Application of spermidine in preparation of medicine for preventing and treating doxorubicin-induced cardiotoxicity and myocardial aging

Abstract

The invention relates to the technical field of spermidine application, in particular to application of spermidine in preparation of medicine for preventing and treating doxorubicin-induced cardiotoxicity and myocardial aging. Cell proliferation experiments and mouse survival rate experiments show that spermidine can reduce death of myocardial cells and mice caused by adriamycin; a q-PCR experiment shows that spermidine can improve abnormal expression conditions of P53, P21, P16, BAX, TNF-alpha, IFN alpha, IL-10 and IL-18 in myocardial cells caused by adriamycin; a beta-galactosidase staining experiment shows that the spermidine can reduce the condition that the expression of a marker beta-galactosidase for myocardial cell senescence induced by adriamycin is increased; a Western Blot experiment shows that the spermidine can improve the condition that the P53 protein is abnormally increased due to the adriamycin; an Elisa experiment shows that spermidine can improve the condition that myocardial injury markers cTn-I and AST and inflammatory factors IL-1beta are abnormally increased due to adriamycin, and the myocardial injury markers cTn-I and AST and inflammatory factors IL-1beta are abnormally increased due to adriamycin. Therefore, spermidine can improve cardiotoxicity and myocardial aging induced by adriamycin.

Description

Technical Field

[0001] The invention relates to the technical field of spermidine application, and in particular to application of spermidine in preparing medicines for preventing and treating adriamycin-induced cardiotoxicity and myocardial aging. Background Art

[0002] Doxorubicin (DOX) is a broad-spectrum anti-tumor drug with dose-dependent cardiotoxicity, including various types of arrhythmias, ventricular dilatation, and ultimately congestive heart failure, which greatly limits the clinical use of DOX. Currently, most studies on doxorubicin cardiotoxicity focus on the mechanism of mitochondrial damage, including mitochondrial DNA damage, mitochondrial oxidative stress, anthracycline binding to mitochondrial inner membrane cardiolipin, mitochondrial transition pore opening, and apoptosis. Despite extensive efforts, drugs that alleviate mitochondrial damage in cardiomyopathy, such as mitochondrial antioxidants, have not yet been clinically approved. Therefore, it is necessary to develop new drugs to treat doxorubicin-induced cardiotoxicity and myocardial aging. Summary of the invention

[0003] In order to solve the above problems, the present invention provides the use of spermidine in preparing a drug for preventing and treating adriamycin-induced cardiotoxicity and myocardial aging.

[0004] The present invention is achieved through the following technical solutions:

[0005] Application of spermidine in the preparation of drugs for preventing and treating doxorubicin-induced cardiotoxicity and myocardial senescence.

[0006] Preferably, spermidine is the only active ingredient in the drug for preventing and treating adriamycin-induced cardiotoxicity and myocardial senescence.

[0007] Preferably, the concentration of spermidine in the drug for preventing and treating doxorubicin-induced cardiotoxicity and myocardial senescence is 10 mg / kg.

[0008] Preferably, the drug includes spermidine and also includes pharmaceutically acceptable excipients.

[0009] Preferably, the pharmaceutically acceptable excipient is one or more of a diluent, a disintegrant, a precipitation inhibitor, a glidant, a binder, a dispersant, a suspending agent, an isotonic agent, a thickener, an emulsifier, a preservative and a stabilizer.

[0010] Preferably, the dosage form of the drug includes enema, tablet, capsule, granule, oral suspension, powder or paste.

[0011] Preferably, the spermidine exerts its preventive and therapeutic effects by reducing the expression of one or more of P53, P21, P16, BAX, TNF-α, IFNα, IL-10, IL-18, cTn-I, AST, IL-1β and β-galactosidase in cardiomyocytes.

[0012] A pharmaceutical composition for preventing and treating adriamycin-induced cardiotoxicity and myocardial senescence, comprising spermidine.

[0013] Preferably, the pharmaceutical composition is in the form of an oral or injectable preparation.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] The present invention provides an application of spermidine in preparing a drug for preventing and treating doxorubicin-induced cardiotoxicity and myocardial aging. The present invention shows through cell proliferation experiments and mouse survival rate experiments that spermidine can reduce the death of myocardial cells and mice caused by doxorubicin; through q-PCR experiments, it is shown that spermidine can improve the abnormal expression of P53, P21, P16, BAX, TNF-α, IFNα, IL-10, and IL-18 in myocardial cells caused by doxorubicin; through β-galactosidase staining experiments, it is shown that spermidine can reduce the increase in the expression of aging marker β-galactosidase in myocardial cells induced by doxorubicin; through Western Blot experiments, it is shown that spermidine can improve the abnormal increase of P53 protein caused by doxorubicin; through Elisa experiments on mouse heart tissue, it is shown that spermidine can improve the abnormal increase of myocardial injury markers cTn-I, AST and inflammatory factor IL-1β caused by doxorubicin. It is the first time to find that spermidine can improve doxorubicin-induced cardiotoxicity and myocardial aging. Therefore, spermidine can be used to develop its antioxidant activity, energy metabolism regulation, growth and development and immune regulation, autophagy inducer, anti-inflammatory properties, anti-aging and other functions in the preparation of drugs for preventing and treating doxorubicin-induced cardiotoxicity and myocardial aging. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0017] Figure 1The figure is a schematic diagram of the effect of spermidine and doxorubicin on the proliferation ability of H9C2 cells. Starting from 5uM, spermidine can significantly improve the decrease in the survival rate of H9C2 cells caused by doxorubicin; *P<0.05, **P<0.01, ***P<0.001.

[0018] Figure 2 This is a schematic diagram of the expression of P53, P16, P21, BAX, TNF-α, IFNα, IL-18 and IL-10 in cells treated with doxorubicin and spermidine compared with cells treated with doxorubicin alone; *P<0.05, **P<0.01, ***P<0.001.

[0019] Figure 2 In the figure, A is a schematic diagram of the expression of P53; B is a schematic diagram of the expression of P16; C is a schematic diagram of the expression of P21; D is a schematic diagram of the expression of BAX; E is a schematic diagram of the expression of TNF-α; F is a schematic diagram of the expression of IFNα; G is a schematic diagram of the expression of IL-18; and H is a schematic diagram of the expression of IL-10.

[0020] Figure 3 The electron microscopy results of β-galactosidase staining of senescent cells of the present invention and the image J image analysis schematic diagram are shown. The blue part in the figure represents β-galactosidase, and it can be seen that the expression of β-galactosidase in the adriamycin group is significantly increased, and spermidine can significantly reduce its expression.

[0021] Figure 3 In the figure, A is the electron microscopy result of β-galactosidase staining of senescent cells in the blank group; B is the electron microscopy result of β-galactosidase staining of senescent cells in the doxorubicin group; C is the electron microscopy result of β-galactosidase staining of senescent cells in the spermidine + doxorubicin group; D is a schematic diagram of image J image analysis.

[0022] Figure 4 It is a diagram showing the Western Blot result of P53 of the present invention and a schematic diagram of image J image analysis.

[0023] Figure 4 In the figure, A is the Western Blot result of P53; B is the image analysis diagram of Image J; *P<0.05, **P<0.01, ***P<0.001.

[0024] Figure 5 It is the survival rate curve diagram of mice of the present invention.

[0025] Figure 6 This is a schematic diagram of the Elisa test results after mouse heart tissue extraction in the present invention.

[0026] Figure 6In the figure, A is a schematic diagram of the Elisa detection results of IL-1β after extraction from mouse heart tissue; B is a schematic diagram of the Elisa detection results of cTn-I after extraction from mouse heart tissue; C is a schematic diagram of the Elisa detection results of AST after extraction from mouse heart tissue; *P<0.05, **P<0.01, ***P<0.001. DETAILED DESCRIPTION

[0027] In order to facilitate the understanding of the present invention, the present invention will be described more comprehensively below, and preferred embodiments of the present invention are provided. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0029] The inventive concept of the present invention is as follows:

[0030] Spermidine is widely present in nature, including organisms such as bacteria, mammals, birds, insects and plants. In the human body, spermidine is found in multiple organs such as the kidney, liver, intestines and stomach. The functions of spermidine include antioxidant activity, energy metabolism regulation, growth and development, and immune regulation. Currently, many experimental studies have shown that spermidine can maintain mitochondrial function, show anti-inflammatory properties, and prevent stem cell aging. It has potential application value in liver protection, neurodegenerative diseases, and delaying aging. Therefore, it is necessary to develop the application of spermidine in the preparation of drugs for the prevention and treatment of doxorubicin-induced cardiotoxicity and myocardial aging.

[0031] Based on this, the present invention provides the use of spermidine in the preparation of drugs for preventing and treating doxorubicin-induced cardiotoxicity and myocardial aging. The present invention shows through cell proliferation experiments and mouse survival rate experiments that spermidine can reduce the death of myocardial cells and mice caused by doxorubicin; through q-PCR experiments, it is shown that spermidine can improve the abnormal expression of P53, P21, P16, BAX, TNF-α, IFNα, IL-10, and IL-18 in myocardial cells caused by doxorubicin; through β-galactosidase staining experiments, it is shown that spermidine can reduce the increase in the expression of aging marker β-galactosidase in myocardial cells induced by doxorubicin; through Western Blot experiments, it is shown that spermidine can improve the abnormal increase of P53 protein caused by doxorubicin; through Elisa experiments on mouse heart tissue, it is shown that spermidine can improve the abnormal increase of myocardial injury markers cTn-I, AST and inflammatory factor IL-1β caused by doxorubicin. It is the first time to find that spermidine can improve doxorubicin-induced cardiotoxicity and myocardial aging. Therefore, spermidine can be used to develop its antioxidant activity, energy metabolism regulation, growth and development and immune regulation, autophagy inducer, anti-inflammatory properties, anti-aging and other functions in the preparation of drugs for preventing and treating doxorubicin-induced cardiotoxicity and myocardial aging.

[0032] The beneficial effects of the present invention are described below by means of specific embodiments:

[0033] Example 1

[0034] 1. Cell experiments

[0035] 1. Cell proliferation assay

[0036] 1.1 Inoculate H9C2 cell suspension in a 96-well plate at 100 ul / well and pre-culture for 24 h in a humidified incubator at 37°C and 5% CO2.

[0037] 1.2 Dissolve spermidine in culture medium to prepare spermidine solutions with different concentration gradients. Add 50ul of the prepared solution and 50ul of 1uM doxorubicin solution to each well. Incubate in a humid incubator at 37°C and 5% CO2 for 24h.

[0038] 1.3 Aspirate the drug solution in each well, add 100ul serum-free culture medium and 10ul CCK-8 solution to each well of the plate, and incubate the culture plate in an incubator for 1 hour.

[0039] The absorbance at 450 nm was measured using an enzyme reader for data analysis.

[0040] Cell survival rate (%) = [(As-Ab) / (Ac-Ab)]*100.

[0041] As = absorbance of experimental wells (the absorbance of wells containing cells, culture medium, CCK-8 and test compound). Ab = absorbance of blank wells (the absorbance of wells containing culture medium and CCK-8).

[0042] Ac = absorbance of control wells (absorbance of wells containing cells, medium and CCK-8).

[0043] 1.4 Experimental results Figure 1 As shown, starting from 5uM, spermidine can significantly improve the decreased survival rate of H9C2 cells induced by doxorubicin.

[0044] 2. qPCR

[0045] 2.1 According to the experimental results in 1, 10uM spermidine was selected as the optimal dose to improve doxorubicin-induced cardiotoxicity. H9C2 cells were divided into three groups: blank group, doxorubicin group and spermidine + doxorubicin group. 5ml of culture medium containing 1% serum was added to the blank group, 5ml of 0.5uM doxorubicin solution prepared with 1% culture medium was added to the doxorubicin group, 2.5ml of 1uM doxorubicin solution and 2.5ml of 20uM spermidine solution were added to the spermidine + doxorubicin group, and incubated in the incubator for 24h. The solvents for the configured drugs were all selected from culture medium containing 1% serum.

[0046] 2.2 Collect cell suspension and extract RNA

[0047] 2.3 Configure the RT system to reverse transcribe RNA into DNA.

[0048] 2.4 Configure the Real Time PCR system and perform the Real Time PCR reaction.

[0049] 2.5 Experimental results Figure 2 As shown, spermidine can improve the increase of P53, P21, P16, BAX, TNF-α, IFNα, IL-10, and IL-18 caused by doxorubicin.

[0050] 3. β-Galactosidase staining

[0051] 3.1 Inoculate the cells in the culture dish into a 6-well plate and treat the cells as in 2.1.

[0052] 3.2 Aspirate the cell culture medium, wash twice with PBS, add 1 ml of β-galactosidase staining fixative, and fix at room temperature for 15 minutes.

[0053] 3.3 Discard the fixative and wash with PBS three times, 2 min each time.

[0054] 3.4 Aspirate PBS, add 1 ml of β-galactosidase staining solution to each well, and incubate at 37°C for 2 h to overnight.

[0055] 3.5 After positive cells are observed to be colored under an ordinary optical microscope, remove the staining working solution and add 2 ml of PBS to cover the cells.

[0056] 3.6 Experimental results Figure 3 As shown, it can be clearly seen that the blue part in the doxorubicin group increased significantly compared with the blank group, and spermidine significantly reduced the expression of β-galactosidase.

[0057] 4. Western Blot

[0058] 4.1 Protein extraction: After treating the cells according to the method in 2.1, collect the suspended cells by centrifugation. Discard the original culture medium in the centrifuge tube, add 600ul pre-cooled PBS to resuspend the cells, and then transfer to a 4℃ high-speed centrifuge for centrifugation at a speed of 3000rpm for 5min. After the centrifugation, aspirate the supernatant, add an appropriate amount of pre-configured protein lysis buffer, which is prepared by RIPA lysis buffer and Protease Inhibitor Cockt ail at a volume ratio of 100:1, vortex mix, and lyse at 4℃ for 30min. After the lysis is completed, centrifuge at 4℃ at a speed of 12000rpm for 15min. After centrifugation, transfer the supernatant to a new EP tube, add 5× loading buffer in proportion, mix well, heat in a 100℃ metal bath for 10min, and cool to room temperature after heating.

[0059] 4.2 Electrophoresis: Prepare separation gel and concentrated gel in proportion, insert the comb after filling, pull out the comb after the gel solidifies, suck out the residual gel with a syringe, add the sample to the spotting well, and perform constant voltage electrophoresis at 80V for concentrated gel and 120V for separation gel.

[0060] 4.3 Transfer: Transfer the membrane at a constant current of 350 mA for 40 minutes.

[0061] 4.4 Antibody incubation: Wash the transferred membrane with TBST three times at a speed of 70 rpm for 5 minutes, add blocking solution and block at room temperature at a speed of 60 rpm for 10 minutes. After blocking, remove the blocking solution, wash three times with TBST at a speed of 70 rpm for 5 minutes, add the diluted primary antibody, shake overnight at 4°C at a speed of 60 rpm. Recover the primary antibody, wash three times with TBST at a speed of 70 rpm for 5 minutes, add the diluted secondary antibody, incubate at room temperature for 60 minutes at a speed of 60 rpm, recover the secondary antibody after incubation, wash three times with TBST at a speed of 60 rpm.

[0062] 4.5 Chemiluminescence detection: Take out the membrane, absorb TBST with filter paper, add freshly prepared ECL mixed solution in the developing box to the protein side of the membrane, and expose in a dark room.

[0063] 4.6 Experimental results Figure 4 As shown in the figure, it can be seen that the expression of P53 in the doxorubicin group was significantly increased, and spermidine could improve the increase of P53 expression induced by doxorubicin.

[0064] 2. Animal Experiments

[0065] 1. Mouse survival rate experiment

[0066] 1.1 Modeling: Kunming mice were divided into five groups; blank group, doxorubicin group, oral 3mM spermidine group, oral 30mM spermidine group, oral 15mM + intraperitoneal injection of 50mg / kg spermidine group. On the third day of the experiment, except for the blank group, a single intraperitoneal injection of doxorubicin 15mg / kg was given, and the blank group was injected with normal saline as a control. In this experiment, oral administration was performed by dissolving spermidine in drinking water, and drinking water was recorded and supplemented daily. The drinking water of the other groups was normal. In addition to adding spermidine to the daily drinking water, the oral 15mM + intraperitoneal injection of 50mg / kg spermidine group also injected 50mg / kg spermidine intraperitoneally every other day. Daily body weight changes and mouse deaths were recorded.

[0067] 1.2 Experimental results Figure 5 As shown, oral administration of spermidine can improve the mortality of mice induced by intraperitoneal injection of high-dose doxorubicin to some extent.

[0068] 2. Formal Experiment on Mice

[0069] 2.1 Modeling: The mice were divided into four groups: blank group, doxorubicin group, spermidine intraperitoneal injection group, and spermidine oral group. Except for the blank group, doxorubicin 0.5 mg / ml was intraperitoneally injected on the 1st, 8th, 15th, and 22nd days of the experiment, and the blank group was injected with normal saline as a control. Starting from the 23rd day, the intraperitoneal injection group was intraperitoneally injected with 10 mg / kg twice a week; the oral group was gavaged with 10 mg / kg once a day.

[0070] 2.2 Elisa detection indicators: troponin I (cTn-I), aspartate aminotransferase (AST), interleukin 1β (IL-1β).

[0071] 2.3 Experimental results Figure 6 As shown, whether it is oral or injected spermidine, it can improve the abnormal increase of IL-1β, cTn-I, and AST caused by doxorubicin to a certain extent.

[0072] Conclusion: From the experimental research data, it can be seen that doxorubicin can induce the death of H9C2 cells and mice, and increase the expression of P53, P21, P16, BAX, TNF-α, IFNα, IL-10, IL-18, cTn-I, AST, IL-1β and β-galactosidase, while spermidine can improve the survival rate of cardiomyocytes and the death of mice, reduce the expression of the above-mentioned proteins, and significantly improve doxorubicin-induced cardiotoxicity and myocardial aging.

[0073] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0074] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. Application of spermidine in the preparation of drugs for preventing and treating doxorubicin-induced cardiotoxicity and myocardial aging.

2. The use according to claim 1, characterized in that Spermidine is the only active ingredient of the drug for preventing and treating doxorubicin-induced cardiotoxicity and myocardial senescence.

3. The use according to claim 1, characterized in that The concentration of spermidine in the drug for preventing and treating adriamycin-induced cardiotoxicity and myocardial senescence is 5 mg / kg to 15 mg / kg.

4. The use according to claim 1, characterized in that The drug also includes pharmaceutically acceptable excipients.

5. The use according to claim 4, characterized in that The pharmaceutically acceptable drug excipient is one or more of a diluent, a disintegrant, a precipitation inhibitor, a glidant, a binder, a dispersant, a suspending agent, an isotonic agent, a thickener, an emulsifier, a preservative and a stabilizer.

6. The use according to claim 1, characterized in that The dosage form of the drug includes enema, tablet, capsule, granule, oral suspension, powder or ointment.

7. The use according to claim 1, characterized in that The spermidine exerts a preventive and therapeutic effect by reducing the expression of one or more of P53, P21, P16, BAX, TNF-α, IFNα, IL-10, IL-18, cTn-I, AST, IL-1β and β-galactosidase in cardiomyocytes.

8. A pharmaceutical composition for preventing and treating adriamycin-induced cardiotoxicity and myocardial senescence, characterized in that: The pharmaceutical composition comprises the spermidine according to claim 1.

9. The pharmaceutical composition according to claim 8, characterized in that The dosage form of the pharmaceutical composition is oral or injection preparation.

Images