Application of ferrous ions in the preparation of products for treating bacterial infections

Abstract

The present invention discloses the use of ferrous ions in the preparation of products for treating bacterial infections, belonging to the field of medical technology. Ferrous ions were used in antibacterial activity tests on Staphylococcus aureus, demonstrating a strong bactericidal effect. When sterilizing Staphylococcus aureus, the survival rate was less than 0.001%, and when sterilizing methicillin-resistant Staphylococcus aureus (MRSA), the survival rate was less than 0.01%. The use of ferrous ion-containing hydrogels in the treatment of keratitis and skin wound infections significantly reduced the risk of MRSA lung infection and effectively and timely prevented the reproduction and spread of microorganisms in the body. This indicates that drugs containing ferrous compounds can be used to treat Staphylococcus aureus infections, including MRSA.

Description

[0001] This invention patent application is a divisional application of the invention patent application with application date of April 25, 2022, application number CN202210438990.1, and the invention name is "Application of ferrous ions in the preparation of products for the treatment of bacterial infections." Technical Field

[0002] The present invention relates to the field of medical technology, and in particular to the application of ferrous ions in preparing products for treating bacterial infections. Background Art

[0003] Keratitis is a common ophthalmic disease and a leading cause of blindness in my country. Bacterial, fungal, and viral infections are among the most direct contributing factors to keratitis. Damage to the corneal epithelium can easily lead to infectious inflammation, particularly with improper contact lens use and contact with contaminated medications or water. It is noteworthy that, according to current authoritative clinical surveys, bacterial keratitis caused by Staphylococcus aureus and Pseudomonas aeruginosa remains the most common cause of clinical keratitis, with an increasing incidence rate year by year. Therefore, the prevention and treatment of this type of keratitis warrants attention.

[0004] Staphylococcus aureus is a common cause of corneal and epidermal infections. Currently, antibiotic therapy remains the most common treatment for S. aureus infections. However, the misuse and abuse of antibiotics has led to increased antibiotic resistance in humans, and the emergence of multidrug-resistant bacteria (such as methicillin-resistant Staphylococcus aureus, MRSA) poses a serious threat to human health. Therefore, new antimicrobial therapies are urgently needed to address this crisis.

[0005] The current treatment strategies for keratitis and epidermal tissue infections caused by Staphylococcus aureus are mainly external antibiotic eye drops, medical antibiotic hydrogels, and some necessary topical steroid ointments, such as ofloxacin eye drops, vancomycin hydrogel, erythromycin hydrogel, chlortetracycline ointment, etc. Existing studies have shown that although antibiotics have high efficiency and low-dose bactericidal efficacy, they cannot completely eliminate bacteria. However, the large-scale use of these antibiotic-containing eye drops and ointments can increase the risk of bacterial resistance and cause the human body to develop drug dependence, forcing the increase of drug dosage, resulting in a vicious cycle. When the human body is repeatedly infected with bacteria, the human body's resistance to these antibiotic ointments will make the antibiotics ineffective for the human body. Moreover, this type of treatment cannot effectively prevent the migration and colonization of bacteria in the body, and is prone to causing lesions in other tissues and organs. Therefore, there is an urgent need to find new antibacterial materials to treat Staphylococcus aureus infections, particularly methicillin-resistant Staphylococcus aureus (MRSA) infections. Summary of the Invention

[0006] In order to solve the above technical problems, the present invention provides the use of ferrous ions in the preparation of products for treating bacterial infections.

[0007] Application of ferrous ions in the preparation of products for treating bacterial infections.

[0008] Preferably, the bacterial infection is an eyeball infection or an epidermal infection.

[0009] Preferably, the bacterial infection is caused by Staphylococcus aureus.

[0010] Preferably, the ferrous ions are derived from any one of ferrous sulfate, ferrous chloride, ferrous gluconate and ferrous lactate.

[0011] Preferably, a hydrogel prepared with ferrous compounds, ascorbic acid and compound A as raw materials is used to treat the bacterial infection; wherein the compound A is selected from hyaluronic acid or sodium alginate.

[0012] Preferably, the concentration of the ferrous ions is ≥1 μM.

[0013] Preferably, the concentration of the ferrous ions is ≥16 μM.

[0014] More preferably, the concentration of the ferrous ions is 16-100 μM.

[0015] Preferably, the method for preparing the hydrogel comprises the following steps:

[0016] S1, dissolving the ferrous compound and ascorbic acid in water to prepare a mixed solution, heating the solution to 45-55° C., wherein the concentration ratio of ascorbic acid to the ferrous compound is 1:1;

[0017] S2. Adding compound A during the heating process and stirring until a stable colloid is formed to obtain the hydrogel, wherein the amount of compound A added is 2% of the mass of the mixed solution;

[0018] Preferably, the concentration of the ferrous compound is 1 mM.

[0019] A hydrogel prepared by the above method.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1. The hydrogel provided by the present invention has a highly effective bactericidal effect on Staphylococcus aureus and MRSA, indicating that ferrous compounds can be used as a potential drug to combat the current antibiotic crisis.

[0022] 2. The present invention uses ferrous sulfate, a commonly used iron supplement, as a typical representative of ferrous compounds to study drugs with ferrous ions as the core to replace antibiotic drugs. Hydrogel is also used as a drug carrier to successfully prepare ferrous ion hydrogel. The present invention uses ferrous sulfate as the core drug to replace antibiotic drugs. The antibacterial hydrogel of ferrous ions prepared by the present invention can effectively treat epidermal and eye infections, and can significantly reduce the risk of MRSA infection in the lungs, and effectively and promptly prevent the reproduction and spread of microorganisms in the body.

[0023] 3. The preparation method of the hydrogel of the present invention is simple, but has strong material adaptability. It can be combined with various materials such as carrageenan, xanthan gum, low acyl colloid, hyaluronic acid, carbomer, etc. to exert strong bactericidal potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 TEM image (A) of the hydrogel of Example 1, element distribution diagram of the hydrogel of Example 1 (B), SEM image of a hyaluronic acid hydrogel not containing ferrous sulfate (C), SEM image of the hydrogel of Example 1 (D), rheological properties diagram of a hyaluronic acid hydrogel not containing ferrous sulfate (E), and rheological properties diagram of the hydrogel of Example 1 (F);

[0025] Figure 2 SEM images (AB) of a sodium alginate hydrogel not containing ferrous sulfate, SEM images (CD) of the hydrogel of Example 2; TEM images (EF) of the hydrogel of Example 2, element distribution diagram of the hydrogel of Example 2 (G), rheological properties diagram of a sodium alginate hydrogel not containing ferrous sulfate (H), and rheological properties diagram of the hydrogel of Example 2 (I);

[0026] Figure 3 Schematic diagram of the ferrous ion release rate of the hydrogel in Example 1 (A) and a schematic diagram of the ferrous ion release rate of the hydrogel in Example 2 (B);

[0027] Figure 4 The following are the bactericidal distribution diagram of the hydrogel in Example 1 on MRSA cells stained with PI and Syto 9 (A), the bactericidal activity effect diagram of the hydrogel in Example 1 (B), the bactericidal distribution diagram of the hydrogel in Example 2 on MRSA cells stained with PI and Syto 9 (C), and the bactericidal activity effect diagram of the hydrogel in Example 2 (D);

[0028] Figure 5 Schematic diagram of the survival rate of mice in different treatment groups (A), schematic diagram of the changes in the inflammatory factor IL-1 of mice in different treatment groups (B), schematic diagram of the changes in the inflammatory factor IL-6 of mice in different treatment groups (C);

[0029] Figure 6The results of mouse eyeball histology sections and the gross images after treatment in each group (A) and the results of mouse lung histology sections and the gross images after treatment in each group (B);

[0030] Figure 7 Schematic diagram of the number of bacteria in the lungs of infected mice 3 days after hydrogel treatment (A) and a general diagram of mouse wound healing (B);

[0031] Figure 8 Schematic diagram of the antibacterial effects of nine iron ions on Staphylococcus aureus (A), schematic diagram of the antibacterial effects of ferrous sulfate on six pathogenic bacteria (B), and schematic diagram of the antibacterial effects of ferrous sulfate on Staphylococcus aureus (C). DETAILED DESCRIPTION

[0032] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited by the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are all conventional methods unless otherwise specified.

[0033] Example 1

[0034] A method for preparing a hydrogel comprises the following steps:

[0035] S1. Dissolving ferrous sulfate and ascorbic acid in sterile ultrapure water to prepare a mixed solution, and heating the solution to 50° C. wherein the concentration of ferrous sulfate in the mixed solution is 1 mM, and the concentration ratio of ascorbic acid to ferrous sulfate is 1:1;

[0036] S2. Add hyaluronic acid powder during the heating process and stir until the hyaluronic acid powder dissolves to form a stable colloid to obtain the hydrogel. The amount of hyaluronic acid added is 2% of the mass of the mixed solution.

[0037] The hydrogel prepared by the above method.

[0038] The hydrogel is used for treating bacterial keratitis.

[0039] Example 2

[0040] A method for preparing a hydrogel comprises the following steps:

[0041] S1. Dissolving ferrous sulfate and ascorbic acid in sterile ultrapure water to prepare a mixed solution, and heating the solution to 50° C. wherein the concentration of ferrous sulfate in the mixed solution is 1 mM, and the concentration ratio of ascorbic acid to ferrous sulfate is 1:1;

[0042] S2. During the heating process, sodium alginate powder is added and stirred until the sodium alginate powder is dissolved to form a stable colloid, thereby obtaining the hydrogel. The amount of sodium alginate added is 2% of the mass of the mixed solution.

[0043] The hydrogel prepared by the above method.

[0044] The hydrogel is used for treating skin wound infection.

[0045] Example 3

[0046] The difference between Example 3 and Example 1 is that the ferrous compound is ferrous chloride.

[0047] Example 4

[0048] The difference between Example 4 and Example 1 is that the ferrous compound is ferrous gluconate.

[0049] Example 5

[0050] The difference between Example 5 and Example 1 is that the ferrous compound is 1 part of ferrous lactate.

[0051] To verify the antibacterial activity of ferrous ions against Staphylococcus aureus, the following test was performed

[0052] 1. Prepare aqueous solutions of 5 ferrous salts, including ferrous sulfate, ferrous chloride, ferrous gluconate, ferrous lactate and potassium ferrocyanide, and 4 iron salts, including ferric chloride, ferric citrate, ferric dextran and potassium ferrocyanide, to a final concentration of 100 μM. 8 The drug susceptibility of Staphylococcus aureus to nine iron salts was tested at 100 CFU / mL. After 12 hours of drug treatment, the total bacterial count was determined in a drop plate assay to further explore the antibacterial effect of iron ions on Staphylococcus aureus.

[0053] Result: Passed Figure 8 A results show that at the same concentration (100μM), 5 different Fe 2+ The antibacterial effect of salt on Staphylococcus aureus is much higher than that of Fe 3+ Salts show that ferrous ions play an important role in killing Staphylococcus aureus. In particular, ferrous sulfate (FeSO4) can inhibit the growth of Staphylococcus aureus by almost 100%. This shows that Fe 2+ It can effectively inhibit the growth of Staphylococcus aureus.

[0054] 2. Prepare ferrous sulfate aqueous solution to a final concentration of 16 μM; and prepare 10 8The six pathogens were tested for ferrous sulfate susceptibility using a 96-well plate assay. After 12 hours of drug treatment, the total bacterial count was determined using a drop plate assay to further investigate the antibacterial effect of ferrous ions on these bacteria.

[0055] Results: Ferrous sulfate with the best bactericidal effect was further selected as the iron agent used in the study, and drug sensitivity tests were performed on 6 different pathogens. Figure 8 B Results showed that 16μM ferrous sulfate exhibited a strong bactericidal effect against all six pathogens, indicating that ferrous ions have a broad-spectrum bactericidal effect and can effectively kill common infection-inducing pathogens. It is worth noting that ferrous sulfate has a significantly better antibacterial effect against Staphylococcus aureus than other pathogens (Escherichia coli, Listeria monocytogenes, Vibrio parahaemolyticus, and Enterobacter sakazakii), with a survival rate of less than 0.001%. It is also worth noting that ferrous sulfate also exhibited good antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA), with a survival rate of less than 0.01% after treatment with 16μM ferrous sulfate.

[0056] 3. Prepare aqueous solutions of ferrous sulfate to final concentrations of 1, 4, 16, and 64 μM, respectively. Prepare 100 μg / mL of ampicillin and vancomycin as positive controls, and set up ultrapure water without ferrous sulfate as a blank control group. 8 CFU / mL of Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA). The 96-well plate assay tested the drug susceptibility of S. aureus to different concentrations of ferrous sulfate. After 12 hours of drug treatment, the total bacterial count was determined using a drop plate assay to further explore the minimum bactericidal concentration (MBC) of ferrous sulfate.

[0057] Results: As Figure 8 C results showed that re-culturing of Staphylococcus aureus treated with ferrous sulfate did not allow them to grow, indicating that the bacteria were likely dead, and the degree of death was closely related to the ferrous sulfate concentration. The MBC value of ferrous sulfate against Staphylococcus aureus was 16 μM. At this concentration, the bactericidal rate reached 99.99%, demonstrating promising potential for its application in sterilization.

[0058] In order to verify the performance of the hydrogel of the present invention, the following analysis was performed:

[0059] Ampicillin and Vancomycin used in the experiment were purchased from Sigma-Aldrich.

[0060] 1. Distribution analysis of ferrous ions in hydrogel

[0061] The hydrogel of Example 1 and the hydrogel of Example 2 were placed in a disposable culture dish for 6 hours, frozen (-20°C), and then placed in a freeze dryer for 48 hours. After drying, they were ground into powder with liquid nitrogen, and the powder samples were taken for testing.

[0062] (1) Analyze the active elements in the hydrogel by X-ray photoelectron spectroscopy (XPS);

[0063] (2) Analyze the phenotypic structure of the hydrogel using field emission scanning electron microscopy (SEM);

[0064] (3) The rheological properties of the hydrogels were analyzed by rotational rheometry: a 40 mm plate geometry with a gap of 1000 μm was used. Before the test, a thin film of silicone oil was applied around the trap to eliminate the effect of water evaporation. The equilibrium time was set to 7 minutes to allow the hydrogel to reach a stable state. The viscosity was tested at a shear rate of 0.1-100 1 / s. The yield performance was tested in the range of 1-2000 Pa at an oscillation frequency of 1 Hz. The oscillatory strain behavior was performed in the strain range of 0.01% to 100% at 1 Hz. Creep and recovery tests were performed within the linear viscoelastic range of 180 degrees and under a constant stress of 10 Pa.

[0065] Results: As Figure 1 and Figure 2 As shown in Figure 2, ferrous ions were successfully loaded into both hydrogel matrices and were evenly distributed in the hydrogels. SEM images showed that the hydrogels loaded with ferrous ions had a heterogeneous surface and porous structure, which were beneficial to the preservation of water in the hydrogels and also to the Fe 2+ Diffusion and release from the hydrogel; the comparison of the rheological properties of the two hydrogels showed that the G' (elastic modulus) and G" (loss modulus) of the ferrous ion-loaded hydrogel could recover to 100% within a few seconds at high shear rates, indicating that the FeSO4-loaded hydrogel has potential fluidity and resilience that are beneficial to drug diffusion.

[0066] The above results indicate that hydrogels loaded with ferrous ions have potential medical application prospects.

[0067] 2. Analysis of ferrous ion release rate in hydrogel

[0068] (1) 10 ml of water was injected into the hydrogel of Example 1 and the hydrogel of Example 2 respectively and placed into dialysis bags (8000-14000 Da).

[0069] (2) Place 100 ml of ultrapure water in the dialysis bag and stir it with a magnetic stirrer at 37°C and 200 rpm for 12 h.

[0070] (3) The concentration of ferrous ions was determined using the o-phenanthroline molecular absorption spectrum colorimetry method, with measurements taken every 30 minutes for a total of 12 hours.

[0071] Results: Ferrous ion release experiment confirmed the results as follows Figure 3 As shown, Fe in the hydrogel 2+ They can be released from the hydrogel in a time-dependent manner, and the FeSO4 hydrogel has the characteristics of slow and long-term release of iron ions. After 12 hours, the Fe 2+ The release rates were approximately 78.3% and 73.6% respectively.

[0072] 3. Analysis of the antibacterial activity of hydrogel in vitro

[0073] (1) Pour the hydrogel of Example 1, the hydrogel of Example 2, the ferrous sulfate hyaluronic acid hydrogel, the ascorbic acid hyaluronic acid hydrogel, the ampicillin hyaluronic acid hydrogel, the vancomycin hyaluronic acid hydrogel, the ferrous sulfate sodium alginate hydrogel, the ascorbic acid sodium alginate hydrogel, the ampicillin sodium alginate hydrogel and the vancomycin sodium alginate hydrogel into a sterile disposable culture dish for use;

[0074] (2)10 8 CFU / mL of S. aureus cells were washed twice with saline and collected onto 1 cm sterile filter paper;

[0075] (3) The filter paper with the collected cells is placed on each hydrogel with the front side facing up, and then sealed and placed in a 37°C incubator for culture;

[0076] (4) After 12 h of incubation, the filter paper was carefully removed with sterile tweezers. The excess hydrogel on the back of the filter paper was wiped off with a sterile cotton swab and placed in a sterile EP tube. 1 mL of sterile saline was added to the tube and sonicated for 30 s to collect the cells on the filter paper.

[0077] (6) The total bacterial colony count was determined using the drop plate method.

[0078] To determine the antibacterial effect of FeSO4-loaded hydrogel, 100 μL of hydrogel from Example 1 and 100 μL of hydrogel from Example 2 were added to the center of the slide, and 5 μL of washed Staphylococcus aureus cells (108 CFU / mL) were vertically dripped onto the surface of the FeSO4 hydrogel and incubated at 37°C for 3 hours. Then, PI (final concentration of 1 μg / mL) was dripped onto the FeSO4 hydrogel containing Staphylococcus aureus cells and incubated at 37°C for 20 minutes. The antibacterial activity of the FeSO4 hydrogel was determined using confocal laser scanning microscopy.

[0079] Results: Through the in vitro surface contact experiment simulating MRSA and ferrous ion hydrogel, it was found that the hydrogel of Example 1 and the hydrogel of Example 2 both had good bactericidal effects. Figure 4 A is the distribution of MRSA cell death stained with PI and Syto 9 under a laser confocal microscope after the hydrogel in Example 1 was treated. The results show that the hydrogel containing ASC and FeSO4 has a greater bactericidal activity, with a bactericidal rate of almost 100%. Figure 4 As shown in Figure B, treatment with a hydrogel containing only FeSO4 can only reduce 31 g CFU, and the hydrogel containing only ASC has no bactericidal effect on MRSA, indicating that the addition of ASC can maximize the bactericidal activity of the hydrogel loaded with ferrous ions. Importantly, compared with the hydrogel in Example 1, the ability of the hydrogel containing Ampicillin to kill MRSA is much lower.

[0080] Figure 4 C is the death distribution of MRSA cells stained with PI and Syto 9 under laser confocal microscopy after treatment with the hydrogel in Example 2. The results show that the hydrogel also has a large bactericidal activity. Figure 4 D It can be seen that the ability of the hydrogel containing Vancomycin to kill MRSA is lower than that of the hydrogel in Example 2. These results show that the hydrogel containing both ferrous sulfate and ascorbic acid can be used to treat MRSA infection.

[0081] 4. Conduct the following animal experiments to further illustrate its effects:

[0082] 1. Verification of the effect of Example 1

[0083] (1) Preparation of Staphylococcus aureus suspension

[0084] MRSA (from the Chinese Academy of Sciences) overnight bacteria transfer (1:200), grow to OD 600 =0.3, collect 1×10 8 ~1×10 10 CFU / mL, remove the culture medium and resuspend in normal saline for later use.

[0085] (2) Construction of a mouse model of Staphylococcus aureus keratitis

[0086] Mouse preparation: 40 BALB / c male mice, approximately 8 weeks old.

[0087] Model construction:

[0088] Anesthesia: Each mouse was anesthetized intramuscularly with 0.125 mL of a 1:1 mixture of ketamine hydrochloride (100 mg / mL): oxazine (20 mg / mL). After approximately 3-5 minutes, when mild anesthesia (absence of eyelid reflex and weak corneal reflex) appeared in the eye, one drop of 1% tetracaine hydrochloride was instilled into the cornea.

[0089] b. Incision: Using a 25-30 gauge needle, make three parallel scratches, each 2 mm in length, in the center of the corneal epithelium, without penetrating the surface stroma, to injure the mouse cornea.

[0090] c. Inoculation: Aim at the mouse eyeball and add two drops of the prepared Staphylococcus aureus suspension (10 8 CFU / mL, 50 μL per drop, 100 μL in total), press the eyelid lightly for 10 seconds to allow full contact between the bacterial solution and the cornea, and then gently restore the eyelid;

[0091] d. Identification: 24 / 48 hours after inoculation (depending on the infection status of the mouse eye), observe the external eye with a flashlight. If there is vascular congestion, corneal opacity, and pus in the eye, the model is considered to be successful.

[0092] e. Grading: There are two levels: mild and severe. Mild: slight opaque discharge, partially or completely covering the pupil and anterior segment of the eye; severe: dense opaque discharge, partially or completely covering the pupil or the anterior segment of the eye, or even corneal perforation.

[0093] (3) Animal grouping and drug administration

[0094] a. Grouping: After the Staphylococcus aureus keratitis model in mice was successfully established, the mice were divided into four groups with 8 mice in each group, namely:

[0095] Blank group (8 mice): The eyes of mice were scratched but not inoculated with bacteria, and sterile saline was dripped into the eyes, totaling 100 μL;

[0096] Model control group (8 mice): The mouse model was established, and 100 μL of sterile saline was dripped into the eyes, followed by treatment with hyaluronic acid hydrogel (1 mL in total);

[0097] Treatment group 1 (8 mice): The eyes of the established mouse model were treated with 100 μL of 1 mM ASC aqueous solution and 1 mM FeSO4 aqueous solution, and then loaded with 1 mL of the hydrogel of Example 1;

[0098] Treatment group 2 (8 mice): The eyes of the established mouse model were treated with 100 μL of 1 mM ASC aqueous solution, followed by treatment with 1 mL of hyaluronic acid hydrogel loaded with ASC (1 mM);

[0099] Positive control group (8 mice): The eyes of the established mouse model were treated with 100 μL of 1 mg / mL ampicillin, followed by treatment with 1 mL of hyaluronic acid hydrogel loaded with ampicillin (1 mg / mL);

[0100] b. Drug administration: Each group of mice was treated with drug according to the designated groups twice daily. Each treatment was first washed with saline containing the drug, followed by application of the drug to the hydrogel, with a 10-minute interval between the two application times, for a total of 7 days.

[0101] c. Observation: During the experiment, each eye should be observed and recorded using a handheld flashlight before administration every day. The eye condition of the mice should also be recorded daily by taking photos.

[0102] d. Ocular colony count: Gently smear the upper and lower eyelids with a cotton ball moistened with sterile saline, then place the ball into 5 mL of sterile saline. Mix thoroughly, aspirate 100 μL, perform serial dilutions, plate, culture, and count.

[0103] e. Blood index test: detect and identify the type and quantity of bacteria in the blood, and test related blood indicators

[0104] f. Lung colony count: Mice treated for 7 days were sacrificed, and the lungs were removed by dissection and placed in 5 mL of PBS. The lungs were sonicated and mixed. 100 μL of the supernatant was aspirated, serially diluted, plated, cultured, and counted.

[0105] g. Ocular histopathological examination: The mice were sacrificed after 7 days of treatment, and the eyeballs were removed, fixed in 10% formalin, embedded in paraffin, and stained with HE. The eyeballs were examined under a light microscope to observe the pathological histological changes.

[0106] Results: 48 hours after infection, the hydrogel of Example 1 was used to treat the eyes of MRSA-infected mice for 7 days. The bactericidal activity of the self-assembled ferrous hydrogel was evaluated by analyzing the survival rate, cellular inflammatory factors, and pathological sections of the eyes and lungs. The results are as follows: Figure 5As shown in A, compared with the 35% survival rate of mice in the control hydrogel group, the survival rate of mice in the FeSO4+ASC hydrogel group was significantly improved, with a survival rate of 100%. At the same time, the serum cytokine IL-1 and IL-6 levels of the uninfected control group were 25pg / mL and 35pg / mL, respectively. The serum cytokine IL-1 and IL-6 levels of the blank hydrogel group were 30pg / mL and 40pg / mL, respectively. The IL-1 and IL-6 levels of the ASC hydrogel-treated group were slightly lower than those of the blank hydrogel-treated group. The serum IL-1 and IL-6 levels of the ampicillin hydrogel group and the FeSO4+ASC hydrogel group were significantly lower than those of the blank hydrogel group, and the decrease in IL-6 and IL-1 in the FeSO4+ASC hydrogel group was the most significant. This suggests that FeSO4+ASC hydrogel can reduce the inflammatory response during Staphylococcus aureus infection, and its effect is greater than that of ampicillin hydrogel ( Figure 5 B, C). HE staining microscope images and histological analysis of mouse eyeballs and lungs after 7 days of treatment with different hydrogels showed that, for mouse eyeballs, Figure 6 As shown in A, the blank hydrogel group mice had pus and blood secretion in their eyeballs, obvious corneal edema, and a large number of neutrophils. The ampicillin hydrogel group mice had significantly fewer corneal inflammatory cells and the corneal thickness returned to normal. However, the mouse cornea still had some repair defects. In the self-assembled ferrous hydrogel group, the mouse corneal tissue basically returned to normal: membrane edema disappeared; there was no inflammatory reaction, and new blood vessels decreased. The results show that the hydrogel of Example 1 has a good therapeutic effect on the Staphylococcus aureus infection of mouse keratitis. Figure 6 As shown in Figure B, mice treated with the FeSO4+ASC hydrogel showed significantly less lung damage compared to the other treatment groups. Their bronchial tubes remained normal in size, their alveoli were intact, and inflammatory cells such as neutrophils and phagocytes were absent in the lungs, essentially the same as those in the uninfected group. These results demonstrate that the hydrogel in Example 1 not only has potential advantages in treating keratitis, but also effectively prevents the spread of corneal infection to the lungs.

[0107] 2. Verification of the effect of Example 2

[0108] (1) Preparation of Staphylococcus aureus suspension

[0109] MRSA (from the Chinese Academy of Sciences) overnight bacteria transfer (1:200), grow to OD 600 =0.3, collect 1×10 8 ~1×10 10 CFU / mL, remove the culture medium and resuspend in normal saline for later use.

[0110] (2) Construction of a mouse model of epidermal trauma infected with Staphylococcus aureus

[0111] Mouse preparation: 30 BALB / c male mice, approximately 8 weeks old.

[0112] Model construction:

[0113] Anesthesia: Each mouse was anesthetized intramuscularly with 0.125 mL of a 1:1 mixture of ketamine hydrochloride (100 mg / mL) and oxyperazine (20 mg / mL). The mouse's respiratory status and heart rate were observed for approximately 3-5 minutes (slowing of the respiratory rhythm and decreasing of the heart rate) to determine whether the anesthesia was successful.

[0114] b. Back wound incision: The mouse's back was depilated with depilatory cream, and the back was scraped with saline gauze and wiped with 75% ethanol. A full-thickness wound (10 mm x 10 mm) was created on the back skin of each mouse, extending deep into the subcutaneous tissue and maintained open, resulting in a loss of skin damage on the mouse's back.

[0115] c. Inoculation: Add two drops of prepared Staphylococcus aureus suspension (10 8 CFU / mL, 50 μL per drop, 100 μL in total), and the wound was bandaged with sterile gauze;

[0116] d. Infection: 24 / 48 hours after inoculation, observe the abscess on the surface of the mouse wound;

[0117] (3) Animal grouping and drug administration

[0118] a. Grouping: After the mouse model of epidermal wound infection with Staphylococcus aureus was successfully established, the mice were divided into 5 groups with 6 mice in each group, namely:

[0119] Blank group (6 mice): Wounds were made on the back of mice, but no bacteria were inoculated. Sterile saline was dripped into the wound, totaling 100 μL.

[0120] Model control group (6 mice): The mouse model was established, no drug treatment was added, 100 μL of sterile saline was dripped into the wound, and then sodium alginate hydrogel was treated, a total of 1 mL;

[0121] Treatment group 1 (6 mice): The wounds of the established mouse model were treated with 100 μL of 1 mM ASC aqueous solution and 1 mM FeSO4 aqueous solution, and then loaded with 1 mL of the hydrogel of Example 2;

[0122] Treatment group 2 (6 mice): The wounds of the established mouse model were treated with 100 μL of 1 mM ASC aqueous solution, followed by 1 mL of sodium alginate hydrogel loaded with ASC (1 mM).

[0123] Positive control group (6 mice): The wounds of the established mouse model were treated with 100 μL of 1 mM vancomycin aqueous solution, followed by treatment with 1 mL of sodium alginate hydrogel loaded with vancomycin;

[0124] b. Dosing: Each group of mice received drug treatment twice daily. Each treatment was first washed with saline containing the drug, followed by hydrogel application, with a 10-minute interval between each treatment. The wound was then bandaged again with sterile gauze for a total of 3 days.

[0125] c. Observation: During the trial, each mouse's wound should be observed and recorded using a handheld flashlight before dosing each day. Photos should also be taken daily to record the wound healing status of the mice.

[0126] d. Count the colonies on the wound surface: Gently smear the wound surface with a cotton ball moistened with sterile saline and place it in 5 mL of sterile saline. Mix thoroughly, aspirate 100 μL of the solution, perform serial dilutions, plate, culture, and count.

[0127] e. Blood index test: detect and identify the type and quantity of bacteria in the blood, and test related blood indicators

[0128] f. Lung colony count: Mice treated for 7 days were sacrificed, and the lungs were removed by dissection and placed in 5 mL of PBS. The lungs were sonicated and mixed. 100 μL of the supernatant was aspirated, serially diluted, plated, cultured, and counted.

[0129] Results: Staphylococcus aureus is a common human pathogen, and bacterial infection is the most common clinical condition. In particular, methicillin-resistant Staphylococcus aureus infection can cause suppurative skin infections, which are seriously life-threatening. Therefore, the important indicators for evaluating the potential application of FeSO4 are its ability to remove MRSA cells from wounds and its ability to promote wound healing. Figure 7 As shown in Figure B, the wound healing ability of the mouse epidermal infection model in the hydrogel treatment group of Example 2 was better than that of the other groups on the 3rd day, and a small amount of pus was still present in the wound of the uninfected treatment group. Among the other 5 MRSA infection groups, the sodium alginate hydrogel treatment group containing only ASC and the hydrogel treatment group containing vancomycin failed to effectively alleviate wound healing, and the wound was seriously pus-bloody. Compared with the vancomycin hydrogel group, the hydrogel of Example 2 was beneficial to wound healing, and no pus and blood were seen on the wound, and the wound area was smaller than the wound area on the 0th day. The lungs of mice in each treatment group were dissected for Staphylococcus aureus detection, and it was found that Figure 7As shown in A, FeSO4+ASC hydrogel can eliminate at least 99.9% of MRSA cells, while the bactericidal effects of the other treatment groups are significantly weaker than that of ferrous hydrogel. FeSO4 hydrogel epidermal treatment has a potential preventive effect on the infection of MRSA cells to the lungs.

[0130] Therefore, it was confirmed that the FeSO4-containing hydrogel had potential antibacterial activity to combat drug resistance of microorganisms in vivo.

[0131] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes preferred embodiments.

[0132] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. 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.

[0133] 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 such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. Use of a ferrous compound in the preparation of a product for treating bacterial infection, characterized in that: The ferrous compound is ferrous sulfate, the concentration of ferrous ions in the ferrous sulfate is 16 μM, and the bacterial infection is caused by Staphylococcus aureus.

2. The use according to claim 1, characterized in that The Staphylococcus aureus is methicillin-resistant Staphylococcus aureus.

3. The use according to claim 1, characterized in that The survival rate of Staphylococcus aureus treated with 16 μM ferrous sulfate was less than 0.001%.

4. The use according to claim 2, characterized in that The survival rate of 16 μM ferrous sulfate against methicillin-resistant Staphylococcus aureus was less than 0.01%.

5. The use according to any one of claims 1 to 4, characterized in that The bacterial infection is an eyeball infection or an epidermal infection.

6. The use according to any one of claims 1 to 4, characterized in that The bacterial infection is Staphylococcus aureus keratitis or Staphylococcus aureus infected epidermal wound.

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