Use of thiolated hyaluronic acid or its salt in myocardial ischemia-reperfusion injury

Abstract

The present application discloses the use of thiolated hyaluronic acid or its salt in myocardial ischemia-reperfusion injury, and belongs to the field of biomedical technology. In this use, the degree of substitution of the synthesized thiolated hyaluronic acid or its salt thiol can reach 10%-40%. The antioxidant capacity of 5mg / ml thiolated hyaluronic acid or its salt can reach more than 50%; in the H9C2 cardiomyocyte SI / RI model, HA-SH can protect H9C2 cardiomyocytes, and can significantly reduce the oxidative stress damage in the SI / RI model; in the constructed rat myocardial ischemia-reperfusion injury model, HA-SH can reduce the oxidative stress damage, apoptosis and inflammatory response caused by myocardial ischemia-reperfusion by regulating the Nrf2 signaling pathway and the HO-1 signaling pathway, reduce the iron mortality rate, and play a role in protecting the heart. The present application provides a new drug development approach, which is of great significance in the field of biomedical technology.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to use of thiolated hyaluronic acid or its salt in myocardial ischemia-reperfusion injury. Background Art

[0002] Acute myocardial infarction seriously endangers human life and health. The "China Cardiovascular Health and Disease Report 2022" shows that the mortality rate of acute myocardial infarction in my country has increased year by year, and about 1 million patients suffer from acute myocardial infarction each year. Reperfusion methods such as drug thrombolysis, intervention, and bypass can reduce the mortality rate of patients, but a considerable number of people cannot avoid myocardial ischemia-reperfusion injury, which then develops into arrhythmias and heart failure. The pathogenesis of myocardial ischemia-reperfusion injury (MI / RI) is very complex. After partial or complete acute occlusion of the coronary artery, when it is recanalized within a certain period of time, the ischemic myocardium can restore normal perfusion, but its tissue damage is a progressively aggravated pathological process. A series of damaging changes in myocardial ultrastructure, energy metabolism, cardiac function, and electrophysiology caused by the ischemic period are more prominent after the blood vessels are recanalized, and even severe arrhythmias may occur, leading to sudden death. Free radical damage is one of the main mechanisms of myocardial ischemia-reperfusion injury. Free radicals are the general term for atoms, atomic groups and molecules that contain a single unpaired electron in the outer electron orbit, including oxygen free radicals (OFR), such as superoxide anions; lipid free radicals, such as alkyl free radicals and alkoxy free radicals; and other free radicals.

[0003] The clinical prevention and treatment principles of ischemia-reperfusion injury include: (1) Prevent reperfusion or significant reduction of blood flow to tissues and organs: Tissue oxygen consumption should be reduced as much as possible during the ischemic period (such as lowering the temperature); calcium blockers should be used before reperfusion, and drugs that scavenge oxygen free radicals should be used at the beginning of reperfusion. Try to shorten and reduce the time and degree of tissue ischemia. (2) Use enzymes that scavenge oxygen free radicals: including superoxide dismutase, catalase, and glutathione peroxidase. (3) Supplement vitamin C and vitamin E: scavenge free radicals. (4) Coenzyme Q10: an antioxidant and membrane stabilizer that has multiple functions such as scavenging free radicals generated by lipid peroxidation, preventing mitochondrial damage during ischemia, and maintaining the integrity of myocardial calcium ion channels. (5) Deferoxamine: Deferoxamine can chelate with iron ions to form complexes, reduce Fe ion concentration, and thus reduce the generation of oxygen free radicals. (6) Mannitol, allopurinol, glucose, magnesium sulfate, etc. all have the function of scavenging free radicals.

[0004] It can be seen that there is currently no drug that can specifically improve clinical myocardial ischemia-reperfusion injury. Therefore, a better intervention strategy needs to be developed. Summary of the invention

[0005] In order to solve the problems existing in the prior art, the present invention provides the following technical solutions.

[0006] The first aspect of the present invention provides the use of thiolated hyaluronic acid or its salt in the preparation of a medicament for preventing or treating myocardial ischemia-reperfusion injury.

[0007] Preferably, myocardial ischemia-reperfusion injury includes oxidative stress response, cardiomyocyte apoptosis, cardiomyocyte ferroptosis and inflammatory response.

[0008] Preferably, the substitution degree of the thiol group of the thiolated hyaluronic acid or its salt is 10%-40%, and the average substitution degree is 24.4%.

[0009] Preferably, the antioxidant capacity of 5 mg / ml of thiolated hyaluronic acid or its salt is 57.3-78.2%, and the average antioxidant capacity is 67.8%.

[0010] Preferably, thiolated hyaluronic acid or its salt regulates the Nrf2 signaling pathway and the HO-1 signaling pathway. Among them, Nrf2 is a transcription factor that is very important for oxidative stress response, and it binds to the antioxidant response element (ARE) located in the promoter region of many cell protection genes. HO-1 is a protease that plays a key role in cellular defense against oxidative stress and inflammation.

[0011] The second aspect of the present invention provides a drug for preventing or treating myocardial ischemia-reperfusion injury, which contains thiolated hyaluronic acid or its salt as an active ingredient and further includes pharmaceutically acceptable excipients.

[0012] Preferably, the pharmaceutically acceptable excipient is selected from at least one of a buffer, an encapsulating agent, a filler, a binder, a transdermal absorbent, a wetting agent, a disintegrant, an absorption enhancer, a surfactant, a colorant, a flavoring agent and an adsorption carrier.

[0013] Preferably, the prevention or treatment comprises at least one of the following:

[0014] (1) Improve myocardial cell apoptosis after myocardial ischemia-reperfusion;

[0015] (2) Reduce the iron death rate of cardiomyocytes after myocardial ischemia-reperfusion;

[0016] (3) Reduce oxidative stress damage caused by myocardial ischemia-reperfusion;

[0017] (4) Reduce the inflammatory response caused by myocardial ischemia-reperfusion.

[0018] Preferably, the dosage form of the drug includes tablets, powders, granules, capsules, injections, sprays, films, suppositories, nasal drops or pills; and / or, the route of administration of the drug includes intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, nasal administration or transdermal administration.

[0019] The beneficial effects of the present invention are as follows: the present invention provides an application of thiolated hyaluronic acid (HA-SH) or its salt in the preparation of a drug for preventing or treating myocardial ischemia-reperfusion injury. In the embodiment of the present invention, it is experimentally confirmed that the degree of substitution of the thiol group of the synthesized thiolated hyaluronic acid or its salt can reach 10%-40%, and the average degree of substitution can be 24.4%. The antioxidant capacity of 5mg / ml thiolated hyaluronic acid or its salt can reach more than 50%; in the H9C2 myocardial cell hypoxia-reoxygenation model (SI / RI model), HA-SH can protect H9C2 myocardial cells, and can significantly reduce the oxidative stress damage in the H9C2 myocardial cell SI / RI model; in the constructed rat myocardial ischemia-reperfusion injury model, HA-SH can reduce the oxidative stress damage, cell apoptosis and inflammatory response caused by myocardial ischemia-reperfusion by regulating the Nrf2 signaling pathway and the HO-1 signaling pathway, reduce the iron mortality of myocardial cells after myocardial ischemia-reperfusion, and play a protective role in the heart. The present invention proposes for the first time that thiolated hyaluronic acid or its salt can improve myocardial ischemia-reperfusion injury, provides a new drug development approach for the prevention and / or treatment of myocardial ischemia-reperfusion injury, and has important significance in the field of biomedical technology. DETAILED DESCRIPTION

[0020] In order to better understand the above technical solution, the above technical solution will be described in detail below in conjunction with specific implementation methods.

[0021] It should be noted that the concentrations mentioned in the present invention are all mass percentage concentrations.

[0022] The present invention intends to achieve the improvement of myocardial ischemia-reperfusion injury by deeply exploring the molecular mechanism and key therapeutic targets of myocardial ischemia-reperfusion injury. Studies have found that excessive generation of free radicals and oxidative stress are important factors in the occurrence and development of MI / RI. They are activated at the beginning of reperfusion and can trigger a series of pathophysiological processes. Therefore, anti-oxidative stress may be one of the most promising therapeutic strategies to alleviate MI / RI. Ferroptosis is an important form of myocardial cell death in MI / RI. Ferroptosis is closely related to oxidative stress, which is characterized by excessive production of ROS, iron accumulation and lipid peroxidation, followed by cell membrane integrity destruction leading to cell death. In the present invention, thiolated hyaluronic acid (HA-SH) or its salt is used to inhibit ferroptosis, improve cell apoptosis and oxidative reactions caused by MI / RI, and play a cardioprotective role. In the ischemic hypoxia model of mouse myocardial cell model, it was observed that HA-SH or its salt can improve cardiac damage caused by MI / RI, reduce infarct area and cell apoptosis, and has a cardioprotective effect.

[0023] The embodiment of the present invention exemplarily provides an application of thiolated hyaluronic acid or its salt in the preparation of a drug for preventing or treating myocardial ischemia-reperfusion injury. Wherein, myocardial ischemia-reperfusion injury includes oxidative stress response, myocardial cell apoptosis, myocardial cell ferroptosis and inflammatory response. The degree of substitution of the thiol group of thiolated hyaluronic acid or its salt is 10%-40%, and the average degree of substitution is 24.4%. The antioxidant capacity of 5mg / ml of thiolated hyaluronic acid or its salt is 57.3-78.2%, and the average antioxidant capacity is 67.8%. Thiolated hyaluronic acid or its salt regulates the Nrf2 signaling pathway and the HO-1 signaling pathway.

[0024] The embodiment of the present invention also exemplarily provides a drug for preventing or treating myocardial ischemia-reperfusion injury, with thiolated hyaluronic acid or its salt as an active ingredient, and also includes pharmaceutically acceptable excipients. Wherein, the pharmaceutically acceptable excipients can be selected from at least one of a buffer, an encapsulating agent, a filler, an adhesive, a transdermal absorbent, a wetting agent, a disintegrant, an absorption promoter, a surfactant, a colorant, a flavoring agent and an adsorption carrier. The prevention or treatment includes at least one of the following: (1) improving myocardial cell apoptosis after myocardial ischemia-reperfusion; (2) reducing the iron mortality rate of myocardial cells after myocardial ischemia-reperfusion; (3) reducing oxidative stress damage caused by myocardial ischemia-reperfusion; (4) reducing the inflammatory response caused by myocardial ischemia-reperfusion. The dosage form of the drug may include tablets, powders, granules, capsules, injections, sprays, films, suppositories, nasal drops or pills; and / or, the route of administration of the drug may include intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, nasal administration or transdermal administration.

[0025] The present invention is described with respect to thiolated hyaluronic acid, and the methods and uses involved in the relevant contents of the corresponding thiolated hyaluronate are the same and will not be described in detail.

[0026] The main technical solutions and technical effects of the present invention include:

[0027] 1. Synthesis of thiolated hyaluronic acid and determination of its antioxidant capacity.

[0028] Hyaluronic acid with molecular weights of 100KD, 500KD, 1000KD, 1500KD, and 2500KD (purchased from Huaxi Biotechnology Co., Ltd. or Beijing Solebow Technology Co., Ltd.) is used, and thiol treatment is performed by conventional methods. Part of the hydroxyl groups of the hyaluronic acid undergoes amidation reaction under the action of EDC (1-ethyl-(3-dimethylaminopropyl) carbodiimide) and is replaced with an ester group containing a thiol group, that is, the amino group on cystamine undergoes amidation reaction with the carboxyl group on the hyaluronic acid to cross-link to obtain hyaluronic acid connected by a stable disulfide bond, and then the strong reducing property of dithiothreitol is used to break the disulfide bond to form a thiol group. The thiolated hyaluronic acid does not change the biocompatibility and biodegradability of the hyaluronic acid itself. The substitution degree of the thiol group of the thiolated hyaluronic acid detected by an elemental analyzer is 10%-40%, and the average substitution degree is 24.4%. Antioxidant capacity determination: By detecting the ability to scavenge hydroxyl free radicals, the antioxidant capacity of thiolated hyaluronic acid was determined as follows: 5 mg / ml of thiolated hyaluronic acid can scavenge more than 50% (57.3-78.2%) of hydroxyl free radicals, and an average of 67.8% of hydroxyl free radicals.

[0029] 2. In the SI / R cell model, HA-SH can protect cardiomyocytes and reduce oxidative stress damage in the cardiomyocyte SI / RI model.

[0030] 1) The SI / RI cell model was treated with HA-SH (molecular weight 1500KD) at different concentrations (0, 0.02%, 0.05%, 0.1%, 0.2%, 0.5%) for 6 hours, and the cell viability of each group of cardiomyocytes was detected by MTT method. The results showed that the SI / RI cell model group significantly inhibited the cell viability of cardiomyocytes compared with the control group (P<0.05). Compared with the SI / RI cell model group, different concentrations (0.02%, 0.05%, 0.1%, 0.2%, 0.5%) of HA-SH (molecular weight 1500KD) pretreatment for 6 hours can improve the cell viability of cardiomyocytes, and the cell viability increases with the increase of HA-SH concentration, in a dose-dependent manner. Compared with the SI / RI cell model group, there is statistical significance.

[0031] 2) Compared with the control group, the amount of LDH released in the supernatant of myocardial cells in the SI / RI cell model group was significantly increased. Flow cytometry was used to detect the effect of HA-SH on ferroptosis of myocardial cells damaged by SI / RI cell model. The results showed that compared with the control group, the proportion of myocardial cell ferroptosis in the SI / RI group was significantly increased. Compared with the SI / R cell model group, HA-SH pretreatment can significantly reduce the proportion of cell ferroptosis in the SI / RI cell model. This shows that HA-SH can inhibit cell ferroptosis in the SI / RI cell model.

[0032] 3) RT-qPCR was used to detect the effect of HA-SH on hypoxia-reoxygenation-induced cellular inflammatory response. Compared with the control group, the mRNA transcription levels of TNF-α, IL-6 and IL-1β in the SI / RI cell model group were significantly increased. Compared with the SI / RI group, the mRNA transcription levels of TNF-α, IL-6 and IL-1β in the SI / RI cell model pretreated with HA-SH were significantly reduced, indicating that HA-SH has an anti-inflammatory effect in the SI / RI cell model. At the same time, the ELISA determination of TNF-α, IL-6 and IL-1β levels in the supernatant of SI / RI injured cardiomyocytes was consistent with the results obtained by RT-qPCR.

[0033] 4) The ROS level in the SI / RI cell model group was significantly higher than that in the control group. Compared with the SI / RI cell model group, the ROS level in the SI / RI model of cardiomyocytes pretreated with HA-SH was significantly reduced.

[0034] 5) It can inhibit MDA and enhance SOD activity. The MDA content in the SI / RI cell model group was significantly higher than that in the control group, and the SOD activity was significantly reduced. Compared with the SI / RI cell model group, the MDA content in the myocardial cell SI / RI model pretreated with HA-SH was significantly reduced, and the SOD activity was significantly increased.

[0035] 6) RT-qPCR was used to detect the levels of Nrf2 and HO-1 mRNA in the cardiomyocyte SI / RI model. Compared with the control group, the levels of Nrf2 and HO-1 mRNA in the SI / RI cell model group were increased. Compared with the SI / RI cell model group, the levels of Nrf2 and HO-1 mRNA in the cardiomyocyte SI / RI model pretreated with HA-SH were significantly increased.

[0036] 3. A rat myocardial ischemia-reperfusion injury model was established, and it was found that HA-SH can reduce the oxidative stress, cell apoptosis and inflammatory response caused by myocardial ischemia-reperfusion by regulating the Nrf2 and HO-1 signaling pathways, thereby playing a protective role against the heart.

[0037] 1) It can prolong the latency of arrhythmia after myocardial ischemia-reperfusion, reduce ST segment potential difference, reduce serum LDH and CK-MB levels, reduce myocardial infarction area, increase the proportion of drug protection area, reduce myocardial edema and inflammatory cell infiltration, and have a myocardial protective effect on MI / RI rats. 2) It can improve the ability of MI / RI rats to resist oxidative stress, significantly increase the SOD activity of MI / RI rats, and reduce the content of MDA and ROS. It can significantly improve the degree of myocardial cell ferroptosis in MI / RI rats and reduce the cell ferroptosis rate. 3) It can upregulate the expression levels of Nrf2 and HO-1 in myocardial tissue of MI / RI rats, and play an anti-MI / RI role by activating the Nrf2 and HO-1 signaling pathways to regulate oxidative stress and cell apoptosis.

[0038] Example 1. Pretreatment of H9C2 cardiomyocytes with HA-SH at different concentrations can increase the cell viability of the SI / RI cell model.

[0039] 1. Synthesis of thiolated hyaluronic acid and determination of its antioxidant capacity.

[0040] 1) Using hyaluronic acid with a molecular weight of 1500KD (purchased from Huaxi Biotechnology Co., Ltd. or Beijing Solebow Technology Co., Ltd.), a conventional method was used for thiol treatment, and part of the hydroxyl groups of the hyaluronic acid under the action of EDC were amidated and replaced with ester groups containing thiol groups, that is, the amino group on cystamine and the carboxyl group on the hyaluronic acid were amidated and cross-linked to obtain hyaluronic acid connected by stable disulfide bonds, and then the strong reducing property of dithiothreitol was used to break the disulfide bonds to form thiol groups.

[0041] 2) The substitution degree of the thiol groups of the thiol-modified hyaluronic acid was detected to be 20.2% by an element analyzer.

[0042] The synthesized HA-SH was ground into fine powder, placed in a 105°C oven to constant weight until dry, and placed in a drying dish to cool for 20 minutes. Weigh 3 portions of 2,5-bis(5-tert-butyl-1,3-benzoxazol-2-yl)thiophenol (standard) and the synthesized HA-SH sample (2.5±0.5) mg, and placed in an elemental analyzer for sampling and detection.

[0043] 3) Antioxidant capacity determination: The antioxidant capacity of thiolated hyaluronic acid was determined by testing the ability to scavenge hydroxyl radicals. The results showed that 5 mg / ml HA-SH could scavenge 63.7% of hydroxyl radicals (greater than 50%).

[0044] Prepare 5.0 mg / ml of the above synthesized thiolated hyaluronic acid and dilute it to 5.0 mg / ml, 2.5 mg / ml, 1.0 mg / ml, 0.1 mg / ml, and 0 mg / ml. Take 1 ml of samples of different concentrations and add 1 ml of 1,10-phenanthroline (1 mM), 1 m phosphate buffer (20 mM, pH 7.4), 1 ml of ammonium ferrous sulfate [FeSO 4 ·(NH 4 ) 2 SO 4 ·6H2O, 0.75mM] and 1ml hydrogen peroxide (0.01%). After the reaction solution is mixed, keep it at 37℃ for 1h. After the reaction solution is cooled to room temperature, the light absorbance at 536nm is measured. The decrease in the light absorbance of the reaction mixture indicates that the test compound has the ability to scavenge hydroxyl free radicals.

[0045] 2. H9C2 cardiomyocyte recovery.

[0046] 1) After thorough disinfection and sterilization under ultraviolet light in the clean bench, place the cryopreserved tubes containing H9C2 cardiomyocytes in a constant temperature water bath, shake gently by hand, and observe the cell thawing. Note that the total time should be controlled within 1 min.

[0047] 2) After most of the cells have thawed, quickly use a pipette to add the cell fluid into a centrifuge tube containing fresh complete DMEM medium, centrifuge at 300g to wash twice, and discard the supernatant.

[0048] 3) Resuspend the cells in complete DMEM medium and inoculate them into culture flasks. 2 Incubator at 37°C, CO 2 The concentration was 5%.

[0049] 4) When the density of cardiomyocytes reaches 80% to 90% confluence, cell subculture is performed: 1 mL of 0.025% trypsin-EDTA (ethylenediaminetetraacetic acid) is added to the culture flask to digest the cells for 1 min. The digestion is terminated as soon as the cells fall off the flask wall; the digested cells are washed twice by centrifugation and subcultured with complete culture medium at a ratio of 1:6.

[0050] 5) Cell counting: Add 10 μL of cell suspension to one side of the glass cell counter and count the total number of cells in the four squares under a microscope. Calculate the cell concentration according to the following formula: Cell concentration (cells / mL) = total number of cells / (4×10 4 ). According to the cell number required for the experiment, the cells were seeded in new culture flasks or six-well plates to complete cell passaging.

[0051] 3. Cell culture and treatment with different concentrations of HA-SH.

[0052] 1) Inoculate 96-well plates with 2000 cardiomyocytes in the logarithmic growth phase per well (100 μL); place in a CO 2 Incubator 37°C, CO 2 Culture was carried out under the condition of 5% concentration;

[0053] 2) Observe the cardiomyocytes under a microscope and observe that the confluence of the cardiomyocytes reaches 70-80%. Treat the cells with different concentrations (0.02%, 0.05%, 0.1%, 0.2%, 0.5%) of HA-SH (molecular weight 1500KD) and continue to culture. At the same time, set up a control group without HA-SH.

[0054] 4.MTT method was used to detect cell activity.

[0055] 1) After HA-SH treatment for 24 h, add 20 μL of MTT solution to each well and place the cells in a 37°C, CO 2 Incubate in a dark incubator for 4 h;

[0056] 2) The culture medium was discarded, and 100 μL of formazan complex dissolved in dimethyl sulfoxide (DMSO) was added to each well. The wavelength of the microplate reader was set to 570 nm, and the OD value of each well was measured.

[0057] 5.SI / RI cell model group experiment.

[0058] 1) Following the same steps as in "3. Cell culture and treatment with different concentrations of HA-SH" above, cells were treated with different concentrations of HA-SH (molecular weight 1500KD) (0.02%, 0.05%, 0.1%, 0.2%, 0.5%), including the SI / RI cell model group without HA-SH. 2 Incubator 37°C, CO 2 The concentration was 5% and the culture was carried out for 6 h.

[0059] 2) Place the cells in a three-gas hypoxic incubator for 4 hours of hypoxia followed by 8 hours of reoxygenation. Continue CO 2 Incubator 37°C, CO 2 The concentration was 5% and the culture was carried out for 6 h.

[0060] 3) Following the same steps as in “4. MTT assay for cell activity”, the cell viability of the SI / RI cell model group was determined.

[0061] 6. Results: Effects of preconditioning on cardiomyocyte viability

[0062] 1) After pre-treatment of cardiomyocytes with HA-SH (molecular weight 1500KD) at different concentrations (0.02%, 0.05%, 0.1%, 0.2%, 0.5%) for 24 hours, the cell viability was detected by MTT method. Compared with the control group, the viability of cardiomyocytes pre-treated with HA-SH at different concentrations did not decrease significantly, indicating that HA-SH has little toxicity to cardiomyocytes.

[0063] 2) After treating cardiomyocytes with HA-SH (molecular weight 1500KD) of different concentrations (0.02%, 0.05%, 0.1%, 0.2%, 0.5%) for 6 hours, the cells were hypoxic for 4 hours and reoxygenated for 8 hours to establish SI / RI cell model, and the cell viability of cardiomyocytes in each group was detected by MTT method. Results Compared with the control group, the SI / RI cell model group significantly inhibited the cell viability of cardiomyocytes. Compared with the SI / RI cell model without HA-SH pretreatment, the SI / RI cell model pretreated with HA-SH (molecular weight 1500KD) of different concentrations (0.02%, 0.05%, 0.1%, 0.2%, 0.5%) can improve the cell viability of hypoxic and reoxygenated cardiomyocytes, and the cell viability increases with the increase of HA-SH concentration, which is dose-dependent. The test results are shown in Table 1.

[0064] Table 1 Cell viability assay results of H9C2 cardiomyocytes and hypoxia-reoxygenation cardiomyocytes after HA-SH treatment

[0065]

[0066] Example 2. Protective effects of HA-SH of different molecular weights and concentrations on cardiomyocytes.

[0067] 1. The synthesis of thiolated hyaluronic acid and the determination of its antioxidant capacity, the culture of H9C2 cardiomyocytes, and the construction of SI / RI cell model are all the same as those in Example 1.

[0068] The molecular weights of hyaluronic acid were 100K, 500K, 1000K, and 2500K (purchased from Bloomage Biotechnology Co., Ltd. or Beijing Solebow Technology Co., Ltd.), and the substitution degree of thiol groups were 34.2%, 26.7%, 24.3%, and 16.6%, respectively, as shown in Table 2. The antioxidant capacity of 5 mg / mL was greater than 50%.

[0069] Table 2 Degree of substitution and antioxidant capacity of thiol-containing hyaluronic acid

[0070] HA-SH molecular weight 100KD 500KD 1000KD 1500KD 2500KD average value Degree of thiol substitution 34.2％ 26.7％ 24.3％ 20.2％ 16.6％ 24.4％ Antioxidant Capacity 78.2％ 69.3％ 68.4％ 63.7％ 59.4％ 67.80％

[0071] 2. Treat with HA-SH of different concentrations (0.02%, 0.1%, 0.5%) and different molecular weights (100KD, 500KD, 1000KD, 2500KD), and the control group without HA-SH (the concentration of HA-SH added was 0%) was the same as Example 1. After pre-treating the cardiomyocytes for 24 hours, the cell viability was detected by MTT method. Compared with the control group, the viability of the cardiomyocytes pre-treated with HA-SH of different concentrations and different molecular weights did not decrease significantly. It can be seen that HA-SH has little toxicity to cardiomyocytes.

[0072] 3. After treating cardiomyocytes with HA-SH of different concentrations (0.02%, 0.1%, 0.5%) and different molecular weights (100KD, 500KD, 1000KD, 2500KD) for 6 hours, the cells were hypoxic for 4 hours and reoxygenated for 8 hours to establish the SI / RI model. The SI / RI cell model without HA-SH was the same as in Example 1. The cell viability of each group of cardiomyocytes was detected by MTT method. Results Compared with the control group, the cell viability of cardiomyocytes in the SI / RI cell model group was significantly inhibited. Compared with the SI / RI cell model group without HA-SH pretreatment (the concentration of added HA-SH was 0%), the SI / RI cell model pretreated for 6 hours with HA-SH at concentrations of 0.02%, 0.1%, and 0.5% and molecular weights of 100KD, 500KD, 1000KD, and 2500KD was able to improve the cell viability of hypoxic and reoxygenated cardiomyocytes. The cell viability increased with the increase of HA-SH concentration in a dose-dependent manner. The test results are shown in Table 3.

[0073] Table 3 Cell viability assay results of H9C2 cardiomyocytes and hypoxia-reoxygenation cardiomyocytes after pretreatment with HA-SH of different molecular weights and concentrations

[0074]

[0075] Example 3. 100KD HA-SH can reduce oxidative stress damage of cardiomyocytes in SI / RI cell model.

[0076] 1. Cultivation of H9C2 cardiomyocytes, construction of SI / RI cell model, and treatment with 100KD HA-SH at different concentrations were the same as in Example 2.

[0077] 2. Detection samples: After 24 hours of cardiomyocyte culture or HA-SH pretreatment, and 6 hours of hypoxia and reoxygenation treatment, the supernatant of each group of cell culture was retained for detection of LDH, inflammatory factors TNF-α, IL-6, IL-1β protein levels and other items. The cells of each group were washed with phosphate buffered saline (PBS) and used for detection of ferroptosis, inflammatory factors TNF-α, IL-6, IL-1β mRNA levels, ROS, MDA, SOD, Nrf2 and HO-1 mRNA levels and other items.

[0078] 3. Determination of lactate dehydrogenase (LDH).

[0079] The test was performed using a kit from Biotech Co., Ltd.

[0080] (1) Set up a blank control group, a cell control group, a maximum enzyme activity control group, an SI / RI cell model group, and a 100KD HA-SH treatment group. The blank control group is a culture medium without cells, the cell control group is a cell culture supernatant that is not treated with HA-SH, the SI / RI model group is a cell culture supernatant that is treated with hypoxia and reoxygenation, and the 100KD HA-SH treatment groups are cell culture supernatants that are treated with different concentrations of HA-SH and then subjected to hypoxia and reoxygenation.

[0081] (2) Maximum enzyme activity control group: cardiomyocytes not treated with HA-SH were cultured for 24 h (the method was the same as in Example 1), 250 μL of LDH release reagent was added, and the mixture was mixed by pipetting; CO 2 Incubate the cells in a cell culture incubator for 1 h and collect the supernatant.

[0082] (3) Add 120 μL of all the supernatants mentioned in (1) and (2) to a 96-well plate; add 60 μL of LDH detection working solution to each well.

[0083] (4) Incubate at room temperature in the dark for 30 min and measure the OD value at a wavelength of 490 nm.

[0084] (5) LDH release amount (%) = (sample OD value - blank control well OD value) / (maximum enzyme activity OD value - blank control well OD value) × 100%; wherein the sample refers to the mixture of all supernatants of the cell control group, SI / RI cell model group, and HA-SH treatment groups with different concentrations of 100KD mentioned in (1).

[0085] (6) Results: Compared with the cell control group, the amount of LDH released in the supernatant of myocardial cells in the SI / RI cell model group was significantly increased. Compared with the SI / RI cell model group without HA-SH treatment, the amount of LDH released in the supernatant of myocardial cells in the SI / RI cell model group after HA-SH pretreatment was significantly reduced. The results are shown in Table 4.

[0086] Table 4 Comparison of LDH release in the supernatant of cardiomyocytes in each group

[0087]

[0088] 4. Flow cytometry was used to detect the effect of HA-SH on ferroptosis of damaged cardiomyocytes in the SI / RI cell model group.

[0089] 1) Take each group of cells collected in step 2 and resuspend the cells in 500 μL of cell ferroptosis reagent binding buffer (Annexin V Binding buffer).

[0090] 2) Add 10 μL of fluorescein isothiocyanate-labeled ferroptosis reagent (Annexin V-FITC) and mix gently.

[0091] 3) Add 5 μL of propidium iodide staining solution and mix gently.

[0092] 4) Incubate at room temperature in the dark for 15 minutes, repeatedly mix the cell suspension during this period, and then place in an ice bath. Use aluminum foil to protect from light. Detect using flow cytometry within 1 hour.

[0093] 5) Results: Compared with the control group, the proportion of cardiomyocyte ferroptosis in the SI / RI model group was significantly increased. Compared with the SI / RI cell model group without HA-SH treatment, HA-SH pretreatment can significantly reduce the proportion of cell ferroptosis, as shown in Table 5. This indicates that HA-SH can inhibit cell ferroptosis in the SI / RI cell model group of cardiomyocytes.

[0094] Table 5 The proportion of myocardial cell ferroptosis in each group

[0095]

[0096] 5. RT-qPCR and ELISA were used to detect the effect of HA-SH on hypoxia-reoxygenation-induced cellular inflammatory response.

[0097] 1) Detection of mRNA levels of TNF-α, IL-6, and IL-1β related to inflammatory response.

[0098] All the experiments were performed using RT-qPCR reagents provided by Applied Biosystems and Shanghai Shenggong Biotechnology Co., Ltd.

[0099] (1) Take each group of cells prepared in step 2 and extract RNA respectively.

[0100] (2) After RNA extraction, quantify the amount and take 1 μg for cDNA synthesis.

[0101] (3) RT-qPCR was used to detect the mRNA levels of TNF-α, IL-6, and IL-1β in each group.

[0102] 2) ELISA was used to determine the protein levels of TNF-α, IL-6, and IL-1β in the cell supernatant.

[0103] All the tests were performed using ELISA kits from R&D Systems, UK.

[0104] (1) Take the cell supernatant prepared in step 2 and centrifuge it for measurement.

[0105] (2) Prepare blank control, standard substance, reference substance, test samples and reagents according to the kit instructions.

[0106] (3) Add diluent, standard, control or sample in sequence. Mix for 1 min. Incubate at room temperature for 2 h. Repeat the washing process 5 times.

[0107] (4) Add binding agent. Incubate at room temperature for 2 h. Repeat washing 5 times.

[0108] (5) Add substrate solution and incubate at room temperature in the dark for 30 min.

[0109] (6) Add stop solution to terminate the reaction.

[0110] (7) Measure the OD value at a wavelength of 450 nm.

[0111] (8) Establish a standard curve and calculate the concentration of each test.

[0112] 3) Results: Compared with the control group, the mRNA transcription levels of TNF-α, IL-6 and IL-1β in the SI / RI cell model group were significantly increased. Compared with the SI / RI cell model group without HA-SH treatment, the mRNA transcription levels of TNF-α, IL-6 and IL-1β in the SI / RI cell model group pretreated with HA-SH were significantly reduced. ELISA measured the levels of TNF-α, IL-6 and IL-1β in the supernatant of SI / RI injured cardiomyocytes, which were significantly reduced, consistent with the trend of the results obtained by RT-qPCR, as shown in Table 6. These results indicate that 100KD HA-SH pretreatment has a significant anti-inflammatory effect in hypoxia / reoxygenation injured cardiomyocytes.

[0113] Table 6 Detection results of cellular inflammatory factors in each group

[0114]

[0115] 6. Detect the ROS level in cardiomyocytes.

[0116] The ROS level in cardiomyocytes was detected using the ROS kit produced by Invitrogen, USA.

[0117] (1) Thaw the fluorescent probe 2,7-dichlorofluorescein diacetate (DCFH-DA) for detecting reactive oxygen species, add it to serum-free DMEM medium at a ratio of 1:1000 to prepare the probe, and store it in the dark.

[0118] (2) Take each group of cells prepared in step 2, wash them twice with phosphate buffered saline (PBS), count the cells, and take the same number of cells from each group for detection.

[0119] (3) Add the prepared DCFH-DA to each group of cells, mix thoroughly, protect from light with tin foil, and place in a CO 2 Incubate at 37°C for 30 min.

[0120] (4) Centrifuge at 300 g for 5 min, discard the supernatant, wash twice with PBS, and resuspend again with PBS.

[0121] (5) Microplate reader readings: measured by a microplate reader at 493 nm (excitation wavelength) and 522 nm (emission wavelength). Intracellular fluorescence was observed using a fluorescence microscope.

[0122] (6) Results: The ROS level in the SI / RI model group was significantly higher than that in the control group, while the ROS level in the SI / RI model group pretreated with HA-SH was significantly lower than that in the SI / RI model group not pretreated with HA-SH, as shown in Table 7.

[0123] Table 7 Detection of reactive oxygen species (ROS) in each group of cells

[0124] concentration Control group SI / RI Model Group SI / RI+0.02% SI / RI+0.1% SI / RI+0.5% ROS generation 0.96 2.37 1.42 1.26 1.05

[0125] 7. Detection of oxidative stress indicators.

[0126] 1) MDA detection.

[0127] The MDA kit from Nanjing Jiancheng Bioengineering Institute was used.

[0128] (1) Take each group of cells prepared in step 2, wash them twice with phosphate buffered saline (PBS), count the cells, and make sure the number of cells in each group is consistent before testing.

[0129] (2) Set up blank, control, standard, and test samples according to the instructions.

[0130] (3) Add detection reagents in sequence.

[0131] (4) Cover the top of the test tube with tin foil, poke holes in it, mix the liquid inside, and place it in a 95°C water bath for 80 min.

[0132] (5) Cool the tube with running water, centrifuge at 400 g for 10 min, and collect 200 μL of the supernatant.

[0133] (6) Read the results using an ELISA reader at 532 nm.

[0134] 2) SOD activity detection.

[0135] The SOD activity detection kit from Randox Company of the United Kingdom was used.

[0136] (1) Take each group of cells prepared in step 2, wash them twice with phosphate buffered saline (PBS), count the cells, and make sure the number of cells in each group is consistent before testing.

[0137] (2) Preparation of enzyme working solution: Mix the enzyme stock solution and enzyme diluent in a ratio of 1:10.

[0138] (3) Preparation of substrate application solution: Mix substrate stock solution and buffer solution at a ratio of 1:200.

[0139] (4) Add cells, enzyme working solution, substrate application solution, etc. in sequence, mix well, and incubate at 37°C for 20 min.

[0140] (5) Read the sample at a wavelength of 450 nm using an ELISA reader, measure the OD value, and calculate the results as shown in Table 8.

[0141] 3)The results are shown in Table 8.

[0142] Table 8 Detection results of MDA and SOD in each group of cells

[0143] concentration Control group SI / RI Model Group SI / RI+0.02% SI / RI+0.1% SI / RI+0.5% MDA 2.06 6.68 3.82 3.15 2.24 SOD 96.8 44.2 73.6 86.4 95.2

[0144] 8. RT-qPCR was used to detect the levels of Nrf2 and HO-1 mRNA in the SI / RI model group of cardiomyocytes. Compared with the control group, the levels of Nrf2 and HO-1 mRNA in the SI / RI model group of cardiomyocytes were slightly increased. Compared with the SI / RI model group, the levels of Nrf2 and HO-1 mRNA in the SI / RI model group of cardiomyocytes pretreated with HA-SH were significantly increased.

[0145] All the experiments were performed using RT-qPCR reagents provided by Applied Biosystems and Shanghai Shenggong Biotechnology Co., Ltd.

[0146] (1) Take each group of cells prepared in step 2 and extract RNA respectively.

[0147] (2) After RNA extraction, quantify the amount and take 1 μg for cDNA synthesis.

[0148] (3) RT-qPCR was used to detect the mRNA levels of TNF-α, IL-6, and IL-1β in each group.

[0149] (4)The results are shown in Table 9.

[0150] Table 9 Detection results of Nrf2 and HO-1 mRNA levels in each group of cells

[0151] concentration Control group SI / RI Model Group SI / RI+0.02% SI / RI+0.1% SI / RI+0.5% Nfr2 0.92 1.38 2.82 2.88 2.96 HO-1 0.98 1.24 2.76 2.82 2.84

[0152] Therefore, these findings indicate that different concentrations of 100KD HA-SH can protect cardiomyocytes and alleviate oxidative stress injury in the cardiomyocyte SI / RI model group.

[0153] Example 4. Different concentrations of HA-SH (500KD) can alleviate oxidative stress damage in the myocardial cell SI / RI model group.

[0154] 1. H9C2 cardiomyocyte culture, SI / RI model group, different concentrations of HA-SH treatment, collection of test samples, and detection method were the same as in Example 3.

[0155] 2. Results of determination of LDH release

[0156] Compared with the control group, the amount of LDH released in the supernatant of myocardial cells in the SI / RI model group was significantly increased. Compared with the SI / RI model group, the amount of LDH released in the supernatant of myocardial cells in the SI / RI model group after HA-SH pretreatment was significantly reduced, as shown in Table 10.

[0157] Table 10 Results of LDH release in the supernatant of cardiomyocytes in each group

[0158] concentration Control group SI / RI Model Group SI / RI+0.02% SI / RI+0.1% SI / RI+0.5% LDH release 17.8％ 64.2％ 25.6％ 17.4％ 16.2％

[0159] 3. Ferroptosis detection results

[0160] Compared with the control, the proportion of cardiomyocyte ferroptosis in the SI / RI model group was significantly increased. Compared with the SI / RI model group, HA-SH pretreatment can significantly reduce the proportion of cell ferroptosis, as shown in Table 11. This indicates that HA-SH can inhibit cardiomyocyte ferroptosis in the SI / RI model group.

[0161] Table 11 The proportion of cell ferroptosis in each group was significantly reduced

[0162] concentration Control group SI / RI Model Group SI / RI+0.02% SI / RI+0.1% SI / RI+0.5% Cell ferroptosis rate 6.2％ 37.3％ 13.6％ 10.1％ 6.8％

[0163] 4. Test results of inflammatory factors.

[0164] Compared with the control group, the mRNA transcription levels of TNF-α, IL-6 and IL-1β in the SI / RI model group were significantly increased. Compared with the SI / RI model group, the mRNA transcription levels of TNF-α, IL-6 and IL-1β in SI / RI cells pretreated with HA-SH were significantly reduced. ELISA measured the levels of TNF-α, IL-6 and IL-1β in the supernatant of SI / RI injured cardiomyocytes, which were significantly reduced, consistent with the trend of the results obtained by RT-qPCR. The results are shown in Table 12. These results indicate that HA-SH has an anti-inflammatory effect in cardiomyocytes SI / RI.

[0165] Table 12 Detection of inflammatory factors in each group

[0166]

[0167] 5. Detection results of reactive oxygen species (ROS).

[0168] The ROS level in the SI / RI model group was significantly higher than that in the control group, while the ROS level in the SI / RI model group pretreated with HA-SH was significantly lower than that in the SI / RI model group. The results are shown in Table 13.

[0169] Table 13 Detection results of reactive oxygen species (ROS) in each cell

[0170] concentration Control group SI / RI Model Group SI / RI+0.02% SI / RI+0.1% SI / RI+0.5% ROS generation 0.96 2.37 1.42 1.28 1.12

[0171] 6. Oxidative stress test results

[0172] It can inhibit MDA and enhance SOD activity. The MDA content of the SI / RI model group was significantly higher than that of the control group, and the SOD activity was significantly reduced. The MDA content of the SI / RI model group pretreated with HA-SH was significantly reduced, and the SOD activity was significantly increased compared with the SI / RI model group. The results are shown in Table 14.

[0173] Table 14 Detection results of MDA and SOD in each group of cells

[0174] concentration Control group SI / RI Model Group SI / RI+0.02% SI / RI+0.1% SI / RI+0.5% MDA 2.06 6.68 3.44 2.76 2.04 SOD 96.8 44.2 77.8 88.6 96.6

[0175] 7. Detection of Nrf2 and HO-1 mRNA levels.

[0176] Compared with the control group, the Nrf2 and HO-1 mRNA levels in the SI / RI model group were slightly increased. Compared with the SI / RI model group, the Nrf2 and HO-1 mRNA levels in the hypoxia-reoxygenation cardiomyocytes pretreated with HA-SH were significantly increased, as shown in Table 15.

[0177] Table 15 Detection results of Nrf2 and HO-1 mRNA levels in each group of cells

[0178] concentration Control group SI / RI Model Group SI / RI+0.02% SI / RI+0.1% SI / RI+0.5% Nfr2 0.92 1.38 2.51 2.76 2.86 HO-1 0.98 1.24 2.42 2.63 2.72

[0179] Therefore, these findings suggest that different concentrations of 500KD HA-SH can protect cardiomyocytes and alleviate oxidative stress injury in the cardiomyocyte SI / RI model.

[0180] Example 5. Different concentrations of HA-SH (1000KD) can alleviate oxidative stress damage in the SI / RI model of cardiomyocytes.

[0181] 1. Cultivation of H9C2 cardiomyocytes, SI / RI cell model, treatment with different concentrations of HA-SH, collection of test samples, and detection method are the same as those in Example 3.

[0182] 2. Results of determination of LDH release

[0183] Compared with the control group, the amount of LDH released in the supernatant of myocardial cells in the SI / RI model group was significantly increased. Compared with the SI / RI model group, the amount of LDH released in hypoxia-reoxygenation myocardial cells pretreated with HA-SH was significantly reduced, as shown in Table 16.

[0184] Table 16 LDH release in the supernatant of myocardial cells in each group was significantly reduced

[0185]

[0186] 3. Detection of ferroptosis.

[0187] The results showed that compared with the control, the proportion of cardiomyocyte ferroptosis in the SI / RI model group was significantly increased. Compared with the SI / RI model, the proportion of cardiomyocyte ferroptosis in the SI / RI model group after HA-SH pretreatment was significantly reduced, as shown in Table 17. This indicates that HA-SH can inhibit the ferroptosis of cardiomyocytes in the SI / RI model.

[0188] Table 17 Cell ferroptosis detection results in each group

[0189]

[0190] 4. Detection of inflammatory factors.

[0191] Compared with the control group, the mRNA transcription levels of inflammatory factors TNF-α, IL-6 and IL-1β in the SI / RI model group were significantly increased. Compared with the SI / RI model group, the mRNA transcription levels of inflammatory factors TNF-α, IL-6 and IL-1β in hypoxia-reoxygenation cardiomyocytes pretreated with HA-SH were significantly reduced. ELISA measured the levels of TNF-α, IL-6 and IL-1β in the supernatant of hypoxia-reoxygenation injured cardiomyocytes, which were significantly reduced, consistent with the trend of the results obtained by RT-qPCR, as shown in Table 18. These results indicate that HA-SH has an anti-inflammatory effect in cardiomyocyte SI / RI.

[0192] Table 18 Detection results of cellular inflammatory factors in each group

[0193]

[0194]

[0195] 5. Detection of ROS levels in cardiomyocytes.

[0196] The ROS level in the SI / RI model group was significantly higher than that in the control group, while the ROS level in the hypoxia-reoxygenation injured cardiomyocytes pretreated with HA-SH was significantly lower than that in the SI / RI model group. The results are shown in Table 19.

[0197] Table 19 Detection of reactive oxygen species (ROS) in each group of cells

[0198] concentration Control group SI / RI Model Group SI / RI+0.02% SI / RI+0.1% SI / RI+0.5% ROS generation 0.96 2.37 1.46 1.24 1.06

[0199] 6. Detection of oxidative stress.

[0200] The MDA content in the SI / RI model group was significantly higher than that in the control group, and the SOD activity was significantly lower. However, the MDA content in the cells pretreated with HA-SH was significantly lower than that in the SI / RI model group, and the SOD activity was significantly higher. The results are shown in Table 20.

[0201] Table 20 Detection results of MDA and SOD in each group of cells

[0202] concentration Control group SI / RI Model Group SI / RI+0.02% SI / RI+0.1% SI / RI+0.5% MDA 2.06 6.68 3.46 2.73 2.08 SOD 96.8 44.2 77.2 88.5 97.6

[0203] 7. Detection of Nrf2 and HO-1 mRNA levels.

[0204] Compared with the control group, the Nrf2 and HO-1 mRNA levels in the SI / RI model group were slightly increased. Compared with the SI / RI model group, the Nrf2 and HO-1 mRNA levels in the hypoxia-reoxygenation-injured cardiomyocytes pretreated with HA-SH were significantly increased, as shown in Table 21.

[0205] Table 21 Detection results of Nrf2 and HO-1 mRNA levels in each group of cells

[0206] concentration Control group SI / RI Model Group SI / RI+0.02% SI / RI+0.1% SI / RI+0.5% Nfr2 0.92 1.38 2.53 2.72 2.82 HO-1 0.98 1.24 2.48 2.67 2.76

[0207] Therefore, these findings suggest that different concentrations of 1000KD HA-SH can protect cardiomyocytes from oxidative stress injury in the cardiomyocyte SI / RI model.

[0208] Example 6. SA-HA (1500KD) at different concentrations can alleviate oxidative stress damage in the SI / RI model of cardiomyocytes.

[0209] 1. Cultivation of H9C2 cardiomyocytes, SI / RI cell model, treatment with different concentrations of HA-SH, collection of test samples, and detection method are the same as those in Example 3.

[0210] 2. Results of determination of LDH release

[0211] Compared with the control group, the amount of LDH released in the supernatant of myocardial cells in the SI / RI model group was significantly increased. Compared with the SI / RI model group, the amount of LDH released in hypoxia-reoxygenation myocardial cells after HA-SH pretreatment was significantly reduced, as shown in Table 22.

[0212] Table 22 LDH release in the supernatant of cardiomyocytes in each group was significantly reduced

[0213]

[0214] 3. Ferroptosis detection results

[0215] The results showed that compared with the control group, the proportion of cardiomyocyte ferroptosis in the SI / RI model group was significantly increased. Compared with the SI / RI model group, HA-SH pretreatment could significantly reduce the proportion of cell ferroptosis, as shown in Table 23. This indicates that HA-SH can inhibit the ferroptosis of cardiomyocytes in the SI / RI model.

[0216] Table 23 The proportion of cell ferroptosis in each group was significantly reduced

[0217]

[0218] 4. Detection of inflammatory factors.

[0219] Compared with the control group, the mRNA transcription levels of inflammatory factors TNF-α, IL-6 and IL-1β in the SI / RI model group were significantly increased. Compared with the SI / RI model group, the mRNA transcription levels of inflammatory factors TNF-α, IL-6 and IL-1β in hypoxia-reoxygenation cardiomyocytes pretreated with HA-SH were significantly reduced. ELISA measured the levels of TNF-α, IL-6 and IL-1β in the supernatant of hypoxia-reoxygenation injured cardiomyocytes, which were significantly reduced, consistent with the trend of the results obtained by RT-qPCR, as shown in Table 24. These results indicate that HA-SH has an anti-inflammatory effect in SI / RI cardiomyocytes.

[0220] Table 24 Detection results of cellular inflammatory factors in each group

[0221]

[0222] 5. Detection of ROS levels in cardiomyocytes.

[0223] The ROS level in the SI / RI model group was significantly higher than that in the control group, while the ROS level in the hypoxia-reoxygenation injured cardiomyocytes pretreated with HA-SH was significantly lower than that in the SI / RI model group. The results are shown in Table 25.

[0224] Table 25 Detection results of reactive oxygen species (ROS) in each group of cells

[0225]

[0226] 6. Oxidative stress detection.

[0227] The MDA content in the SI / RI model group was significantly higher than that in the control group, and the SOD activity was significantly lower. However, the MDA content in the cells pretreated with HA-SH was significantly lower than that in the SI / RI model group, and the SOD activity was significantly higher. The results are shown in Table 26.

[0228] Table 26 Detection results of MDA and SOD in each group of cells

[0229]

[0230] 7. Detection of Nrf2 and HO-1 mRNA levels.

[0231] Compared with the control group, the Nrf2 and HO-1 mRNA levels in the SI / RI model group were slightly increased. Compared with the SI / RI model group, the Nrf2 and HO-1 mRNA levels in the hypoxia-reoxygenation-injured cardiomyocytes pretreated with HA-SH were significantly increased, as shown in Table 27.

[0232] Table 27 Detection of Nrf2 and HO-1 mRNA levels in each group

[0233]

[0234] Therefore, these findings suggest that different concentrations of 1500KD HA-SH can protect cardiomyocytes from oxidative stress injury in the cardiomyocyte SI / RI model.

[0235] Example 7. SA-HA (2500 KD) at different concentrations can alleviate oxidative stress damage in the SI / RI model of cardiomyocytes.

[0236] 1. Cultivation of H9C2 cardiomyocytes, SI / RI cell model, treatment with different concentrations of HA-SH, collection of test samples, and detection method are the same as those in Example 3.

[0237] 2. Determination of LDH release.

[0238] Compared with the control group, the amount of LDH released in the supernatant of myocardial cells in the SI / RI model group was significantly increased. Compared with the SI / RI model group, the amount of LDH released in hypoxia-reoxygenation myocardial cells after HA-SH pretreatment was significantly reduced, as shown in Table 28.

[0239] Table 28 LDH release in the supernatant of myocardial cells in each group was significantly reduced

[0240]

[0241] 3. Detection of ferroptosis.

[0242] The results showed that compared with the control group, the proportion of cardiomyocyte ferroptosis in the SI / RI model group was significantly increased. Compared with the SI / RI model group, HA-SH pretreatment could significantly reduce the proportion of cell ferroptosis, as shown in Table 29. This indicates that HA-SH can inhibit the ferroptosis of cardiomyocytes in the SI / RI model.

[0243] Table 29 The proportion of cell ferroptosis in each group was significantly reduced

[0244]

[0245] 4. Detection of inflammatory factors.

[0246] Compared with the control group, the mRNA transcription levels of inflammatory factors TNF-α, IL-6 and IL-1β in the SI / RI model group were significantly increased. Compared with the SI / RI model group, the mRNA transcription levels of inflammatory factors TNF-α, IL-6 and IL-1β in hypoxia-reoxygenation cardiomyocytes pretreated with HA-SH were significantly reduced. ELISA assay showed that the levels of TNF-α, IL-6 and IL-1β in the supernatant of hypoxia-reoxygenation injured cardiomyocytes were significantly reduced, which was consistent with the trend of the results obtained by RT-qPCR, as shown in Table 30. These results indicate that HA-SH has an anti-inflammatory effect in cardiomyocyte SI / RI.

[0247] Table 30 Detection results of inflammatory factors in each group

[0248]

[0249] 5. Detection of ROS levels in cardiomyocytes.

[0250] The ROS level in the SI / RI model group was significantly higher than that in the control group, while the ROS level in the hypoxia-reoxygenation injured cardiomyocytes pretreated with HA-SH was significantly lower than that in the SI / RI model group. The results are shown in Table 31.

[0251] Table 31 Detection results of reactive oxygen species (ROS) in each group of cells

[0252] concentration Control group SI / RI Model Group SI / RI+0.02% SI / RI+0.1% SI / RI+0.5% ROS generation 0.96 2.37 1.38 1.12 1.00

[0253] 6. Detection of oxidative stress.

[0254] The MDA content in the SI / RI model group was significantly higher than that in the control group, and the SOD activity was significantly lower. However, the MDA content in the cells pretreated with HA-SH was significantly lower than that in the SI / RI model group, and the SOD activity was significantly higher. The results are shown in Table 32.

[0255] Table 32 Detection results of MDA and SOD in each group of cells

[0256] concentration Control group SI / RI Model Group SI / RI+0.02% SI / RI+0.1% SI / RI+0.5% MDA 2.06 6.68 3.44 2.52 2.12 SOD 96.8 44.2 77.6 89.8 97.2

[0257] 7. Detection of Nrf2 and HO-1 mRNA levels.

[0258] Compared with the control group, the Nrf2 and HO-1 mRNA levels in the SI / RI model group were slightly increased. Compared with the SI / RI model group, the Nrf2 and HO-1 mRNA levels in the hypoxia-reoxygenation-injured cardiomyocytes pretreated with HA-SH were significantly increased, as shown in Table 33.

[0259] Table 33 Detection results of Nrf2 and HO-1 mRNA levels in each group

[0260] concentration Control group SI / RI Model Group SI / RI+0.02% SI / RI+0.1% SI / RI+0.5% Nfr2 0.92 1.38 2.56 2.75 2.88 HO-1 0.98 1.24 2.42 2.66 2.74

[0261] Therefore, these findings suggest that different concentrations of 2500KD HA-SH can protect cardiomyocytes from oxidative stress injury in the cardiomyocyte SI / RI model.

[0262] Example 8. The therapeutic effects of different concentrations of HA-SH on the rat myocardial ischemia-reperfusion injury model.

[0263] 1. Establish a rat myocardial ischemia-reperfusion injury model.

[0264] 1) SPF Wistar rats were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd., female, 49-55 days old, weighing 150-170 g.

[0265] 2) Preparation of rat cardiac ischemia-reperfusion model (MI / RI).

[0266] (1) Rats were fasted for 12 h before surgery and anesthetized with 10% chloral hydrate (150 mg / kg) by intraperitoneal injection.

[0267] (2) Artificial respiration was performed by oral intubation and connected to a ventilator; an electrocardiogram was recorded throughout the process.

[0268] (3) Routinely disinfect the left anterior chest area of ​​the rat, make an incision between the 3rd and 4th intercostal space, expose the heart, and cut open the pericardium.

[0269] (4) The left anterior descending coronary artery is ligated at the upper 1 / 3 of the left anterior descending branch. The anterior cardiac wall at the distal end of the ligation turns purple-red. The successful operation is marked by a significant ST segment elevation of 0.1 mV on electrocardiogram monitoring.

[0270] (5) 30 minutes after ligation, the chest was opened again to open the ligature knot, and reperfusion was performed for 4 hours. The electrocardiogram was recorded throughout the process.

[0271] (6) After the reperfusion time is over, 2 mL of blood is collected from the right ventricle, and the supernatant is collected and marked for use in LDH, CK-MB and other tests.

[0272] (7) The heart was quickly cut out and placed in pre-cooled PBS. The myocardial tissue of the left ventricle was cut out and frozen in liquid nitrogen for later use. The myocardial infarction area was measured by triphenyltetrazolium chloride and Evans blue. The area of ​​the myocardial ischemic drug protection zone, the area of ​​the myocardial infarction zone, the area of ​​the myocardial ischemic risk zone, and the area of ​​the entire heart were calculated respectively. The percentage of the myocardial ischemic risk zone, the percentage of the infarction zone within the myocardial ischemic risk zone, and the percentage of the myocardial ischemic drug protection zone were calculated.

[0273] (8) The remaining left ventricle was added with tissue lysis solution containing protease inhibitors, homogenized, and ultrasonically disrupted to prepare a 10% tissue supernatant, which was frozen at -80°C for later use. It was used to detect MDA, SOD, ROS, Nrf2, HO-1 mRNA levels, ferroptosis, etc.

[0274] 2. Drug use: According to body weight, HA-SH is prepared into solutions at different concentrations and administered orally at 2 ml / kg 24 hours in advance.

[0275] 3. Arrhythmia data processing and analysis.

[0276] (1) The electrocardiogram recorded the time of coronary artery ligation ischemia, reperfusion, arrhythmia and normal rhythm. The ST segment elevation (potential difference ≥ 0.1 mV) of the rat electrocardiogram was used as a marker for the successful establishment of MI / RI model.

[0277] (2) Calculation

[0278] Arrhythmia latency time (s) = arrhythmia occurrence time point - reperfusion time point;

[0279] Arrhythmia duration (s) = time point when heart rhythm returns to normal - time point when arrhythmia occurs.

[0280] 4. Detection of LDH and CK-MB.

[0281] 1) Determination of serum LDH in rats.

[0282] (1) Sample dilution: Thaw the serum on ice and dilute the serum sample with physiological saline at a volume ratio of 1:9 and keep on ice until ready for use.

[0283] (2) Add buffer and coenzyme I in sequence, mix well, and incubate at 37°C for 15 min.

[0284] (3) Add the substrate 2,4-dinitrophenylhydrazine (2,4-C6H6N4O4), mix well, and incubate at 37°C for 15 min.

[0285] (4) Add stop solution (0.4 mol / L NaOH solution), mix well, and leave at room temperature for 5 min. Read the result at 450 nm using a microplate reader.

[0286] 2) Determination of rat serum CK-MB.

[0287] (1) The blood to be tested is diluted 6 times with diluent, added to the ELISA plate, covered with a sealing film, incubated at 37°C for 1 hour, and then the sealing film is removed and the liquid is discarded.

[0288] (2) Add biotinylated antibody and incubate at 37°C for 1 h.

[0289] (3) Add detergent and let stand for 1 min. Shake dry, pat dry, and repeat 3 times.

[0290] (4) Add enzyme conjugate working solution and incubate at 37°C for 30 min.

[0291] (5) Add chromogenic substrate and incubate at 37°C in the dark for 15 min.

[0292] (6) Add stop solution.

[0293] (7) Immediately measure the OD value at 450 nm using an ELISA reader and calculate the CK-MB content.

[0294] 5. Detection of MDA, SOD, ROS, Nrf2, HO-1 mRNA levels, and ferroptosis.

[0295] The detection of MDA, SOD and ROS in cardiac tissue cells, mRNA extraction and RT-PCR detection of Nrf2 and HO-1 mRNA levels were the same as in Example 3.

[0296] 6. The therapeutic effect of HA-SH on rat MI / RI model.

[0297] 1) Effect on arrhythmia in rat MI / RI model: Based on the real-time recorded electrocardiogram of rats, the arrhythmia latency and duration of arrhythmia in each group of rats were analyzed. Compared with the MI / RI model group, the MI / RI model treated with different concentrations of HA-SH can significantly delay the latency of arrhythmia in rats with MI / RI injury, and has a certain improvement effect on arrhythmia after MI / RI in rats. In the monitoring of the duration of arrhythmia, different concentrations of HA-SH also reduced the duration of arrhythmia in rats with MI / RI injury, and the results are shown in Table 34.

[0298] Table 34 Effects on arrhythmia in rat MI / RI model

[0299]

[0300] 2) Reduce the levels of serum LDH and CK-MB.

[0301] Compared with the control group, the serum LDH and CK-MB levels of rats with ischemic injury in the MI / RI model were significantly increased, and the serum LDH and CK-MB levels of rats treated with HA-SH were reduced, as shown in Table 35. This indicates that it has a certain protective effect on myocardial injury.

[0302] Table 35 Effects on serum LDH and CK-MB levels in rats

[0303]

[0304] 3) Protective effect on myocardial damage.

[0305] The area of ​​myocardial infarction (IA), the area of ​​myocardial ischemic drug protection zone (PA), the area of ​​myocardial ischemic risk zone (AAR) and the total area of ​​the heart were calculated for each group. The results showed that different concentrations of HA-SH increased the proportion of AAR area to cross-sectional area, increased the proportion of PA area to cross-sectional area, and significantly reduced the IA / AAR ratio. This shows that HA-SH can reduce the area of ​​myocardial infarction, increase the proportion of drug protection zone area, and has a myocardial protective effect on MI / RI rats. The results are shown in Table 36.

[0306] Table 36 Effect on myocardial infarction area

[0307]

[0308] 4) Effect on the ability of rats to resist oxidative stress.

[0309] It can improve the ability of MI / RI rats to resist oxidative stress, significantly increase the SOD activity of MI / RI rats, and reduce the content of MDA and ROS. It can significantly improve the degree of myocardial cell ferroptosis in MI / RI rats and reduce the cell ferroptosis rate. The results are shown in Table 37.

[0310] Table 37 Improves the ability of MI / RI rats to resist oxidative stress

[0311]

[0312] 5) Effects on Nrf2 and HO-1 mRNA levels in rat myocardial tissue.

[0313] After HA-SH treatment, the levels of Nrf2 and HO-1 mRNA in rat myocardial tissue increased, suggesting that it exerts an anti-MI / RI effect by regulating oxidative stress and cell apoptosis through activating the Nrf2 / HO-1 signaling pathway. The results are shown in Table 38.

[0314] Table 38 Effect of Nrf2 and HO-1 mRNA levels in rat myocardial tissue

[0315] concentration Control group MI / RI model group MI / RI+0.02% MI / RI+0.1% MI / RI+0.5% Nfr2 0.94 1.34 2.78 3.05 3.28 HO-1 0.98 1.56 3.12 3.42 3.47

[0316] The above results show that HA-SH can reduce myocardial ischemia-reperfusion injury by inhibiting cell apoptosis, resisting oxidative stress, and reducing inflammatory response, and improve myocardial ischemia-reperfusion injury by activating the Nrf2 / HO-1 signaling pathway.

[0317] Example 9. The therapeutic effects of HA-SH with different molecular weights on rat myocardial ischemia-reperfusion injury model.

[0318] 1. Construct a rat myocardial ischemia-reperfusion injury model, the same as in Example 8.

[0319] 2. Drug use: According to body weight, HA-SH with different molecular weights is prepared into solutions according to concentrations and administered intragastrically at 2 ml / kg. This should be done 24 hours in advance.

[0320] 3. Arrhythmia data processing and analysis: same as Example 8.

[0321] 4. Detection of LDH, CK-MB, etc.: Same as Example 8.

[0322] 5. MDA, SOD, ROS, Nrf2, HO-1 mRNA levels, and ferroptosis detection: Same as Example 8.

[0323] 6. Therapeutic effect on rat MI / RI model.

[0324] 1) Effect on arrhythmia in rat MI / RI model: Compared with the model group, HA-SH with different molecular weights can significantly delay the incubation period of arrhythmia in rats with MI / R injury, and have a certain improvement effect on arrhythmia in rats after MI / RI. In the monitoring of the duration of arrhythmia, HA-SH with different molecular weights also reduced the duration of arrhythmia in rats with MI / R injury, as shown in Table 39.

[0325] Table 39 Effects of different molecular weight HA-SH on arrhythmias in rat MI / RI model

[0326]

[0327]

[0328] 2) Reduce serum LDH and CK-MB levels.

[0329] Compared with the control group, the serum LDH and CK-MB levels of rats with ischemic injury in the MI / RI model were significantly increased, and the serum LDH and CK-MB levels of rats treated with HA-SH were reduced, indicating that it has a certain protective effect on myocardial injury. The results are shown in Table 40.

[0330] Table 40 Effects of different molecular weight HA-SH on the levels of LDH and CK-MB in rat serum

[0331]

[0332] 3) Protective effect on myocardial damage.

[0333] HA-SH with different molecular weights increased the ratio of AAR area to cross-sectional area, increased the ratio of PA area to cross-sectional area, and significantly reduced the ratio of IA / AAR. It reduced the area of ​​myocardial infarction, increased the ratio of drug protection area, and had a myocardial protective effect on MI / RI rats. The results are shown in Table 41.

[0334] Table 41 Effects of different molecular weight HA-SH on myocardial infarction area in rats

[0335]

[0336]

[0337] 4) Effect on the ability of rats to resist oxidative stress.

[0338] It can improve the ability of MI / RI rats to resist oxidative stress, significantly increase the SOD activity of MI / RI rats, and reduce the content of MDA and ROS. It can significantly improve the degree of myocardial cell ferroptosis in MI / RI rats and reduce the cell ferroptosis rate. The results are shown in Table 42.

[0339] Table 42 Different molecular weight HA-SH can improve the ability of MI / RI rats to resist oxidative stress

[0340]

[0341]

[0342] 5) Effects on Nrf2 and HO-1 mRNA levels in rat myocardial tissue.

[0343] After HA-SH treatment, the levels of Nrf2 and HO-1 mRNA in rat myocardial tissue increased, suggesting that it exerts an anti-MI / RI effect by regulating oxidative stress and cell apoptosis through activating the Nrf2 / HO-1 signaling pathway. The results are shown in Table 43.

[0344] Table 43 Different molecular weight HA-SH can increase the level of Nrf2 and HO-1 mRNA in myocardial tissue of MI / RI rats

[0345]

[0346] Although preferred embodiments of the present invention have been described, additional changes and modifications may be made to these embodiments by those skilled in the art once the basic inventive concepts are known. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention. Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. Use of thiolated hyaluronic acid or its salt in the preparation of a medicament for preventing or treating myocardial ischemia-reperfusion injury.

2. The use according to claim 1, characterized in that: Myocardial ischemia-reperfusion injury includes oxidative stress response, cardiomyocyte apoptosis, cardiomyocyte ferroptosis and inflammatory response.

3. The use according to claim 1, characterized in that: The degree of substitution of the thiol group of the thiolated hyaluronic acid or its salt is 10%-40%, and the average degree of substitution is 24.4%.

4. The use according to claim 3, characterized in that: The antioxidant capacity of 5 mg / ml of thiolated hyaluronic acid or its salt is 57.3~78.2%, and the average antioxidant capacity is 67.8%.

5. The use according to claim 1, characterized in that: Thiolated hyaluronic acid or its salts regulate Nrf2 signaling pathway and HO-1 signaling pathway.

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