Preparation method of nano chitosan antibacterial synergist

Abstract

The invention discloses a preparation method of a nano chitosan antibacterial synergist, which comprises the following steps: dissolving chitosan in acetic acid, and standing overnight after dissolving until no obvious particles exist to obtain a chitosan solution; adding an antibacterial drug into the chitosan solution, performing magnetic stirring, and then adding a cross-linking agent to obtain the nano chitosan antibacterial synergist. The nano chitosan conjugated penicillin is used for the first time, the effect of the nano chitosan conjugated penicillin on clinical drug-resistant escherichia coli is verified, application possibility is provided for reducing use and drug residues of clinical drugs, and compared with a current common antibiotic treatment method, the nano chitosan conjugated penicillin has the advantages that the application range is wide; the preparation method has the advantages that the antibacterial effect is improved, the drug use amount is reduced, drug residues are reduced, the drug resistance problem is solved, and the preparation method is simple and convenient. According to the research, on the basis of drug-resistant escherichia coli, nano chitosan antibacterial synergists with different concentrations are added, and the antibacterial effect of the nano chitosan antibacterial synergists on the drug-resistant escherichia coli is evaluated. And a certain theoretical and experimental basis is provided for clinical medication.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine and animal husbandry and veterinary medicine, and specifically relates to a method for preparing a nano-chitosan antibacterial synergist. Background Art

[0002] The application of nanotechnology in the field of medicine has attracted widespread attention, especially in the field of precise drug delivery and enhanced efficacy. Nanomedicines can improve the solubility and bioavailability of drugs through their nanoscale particle size, which can theoretically significantly improve the therapeutic effect. However, in the clinical treatment of aquaculture, these nanomedicines face multiple challenges and limitations.

[0003] The stability and biocompatibility of nanomedicines are difficult to maintain in complex aquaculture environments. For example, since mastitis is often accompanied by an inflammatory response, this may change the pH value or enzyme activity of the local environment, causing the nanocarriers to quickly decompose or aggregate, thereby affecting the effective release and therapeutic effect of the drug. Secondly, the targeted release technology of existing nanomedicines is often difficult to accurately control in the treatment of mastitis, and it is difficult for the drug to reach the deep layers of the inflamed tissue, thereby reducing its antibacterial effect. In addition, the existing nanomedicine preparation process usually involves high-cost materials and complex technologies, which is particularly prominent in the economic benefit-driven aquaculture industry, limiting its widespread application.

[0004] Nano-chitosan has shown certain potential in the field of antibacterial due to its unique physical and chemical properties and biological activity. However, in the face of complex clinical environments and diverse pathogens, the antibacterial effect of existing nano-chitosan is often unsatisfactory. For example, existing nano-chitosan has a good inhibitory effect on certain specific strains, but it is difficult to deal with complex mixed infections or drug-resistant strains.

[0005] At the same time, traditional antibiotics such as penicillin are facing the challenge of increasing drug resistance in clinical applications. Simply increasing the dosage of antibiotics not only fails to effectively solve the problem of drug resistance, but may also lead to more serious problems of drug residues and environmental pollution. Therefore, it is of great significance to develop a preparation that can improve the effect of antibiotics and reduce the dosage.

[0006] In view of the above problems, it is necessary to develop a nano-chitosan antibacterial enhancer to solve the limitations of the existing technology. Summary of the invention

[0007] The purpose of this section is to summarize some aspects of embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the specification abstract and the invention title of this application to avoid blurring the purpose of this section, the specification abstract and the invention title, and such simplifications or omissions cannot be used to limit the scope of the present invention.

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

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

[0010] In order to solve the above technical problems, the present invention provides the following technical solutions: A method for preparing a nano-chitosan antibacterial synergist, characterized in that: it comprises:

[0011] The chitosan is dissolved in acetic acid, and after being dissolved, it is allowed to stand overnight until no obvious particles are present, thereby obtaining a chitosan solution; an antibacterial drug is added to the chitosan solution for magnetic stirring, and then a cross-linking agent is added to obtain a nano-chitosan antibacterial enhancer.

[0012] As a preferred embodiment of the preparation method of the present invention, the molecular weight of the chitosan solution is 5-150 kDa, and the concentration is 2 mg / mL.

[0013] As a preferred embodiment of the preparation method of the present invention, the concentration of the acetic acid is 0.5-1%.

[0014] As a preferred embodiment of the preparation method of the present invention, the antibacterial drugs include penicillin and disulfiram.

[0015] As a preferred embodiment of the preparation method of the present invention, the cross-linking agent is sodium tripolyphosphate.

[0016] As a preferred embodiment of the preparation method of the present invention, the addition ratio of the chitosan solution, the antibacterial drug and the cross-linking agent is 150-250:3-7:50-80.

[0017] As a preferred embodiment of the preparation method of the present invention, the concentration of the cross-linking agent is 1.1 mg / mL.

[0018] As a preferred embodiment of the preparation method of the present invention, the addition ratio of chitosan to acetic acid is 80-120:0.5-1.5.

[0019] Another object of the present invention is to overcome the deficiencies in the prior art and provide a nano-chitosan antibacterial synergist.

[0020] Another object of the present invention is to overcome the deficiencies in the prior art and provide a nano-chitosan antibacterial synergist for use in veterinary treatment of bacterial infections.

[0021] Beneficial effects of the present invention:

[0022] (1) The present invention discloses a method for preparing a nano-chitosan antibacterial synergist, including a method for preparing nano-chitosan and a method for conjugating an antibacterial drug with 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 to drug-resistant strains is enhanced; compared with the use of penicillin alone, the synergist can achieve the same or better antibacterial effect at a lower concentration, which helps to reduce the excessive use of antibiotics; due to the reduced dosage, the drug residue in the environment can be effectively reduced, reducing the potential impact on the ecosystem.

[0023] (2) The present invention adopts simple cross-linking and conjugation technology, the preparation process is simple and convenient, the cost is low, and it is conducive to large-scale production and application.

[0024] (3) The present invention uses nano-chitosan conjugated penicillin for the first time, and its effect has been verified on clinical drug-resistant Escherichia coli, providing a possible application for reducing the use of clinical drugs and drug residues. Compared with the currently commonly used antibiotic treatment methods, the present invention has the advantages of improving antibacterial effect, reducing drug usage, reducing drug residues, coping with drug resistance problems and simple preparation methods. This study is based on drug-resistant Escherichia coli, and by adding different concentrations of nano-chitosan antibacterial enhancers, its antibacterial effect on drug-resistant Escherichia coli is evaluated. It is expected to provide a certain theoretical and experimental basis for clinical medication. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor. Among them:

[0026] Figure 1 This is a transmission electron microscope TEM image of the nano-chitosan antibacterial synergist in Example 1 of the present invention.

[0027] Figure 2 This is the Fourier transform infrared spectrum of the nano-chitosan antibacterial synergist in Example 1 of the present invention.

[0028] Figure 3 This is the antibacterial MIC graph of the nano-chitosan antibacterial synergist in Example 3 of the present invention against penicillin-resistant Escherichia coli.

[0029] Figure 4 This is a 24-hour growth curve of the nano-chitosan antibacterial synergist against penicillin-resistant Escherichia coli in Example 3 of the present invention.

[0030] Figure 5This is a graph showing the mRNA expression of inflammatory factors in the liver of mice after treatment with the nano-chitosan antibacterial enhancer in Example 5 of the present invention.

[0031] Figure 6 This is a HE slice image of mouse liver after treatment with nano-chitosan antibacterial synergist in Example 6 of the present invention.

[0032] Figure 7 This is the antibacterial MIC graph of penicillin-resistant Escherichia coli of the nano-chitosan antibacterial synergist (CS-T-disulfiram) in Example 2 of the present invention.

[0033] Figure 8 The antibacterial MIC graph of the nano-chitosan antibacterial synergist in Comparative Examples 3, 4, and 5 of the present invention against drug-resistant Escherichia coli (resistant to penicillin / ceftiofur). DETAILED DESCRIPTION

[0034] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0035] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0036] 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 term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0037] Unless otherwise specified, all raw materials used in the examples of the present invention are commercially available, as shown in Table 1 for details.

[0038] Table 1

[0039]

[0040]

[0041] Example 1

[0042] The present invention provides a method for preparing a nano-chitosan antibacterial synergist:

[0043] (1) Preparation of synergist: 100 mg of chitosan with a molecular weight of 50 kDa was dissolved in 1% acetic acid, and the solution was allowed to stand overnight to allow the particles to fully dissolve in water until no obvious particles were left, thereby preparing a 2 mg / mL chitosan solution;

[0044] Under continuous magnetic stirring, add 2.56 mL of 5 mg / mL penicillin to 100 mL of chitosan solution, continue stirring, and then slowly add 1.1 mg / mL sodium tripolyphosphate solution in a volume ratio of 3:1. The solution changes from transparent to turbid, and then adjust the pH of the solution to 4.6 using 1 mol / mL NaOH.

[0045] (2) Sample preparation: The prepared nano-chitosan antibacterial synergist was placed in a vacuum freeze dryer to obtain freeze-dried powder. An appropriate amount of freeze-dried powder was taken and the morphology was observed using a Tecnai 12 transmission electron microscope (TEM).

[0046] (3) Image analysis: Transmission electron microscopy results are as follows: Figure 1 As shown in the figure, the nano-chitosan antibacterial synergist exhibits an obvious granular structure, with an average particle size of about 150nm, successfully reaching the nanoscale.

[0047] Example 2

[0048] The difference from Example 1 is that penicillin is replaced by disulfiram.

[0049] (1) Preparation of synergist: 100 mg of chitosan with a molecular weight of 50 kDa was dissolved in 1% acetic acid, and the solution was allowed to stand overnight to allow the particles to fully dissolve in water until no obvious particles were left, thereby preparing a 2 mg / mL chitosan solution;

[0050] Under continuous magnetic stirring, add 2.56 mL of 5 mg / mL disulfiram to 100 mL of chitosan solution, continue stirring, and then slowly add 1.1 mg / mL sodium tripolyphosphate solution. The volume ratio of the two is 3:1. The solution changes from transparent to turbid. Then, use 1 mol / mL NaOH to adjust the pH of the solution to 4.6.

[0051] (2) MIC: dilute the resistant E. coli to 1×10 5 CFU / mL, inoculated into a 96-well plate. The synergist (CS-T-disulfiram) and other antibacterial drugs were diluted step by step using the two-fold dilution method and added to the bacterial solution and mixed thoroughly. The 96-well plate was placed in a 37°C biological incubator for 12 hours and the OD was measured using an enzyme reader. 600 Absorbance value.

[0052] (3) Image analysis: OD 600 The absorbance value is plotted as a MIC trend graph, and the results are as follows Figure 7 As shown, the control group (MH broth) OD 600The absorbance value is about 0.05. Disulfiram has no significant inhibitory effect on drug-resistant Escherichia coli, even at a concentration of 64 μg / mL. CS-T-disulfiram represents a nano-chitosan antibacterial enhancer conjugated with disulfiram. The results showed that the minimum inhibitory concentration of CS-T-disulfiram (16 μg / mL) was consistent with that of CS-T, and the OD 600 The absorbance value is lower than that of CS-T, which proves that the nano-chitosan antibacterial synergist has a stronger antibacterial effect than using disulfiram and nano-chitosan alone.

[0053] Example 3

[0054] (1) Sample preparation: 0.1 g of the freeze-dried powder prepared in Example 1 was taken and subjected to infrared spectroscopy analysis of the sample using a potassium bromide (KBr) tabletting method and a Cary 610 / 670 infrared microspectrometer (IR).

[0055] (2) Image analysis: The results obtained by infrared microscopy are plotted using origin. The results are as follows: Figure 2 As shown in Figure 2, infrared spectroscopy analysis reveals the structural evolution of chitosan from its original state to nano-scale and then to drug conjugation. Chitosan exhibits typical -OH, -NH2 and C=O stretching vibration peaks at 3434cm-1, 2871cm-1 and 1651cm-1.

[0056] After cross-linking with sodium tripolyphosphate, the spectrum of nano-chitosan showed significant changes: the peak intensities of -OH and -NH2 weakened, indicating that these groups participated in the cross-linking reaction; at the same time, P=O and POC vibration peaks appeared at 1068cm-1 and 885cm-1, confirming the successful introduction of sodium tripolyphosphate. The nano-chitosan after penicillin conjugation retained the characteristic peaks of the cross-linked structure, but there were slight changes in the 1600-1500cm-1 and 1100-1000cm-1 regions, especially the COC and CO stretching vibration peaks at 1054cm-1 increased intensities, which can be attributed to the successful conjugation of penicillin. This series of spectral changes not only confirmed the successful preparation of nano-chitosan, but also provided strong spectroscopic evidence for the effective loading of penicillin.

[0057] Comparative Example 1 (Penicillin)

[0058] (1) 100 mg of chitosan with a molecular weight of 50 kDa was dissolved in 1% acetic acid, and the solution was allowed to stand overnight to allow the particles to fully dissolve in water until no obvious particles were left, thereby preparing a 2 mg / mL chitosan solution; under the action of continuous magnetic stirring, 2.56 mL of penicillin with a concentration of 5 mg / mL was added to 100 mL of the chitosan solution, and the solution was stirred continuously until completely mixed, and then the pH of the solution was adjusted to 4.6 using 1 mol / M NaOH to obtain a penicillin enhancer.

[0059] Comparative Example 2 (CS-T)

[0060] (1) 100 mg of chitosan with a molecular weight of 50 kDa was dissolved in 1% acetic acid, and the solution was allowed to stand overnight to allow the particles to fully dissolve in water until no obvious particles were left, thereby preparing a 2 mg / mL chitosan solution; under the action of continuous magnetic stirring, 33 mL of sodium tripolyphosphate with a concentration of 1.1 mg / mL was added to 100 mL of the chitosan solution, and the solution was continuously stirred until it changed from transparent to turbid, and then the pH of the solution was adjusted to 4.6 using 1 mol / mL NaOH to obtain a nano-chitosan synergist.

[0061] Example 4

[0062] (1) Bacterial culture: Escherichia coli was isolated from milk samples of patients with clinical mastitis. The isolated strain was inoculated into sterile MH medium and cultured until the bacterial solution concentration reached 1×10 8 CFU / mL, the cultured bacteria were spread on MH agar plates and penicillin-resistant E. coli strains were screened using the KB disk diffusion method according to the CLSI guidelines.

[0063] (2) MIC: dilute the resistant E. coli to 1×10 5 CFU / mL, inoculate into 96-well plates. Use the two-fold dilution method to dilute the enhancer step by step and add it to the bacterial solution, mix thoroughly. Place the 96-well plate in a 37℃ biological incubator for 12 hours, and use an enzyme reader to measure the OD 600 Absorbance value.

[0064] (3) 24-hour growth curve: dilute the resistant E. coli to 1×10 5 After CFU / mL, place in a shaker at 37°C and measure OD with an enzyme marker every 2 hours. 600 The absorbance values ​​were measured when penicillin, nano-chitosan and nano-chitosan antibacterial enhancer were added at 4h and 8h of culture, respectively.

[0065] (4) Image analysis: OD 600 The absorbance value is plotted as a MIC trend graph, and the results are as follows Figure 3As shown, the OD value of the control group (MH broth) was about 0.05. The inhibitory effect of penicillin on drug-resistant Escherichia coli was not significant, even at a concentration of 64 μg / mL, the inhibitory effect was not significant; when the concentration was lower than 32 μg / mL, there was almost no inhibitory effect. According to the CLSI standard, this strain meets the characteristics of penicillin-resistant Escherichia coli. CS-T represents nano-chitosan cross-linked with TPP, and CS-T-penicillin represents nano-chitosan antibacterial enhancer conjugated with penicillin. The results showed that the minimum inhibitory concentration of CS-T-penicillin (8 μg / mL) was better than that of CS-T, proving that the nano-chitosan antibacterial enhancer has a stronger antibacterial effect than penicillin and nano-chitosan alone.

[0066] OD 600 The absorbance value is used to draw a 24h growth curve trend chart, and the results are as follows Figure 4 As shown, the control group was the natural growth group of bacteria. 8 μg / mL of penicillin was added to the resistant Escherichia coli during the 4h growth phase and the 8h logarithmic phase, respectively. It was found that the growth of the resistant Escherichia coli was not effectively inhibited. After adding 8 μg / mL CS-T-penicillin, the growth of the resistant Escherichia coli was inhibited. CS-T did not have an ideal effect in the 8h bacterial logarithmic growth phase.

[0067] Comparative Example 3

[0068] The difference from Example 1 is that penicillin is replaced by ceftiofur.

[0069] The KB disk diffusion method was used to screen out ceftiofur-resistant Escherichia coli strains according to the CLSI guidelines.

[0070] (1) Image analysis: OD 600 The absorbance value is plotted as a MIC trend graph, and the results are as follows Figure 8 As shown, the OD value of the control group (MH broth) was about 0.05, and the inhibitory effect was not significant.

[0071] Comparative Example 4

[0072] The difference from Example 1 is that penicillin is replaced by epigallocatechin gallate.

[0073] (1) Image analysis: OD 600 The absorbance value is plotted as a MIC trend graph, and the results are as follows Figure 8 As shown, the OD value of the control group (MH broth) was about 0.05, and the inhibitory effect was not significant.

[0074] Comparative Example 5

[0075] The difference from Example 1 is that penicillin is replaced by metformin.

[0076] (1) Image analysis: OD600 The absorbance value is plotted as a MIC trend graph, and the results are as follows Figure 8 As shown, the OD value of the control group (MH broth) was about 0.05, and the inhibitory effect was not significant.

[0077] Comparative Example 6

[0078] The difference from Example 1 is that the cross-linking agent is genipin.

[0079] (1) Preparation of synergist: Weigh 100 mg of genipin powder, dissolve it in 50 mL of 20% anhydrous ethanol solution, shake it thoroughly to obtain a 2 mg / mL genipin solution. Add 2.56 mL of 5 mg / mL penicillin solution to 100 mL of chitosan solution and stir evenly. Subsequently, 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 to react for 12 hours until the solution turns transparent blue, and obtain the nano-chitosan antibacterial synergist CSNP-penicillin cross-linked by genipin.

[0080] Meanwhile, the genipin solution was mixed with the pure chitosan solution at a volume ratio of 0.1:1 and reacted for 12 h under the same conditions to prepare the control sample nano-chitosan CSNP.

[0081] (2) MIC: dilute the resistant E. coli to 1×10 5 CFU / mL, inoculated into a 96-well plate. The synergist (CSNP-penicillin) and other antibacterial drugs were diluted step by step using the two-fold dilution method and added to the bacterial solution and mixed thoroughly. The 96-well plate was placed in a 37°C biological incubator for 12 hours and the OD was measured using an enzyme reader. 600 Absorbance value.

[0082] (3) Image analysis: According to OD 600 The antibacterial effect determined by the absorbance value is shown in Table 2. The antibacterial effect of penicillin on drug-resistant Escherichia coli is not significant. The antibacterial effect of genipin solution on drug-resistant Escherichia coli is lost after 16 μg / mL, while the antibacterial effect of CSNP and CSNP-penicillin is consistent with that of genipin solution.

[0083] Table 2

[0084]

[0085]

[0086] Example 5

[0087] (1) Mouse grouping: 15 adult female ICR mice, each weighing about 25 g, were raised and divided into three groups, namely, control group, infection group, and treatment group. The control group was raised normally without any treatment, the infection group was intraperitoneally injected with drug-resistant Escherichia coli to simulate systemic bacterial infection, and the treatment group was treated with nano-chitosan antibacterial enhancer based on the treatment of the infection group.

[0088] (2) Mouse inflammation model: 1×10 6 The resistant Escherichia coli (CFU / mL) was intraperitoneally injected at 300 μL / mouse to simulate the bacterial infection inflammation model. The mice successfully infected showed reduced activity, decreased appetite, erect hair and accelerated breathing.

[0089] (3) Treatment of mice: The mice in the treatment group were treated with 8 μg / mL nano-chitosan antibacterial enhancer (300 μL / mouse) by intraperitoneal injection, twice a day, for a course of three days. The mice in the infection group were treated with normal saline injection.

[0090] (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 fully ground using a tissue homogenizer, and then the homogenate was centrifuged at 4°C and 12,000 rpm to obtain the supernatant. 300 μL of chloroform was added to the supernatant, and after sufficient shaking and mixing, the mixture was centrifuged again at 4°C and 12,000 rpm to collect the aqueous phase. An equal volume of isopropanol was added to the aqueous phase, and the mixture was allowed to stand and then 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 naturally dried, it was resuspended in 20 μL RNase-free ddH2O to complete the extraction of tissue RNA.

[0091] (5) Primer design:

[0092] Interleukin-6 (IL-6):

[0093] Forward primer: 5'-TAGTCCTTCCTACCCCAATTTCC-3'

[0094] Reverse primer: 5'-TTGGTCCTTAGCCACTCCTTC-3'

[0095] Interleukin 8 (IL-8):

[0096] Forward primer: 5'-CTGGGATTCACCTCAAGAACATC-3'

[0097] Reverse primer: 5'-CAGGGTCAAGGCAAGCCTC-3'

[0098] Tumor necrosis factor-α (TNF-α):

[0099] Forward primer: 5'-CCCTCACACTCAGATCATCTTCT-3'

[0100] Reverse primer: 5'-GCTACGACGTGGGCTACAG-3'

[0101] (6) RNA reverse transcription: (1) Genomic DNA removal: Add 4 μL 4×gDNA wiper Mix and 1 μL template RNA to the reaction tube, and add RNase free ddH2O to a total volume of 16 μL. Mix gently with a pipette and incubate at 42°C for 2 min. (2) Reverse transcription: Add 4 μL 5×No RT Control Mix directly to the above reaction tube, mix gently, incubate at 50°C for 15 min, and then incubate at 85°C for 2 min to complete reverse transcription.

[0102] (7) Real-time fluorescence quantitative PCR: Prepare the following mixture: 10.0 μL 2×AceQ qPCR SYBR Green Master 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°C pre-denaturation for 5 min; followed by 40 cycles of 95°C denaturation for 10 s, 60°C annealing and extension for 30 s; and finally, melting curve analysis was performed at 95°C for 15 s, 60°C for 60 s, and 95°C for 15 s.

[0103] (8) Image analysis: The acquired mouse liver fluorescence quantitative PCR images were analyzed. The results are as follows: Figure 5 The data showed that after treatment with nano-chitosan antibacterial enhancer, the gene expression levels of inflammatory factors IL-6, IL-8, and TNF-α in the mouse liver were significantly downregulated. This significant effect confirmed that nano-chitosan antibacterial enhancer can effectively reduce the inflammatory response in mice, thereby achieving the expected therapeutic effect.

[0104] Example 6

[0105] (1) Tissue fixation: After the experiment in Example 8, a portion of the mouse livers from each group were taken and immersed in 5% paraformaldehyde tissue fixative and stored at room temperature.

[0106] (2) HE staining of tissue: The fixed tissue was sent to Yanxuan Biotechnology (Hangzhou) Co., Ltd. for HE staining.

[0107] (3) Image analysis: HE staining results are as follows Figure 6 As shown in the figure, by comparing and analyzing the HE sections of mouse livers in 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, while the infection group showed obvious inflammatory characteristics, including disordered arrangement of hepatocytes, cell swelling, dilation of hepatic sinusoids, and infiltration of inflammatory cells. The treatment group showed significant improvement compared with the infection group, with reduced inflammation and closer hepatocyte morphology and arrangement to normal. This series of changes showed that E. coli infection did cause significant liver inflammation and tissue damage, and the treatment methods adopted alleviated these pathological changes to a certain extent and promoted the repair of liver tissue.

[0108] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the present invention.

Claims

1. A method for preparing a nano-chitosan antibacterial synergist, characterized in that: include, Dissolving chitosan in acetic acid, and leaving it to stand overnight until no obvious particles remain, to obtain a chitosan solution; Antibacterial drugs are added into the chitosan solution for magnetic stirring, and then a cross-linking agent is added to obtain a nano-chitosan antibacterial synergist.

2. The preparation method according to claim 1, characterized in that: The molecular weight of the chitosan solution is 5-150 kDa, and the concentration is 2 mg / mL.

3. The preparation method according to claim 1, characterized in that: The concentration of the acetic acid is 0.5-1%.

4. The preparation method according to claim 1, characterized in that: The antibacterial drugs include penicillin and disulfiram.

5. The preparation method according to claim 1, characterized in that: The cross-linking agent is sodium tripolyphosphate.

6. The preparation method according to claim 1, characterized in that: The addition ratio of the chitosan solution, the antibacterial drug and the cross-linking agent is 150-250:3-7:50-80.

7. The preparation method according to claim 5, characterized in that: The concentration of the cross-linking agent was 1.1 mg / mL.

8. The preparation method according to claim 3, characterized in that: The addition ratio of chitosan to acetic acid is 80-120:0.5-1.

5.

9. The nano-chitosan antibacterial synergist prepared by the preparation method according to claims 1 to 8.

10. Use of the nano-chitosan antibacterial synergist as claimed in claim 9 in veterinary treatment of bacterial infection diseases.

Images