Preparation method of nanochitosan bacteriostatic synergist

Abstract

The application discloses a preparation method of a nano-chitosan bacteriostatic synergist, which comprises the following steps: dissolving chitosan in acetic acid, standing overnight after dissolving until no obvious particles are generated, and obtaining a chitosan solution; adding a bacteriostatic drug into the chitosan solution and performing magnetic stirring, and then adding a crosslinking agent to obtain the nano-chitosan bacteriostatic synergist. The nano-chitosan conjugated penicillin is used for the first time, and the effect verification is obtained on clinically drug-resistant escherichia coli, which provides the application possibility for reducing the use of clinical drugs and drug residues. Compared with the current commonly used antibiotic treatment method, the application has the advantages of improving the bacteriostatic effect, reducing the drug usage, reducing the drug residues, coping with the drug resistance problem and the simple preparation method. In the research, the drug-resistant escherichia coli is taken as the basis, different concentrations of the nano-chitosan bacteriostatic synergist are added, and the bacteriostatic effect of the nano-chitosan bacteriostatic synergist on the drug-resistant escherichia coli is evaluated. The research aims to provide certain theoretical and experimental basis for clinical medication.

Description

Technical Field

[0001] This invention belongs to the fields of biomedicine and animal husbandry and veterinary technology, and specifically relates to a method for preparing a nano-chitosan antibacterial synergist. Background Technology

[0002] The application of nanotechnology in the medical field has attracted widespread attention, particularly for its unique advantages in precise drug delivery and enhanced therapeutic efficacy. Nanomedicines, through their nanoscale particle size, improve drug solubility and bioavailability, theoretically potentially significantly enhancing therapeutic effects. However, in clinical treatment within aquaculture, these nanomedicines face several challenges and limitations.

[0003] Maintaining the stability and biocompatibility of nanomedicines is challenging in complex aquaculture environments. For example, since mastitis is often accompanied by an inflammatory response, this can alter the local pH or enzyme activity, leading to rapid decomposition or aggregation of nanocarriers, thus affecting effective drug release and therapeutic efficacy. Secondly, existing targeted release technologies for nanomedicines are often difficult to control precisely in mastitis treatment, making it hard for drugs to reach deep into inflamed tissues, thereby reducing their antibacterial effect. Furthermore, existing nanomedicine preparation processes typically involve high-cost materials and complex technologies, which is particularly problematic in the profit-driven aquaculture industry, limiting their widespread application.

[0004] Nano-chitosan has shown potential in the field of antibacterial applications due to its unique physicochemical properties and bioactivity. However, the antibacterial effects of existing nano-chitosan are often unsatisfactory when faced with complex clinical environments and diverse pathogens. For example, existing nano-chitosan exhibits good inhibitory effects against certain specific bacterial species, but struggles to address complex mixed infections or drug-resistant strains.

[0005] Meanwhile, traditional antibiotics such as penicillin face the challenge of increasing drug resistance in clinical applications. Simply increasing antibiotic dosage not only fails to effectively solve the resistance problem but may also lead to more serious drug residues and environmental pollution. Therefore, developing a formulation that can improve antibiotic efficacy while reducing dosage is of great significance.

[0006] In view of the above problems, it is necessary to develop a nano-chitosan antibacterial synergist to overcome the limitations of existing technologies. Summary of the Invention

[0007] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0008] In view of the problems existing in the above and / or prior art, the present invention is proposed.

[0009] Therefore, the purpose of this invention is to overcome the shortcomings of the prior art and provide a method for preparing a nano-chitosan antibacterial synergist.

[0010] To solve the above-mentioned technical problems, the present invention provides the following technical solution: A method for preparing a nano-chitosan antibacterial synergist, characterized in that it includes: Chitosan was dissolved in acetic acid and allowed to stand overnight until no obvious particles were visible, thus obtaining a chitosan solution. An antibacterial agent was added to the chitosan solution and the mixture was magnetically stirred. A cross-linking agent was then added to obtain a nano-chitosan antibacterial synergist.

[0011] In a preferred embodiment of the preparation method described in this invention, the chitosan solution has a molecular weight of 5-150 kDa and a concentration of 2 mg / mL.

[0012] In a preferred embodiment of the preparation method described in this invention, the concentration of the acetic acid is 0.5-1%.

[0013] In a preferred embodiment of the preparation method described in this invention, the antibacterial drug includes penicillin and disulfiram.

[0014] In a preferred embodiment of the preparation method described in this invention, the crosslinking agent is sodium tripolyphosphate.

[0015] In a preferred embodiment of the preparation method described in this invention, the ratio of the chitosan solution, the antibacterial drug, and the crosslinking agent is 150–250:3–7:50–80.

[0016] In a preferred embodiment of the preparation method described in this invention, the concentration of the crosslinking agent is 1.1 mg / mL.

[0017] In a preferred embodiment of the preparation method described in this invention, the ratio of chitosan to acetic acid is 80-120:0.5-1.5.

[0018] Another objective of this invention is to overcome the shortcomings of the prior art and provide a nano-chitosan antibacterial synergist.

[0019] Another objective of this invention is to overcome the shortcomings of the prior art and provide an application of a nano-chitosan antibacterial synergist in the treatment of bacterial infections in veterinary medicine.

[0020] Beneficial effects of this invention: (1) This invention discloses a method for preparing a nano-chitosan antibacterial synergist, including a method for preparing nano-chitosan and a method for conjugating antibacterial drugs and nano-chitosan. Through the synergistic effect of nano-chitosan and penicillin, the minimum inhibitory concentration (MIC) of drug-resistant Escherichia coli is significantly reduced, and the inhibitory ability against drug-resistant strains is enhanced. Compared with the use of penicillin alone, this synergist can achieve the same or better antibacterial effect at a lower concentration, which helps to reduce the overuse of antibiotics. Due to the reduced dosage, the drug residue in the environment can be effectively reduced, and the potential impact on the ecosystem can be reduced.

[0021] (2) The present invention uses simple cross-linking and conjugation technology, the preparation process is simple to operate and has low cost, which is conducive to large-scale production and application.

[0022] (3) This invention is the first to use nano-chitosan conjugated penicillin, and its effectiveness has been verified against clinically resistant Escherichia coli. This provides a potential application for reducing clinical drug use and drug residues. Compared to currently used antibiotic treatments, this invention has advantages such as improved antibacterial effect, reduced drug usage, reduced drug residues, addressing drug resistance issues, and simple preparation method. This study, based on drug-resistant Escherichia coli, evaluated its antibacterial effect against drug-resistant Escherichia coli by adding different concentrations of nano-chitosan antibacterial synergists. The aim is to provide a certain theoretical and experimental basis for clinical drug use. Attached Figure Description

[0023] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein: Figure 1 This is a transmission electron microscope (TEM) image of the nano-chitosan antibacterial synergist in Example 1 of the present invention.

[0024] Figure 2 This is the Fourier transform infrared spectrum of the nano-chitosan antibacterial synergist in Example 1 of the present invention.

[0025] Figure 3 The image shows the inhibitory MIC of the nano-chitosan antibacterial synergist against penicillin-resistant Escherichia coli in Example 1 of this invention.

[0026] Figure 4 This is a 24-hour growth curve of the nano-chitosan antibacterial synergist against penicillin-resistant Escherichia coli in Example 1 of the present invention.

[0027] Figure 5This is a graph showing the expression levels of inflammatory factor mRNA in the liver of mice after treatment with nano-chitosan antibacterial synergist in Example 5 of this invention.

[0028] Figure 6 This is an HE slice of mouse liver after treatment with nano-chitosan antibacterial synergist in Example 6 of the present invention.

[0029] Figure 7 The image shows the inhibitory MIC of the nano-chitosan antibacterial synergist (CS-T-disulfiram) on penicillin-resistant Escherichia coli in Example 2 of this invention.

[0030] Figure 8 The MIC diagrams show the antibacterial activity of nano-chitosan synergists against drug-resistant Escherichia coli (resistant to penicillin / cefotaxime) in Comparative Examples 3, 4, and 5 of this invention. Detailed Implementation

[0031] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0032] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0033] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0034] Unless otherwise specified, all raw materials used in the embodiments of this invention are commercially available. See Table 1 for details.

[0035] Table 1 Example 1

[0036] This invention provides a method for preparing a nano-chitosan antibacterial synergist: (1) Preparation of synergist: Take 100 mg of chitosan with a molecular weight of 50 kDa and dissolve it in 1% acetic acid. After dissolving, let it stand overnight so that the particles can be fully dissolved in water until there are no obvious particles, and prepare a chitosan solution of 2 mg / mL. Under continuous magnetic stirring, 2.56 mL of penicillin with a concentration of 5 mg / mL was added to 100 mL of chitosan solution. After continuous stirring, sodium tripolyphosphate solution with a concentration of 1.1 mg / mL was slowly added. The volume ratio of chitosan solution to sodium tripolyphosphate solution was 3:1. The solution changed from transparent to turbid. Then, the pH of the solution was adjusted to 4.6 using 1 mol / mL NaOH.

[0037] (2) Sample preparation: The prepared nano-chitosan antibacterial synergist was placed in a vacuum freeze dryer to obtain lyophilized powder. An appropriate amount of lyophilized powder was taken and its morphology was observed using a Tecnai 12 transmission electron microscope (TEM).

[0038] (3) Image analysis: The results of transmission electron microscopy are as follows Figure 1 As shown, the nano-chitosan antibacterial synergist exhibits a distinct particulate structure with an average particle size of approximately 150 nm, successfully reaching the nanoscale. Example 2

[0039] The difference from Example 1 is that penicillin is replaced with disulfiram.

[0040] (1) Preparation of synergist: Take 100 mg of chitosan with a molecular weight of 50 kDa and dissolve it in 1% acetic acid. After dissolving, let it stand overnight so that the particles can be fully dissolved in water until there are no obvious particles, and prepare a chitosan solution of 2 mg / mL. Under continuous magnetic stirring, 2.56 mL of disulfiram with a concentration of 5 mg / mL was added to 100 mL of chitosan solution. After continuous stirring, sodium tripolyphosphate solution with a concentration of 1.1 mg / mL was slowly added. The volume ratio of chitosan solution to sodium tripolyphosphate solution was 3:1. The solution changed from transparent to turbid. Then, the pH of the solution was adjusted to 4.6 using 1 mol / mL NaOH.

[0041] (2) MIC: The drug-resistant Escherichia coli was diluted to 1×10⁵ CFU / mL and inoculated into 96-well plates. The synergist (CS-T-disulfiram) and other antibacterial drugs were serially diluted using the two-fold dilution method and added to the bacterial culture, and then thoroughly mixed. After incubating the 96-well plates in a 37℃ biological incubator for 12 h, the OD600 absorbance value was measured using an ELISA reader.

[0042] (3) Image analysis: A trend graph of MIC was plotted using the OD600 absorbance values, and the results are as follows. Figure 7As shown, the OD600 absorbance of the control group (MH broth) was approximately 0.05. Disulfiram showed no significant inhibitory effect on drug-resistant E. coli, even at a concentration of 64 μg / mL. CS-T-disulfiram represents a disulfiram-conjugated nano-chitosan antibacterial synergist. Results showed that the minimum inhibitory concentration (MIC) of CS-T-disulfiram (16 μg / mL) was consistent with CS-T, but the OD600 absorbance was lower, demonstrating that the nano-chitosan antibacterial synergist had a stronger antibacterial effect than disulfiram and nano-chitosan alone. Example 3

[0043] (1) Sample preparation: Take 0.1g of the lyophilized powder prepared in Example 1, use potassium bromide (KBr) tableting method, and use Cary 610 / 670 micro infrared spectrometer (IR) to perform infrared spectral analysis of the sample.

[0044] (2) Image analysis: The results obtained from the micro-infrared spectroscopy were plotted using Origin, and the results are as follows: Figure 2 As shown, infrared spectroscopy analysis revealed the structural evolution of chitosan from its original state to its nanoscale form and then to its drug-conjugated state. Chitosan exhibited typical -OH, -NH2, and C=O stretching vibration peaks at 3434 cm⁻¹, 2871 cm⁻¹, and 1651 cm⁻¹.

[0045] After crosslinking with sodium tripolyphosphate, the spectrum of nano-chitosan showed significant changes: the intensity of the -OH and -NH2 peaks decreased, indicating that these groups participated in the crosslinking reaction; simultaneously, P=O and POC vibrational peaks appeared at 1068 cm⁻¹ and 885 cm⁻¹, confirming the successful introduction of sodium tripolyphosphate. The penicillin-conjugated nano-chitosan retained the characteristic peaks of the crosslinked structure, but showed slight changes in the 1600–1500 cm⁻¹ and 1100–1000 cm⁻¹ regions, particularly with enhanced intensity of the COC and CO stretching vibrational peaks at 1054 cm⁻¹. These changes can be attributed to the successful conjugation of penicillin. This series of spectral changes not only confirms the successful preparation of nano-chitosan but also provides strong spectroscopic evidence for the effective loading of penicillin.

[0046] Comparative Example 1 (Penicillin) (1) Dissolve 100 mg of chitosan with a molecular weight of 50 kDa in 1% acetic acid. After dissolving, let it stand overnight to allow the particles to fully dissolve in the water until there are no obvious particles, and prepare a chitosan solution with a concentration of 2 mg / mL. Under the action of continuous magnetic stirring, add 2.56 mL of penicillin with a concentration of 5 mg / mL to 100 mL of chitosan solution and continue stirring until completely mixed. Then use 1 mol / M NaOH to adjust the pH of the solution to 4.6 to obtain the penicillin synergist.

[0047] Comparative Example 2 (CS-T) (1) Dissolve 100 mg of chitosan with a molecular weight of 50 kDa in 1% acetic acid. After dissolving, let it stand overnight to allow the particles to fully dissolve in the water until there are no obvious particles, and prepare a chitosan solution with a concentration of 2 mg / mL. Under the action of continuous magnetic stirring, add 33 mL of sodium tripolyphosphate with a concentration of 1.1 mg / mL to 100 mL of chitosan solution and continue stirring until the solution changes from transparent to turbid. Then use 1 mol / mL NaOH to adjust the pH of the solution to 4.6 to obtain the nano-chitosan synergist. Example 4

[0048] (1) Bacterial culture: Escherichia coli was isolated from milk samples of patients with clinical mastitis. The isolated strains were inoculated into sterile MH medium and cultured until the bacterial concentration reached 1×10⁸ CFU / mL. The cultured bacteria were then spread on MH agar plates, and penicillin-resistant Escherichia coli strains were screened using the KB disk diffusion method according to CLSI guidelines.

[0049] (2) MIC: Dilute the drug-resistant Escherichia coli to 1×10⁵ CFU / mL and inoculate it into 96-well plates. The synergist was serially diluted using the two-fold dilution method and added to the bacterial suspension, and then mixed thoroughly. After incubating the 96-well plates in a 37℃ biological incubator for 12 h, the OD600 absorbance value was measured using an ELISA reader.

[0050] (3) 24h growth curve: After diluting the drug-resistant Escherichia coli to 1×105 CFU / mL, it was placed in a shaker at 37℃ and the OD600 absorbance value was measured every 2h using an enzyme-linked immunosorbent assay reader. Penicillin, nano-chitosan and nano-chitosan antibacterial synergist were added at 4h and 8h of culture, respectively.

[0051] (4) Image analysis: A trend graph of MIC was plotted using the OD600 absorbance values, and the results are as follows. Figure 3 As shown, the OD value of the control group (MH broth) was approximately 0.05. Penicillin showed no significant inhibitory effect on drug-resistant E. coli; even at a concentration of 64 μg / mL, the antibacterial effect was not significant; below 32 μg / mL, there was almost no antibacterial effect. According to CLSI standards, this strain meets the characteristics of penicillin-resistant E. coli. CS-T represents TPP-crosslinked nano-chitosan, and CS-T-penicillin represents a penicillin-conjugated nano-chitosan antibacterial synergist. The results showed that the minimum inhibitory concentration (MIC) of CS-T-penicillin (8 μg / mL) was superior to that of CS-T, demonstrating that the nano-chitosan antibacterial synergist has a stronger antibacterial effect than penicillin and nano-chitosan alone.

[0052] A 24-hour growth curve trend chart was plotted using OD600 absorbance values, and the results are as follows: Figure 4As shown, the control group was the group where bacteria grew naturally. When 8 μg / mL of penicillin was added to the 4-hour growth phase and the 8-hour logarithmic growth phase of drug-resistant Escherichia coli, its growth was not effectively inhibited. However, after adding 8 μg / mL of CS-T-penicillin, the growth of drug-resistant Escherichia coli was inhibited. CS-T did not have the desired effect during the 8-hour logarithmic growth phase of bacteria.

[0053] Comparative Example 3 The difference from Example 1 is that penicillin is replaced with ceftiofur.

[0054] Ceftiofur-resistant Escherichia coli strains were screened using the KB disk diffusion method, guided by CLSI.

[0055] (1) Image analysis: A trend graph of MIC was plotted using the OD600 absorbance value, and the results are as follows. Figure 8 As shown, the OD value of the control group (MH broth) was approximately 0.05. The inhibitory effect was not significant.

[0056] Comparative Example 4 The difference from Example 1 is that penicillin is replaced with epigallocatechin gallate.

[0057] (1) Image analysis: A trend graph of MIC was plotted using the OD600 absorbance value, and the results are as follows. Figure 8 As shown, the OD value of the control group (MH broth) was approximately 0.05. The inhibitory effect was not significant.

[0058] Comparative Example 5 The difference from Example 1 is that penicillin is replaced with metformin.

[0059] (1) Image analysis: A trend graph of MIC was plotted using the OD600 absorbance value, and the results are as follows. Figure 8 As shown, the OD value of the control group (MH broth) was approximately 0.05. The inhibitory effect was not significant.

[0060] Comparative Example 6 The difference from Example 1 is that the crosslinking agent is genipin.

[0061] (1) Preparation of synergist: Weigh 100 mg of genipin powder and dissolve it in 50 mL of 20% anhydrous ethanol solution. Shake well to obtain a 2 mg / mL genipin solution. Add 2.56 mL of a 5 mg / mL penicillin solution to 100 mL of chitosan solution and stir well. Then, slowly add the genipin solution to the chitosan-penicillin mixture at a volume ratio of 0.1:1. Place the mixture in a 37°C water bath and react for 12 hours until the solution turns transparent blue, thus obtaining the genipin-crosslinked nano-chitosan antibacterial synergist CSNP-penicillin.

[0062] Meanwhile, genipin solution and pure chitosan solution were mixed at a volume ratio of 0.1:1 and reacted under the same conditions for 12 hours to prepare control sample nano-chitosan CSNP.

[0063] (2) MIC: Dilute drug-resistant Escherichia coli to 1×10⁵ CFU / mL and inoculate into 96-well plates. The synergist (CSNP-penicillin) and other antibacterial drugs were serially diluted using the two-fold dilution method and added to the bacterial culture, and then thoroughly mixed. After incubating the 96-well plates in a 37℃ biological incubator for 12 h, the OD600 absorbance value was measured using an ELISA reader.

[0064] (3) Image analysis: The antibacterial effect was judged based on the OD600 absorbance value as shown in Table 2. The antibacterial effect of penicillin on drug-resistant Escherichia coli was not significant. Genipin solution lost its antibacterial effect on drug-resistant Escherichia coli after 16 μg / mL. The antibacterial effect of CSNP and CSNP-penicillin was consistent with that of genipin solution.

[0065] Table 2 Example 5

[0066] (1) Mouse grouping: Fifteen adult female ICR mice, each weighing approximately 25g, were fed and divided into three groups: control group, infection group, and treatment group. The control group was fed normally without any treatment. The infection group was treated with intraperitoneal injection of drug-resistant Escherichia coli to simulate systemic bacterial infection. The treatment group was treated with nano-chitosan antibacterial synergist in addition to the treatment given to the infection group.

[0067] (2) Mouse inflammation model: Mice in the infection group and the treatment group were injected intraperitoneally with 300 μL of drug-resistant Escherichia coli (1×106 CFU / mL) to simulate a bacterial infection inflammation model. After successful infection, the mice showed reduced activity, decreased appetite, bristling hair and rapid breathing.

[0068] (3) Mouse treatment: Mice in the treatment group were treated with intraperitoneal injection of 300 μL / mouse of 8 μg / mL nano-chitosan antibacterial synergist, twice a day, for three days as one course of treatment. Mice in the infection group were treated with physiological saline.

[0069] (4) Tissue RNA extraction: After the experiment, 100 mg of liver tissue was taken from each of the three groups of mice and placed in 1 mL of Trizol reagent. The tissue was thoroughly homogenized using a tissue homogenizer, and then the homogenate was centrifuged at 4 °C and 12,000 rpm, and the supernatant was collected. 300 μL of chloroform was added to the supernatant, and the mixture was thoroughly shaken and centrifuged again at 4 °C and 12,000 rpm, and the aqueous phase was collected. An equal volume of isopropanol was added to the aqueous phase, and after standing, the mixture was centrifuged and the supernatant was discarded. The precipitate was washed with 1 mL of 75% anhydrous ethanol, centrifuged at 4 °C and 7,500 rpm, and the supernatant was discarded. After the precipitate was air-dried, it was resuspended in 20 μL of RNase-free ddH2O to complete the extraction of tissue RNA.

[0070] (5) Primer design: Interleukin-6 (IL-6): Forward primer: 5'-TAGTCCTTCCTACCCCAATTTCC-3' Reverse primer: 5'-TTGGTCCTTAGCCACTCCTTC-3' Interleukin-8 (IL-8): Forward primer: 5'-CTGGGATTCACCTCAAGAACATC-3' Reverse primer: 5'-CAGGGTCAAGGCAAGCCTC-3' Tumor necrosis factor-α (TNF-α): Forward primer: 5'-CCCTCACACTCAGATCATCTTCT-3' Reverse primer: 5'-GCTACGACGTGGGCTACAG-3' (6) RNA reverse transcription: (1) Genomic DNA removal: Add 4 μL of 4×gDNA wiper Mix and 1 μL of template RNA to the reaction tube, and supplement with RNase-free ddH2O to a total volume of 16 μL. Mix gently with a pipette and incubate at 42℃ for 2 min. (2) Reverse transcription reaction: Add 4 μL of 5×No RT Control Mix directly to the above reaction tube, mix gently, incubate at 50℃ for 15 min, and then incubate at 85℃ for 2 min to complete reverse transcription. (7) Real-time quantitative PCR: Prepare the following mixture: 10.0 μL 2×AceQ qPCR SYBR GreenMaster Mix, 0.4 μL each of forward and reverse primers (10 μM), 0.4 μL 50×ROX Reference Dye 2, 0.2 μL cDNA template, and finally add ddH2O to 20 μL. The PCR amplification program was set as follows: 95℃ pre-denaturation for 5 min; followed by 40 cycles of 95℃ denaturation for 10 s and 60℃ annealing extension for 30 s; finally, perform melting curve analysis at 95℃ for 15 s, 60℃ for 60 s, and 95℃ for 15 s.

[0071] (8) Image analysis: The acquired mouse liver images were analyzed, and the results are as follows: Figure 5 As shown in the figure, the data indicate that treatment with the nano-chitosan antibacterial synergist significantly downregulated the gene expression levels of inflammatory factors IL-6, IL-8, and TNF-α in the liver of mice. This significant effect confirms that the nano-chitosan antibacterial synergist can effectively reduce the inflammatory response in mice, thereby achieving the expected therapeutic effect. Example 6

[0072] (1) Tissue fixation: Take several mouse livers from each group after the experiment in Example 5, soak them in 5% paraformaldehyde tissue fixation solution, and store them at room temperature.

[0073] (2) HE staining of tissue: The fixed tissue was sent to Yanxuan Biotechnology (Hangzhou) Co., Ltd. for HE staining. (3) Image analysis: HE staining results are as follows Figure 6 As shown, by comparing and analyzing hematoxylin and eosin (HE) sections of mouse livers from the control group, infection group, and treatment group, the effects of *E. coli* infection on liver tissue and the recovery after treatment can be clearly observed. The control group showed normal liver tissue structure, with neatly arranged cells and no inflammatory response. The infection group showed obvious inflammatory features, including disordered hepatocyte arrangement, cell swelling, sinusoidal dilation, and inflammatory cell infiltration. The treatment group showed significant improvement compared to the infection group, with reduced inflammation and hepatocyte morphology and arrangement closer to normal. These changes indicate that *E. coli* infection does indeed cause significant liver inflammation and tissue damage, and the treatment method alleviated these pathological changes to some extent and promoted liver tissue repair.

[0074] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the present invention.

Claims

1. A method for preparing a nano-chitosan antibacterial synergist, characterized in that: include, Dissolve 100 mg of chitosan with a molecular weight of 50 kDa in 1% acetic acid. After dissolution, let it stand overnight to allow the particles to fully dissolve in the water until there are no obvious particles, thus preparing a 2 mg / mL chitosan solution. Under continuous magnetic stirring, 2.56 mL of disulfiram with a concentration of 5 mg / mL was added to 100 mL of chitosan solution. After continuous stirring, sodium tripolyphosphate solution with a concentration of 1.1 mg / mL was slowly added. The volume ratio of chitosan solution to sodium tripolyphosphate solution was 3:

1. The solution changed from transparent to turbid. The pH of the solution was then adjusted to 4.6 using 1 mol / mL NaOH to obtain the nano-chitosan antibacterial synergist.

2. The nano-chitosan antibacterial synergist prepared by the preparation method according to claim 1.

3. The application of the nano-chitosan antibacterial synergist as described in claim 2 in the preparation of veterinary drugs for treating penicillin-resistant Escherichia coli infections.

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