Application of iron sulfide nanozymes in the preparation of drugs for the treatment of acute kidney injury-related diseases

Abstract

The present invention discloses the use of iron sulfide nanozymes (GFeSNs) in the preparation of drugs for treating diseases related to acute kidney injury. In vitro experimental results show that GFeSNs can effectively scavenge free radicals and exhibit GFeSN concentration dependence. In vivo experiments using glycerol injection to establish an acute kidney injury model show that GFeSNs can effectively alleviate acute kidney injury and protect renal function, with broad application prospects. Depending on clinical medication needs, GFeSNs can be used as a single active ingredient in a drug or combined with other drugs to form a compound preparation. GFeSNs have the advantages of being safe, non-toxic, effective, economical, and practical.

Description

Technical Field

[0001] The present invention relates to the application of iron sulfide nanozyme in preparing medicine for treating diseases related to acute kidney injury, and belongs to the field of pharmaceutical industry. Background Art

[0002] Inflammation is closely related to oxidative stress. Reactive oxygen species (ROS) in excessive inflammatory responses can aggravate local tissue damage and lead to diseases such as acute kidney injury (AKI). Broad-spectrum antioxidants such as N-acetyl-L-cysteine ​​and acetyl-L-carnitine are commonly used clinically to remove ROS and achieve the purpose of treating AKI. In addition, fluid rehydration and renal dialysis are also commonly used clinical methods for treating AKI. However, these treatments have the disadvantages of low bioavailability, high toxicity and low efficacy, and patients suffer greater pain, which limits their clinical application in the treatment of ROS-related diseases.

[0003] Studies have shown that iron-based nanomaterials have been widely used in biomedicine due to their high biocompatibility and biological functions. Iron sulfide nanozymes (GFeSNs) have the activity of scavenging a variety of free radicals, can continuously and stably release polysulfide hydrogen, and exhibit strong anti-inflammatory and antioxidant properties. The synthesized iron sulfide nanozymes have high free radical scavenging activity and can continuously and stably release polysulfide hydrogen. This synergistic effect makes iron sulfide nanozymes have a broad spectrum of ROS scavenging activity, including OH, O2 .- and H2O2. Summary of the Invention

[0004] Purpose of the invention: The first purpose of the present invention is to provide the use of iron sulfide nanozymes (GFeSNs) in the preparation of drugs for treating diseases related to acute kidney injury; the second purpose of the present invention is to provide the use of GFeSNs in the preparation of drugs for clearing reactive oxygen species related diseases.

[0005] Technical solution: In order to solve the above problems, the present invention provides the use of GFeSNs in the preparation of drugs for treating diseases related to acute kidney injury and the use of GFeSNs in the preparation of drugs for diseases related to scavenging reactive oxygen species.

[0006] Wherein, the drug is a single-component preparation or a compound preparation.

[0007] The drug is in the form of tablets, capsules, granules, injections or oral liquid preparations.

[0008] The preparation method of GFeSNs of the present invention comprises the following steps:

[0009] Ferric chloride was dissolved in ethylene glycol (EG), ultrasonically stirred, sodium acetate and glutathione (GSH) were added, ultrasonically stirred, heated for reaction, washed, and freeze-dried to obtain GFeSNs.

[0010] Wherein, the molar ratio of the ferric chloride, ethylene glycol, sodium acetate and glutathione is 1-10:20-40:20-40:1-10.

[0011] Preferably, the molar ratio of ferric chloride, ethylene glycol, sodium acetate and glutathione is 4-8:30-40:20-30:3-6.

[0012] The ultrasonic stirring time is 20-30 min, and the continued ultrasonic stirring time is 20-30 min.

[0013] The temperature of the heating reaction is 180-220° C., and the reaction time is 10-24 hours.

[0014] Wherein, the washing is performed by washing with anhydrous ethanol and ultrapure water for more than three times respectively.

[0015] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0016] The experiments of the present invention confirmed that GFeSNs can effectively alleviate acute kidney injury and protect renal function. Its mechanism of action is related to GFeSNs alleviating oxidative stress, and it has broad application prospects.

[0017] According to clinical medication needs, GFeSNs can be used as a single active ingredient to make drugs, or combined with other drugs to make compound preparations. GFeSNs have the advantages of being safe, non-toxic, effective, economical, and practical. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the SEM image of GFeSNs prepared in Example 1;

[0019] Figure 2 The figure shows the ability of GFeSNs at different concentrations to scavenge ROS.

[0020] Figure 3 The fluorescence intensity diagram of hydrogen sulfide released by GFeSNs with different concentrations;

[0021] Figure 4 GFeSNs protect NRK-52E cells from H2O2 damage;

[0022] Figure 5 is the cytotoxicity graph of GFeSNs on NRK-52E cells;

[0023] Figure 6 This is a graph to detect the level of hydrogen polysulfide in NRK-52E cells;

[0024] Figure 7This figure shows the in vivo therapeutic effect study of GFeSNs on AKI mice. DETAILED DESCRIPTION

[0025] The technical solution of the present invention is further described in detail below with reference to the accompanying drawings.

[0026] Example 1 Preparation of GFeSNs

[0027] FeCl₃ (5 mmol) was dissolved in EG (40 mL) and stirred with ultrasonic magnetic stirring for 15 minutes. NaAc·3H₂O (40 mmol) and GSH (4 mmol) were then slowly added to the solution, with continuous ultrasonic stirring until the solution became transparent. The transparent solution was then transferred to a polytetrafluoroethylene reactor and reacted at 200°C for 12 hours. The product was then washed three times with anhydrous ethanol and then three times with ultrapure water, and then lyophilized to obtain GFeSNs, which were stored at 4°C for subsequent use.

[0028] Example 2 Preparation of GFeSNs

[0029] FeCl₃ (3 mmol) was dissolved in EG (30 mL) and stirred with ultrasonic magnetic stirring for 15 minutes. NaAc·3H₂O (30 mmol) and GSH (5 mmol) were then slowly added to the solution, with continuous ultrasonic stirring until the solution became transparent. The transparent solution was then transferred to a polytetrafluoroethylene reactor and reacted at 200°C for 10 hours. The product was then washed three times with anhydrous ethanol and then three times with ultrapure water, and then lyophilized to obtain GFeSNs, which were stored at 4°C for subsequent use.

[0030] Example 3 Preparation of GFeSNs

[0031] FeCl₃ (8 mmol) was dissolved in EG (35 mL) and stirred with ultrasonic magnetic stirring for 15 minutes. NaAc·3H₂O (25 mmol) and GSH (6 mmol) were then slowly added to the solution, with continuous ultrasonic stirring until the solution became transparent. The transparent solution was then transferred to a polytetrafluoroethylene reactor and reacted at 200°C for 18 hours. The product was then washed three times with anhydrous ethanol and then three times with ultrapure water, and then lyophilized to obtain GFeSNs, which were stored at 4°C for subsequent use.

[0032] Example 4 Preparation of GFeSNs

[0033] FeCl₃ (10 mmol) was dissolved in EG (35 mL) and stirred with ultrasonic magnetic stirring for 15 minutes. NaAc·3H₂O (35 mmol) and GSH (8 mmol) were then slowly added to the solution, with continuous ultrasonic stirring until the solution became transparent. The transparent solution was then transferred to a polytetrafluoroethylene reactor and reacted at 200°C for 24 hours. The product was then washed three times with anhydrous ethanol and then three times with ultrapure water, and then lyophilized to obtain GFeSNs, which were stored at 4°C for subsequent use.

[0034] The GFeSNs prepared in Example 1 were analyzed by scanning electron microscopy. Figure 1 shown. Figure 1 The SEM image of GFeSNs prepared in Example 1 (scale bar: 100 nm) is shown. Figure 1 It can be seen that GFeSNs exhibit an irregular lamellar structure with a size of about 200 nm.

[0035] Example 5 Detection of the Reactive Oxygen Scavenging Ability of GFeSNs at Different Concentrations

[0036] The ABTS detection kit, ·OH detection kit, H2O2 detection kit, and 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) were used to detect the ability of GFeSNs (2.5-80 μg / mL) to scavenge reactive oxygen species.

[0037] The GFeSNs prepared in Example 1 were prepared into GFeSNs solutions with concentrations of 2.5 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL, and 80 μg / mL, respectively, using double distilled water or anhydrous ethanol.

[0038] The ABTS, ·OH, and H2O2 scavenging abilities were tested according to the manufacturer's instructions for the ABTS, ·OH, and H2O2 test kits. 0.2 mM DPPH was mixed with GFeSNs solutions of varying concentrations, and after fading in a water bath, the absorbance was measured at 517 nm using a microplate reader. The results are shown in Figure 2. Figure 2 As shown, Figure 2 Figure 2 is a graph showing the ability of GFeSNs at different concentrations to scavenge ROS, where (a) is the efficiency of GFeSNs at different concentrations to scavenge ABTS free radicals; (b) is the efficiency of GFeSNs at different concentrations to scavenge DPPH; (c) is the efficiency of GFeSNs at different concentrations to scavenge ·OH free radicals; (d) is the efficiency of GFeSNs at different concentrations to scavenge H2O2. Figure 2 It can be seen that the higher the concentration of GFeSNs, the better the free radical scavenging ability of GFeSNs, and it shows concentration dependence.

[0039] Example 6 Evaluation of the Effect of GFeSNs on Alleviating Cellular H2O2 Damage

[0040] The sulfane sulfur detection reagent SSP4 was mixed with GFeSNs solutions of different concentrations (GFeSNs prepared in Example 1 were prepared with double distilled water to prepare GFeSNs solutions with concentrations of 2.5 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL, and 80 μg / mL, respectively) at a ratio of 1:10. After reacting in the dark for 30 minutes, the supernatant was aspirated and a black light-proof ELISA plate was used with an excitation wavelength of 470 nm and an emission wavelength of 515 nm. The fluorescence intensity was scanned and zeroed with double distilled water. The results are shown in FIG. Figure 3 shown. Figure 3 The fluorescence intensity diagram of polysulfide released by GFeSNs at different concentrations is shown in Figure 2. Figure 3 It can be seen that the higher the concentration of GFeSNs, the more polysulfide is generated, showing a concentration-dependent manner.

[0041] NRK-52E cells were cultured at 1×10 4 The cells were plated in a 96-well plate at a density of 100 μg / mL and cultured in a 37°C, 5% CO2 incubator for 24 hours. The above-mentioned GFeSNs solutions of different concentrations (0 μg / mL, 2.5 μg / mL, 5 μg / mL, and 10 μg / mL) were added respectively. After incubation in the incubator for 1 hour, 0.3 mM H2O2 was added. The cells were cultured in a 37°C, 5% CO2 incubator for another 24 hours. The cell viability was detected by MTT. The results are shown in Table 1. Figure 4 shown. Figure 4 GFeSNs protect NRK-52E cells from H2O2 damage. Figure 4 It can be seen that GFeSNs can alleviate cell damage caused by H2O2, and the cell viability can be significantly increased to more than 80% when the GFeSNs concentration is only 10 μg / mL.

[0042] NRK-52E cells were cultured at 1×10 4 The cells were plated in a 96-well plate at a density of 100 μg / mL and cultured in a 37°C, 5% CO2 incubator for 24 hours. After that, the above-mentioned GFeSNs solutions of different concentrations (0 μg / mL, 2.5 μg / mL, 5 μg / mL, 10 μg / mL, and 20 μg / mL) were added. After further culture for 24 hours, the cell viability was detected by MTT. The results are shown in the figure. Figure 5 shown. Figure 5 The cytotoxicity of GFeSNs to NRK-52E cells is shown in Figure 2. Figure 5 It can be seen that GFeSNs have no toxicity to NRK-52E cells at a high concentration of 20 μg / mL and can promote cell growth.

[0043] NRK-52E cells were cultured at 2×10 5The cells were plated in a 24-well plate at a density of 100 μg / mL and cultured in a 37°C, 5% CO2 incubator for 24 hours. After that, different concentrations of GFeSNs (0 μg / mL, 5 μg / mL, and 10 μg / mL) were added. The cells were cultured in a 37°C, 5% CO2 incubator for 6 hours. After that, SSP4 and DAPI were added for staining, and the cells were imaged using a fluorescence microscope. The results are shown in Figure 2. Figure 6 shown. Figure 6 To detect the level of polysulfide hydrogen in NRK-52E cells (scale bar: 20 μm), Figure 6 It can be seen that the polysulfides released by GFeSNs can enter the cells. The higher the concentration of GFeSNs, the more polysulfides enter the cells and the stronger the fluorescence.

[0044] Example 7: In vivo therapeutic effect study of GFeSNs on AKI mice

[0045] BALB / C mice aged 6-8 weeks were adaptively cultured for 1-2 days and divided into a normal control group (PBS group), an AKI group, and an AKI administration group (AKI+GFeSNs group), with 6-10 mice in each group. Water was cut off for 15 hours before the experiment, and the mice were intramuscularly injected with 8mL / kg 50% glycerol to establish an acute kidney injury model. 2 hours later, 0.5mg / kg GFeSNs solution was injected into the tail vein. PBS was injected into the tail vein of the PBS group and the AKI group. All mice ate and drank water normally. One day later, blood was collected from the mouse eyeballs to separate the serum to detect blood creatinine and urea nitrogen levels. A portion of the kidney tissue was fixed in 4% paraformaldehyde and stained with H&E. At the same time, some mice were injected with up to 1mg / kg of GFeSNs into the tail vein. The mice were killed on the 7th and 30th days, and the main organs such as the heart, liver, spleen, lungs and kidneys were taken for H&E staining. The experimental results are shown as follows: Figure 7 shown. Figure 7 Figure 2 is a study on the in vivo therapeutic effect of GFeSNs on AKI mice, where (a) is the blood creatinine (CRE) level of each group of mice; (b) is the blood urea nitrogen (BUN) level of each group of mice; (c) is the H&E staining of the kidney tissue of each group of mice (scale bar: 50μm); (d) is the H&E staining of the main organs of each group of mice (scale bar: 50μm). Figure 7 As can be seen from (a) and (b), the blood creatinine and urea nitrogen levels in the glycerol-induced AKI group increased significantly, and renal function worsened, while the AKI administration group effectively reduced the blood creatinine and urea nitrogen levels in the AKI group. Figure 7 (c) H&E staining results showed that AKI mice had a large number of apoptosis in renal cortical cells and loss of renal tubular structure, while the renal damage of mice in the AKI treatment group was significantly reduced, further confirming that GFeSNs have a protective effect on renal function in acute kidney injury. Figure 7(d) H&E staining results showed that there was no significant change in the heart, liver, spleen, lung, kidney and other parts of the body compared with the normal group at different time points, indicating that GFeSNs have high biosafety.

Claims

1. Use of GFeSNs in preparing a drug for treating acute kidney injury, wherein the preparation method of GFeSNs comprises the following steps: Ferric chloride is dissolved in ethylene glycol, ultrasonically stirred, sodium acetate and glutathione are added, ultrasonic stirring is continued, heating reaction is carried out, washing, and freeze-drying are performed to obtain GFeSNs; the molar ratio of the ferric chloride, ethylene glycol, sodium acetate and glutathione is 1-10:20-40:20-40:1-10.

2. The use according to claim 1, characterized in that The molar ratio of the ferric chloride, ethylene glycol, sodium acetate and glutathione is 4-8:30-40:20-30:3-6.

3. The use according to claim 1, characterized in that The medicine is a single-component preparation or a compound preparation.

4. The use according to claim 1, characterized in that The dosage form of the medicine is tablet, capsule, granule, injection or oral liquid preparation.

5. The use according to claim 1, characterized in that The ultrasonic stirring time is 20-30 min, and the continued ultrasonic stirring time is 20-30 min.

6. The use according to claim 1, characterized in that The temperature of the heating reaction is 180-220° C., and the reaction time is 10-24 h.

7. The use according to claim 1, characterized in that The washing is performed by washing with anhydrous ethanol and ultrapure water for more than three times.

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