Hydrogel carrying isoquercitrin, preparation method of hydrogel and application of hydrogel in myocardial oxidative damage resistance

Abstract

The invention provides hydrogel carrying isoquercitrin, a preparation method of the hydrogel and application of the hydrogel in myocardial oxidative damage resistance, and belongs to the technical field of biological medicine. The invention provides a hydrogel loaded with isoquercitrin, and particularly provides a preparation method of the hydrogel. According to the hydrogel, the pharmacological activity of the isoquercitrin is reserved, the problem that the water solubility of the isoquercitrin is poor is remarkably solved, the biocompatibility and the solubility of the isoquercitrin are enhanced, a better treatment effect is shown on the cardiomyopathy induced by oxidative stress, and the hydrogel can be applied to the treatment of the oxidative stress induced cardiomyopathy. And the application of the isoquercitrin in the treatment direction of cardiomyopathy-related diseases is expanded.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a hydrogel loaded with isoquercetin, a preparation method thereof, and an application thereof in resisting myocardial oxidative damage. Background Art

[0002] Oxidative stress-induced cardiomyopathy (OIC) is a serious threat to human health. During cancer chemotherapy, OIC induced by drugs such as doxorubicin (DOX) is a particularly prominent problem. Clinical studies have shown that some cancer patients receiving DOX chemotherapy experience varying degrees of cardiotoxicity, which can lead to OIC, severely impacting their quality of life and prognosis. Therefore, the development of effective therapeutics and treatments for OIC is urgent.

[0003] Isoquercetin (IQ) is a flavonoid compound found in a wide variety of plants, such as ginseng. In traditional medicine, herbal remedies containing IQ are often used to treat cardiovascular-related symptoms. IQ's chemical structure gives it the potential to interact with a variety of biomolecules, scavenging excess reactive oxygen species (ROS) in the body and reducing oxidative stress-induced cell damage. It can also regulate inflammatory signaling pathways and inhibit inflammatory responses. However, isoquercetin has poor water solubility and low bioavailability, necessitating the development of drug delivery systems to improve its properties and enhance its anti-inflammatory effects.

[0004] Carboxymethyl chitosan (CMCS) is a water-soluble chitosan derivative and an amphoteric polyelectrolyte. It is formed by introducing the hydrophilic group -CH2COOH into the chitosan polymer chain. Hyaluronic acid (HA) is an acidic mucopolysaccharide that exhibits multiple important physiological functions in the body due to its unique molecular structure and physicochemical properties. CMCS and oxidized hyaluronic acid (OHA) have been studied as biocompatible materials for drug delivery (such as anticancer drugs), but their use in IQ delivery systems for the treatment of cardiomyopathy has not been reported. Summary of the Invention

[0005] In order to solve the problems existing in the prior art, the purpose of the present invention is to provide a hydrogel loaded with isoquercetin and a preparation method thereof, which retains the pharmacological activity of isoquercetin itself and enhances the biocompatibility and solubility of isoquercetin, showing a better therapeutic effect on oxidative stress-induced cardiomyopathy.

[0006] The present invention also aims to provide the use of the isoquercetin-loaded hydrogel in the preparation of drugs for inhibiting inflammatory responses, alleviating and / or inhibiting myocardial cell apoptosis, resisting myocardial oxidative damage, and treating oxidative stress-induced cardiomyopathy.

[0007] Another object of the present invention is to provide a drug for alleviating and / or treating oxidative stress-induced cardiomyopathy.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] The present invention provides a method for preparing a hydrogel loaded with isoquercetin, comprising the following steps:

[0010] A sodium periodate aqueous solution is added to a hyaluronic acid aqueous solution, and the mixture is stirred in the dark. Ethylene glycol is added to terminate the reaction, and the reaction product is dialyzed and freeze-dried to obtain oxidized hyaluronic acid; the oxidized hyaluronic acid is dissolved in water to obtain an oxidized hyaluronic acid aqueous solution; the oxidized hyaluronic acid aqueous solution is mixed with an isoquercetin mixture to obtain a precursor solution containing oxidized hyaluronic acid and isoquercetin; the precursor solution containing oxidized hyaluronic acid and isoquercetin is added to a carboxymethyl chitosan aqueous solution under vortex conditions, and the vortex is continued for 10 to 30 seconds, and the mixture is allowed to stand for 3 to 5 minutes to obtain a hydrogel.

[0011] Preferably, the concentration of the hyaluronic acid aqueous solution is 10 mg / mL, the concentration of the sodium periodate aqueous solution is 0.1 g / mL; the volume ratio of the hyaluronic acid aqueous solution, the sodium periodate aqueous solution and ethylene glycol is 40:1:0.04; and the stirring time in the dark is 6 to 8 hours.

[0012] Preferably, the molecular weight of the dialyzed solution is 8000 to 14000 Da.

[0013] Preferably, in the precursor solution containing oxidized hyaluronic acid and isoquercetin, the mass fraction of oxidized hyaluronic acid is 1.9-2.1%, and the final concentration of isoquercetin is 250 μM.

[0014] Preferably, the mass fraction of carboxymethyl chitosan in the carboxymethyl chitosan aqueous solution is 4.5-5.0%; and the volume ratio of the precursor solution containing oxidized hyaluronic acid and isoquercetin to the carboxymethyl chitosan aqueous solution is 1:0.4.

[0015] The present invention also provides a hydrogel prepared by the above preparation method.

[0016] The present invention also provides the use of the hydrogel in any of the following:

[0017] (1) preparing drugs for inhibiting inflammatory reactions;

[0018] (2) preparing drugs for alleviating and / or inhibiting myocardial cell apoptosis;

[0019] (3) Preparation of drugs for preventing myocardial oxidative damage;

[0020] (4) Prepare drugs for treating oxidative stress-induced cardiomyopathy.

[0021] Preferably, the cardiomyopathy includes tumor heart disease.

[0022] The present invention also provides a drug for preventing and / or treating oxidative stress-induced cardiomyopathy, wherein the active ingredient of the drug includes the hydrogel.

[0023] Preferably, the drug further comprises a pharmaceutically acceptable carrier.

[0024] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0025] The present invention provides a novel isoquercetin drug delivery method, using carboxymethyl chitosan and oxidized hyaluronic acid as biocompatible materials to prepare a hydrogel loaded with isoquercetin. While retaining the pharmacological activity of isoquercetin itself, the problem of poor water solubility of isoquercetin is significantly solved, the biocompatibility and solubility of isoquercetin are enhanced, a suitable isoquercetin release rate is provided, the bioavailability of isoquercetin is improved, and it has a higher myocardial protective effect. It shows a better therapeutic effect on oxidative stress-induced cardiomyopathy, can be used for the prevention and treatment of tumor heart disease, and expands the application of isoquercetin in the treatment of cardiomyopathy-related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 :H NMR spectra of hyaluronic acid (HA) and oxidized hyaluronic acid (OHA);

[0027] Figure 2 : FT-IR spectra of HA, OHA, CMCS, IQ, and IQ@gel;

[0028] Figure 3 :Scanning electron microscopy results of IQ@gel;

[0029] Figure 4 : IQ@gel frequency sweep test results;

[0030] Figure 5 : IQ@gel injectability test results;

[0031] Figure 6 :pH responsiveness results of IQ@gel;

[0032] Figure 7 :IQ@gel drug release profile;

[0033] Figure 8 :IQ@gel biocompatibility results;

[0034] Figure 9 :The protective effect of IQ@gel on H9c2 cells;

[0035] Figure 10 :Effects of IQ@gel on ROS levels in H9c2 cells;

[0036] Figure 11 : Daily body weight fluctuations and comparison results of mice in each group;

[0037] Figure 12 : Echocardiogram, LVEF and LVFS statistical results of mice in each group;

[0038] Figure 13 : Comparison of FFA, ATP, LA, and LDH contents in each group of mice;

[0039] Figure 14 : HE staining of the hearts of mice in each group;

[0040] Figure 15 : PSR staining of the hearts of mice in each group. DETAILED DESCRIPTION

[0041] The present invention provides a method for preparing a hydrogel loaded with isoquercetin, comprising the following steps:

[0042] A sodium periodate aqueous solution is added to the hyaluronic acid aqueous solution, and the mixture is stirred in the dark. Ethylene glycol is added to terminate the reaction, and the reaction product is dialyzed and freeze-dried to obtain oxidized hyaluronic acid; the oxidized hyaluronic acid is dissolved in water to obtain an oxidized hyaluronic acid aqueous solution; the oxidized hyaluronic acid aqueous solution is mixed with an isoquercetin mixture to obtain a precursor solution containing oxidized hyaluronic acid and isoquercetin; the precursor solution containing oxidized hyaluronic acid and isoquercetin is added to a carboxymethyl chitosan aqueous solution under vortex conditions, and the vortex is continued for 10 to 30 seconds, and the mixture is allowed to stand for 3 to 5 minutes to obtain a hydrogel.

[0043] In the present invention, the hyaluronic acid aqueous solution can be prepared by dissolving hyaluronic acid in distilled water, with a concentration of 10 mg / mL; the sodium periodate aqueous solution can be prepared by dissolving sodium periodate in distilled water, with a concentration of 0.1 g / mL; the dark stirring is preferably light-shielded stirring, and the dark stirring time is 6 to 8 hours, preferably 7 hours. In the present invention, ethylene glycol is added after stirring to terminate the reaction. The volume ratio of the hyaluronic acid aqueous solution, sodium periodate aqueous solution, and ethylene glycol in the present invention is 40:1:0.04.

[0044] The reaction product of the present invention is subjected to a dialysis membrane for impurity removal treatment, wherein the molecular weight of the dialysis is 8000 to 14000 Da, preferably 9000 Da, 10000 Da, 11000 Da, 12000 Da or 13000 Da. The dialysis step of the present invention is a conventional dialysis operation step in the art.

[0045] In the present invention, after dialysis and impurity removal, freeze drying is preferably performed to obtain oxidized hyaluronic acid. The freeze drying of the present invention is preferably performed at a cold trap temperature of -40°C for 72 to 96 hours.

[0046] The present invention dissolves the prepared oxidized hyaluronic acid in water to obtain an oxidized hyaluronic acid aqueous solution. As an optional embodiment, the present invention adds the oxidized hyaluronic acid into distilled water and stirs it at 37° C. for 3 to 5 hours until it is completely dissolved to obtain an oxidized hyaluronic acid aqueous solution.

[0047] The isoquercetin mixed solution of the present invention can be prepared by adding isoquercetin into distilled water and thoroughly vortex mixing to obtain the isoquercetin mixed solution.

[0048] The present invention mixes an aqueous solution of oxidized hyaluronic acid with a mixture of isoquercetin to obtain a precursor solution containing oxidized hyaluronic acid and isoquercetin. As an alternative embodiment, the present invention adds the isoquercetin mixture to the aqueous solution of oxidized hyaluronic acid and thoroughly vortexes to obtain a precursor solution containing oxidized hyaluronic acid and isoquercetin. In the precursor solution containing oxidized hyaluronic acid and isoquercetin, the mass fraction of oxidized hyaluronic acid is 1.9-2.1%, and the final concentration of isoquercetin is 250 μM.

[0049] The present invention dissolves carboxymethyl chitosan in water to obtain a carboxymethyl chitosan aqueous solution. As an optional embodiment, the present invention adds carboxymethyl chitosan to distilled water and stirs it at 37°C for 3 to 5 hours until it is completely dissolved to obtain a carboxymethyl chitosan aqueous solution. The mass fraction of carboxymethyl chitosan in the carboxymethyl chitosan aqueous solution of the present invention is 4.5 to 5.0%;

[0050] The present invention adds a carboxymethyl chitosan aqueous solution to a container, and then adds a precursor solution containing oxidized hyaluronic acid and isoquercetin under vortex conditions. The carboxymethyl chitosan and the oxidized hyaluronic acid / isoquercetin react through a Schiff base reaction to form an isoquercetin-loaded hydrogel. The volume ratio of the precursor solution containing oxidized hyaluronic acid and isoquercetin to the carboxymethyl chitosan aqueous solution is 1:0.4.

[0051] The present invention also provides a hydrogel prepared by the above-mentioned preparation method. By selecting suitable gel-forming compounds, the gel-forming concentrations and proportions of the compounds, etc., the prepared isoquercetin-loaded hydrogel has an optimal isoquercetin release curve and myocardial antioxidant activity, good biocompatibility, and an appropriate drug release rate. Thus, it can significantly improve symptoms such as heart failure and myocardial infarction caused by tumor heart disease, and the effect is significantly better than that of isoquercetin alone.

[0052] The present invention also provides the use of the hydrogel in any of the following:

[0053] (1) preparing drugs for inhibiting inflammatory reactions;

[0054] (2) preparing drugs for alleviating and / or inhibiting myocardial cell apoptosis;

[0055] (3) Preparation of drugs for preventing myocardial oxidative damage;

[0056] (4) Preparing a drug for treating oxidative stress-induced cardiomyopathy; preferably, the cardiomyopathy includes tumor heart disease.

[0057] The present invention also provides a drug for preventing and / or treating oxidative stress-induced cardiomyopathy, wherein the active ingredient of the drug includes the above-mentioned hydrogel. The drug of the present invention may use the above-mentioned hydrogel loaded with isoquercetin as the sole active ingredient, or may be combined with other feasible active ingredients.

[0058] The drug of the present invention also includes a pharmaceutically acceptable carrier, and the present invention does not limit the specific carrier. The drug of the present invention is preferably administered orally or by injection, and the present invention does not limit the specific drug dosage form and excipient selection.

[0059] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0060] In the following examples, unless otherwise specified, all methods are conventional.

[0061] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0062] Example 1

[0063] Preparation method of isoquercetin-loaded hydrogel (IQ@gel):

[0064] (1) Preparation of oxidized hyaluronic acid (OHA):

[0065] Dissolve hyaluronic acid in distilled water to obtain a hyaluronic acid aqueous solution with a concentration of 10 mg / mL; dissolve sodium periodate in distilled water to obtain a sodium periodate aqueous solution with a concentration of 0.1 g / mL; add sodium periodate aqueous solution to the hyaluronic acid aqueous solution (the volume ratio of the hyaluronic acid aqueous solution to the sodium periodate aqueous solution is 40:1) and stir in the dark for 6 hours; add ethylene glycol to the system (the volume ratio of the hyaluronic acid aqueous solution, the sodium periodate aqueous solution and the ethylene glycol is 40:1:0.04) to terminate the reaction; the reaction product is passed through a dialysis membrane (10000 Da) to remove impurities, and dried at a cold trap temperature of -40°C for 72 hours to obtain oxidized hyaluronic acid.

[0066] (2) Preparation of a precursor solution containing oxidized hyaluronic acid and isoquercetin (IQ / OHA):

[0067] The oxidized hyaluronic acid of step (1) is added into distilled water and stirred at 37° C. for 4 h until completely dissolved to obtain an oxidized hyaluronic acid aqueous solution; isoquercetin is added into distilled water and thoroughly vortexed to obtain an isoquercetin mixed solution; the isoquercetin mixed solution is added into the oxidized hyaluronic acid aqueous solution and thoroughly vortexed to obtain a precursor solution containing oxidized hyaluronic acid and isoquercetin (the mass fraction of oxidized hyaluronic acid is 1.9-2.1%, and the final concentration of isoquercetin is 250 μM).

[0068] (3) Preparation of carboxymethyl chitosan (CMCS) aqueous solution:

[0069] Carboxymethyl chitosan was added into distilled water and stirred at 37° C. for 4 h until completely dissolved, to obtain a carboxymethyl chitosan aqueous solution with a mass fraction of 5%.

[0070] (4) IQ / OHA reacts with CMCS via Schiff base to form isoquercetin-loaded hydrogel (IQ@gel):

[0071] The carboxymethyl chitosan aqueous solution obtained in step (3) was added to a 5 mL centrifuge tube. The precursor solution containing oxidized hyaluronic acid and isoquercetin obtained in step (2) was added at a volume ratio of 1:0.4 while vortexing. After addition, the mixture was vortexed for 10 seconds and allowed to stand for 3 minutes to obtain the hydrogel (IQ@gel).

[0072] Test Example 1

[0073] 1. Adoption 1 H NMR structural evaluation of hyaluronic acid (HA) and oxidized hyaluronic acid (OHA):

[0074] use 1 H NMR spectrometer (600 MHz NMR spectrometer, Bruker Plus, Germany). 3 mg of OHA or 3 mg of HA from step (1) of Example 1 were dissolved in 1 mL of deuterium oxide (DO) solvent and then transferred to an NMR tube. Data were analyzed using Mestrelab MNova software (version 14.2.2).

[0075] The results of H NMR spectroscopy are as follows Figure 1 As shown, the results showed that HA was successfully oxidized to OHA.

[0076] 2. Detection of HA, OHA, CMCS, IQ and IQ@gel using FT-IR spectroscopy:

[0077] Thin films were prepared by mixing potassium bromide with the samples. Spectra were recorded using a Fourier transform infrared spectrometer (Nicoleti S5, Thermo Fisher Scientific, USA). The scan range was set between 4000 and 500 cm -1 between.

[0078] The infrared spectrum results are as follows Figure 2 As shown, the results indicate that a Schiff base reaction occurs between CMCS and OHA, and the hydrogel prepared in the present invention is cross-linked via the Schiff base reaction.

[0079] 3. Characterization of IQ@gel using SEM:

[0080] The surface and cross-sectional morphologies of dried IQ@gel were characterized using a scanning electron microscope (SEM) (ZEISS Sigma 360, Germany) at 3.00 KV. Before the experiments, IQ@gel was freeze-dried and spray-coated with gold.

[0081] Scanning electron microscopy results Figure 3 As shown in the figure, the scale of the left figure is 100μm, the scale of the right figure is 50μm, and the right figure is an enlargement of the box in the left figure. The results show that IQ@gel has a relatively uniform porous and interconnected structure, which can well carry IQ.

[0082] 4. IQ@gel frequency sweep test:

[0083] The Matrigel precursor solutions (IQ / OHA and CMCS) were removed from a 4°C refrigerator and placed in a 2cm diameter, 3mm thick circular mold. The mold was then allowed to crosslink at room temperature to form a gel. The Matrigel was then placed on the measurement platform of a rotational rheometer, and the probe was pressed down until the normal force reached 0.2N. Finally, a frequency sweep was selected on the computer interface to measure the viscoelasticity and strength of the hydrogel.

[0084] IQ@gel frequency sweep test results are as follows Figure 4 As shown, the results show that IQ@gel has moderate strength and low injection resistance, making it suitable for injection.

[0085] 5. IQ@gel injectability test:

[0086] Use a syringe to absorb the hydrogel and push the syringe to observe the injectability of the hydrogel. Figure 5 As shown, Figure 5 The left image shows the initial injection, and the right image shows the plunger pushed a certain distance. IQ@gel gelation and stability were observed through the syringe, demonstrating its potential feasibility for gelation in subcutaneous tissue and the peritoneal cavity.

[0087] 6. pH responsiveness of IQ@gel:

[0088] Observe the drug release effect of the hydrogel under different pH conditions: the temperature was set to 37°C, and it was placed in a buffer solution containing different pH values (pH 3, pH 5, pH 8), and oscillated in a constant temperature water bath at 37°C for 72 hours. At fixed time intervals (0h, 8h, 16h, 24h, 32h, 40h, 48h, 56h, 64h, 72h), 5mL of drug-released solution was taken out and the same volume of the same buffer solution was added. The absorbance value was measured at 360nm using an enzyme reader, and an equal volume of the corresponding buffer solution was added to the reaction bottle. The cumulative release percentage of isoquercetin in different release systems was calculated based on the standard curve of isoquercetin, and the release curve was drawn. The drug release in different pH environments was calculated and analyzed based on the absorbance at different time periods. The results are shown in Figure 2. Figure 6 shown.

[0089] The results showed that IQ@gel is pH-responsive and has good drug release under acidic conditions, and can quickly release a large amount of drugs to achieve an effective drug dose.

[0090] 7. IQ@gel drug release curve drawing:

[0091] The water bath temperature was set to 37°C, the hydrogel was placed in a PBS buffer solution, and oscillated in a constant temperature water bath at 37°C for 72 hours. At fixed intervals (0h, 8h, 16h, 24h, 32h, 40h, 48h, 56h, 64h, 72h), 5mL of drug-released solution was taken out and the same volume of the same buffer solution was added. The absorbance value was measured at 360nm using an enzyme marker, and an equal volume of the corresponding buffer was added to the reaction bottle. The cumulative release percentage of isoquercetin in different release systems was calculated based on the standard curve of isoquercetin, and the release curve was drawn. The results are shown in Figure 2. Figure 7 shown.

[0092] The results showed that the isoquercetin carried by IQ@gel was well released under physiological pH conditions and had a certain sustained-release effect, which could maintain the drug concentration unchanged for a certain period of time.

[0093] 8. IQ@gel biocompatibility testing:

[0094] IQ@gel was freeze-dried and sterilized. The sterilized samples were incubated in DMEM medium for 24 h to prepare the extract. H9c2 cells (1×10 cells per well) 4Cells (100 cells) were seeded in 96-well plates and cultured at 37°C for 24, 48, and 72 hours. The extract was diluted to concentrations of 25 μM, 50 μM, 100 μM, 150 μM, and 200 μM, respectively. The culture medium was removed and replaced with the extract from IQ@gel. MTT and DMSO were added, and absorbance at 540 nm was measured (MTT assay) to assess cytocompatibility.

[0095] The results are as follows Figure 8 As shown, the results showed that the hydrogel loaded with 25μM to 100μM isoquercetin had good biocompatibility and low toxicity to H9c2 cells, and IQ@gel was suitable for subsequent drug delivery therapy.

[0096] 9. Evaluation of the protective effect of IQ@gel on H9c2 cells:

[0097] H9c2 cells were plated at 1×10 4 Cells were plated at a density of 100 μM in 96-well plates and divided into four treatment groups: control, DOX, DOX+IQ, and DOX+IQ@gel. After 24 hours of incubation at 37°C and 5% CO2, IQ@gel extract (final concentration: 100 μM), DOX (final concentration: 5 μM), and IQ (final concentration: 100 μM) were added according to the group and incubated for another 24 hours. Apoptotic bodies and cells at different stages of apoptosis were observed using Hoechst 33342 and PI staining. Cell morphology was observed under blue and red fluorescence inverted microscopes, respectively.

[0098] The results are as follows Figure 9 As shown in the figure, the scale bar is 200 μm. PI and Hoechst 33342 staining results show that isoquercetin (IQ) can significantly ameliorate doxorubicin (DOX)-induced cardiomyocyte apoptosis. The therapeutic effect of IQ@gel-loaded isoquercetin (IQ) on H9c2 cells is similar to that of direct administration.

[0099] 10. Effect of IQ@gel on ROS levels in H9c2 cells:

[0100] H9c2 cells were plated at 1×10 4 Cells were plated at a density of 100 μM in 96-well plates and cultured at 37°C and 5% CO2. The cells were divided into four treatment groups: control, DOX, DOX+IQ, and DOX+IQ@gel. After 24 hours of culture at 37°C and 5% CO2, IQ@gel extract (final concentration: 100 μM), DOX (final concentration: 5 μM), and IQ (final concentration: 100 μM) were added to the cells according to the group and incubated for another 24 hours. After 24 hours of treatment, ROS levels in H9c2 cells were measured using the DCFH-DA fluorescent probe.

[0101] The results of DCFH-DA fluorescence detection are as follows Figure 10 The above experimental results show that IQ@gel can significantly reduce the level of reactive oxygen species in H9c2 cells induced by DOX, and exhibits a more powerful anti-oxidative stress effect compared to direct administration of IQ.

[0102] 11. Mouse experiments:

[0103] Mice were randomly divided into three groups, six in each: control (Con), DOX, DOX+IQ, and DOX+IQ@gel. The control group received intraperitoneal injections of normal saline; the DOX group received intraperitoneal injections of DOX (2 mg / kg); the DOX+IQ group received intraperitoneal injections of DOX (2 mg / kg) and 20 mg / kg of IQ; and the DOX+IQ@gel group received intraperitoneal injections of DOX (2 mg / kg) and IQ@gel (equivalent to 20 mg / kg of IQ). The mice were administered intraperitoneally every other day for 14 consecutive days. Mouse body weights were recorded daily.

[0104] The daily weight fluctuations of mice were counted, and the results were as follows Figure 11 As shown in Figure 3, the results showed that intraperitoneal injection of IQ@gel could significantly improve the body weight loss of mice caused by DOX-induced heart failure.

[0105] After the administration, the mice were anesthetized with 2% isoflurane and placed in the supine position. A high-frequency ultrasound machine (EPIQ 5, Philips) was used to obtain images from different cardiac angles. Vital signs were monitored throughout the process, and the following parameters were measured: left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), left ventricular end-systolic diameter (LVIDd), and left ventricular end-diastolic diameter (LVIDs). LVIDd and LVIDs were not significantly affected by the modeling. The results of cardiac images and LVEF (expressed as EF in the figure) and LVFS (expressed as FS in the figure) of each group are shown in the figure. Figure 12 As shown, the results showed that injection of IQ@gel significantly alleviated the decline in ejection fraction induced by DOX, indicating the cardioprotective function of IQ@gel.

[0106] After the administration, the FFA content detection kit (Beijing Solebow Technology Co., Ltd.; BC0590) was used to measure the FFA in the mouse serum and the detection was carried out at a wavelength of 550 nm using an enzyme reader. The ATP content was measured using the ATP content detection kit (Biyuntian Biotechnology Co., Ltd.; S0026B) according to the instructions. The LA content was measured using the L-lactic acid detection kit (Biyuntian Biotechnology Co., Ltd.; S0208S). The LDH content in the mouse serum was measured using a mouse lactate dehydrogenase (LDH) ELISA kit. The results are shown in Figure 2. Figure 13FFA, ATP, LA, and LDH are mitochondrial markers. The results showed that IQ@gel effectively alleviated DOX-induced mitochondrial damage in cardiomyocytes and improved cardiomyocyte energy metabolism.

[0107] The mice were euthanized and the hearts were fixed with 4% paraformaldehyde for HE staining and PSR staining. Figure 14 As shown, the PSR staining results are as follows Figure 15 PSR staining results showed that IQ@gel had a protective effect on DOX-induced myocardial fibrosis, and HE staining results showed that IQ@gel had a significant therapeutic effect on DOX-induced pathological damage.

[0108] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for preparing a hydrogel loaded with isoquercetin, characterized in that: The following steps are involved: A sodium periodate aqueous solution is added to a hyaluronic acid aqueous solution, and the mixture is stirred in the dark. Ethylene glycol is added to terminate the reaction, and the reaction product is dialyzed and freeze-dried to obtain oxidized hyaluronic acid; the oxidized hyaluronic acid is dissolved in water to obtain an oxidized hyaluronic acid aqueous solution; the oxidized hyaluronic acid aqueous solution is mixed with an isoquercetin mixture to obtain a precursor solution containing oxidized hyaluronic acid and isoquercetin; the precursor solution containing oxidized hyaluronic acid and isoquercetin is added to a carboxymethyl chitosan aqueous solution under vortex conditions, and the vortex is continued for 10 to 30 seconds, and the mixture is allowed to stand for 3 to 5 minutes to obtain a hydrogel.

2. The preparation method according to claim 1, characterized in that The concentration of the hyaluronic acid aqueous solution is 10 mg / mL, and the concentration of the sodium periodate aqueous solution is 0.1 g / mL; the volume ratio of the hyaluronic acid aqueous solution, the sodium periodate aqueous solution and ethylene glycol is 40:1:0.04; and the stirring time in the dark is 6 to 8 hours.

3. The preparation method according to claim 1, characterized in that The molecular weight of the dialyzed protein is 8000-14000 Da.

4. The preparation method according to claim 1, characterized in that In the precursor solution containing oxidized hyaluronic acid and isoquercetin, the mass fraction of oxidized hyaluronic acid is 1.9-2.1%, and the final concentration of isoquercetin is 250 μM.

5. The preparation method according to claim 1, characterized in that The mass fraction of carboxymethyl chitosan in the carboxymethyl chitosan aqueous solution is 4.5-5.0%; the volume ratio of the precursor solution containing oxidized hyaluronic acid and isoquercetin to the carboxymethyl chitosan aqueous solution is 1:0.

4.

6. The hydrogel prepared by the preparation method according to any one of claims 1 to 5.

7. Use of the hydrogel according to claim 6 in any of the following: (1) preparing drugs for inhibiting inflammatory reactions; (2) preparing drugs for relieving and / or inhibiting myocardial cell apoptosis; (3) Preparation of drugs for preventing myocardial oxidative damage; (4) Prepare drugs for treating oxidative stress-induced cardiomyopathy.

8. The use according to claim 7, characterized in that The cardiomyopathy includes tumor heart disease.

9. A drug for preventing and / or treating oxidative stress-induced cardiomyopathy, characterized in that: The active ingredient of the medicine comprises the hydrogel according to claim 6.

10. The drug according to claim 9, characterized in that The drug further includes a pharmaceutically acceptable carrier.

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