Application of naringenin in preparation of medicine for preventing and treating tacrolimus-induced cardiotoxicity

Abstract

The invention discloses application of naringenin in preparation of a medicine for preventing and treating cardiotoxicity induced by tacrolimus. The naringenin disclosed by the invention is a natural active matter, is non-toxic to cells, and has a potential heart protection effect of reversing, preventing and treating tacrolimus-induced cardiotoxicity; naringenin reduces expression of Cav1.2 on a membrane so as to influence a calcium signal channel and reverse calcium overload and cell apoptosis caused by tacrolimus; cav1.2 can be used as a target spot for preventing and treating cardiotoxicity induced by FK506, and the invention also prompts that overload calcium can be treated by using a corresponding calcium chelating agent, so that a theoretical basis is provided for the naringenin as a potential auxiliary medicine for tacrolimus application; the further deep research is expected to be beneficial to deep understanding of the action mechanism of the naringenin and provide more support for clinical application of the naringenin.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and particularly relates to the use of naringenin in preparing a drug for preventing and treating tacrolimus-induced cardiotoxicity. Background Art

[0002] Tacrolimus (FK506, F) is a potent calcineurin inhibitor, which is widely used in anti-rejection therapy after organ transplantation. It exerts immunomodulatory effects by inhibiting T cell activation and lymphokine production. However, its clinical application is limited by a variety of toxic side effects, especially kidney and heart toxicity issues are increasingly concerned. The present invention focuses on cardiac toxicity. Studies have shown that tacrolimus can cause positive inotropic effects, enhance myocardial contractility and heart rate, and long-term use may cause cardiac overload, myocardial damage and even heart failure. A compassionate use study conducted in patients with end-stage pulmonary hypertension showed that 3 patients received low-dose FK506 (trough concentration 1.5-2.5 ng mL -1 ) After 12 months of treatment, their heart failure symptoms, New York Heart Association functional class, 6-minute walk distance, and N-terminal pro-brain natriuretic peptide level were improved, and the expression of bone morphogenetic protein receptor 2 in peripheral blood mononuclear cells was upregulated.

[0003] Calcium ions ([Ca 2+ i) Homeostasis is crucial for myocardial cell function. Tacrolimus may disrupt calcium homeostasis through indirect mechanisms, such as: 1. Reactive oxygen species (ROS)-mediated calcium imbalance: ROS generated by tacrolimus metabolism can damage sarcoplasmic reticulum function, leading to [Ca 2+ Abnormal release of cytoplasm leads to calcium overload, which in turn activates apoptosis signaling pathways (such as caspase -3 , Ba× / Bcl -2 2. Mitochondrial dysfunction: Calcium overload can impair mitochondrial membrane potential, inhibit ATP synthesis, and exacerbate oxidative stress, creating a vicious cycle. This pathological process is similar to the mechanism of cardiotoxicity induced by anthracyclines such as doxorubicin, suggesting that calcium overload may be a core component of tacrolimus cardiotoxicity.

[0004] Currently, the management of tacrolimus-induced cardiotoxicity primarily relies on dose adjustment and regular monitoring, with a lack of specific therapeutic agents. While traditional antioxidants (such as vitamin E) can partially alleviate oxidative stress, their effects on calcium homeostasis and apoptosis pathways are limited. Therefore, screening for natural compounds with multi-target protective effects has become an important research direction.

[0005] In addition, interventions for tacrolimus cardiotoxicity still have the following core problems: 1. Single-target limitations: Existing drugs (such as antioxidants, calcium regulators) only target a single pathological link and cannot cover the multi-mechanism toxicity network of tacrolimus. 2. Imbalance between efficacy and safety: Some protective agents (such as cyclosporine A) may aggravate immunosuppression or induce toxicity in other organs. 3. Lack of cross-model effectiveness: Drugs that are effective in doxorubicin or isoproterenol models (such as quercetin) have not yet been verified for their applicability to tacrolimus toxicity, and the mechanism speculation lacks experimental support. In addition, the application of natural flavonoids in cardiovascular protection is limited by the low delivery efficiency caused by their low solubility. For example, the quercetin nanoformulation reported in the literature has improved bioavailability, but the toxicity of the carrier material has limited clinical translation. How to develop a cardioprotective agent with multi-target synergistic effects, high safety and tissue accumulation is still a technical problem that needs to be broken through in this field. Summary of the Invention

[0006] The purpose of this section is to summarize some aspects of the embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the abstract and title of this application to avoid obscuring the purpose of this section, the abstract and the title of the invention, and such simplifications or omissions should not be used to limit the scope of the present invention.

[0007] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0008] Therefore, the purpose of the present invention is to overcome the deficiencies in the prior art and provide a method for preparing a drug for preventing and treating tacrolimus-induced cardiotoxicity.

[0009] To solve the above technical problems, the present invention provides the following technical solution: a use of naringenin in the preparation of a drug for preventing and treating tacrolimus-induced cardiotoxicity.

[0010] As a preferred embodiment of the application of the present invention, the concentration of naringenin in the drug is 40-80 μM.

[0011] As a preferred embodiment of the application of the present invention, the naringenin downregulates the expression of the L-type calcium channel Cav1.2 on the cell membrane, thereby restoring calcium overload to normal levels and affecting the calcium signaling pathway, thereby reversing the myocardial toxicity caused by tacrolimus-induced calcium overload.

[0012] As a preferred embodiment of the application of the present invention, the naringenin pre-incubation can prevent the cardiac toxicity caused by calcium overload due to the increased expression of Cav1.2 induced by tacrolimus.

[0013] As a preferred embodiment of the application of the present invention, the naringenin inhibits the calcium signaling pathway by restoring calcium homeostasis, thereby reversing the calcium overload and cell apoptosis caused by the increased expression of Cav1.2 on the cell membrane induced by tacrolimus.

[0014] As a preferred embodiment of the application of the present invention, the naringenin pre-incubation can stabilize the expression of Cav1.2 and prevent tacrolimus-induced cell apoptosis.

[0015] As a preferred embodiment of the application of the present invention, the Cav1.2 can serve as a key therapeutic target for calcium overload, alleviating the cardiotoxicity caused by calcium overload induced by FK506.

[0016] As a preferred embodiment of the application of the present invention, the Cav1.2 can be used as a screening marker for alleviating cardiac toxicity caused by calcium overload induced by FK506.

[0017] Another object of the present invention is to overcome the deficiencies in the prior art and provide a pharmaceutical composition, characterized in that: the pharmaceutical composition comprises the naringenin according to claim 2 and a pharmaceutically acceptable carrier or preparation.

[0018] As a preferred embodiment of the pharmaceutical composition of the present invention, the dosage form includes one of tablets, hard capsules, soft capsules, aqueous suspensions, oily suspensions, granules, emulsions, and syrups.

[0019] Beneficial effects of the present invention:

[0020] (1) The present invention demonstrates that the natural active ingredient naringenin is harmless to cells and has the effect of removing the toxicity of tacrolimus to protect the heart;

[0021] (2) Naringenin reversed tacrolimus-induced calcium overload and cell apoptosis by inhibiting the calcium signaling pathway by downregulating the expression of L-type calcium channel Cav1.2 protein on the cell membrane;

[0022] (3) The present invention demonstrates that Cav1.2 protein can be used as a potential target for the treatment of FK506-induced cardiotoxicity and can be used as a potential screening marker for FK506-induced cardiotoxicity;

[0023] (4) This invention provides a theoretical basis for naringenin as a potential antidote for tacrolimus-induced cardiotoxicity. Further research will help to gain a deeper understanding of the mechanism of action of naringenin and provide more theoretical support for its clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. Those skilled in the art can also derive other drawings based on these drawings without inventive effort. Among them:

[0025] Figure 1 This is the result of FK506-induced H9c2 cell cardiotoxicity model.

[0026] Figure 2 The figure shows the results of naringenin treatment of FK506-induced cardiotoxicity.

[0027] Figure 3 The figure shows the results of naringenin preventing FK506-induced cardiotoxicity.

[0028] Figure 4 The graph shows the effects of different concentrations of FK506 on calcium overload and L-type calcium channel protein expression in H9c2 cells.

[0029] Figure 5 The figure shows the results that naringenin alleviates FK506-induced calcium overload by inhibiting the high expression of Cav1.2.

[0030] Figure 6 This is the result of naringenin preventing tacrolimus-induced Cav1.2 overexpression and activation of calcium signaling pathway.

[0031] Figure 7 This is an experiment to reverse FK506-induced cell apoptosis by naringenin. DETAILED DESCRIPTION

[0032] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0033] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways different from those described herein. Those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0034] Secondly, the term "one embodiment" or "embodiment" herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in various places throughout this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive of other embodiments.

[0035] The structural formula of naringenin in the present invention is shown in Formula 1:

[0036]

[0037] The reagents used in the examples of the present invention are shown in Table 1:

[0038] Table 1

[0039]

[0040]

[0041]

[0042] Example 1 Establishment of a Tacrolimus / FK506-Induced Cardiotoxicity Model in Rat Cardiomyocytes H9c2

[0043] 1. CCK-8 assay

[0044] (1) Take rat cardiomyocytes H9c2 in the logarithmic growth phase and inoculate 5×10 3 ~1×10 4 cells in 96-well plates;

[0045] (2) After overnight incubation, 0, 2.5, 5, 10, and 20 μM tacrolimus and the same volume of solvent (anhydrous ethanol) were added for 12 h or 0, 10, 20, 40, and 80 μM naringenin and the same volume of solvent (DMSO) were added for 24 h;

[0046] (3) Incubate with FK506 (10 μM) for 12 h, incubate with naringenin (40 μM) for 24 h, and incubate with FK506 (10 μM) for 12 h followed by adding naringenin (40 μM) for 24 h. The diluted mixture was added to each well at a ratio of 100 μl of culture medium per 10 μl of CCK-8 reagent.

[0047] (4) After incubation at 37°C for 1.5 h, the absorbance at 450 nm was measured using a microplate reader.

[0048] 2. ELISA test

[0049] (1) Collect the cell culture supernatant: centrifuge at 4000 rpm for 20 min, remove the cell pellet, and store the supernatant below -20°C to avoid repeated freezing and thawing;

[0050] (2) Reagent preparation: All reagents and components are first returned to room temperature; dilute the concentrated washing solution taken out of the refrigerator and dissolve it in a water bath at a ratio of 1:20, that is, 1 part concentrated washing solution is added to 19 parts distilled water; substrate solution A and B are thoroughly mixed at a volume ratio of 1:1 and used within 15 minutes after mixing;

[0051] (3) Preparation of slats: Take out the required slats from the aluminum foil bag, seal the remaining slats in a ziplock bag and put them back into the refrigerator.

[0052] (4) Adding standards and samples: Set up standard wells, 0 value wells (add 50 μL of sample diluent), blank wells (no addition), and sample wells (add 50 μL of the sample to be tested); add 50 μL of standards of different concentrations to each standard well.

[0053] (5) Add enzyme-labeled antibody for incubation: Add 100 μL of HRP-labeled detection antibody to the standard wells, 0 value wells, and sample wells, except for the blank wells. Block with 5% skim milk or 5% BSA for 1 hour; cover the reaction plate with sealing film and incubate in a 37°C water bath or incubator in the dark for 60 minutes;

[0054] (6) Washing: Remove the sealing film, discard the liquid, and pat dry on absorbent paper. Fill each well with washing solution, let it stand for 20 seconds, shake off the washing solution, and repeat 5 times;

[0055] (7) Color development: Mix substrates A and B at a 1:1 volume ratio and add 100 μL of the substrate mixture to all wells. Incubate in a thermostat in the dark for 15 minutes.

[0056] (8) Stop reaction: add 50 μL of stop solution to all wells;

[0057] (9) Determine absorbance: Read the absorbance (OD value) of each well at a wavelength of 450 nm on a microplate reader;

[0058] (10) Calculation of results: Standard curve fitting - Use the standard concentration as the horizontal axis and the corresponding absorbance (OD value) as the vertical axis to create a standard curve equation using a four-parameter logistic curve fit. Using the standard curve equation, calculate the absorbance (OD value) of the sample.

[0059] The experimental results are as follows Figure 1 As shown, Figure 1 a is the chemical structural formula of tacrolimus.

[0060] Figure 1 b shows the CCK8 cell viability assay of H9c2 cells after incubation with different concentrations of FK506 (0μM, 2.5μM, 5μM, 10μM, and 20μM) for 12 hours. In the CCK8 assay, the cell viability of H9c2 cells gradually decreased with increasing FK506 concentrations. Compared with the control group, there was no significant difference in cell viability in the low-concentration 2.5μM and 5μM FK506-treated groups (P>0.05), while the cell viability in the medium-concentration 10μM and high-concentration 20μM FK506-treated groups was significantly reduced, and the reduction is expressed as a numerical value (P<0.05). This indicates that FK506 exerts a dose-dependent toxic effect on H9c2 cells.

[0061] Figure 1 cf is an ELISA kit to test the effects of different concentrations (0μM, 2.5μM, 5μM, 10μM, 20μM) of FK506 on the cardiotoxicity indicators LDH (lactate dehydrogenase), CK-MB (creatine kinase isoenzyme), cTn-T (cardiac troponin T), and BNP (brain natriuretic peptide) to determine the dosage concentration of FK506 that causes cardiotoxicity.

[0062] As the FK506 treatment time increased, the cell viability of H9c2 cells gradually decreased. Compared with the control group, the amount of LDH released by the FK506 treatment groups at all concentrations was significantly increased (P<0.05). In the ELISA experiment, LDH is lactate dehydrogenase, an intracellular enzyme that is released outside the cell when the cell membrane is damaged. It can be used as an important marker of cell damage. The ELISA results showed that ( Figure 1 c) There was no significant change in the LDH release in the low concentration (2.5μM, 5μM) FK506 treatment groups compared with the control group (P>0.05). The LDH release in the 10μM and 20μM FK506 treatment groups was significantly increased compared with the control group (P<0.05). The increase in the release indicated that H9c2 cells were significantly damaged by the toxicity of FK506 starting from the 10μM FK506 treatment.

[0063] CK-MB is a creatine kinase isoenzyme. When myocardial cells are damaged, CK-MB will be released into the blood, causing a significant increase in serum levels. It has certain specificity. The results of the ELISA experiment showed that ( Figure 1 d), CK-MB concentration also increased in a dose-dependent manner with the increase of FK506 concentration. Compared with the control group, the CK-MB concentration in the 10μM and 20μM FK506 treatment groups was significantly increased (P<0.05). The increase in CK-MB concentration indicated that FK506 damaged H9c2 cells.

[0064] cTn-T is cardiac troponin T, a highly sensitive and specific marker of myocardial injury and a core diagnostic indicator of myocardial injury. Under normal circumstances, its concentration in the blood is extremely low, and its release into the blood indicates myocardial cell damage or necrosis. ELISA test results show that ( Figure 1 e). cTn-T concentrations also increased in a dose-dependent manner with increasing FK506 concentrations. Compared with the control group, the release levels in the 10μM FK506-treated group were 180pg / ml, reaching a significant level (P<0.05), and the 20μM FK506-treated group released 190pg / ml, reaching a significant level, indicating that FK506 significantly damaged H9c2 cells.

[0065] BNP is a brain natriuretic peptide, a hormone secreted by the ventricles. When the heart is stressed or damaged, the ventricular wall is dilated or stretched, stimulating myocardial cells to release BNP into the blood. Increased levels of BNP are usually associated with heart failure and are an important marker for heart failure. ELISA test results show that ( Figure 1 .f) With the increase of FK506 concentration, the secretion of BNP gradually increased. Compared with the control group, the secretion of BNP in the FK506 treatment groups at all concentrations was significantly increased (P<0.05), increasing to 160pg / ml at 10μM and 180pg / ml at 20μM. The increase in secretion indicates that FK506 at 10μM can cause damage to H9c2 cells and impair cardiac function.

[0066] In summary, the FK506 modeling concentration was determined to be 10 μM based on comprehensive consideration of the clinical dosage.

[0067] Figure 1 g: H9c2 cells were stimulated with 10 μM FK506 at different times (0 h, 12 h, and 24 h), and CCK8 activity was detected.

[0068] Figure 1 hk is the changes in cardiac toxicity indicators LDH (lactate dehydrogenase), CK-MB (creatine kinase isoenzyme), cTn-T (cardiac troponin T), and BNP (brain natriuretic peptide) after H9c2 was stimulated with an equal amount of 10 μM FK506 at different times (0h, 12h, 24h, 48h) to determine the FK506 modeling time.

[0069] FK506 induces cardiotoxicity in H9c2 cells in a concentration-dependent manner, and 10 μM can produce significant cardiotoxicity. 10 μM was determined as the concentration for establishing the FK506-induced H9c2 cardiotoxicity model. 0, 12, 24, and 48 h FK506 treatment times were set to determine the duration of FK506-induced cardiotoxicity. ELISA results showed that ( Figure 1 .h- Figure 1 .k), with the increase of 10μM FK506 treatment time, the cardiotoxicity indicators such as LDH, CK-MB, cTn-T and BNP increased in a time-dependent manner. Compared with the control group, the above indicators reached significant levels after 12h treatment with 10μM FK506 (P<0.05).

[0070] In summary, it was determined that treating H9c2 cells with 10 μM FK506 for 12 h could successfully establish its induced cardiotoxicity model.

[0071] Example 2 Naringenin reverses tacrolimus-induced cardiotoxicity

[0072] 1. CCK-8 detection is the same as in Example 1.

[0073] 2. Cell Seeding and Culture: H9c2 cells in the logarithmic growth phase were seeded into six-well plates at a confluence of 35%. Twenty-four hours after seeding, cells in the naringenin pre-incubation group were treated with 0, 10, 20, 40, or 80 μM naringenin and incubated for 24 hours. Except for the control group, 10 μM tacrolimus was added to each well for 12 hours. Twenty-four hours after seeding, cells in the naringenin treatment group were treated with 10 μM tacrolimus and incubated for 12 hours. Then, cells in the naringenin treatment group were treated with 0, 10, 20, 40, or 80 μM naringenin and incubated for 24 hours. Immunoblotting was performed 36 hours after drug treatment.

[0074] 3. ELISA detection is the same as in Example 1.

[0075] The experimental results are as follows Figure 2 As shown, Figure 2 a is the chemical structural formula of naringenin.

[0076] Figure 2 b is a CCK8 experiment in which H9c2 cells were treated with different concentrations of naringenin (0, 10 μM, 20 μM, 40 μM, 80 μM) for 24 hours. The results showed that in rat cardiomyocytes H9c2, treatment with different concentrations of naringenin (0, 10, 20, 40, 80 μM) for 24 hours had no significant effect on H9c2 cell viability, indicating that naringenin has no cardiotoxic effect on H9c2 cells.

[0077] Figure 2 c is the CCK8 assay after treatment with 10 μM FK506 for 12 hours and then with different concentrations (0 μM, 10 μM, 20 μM, 40 μM, 80 μM) of naringenin for 24 hours. The cell viability in the FK506 treatment group was significantly reduced (P < 0.05). Figure 2 As shown in the fifth well of Figure c, the cardiotoxicity model was successfully induced, and treatment with different concentrations of naringenin increased cell viability in a dose-dependent manner. When the concentration of naringenin was 40 μM, cell viability was restored to physiological levels.

[0078] Figure 2dj is the use of ELISA experiments to test LDH, CK-MB, cTn-T and BNP indicators to evaluate the effect of different concentrations (0μM, 10μM, 20μM, 40μM, 80μM) of naringenin in the treatment of FK506-induced cardiotoxicity. The results showed that in the cardiotoxicity model group treated with 10μM FK506 for 12 hours, cardiotoxicity indicators such as LDH, CK-MB, cTn-T and BNP were significantly increased (P<0.05). After treatment with naringenin, the levels of these myocardial injury markers decreased in a dose-dependent manner. When the naringenin concentration was 40μM, the levels of these markers could be restored to physiological levels, that is, there was no significant difference with the control group. Combined with the results of the CCK8 experiment, 40μM was selected as the treatment concentration for subsequent experiments.

[0079] Figure 2 hk used ELISA experiments to test LDH, CK-MB, cTn-T and BNP indicators to evaluate the cardiotoxic effect of 40μM naringenin treatment at different times (0h, 12h, 24h, 48h). The results showed that after 12 hours of naringenin treatment, cardiotoxicity indicators such as LDH, CK-MB, cTn-T and BNP did not change significantly (P>0.05). However, after 24 hours of naringenin treatment, cardiotoxicity indicators such as LDH, CK-MB, cTn-T and BNP were significantly reduced, and there was no difference in the effect between 48 hours and 24 hours of treatment.

[0080] In summary, naringenin can reverse FK506-induced cardiotoxicity, and the therapeutic concentration of naringenin for FK506-induced cardiotoxicity was determined to be 40 μM, and the treatment time was 24 hours.

[0081] Example 3 Naringenin prevents tacrolimus-induced cardiotoxicity

[0082] 1. CCK-8 detection is the same as Example 2.

[0083] 2. ELISA detection is the same as in Example 2.

[0084] Figure 3a is a CCK8 assay for cell viability after H9c2 cells were incubated with different concentrations of naringenin (0μM, 10μM, 20μM, 40μM, and 80μM) for 24 hours and then treated with 10μM FK506 for 12 hours. The results showed that FK506 treatment caused a significant decrease in H9c2 cell viability (P<0.05). After pre-incubation with different concentrations of naringenin for 24 hours and then treatment with 10μM FK506 for 12 hours, naringenin was found to increase cell viability in a dose-dependent manner. The effect of low concentrations of 10μM and 20μM naringenin on H9c2 cells in preventing FK506-induced cardiotoxicity was weak, and there was still a significant difference in cell viability compared with the control group (P<0.05). When the concentration of naringenin was increased to 40μM, cell viability was maintained at a physiological level, which is the same as the effective therapeutic concentration of naringenin.

[0085] Figure 3 be used ELISA experiments to test LDH, CK-MB, cTn-T and BNP indicators to evaluate the effect of naringenin at different concentrations (0μM, 10μM, 20μM, 40μM, 80μM) in preventing FK506-induced cardiotoxicity. The results showed that pre-incubation with low concentrations (10, 20μM) of naringenin could significantly alleviate the cardiotoxicity induced by FK506 (P<0.05), and medium concentration (40μM) of naringenin could significantly reduce the levels of these markers after the addition of FK506 (P<0.01) and restore them to close to physiological levels. There was no significant difference in the effects between high concentration (80μM) and medium concentration (40μM) (P>0.05), both of which had a significant effect in preventing FK506-induced cardiotoxicity (P<0.05). Therefore, 40μM was determined to be the optimal preventive concentration of naringenin.

[0086] Figure 3 fg used ELISA experiments to test LDH, CK-MB, cTn-T and BNP indicators to evaluate the preventive effect of 40μM naringenin at different times (0h, 12h, 24h, 48h) on cardiotoxicity. The results showed that 24h of naringenin preincubation could significantly reduce cardiotoxicity (P<0.01).

[0087] In summary, 40 μM naringenin has a good protective effect against FK506-induced cardiotoxicity.

[0088] Example 4 Naringenin inhibits overexpression of L-type calcium channel Cav1.2 and alleviates tacrolimus-induced calcium overload

[0089] 1. Cell inoculation, culture and treatment are the same as in Example 2

[0090] 2. Calcium flux detection

[0091] (1) Sample preparation: Rat myocardial cells H9c2 in the logarithmic growth phase were inoculated with 5×10 3 -1×10 4 Cells were plated in 96-well plates. After overnight culture, group A (naringenin pre-incubation group) was treated with naringenin at a final concentration of 0, 10, 20, 40, and 80 μM. Group B (naringenin treatment group) was treated with tacrolimus at a final concentration of 10 μM. Group B was treated with a change of medium 12 hours later and naringenin was added to a final concentration of 0, 10, 20, 40, and 80 μM for 24 hours. Group A was treated with a change of medium 24 hours later and tacrolimus was added to a final concentration of 10 μM for 12 hours. Group C was treated with tacrolimus at a final concentration of 0, 2.5, 5, 10, and 20 μM for 12 hours. Three replicate wells were set for each group A, B, and C.

[0092] (2) Calcium indicator loading: Fluo-4 / AM dye was diluted to 2×Fluo-4 / AM concentration with buffer, and 100 μM was added to each well and incubated at 37°C and 5% CO2 for 60 min to allow the dye to fully enter the cells;

[0093] (3) Full spectrum flow cytometry detection: set the total acquisition time to 2 min and the interval time to 2 s; the signals at the first 10 time points were used as the background fluorescence level of the cells (F bkg ), then 30 μL 4×KCL agonist (240 mM) was added to induce calcium flux, and the peak value of the cell calcium signal (F peak ).

[0094] (5) Data calculation: First calculate △F peak =F peak -F bkg , and then perform △F peak / F bkg Calculate and draw a line graph and display the data with time as the horizontal axis and F as the vertical axis.

[0095] 3. Calcium imaging

[0096] (1) Sample preparation: Rat cardiomyocytes H9c2 in the logarithmic growth phase were inoculated with 4×10 5 Cells were plated in six-well plates; after overnight culture, group A (naringenin pre-incubation group) was treated with naringenin at a final concentration of 0, 10, 20, 40, and 80 μM, and group B (naringenin treatment group) was treated with tacrolimus at a final concentration of 10 μM. Group B was treated with a change of medium 12 hours later and naringenin was added to a final concentration of 0, 10, 20, 40, and 80 μM for 24 hours; group A was treated with a change of medium 24 hours later and tacrolimus was added to a final concentration of 10 μM for 12 hours; group C was treated with tacrolimus at a final concentration of 0, 2.5, 5, 10, and 20 μM for 12 hours; three replicate wells were set for each group A, B, and C.

[0097] (2) Calcium indicator loading: Fluo-4 / AM dye was diluted with buffer to a concentration of 2×Fluo-4 / AM reagent, and 100 μM 2×Fluo-4 / AM reagent was added to each well and incubated at 37°C and 5% CO2 for 60 min to allow the dye to fully enter the cells;

[0098] (3) Fluorescence detection: Fluorescence detection was performed using a confocal microscope with an excitation wavelength of 490 nm and an emission wavelength of 520 nm.

[0099] 4. Western Blot Detection:

[0100] (1) Aspirate the cell culture medium, wash three times with pre-chilled PBS, add RIPA lysis buffer containing cocktail protease inhibitors, lyse on ice for 30 minutes, and then collect in EP tubes and centrifuge;

[0101] (2) Aspirate the supernatant and add an equal volume of 5× loading buffer to make the final working concentration 1×, and boil at 37°C for 30 min;

[0102] (3) Protein electrophoresis on SDS-PAGE: 50 μg of total protein was loaded and electrophoresed on 8% SDS-PAGE gel at a voltage of 50 V for the stacking gel. The voltage was adjusted to 100 V after the sample entered the separating gel.

[0103] (4) Transfer: After electrophoresis, the stacking gel was removed and a transfer sandwich was assembled using a methanol-pretreated PVDF membrane and filter paper immersed in electrotransfer buffer. After removing all air bubbles, electrotransfer was performed on ice (300 mA, 60 min).

[0104] (5) Block with 5% skim milk or 5% BSA for 1 h;

[0105] (6) After blocking, add diluted primary antibody and incubate at room temperature for 2 h or at 4°C overnight;

[0106] (7) Wash three times with 1×TBST, 5 min each time, add corresponding secondary antibody after washing, and incubate at room temperature for 1 h;

[0107] (8) ECL detection: Wash three times with 1× TBST, 5 min each time. Mix ECL solution A and solution B at a ratio of 1:1, soak the PVDF membrane, and image using a chemiluminescence imager.

[0108] The experimental results are as follows Figures 4 to 6 shown.

[0109] Figure 4a H9c2 cells were treated with FK506 at different concentrations (0 μM, 2.5 μM, 5 μM, 10 μM, 20 μM) for 12 h, then incubated with Fluo-4 / AM for 30 min. After adding 240 mM KCL agonist, the intracellular [Ca 2+ ]i concentration changes within 1 minute. Figure 4 b: H9c2 cells were treated with FK506 at different concentrations (0 μM, 2.5 μM, 5 μM, 10 μM, 20 μM) for 12 h and then incubated with Fluo-4 / AM for 30 min to detect the intracellular calcium fluorescence intensity. Figure 4 cd are Figure 4 a and Figure 4 Quantitative plot of b.

[0110] In calcium flux and calcium imaging experiments ( Figure 4 ad), compared with the highest value of calcium flux detected by KCL agonist in the control group (0.18 μM), FK506-treated groups induced increased calcium flux, and with the increase of FK506 concentration (2.5, 5, 10, 20 μM), [Ca 2+ The peak value of [Ca]i increased and the time of maintaining high level was prolonged. Starting from 5μM FK506, [Ca 2+ ]i peak value can reach 0.22mM, which can reach a statistically significant level compared with the control group (P<0.05). Subsequent related concentrations can induce intracellular calcium overload, and are concentration-dependent. In the low concentration (5μM) FK506 stimulation group, the intracellular calcium flux increased significantly (P<0.05), and the intracellular calcium flux increase in the 10μM FK506 stimulation group reached an extremely significant level (P<0.01). Compared with the control group, the calcium flux level increased by 1.2 times. At the same time, fluorescence microscopy imaging showed the fluorescence intensity of intracellular calcium after Fluo-4 / AM staining, and as the FK506 concentration increased, the green fluorescence intensity increased, further confirming the intracellular calcium overload phenomenon.

[0111] The L-type calcium channels expressed in H9c2 cells are Cav1.3 and Cav1.2 subunits, with Cav1.2 being predominantly expressed over Cav1.3. After stimulation with a concentration gradient of tacrolimus (2.5, 5, 10, 20 μM) for 12 h, the expression of L-type calcium channel subtypes Cav1.3 and Cav1.2 in H9c2 cells was semi-quantitatively analyzed by Western Blot. Figure 4 e: After H9c2 cells were treated with FK506 at different concentrations (0 μM, 2.5 μM, 5 μM, 10 μM, 20 μM) for 12 h, proteins were collected and Western blot was performed to detect the expression of L-type calcium channel proteins (Cav1.3, Cav1.2). Figure 4fg are the quantitative analysis results of Cav1.3 and Cav1.2 protein expressions, respectively.

[0112] The results showed that when the concentration of FK506 reached 10μM and 20μM, the total protein expression level of Cav1.2 was significantly increased, with significant statistical differences compared with the control group (P<0.05), while there was no significant difference at low concentrations (2.5μM, 5μM) (P>0.05). Taken together, these results indicate that high concentrations (10μM and 20μM) of FK506 can induce calcium overload in H9c2 cells. Western Blot analysis of L-type calcium channel proteins showed that high concentrations (10μM and 20μM) of FK506 can upregulate the protein expression of Cav1.2, but have no effect on the expression of Cav1.3. The calcium overload process induced by FK506 may be related to the upregulation of Cav1.2 protein expression by FK506.

[0113] To investigate whether the reversal of FK506-induced cardiotoxicity by naringenin is related to the alleviation of calcium overload, the 10 μM FK506-stimulated group was treated with different concentrations of naringenin (20 μM and 40 μM). Figure 5 a) The blank control group, 10 μM FK506 stimulation for 12 hours, and 10 μM FK506 stimulation for 12 hours were set up, and then different concentrations (20 μM, 40 μM) of naringenin were treated for 24 hours. The intracellular [Ca 2+ ]i’s changing trend within 1 minute, Figure 5 b is Figure 5 Quantitative analysis of Figure a. The results showed that FK506 induced calcium overload, while naringenin dose-dependently inhibited FK506-induced [Ca 2+ ]i internal flow.

[0114] Figure 5 c shows the blank control group, 10 μM FK506 stimulation for 12 hours, and 10 μM FK506 stimulation for 12 hours followed by treatment with different concentrations (20 μM, 40 μM) of naringenin for 24 hours, after which the intracellular calcium fluorescence intensity was detected under a fluorescence microscope using the Fluo-4 / AM fluorescent probe. Figure 5 d is Figure 5 Quantitative analysis of Figure c showed that the fluorescence intensity of the cells in the control group was low, the fluorescence intensity of the group treated with 10 μM FK506 was significantly enhanced, and the fluorescence intensity of the group treated with naringenin was weakened, confirming the change in calcium concentration.

[0115] Figure 5 d Calcium flow and calcium imaging results showed that the [Ca 2+ ]i was maintained at a low level; 10 μM FK506 (F 10μM) treatment, [Ca 2+ ]i increased rapidly and reached a high peak, then slowly decreased, indicating that FK506-induced cytotoxicity was related to calcium overload, which was consistent with 4a. 10μM +N 20μM ), [Ca 2+ ]i peak value was significantly lower than that of the FK506 group (P<0.05), and with the increase of naringenin concentration, [Ca 2+ ]i peak value decreased more significantly, indicating that naringenin can effectively alleviate FK506-induced calcium overload. 10μM +N 40μM The FK506-induced calcium overload was reduced to the physiological level (0.18 mM) in the control group. 2+ ]i peak value and F 10μM Group ([Ca 2+ ]i is 0.26mM) and reached an extremely significant level compared with the control.

[0116] Figure 5 e is the expression of L-type calcium channel subunits Cav1.2 and Cav1.3 in the above treatment groups detected by Western blot. Figure 5 fg are quantitative analyses of Cav1.3 and Cav1.2 protein expression, respectively. The results showed that treatment with 10 μM FK506 alone significantly increased Cav1.2 protein expression, with statistically significant differences compared to the control group (P<0.05), indicating that FK506 successfully induced a cardiotoxic model, while Cav1.3 protein expression remained unchanged. When naringenin (20 μM, 40 μM, and 80 μM) was added to the 10 μM FK506 treatment, Cav1.2 protein expression levels decreased with increasing naringenin concentration, indicating that naringenin can inhibit the FK506-induced increase in Cav1.2 protein expression, reaching a peak at 40 μM.

[0117] After membrane protein extraction, Western Blot experiment was performed. Figure 5 h is the detection of the expression of Cav1.2 and Cav1.3 in the cell membrane and cytoplasm in different treatment groups, Figure 5 ij represents quantitative analysis of Cav1.3 and Cav1.2 protein expression in the cell membrane and cytoplasm, respectively. The results showed that FK506 treatment alone increased Cav1.2 protein expression, which decreased after the addition of naringenin. There was no significant trend in Cav1.3. There was no clear pattern in the expression of Cav1.2 and Cav1.3 proteins in the cytoplasm across the treatment groups. However, significant differences in the expression of Cav1.2 and Cav1.3 proteins on the cell membrane were observed. This result is consistent with the total protein Western Blot results, indicating that naringenin reverses FK506-induced calcium overload by inhibiting the expression of Cav1.2 on the cell membrane.

[0118] Calcium flow disorder is closely related to the expression of key indicators of the calcium signaling pathway. FK506 is a specific inhibitor of calcineurin, which downregulates the expression of calcineurin, indicating that the FK506 model was successfully established. Western Blot detection was also performed on the key proteins of the calcium signaling pathway, CaM (calmodulin) and CaMKII (calmodulin kinase II). Figure 5 k is the Western Blot detection result of the key proteins of calcium signaling pathway, calcineurin, phosphorylated calmodulin kinase II (Pi-CaMKII), calmodulin kinase II (CaMKII), phosphorylated calmodulin (Pi-CaM) and calmodulin (CaM) in the above treatment groups. Figure 5 ln represents the quantitative analysis of the above protein expressions.

[0119] It was found that the CaM / CaMKII calcium signaling pathway was activated, specifically in the increased expression of Cav1.2 channel protein on the cell membrane in H9c2 cells after FK506 stimulation, which led to more calcium ions flowing into the cell and the increase of intracellular [Ca 2+ ]i concentration increases, free calcium ions [Ca 2+ ]i will bind to calmodulin (CaM), which binds to Ca 2+ After CaM is phosphorylated and activated, it then activates the downstream CaMKII protein. The modified Pi-CaMKII can phosphorylate and modify multiple downstream substrates and regulate various cellular functions.

[0120] In summary, FK506 induces calcium overload in H9c2 cells by upregulating protein expression on the cell membrane of Cav1.2 and activating the phosphorylation of downstream CaM and CaMKII. Naringenin can relieve FK506-induced calcium overload, and the mechanism may be related to the inhibition of Cav1.2 expression and the inhibition of CaM and CaMKII phosphorylation.

[0121] However, the overexpression of Cav1.2 in H9c2 cells was significantly inhibited by the concentration gradient (F 10μM +N 20μM 、F 10μM +N 40μM ) naringenin treatment and (N 20μM +F 10μM 、N 40μM +F 10μM ) pre-incubation( Figure 6 ) group was significantly inhibited.

[0122] Figure 6 This is the result diagram of naringenin preventing tacrolimus-induced Cav1.2 overexpression and activation of calcium signaling pathway, among which, Figure 6 a shows the blank control group, 10 μM FK506 stimulation for 12 hours, and pre-incubation with different concentrations (20 μM, 40 μM) of naringenin for 24 hours, followed by 10 μM FK506 stimulation for 12 hours for Western blot detection of the expression of L-type calcium channel subunits Cav1.2 and Cav1.3. Figure 6 b and Figure 6 c Quantitative analysis of Cav1.2 and Cav1.3 expression; Figure 6 d is the Western Blot detection results of key proteins of calcium signaling pathway, calcineurin, phosphorylated calmodulin kinase II (Pi-CaMKII), calmodulin kinase II (CaMKII), phosphorylated calmodulin (Pi-CaM) and calmodulin (CaM) in the above treatment groups. Figure 6 eg are the quantitative analysis of the above protein expressions.

[0123] The results showed that the inhibition of Cav1.2 protein expression was dose-dependent with naringenin, where a significant therapeutic effect was observed at 20 μM (P<0.05), and 40 μM could restore Cav1.2 protein expression to physiological levels (P<0.01). 2+ ]i influx, especially after treatment with 40μM naringenin, which significantly suppressed calcium overload, reaching physiological levels (P<0.01). This, in turn, hindered the activation of downstream calcium signaling pathways, restoring the relevant indicators of cardiotoxicity to normal levels. In summary, naringenin reverses tacrolimus-induced cardiotoxicity by downregulating Cav1.2 expression on the cell membrane, inhibiting calcium influx, and thus inhibiting the activation of calcium signaling pathways, thereby exerting its anti-cardiotoxic effect.

[0124] Example 5 Naringenin reverses tacrolimus-induced apoptosis

[0125] 1. Cell inoculation is the same as in Example 2;

[0126] 2. Immunoblotting detection was the same as in Example 1.

[0127] 3. Flow cytometry detection: Perform the operation and detection according to the instructions of the Anne×inV-FITC / PI apoptosis detection kit.

[0128] The control group and FK506 stimulation group (F 10μMWestern Blot was performed to detect the key molecules of apoptosis pathway, poly (ADP-ribose) polymerase (PARP) and caspase-3, in the treatment group (10 μM FK506 stimulation for 12 h and then different concentrations (10, 20, 40, 80 μM) naringenin treatment for 24 h), and in the pre-incubation group (different concentrations (10, 20, 40, 80 μM) naringenin pre-incubated for 24 h and then stimulated with 10 μM FK506 for 12 h). Figure 7 a and Figure 7 d shows the Western Blot detection results of key apoptosis signaling molecules, PARP (poly ADP-ribose polymerase) and Caspase3 (caspase 3) and their corresponding cleavage bodies in the naringenin treatment group and the naringenin pre-incubation group, respectively. Figure 7 bc and Figure 7 ef are the quantitative analysis results of Cleaved-PARP / PARP and Cleaved-caspase3 / caspase3 in the above treatment groups, respectively.

[0129] The results show that ( Figure 7 ae), compared with the control group, F 10μM The precursors of PARP and Caspase-3 in the group were significantly cleaved to produce Cleaved-PARP and Cleaved-caspase-3. Western Blot analysis of the groups treated with different concentrations of naringenin showed that naringenin could dose-dependently inhibit the increase of cleaved-PARP and Cleaved-caspase-3 caused by FK506 stimulation. Among them, the low concentration (10μM) naringenin treatment group had no significant inhibitory effect on the cleavage of the key apoptosis proteins PARP and caspase-3, while the inhibitory effect reached a statistically significant level when treated with 20μM naringenin (P<0.05). The inhibitory effect of 40μM naringenin was not significantly different from the physiological level (P<0.01). The effect of high concentration (80μM) naringenin was equivalent to that of 40μM.

[0130] The key concentrations of 20μM and 40μM were selected to explore the therapeutic effect of naringenin. The effect of naringenin on reversing FK506-induced cell apoptosis was detected by flow cytometry. Anne×inV-FITC / PI apoptosis detection kit was used for flow cytometry experiments. The results showed that ( Figure 7(As shown in Figure 5), compared to the control group (Q2+Q3 percentage of 5.28%), H9c2 cells underwent significant apoptosis after FK506 stimulation, with Q2+Q3 accounting for 35.4%, and late apoptosis was the main cause. After treatment with 20μM and 40μM naringenin, cell apoptosis was improved. In the 20μM treatment group, Q2+Q3 accounted for 23.4%, a 12% decrease compared to the apoptosis percentage in the stimulation group. After treatment with 40μM naringenin, the Q2+Q3 percentage decreased to 11.18%, and the number of late apoptotic cells decreased to 8.79%, which is comparable to the normal apoptosis level of cells. This indicates that 40μM naringenin can reverse tacrolimus-induced cell apoptosis and that naringenin has a good protective effect on the heart.

[0131] In summary, FK506 can promote the apoptosis of H9c2 cells by activating PARP and Caspase3, while 40 μM naringenin can reverse these apoptosis-related events induced by FK506, thereby restoring cell apoptosis to physiological levels.

[0132] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, and all of these should be included in the scope of the present invention.

Claims

1. Application of naringenin in the preparation of drugs for preventing and treating tacrolimus-induced cardiotoxicity.

2. The use of naringenin according to claim 1 in the preparation of a drug for preventing and treating tacrolimus-induced cardiotoxicity, characterized in that: The concentration of naringenin in the medicine is 40-80 μM.

3. The use of naringenin according to claim 1 in the preparation of a drug for preventing and treating tacrolimus-induced cardiotoxicity, characterized in that: The naringenin downregulates the expression of L-type calcium channel Cav1.2 on the cell membrane, thereby restoring calcium overload to normal levels and affecting the calcium signaling pathway, thereby reversing the myocardial toxicity caused by tacrolimus-induced calcium overload.

4. The use of naringenin as claimed in claim 1 in the preparation of a drug for preventing and treating tacrolimus-induced cardiotoxicity, characterized in that: Pre-incubation with naringenin can prevent the cardiotoxicity caused by calcium overload induced by tacrolimus-induced increased expression of Cav1.

2.

5. The use of naringenin as claimed in claim 1 in the preparation of a drug for preventing and treating tacrolimus-induced cardiotoxicity, characterized in that: The naringenin inhibits the calcium signaling pathway by restoring calcium homeostasis, thereby reversing the calcium overload and cell apoptosis caused by the increased expression of Cav1.2 on the cell membrane induced by tacrolimus.

6. The use of naringenin in the preparation of a drug for preventing and treating tacrolimus-induced cardiotoxicity according to claim 5, characterized in that: The naringenin pre-incubation can stabilize the expression of Cav1.2 and prevent tacrolimus-induced cell apoptosis.

7. The use of naringenin in the preparation of a drug for preventing and treating tacrolimus-induced cardiotoxicity according to claim 5, characterized in that: The Cav1.2 can serve as a key therapeutic target for calcium overload and alleviate the cardiotoxicity caused by calcium overload induced by FK506.

8. The use of naringenin in the preparation of a drug for preventing and treating tacrolimus-induced cardiotoxicity according to claim 5, characterized in that: The Cav1.2 can be used as a screening marker for screening and alleviating cardiac toxicity caused by calcium overload induced by FK506.

9. A pharmaceutical composition, characterized in that: The pharmaceutical composition comprises the naringenin according to claim 2 and a pharmaceutically acceptable carrier or preparation.

10. The pharmaceutical composition according to claim 9, wherein: The dosage form includes one of tablets, hard capsules, soft capsules, aqueous suspensions, oily suspensions, granules, emulsions, and syrups.

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