centrifuged cell supernatant, the cells were added to complete medium, resuspended by pipetting, then placed in a 100mm dish in an incubator and differential-speed attached for 90 minutes. Next, the culture medium in the petri dish was aspirated into a new 50ml centrifuge tube for counting, plate manipulation was performed as needed, and then the cells were cultured with complete medium and 100. Mu. M bromodeoxyuridine (BrdU) was added to inhibit fibroblast growth. After 48 hours, serum-free medium can be changed to treat cells.

2. Construction of doxorubicin-induced primary myocardial cell injury model

1Mg of doxorubicin was dissolved in 3.68mL of PBS to prepare a 0.5mM doxorubicin stock solution. 1.29mg of 5-hydroxyproline was dissolved in 10mL of PBS to prepare a 1mM stock solution of 5-hydroxyproline. Primary cardiomyocyte seeding Density 2X 10 ⁵/mL was seeded in 96 well cell culture plates. The culture medium was a high-sugar DMEM plate medium containing Brdu and 10% serum two days before culture, and the culture medium was changed to high-sugar DMEM medium the third day. After the cell state is stable, the cell culture solution is equally divided into three groups: control group, DOX group, DOX+5-OXO, each group was as follows:

control group: adding PBS with the same volume;

DOX group: a corresponding volume of doxorubicin stock solution was added up to the working concentration (1 uM);

DOX+5-OXO group: doxorubicin stock and a concentration gradient of 5-oxoproline stock were added in equal amounts to the DOX group up to working concentrations (1 uM, 2uM, 5 uM).

After 24h incubation with constant temperature medium, CCK8 experiments were performed for each group.

3. CCK-8 cell Activity assay

After incubating the above groups of Neonatal Rat Cardiomyocytes (NRCMs) in a constant temperature incubator for 24 hours, the cell surfaces were first washed with PBS, and then the cells were incubated with high sugar DMEM medium containing 10% CCK-8 reagent in a dark environment at a constant temperature of 37 ℃ for 2 hours. After incubation, absorbance was measured at 450nm with a microplate reader and a cell activity curve was plotted, the results of which are shown in FIG. 2.

As can be seen from FIG. 2, 5-OXO effectively reduced DOX-induced myocardial cytotoxicity, while 5-OXO supplementation concentration of 5uM was most significantly improved.

Example 3

Experiment of 5-oxyproline intervention doxorubicin-induced myocardial injury mice cardiac function the instrumentation and reagents used in this example are shown in tables 4 and 5, respectively, below.

TABLE 4 Main instruments and devices of this example

TABLE 5 Main reagents of this example

1. Laboratory animals and groups

SPF-class male C57 mice were supplied from the Shanghai university affiliated Renji Hospital laboratory animal center for 32, 6-8 weeks and approximately 25g in weight, and the experiment was conducted after 3 days of adaptive rearing in the laboratory animal center. During the raising period, the environment of the mouse raising room is kept clean, the temperature and humidity are constant (the temperature is 23+/-1 ℃ and the humidity is 40+/-5%), and the period of 12 hours of light and shade is provided. All experiments were conducted following guidelines for ethics of experimental animals and passed examination by the institutional ethics of animals committee of the Shanghai university of transportation, affiliated with the Shanghai university of Council.

Mice were randomly divided into control groups (Ctrl), 5-oxoproline group (5-OXO), doxorubicin group (DOX), doxorubicin+5-oxoproline group (dox+5-OXO), and each group was dosed as follows:

Group 5-oxyproline (5-OXO): 5-Oxoproline was administered by intraperitoneal injection (0.05 mg/kg) 1 time a day;

Doxorubicin group (DOX): doxorubicin was given by intraperitoneal injection (5 mg/kg), 1 week 1;

Doxorubicin + 5-oxyproline group (DOX + 5-OXO): administering doxorubicin + 5-oxoproline in an amount equivalent to the 5-oxoproline group (5-OXO) and doxorubicin group (DOX);

Control group: administering an equivalent amount of physiological saline solution to doxorubicin + 5-oxoproline group (DOX + 5-oxo);

Each group was dosed continuously for 4 weeks, and the mice were weighed weekly during dosing.

The mice are observed for 1 week after 4 weeks of molding administration, materials are obtained after heart color ultrasound examination (a Vevo ultra-high resolution multi-mode small animal ultrasonic photoacoustic imaging system) is carried out, and the hearts are weighed after moisture is absorbed after the materials are obtained.

The results of the weight change and cardiac weight change after administration of each group of mice are shown in fig. 3. As can be seen from fig. 3, the body weight and heart weight of doxorubicin group (DOX) mice decreased significantly with the prolonged administration time, and the body weight and heart weight of 5-oxyproline group (5-OXO) mice decreased slightly as compared with the control group, and the body weight and heart weight of doxorubicin+5-oxyproline group (dox+5-OXO) mice decreased partially as compared with doxorubicin group (DOX). From this, it can be demonstrated that 5-oxyproline (5-OXO) was able to restore the weight and heart weight of the mice that were reduced.

Pathological tissue detection was then performed on each set of myocardial tissue: HE. Masson staining was used to observe myocardial tissue pathological changes; WGA staining to observe cardiomyocyte size changes; 4-HNE staining is used for detecting the oxidative stress degree of myocardial cells; TUNEL staining detects myocardial apoptosis index.

2. Mouse heart ultrasound detection

The heart functions of the mice after 4 weeks of administration of the control group, the 5-OXO group, the DOX group and the DOX+5-OXO group are respectively detected by using a Vevo ultra-high resolution animal ultrasonic imaging system and a high frequency probe to be applied to heart ultrasonic examination of the mice.

Prior to the experiment, the mice were first dehaired with depilatory cream, half anesthetized by isoflurane inhalation anesthesia, and kept spontaneously breathing. Short axis slice images are then acquired at the parasternal left ventricular papillary muscle locations to assess left ventricular condition. Meanwhile, an M-mode echocardiographic recording of 15 cardiac cycles was recorded using a two-dimensional mode under guidance, and the Left Ventricular End Diastole Volume (LVEDV) and the like were measured. The detection results are shown in fig. 4 and 5.

From the results and functional levels of the left ventricle of the heart examined by the M-ultrasound of figures 4 and 5, it can be seen that 5-oxoproline intervention significantly restores the reduced left ventricular end-diastole volume (LVEDV), reducing the extent of cardiac atrophy after doxorubicin induction; while significantly increasing ejection fraction (EF%), short axis shrinkage (FS%) and stroke volume (CO), improving myocardial contractility and compliance.

2. Pathological tissue detection

2.1 Preparation of paraffin sections of mouse heart

The hearts of the mice after the above-mentioned various models were sampled, excess tissues were cut off by washing with PBS, and then the hearts were fixed in 4% paraformaldehyde for 24 hours, which was stored in a refrigerator at 4 ℃. After fixation, the heart is placed in a tissue embedding cassette for dehydration. After the paraffin of the embedding machine is melted, the embedding box is placed in a preheated embedding machine tissue groove, and is soaked with the paraffin twice in a first groove and is soaked with the paraffin three times in a second groove. After tissue dehydration is completed, placing the sample into a waxed embedding box, dripping a small amount of wax liquid, closing the embedding box, and cooling, wherein embedding is completed.

The embedded wax block is fixed on a precooled paraffin slicer, the slice thickness is adjusted to be 10 mu m, and the slice thickness is 3 mu m. After cutting off wax flakes, spreading the wax flakes in a preheated water tank at 40 ℃ through a writing brush, then taking out the wax flakes by using a glass slide, and then placing the wax flakes on a flake baking machine for flaking for 30 minutes.

2.2 Masson staining

The dewaxed sections were washed 3X 5min with ddH2O on a 40rpm shaker. The heart was then circled around with a framing pen and air dried. The sections were subsequently stained in sequence as follows:

(1) The slices are put into a mordant dye solution for mordant dyeing, and are incubated for 1h in a baking oven at 60 ℃, and ddH2O is washed for 3X 5min after the completion;

(2) Drop dyeing of azure blue staining solution, incubating for 3min at room temperature, and washing for 2X 15s by ddH2O after completion;

(3) Drop-staining the Mayer hematoxylin staining solution for 3min to finish ddH2O washing for 2×15s;

(4) Differentiation of acidic differentiation liquid 5s, ddH2O rinse for 10min;

(5) Ponceau red dye drop-dyed for 10min and ddH2O washed for 2×15s;

(6) The phosphomolybdic acid dye is dripped for 10min;

(7) Directly dripping aniline blue staining solution for 5min after skimming phosphomolybdic acid;

(8) Washing off aniline blue by using acetic acid solution, and then dropwise adding weak acid solution for incubation for 2min;

(9) Dehydrating with 95% ethanol for 30s, dehydrating with anhydrous ethanol for 2×10s, dehydrating with xylene for 2×5s, and sealing with neutral resin.

The staining results are shown in FIG. 6. As can be seen in FIG. 6, masson's staining showed that the DOX group showed significant fibrosis of the myocardial tissue, whereas 5-oxoproline significantly reduced fibrosis of the myocardial tissue.

2.3 HE staining

The slices were dewaxed and then soaked in distilled water for 2min. And then limiting a tissue slice area by a slice organization stroke circle, dripping hematoxylin staining solution for 15min after the organization stroke is dried, and washing off floating color by distilled water. The tabs were differentiated by dropping a differentiation liquid for 30s and rinsed with tap water for 2X 5min. And finally, dripping eosin staining solution for 1min, and rapidly dehydrating after eluting residual staining solution by distilled water after the staining is finished. The dehydration step is as follows:

(1) Soaking in 75% ethanol, 85% ethanol, 95% ethanol and 100% ethanol for 3s;

(2) Soaking in 100% ethanol for 1min, adding xylene, washing to remove impurities for 2×1min, and sealing with neutral resin.

After the sealing is finished, the result is shown in figure 7.

2.4 WGA staining

Paraffin sections were removed, de-waxed and hydrated. Sections were placed in a microplate containing PBS (phosphate buffer), one well for each sample. 0.1% Triton X-100 (in PBS) was added, incubated for 10 min, and washed three times with PBS. The WGA solution was added for staining. The concentration and time of WGA can be optimized according to experimental needs. Typically the WGA concentration is 5-20 mug/mL and the staining time is 30-60 minutes. The washing was performed three times with PBS for 5 minutes. DAPI (4', 6-diamidino-2-phenylindole) staining solution was added, incubated for 10 min, and washed three times with PBS. Reversing the slide, adding a proper amount of anti-browning glue for sealing, and observing fluorescence. The results are shown in FIG. 8.

As can be seen from fig. 7 and 8, the cardiomyocytes of the control group were morphologically normal; the DOX group found significant cardiomyocyte atrophy, and 5-oxyproline could significantly interfere with DOX-induced cardiomyocyte atrophy.

2.5 4-HNE immunohistochemical staining

Heart paraffin sections were deparaffinized, antigen repaired, blocked after permeabilization, and probed with primary antibody (4 ℃ overnight), followed by incubation with secondary antibody for another 1h at room temperature. After SP (streptavidin-peroxidase) is added dropwise, DAB is used for color development for 5-10min, the reaction is stopped, hematoxylin is counterstained for 2min, hydrochloric acid alcohol is differentiated, and the mixture is dehydrated, transparent and sealed. The results are shown in FIG. 9.

As can be seen from FIG. 9, the degree of oxidative stress of the myocardial cells of the mice was detected by 4-HNE immunohistochemical staining, and the degree of oxidative stress of the myocardial tissue of the mice in the DOX group was found to be significantly increased compared with that of the control group, while the level of oxidative stress of the myocardial tissue of the mice after the corresponding 5-oxyproline is dried was significantly decreased.

2.9 Tunel dyeing

Heart paraffin sections were dewaxed, antigen repaired, permeabilized and blocked. An appropriate amount of TUNEL detection solution (TdT enzyme: fluorescent labeling solution=1:9) was prepared, and the mixture was thoroughly mixed. Mu.l TUNEL assay was added to the sample and incubated at 37℃for 60 minutes in the absence of light. The cells were washed three times with PBS and observed under a fluorescence microscope after liquid-sealing with an anti-fluorescence quenching sealing sheet, and the results are shown in FIG. 10.

As can be seen from fig. 10, the apoptosis of mouse cardiac muscle cells was detected by TUNEL, and it was found that the number of apoptosis of cardiac muscle tissue of mice in the DOX group was significantly increased compared with that of the control group, while the number of apoptosis cells in cardiac muscle of mice interfered with by the corresponding 5-oxyproline was significantly decreased compared with that of the DOX group.

From the above experimental results, it is known that 5-oxyproline can partially restore the decreased cardiac function and heart weight, improve myocardial cell atrophy and myocardial tissue fibrosis, and alleviate oxidative stress and myocardial apoptosis of myocardial tissue, and can be used for preventing or treating cardiomyopathy induced by chemotherapy in tumor patients. The inventor of the application discovers that the 5-oxyproline level in the chemotherapy-induced cardiomyopathy is obviously reduced for the first time, and further researches on the mechanism of the chemotherapy-induced cardiomyopathy finally find that the 5-oxyproline has good effect on preventing or treating the chemotherapy-induced cardiomyopathy of a tumor patient, and provides a micromolecular metabolite aiming at the chemotherapy-induced cardiomyopathy of the tumor patient as a therapeutic drug, thereby having important clinical significance.

Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted equally without departing from the spirit and scope of the technical solution of the present invention.

Claims (4)

The invention discloses an application of 1.5-oxyproline in the preparation of a drug for preventing or treating cardiomyopathy, wherein the cardiomyopathy is cardiomyopathy induced by chemotherapy in tumor patients, and the drug used in the chemotherapy is an anthracycline drug. 2. Use of 5-oxoproline in the preparation of a drug for preventing or treating cardiomyopathy according to claim 1, characterized in that the anthracycline drug is doxorubicin. The use of 3.5-oxyproline in the preparation of a drug for restoring decreased cardiac function, restoring decreased heart weight, improving myocardial cell atrophy, improving myocardial tissue fibrosis, reducing myocardial tissue oxidative stress, and reducing myocardial cell apoptosis, wherein the decreased cardiac function, decreased heart weight, myocardial cell atrophy, myocardial tissue fibrosis, myocardial tissue oxidative stress, and myocardial cell apoptosis are induced after chemotherapy in tumor patients, and the chemotherapy drug is an anthracycline drug. 4. The use according to claim 3, characterized in that the anthracycline drug is doxorubicin.