Application of bacteriocin F in the preparation of products for inhibiting Helicobacter pylori

Abstract

The present invention provides an application of bacteriocin F in the preparation of a product for inhibiting Helicobacter pylori, belonging to the technical field of biomedicine. The amino acid sequence of bacteriocin F is shown in SEQ ID NO.1, and bacteriocin F has an obvious inhibitory effect on Helicobacter pylori. At the same time, the present invention also provides a preparation method of a nano-composite hydrogel loaded with bacteriocin F. The prepared nano-composite hydrogel has rheological properties, self-healing properties and injectability, and also has a degradation ability, so as to effectively release bacteriocin F. The nano-composite hydrogel improves the therapeutic efficiency of bacteriocin F on the expression of proteins such as the total number of white blood cells in the whole blood of Hp-infected mice, the ratio of PGⅠ / PGⅡ in serum, inflammatory factor IL-10, gastric tissue NF-κB, IκB-α, p-NF-κB, p-IκB-α, etc. The hydrogel, bacteriocin F, and the nano-composite hydrogel loaded with bacteriocin F effectively relieve the inflammation caused by Hp infection by inhibiting the activation of the TLR4 / Myd88 / NF-κB pathway.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the application of bacteriocin F in the preparation of products for inhibiting Helicobacter pylori. Background Art

[0002] Helicobacter pylori (Hp) is a curved spiral-shaped Gram-negative bacterium colonized on the gastric mucosa. The population infected with Hp in the world may develop into indigestion, chronic gastritis, peptic ulcer and gastric cancer. An effective measure to prevent gastric cancer is to prevent and eradicate Hp infection. At present, the treatment of Hp mainly relies on the triple therapy and quadruple therapy composed of the combined use of antibacterial drugs (such as amoxicillin, clarithromycin, metronidazole and levofloxacin), anti-secretory drugs (proton pump inhibitors) and bismuth agents. However, the use of antibiotics has led to the gradual increase of the drug resistance rate and the continuous emergence of problems such as intestinal flora disorder. Therefore, Hp has problems such as high infection rate, high drug resistance rate and difficult clearance. Finding new therapeutic drugs and reducing the drug resistance rate are still urgent problems to be solved at present.

[0003] Bacteriocin is a small peptide with antibacterial activity synthesized by the ribosomal pathway of bacteria. Most bacteriocins are secreted by probiotics. Because of its high antibacterial activity, green safety and other characteristics, it is considered an ideal alternative to antibiotics. This bacteriocin is rapidly decomposed after entering the human body, will not cause intestinal flora disorder, and plays an antibacterial role against strains with similar genetic relationships, and can specifically inhibit specific pathogenic bacteria. However, more and more bacteriocins with broad-spectrum antibacterial effects have been reported, such as Nisin synthesized by lactic acid bacteria. Such bacteriocins can inhibit food spoilage bacteria, animal and human pathogenic bacteria and even drug-resistant pathogenic bacteria. It has been found that NisinA, lactacin a164 and BH5, etc. all have antibacterial effects on Hp. However, most bacteriocins are sensitive to the environment and proteases, and the sequences and action mechanisms of many bacteriocins are unknown, which limits their application in vivo. And all pathogens tend to continuously adapt to environmental changes, such as biofilm formation. Bacteria targeted by bacteriocin will also form components resistant to bacteriocin when continuously exposed. For example, enzymes secreted by pathogens can degrade bacteriocin, resulting in bacteriocin drug resistance, which is the same as antibiotic resistance and is also a catastrophic phenomenon. And pathogens tend to continuously adapt to environmental changes to produce drug resistance. Whether bacteriocin can stably exist in the complex environment of the gastrointestinal tract is still unknown. Therefore, preventing bacteriocin from producing drug resistance, being able to stably exist in gastric juice, and improving the action efficiency of bacteriocin are the key steps for its application transformation.

[0004] Hydrogels have good biocompatibility and biodegradability due to their unique hydrophilicity and three-dimensional spatial structure, and are widely used in the biomedical field. Hydrogels have stimulus responsiveness to temperature, pH, concentration, or the external environment through different cross-linking methods. Therefore, encapsulating and wrapping bioactive substances in drugs with hydrogels can effectively protect their biological activity in environments such as the oral cavity, stomach, small intestine, colon, and skin and release them at specific targets, thus significantly improving the therapeutic effect of the active substances. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide an application of bacteriocin F in the preparation of products for inhibiting Helicobacter pylori.

[0006] To achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0007] The present invention provides an application of bacteriocin F in the preparation of products for inhibiting Helicobacter pylori, and the amino acid sequence of the bacteriocin F is as shown in SEQ ID NO.1.

[0008] The present invention provides an application of bacteriocin F in the preparation of products for treating diseases caused by Helicobacter pylori infection, and the amino acid sequence of the bacteriocin F is as shown in SEQ ID NO.1.

[0009] Preferably, the disease includes gastritis.

[0010] The present invention provides a preparation method of a nano-composite hydrogel loaded with bacteriocin F, including the following steps:

[0011] Mix and dissolve nano-clay with water, then add dopamine hydrochloride for reaction. After forming polydopamine, add bacteriocin F with the amino acid sequence as shown in SEQ ID NO.1, and mix evenly to obtain a nano-composite hydrogel loaded with bacteriocin F.

[0012] Preferably, the mass-volume ratio of the nano-clay, dopamine hydrochloride to water is 2 - 3 g: 0.2 - 1 g: 90 - 110 mL.

[0013] Preferably, the temperature of the reaction is room temperature, and the reaction time is more than 5 h;

[0014] Preferably, the mass ratio of dopamine hydrochloride to bacteriocin F is 450 - 550: 40 - 60.

[0015] The present invention also provides a nano-composite hydrogel loaded with bacteriocin F prepared by the above preparation method.

[0016] The present invention also provides an application of the above nano-composite hydrogel loaded with bacteriocin F in the preparation of products for inhibiting diseases caused by Helicobacter pylori infection.

[0017] Preferably, the disease includes gastritis.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention provides an application of bacteriocin F in the preparation of a product for inhibiting Helicobacter pylori. It has been found that bacteriocin F has an obvious inhibitory effect on Helicobacter pylori. At the same time, the present invention also provides a preparation method of a nanocomposite hydrogel loaded with bacteriocin F. The prepared nanocomposite hydrogel has rheological properties, self-healing properties and injectability, and also has a degradation ability, so as to effectively release bacteriocin F. The nanocomposite hydrogel improves the treatment efficiency of bacteriocin F on the protein expressions such as the total number of white blood cells in the whole blood of Hp-infected mice, the ratio of PGⅠ / PGⅡ in serum, inflammatory factor IL-10, gastric tissue NF-κB, IκB-α, p-NF-κB, p-IκB-α, etc. The hydrogel, bacteriocin F, and the nanocomposite hydrogel loaded with bacteriocin F effectively relieve the inflammation caused by Hp infection by inhibiting the activation of the TLR4 / Myd88 / NF-κB pathway. The nanocomposite hydrogel of the present invention not only promotes the application transformation of bacteriocin F, but also provides a new strategy for increasing the application fields of hydrogels. And the preparation method of the present invention is simple and easy to operate, and is suitable for industrial production. Description of the Drawings

[0020] Figure 1 Are the characterization results of the nanocomposite hydrogel loaded with bacteriocin F. A is the time scan curve of the nanocomposite hydrogel loaded with bacteriocin F, B is the dynamic strain result of the nanocomposite hydrogel loaded with bacteriocin F at 37 °C, C is the viscosity curve of the nanocomposite hydrogel loaded with bacteriocin F, D is the degradation rate of the nanocomposite hydrogel loaded with bacteriocin F in simulated gastric juice; E is the morphology of the nanocomposite hydrogel loaded with bacteriocin F magnified 1.0k times under a scanning electron microscope; F is the morphology of the nanocomposite hydrogel loaded with bacteriocin F magnified 2.0k times under a scanning electron microscope;

[0021] Figure 2 Are the effects of different concentrations of bacteriocin F on the number of Hp bacteria. *, indicates comparison of each group with the Nc group; **, P < 0.01; ***, P < 0.001;

[0022] Figure 3Effects of different groups on the body weight, liver, spleen and kidney weights of Hp-infected mice. A shows the effect on the body weight of Hp-infected mice treated with different groups, B shows the effect on the liver weight of Hp-infected mice treated with different groups, C shows the effect on the spleen weight of Hp-infected mice treated with different groups, and D shows the effect on the kidney weight of Hp-infected mice treated with different groups. Among them, *, indicates comparison with the Hp group; ns, no significant difference; *, P<0.05; **, P<0.01; ****, P<0.0001

[0023] Figure 4 Results of blood routine and serum ELISA tests on Hp-infected mice treated with different groups. A shows the effect on the total white blood cells in whole blood of Hp-infected mice treated with different groups, B shows the effect on IL-1β in serum of Hp-infected mice treated with different groups, C shows the effect on IL-10 in serum of Hp-infected mice treated with different groups, and D shows the effect on PGⅠ / PGⅡ in serum of Hp-infected mice treated with different groups. Among them, *, indicates comparison with the Hp group; ns, no significant difference; *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001;

[0024] Figure 5 Results of gastric tissue pathological examinations of Hp-infected mice treated with different groups. Among them, A shows representative hematoxylin-eosin staining images of gastric tissues of Hp-infected mice treated with different groups, and B shows the effect on the pathological scores of hematoxylin-eosin staining of gastric tissues of Hp-infected mice treated with different groups;

[0025] Figure 6 Effects of immunohistochemical results on the protein expressions of NF-κB and IκB-α in gastric tissues of Hp-infected mice treated with different groups. A shows representative immunohistochemical staining images of NF-κB protein expression in gastric tissues of Hp-infected mice treated with different groups, B shows representative immunohistochemical staining images of IκB-α protein expression in gastric tissues of Hp-infected mice treated with different groups, C shows the average optical density value of NF-κB protein expression in gastric tissues of Hp-infected mice treated with different groups, and D shows the average optical density value of IκB-α protein expression in gastric tissues of Hp-infected mice treated with different groups. Among them, * indicates comparison with the Hp group; ***, P<0.001; ****, P<0.0001;

[0026] Figure 7In A, it shows the effect of different groups of treatments on the NF-κB mRNA level in the gastric tissue of Hp-infected mice; in B, it shows the effect of different groups of treatments on the Myd88 mRNA level in the gastric tissue of Hp-infected mice; in C, it shows the effect of different groups of treatments on the IL-1β mRNA level in the gastric tissue of Hp-infected mice; in D, it shows the effect of different groups of treatments on the TLR4IL-10 mRNA level in the gastric tissue of Hp-infected mice; in E, it shows the effect of different groups of treatments on the TNF-α mRNA level in the gastric tissue of Hp-infected mice; in F, it shows the effect of different groups of treatments on the IL-10 mRNA level in the gastric tissue of Hp-infected mice, where *, indicates comparison with the Hp group; ns, no significant difference; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Detailed implementation manners

[0027] The present invention provides an application of bacteriocin F in the preparation of a product for inhibiting Helicobacter pylori, and the amino acid sequence of the bacteriocin F is shown as SEQ ID NO.1.

[0028] In the present invention, the bacteriocin F is isolated, purified and identified from Lactobacillus casei, and has good acid-base stability and protease stability. The present invention researches and discovers that bacteriocin F can significantly inhibit Helicobacter pylori. In the present invention, the product includes a reagent, a drug or a vaccine. In the present invention, the bacteriocin F can be used as the sole active ingredient to inhibit Helicobacter pylori, or can act in combination with other active ingredients for inhibiting Helicobacter pylori to inhibit Helicobacter pylori. The drug also includes pharmaceutically acceptable excipients, and the bacteriocin F accounts for more than 40% of the drug. The excipients include one or more of a shaping agent, a flavoring agent, a preservative, a solvent, etc. The product can be used by oral administration or injection.

[0029] The present invention provides an application of bacteriocin F in the preparation of a product for treating diseases caused by Helicobacter pylori infection, and the amino acid sequence of the bacteriocin F is shown as SEQ ID NO.1.

[0030] In the present invention, the product includes a reagent, a drug or a vaccine. The diseases include gastritis.

[0031] The present invention uses the weakly alkaline oxygen-containing environment of the nano-clay dispersion liquid to pre-polymerize dopamine monomers to form polydopamine, and mixes the polydopamine with bacteriocin F to improve the rheological properties of the dopamine / nano-clay aggregates, thereby forming a novel nano-composite hydrogel that can not only depolymerize and release bacteriocin F under acidic conditions, but also stably exist under physiological conditions.

[0032] Based on this, the present invention provides a preparation method of a nano-composite hydrogel loaded with bacteriocin F, comprising the following steps:

[0033] Mix and dissolve nano-clay with water, then add dopamine hydrochloride for reaction. After forming polydopamine, add bacteriocin F with the amino acid sequence shown in SEQ ID NO.1, and mix well to obtain a nano-composite hydrogel loaded with bacteriocin F.

[0034] In the above preparation method, mix and dissolve nano-clay with water, then add dopamine hydrochloride for reaction to form polydopamine. The mass-volume ratio of the nano-clay, dopamine hydrochloride to water is preferably 2 - 3 g: 0.2 - 1 g: 90 - 110 mL, more preferably 2.2 - 2.8 g: 0.3 - 0.7 g: 95 - 105 mL, and even more preferably 2.5 g: 0.5 g: 100 mL. The nano-clay is also called lithium magnesium silicate, and its structural formula is Na 0.7 Si8Mg 5.5 Li 0.3 O 20 (OH)4 is a nano-material with a lamellar structure that has a negatively charged surface and a positively charged edge. The nano-clay dispersion obtained by mixing and dissolving nano-clay with water in the present invention can directly polymerize dopamine in a weakly alkaline oxygen-containing environment, and no other strong oxidants need to be introduced during the reaction process. Moreover, the interaction between the surface charge of the nano-clay and the drug molecules can achieve the controlled release of the drug molecules, and it can easily interact with the charge on the surface of bacteria to enhance the bactericidal effect. There are no special limitations on the sources of the nano-clay and dopamine hydrochloride in the present invention, and known methods in the art or commercially available products can be used.

[0035] In the present invention, the temperature of the reaction is room temperature, and the reaction time is more than 5 h. The mass ratio of dopamine hydrochloride to bacteriocin F is preferably 450 - 550:40 - 60, more preferably 470 - 520:45 - 55, and even more preferably 500:50.

[0036] The present invention also provides a nano-composite hydrogel loaded with bacteriocin F prepared by the above preparation method.

[0037] The present invention also provides an application of the above nano-composite hydrogel loaded with bacteriocin F in the preparation of products for inhibiting diseases caused by Helicobacter pylori infection.

[0038] In the present invention, the products include reagents, drugs or vaccines. The products can be used by oral administration or injection. In the present invention, the nano-composite hydrogel loaded with bacteriocin F can be used as the sole active ingredient to inhibit diseases caused by Helicobacter pylori infection, or can act in combination with other active ingredients that inhibit Helicobacter pylori to inhibit diseases caused by Helicobacter pylori infection. The diseases include gastritis.

[0039] In the present invention, unless otherwise specified, all raw material components are commercially available products well-known to those skilled in the art.

[0040] The technical solutions provided by the present invention will be described in detail below in conjunction with embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0041] In the following embodiments, the Helicobacter pylori is derived from Daoyan Wu # ; Mei Cao # ; Jie Zhou; Shiying Yan; Jingshan Peng; Zhihao Yu; Andong Zhang; Jialin Wu; Xin Yan; Jian Zhao * ; the Helicobacter pylori described in Lactobacillus casei T1 from kurut against Helicobacter pylori-induced inflammation and the gut microbial disorder, Journal of Functional Foods, 2021, 85C: 104611.

[0042] The nano-clay is also called lithium magnesium silicate.

[0043] The animals are SPF-grade healthy adult male BALB / c mice, 6 - 8 weeks old, weighing 18 - 22 g, purchased from the Experimental Animal Center of Guizhou Medical University, and the animal production license number is SYXK(Qian)2018 - 0001. Before the experiment, the mice were adaptively fed for one week, and the mice drank water and ate freely. All the mice were maintained in a controlled environment (20 - 22 °C, 40 - 60% humidity, 12 h light / dark cycle).

[0044] Example 1

[0045] A preparation method of a nano-composite hydrogel loaded with bacteriocin F comprises the following steps:

[0046] Add 100 mL of deionized water to 2.5 g of nano-clay, stir and dissolve completely at room temperature, then add 0.5 g of dopamine hydrochloride and stir and dissolve completely. Stir at room temperature for more than 5 h to make dopamine hydrochloride fully react to form polydopamine. Finally, add 50 mg of bacteriocin F and stir until a homogeneous state is obtained, to obtain a nano-composite hydrogel loaded with bacteriocin F, wherein the amino acid sequence of bacteriocin F is MDSLKTLLVANRGEIV (SEQ ID NO.1).

[0047] Characterization and analysis of the nanocomposite hydrogel loaded with bacteriocin F: When the rheometer parameters reached the set values and were stable, 1-2 g of the nanocomposite hydrogel loaded with bacteriocin F was weighed and placed into a mixer. Data was saved and the motor was stopped when the expected state was obtained by observing and recording the curve. The instrument was cleaned and the equipment was turned off. An appropriate amount of the nanocomposite hydrogel loaded with bacteriocin F was quenched with liquid nitrogen and freeze-dried, and its cross-section was observed and photographed under an electron microscope. 2 mL of hydrochloric acid was taken and 10 g of pepsin was added. After shaking evenly, it was diluted with water to 500 mL to obtain simulated gastric juice. An appropriate amount of hydrogel was placed in the simulated gastric juice and placed in a shaker at 37 °C and 120 rpm to simulate its environment in the stomach. The remaining hydrogel in the simulated gastric juice was weighed at different time points.

[0048] The results are as Figure 1 shown. The time sweep curve, dynamic strain at 37 °C, and viscosity curve of the nanocomposite hydrogel of the present invention indicate that it has rheological properties, self-healing properties, and injectability (see A-C in Figure 1 ). The degradation rate of the nanocomposite hydrogel in simulated gastric juice at the 96th hour was 75% (see D in Figure 1 ). The three-dimensional network structure of the nanocomposite hydrogel could be observed under the electron microscope at 1.0k times and 2.0k times (E-F in Figure 1 ), and it has the ability to carry substances.

[0049] Example 2

[0050] (1) Antibacterial experiment of bacteriocin F with the amino acid sequence shown in SEQ ID NO.1 against Hp

[0051] Single colonies of Hp bacteria growing in the logarithmic phase on the plate were resuspended in a liquid medium containing 10% fetal bovine serum until the absorbance value at OD 600 nm was 0.4-0.5. They were evenly dispensed into sterile shaking tubes, and bacteriocin F with the amino acid sequence shown in SEQ ID NO.1 was added to make its final concentration 0 μg / mL (control group), 50 μg / mL, 200 μg / mL, and 500 μg / mL. After incubation in a microaerophilic constant temperature shaker at 120 rpm, 37 °C, and 10% CO2 for 24 h, the bacterial solution was serially diluted with sterile PBS, and 10 μL was spotted on a sterile blood agar plate. After culturing for 72 h, counting was performed and expressed as CFU / mL.

[0052] Figure 2 The results showed that compared with the control group, bacteriocin F had a significant inhibitory effect on the number of Hp bacteria at concentrations of 50 μg / mL, 200 μg / mL, and 500 μg / mL (P<0.05).

[0053] (2) Establishment and grouping of the Hp-infected mouse model:

[0054] The mice were randomly divided into Nc group, Hp group, HpH group, HpF group, HpHF group, and HpA group, with 12 mice in each group. Before gavage treatment, they were fasted overnight. During the first two-week modeling stage, Nc was the control group and was gavaged with normal saline. In the Hp group, HpH group, HpF group, HpHF group, and HpA group, each mouse was gavaged with 0.25 mL of NaHCO3 with a concentration of 1.8 mg / mL to neutralize gastric acid, which was beneficial for better colonization of Hp in gastric tissue. One hour later, each group was gavaged with 300 μL of Hp live bacteria suspension with a McFarland concentration greater than 5.0. After gavage every other day for 2 weeks, two mice were randomly sacrificed, and the pyloric part of the gastric tissue was taken for urease test paper detection and nested PCR detection to determine the Hp colonization situation in the mice and verify the successful colonization of Hp in the mice. During the next two-week treatment stage, each mouse in the HpH group was gavaged with 300 μL of hydrogel, each mouse in the HpF group was gavaged with 300 μL of bacteriocin F with an amino acid sequence as shown in SEQ ID NO.1 at a concentration of 50 μg / mL, each mouse in the HpHF group was gavaged with 300 μL of the nano-composite hydrogel loaded with bacteriocin F prepared in Example 1, and each mouse in the HpA group was gavaged with 300 μL of 50 μg / mL antibiotic amoxicillin. After gavage every other day for two consecutive weeks, the mice were anesthetized and sacrificed. The weights of the mice in each group were recorded separately. At the same time, the livers, kidneys, and spleens of the mice in each group were collected and weighed. Whole blood, serum, gastric tissue, and intestinal contents of the mice in each group were collected for subsequent detection.

[0055] The preparation method of the hydrogel is as follows: 100 mL of deionized water was added to 2.5 g of nano-clay. After stirring and dissolving completely at room temperature, 0.5 g of hydrochloric acid dopamine was added and stirred and dissolved completely. Stirring was carried out at room temperature for more than 5 h to make hydrochloric acid dopamine react fully to form polydopamine, and the hydrogel was obtained.

[0056] (3) Bacteriocin F and its nano-composite hydrogel can relieve weight loss, and enlargement of the liver, kidneys, and spleen caused by Hp infection

[0057] As Figure 3 shown, compared with the control group, after the mice were infected with Hp, their weights decreased slightly. After treatment with the hydrogel, bacteriocin F, and the nano-composite hydrogel loaded with bacteriocin F, the weight loss situation was alleviated (see Figure 3 A in). Compared with the control group, after the mice were infected with Hp, the ratios of their livers, kidneys, and spleens to their weights increased. After treatment with the hydrogel, bacteriocin F, and the nano-composite hydrogel loaded with bacteriocin F, the enlargement of the liver, kidneys, and spleen was improved (see Figure 3 B-D in).

[0058] (4) Effects of bacteriocin F and its hydrogel on blood routine and serum ELISA test results of Hp-infected mice

[0059] Using a fully automatic blood cell analyzer to analyze the complete blood count of the whole blood of mice after treatment in each group, and detecting the levels of inflammatory markers in the serum of mice after treatment in each group by ELISA: Take out the frozen serum, operate according to the kit instructions, detect the contents of inflammatory markers IL-1β, IL-10, PGⅠ and PGⅡ in the serum of mice, and count the PGⅠ / PGⅡ ratio after treatment in each group.

[0060] Figure 4 The results showed that after mice were infected with Hp, compared with the control group, the total white blood cell count in the whole blood increased. After treatment with hydrogel, bacteriocin F, nano-composite hydrogel loaded with bacteriocin F and amoxicillin, the white blood cell count decreased. Among them, the nano-composite hydrogel loaded with bacteriocin F and amoxicillin had the best effect on alleviating the white blood cell count in mice (P<0.05). Compared with the control group, after mice were infected with Hp, the content of inflammatory factor IL-1β in the serum increased (P<0.05). After treatment, it decreased in each group. Among them, bacteriocin F and amoxicillin treatment significantly decreased the content of IL-1β in the serum (P<0.0001). After mice were infected with Hp, the anti-inflammatory factor IL-10 decreased. After treatment, the concentration of IL-10 in the serum increased in the nano-composite hydrogel loaded with bacteriocin F and amoxicillin group (P<0.001). The PGⅠ / PGⅡ ratio is closely related to the process of Hp infection and the treatment of atrophic gastritis and gastric cancer. When Hp is infected, gastric mucosa atrophy and gastric cancer occur, the PGⅠ / PGⅡ ratio decreases. After mice were infected with Hp, the PGⅠ / PGⅡ ratio decreased significantly (P<0.0001). After treatment in each group, the ratio increased significantly (P<0.01). Among them, bacteriocin F had the best alleviating effect.

[0061] (5) Effects of bacteriocin F and its nano-composite hydrogel on the pathological examination results of the gastric tissues of Hp-infected mice

[0062] HE staining to examine the pathological changes of the gastric tissues of mice: The gastric tissues of mice fixed in 4% paraformaldehyde were embedded in paraffin and cut into 5μm thin sections. After staining with hematoxylin-eosin staining method (HE staining), a section scanner was used to observe the infiltration of inflammatory cells in the gastric mucosa tissue and the morphology of the gastric mucosa.

[0063] Morphological detection of mouse gastric tissues:

[0064] Scoring criteria: There are 5 histological change grades for chronic gastritis, namely Hp, activity, inflammatory reaction, atrophy and intestinal metaplasia, which are divided into 4 grades of none, mild, moderate and severe (0, +, ++, +++). The grading criteria adopt the pathological diagnosis criteria of chronic gastritis in China and the visual analogue scoring method of the new Sydney system.

[0065] Observation of chronic gastritis in Hp-infected mice: According to the chronic inflammation scoring criteria, the degree of inflammation was quantitatively scored by observing the chronic inflammatory cells in the lamina propria of the mucosal field of the pathological section. 0 - no inflammatory cell infiltration in the lamina propria; 1 - a small amount of inflammatory cell infiltration in the lamina propria; 2 - a moderate amount of inflammatory cell infiltration in the lamina propria; 3 - a large amount of inflammatory cell infiltration in the lamina propria.

[0066] Figure 5 The results showed that compared with the control group, after mice were infected with Hp, under the microscope at 100 times magnification, the mucosal wall of the gastric tissue was observed to be thinned, the mucosal epithelium was irregularly arranged, local columnar epithelium was damaged and shed (black arrow), obvious inflammatory cell infiltration was visible in the mucosal layer (blue arrow), connective tissue hyperplasia accompanied by inflammatory cell infiltration was visible in the submucosal layer (yellow arrow), and a small amount of inflammatory cell infiltration was visible in the locally ruptured muscular layer (green arrow). The treatment with hydrogel and amoxicillin did not improve the gastritis caused by Hp infection, and bacteriocin F and the nanocomposite hydrogel loaded with bacteriocin F improved slightly.

[0067] (6) Effects of bacteriocin F and its nanocomposite hydrogel on immunohistochemical results of inflammatory factors in the gastric tissue of Hp-infected mice

[0068] Immunohistochemical detection of mouse gastric tissue:

[0069] Section dewaxing: Place the paraffin sections successively into xylene I, xylene II, xylene III, absolute ethanol I, absolute ethanol II, 95% alcohol, 90% alcohol, 80% alcohol, 70% alcohol, and wash in distilled water for 5 min; Antigen retrieval: Place the dewaxed and hydrated tissue sections on a heat-resistant plastic section rack in a beaker, add an appropriate amount of retrieval solution to the beaker, the liquid level should be higher than the section tissue. First, heat with high gear until the liquid boils, then adjust to medium gear, start timing for 15 min of retrieval. After the sample is cooled to room temperature in cold water, take out the glass slide and rinse it 3 times with PBS with a pH of 7.4, 3 min each time; Block endogenous peroxidase: Drop 3% hydrogen peroxide solution onto the section tissue and incubate at room temperature for 15 min, then rinse with PBS 3 times, 3 min each rinse; Serum blocking: Dry the glass slide with absorbent paper, drop the diluted goat serum, and block at room temperature for 30 min; Primary antibody incubation: Drain the excess liquid, drop the diluted primary antibody (the dilution ratio of IκB-α is 1:200, and the dilution ratio of NF-κB p65 is 1:100), then place the incubation box at 4°C and incubate overnight; Enzyme-labeled secondary antibody incubation: Rinse the sections with PBS 3 times, 3 min each time. After drying the sections with absorbent paper, drop the secondary antibody and incubate at 37°C for 30 min; Rinse the sections with PBS 4 times, 3 min each time, dry the sections, drop the freshly prepared chromogenic solution on each section, observe under the microscope, the positive signal is yellowish-brown or brownish-black, and rinse the sections with tap water to terminate the chromogenesis; Counterstain with mayer hematoxylin for 2 min, then wash and blue with PBS solution; Dehydration: Place the sections successively into 70% alcohol, 80% alcohol, 90% alcohol, 95% alcohol, absolute ethanol I, absolute ethanol II, xylene I, xylene II for dehydration and clearing for 5 min, and finally place the sections in a ventilated place to air dry; Sealing: Drop neutral balsam beside the tissue, cover with a coverslip, and make sure no bubbles are generated. Place the sealed sections flat in a ventilated place to dry; Microscopic examination: The dried sections can be observed and images can be collected under the microscope.

[0070] NF-κB and IκB-α are two key transcription factors that regulate inflammation in the NF-κB signaling pathway.

[0071] Figure 6 The results showed that after mice were infected with Hp, the immunohistochemical results of gastric tissue showed that the NF-κB protein was activated and the expression of IκB-α protein decreased (P < 0.001). After treatment with hydrogel, bacteriocin F, and nano-composite hydrogel loaded with bacteriocin F, the activation of NF-κB protein could be inhibited and the expression of IκB-α protein could be promoted. Among them, the nano-composite hydrogel loaded with bacteriocin F had the best alleviating effect (P < 0.0001). It can be seen that bacteriocin F and the nano-composite hydrogel loaded with bacteriocin F may reduce Hp-induced gastric mucosal inflammatory damage by regulating the NF-κB signaling pathway.

[0072] (6) Effects of bacteriocin F and its nano-composite hydrogel on mRNA and protein levels of inflammatory factors in gastric tissues of Hp-infected mice

[0073] RT-qPCR detection of mouse gastric tissues:

[0074] Gastric tissue RNA was extracted by the traditional TRIzol method, and the extracted RNA was detected for purity and concentration using NanoDrop 2000. Qualified samples were reverse transcribed into cDNA according to the kit instructions. The RT-qPCR system was 10 μL of 2× Mix, 1 μL each of upstream and downstream primers (primer sequences are shown in Table 1), 1 μL of cDNA, and 10 μL of enzyme-free water. The reaction program was 95°C for 30 s, 95°C for 5 s, and 60°C for 30 s, for a total of 40 cycles. Using Gapdh as an internal reference, the expression level was calculated by the 2 ^(-ΔΔCt) method.

[0075] Table 1 Primer sequences

[0076]

[0077] Figure 7 The results showed that the mRNA levels of NF-κB, Myd88, and TNF-α were significantly increased in the gastric tissues of Hp-infected mice (P < 0.05). After treatment with hydrogel, bacteriocin F, nano-composite hydrogel loaded with bacteriocin F, and amoxicillin, the mRNA levels of NF-κB, Myd88, and TNF-α could be inhibited. After mice were infected with Hp, the mRNA levels of IL-1β and TLR4 in gastric tissues were significantly increased (P < 0.05), while after treatment with hydrogel, bacteriocin F, nano-composite hydrogel loaded with bacteriocin F, and amoxicillin, the mRNA levels of IL-1β and TLR4 could be decreased. In addition, the anti-inflammatory factor IL-10 decreased after Hp infection, and the levels of IL-10 increased after treatment with hydrogel, bacteriocin F, nano-composite hydrogel loaded with bacteriocin F, and amoxicillin.

[0078] In summary, the nano-composite hydrogel loaded with bacteriocin F improved the therapeutic efficiency of bacteriocin F on the protein expressions of total white blood cell count in whole blood of Hp-infected mice, the ratio of PGⅠ / PGⅡ in serum, inflammatory factor IL-10, gastric tissue NF-κB, IκB-α, p-NF-κB, p-IκB-α, etc. Hydrogel, bacteriocin F, and nano-composite hydrogel loaded with bacteriocin F effectively alleviated the inflammation caused by Hp infection by inhibiting the activation of the TLR4 / Myd88 / NF-κB pathway and could regulate the intestinal flora disorder caused by Hp infection.

[0079] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Use of a bacteriocin F-loaded nanocomposite hydrogel in the preparation of a product for inhibiting Helicobacter pylori infection, characterized in that, The preparation method of the nano-composite hydrogel loaded with bacteriocin F comprises the following steps: mixing and dissolving nano-clay with water, then adding dopamine hydrochloride for reaction. After forming polydopamine, bacteriocin F with the amino acid sequence shown in SEQ ID NO.1 is added and mixed evenly to obtain the nano-composite hydrogel loaded with bacteriocin F; The mass ratio of the dopamine hydrochloride to the bacteriocin F is 450-550:40-60; The mass-volume ratio of the nano-clay, the dopamine hydrochloride to the water is 2-3 g: 0.2-1 g: 90-110 mL; the temperature of the reaction is room temperature, and the reaction time is more than 5 h.

2. The application according to claim 1, wherein The product is a product for inhibiting gastritis caused by Helicobacter pylori infection.

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