Application of 2 '-fucosyllactose in preparation of medicine for preventing and treating doxorubicin-induced cardiotoxicity and / or myocardial aging

Abstract

The invention relates to the technical field of drug research, in particular to application of 2 '-fucosyllactose (2'-FL) in preparation of a drug for preventing and treating doxorubicin-induced cardiotoxicity and / or myocardial aging. The invention relates to a preparation method of the traditional Chinese medicine. The 2, 2 '-fucosyllactose can reduce cardiotoxicity and myocardial aging induced by doxorubicin, improve abnormal expression conditions, caused by doxorubicin, of P53, P21, P16, IFN alpha, IL-1beta, IL-6, TNF-alpha and IRF3 in rat myocardial cells, reduce expression of doxorubicin-induced rat myocardial cell aging markers, namely-beta-galactosidase, and improve rat myocardial cell aging sensitivity. And the conditions of abnormal increase of P53, P16 and P21 proteins caused by adriamycin, abnormal mitochondrial membrane potential and myocarditis and fibrosis caused by adriamycin are improved.

Description

Technical Field

[0001] The invention relates to the technical field of drug research, in particular to application of 2'-fucosyllactose in preparing drugs for preventing and treating adriamycin-induced cardiotoxicity and / or myocardial senescence. 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.

[0003] 2'-fucosyllactose is an oligosaccharide composed of fucose, lactose and glucose linked by glycosidic bonds. Its name comes from the structural feature that fucose is linked to the hydroxyl group at the 2' position of lactose. It is mainly found in human breast milk in nature and is one of the most abundant nutrients in breast milk. In addition, it can also be obtained by microbial fermentation or chemical synthesis. 2'-fucosyllactose has intestinal flora regulation, immunomodulatory and antibacterial effects. The official evaluation committee for new foods has evaluated this value and confirmed its safety. Other government risk assessment agencies have evaluated the existing safety data and concluded that this ingredient is safe for use in infant formula at a concentration not exceeding 2.0g / L (based on ingestion). The US FDA and the Dutch Medical Evaluation Board (MEB) have both verified its safety. However, more uses of 2'-fucosyllactose are yet to be explored. Summary of the invention

[0004] Based on the above technical problems, the present invention provides the use of 2'-fucosyllactose in the preparation of a drug for improving adriamycin-induced myocardial injury and / or myocardial aging.

[0005] The present invention provides the following technical solutions: In a first aspect, the present invention provides a use of 2'-fucosyllactose in the preparation of a drug for improving adriamycin-induced cardiotoxicity and / or myocardial aging.

[0006] As a preferred embodiment of the present invention, the 2'-fucosyllactose is used to prepare a drug for improving the survival rate of cardiomyocytes.

[0007] As a preferred embodiment of the present invention, the 2'-fucosyllactose is used to prepare a drug for improving myocardial cell damage.

[0008] Furthermore, the drug is prepared by compounding the 2'-fucosyllactose with a pharmaceutically acceptable auxiliary material or carrier.

[0009] As a preferred embodiment of the present invention, the drug is an oral preparation or an injectable preparation.

[0010] It is understood that the medicine of the embodiment of the present invention can be added with different pharmaceutically acceptable excipients to prepare suitable clinical dosage forms, which include but are not limited to the following dosage forms: tablets, capsules, granules, powders, syrups or other oral liquid preparations, pills, injections, preferably granules, capsules or tablets. These pharmaceutically acceptable excipients include but are not limited to diluents, wetting agents, adhesives, disintegrants, lubricants, regulators, solubilizers, cosolvents, emulsifiers, antioxidants, preservatives, pH regulators, isotonic or isotonic regulators, etc.

[0011] Among them, the diluent is selected from starch, sucrose, cellulose, inorganic salts, etc.; the wetting agent is selected from water, ethanol, etc.; the binder is selected from starch slurry, dextrin, sugar, cellulose derivatives, gelatin, povidone, polyethylene glycol, etc.; the disintegrant is selected from starch, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, sodium cross-linked methyl cellulose, cross-linked polyvinylpyrrolidone, surfactant, Paon disintegrant, etc.; the lubricant is selected from talc, calcium stearate, magnesium stearate, magnesium lauryl sulfate, micro powder silica gel, polyethylene glycol, etc.; the regulator is selected from pigments, flavors, sweeteners, mucilage agents, deodorants, etc.; the solubilizer is selected from Tweens, polyoxyethylene fatty alcohol ethers, soaps, sulfates, sulfonates, etc.; the cosolvent is selected from organic acids and their salts, amide and amine compounds, inorganic salts, polyethylene glycol, glycerol, etc.; the emulsifier The antioxidant is selected from Span, Tween, Methylcellulose, benzylglycerol fatty acid ester, higher fatty acid salt, sulfate, sulfonate, gum arabic, tragacanth, gelatin, pectin, phospholipid, agar, sodium alginate, hydroxide, silicon dioxide, bentonite, etc.; the antioxidant is selected from sulfite, pyrosulfite, bisulfite, ascorbic acid, gallic acid and its esters, etc.; the preservative is selected from parabens, organic acids and their salts, quaternary ammonium compounds, chlorhexidine acetate, alcohols, phenols and volatile oils, etc.; the pH adjuster is selected from hydrochloric acid, sulfuric acid, phosphoric acid, tartaric acid, acetic acid, sodium hydroxide, sodium bicarbonate, ethylenediamine, meglumine, phosphate, acetate, citric acid, citrate, etc.; the isotonic or isotonic adjuster is selected from glucose, sodium chloride, sodium citrate, sorbitol and xylitol, etc.

[0012] It can be understood that the drugs involved in the embodiments of the present invention can be prepared in different dosage forms based on different excipients, and accordingly, the administration methods can also be diverse.

[0013] The second aspect of the present invention provides a drug for improving adriamycin-induced cardiotoxicity and / or myocardial aging, wherein the drug contains the 2'-fucosyllactose as the only effective active ingredient.

[0014] The third aspect of the present invention provides a drug for improving the survival rate of cardiomyocytes, which contains the 2'-fucosyllactose as the only effective active ingredient.

[0015] In a fourth aspect, the present invention provides a drug for improving myocardial cell damage, which contains the 2'-fucosyllactose as the only effective active ingredient.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The present invention shows that 2'-fucosyllactose can reduce the cardiotoxicity induced by doxorubicin through cell proliferation experiments and rat body weight experiments; shows that 2'-fucosyllactose can improve the cardiotoxicity caused by doxorubicin through ultrasonic detection and serum detection; shows that 2'-fucosyllactose can improve the abnormal expression of P53, P21, P16, IFNα, IL-1β, IL-6, TNF-α and IRF3 in rat cardiomyocytes induced by doxorubicin through q-PCR experiments; shows that 2'-fucosyllactose can reduce the high expression of β-galactosidase, a marker of rat cardiomyocyte aging induced by doxorubicin through β-galactosidase staining experiments; shows that 2'-fucosyllactose can reduce the high expression of β-galactosidase, a marker of rat cardiomyocyte aging induced by doxorubicin through Western Blot experiments showed that 2'-fucosyllactose could improve the abnormal increase of P53, P16 and P21 proteins caused by doxorubicin; immunofluorescence experiments showed that 2'-fucosyllactose could improve the abnormal mitochondrial membrane potential caused by doxorubicin; HE and Masson staining of rat heart tissue sections showed that 2'-fucosyllactose could improve myocardial inflammation and fibrosis caused by doxorubicin. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a screening diagram for the optimal cell administration concentration of doxorubicin (DOX) and 2'-fucosyllactose (2'-FL), where CON is the blank group. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001. A, DOX concentration screening; B, 2'-fucosyllactose (2'-FL) + DOX concentration screening; C, 2'-fucosyllactose concentration screening;

[0018] Figure 2Schematic diagram of the expression of P53, P16, TNF-α, IFNα, IRF3, IL-1β, and IL-6 after cells were treated with the blank group (CON), doxorubicin (DOX), and 2'-fucosyllactose (2'-FL) + DOX. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001.

[0019] Figure 3 Schematic diagram of the results of β-galactosidase staining after H9C2 cells were treated with the blank group (CON), doxorubicin (DOX), and 2'-fucosyllactose (2'-FL) + DOX.

[0020] Figure 4 Western Blot results and image J analysis diagram of H9C2 cells treated with the blank group (CON), doxorubicin (DOX), and 2'-fucosyllactose (2'-FL) + DOX. * P <0.05, ** P <0.01.

[0021] Figure 5 The graph shows the changes in mitochondrial membrane potential of H9C2 cells after treatment with the blank group (CON), doxorubicin (DOX), and 2'-fucosyllactose (2'-FL) + DOX.

[0022] Figure 6 Schematic diagram of the survival status, body weight changes, heart weight, heart volume ratio and serum test results of rats in the blank group (CON), doxorubicin (DOX), and 2'-fucosyllactose (2'-FL) + DOX treatment.

[0023] Figure 7 These are the ultrasonic detection results of cardiac function indexes in rats after treatment with blank group (CON), doxorubicin (DOX), and 2'-fucosyllactose (2'-FL) + DOX. * P <0.05, ** P <0.01.

[0024] Figure 8Schematic diagram of the results of β-galactosidase staining, HE staining, and MASSON staining of rat heart tissue after treatment with the blank group (CON), doxorubicin (DOX), and 2'-fucosyllactose (2'-FL) + DOX. The black arrows point to the muscle fibers, and the red arrows point to the inflammatory cells.

[0025] Fig. 9 Schematic diagram of the expression of P16, P21, P53, IFNα, IFNβ, and IRF3 in rat heart tissue after treatment with the blank group (CON), doxorubicin (DOX), and 2'-fucosyllactose (2'-FL) + DOX. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001. DETAILED DESCRIPTION

[0026] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0027] Doxorubicin is a broad-spectrum antitumor drug with dose-dependent cardiotoxicity, including various types of arrhythmias, ventricular dilatation, and ultimately congestive heart failure. These cardiotoxicities greatly limit the clinical use of DOX.

[0028] 2'-fucosyllactose is an oligosaccharide composed of fucose, lactose and glucose connected by glycosidic bonds. 2'-fucosyllactose has the effects of regulating intestinal flora, immunomodulation and antibacterial. However, more uses of 2'-fucosyllactose are yet to be explored.

[0029] The present invention proves through experiments that 2'-fucosyllactose can reduce the cardiac toxicity induced by doxorubicin, improve the abnormal expression of P53, P21, P16, IFNα, IL-1β, IL-6, TNF-α, and IRF3 in rat cardiomyocytes induced by doxorubicin, reduce the high expression of β-galactosidase, a marker of rat cardiomyocyte aging induced by doxorubicin, improve the abnormal mitochondrial membrane potential caused by doxorubicin, and improve myocardial inflammation and fibrosis caused by doxorubicin.

[0030] 1. Cell experiments 1. Cell proliferation assay (1) Inoculate the H9C2 cell suspension (100 uL / well) in a 96-well plate and pre-culture in a humidified incubator for 24 h (at 37°C, 5% CO2).

[0031] (2) Dissolve 2'-fucosyllactose (2'-FL) and doxorubicin (DOX) in serum-free culture medium to prepare a final concentration of 0.5 μM DOX and 0.5 μM DOX + 100 μM 2'-FL mixed solution. Discard the original culture medium in the 96-well plate and divide the plate into three groups: CON\DOX\2'-FL+DOX. Add 100 μL of serum-free culture medium, 0.5 μM DOX, and 0.5 μM DOX + 100 μM 2'-FL, respectively, and culture in a humid incubator for 24 h (at 37°C, 5% CO2).

[0032] (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 to 4 hours.

[0033] (4) Use an ELISA reader to measure the absorbance at 450 nm and perform data analysis. Cell viability (%) = [(As-Ab) / (Ac-Ab)]*100

[0034] As = absorbance of the experimental well (luminosity of the well containing cells, culture medium, CCK-8 and test compound) Ab = absorbance of blank well (absorbance of wells containing culture medium and CCK-8) Ac = absorbance of control well (absorbance of wells containing cells, culture medium and CCK-8) 2. qPCR (1) H9C2 cells were divided into three groups: blank group (CON), doxorubicin (DOX), and 2'-fucosyllactose (2'-FL) + DOX. 5 ml of culture medium containing 10% serum was added to the CON group, 5 ml of 0.5uM doxorubicin solution prepared with 10% culture medium was added to the DOX group, and 5 ml of a mixed solution of 0.5μM DOX + 100μM 2'-FL was added to the 2'-FL + DOX group, and the cells were incubated in an incubator for 24 h.

[0035] (2) Collect cells and extract RNA after lysis (3) Configure the RT system to reverse transcribe RNA into DNA.

[0036] (4) Configure the Real Time PCR system and perform the Real Time PCR reaction.

[0037] 3. β-Galactosidase staining (1) Inoculate the cells in the culture dish into a 6-well plate and treat the cells (same as 2.1) (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 min.

[0038] (3) Discard the fixative solution and wash with PBS three times, 2 min each time.

[0039] (4) Aspirate PBS and add 1 ml of β-galactosidase staining solution to each well. Incubate at 37°C for 2 h to overnight.

[0040] (5) After positive cells are observed to be colored under an ordinary optical microscope, remove the staining solution, wash the staining solution with PBS, and finally add 1 ml of PBS to cover the cells and observe.

[0041] 4. Western Blot (1) Protein extraction: After treating the cells according to the qPCR method, collect the suspended cells by centrifugation. Discard the original culture medium in the centrifuge tube, add 1 ml of pre-cooled PBS to resuspend the cells, and then transfer to a 4℃ high-speed centrifuge for centrifugation (3000rpm, 5min). After centrifugation, aspirate the supernatant, add an appropriate amount of pre-configured protein lysis buffer (RIPA lysis buffer: ProteaseInhibitor Cocktail=100:1), vortex to mix, and lyse at 4℃ for 30min. After lysis, centrifuge at 4℃ (12000rpm, 15min), 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.

[0042] (0) Electrophoresis: Prepare separation gel and concentrated gel in proportion, insert the comb after filling, remove 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.

[0043] (1) Transfer: Use fast transfer solution and transfer at a constant current of 350 mA for 30-40 minutes.

[0044] (4) Antibody incubation: Wash the transferred membrane twice with TBST (70 rpm, 5 min), add fast blocking solution and block at room temperature (60 rpm, 10 min). After blocking, remove the blocking solution, wash three times with TBST (70 rpm, 5 min), add the diluted primary antibody, and shake at 4°C overnight (60 rpm). Recover the primary antibody, wash three times with TBST (70 rpm, 5 min), add the diluted secondary antibody, and incubate at room temperature for 60 min (60 rpm). After incubation, recover the secondary antibody and wash three times with TBST (60 rpm).

[0045] (5) Chemiluminescence detection: Remove the membrane, absorb the TBST with filter paper, add freshly prepared ECL mixed solution in the developing box to the protein side of the membrane, and expose it in a dark room.

[0046] 5. Mitochondrial membrane potential detection (1) Treat cells according to the qPCR method.

[0047] (2) Follow the instructions of the JC-1 kit (Biyuntian) and avoid light during the process. After the sample is prepared, observe it with a confocal microscope.

[0048] 6. Results 6.1 Screening of optimal cell administration concentrations of doxorubicin (DOX) and 2'-fucosyllactose (2'-FL) The results are as follows Figure 1 As shown. It can be seen that 2'-fucosyllactose within 10mM has little effect on the survival of H9C2 cells. Starting from 1μM, 2'-fucosyllactose can significantly improve the decrease in H9C2 cell survival rate caused by doxorubicin. Therefore, 0.5μM doxorubicin was screened as the dosing concentration and 100μM 2'-fucosyllactose was screened as the treatment concentration.

[0049] 6.2. q-PCR detection of P53, P16, TNF-α, IFNα, IRF3, IL-1β, and IL-6 expression According to the q-PCR results ( Figure 2 ), the expressions of P53, P16, TNF-α, IFNα, IRF3, IL-1β, and IL-6 increased after cells were treated with doxorubicin, and decreased significantly after the use of 2'-fucosyllactose.

[0050] 6.3. β-Galactosidase staining The results are as follows Figure 3 As shown, the blue part in the figure represents β-galactosidase. It can be seen that the expression of β-galactosidase in the doxorubicin group was significantly increased, and 2'-fucosyllactose could significantly reduce its expression.

[0051] 6.4 Western Blot Detection The results are as follows Figure 4 As shown in the figure, the expression of P16, P21, and P53 in the adriamycin group was significantly increased, and these indicators were reduced after the use of 2'-fucosyllactose.

[0052] 6.5. Mitochondrial membrane potential detection The JC-1 fluorescent probe was used to measure the mitochondrial membrane potential. Under high membrane potential conditions, JC-1 molecules aggregated in the mitochondria and exhibited red fluorescence. Under low membrane potential conditions, JC-1 existed in monomeric form and emitted green fluorescence. Figure 5 As shown in Figure 2, it can be seen that the mitochondrial membrane potential decreased after doxorubicin treatment, and the use of 2'-fucosyllactose can improve this situation.

[0053] 2. Animal Experiments 1. Rat Modeling SD rats were divided into three groups: blank group (CON), doxorubicin (DOX), and 2'-fucosyllactose (2'-FL) + DOX. The DOX group and the 2'-FL + DOX group were intraperitoneally injected with doxorubicin at 2 mg / kg body weight, and the CON group was injected with an equal amount of saline; two hours later, the 2'-FL + DOX group was intraperitoneally injected with 2'-fucosyllactose at 150 mg / kg body weight; twice a week for four consecutive weeks. The weight changes and deaths of the rats were recorded before each administration.

[0054] 2. Rat testing 2.1 Ultrasonic testing On the 30th day of modeling, the rats' chest and abdominal hair were cleaned and ultrasonic testing was performed.

[0055] 2.2 Serum testing and cardiac tissue section staining The next day after the ultrasound test, blood and heart tissue samples were collected from the heart. After recording the heart weight and corresponding number of each rat, the heart was cut into two halves, the apex was frozen with liquid nitrogen and stored in a -80°C refrigerator, and the other half was soaked in paraformaldehyde. The serum extracted from the blood after centrifugation was stored at 4°C and sent for examination on the same day to detect creatine kinase (CK) and lactate dehydrogenase (LDH). After the heart tissue in paraformaldehyde was fixed, HE staining was performed to observe inflammation and Masson staining was performed to observe fibrosis. Frozen tissue sections were then stained for β-galactosidase.

[0056] 2.3. Cardiac frozen tissue testing They were used to extract RNA for qPCR detection and protein for Western Blot experiments.

[0057] 3. Results 3.1 Changes in body weight, heart-to-body weight ratio, creatine kinase (CK), and lactate dehydrogenase (LDH) in mice The results show that ( Figure 6 ), doxorubicin caused a decrease in body weight, an increase in heart-to-body weight ratio, and an increase in creatine kinase (CK) and lactate dehydrogenase (LDH) in rats. These indicators were significantly improved after the use of 2'-fucosyllactose.

[0058] 3.2 Ultrasonic testing from Figure 7 It can be seen that in the doxorubicin group, the left ventricular ejection fraction (LVEF), left ventricular short axis shortening rate (LVFS), interventricular septum end-systolic thickness (IVS;s), left ventricular posterior wall thickness (LVPW) decreased, and the left ventricular end-systolic internal diameter (LVIDs) and left ventricular volume (LV VOL) ​​increased. These indicators can be significantly improved after the use of 2'-fucosyllactose.

[0059] 3.3 Tissue staining The results are as follows Figure 8 As shown in the figure, the expression of β-galactosidase was significantly increased in the doxorubicin group, and the inflammation and fibrosis of cardiac tissue increased, while 2'-fucosyllactose could significantly improve these conditions. Specifically: the muscle fiber structure of the CON group was normal, without congestion, cell degeneration and necrosis, and inflammatory reaction; the muscle fibers of the DOX group were loose, with enlarged gaps, and a small amount of inflammatory cell infiltration was observed in the muscle fiber inflammatory reaction; the muscle fiber structure of the 2'-fucosyllactose+DOX group was normal, without congestion, cell degeneration and necrosis, and inflammatory reaction.

[0060] 3.4 Expression of P16, P21, P53, IFNα, IFNβ, and IRF3 in cardiac tissue The results are as follows Fig. 9 As shown, the expression of these indices increased after cells were treated with doxorubicin, and decreased significantly after the use of 2'-fucosyllactose.

[0061] In summary, doxorubicin can induce cardiotoxicity and senescence in H9C2 cells and SD rats, increase the expression of P53, P21, P16, IFNα, IL-1β, IL-6, TNF-α, IRF3 and β-galactosidase, and cause disturbances in cardiac function indicators, while 2'-fucosyllactose can significantly improve doxorubicin-induced cardiotoxicity and myocardial senescence.

Claims

1. Use of 2'-fucosyllactose in the preparation of drugs for improving doxorubicin-induced cardiotoxicity and / or myocardial aging.

2. The use according to claim 1, characterized in that: The 2'-fucosyllactose is used for preparing medicine for improving the survival rate of myocardial cells.

3. The use according to claim 1, characterized in that: The 2'-fucosyllactose is used for preparing medicine for improving myocardial cell damage.

4. The use according to any one of claims 1 to 3, characterized in that: The drug is prepared by compounding the 2'-fucosyllactose with pharmaceutically acceptable auxiliary materials or carriers.

5. The drug according to claim 4, characterized in that The medicine is an oral preparation or an injection preparation.

6. The drug according to claim 5, characterized in that The medicine is in the form of granules, capsules or tablets.

7. A drug for improving adriamycin-induced cardiotoxicity and / or myocardial aging, characterized in that: The invention uses the 2'-fucosyllactose as claimed in claim 1 as the only effective active ingredient.

8. A drug for improving the survival rate of cardiomyocytes, characterized in that: The invention uses the 2'-fucosyllactose as claimed in claim 1 as the only effective active ingredient.

9. A drug for improving myocardial cell damage, characterized in that: The invention uses the 2'-fucosyllactose as claimed in claim 1 as the only effective active ingredient.

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