Application of 5-oxoproline in preparation of drug for preventing or treating cardiomyopathy

Abstract

The present invention relates to a new application of 5-oxoproline in the preparation of a drug for preventing or treating cardiomyopathy, and particularly in the prevention or treatment of chemotherapy-induced cardiomyopathy in tumor patients. By means of further research on chemotherapy-induced cardiomyopathy in tumor patients, it has found that 5-oxoproline can partially restore reduced cardiac function, alleviate cardiomyocyte atrophy and myocardial tissue fibrosis, reduce myocardial tissue oxidative stress and cardiomyocyte apoptosis, and can be used for preventing or treating chemotherapy-induced cardiomyopathy in tumor patients, which addresses the currrent problem of no treatment means of small molecule metabolites for chemotherapy-induced cardiomyopathy in tumor patients, exhibits the potential to significantly reduce the mortality of tumor paitents due to cardiovascular disease, and has important significance for clinical prevention and treatment.

Description

5-oxoproline for use in the preparation of a medicament for preventing or treating cardiomyopathy TECHNICAL FIELD

[0001] The present application discloses a new application of 5-oxoproline, in particular, the application of 5-oxoproline in the preparation of a medicament for preventing or treating chemotherapy-induced cardiomyopathy in tumor patients. BACKGROUND

[0002] Heart failure is the end stage of various cardiovascular diseases, with high mortality and morbidity, and is the last fortress to be conquered in the field of cardiovascular diseases. Heart failure in tumor patients has become a new and emerging frontier. Various types of chemotherapy drugs bring survival benefits, but also have various adverse reactions, especially chemotherapy-induced cardiac damage. One-third of tumor patients die of cardiovascular diseases rather than the tumor itself, which is the primary cause of non-cancer-related deaths in tumor patients.

[0003] The most classic chemotherapy drug for tumor treatment is anthracycline, which is widely used in clinical treatment due to its strong therapeutic effect. However, its clinical application is largely affected by cumulative and dose-dependent cardiotoxicity, which may eventually lead to irreversible structural changes in the myocardium and progressive heart failure. Currently, dexrazoxane is the only drug approved by FDA for preventing cardiotoxicity caused by anthracyclines. However, due to its serious side effect of bone marrow suppression, it is prohibited for use in children with hematological tumors. In addition, there are studies attempting to use anti-heart failure drugs such as angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, and beta blockers to prevent breast cancer treatment-related heart failure (anthracyclines, trastuzumab). However, such drugs are not targeted, and their efficacy, timing of use, and suitable population are not conclusive.

[0004] Therefore, specifically and effectively preventing or reversing the aggravation of chemotherapy-induced myocardial damage in tumor patients can effectively reduce non-cancer cardiovascular mortality and improve the overall survival prognosis of tumor chemotherapy patients. SUMMARY

[0005] The purpose of the present application is to provide the application of 5-oxoproline (5-oxoproline, 5-OXO) in the preparation of a medicament for preventing or treating chemotherapy-induced cardiomyopathy in tumor patients.

[0006] To achieve the purpose of the present application, the technical solution adopted by the present application is: the application of 5-oxoproline in the preparation of a medicament for preventing or treating cardiomyopathy.

[0007] The present inventors found in the research that 5-oxoproline has a new application in preventing or treating cardiomyopathy. 5-oxoproline can be combined with other active ingredients or excipients to prepare a medicament for preventing or treating cardiomyopathy, thereby providing a new treatment option for the treatment of cardiomyopathy.

[0008] As a preferred embodiment of the use of 5-oxoproline in the preparation of a drug for preventing or treating cardiomyopathy according to the present application, the cardiomyopathy is cardiomyopathy induced after chemotherapy of tumor patients. The new application of 5-oxoproline in preventing or treating cardiomyopathy found in the present application refers to cardiomyopathy induced after chemotherapy of tumor patients, in particular, irreversible structural changes in myocardium, progressive heart failure, and other cardiomyopathies caused by the cumulative effect and dose-dependent cardiotoxicity of chemotherapy drugs during chemotherapy.

[0009] As a preferred embodiment of the use of 5-oxoproline in the preparation of a drug for preventing or treating cardiomyopathy according to the present application, the drug used in the chemotherapy is an anthracycline drug. More preferably, the anthracycline drug is doxorubicin. The chemotherapy drug is preferably but not limited to an anthracycline drug, such as doxorubicin, and 5-oxoproline has good effects on the prevention or treatment of cardiomyopathy, especially on cardiomyopathy induced by the classic drug anthracycline in the current treatment of tumor patients.

[0010] In addition, the present application also provides the use of 5-oxoproline in the preparation of a drug for any one of 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. The present inventors found in the research that 5-oxoproline has certain recovery effect on decreased cardiac function and heart weight, can improve myocardial cell atrophy and myocardial tissue fibrosis, and can also reduce myocardial tissue oxidative stress and myocardial cell apoptosis, and can be used for the preparation of a drug having any one of the functions of restoring cardiac function and heart weight, improving myocardial cell atrophy, improving myocardial tissue fibrosis, reducing myocardial tissue oxidative stress, and reducing myocardial cell apoptosis.

[0011] Preferably, the above-mentioned decrease in cardiac function, decrease in heart weight, myocardial cell atrophy, myocardial tissue fibrosis, myocardial tissue oxidative stress, and myocardial cell apoptosis are induced after chemotherapy of tumor patients. In the new application of 5-oxoproline, especially for the decrease in cardiac function, decrease in heart weight, myocardial cell atrophy, myocardial tissue fibrosis, myocardial tissue oxidative stress, and myocardial cell apoptosis caused by chemotherapy of tumor patients, 5-oxoproline has good recovery and improvement effects on these symptoms caused by chemotherapy drugs.

[0012] Preferably, the chemotherapy employs an anthracycline drug. More preferably, the anthracycline drug includes common drugs currently clinically available for chemotherapy, such as, but not limited to, doxorubicin. The above symptoms are generally caused by accumulation and dosage of the chemotherapy drugs, especially irreversible structural changes and damage to heart function caused by accumulation and dosage of anthracycline chemotherapy drugs, such as, but not limited to, doxorubicin, etc. The 5-oxoproline has good preventive and therapeutic effects on the above myocardial irreversible structural changes and damage induced by these chemotherapy drugs.

[0013] Finally, the present application also provides a drug for preventing or treating cardiomyopathy induced after chemotherapy of a tumor patient, which comprises 5-oxoproline and a pharmaceutically acceptable carrier. The 5-oxoproline described in the present application can be prepared into a drug for preventing or treating cardiomyopathy induced after chemotherapy of a tumor patient together with a pharmaceutically acceptable carrier.

[0014] Preferably, the drug can be prepared into various dosage forms in the art, such as, but not limited to, injections.

[0015] The present application solves the problem that there is no small molecule metabolite treatment for cardiomyopathy induced by chemotherapy of a tumor patient by further research on cardiomyopathy induced by chemotherapy of a tumor patient, and finds that 5-oxoproline can partially restore decreased heart function and heart weight, improve myocardial cell atrophy and myocardial tissue fibrosis, reduce myocardial tissue oxidative stress and myocardial cell apoptosis, and can be used for preventing or treating cardiomyopathy induced by chemotherapy of a tumor patient, which is expected to significantly reduce the mortality of tumor patients due to cardiovascular diseases, and has important clinical preventive and therapeutic significance. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 is a graph showing the changes of 5-oxoproline in serum and myocardial tissue of mice and its correlation with early heart function decline.

[0017] Figure 2 is a cell curve graph of the control group, the DOX group, the DOX+5-OXO group;

[0018] Figure 3 is a graph showing the body weight changes and heart weight / body weight of mice in the control group, the 5-OXO group, the DOX group, and the DOX+5-OXO group after administration.

[0019] Figure 4 is an ultrasound detection graph of the left ventricle of the control group, the 5-OXO group, the DOX group, and the DOX+5-OXO group.

[0020] Figure 5 is a comparison graph of changes in left ventricular end systolic volume (LVEDV), ejection fraction (EF%), fractional shortening (FS%), and stroke volume (CO) of the control group, the 5-OXO group, the DOX group, and the DOX+5-OXO group.

[0021] Figure 6 is a Masson staining diagram of the control group, 5-OXO group, DOX group, DOX+5-OXO group and a quantitative statistical diagram of myocardial collagen area.

[0022] Figure 7 is a general diagram of the heart and a HE staining diagram of the control group, 5-OXO group, DOX group, DOX+5-OXO group.

[0023] Figure 8 is a WGA staining diagram of the control group, 5-OXO group, DOX group, DOX+5-OXO group and a statistical diagram of myocardial cell cross-sectional area.

[0024] Figure 9 is the result of myocardial tissue 4-HNE immunohistochemical staining of the control group, 5-OXO group, DOX group, DOX+5-OXO group.

[0025] Figure 10 is the myocardial cell apoptosis and statistical analysis of apoptotic cells of the control group, 5-OXO group, DOX group, DOX+5-OXO group. DETAILED DESCRIPTION

[0026] In order to better illustrate the purpose, technical scheme and advantages of the present application, the present application will be further described below in combination with the drawings and specific examples.

[0027] Example 1

[0028] Early changes in the content of 5-oxoproline in blood induced by anthracycline-induced cardiomyopathy

[0029] 1. Experimental animals and grouping

[0030] Sixteen 6-8 week old C57 mice were selected and randomly divided into an experimental group and a control group, 8 mice in each group.

[0031] The mice in the experimental group were subjected to tumor cardiomyopathy modeling (doxorubicin, 5 mg / kg, once), and the heart function was evaluated by cardiac color Doppler ultrasound after 1 week.

[0032] 2. Extraction of metabolites in blood

[0033] (1) The experimental group mice and the control group mice after tumor heart disease modeling were anesthetized, the heart was removed, the heart was washed with pre-cooled 4℃ physiological saline, the residual blood was discharged, and part of the apex was quickly frozen with liquid nitrogen for myocardial tissue metabolomics analysis;

[0034] (2) The myocardial tissue frozen with liquid nitrogen was slowly thawed at 4°C, and then a proper amount of sample was added into a pre-cooled methanol / acetonitrile / water solution (2:2:1, v / v), vortex mixed, ultrasonicated at low temperature for 30 min, placed at -20°C for 10 min, centrifuged at 14000g at 4°C for 20 min, and then the supernatant was vacuum dried. When mass spectrometry was performed, 100 μL of acetonitrile / water solution (acetonitrile:water = 1:1, v / v) was added to re-dissolve the sample, vortexed, centrifuged at 14000g at 4°C for 15 min, and then the supernatant was taken, to obtain the metabolic extract.

[0035] 3. Detection of 5-oxoproline content

[0036] The metabolic extract was used as a sample to detect the 5-oxoproline content therein, and the detection method was as follows:

[0037] (1) Sample pretreatment

[0038] 5-oxoproline internal standard synthesis: a glutamic acid-d5 solution with a concentration of 1 mg / mL was prepared by using 0.1N hydrochloric acid, and then 5-oxoproline-d5 internal standard solution was synthesized by heating at 80°C for 15 hours. The pH was adjusted to neutral by using ammonia water, to obtain the 5-oxoproline internal standard.

[0039] Internal standard working solution preparation: the 5-oxoproline internal standard was diluted 200 times by using a 75% acetonitrile / water solution containing 0.2% formic acid, to prepare the 5-oxoproline internal standard working solution.

[0040] Sample treatment: 50 μL of the standard curve, quality control sample and sample to be tested were taken into a 1.5 mL centrifuge tube, 400 μL of the internal standard working solution was added, vortexed for 60 seconds, and then centrifuged at 13000 rpm for 10 min at 4°C. 200 μL of the supernatant was transferred into a 2.2 mL 96-well plate, and then the sample was ready for machine testing.

[0041] (2) Liquid phase method

[0042] The metabolites in the sample were separated by using a chromatographic column Phenomenex Kinetex F5 (2.6um, 2.1x100mm), and the specific chromatographic conditions were as follows:

[0043] The mobile phase A was 0.1% formic acid water;

[0044] The mobile phase B was 0.1% formic acid acetonitrile;

[0045] The flow rate was 0.25 ml / min;

[0046] The column temperature was 25°C;

[0047] The gradient elution conditions are shown in Table 1.

[0048] Table 1 Gradient elution conditions

[0049] (3) Mass spectrometry method

[0050] AB SCIEX Triple Quad TM 4500MD liquid chromatography tandem mass spectrometry detection system, using ESI+, multi-reaction monitoring (MRM) mode for mass spectrometry scanning; curtain gas (CUR) is 35 psi; collision gas (CAD) is 9 psi; voltage is +5500V; desolvation gas temperature is 500℃; heating gas (GS1) is 50 psi; auxiliary gas (GS2) is 50 psi. The mass spectrometry channel parameters are shown in Table 2.

[0051] Table 2 Mass spectrometry channel parameters

[0052] (4) Calibration and quality control

[0053] The 5-oxoproline standard was prepared into a 50mM primary stock solution with water, diluted into a 4mM secondary stock solution with water, and then diluted with water to obtain different concentrations of standard curves and quality control, as shown in Table 3.

[0054] Table 3 Standard curves and quality controls of different concentrations

[0055] The detection results are shown in Figure 1. As can be seen from Figure 1, the content of 5-oxoproline in the blood of doxorubicin-induced cardiomyopathy mice decreased significantly, and was negatively correlated with the early heart function decline indicator, global longitudinal strain (GLS).

[0056] The inventors of the present application first discovered that the content of 5-oxoproline decreased significantly in chemotherapy-induced cardiomyopathy, and through this discovery, further research on tumor patient chemotherapy-induced cardiomyopathy was conducted, and it was found that 5-oxoproline could partially restore the decreased heart function and heart weight, improve myocardial cell atrophy and myocardial tissue fibrosis, and reduce myocardial tissue oxidative stress and myocardial cell apoptosis.

[0057] Example 2

[0058] Effect of 5-oxoproline on doxorubicin-induced toxicity of rat primary myocardial cells

[0059] 1. Extraction and culture of rat neonatal primary myocardial cells (NRCMs)

[0060] Twenty 1-3 day old Sprague-Dawley rats (purchased from Renji Hospital Animal Experimental Center) were first soaked in 70% alcohol for a few seconds for disinfection, then a vertical incision was made from both axillary under the chest using instruments, and the heart was taken out with a curved forceps. Then the heart was washed clean in PBS, placed in a conical flask, and about 8 ml of the digestion solution (25 ml 2 mg / ml collagenase II, 23 ml 0.25% trypsin without EDTA, 2 ml PBS) was injected each time. The conical flask was placed on a constant temperature magnetic stirrer, set at 37°C, and the stirring speed was set at 200 rpm. The first stirring digestion was performed for 20 minutes, the supernatant was collected, the residue was discarded, and the digestion solution was added again for the second digestion for 15 minutes. The supernatant was collected again, the residue was discarded, and mechanical digestion was performed on the stirrer for about 10 minutes each time. At the third time of digestion, the supernatant was used to wash the heart in 5% serum DMEM medium, and the heart was treated with strong blowing and neutralization of the digestion solution. This process was continuously repeated until the heart was completely digested or only fibrous tissue remained.

[0061] Then, the treated heart supernatant was centrifuged at 1200 rpm, and the supernatant was discarded. Each tube of cells was added with 2 ml of red blood cell lysis solution, blown, and after standing in the lysis solution for 2 minutes, medium was added and the cells were aggregated.

[0062] Finally, centrifugation was performed again, and the centrifugation conditions were adjusted to 1000 rpm and 5 minutes. After centrifugation, the cell supernatant was discarded, the cells were added to complete medium, resuspended by blowing, then placed in a 100 mm culture dish in an incubator, and subjected to differential adhesion for 90 minutes. Then, the medium in the culture dish was aspirated into a new 50 ml centrifuge tube for counting, and the cells were cultured as needed, and 100 μM bromodeoxyuridine (Brdu) was added to inhibit the growth of fibroblasts. After 48 hours, the cells were treated with serum-free medium.

[0063] 2. Construction of a doxorubicin-induced primary myocardial cell injury model

[0064] 1 mg of doxorubicin was dissolved in 3.68 mL of PBS solution to prepare a 0.5 mM doxorubicin stock solution. 1.29 mg of 5-oxoproline was dissolved in 10 mL of PBS solution to prepare a 1 mM 5-oxoproline stock solution. The primary myocardial cells were seeded at a density of 2 x 10 5 / mL density inoculated in 96-well cell culture plates. Two days before culture, the medium was high-sugar DMEM plating medium containing Brdu and 10% serum, and on the third day, it was replaced with high-sugar DMEM medium. After the cell state was stable, the cell culture solution was evenly divided into three groups: control group, DOX group, DOX+5-OXO group, each group as follows:

[0065] Control group: add the same volume of PBS;

[0066] DOX group: add the corresponding volume of doxorubicin stock solution to the working concentration (1uM);

[0067] DOX+5-OXO group: add the same amount of doxorubicin stock solution as the DOX group and 5-oxoproline stock solution with gradient concentration to the working concentration (1uM, 2uM, 5uM).

[0068] Each group was incubated in a constant temperature medium for 24h, and then CCK8 experiment was performed.

[0069] 3、CCK-8 cell activity detection

[0070] After incubating the above-mentioned each group of neonatal rat myocardial cells (NRCMs) in a constant temperature incubator for 24h, first wash the cell surface with PBS, then incubate the cells with high-sugar DMEM medium containing 10% CCK-8 reagent in a light-proof environment at 37℃ constant temperature for 2h. After incubation, measure the absorbance at 450nm with a microplate reader, and draw a cell activity curve, as shown in Figure 2.

[0071] As shown in Figure 2, 5-OXO can effectively reduce the myocardial cell toxicity caused by DOX, and the 5uM 5-OXO supplementation concentration has the most obvious improvement effect.

[0072] Example 3

[0073] 5-oxoproline intervention of doxorubicin-induced myocardial injury in mice

[0074] The instruments and reagents used in this example are shown in Tables 4 and 5, respectively.

[0075] Table 4 Main instruments and equipment of this example

[0076] Table 5 Main reagents of this example

[0077] 1. Experimental animals and grouping 32 SPF male C57 mice, 6-8 weeks old, weighing about 25 g, were provided by the Experimental Animal Center of Renji Hospital, Shanghai Jiaotong University, and were adaptively fed for 3 days before the experiment. During the feeding period, the mouse feeding room was kept clean and tidy, with constant temperature and humidity (temperature 23±1℃, humidity 40±5%), and with a 12-hour light-dark cycle. All experiments were conducted in accordance with the guidelines for the ethics of experimental animals and were approved by the Animal Ethics Committee of Renji Hospital, Shanghai Jiaotong University.

[0078] The mice were randomly divided into control group (Ctrl), 5-oxoproline group (5-OXO), doxorubicin group (DOX), and doxorubicin + 5-oxoproline group (DOX+5-OXO), and the dosing of each group was as follows:

[0079] 5-oxoproline group (5-OXO): 5-oxoproline was given intraperitoneally (0.05 mg / kg), once a day

[0080] Doxorubicin group (DOX): doxorubicin was given intraperitoneally (5 mg / kg), once a week;

[0081] Doxorubicin + 5-oxoproline group (DOX+5-OXO): doxorubicin + 5-oxoproline was given, with the same dosage as the 5-oxoproline group (5-OXO) and the doxorubicin group (DOX);

[0082] Control group: the same amount of normal saline solution as the doxorubicin + 5-oxoproline group (DOX+5-OXO) was given;

[0083] Each group was given medication continuously for 4 weeks, and the body weight of the mice was measured every week during the medication process.

[0084] After 4 weeks of modeling and medication, the mice were observed for 1 week, and then the heart was taken after the cardiac color Doppler ultrasound examination (Vevo ultrahigh resolution multi-modal small animal ultrasound photoacoustic imaging system). After the sample was taken, the heart was weighed after the water was absorbed.

[0085] The results of the body weight change and heart weight change of the mice in each group after medication are shown in Figure 3. As can be seen from Figure 3, with the extension of the medication time, the body weight and heart weight of the mice in the doxorubicin group (DOX) decreased significantly, the body weight of the mice in the 5-oxoproline group (5-OXO) decreased slightly compared with the control group, and the body weight and heart weight of the mice in the doxorubicin + 5-oxoproline group (DOX+5-OXO) decreased compared with the doxorubicin group (DOX). Thus, it can be shown that 5-oxoproline (5-OXO) can restore the decreased body weight and heart weight of the mice.

[0086] Then the myocardial tissue of each group was subjected to pathological tissue detection: HE, Masson staining was used to observe the pathological changes of myocardial tissue; WGA staining was used to observe the size changes of myocardial cells; 4-HNE staining was used to detect the degree of oxidative stress of myocardial cells; TUNEL staining was used to detect the apoptosis index of myocardial cells.

[0087] 2. Mouse heart ultrasound detection

[0088] Vevo ultrahigh-resolution small animal ultrasound imaging system and high-frequency probe were used for mouse heart ultrasound examination to detect the heart function of the control group, 5-OXO group, DOX group and DOX+5-OXO group after 4 weeks of administration.

[0089] Before the experiment, the mouse's chest hair was first removed with depilatory cream, and half of the mice were anesthetized by isoflurane inhalation and maintained spontaneous breathing. Subsequently, short-axis section images were obtained at the left ventricular papillary muscle position on the side of the sternum to evaluate the left ventricle. At the same time, 15 cardiac cycles of M-mode echocardiogram were recorded using guided two-dimensional mode, and left ventricular end-diastolic volume (LVEDV) was measured. The detection results are shown in Figures 4 and 5.

[0090] As can be seen from the results of M-mode ultrasound examination of the left ventricle of the heart and the functional level in Figures 4 and 5, 5-oxoproline intervention significantly restored the reduced left ventricular end-diastolic volume (LVEDV), reduced the degree of cardiac atrophy induced by doxorubicin; at the same time, significantly increased the ejection fraction (EF%), fractional shortening (FS%) and stroke volume (CO), improved myocardial contractile function and compliance.

[0091] 2. Pathological tissue detection

[0092] 2.1 Preparation of mouse heart paraffin sections

[0093] The hearts of the mice in the above groups after modeling were sampled, washed with PBS and the excess tissue was cut off. Then the heart was fixed in 4% paraformaldehyde for 24 h and stored in a 4°C refrigerator. After fixation, the heart was placed in a tissue embedding box for dehydration. After the embedding machine paraffin was melted, the embedding box was placed in the preheated embedding machine tissue tank, and the first tank was immersed in wax for two times and the second tank was immersed in wax for three times. After the tissue was dehydrated, the sample was placed in the wax- immersed embedding box, a small amount of wax was added, the embedding box was closed and cooled, and the embedding was completed at this time.

[0094] The embedded wax block was fixed on a pre-cooled paraffin sectioning machine, the section thickness was adjusted to 10 μm and the thickness of the formed section was 3 μm. After the wax section was cut, it was spread in a 40°C preheated water tank with a brush, then it was lifted with a glass slide and placed on a sectioning machine for 30 min.

[0095] 2.2 Masson staining

[0096] After dewaxing, the sections were washed with ddH2O for 3x5min on a 40rpm shaker. Then the sections were circled with a histological pen at the periphery of the heart and allowed to dry. Subsequently, the sections were stained in the following order:

[0097] (1) The sections were placed in the mordant solution for mordanting, incubated in a 60°C oven for 1h, and then washed with ddH2O for 3x5min;

[0098] (2) The sections were stained with azure blue solution for 3min at room temperature, and then washed with ddH2O for 2x15s;

[0099] (3) The sections were stained with Mayer's hematoxylin solution for 3min, and then washed with ddH2O for 2x15s;

[0100] (4) The sections were differentiated with acid differentiation solution for 5s, and then washed with ddH2O for 10min;

[0101] (5) The sections were stained with ponceau red solution for 10min, and then washed with ddH2O for 2x15s;

[0102] (6) The sections were treated with phosphomolybdic acid solution for 10min;

[0103] (7) After removing the phosphomolybdic acid, aniline blue solution was added directly for 5min;

[0104] (8) After removing the aniline blue with acetic acid solution, weak acid solution was added for 2min;

[0105] (9) The sections were dehydrated with 95% ethanol for 30s, anhydrous ethanol for 2x10s, xylene for 2x5s, and neutral balsam for mounting.

[0106] The staining results are shown in FIG. 6. As shown in FIG. 6, Masson staining showed that the myocardial fibrosis in the DOX group was significantly reduced by 5-oxoproline.

[0107] 2.3 HE Staining

[0108] After dewaxing, the sections were immersed in distilled water for 2min. Then the sections were circled with a histological pen to define the tissue section area, and after the histological pen mark was dry, hematoxylin staining solution was added for 15min, and then the sections were immersed in distilled water to remove the excess dye. Differentiation solution was added to the sections for 30s, and then the sections were immersed in tap water for 2x5min. Finally, eosin staining solution was added for 1min, and then the sections were washed with distilled water to remove the residual dye, and then the sections were rapidly dehydrated. The dehydration steps were as follows:

[0109] (1) 75% ethanol, 85% ethanol, 95% ethanol, and 100% ethanol were added for 3s;

[0110] (2) 100% ethanol was added for 1min, and then xylene was added to remove impurities for 2x1min, and then the sections were mounted with neutral balsam.

[0111] After the end of the sealing, the results were observed under the microscope and are shown in Figure 7.

[0112] 2.4 WGA staining

[0113] The paraffin sections were removed, de-waxed and hydrated. The sections were placed in a micro-well plate containing PBS (phosphate buffered saline), one well for each sample. 0.1% Triton X-100 (in PBS) was added and incubated for 10 minutes, and then washed with PBS three times. WGA solution was added for staining. The concentration of WGA and the time can be optimized according to the experiment. Generally, the concentration of WGA is 5-20 μg / mL, and the staining time is 30-60 minutes. Washed with PBS three times, each time for 5 minutes. DAPI (4', 6-diamidino-2-phenylindole) staining solution was added and incubated for 10 minutes, and then washed with PBS three times. The slide was inverted, and an appropriate amount of anti-browning glue was added to cover it, and the fluorescence was observed. The results are shown in Figure 8.

[0114] As can be seen from Figure 7 and Figure 8, the myocardial cells in the control group were normal in shape; myocardial cell atrophy was found in the DOX group, and 5-oxoproline could significantly intervene in the myocardial cell atrophy caused by DOX.

[0115] 2.5 4-HNE immunohistochemical staining

[0116] After the heart paraffin sections were de-waxed, antigen repaired, and permeated, they were blocked and probed with a primary antibody (4°C, overnight), and then incubated with a secondary antibody at room temperature for another 1 h. After adding SP (streptavidin-peroxidase), DAB was developed for 5-10 min, the reaction was terminated, hematoxylin was re-stained for 2 min, hydrochloric acid alcohol was differentiated, and conventional dehydration, transparency, and sealing were performed. The results are shown in Figure 9.

[0117] As can be seen from Figure 9, the degree of oxidative stress of mouse myocardial cells was detected by 4-HNE immunohistochemical staining, and it was found that the degree of oxidative stress of mouse myocardial tissue in the DOX group was significantly increased compared with the control group, and the oxidative stress level of mouse myocardial tissue after the corresponding 5-oxoproline intervention was significantly decreased.

[0118] 2.9 Tunel staining

[0119] After the heart paraffin sections were de-waxed, antigen repaired, and permeated, they were blocked. An appropriate amount of TUNEL detection solution (TdT enzyme: fluorescent labeling solution = 1:9) was prepared and mixed thoroughly. 50 μl of TUNEL detection solution was added to the sample, and incubated at 37°C in the dark for 60 minutes. After washing with PBS three times, the sample was observed under a fluorescence microscope after being sealed with an anti-fluorescence quenching sealing solution, and the results are shown in Figure 10.

[0120] As shown in Figure 10, the apoptosis of myocardial cells in mice was detected by TUNEL, and it was found that the number of apoptotic myocardial cells in the DOX group was significantly increased compared with the control group, while the number of apoptotic myocardial cells in the 5-oxoproline intervention group was significantly reduced compared with the DOX group.

[0121] As can be seen from the above experimental content, 5-oxoproline can partially restore the decreased heart function and heart weight, improve myocardial cell atrophy and myocardial tissue fibrosis, reduce myocardial tissue oxidative stress and myocardial cell apoptosis, and can be used for preventing or treating chemotherapy-induced cardiomyopathy in tumor patients. The inventors of the present application first discovered that the level of 5-oxoproline was significantly decreased in chemotherapy-induced cardiomyopathy, and further studied the mechanism of chemotherapy-induced cardiomyopathy, and finally found that 5-oxoproline had good effect on preventing or treating chemotherapy-induced cardiomyopathy in tumor patients, and provided a small molecule metabolite for chemotherapy-induced cardiomyopathy in tumor patients as a therapeutic drug, which has important clinical significance.

[0122] Finally, it should be explained that the above examples are only used to illustrate the technical solutions of the present application, but not to limit the protection scope of the present application. Although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present application.

Claims

1. Use of 5-oxoproline for the preparation of a medicament for the prevention or treatment of cardiomyopathy induced in a patient with cancer after chemotherapy with an anthracycline.

2. The use of 5-oxoproline according to claim 1 for the preparation of a medicament for the prevention or treatment of cardiomyopathy, characterized in that, The anthracycline is doxorubicin.

3. Use of 5-oxoproline for the preparation of a medicament for the restoration of decreased cardiac function, the restoration of decreased heart weight, the improvement of cardiomyocyte atrophy, the improvement of myocardial tissue fibrosis, the reduction of myocardial tissue oxidative stress, the reduction of cardiomyocyte apoptosis, any one of which is induced in a patient with cancer after chemotherapy with an anthracycline.

4. Use according to claim 3, wherein the compound is ###0002### The anthracycline is doxorubicin.

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