CN112080444A

CN112080444A - Lactic acid bacteria for preventing and treating gastritis caused by helicobacter pylori and application thereof

Info

Publication number: CN112080444A
Application number: CN202010841680.5A
Authority: CN
Inventors: 顾青; 郦萍; 周青青; 薛冰尧; 洪维鍊; 赵雯; 刘福东
Original assignee: Zhejiang Gongshang University
Current assignee: Zhejiang Gongshang University
Priority date: 2020-08-20
Filing date: 2020-08-20
Publication date: 2020-12-15

Abstract

The lactobacillus for preventing and treating gastritis caused by helicobacter pylori is lactobacillus plantarum ZJ316 with the deposit number of CCTCC No: m208077.

Description

Lactic acid bacteria for preventing and treating gastritis caused by helicobacter pylori and application thereof

Technical Field

The invention relates to lactic acid bacteria, in particular to lactic acid bacteria for preventing and treating gastritis caused by helicobacter pylori and application thereof.

Background

Helicobacter pylori generally has two morphologies in nature. One is rod-like or spiral, which is mostly in the growth phase, and the other is globular, which is in the aging or death phase. The pathogenicity of pyrori can be divided into high virulence strains and low virulence strains according to their virulence characteristics. The first is H⁺VacA⁺It is highly toxic and pathogenic, and can easily cause various stomach disorders and even gastric cancer, and the second type is H^-VacA^-It has weak toxicity or no toxicity, no special symptom and no pathogenic ability. Long-term infection with H.pylori causes chronic gastritis, which, if left untreated, can ultimately lead to gastrointestinal ulcerationAnd the occurrence of gastric cancer, it is important to pay attention to^[9]。

Helicobacter pylori can colonize and cause diseases in the stomach for a long time, and has a great relationship with the physiological characteristics of helicobacter pylori. Because helicobacter pylori itself has characteristics of motility, acid resistance, adhesion, generation of virulence factors, and the like. Pyrori has motility due to its flagellar structure. The formation of flagella, the synthesis of Lipopolysaccharide (LPS) and the glycosylation of proteins are closely related to two genes, flaA 1 and WbpB, on terminal flagella. Pyhori, a mutant with some gene variation due to special reasons, cannot colonize the gastric mucosal surface in animals and plants because of its nonflagellation. Human gastrointestinal tract is always in a highly acidic environment, and h. A number of experimental reports have demonstrated that h.pyrori can produce a urease agent which hydrolyses urea in the stomach and reacts to produce CO₂And ammonia, so that the pH value around the thalli is increased, a neutral microenvironment is formed around the H.pyrori, the effect of neutralizing gastric acid is achieved, and the H.pyrori is protected from being invaded by gastric acid or dying^[10]. Pylori is able to colonize the stomach for a long period of time and is not excreted out of the body due to peristalsis of the stomach, because it is adhesive. Pylori's surface is attached with many proteins that can generate a variety of adhesion molecules. Such as: neutrophil activating protein, neuraminyl lactose binding fibril hemagglutinin (h. pyroria a), AlpA, AlpB, BabA, lipopolysaccharide linked Lewis antigens. The most direct cause of H.pyrori is due to its important role played by virulence factors. Virulence factors include mainly vacuolar toxin (VacA) and vacuolar toxin-associated protein (CagA protein). Vacuolar toxins can cause damage to the gastric mucosa because they can bind to specific receptors, causing vacuoles and even membrane perforation of cells. Vacuolar toxin can also cause apoptosis because it can affect cell surface signaling, preventing repair functions of surface epithelial cells. The vacuolating toxin related protein does not express the related ability of damaging cell membranes and even apoptosis toxin, but has a certain correlation with the expression of the vacuolating toxinIt is favorable for H.pyrori to be planted on gastric mucosa for a long time and destroy gastric epithelial cells, and is related to gastritis, duodenal ulcer and gastric epithelial metaplasia

After the body is infected by Helicobacter pylori (Helicobacter pylori), various stomach diseases can be caused. The infection rate of H.pyri in adults is up to 40-60%, and the health and life quality of people are seriously threatened. Therefore, there is an urgent need to find a prophylactic or therapeutic regimen for H.pylori infection without side effects.

Disclosure of Invention

An object of the present invention is to provide a lactic acid bacterium for preventing and treating helicobacter pylori-induced gastritis and use thereof, wherein the lactic acid bacterium can be used for preventing or treating helicobacter pylori-induced gastritis.

Another object of the present invention is to provide a lactic acid bacterium for preventing and treating helicobacter pylori-induced gastritis, wherein the lactic acid bacterium can be prepared into food, and use thereof.

According to one aspect of the present invention, there is provided a lactic acid bacterium for the prevention and treatment of helicobacter pylori-induced gastritis, which is lactobacillus plantarum ZJ316 deposited under the accession number CCTCC No: m208077.

According to an embodiment of the present invention, the lactic acid bacteria can reduce infiltration of inflammatory cells.

According to an embodiment of the invention, the lactic acid bacteria are capable of increasing the abundance of beneficial bacterial groups in the treatment of helicobacter pylori induced gastritis.

According to an embodiment of the invention, the lactic acid bacteria are capable of increasing the abundance of beneficial bacterial groups in the prevention of helicobacter pylori induced gastritis.

According to an embodiment of the present invention, the lactic acid bacteria are capable of reducing the abundance of pathogenic bacteria in the prevention of helicobacter pylori-induced gastritis.

According to an embodiment of the invention, the lactic acid bacteria are capable of reducing the abundance of the pathogenic flora in the treatment of helicobacter pylori induced gastritis.

According to an embodiment of the present invention, the lactic acid bacteria can increase glutathione content in the liver in the prevention of helicobacter pylori-induced gastritis.

According to an embodiment of the present invention, the lactic acid bacteria are capable of increasing glutathione content in the liver in the treatment of helicobacter pylori-induced gastritis.

According to one embodiment of the present invention, the lactic acid bacteria are capable of up-regulating the expression levels of proinflammatory cytokines IL-6 and IFN- γ in the gastric mucosa in the prevention of helicobacter pylori-induced gastritis.

According to one embodiment of the invention, the lactic acid bacteria are capable of up-regulating the expression levels of the proinflammatory cytokines IL-6 and IFN-gamma in the gastric mucosa in the treatment of helicobacter pylori-induced gastritis.

According to an embodiment of the present invention, the lactic acid bacteria refer to a culture solution containing a combination of one or both of a cell surface substance and a cell secretion substance.

According to another aspect of the present invention, the present invention provides a use of lactobacillus plantarum ZJ316 for preventing and treating helicobacter pylori-induced gastritis, CCTCC No: m208077.

According to an embodiment of the invention, the lactic acid bacteria are used for increasing the abundance of beneficial bacterial groups.

According to one embodiment of the invention, the lactic acid bacteria are used to up-regulate the expression of the proinflammatory cytokines IL-6 and IFN-gamma in the gastric mucosa.

According to an embodiment of the present invention, the lactic acid bacteria are used for increasing glutathione content in the liver.

According to another aspect of the present invention, there is provided a food product comprising a lactic acid bacterium and an edible material, the lactic acid bacterium being lactobacillus plantarum ZJ316, CCTCC No: m208077.

According to an embodiment of the present invention, the edible material is selected from one or more of a group consisting of combined water, fluid milk, condensed milk, yogurt, frozen yogurt, lactobacillus fermented beverage, milk powder, ice cream, cheese, soy milk, fermented soy milk, vegetable juice, fruit juice, sports drink, dessert, jelly, candy, baby food, health food, animal feed, herbal composition, and dietary supplement.

According to an embodiment of the invention, the lactic acid bacteria are live bacteria or dead bacteria.

Drawings

Figure 1 is the treatment time point for the preventive group.

Figure 2 is a graph of body weight dynamics of mice in different groups of the prevention group.

FIG. 3 shows the results of HE staining of gastric mucosa in mice in the prevention group.

Fig. 4 shows the change in gastric mucositis factor in mice, indicating that P <0.05 is significant compared to YH.

Fig. 5 is an OTU cumulative graph.

Fig. 6 is an OUT Rank curve.

FIG. 7 is a graph of the distribution of mouse gastric microflora at the "phylum" level.

FIG. 8 is a plot of the distribution of mouse gastric microflora at the "genus" level.

FIGS. 9A, 9B are beta-diversity heat maps, respectively: fig. 9A is based on unweighted distance and fig. 9B is based on weighted distance.

Fig. 10A and 10B are the LEfse analysis notes for the prevention group, respectively: FIG. 10(A) is a clustering tree diagram, and FIG. 10(B) is a LAD score obtained by LAD analysis of statistically significant microorganism groups in different groups, with different colors representing different groups.

FIG. 11 is a graph of prophylaxis of helicobacter pylori anemometry.

Fig. 12 is treatment group treatment time points.

Figure 13 is the body weight dynamics of mice in different groups of the treatment group.

FIG. 14 shows the results of HE staining of gastric mucosa in mice in the treatment group.

Fig. 15 shows the change in gastric mucositis factor in mice, indicating that P <0.05 is significant compared to YH.

Fig. 16 is an OTU cumulative graph.

Fig. 17 is an OUT Rank curve.

FIG. 18 is a plot of the distribution of mouse gastric microflora at the "phylum" level.

FIG. 19 is a plot of the distribution of mouse gastric microflora at the "genus" level.

FIGS. 20A, 20B are beta-diversity heat maps, respectively: fig. 20A is based on unweighted distance and fig. 20B is based on weighted distance.

Fig. 21A and 21B are the LEfse analysis notes for the prevention group, respectively: fig. 21(a) is a clustering tree diagram. FIG. 21(B) is a LAD score obtained by LAD analysis of statistically significant microorganism groups in different groups, with different colors representing different groups.

FIG. 22 is a graph of the treatment group helicobacter pylori grade.

Detailed Description

The following description is presented to disclose the invention so as to enable any person skilled in the art to practice the invention. The preferred embodiments in the following description are given by way of example only, and other obvious variations will occur to those skilled in the art. The basic principles of the invention, as defined in the following description, may be applied to other embodiments, variations, modifications, equivalents, and other technical solutions without departing from the spirit and scope of the invention.

It is understood that the terms "a" and "an" should be interpreted as meaning that a number of one element or element is one in one embodiment, while a number of other elements is one in another embodiment, and the terms "a" and "an" should not be interpreted as limiting the number.

According to one aspect of the present invention, there is provided a lactic acid bacterium, wherein said lactic acid bacterium can be further added with an edible material to prepare a food product, wherein the edible material includes, but is not limited to: water (water), fluid milk products (fluid milk products), milk (milk), concentrated milk (concentrated milk); fermented milks (fermented milks), such as yogurt (yogurt), yogurt (sour milk), frozen yogurt (yogurt), fermented milk-based beverages (lactic acid bacteria-fermented beverages); milk powder (milk powder); ice cream (icecream); cheese (cheese); cheese (dry cheese); soymilk (soy milk); fermented soybean milk (fermented soybean milk); vegetable and fruit juices (vegetable-fruit juices); fruit juices (juices); sports drinks (sports drinks); dessert (confection); jelly (jellies); candies (confections); infant food (infant formula); health foods (health foods); animal feeds (animal feeds); chinese herbal medicines (Chinese herbases); dietary supplements (dietary supplements), and the like.

The edible material used in the present invention may be a dietary supplement, which may be administered to a user in the following manner: mixing with a suitable drinkable liquid, such as water, yogurt, milk or fruit juice; or may be mixed with solid or liquid food products. In the present specification, the dietary supplement may be in the form of tablets, pills, capsules, dragees (lozenes), granules, powders, suspensions, sachets, pastilles, candies, syrups and corresponding administration forms, typically in unit dose form, and manufactured in a conventional manner for preparing dietary supplements.

The lactic acid bacteria provided by the invention can be culture solution of dead bacteria or live bacteria.

The following examples are illustrative only, and dosages may vary depending on variations, without limitation, the activity of the compound employed, the disease or physiological state being treated, the mode of administration, the individual requirements, the severity of the disease, and the judgment of the practitioner.

According to another aspect of the invention, the invention is based on the research on the effect of the lactobacillus plantarum ZJ316 on the helicobacter pylori infection C57BL/6J mouse model through the prevention effect, and the improvement effect of the lactobacillus plantarum ZJ316 on the gastritis symptoms of the mice is evaluated by detecting various indexes of immune response and gastric microecology, and the main conclusion is as follows:

1. the lactobacillus plantarum ZJ316 has no influence on the weight, normal diet and drinking water and activity of mice infected by helicobacter pylori through the prevention effect.

2. The lactobacillus plantarum ZJ316 reduces the infiltration condition of inflammatory cells through a preventive effect, remarkably reduces the expression level of proinflammatory cytokines (IL-6 and IFN-gamma) in the gastric mucosa of a mouse, and up-regulates the expression level of the inflammatory cytokines (IL-10).

3. The Lactobacillus plantarum ZJ316 can increase the diversity and abundance of beneficial flora such as Lactobacillus in gastric mucosa of the gastritis mice through a prevention effect, and simultaneously reduce the abundance of pathogenic flora such as Helicobacter and Clostridium, thereby being beneficial to maintaining the balanced and stable state of the gastric microecological flora of the mice.

4. The content of glutathione in the liver can be reduced after helicobacter pylori infects mice, and the lactobacillus plantarum ZJ316 can improve the content of glutathione in the liver through a preventive effect and reduce the oxidative damage of a host caused by helicobacter pylori infection.

According to another aspect of the invention, the invention is based on the research on the effect of the Lactobacillus plantarum ZJ316 on the helicobacter pylori infection C57BL/6J mouse model through treatment effect, and the improvement effect of the Lactobacillus plantarum ZJ316 on the gastritis symptoms of the mice is evaluated by detecting various indexes of immune response and gastric microecology, and the main conclusion is as follows:

1. the Lactobacillus plantarum ZJ316 has no influence on the weight, normal diet and drinking water and activity of mice infected by helicobacter pylori through treatment.

2. The lactobacillus plantarum ZJ316 reduces the infiltration condition of inflammatory cells through a treatment effect, and remarkably reduces the expression level of proinflammatory cytokines (IL-6 and IFN-gamma) in the gastric mucosa of the mouse.

3. The Lactobacillus plantarum ZJ316 can increase the diversity and abundance of beneficial flora such as Lactobacillus and Roseburia in gastric mucosa of the gastritis mice through treatment, and simultaneously reduce the abundance of pathogenic flora such as Prevotella and Helicobacter, thereby being beneficial to maintaining the balanced and stable state of gastric microecological flora of the mice.

4. The helicobacter pylori can cause the reduction of the glutathione content in the liver after infecting mice, and the lactobacillus plantarum ZJ316 can improve the glutathione content in the liver through the treatment effect and reduce the oxidative damage of a host caused by the helicobacter pylori infection.

The Lactobacillus plantarum ZJ316(Lactobacillus plantarum ZJ316) is preserved in China center for type culture Collection with the address: china Wuhan university, preservation number CCTCC No: m208077, deposited on 2008/5/23 days, isolated from infant faeces.

The specific research method and technical effects are as follows:

research on prevention of mouse infection with helicobacter pylori by lactobacillus plantarum ZJ316Experimental materials and methods

Experimental strains

Lactobacillus plantarum ZJ316 and Lactobacillus rhamnosus LGG were stored in the laboratory. Helicobacter pylori (Helicobacter pylori ZJC03) is isolated from gastric mucosal tissue of patients with gastritis and peptic ulcer, who are treated by Shore Hospital of medical college of Zhejiang university.

Laboratory animal

Female SPF (Specific pathogen free) grade C57BL/6J mice of 6 weeks of age were selected, 48 mice were purchased from Shanghai Sphere-BiKai laboratory animals Co., Ltd, and were bred in animal houses of laboratory animal departments of public health clinical centers in Shanghai city after purchase. The temperature of the animal room is 24 +/-2 ℃, and padding, feed and drinking water need to be replaced every week under the environment of 12 hours of day and night alternation. Wherein the drinking water and the padding need to be sterilized at high temperature and high pressure. The feed was purchased from shanghai shilin biotechnology limited.

Main reagent and consumable

TABLE 1 Experimental reagents and consumables

Tab.1 List of main reagents and consumables

Laboratory apparatus and device

TABLE 2 Main instruments and Equipment

Tab.2 List of main equipments

Preparation of culture medium

Preparation of MRS liquid culture medium: 52.4g of the medium was weighed, dissolved in 1000mL of ultrapure water by heating, and autoclaved at 121 ℃ for 15 minutes for use.

Preparation of MRS solid culture medium: 52.4g of the medium was weighed, 1.5% agar was added thereto, dissolved in 1000mL of ultrapure water by heating, and autoclaved at 121 ℃ for 15 minutes for use.

Columbia blood agar base (CAB) medium: 9g of the medium was weighed, dissolved in 1000mL of ultrapure water by heating, and autoclaved at 121 ℃ for 15 minutes for use.

Brain Heart Infusion (BHI) broth culture: weighing 25g of the culture medium, heating and dissolving in 500mL of ultra-0 pure water, and autoclaving at 121 ℃ for 15 minutes for later use.

Experimental methods and contents

Activation and culture of bacterial species

(1) Activation and culture of lactic acid bacteria

Thawing lactic acid bacteria frozen in a glycerol tube at the temperature of minus 80 ℃ at room temperature, picking bacterial liquid in the glycerol tube by using an inoculating loop, streaking and inoculating on a sterilized MRS solid plate, sealing the plate by using a sealing film, putting the plate into an incubator at the temperature of 37 ℃ for 24h for culture and resuscitation, repeating the steps, after twice subculture activation, picking single bacterial colony of the lactic acid bacteria into an MRS liquid culture medium, carrying out static culture at the constant temperature of 37 ℃ for 16-18h, centrifuging the bacterial liquid for 10min at the speed of 8000r/min, and collecting supernatant for later use. Washing the bacterial sludge twice with normal saline, and re-suspending with normal saline to reach viable count of about 1 × 10⁹CFU/mL. And (5) standby.

(2) Activation and culture of helicobacter pylori

Freezing helicobacter pylori in glycerol tube at-80 deg.C, thawing at room temperature, streaking and inoculating on CAB blood agar plate containing antibiotic and 7% sterile sheep blood, and placing into anaerobic box (85% N) containing micro-nutrition bag₂、10％CO₂、5％O₂) Culturing at 37 deg.C for 48-72 h for resuscitation, activating for two generations, and washing with BHI liquid culture medium to obtain 1 × 10⁸CFU/ml bacterial suspension for use.

Establishment of gastritis model of helicobacter pylori-preventing mouse

48C 57BL/6J mice, after 1 week of acclimatization, were divided into 6 groups of 8 mice each, and the mice were treated under normal diet conditions, specifically as follows:

(1) YH group (n ═ 8): the normal saline of the stomach is 400 mu L/one per day for 21 days, and the BHI of the stomach is 400 mu L/one three times after 21 days, and the times are separated every other day.

(2) YGH group (n ═ 8): the lactobacillus plantarum ZJ 316400 mu L/one stomach is used for 21 days, and the stomach is irritated BHI 400 mu L/one stomach for three times after 21 days, and the times are separated every other day.

(3) YTH group (n ═ 8): the lactobacillus rhamnosus LGG is administrated at a dose of 400 muL/piece/day for 21 days, and the BHI is administrated at a dose of 400 muL/piece three times after 21 days, once every other day.

(4) YG group (n ═ 8): gavage of normal saline 400. mu.L/body/day for 21 days, and after 21 days, suspension of H.pyri bacteria 400. mu.L/body three times, once every other day.

(6) YT group (n ═ 8): the lactobacillus plantarum ZJ 316400 mu L/one stomach for 21 days, and the H.pyri bacterial suspension 400 mu L/one stomach for three times after 21 days, once every other day.

(7) YC group (n ═ 8): the lactobacillus rhamnosus LGG is 400 mu L/piece/day for 21 days, and the H.pyrori bacterial suspension is 400 mu L/piece three times after 21 days and is used once every other day.

Mice were weighed once a week during the animal experiments and observed for active growth status. After the molding is finished, feeding is carried out for 35 days without treatment of normal diet, the mice are killed by a vertebral dislocation method, and the mice are dissected in a biological safety cabinet. Aseptically excising gastric fundus, washing the remaining gastric mucosa tissue with sterile normal saline, dividing into three parts, placing in a freezing tube, treating with liquid nitrogen, refrigerating at-80 deg.C, and measuring the experimental index. Meanwhile, taking out the liver to prepare tissue homogenate for measuring the glutathione index. Wherein, the gastric mucosa of the H.pylori molding group (YG group) needs to be immediately put into a urease indicator to confirm whether the H.pylori infection mouse model is successfully established. The treatment time of the preventive group can be referred to as shown in FIG. 1.

Gastric mucosal tissue section, H&E staining and tissue damage evaluation

(1) Preparation of Paraffin section of gastric mucosa

The preparation process of the paraffin section mainly comprises the steps of sampling, fixing, removing fixing liquid, dehydrating, transparentizing, waxing, finishing and slicing.

Sampling and fixing: about 50mg of gastric mucosa is taken and fixed in a general tissue fixing solution (neutral) for 18-24 h.

Removing the stationary liquid: the fixed tissue was then rinsed with running water for 24h to remove the fixative.

And (3) dehydrating: ethanol with different concentration gradients is selected as a dehydrating agent to dehydrate gastric mucosa tissues. 70% ethanol (30min), 80% ethanol (30min), 90% ethanol (30min), 95% ethanol (20min) and 100% ethanol (20min)

And (3) transparency: placing the dehydrated colon tissue in mixed solution of alcohol and xylene at equal ratio for 15min, and soaking in xylene I and xylene II for 15min to complete transparency;

waxing, trimming and slicing: the colon tissue after the transparency was paraffin-embedded by EG1160 paraffin embedding machine (come card). The embedded colon tissue is solidified by paraffin, and the paraffin block is trimmed according to the position of the small tissue block; the trimmed paraffin blocks were sliced (5 μm) using an RM2235 rotary slicer (come card).

(2) H & E staining of gastric mucosa sections

The H & E staining step of the gastric mucosa slice mainly comprises the steps of spreading and fishing slices, baking slices, hydrating, dehydrating, hematoxylin staining, differentiating, rinsing, eosin counterstaining, dehydrating, transparentizing and sealing slices, and the specific operation is as follows:

and (3) displaying and fishing pieces: placing the cut gastric mucosa slices in a constant-temperature water bath at 45 ℃ for spreading, and carefully fishing out the spread slices by using a glass slide;

baking slices: placing the slices in an oven at 37 ℃ for baking overnight;

hydration: xylene I15min, xylene II 5min, xylene: anhydrous ethanol (1:1) for 2min

And (3) dehydrating: 100% ethanol I for 5min, 100% ethanol II for 5min, 80% ethanol for 5min, and distilled water for 5min

Staining sappan wood semen: washing with hematoxylin staining solution for 5min and 30min

Differentiation and rinsing: 1% hydrochloric acid ethanol 30s, water washing 30s, and distilled water washing 5s

Eosin counterstaining: dyeing with 0.5% eosin solution for 1-3min

And (3) dehydrating: washing with distilled water for 30s, 80% ethanol for 30s, 95% ethanol I for 1min, 95% ethanol II for 1min, anhydrous ethanol I for 3min, and anhydrous ethanol II for 3min

And (3) transparency: xylene I3 min, xylene II 3min

Sealing: encapsulating with neutral gum. After the end of mounting, the plate was observed by an optical microscope with NIKON Eclipse Ci and photographed. The imaging system is as follows: NIKON digital sight DS-FI2, photographic magnification: 400X and 200X. 4 groups of the tissue are randomly selected for tissue damage evaluation of gastric mucosa tissue sections.

Determination of inflammatory factors in gastric mucosal tissue

(1) Extraction of Total RNA from gastric mucosal tissue

1) A homogenate tube was taken, 1mL of Trizol Reagent was added, and the mixture was placed on ice for precooling.

2) 100mg of tissue was taken and added to the homogenizer tube.

3) The homogenizer is ground thoroughly until no tissue mass is visible.

4) The supernatant was centrifuged at 12000rpm for 10 min.

5) Adding 250 μ L of chloroform, inverting the centrifuge tube for 15s, mixing well, and standing for 3 min.

6) Centrifuge at 12000rpm for 10min at 4 ℃.

7) The supernatant was transferred to a new centrifuge tube, 0.8 times the volume of isopropanol was added, and the mixture was mixed by inversion.

8) Standing at-20 deg.C for 15 min.

9) Centrifuging at 12000rpm at 4 deg.C for 10min to obtain white precipitate as RNA.

10) The liquid was removed by suction and 1.5mL of 75% ethanol was added to wash the precipitate.

11) Centrifuge at 12000rpm for 5min at 4 ℃.

12) The liquid was aspirated off and the centrifuge tube was placed on a clean bench and blown for 3 min.

13) Add 15. mu.L of RNase free water to dissolve the RNA.

14) Incubate at 55 ℃ for 5 min.

15) RNA concentration and purity were determined using Nanodrop 2000: after the blank of the instrument is zeroed, 2.5 mu L of RNA solution to be detected is placed on a detection base, a sample arm is put down, and light absorption value detection is started by using software on a computer.

16) The RNA with the excessive concentration is diluted with a proper proportion, so that the final concentration is 100-500 ng/. mu.L.

(2) Reverse transcription of RNA into CDNA in gastric mucosal tissue

1) A PCR tube was taken and a solution containing 10. mu.L of RNA was added thereto.

2) Add 1. mu.L oligo (dT) 18.

3) Make up to 12 μ L with nuclease-free deionized water.

4) Preserving the temperature for 5min at 65 ℃ on a PCR instrument, and quickly cooling on ice.

5) mu.L of 5 × Reaction Buffer, 2. mu.L of 10mM dNTP Mix, 1. mu.L of RiboLock RNAase inhibitor (20U/. mu.L)) and 1. mu.L of RevertAI M-MuLV reverse transcriptase (200U/. mu.L) were added in this order and mixed by pipetting.

6) Keeping the temperature of the PCR sample at 42 ℃ for 60min, and keeping the temperature of the PCR sample at 80 ℃ for 5min after the completion of the reaction to inactivate the reverse transcriptase.

(3) Real-time fluorescent quantitative PCR

Preparing a real-time fluorescent quantitative PCR (RT-qPCR) system according to requirements, performing RT-qPCR by using a fluorescent quantitative PCR instrument Stepone plus, and evaluating the data quality of the real-time fluorescent quantitative PCR by a dissolution curve and an amplification curve. The levels of the respective genes were normalized by using GAPDH as an internal reference gene as shown in the reaction system of Table 3. The RT-qPCR amplification system is as follows: the pre-denaturation is carried out for 40 times at 95 ℃, 10min, 15s at 95 ℃ and 30s at 60 ℃, the melting curve is from 60 ℃ to 95 ℃, and the temperature is increased by 0.3 ℃ every 15 s. RT-qPCR primer sequences are shown in Table 4. By using 2^-ΔΔCtThe method analyzes the data.

TABLE 3 RT-qPCR reaction System

Tab.3 RT-qPCR reaction system

TABLE 4 RT-qPCR primer sequences

Tab.4 Primer sequences for quantitative Real-Time polymerase chain reaction

High-throughput sequencing detection of mouse gastric microecological composition and change condition of mouse gastric microecological composition by 16SrRNA gene

3.2.6.2 liquid nitrogen treated, frozen tube gastric mucosa tissue, DNA extracted from Huada gene and sequenced.

Detection of glutathione in liver tissue

The content of the GSH is calculated by a formula through measuring according to the specification of a micro reduced Glutathione (GSH) test box (Nanjing institute of bioengineering).

Data processing and analyzing method

Data analysis and mapping were performed using SPSS 23.0 statistical software and OriginPro 9.0 mapping software. The analysis and comparison of the significance difference between groups adopts a one-way analysis of variance (AVOVA), and is expressed as P <0.05, and has statistical significance.

Analysis and discussion of results

Dynamic change of mouse body weight and growth activity condition

After the molding is finished, the gastric mucosa of the helicobacter pylori molding group (YG group) is immediately put into a urease indicator, and the color changes instantly, which indicates that a helicobacter pylori infection mouse model is successfully established. In an experiment in which the prophylactic effect of Lactobacillus plantarum ZJ316 on H.pylori-infected mice was investigated for 68 days, the body weight change of each group was as shown in FIG. 2. After one week of acclimatization, the weight of the mice was weighed, and the weight average of the mice reached 17.71. + -. 1.03 g. After 68 days of culture and molding, the weight of the product finally reaches 22.54 plus or minus 0.93 g. At week 4, the body weight of individual mice slightly decreased, and it was possible that continuous gavage had some effect on the growth of the mice, and also that the body weight fluctuation was within the normal range. In the experiment process of the period of 68 days, the diet drinking water condition of each group of mice has no obvious difference during the molding period, so that the H.pyrori infected mice have no influence on the weight, normal diet drinking water and activity condition of the H.pyrori infected mice. Pylori infection is a slow disease process and has no major impact on the body weight and diet of the animal. Consistent with the experimental results of this study.

Gastric mucosa H&E pathological staining observation and tissue damage evaluation

Histopathological staining observation and tissue damage evaluation are currently effective means for diagnosing the severity of gastritis, and are widely used in clinical examination. The results of gastric mucosal staining of mice in different prophylactic treatment groups are shown in FIG. 3. YH group, YGH group and YTH group have no gastric lavage helicobacter pylori, and the staining result shows that the tissue mucosa layer has clear structure, complete epithelium, closely arranged mucosa gland body and normal shape, and no obvious inflammatory reaction is seen. The YG group only perfused with helicobacter pylori, and the staining result showed that although the tissue structure was intact, the mucosal layer glands were closely arranged, but a small amount of glandular epithelial cells were swollen, the cytoplasm was vacuolated (black arrow), and the local mucosal layer and submucosa were more infiltrated with inflammatory cells, granulocytes, lymphocytes and the like (red arrow). The YT group prevents the infection of helicobacter pylori by feeding the lactobacillus plantarum ZJ316 in advance, and the staining result shows that the tissue structure is complete, the arrangement of mucosal glands is compact, a small amount of lymphocyte infiltration (red arrow) is carried out on submucosa, and other obvious abnormalities are not seen. And the YC group prevents the infection of the helicobacter pylori by irrigating the stomach of the lactobacillus rhamnosus LGG in advance, and the staining result shows that the structure of a tissue mucosa is clear, the arrangement of mucosa is compact, the shape is normal, a small amount of inflammatory cell infiltration can be seen in a submucosa layer, and lymphocytes, mast cells and the like (black arrows) exist. From the above results, it can be seen that both lactobacillus plantarum ZJ316 and lactobacillus rhamnosus LGG can play a role in preventing inflammatory response caused by helicobacter pylori infection, reducing infiltration of inflammatory cells, but the prevention effect of lactobacillus plantarum ZJ316 is more obvious.

Changes in gastric mucositis factor in mice

Helicobacter pylori colonizes the gastric mucosa and produces metabolites that are continuously stimulated to elicit a locally specific immune response. A large number of in vivo experiments prove that the gastric mucosa is infected by helicobacter pylori, and then immune reaction mainly takes place based on Th1 type, and inflammatory factors IL-6, interferon IFN-gamma and Th2 cell response are induced to generate anti-inflammatory factors IL-10. The changes of gastric mucositis factors in mice of different prevention groups are shown in fig. 4. Compared with YH group, after 68 days of experiment, the YG group had obvious increase of IFN-gamma and IL-6 and obvious decrease of IL-10 without any prevention and treatment measures after being infected by helicobacter pylori, which indicates that helicobacter pylori can stimulate the inflammatory reaction of the organism, leading to increase of inflammatory factors and reduction of inflammatory factors. However, compared with YG groups, the YT group and the YC group for preventing the helicobacter pylori through the lactobacillus plantarum ZJ316 and the lactobacillus rhamnosus LGG for intragastric administration have obvious reduction of IFN-gamma and IL-6 and obvious increase of IL-10, which indicates that the lactobacillus plantarum ZJ316 and the lactobacillus rhamnosus LGG for intragastric administration have certain prevention effect on inflammatory response caused by helicobacter pylori infection. The YGH group and YTH group of only the lactobacillus for gastric lavage showed no significant change in the inflammatory factor index and YH group, indicating that the lactobacillus for gastric lavage did not affect the gastric mucosal immunity of mice. The results are similar to the H & E pathological staining observation results of the gastric mucosa of the mouse, and the fact that the lactobacillus plantarum ZJ316 can prevent inflammatory reaction caused by helicobacter pylori infection through intragastric lavage is proved.

Analysis of mouse stomach microecological change condition by 16SrRNA gene high-throughput sequencing detection

16SrRNA gene high-throughput sequencing result

16SrRNA (V4 hypervariable region) is sequenced in microbial communities in 48 stomach samples of 6 groups of mice by adopting an Illumina MiSeq sequencing platform, software FLASH (Fast Length Adjustment of Short reads, V1.2.11) is used for sequence splicing, paired reads obtained by sequencing at two ends are assembled into a sequence by utilizing an overlapping relation, and the Tags of the hypervariable region is obtained. The obtained data are spliced to obtain the final data as shown in table 5. As can be seen, the total number of effective Tags sequences obtained from the sequencing data of 48 stomach samples is 2472780, wherein 50829 + -1342 samples are obtained from YC group samples, 51265 + -673 samples are obtained from YG group samples, 52186 + -130 samples are obtained from YGH group samples, 52078 + -74 samples are obtained from YH group samples, 50690 + -1339 samples are obtained from YT group samples, 52047 + -49 samples are obtained from YTH group samples, and the Tags splicing rate obtained from the sequencing result is more than 97%, which indicates that the sequencing result is more ideal. OTUs (operational Taxonomic units) are phylogenetically or population genetics studies that group sequences into groups according to their similarity to each other, each group being an OTU. Tags with similarity above 97% are typically clustered into an OTU using the software usearc (v7.0.1090).

TABLE 5 high throughput sequencing sequence basis information

Tab.5 Information of Illumina sequencing

(1) Species accumulation plot

The species accumulation curve chart is mainly used for reflecting whether the sequencing data quantity meets the requirements, the abscissa represents the number of samples, the ordinate represents the number of OTUs (detected number of species), and the species accumulation curve reflects the influence of the sampling number on the species diversity; when the number of the samples is less, the discovered species are not comprehensive and cannot represent the whole community structure, and as the number of the samples rises, the discovered species are more and can also represent the community structure. In practical analysis, if the end part of the curve shows a sharp rising trend, the sampling amount is insufficient, the sequencing depth is insufficient, the sample amount needs to be increased, and new species can be continuously found; when the ascending trend at the end of the curve tends to be flat, the sampling quantity is enough, namely the sequencing depth meets the experimental requirements. Fig. 3.5 can see that when the ascending trend at the end of the curve is flat, the sampling amount is sufficient, i.e. the sequencing depth meets the experimental requirements.

(2) Rich grade curve

The difference of OTU Rank curves and the cumulative species curve are a form of showing the diversity of species in a sample, and can simultaneously explain two aspects of the diversity of the sample, namely the abundance and uniformity of the species contained in the sample. The relative abundance of each OTU in each sample was calculated and then plotted from large to small, with the scale of OTUs as the abscissa and the relative abundance of OTUs as the ordinate. The abscissa is ranked by OTU abundance of the sample (from high to low), and the ordinate is OTU abundance. The abundance of species in the sample is reflected by the length of the horizontal axis of the curve, with a wider curve indicating a richer composition of species in the sample. The uniformity of species in the sample is reflected by the shape of the vertical axis of the curve, with flatter curves indicating greater uniformity of species composition in the sample. As can be seen from fig. 6, as the abundance of the sample OUT increases, the curve becomes wider in the horizontal direction and tends to be smooth in the vertical direction, which indicates that the species is abundant and the distribution is more uniform.

Influence of lactobacillus plantarum ZJ316 on structural composition of gastric mucosa flora of mice through prevention effect

(1) Effect of Lactobacillus plantarum ZJ316 on the structural composition of mouse gastric mucosal flora at the "phylum" level

According to the results of the species annotation of OTU, the flora in the gastric mucosa samples of 6 groups of mice were analyzed separately on the basis of the bacterial "phylum" level and plotted as a bar graph as shown in fig. 7. The dominant flora in the gastric mucosa of each group of mice were Firmicutes, Bacteroidetes and Proteobacteria, respectively, wherein Firmicutes and Bacteroidetes are the highest flora and account for an average of about 70% of the total OUT of each sample. The bacterial cell wall of the firmicutes is high in peptidoglycan content and is mostly spherical or rod-shaped, wherein the peptidoglycan content mainly comprises bacilli, clostridia, erysipelothrix and the like. The longstalactites have strong vitality and can resist extreme environments such as dehydration and the like. Wherein the Lactobacillus (Lactobacillus) of the class Bacillus (Bacillus) has probiotic functions of promoting gastrointestinal digestion and maintaining gastrointestinal health. Bacteroides (Bacteroides) bacteria in Bacteroides can help host decompose polysaccharide to improve polysaccharide nutrient utilization rate, accelerate blood vessel formation of gastrointestinal mucosa, promote immune system development to improve immunity, and maintain microecological balance in gastrointestinal tract. Proteobacteria (Proteobacteria) is the largest of bacteria, including pathogenic bacteria such as Escherichia coli, Salmonella, helicobacter pylori, and the like, and is the beginning of main gastrointestinal diseases. As can be seen from FIG. 7, no control was taken in the YG group because of infection with H.pylori, in which the Proteobacteria (Proteobacteria) ratio was significantly increased as compared with the other groups. The YGH group and YTH group only irrigated with Lactobacillus plantarum 316 and Lactobacillus rhamnosus LGG, respectively, and the sum of Firmicutes and bacteroides in the two groups accounts for the highest ratio in all groups, indicating that intake of lactic acid bacteria is beneficial to increase the number of probiotics in the stomach and intestine and maintain the health of the organism. The number of Proteobacteria (Proteobacteria) in the lactobacillus plantarum ZJ316 prevention post-treatment group (YT group) is obviously reduced compared with that in the YG group, and the number of Firmicutes and Bacteroidetes is also increased, so that the lactobacillus plantarum ZJ316 can play a certain role in inhibiting the propagation of pathogenic bacteria through the prevention effect on the structural change of mouse gastric mucosa flora on the 'phylum' level.

(2) Effect of Lactobacillus plantarum ZJ316 on the composition of the gastric mucosal flora in mice at the "genus" level

A bar chart is drawn on flora 15 before relative abundance in gastric mucosa samples of mice of 6 groups at the genus level as shown in FIG. 8, and the flora of the 6 groups of samples at the genus level is analyzed, wherein the flora mainly comprises Lactobacillus, Helicobacter, Staphylococcus, and Veillonella, and the like, and the Lactobacillus content is the highest, so that the Lactobacillus is laterally the main flora in the gastrointestinal tract and is responsible for maintaining the microecological balance of the gastrointestinal tract of a host. As can be seen from FIG. 8, the pathogenic bacteria content in the post-treatment groups (YT group and YC group) for the prevention of Lactobacillus plantarum ZJ316 and Lactobacillus rhamnosus LGG was significantly lower than that in the H.pylori-producing group (YG), wherein the reduction of Helicobacter was most significant, and the effect of YT group was more significant than that of YC group. The structural change of mouse gastric mucosa flora on the genus level shows that the lactobacillus plantarum ZJ316 can play a certain role in inhibiting helicobacter pylori through the prevention effect.

Beta-diversity analysis for preventing flora in gastric mucosa of mice in group

The Beta diversity (Beta diversity) analysis was used to compare the magnitude of the difference between a pair of samples in terms of species diversity. UniFrac is a method for comparing species population differences among samples by using phylogenetic information and is divided into 2 types, namely weighted UniFrac (weighted UniFrac) and non-weighted UniFirac (unweighted UniFrac), wherein weighted UniFrac considers sequence abundance and unweighted UniFrac does not consider sequence abundance. FIGS. 9A and 9B are Beta diversity matrix heatmap, respectively. The larger the index of the Beta diversity heatmap, the larger the inter-sample differences. The closer the evolutionary tree distance, the greater the sample similarity. As can be seen in fig. 9A and 9B, parallel samples in the same group are better clustered together except for individual samples, indicating that the samples are better sequenced in parallel. From the figure, it can be seen that weighted unifrac (weighted unifrac) and non-weighted unifiac (unweighted unifrac) are less effective in clustering samples of the same group, and when species abundance is taken into consideration, the gastric microbial flora among mouse individuals has larger difference, but it can be seen that the samples of different groups have obviously larger difference. The blank control group (YH group) and the helicobacter pylori model group (YG group) have large difference in heat indexes and obvious difference in heat colors, which indicates that the two groups of mouse gastric mucosa microbial flora have large difference on the whole. Meanwhile, the lactobacillus plantarum 316 preventive treatment group (YT group) and the helicobacter pylori artificial model group (YG group) have certain heat index difference, so that the change of the gastric microbial flora caused by the growth of the helicobacter pylori can be inhibited to a certain extent by the preventive treatment of the lactobacillus plantarum ZJ316 for intragastric gavage of the mice from the side.

Analysis of species significant differences in gastric mucosa of mice in each prevention treatment group

In order to achieve the purpose of specifically analyzing the species difference (Biomarke) with obvious abundance among groups, LEfSe is selected for analysis. The LEfse analysis, namely LDA Effect Size analysis, is a linear discriminant analysis method used for finding high-dimensional biological markers and software for revealing genome features, and is used for distinguishing two or more biological conditions (or clusters). One color circle represents one biorarker, and the yellow nodes represent the microbial groups that do not play a significant role in the different groups. From inside to outside, the circles are species at phylum, class, order, family and genus levels.

For comparative analysis of significant differences in the groups of H.pylori infection building blocks and Lactobacillus plantarum treatment prevention groups, the relative abundance of the groups of six groups, YG, YC, YTH, YGH and YH, was analyzed by LEfSe to find significantly changed groups. As shown in FIGS. 10A and 10B, the groups in which significant increases occur in the gastric microbial flora of the Helicobacter pylori colonization group (YG) are Bacteroides at the "phylum" level, Epsilonproteobacteria, Bacteroides at the "class" level, Camphorobacteres at the "order" level, Bacteroides, Helicobacter, Prevotella, Porphyromonadeaceae, Odoribacteriaceae, Eubacter, Clostridiae, Rhizobiaceae, Helicobacter, Parabarides, Clostridia, Odoribacterium, Roseburia, Candidatus, Arthromitus, Anarofustis, Agrobacterium, and the like at the "genus" level. Wherein, Helicobacter can cause various digestive tract diseases such as chronic active gastritis, peptic ulcer, gastric cancer and the like, and belongs to one of pathogenic bacteria; clostridium can break down sugar in muscle and connective tissues, produce exotoxin, cause severe emphysema of tissues, affect blood supply, and cause large-area necrosis of tissues; parabacteroides and Odoribacter are common bacteria for maintaining the intestinal health of animals, and are beneficial to the regulation of the blood pressure of overweight and obese pregnant women. Roseburia is a conditional pathogen which is usually parasitic on skin, oral cavity, gastrointestinal mucosa and the like of a human body, is easy to cause candidiasis when the immune function of the body is low or the micro-ecological environment of a normal living part is disordered, and is a pathogenic bacterium.

The groups in which a significant increase in the Lactobacillus plantarum ZJ316 preventive treatment group (YT) gastric microflora occurred were Dehalobacter iaceae, Geodermatophiaceae at the "family" level and Proteus, Geodermatophilus, Dehalobacter at the "genus" level. Proteus is a parasitic and pathogenic bacterium of humans and animals, and exists in the intestinal tracts of humans and animals, but is generally not pathogenic. Geodermatophilus and Dehalobacter are also non-pathogenic. The group in which a significant increase occurred in the LGG preventive treatment group (YC) gastric microbial flora of Lactobacillus rhamnosus was Verrucomicrobia at the "phylum" level, Verrucomicrobiae at the "class" level, Verrucomicrobiales at the "order" level, Streptococcaceae at the "family" level, Streptococcus, Jeotgaliccus, Akkermansia at the "genus" level. Streptococcus is mostly non-pathogenic but may cause suppurative inflammation, toxic diseases and the like. Jeotgalicoccus belongs to the phylum firmicutes and is not pathogenic. Akkermansia is the only member of the phylum verrucomicrobia. The content of the extract in human body is rich, and the extract is distributed on the surface of gastrointestinal tract of animals and can be up to 1-5%. The more Akkermansia in the intestinal tract, the less obesity, inflammation and type 2 diabetes, and the higher temperature resistant bacterial protein on the membrane can promote the barrier function of the intestinal tract. The result shows that the Lactobacillus plantarum ZJ316 improves the abundance of probiotics such as Lactobacillus and the like in the gastric mucosa of the mouse through a prevention effect, and reduces the abundance of pathogenic bacteria such as Helicobacter, Clostridium and the like.

As can be seen from FIG. 11, the average relative abundance of Helicobacter pylori (Helicobacter pylori) in the YG group was 0.190, whereas the abundance of Helicobacter pylori in the YT group was reduced to 0.090 and the abundance of Helicobacter pylori in the YC group was reduced to 0.016, indicating that the mice prophylactically treated with lactic acid bacteria were infected with Helicobacter pyloriThe obvious difference of the stomach micro-ecological flora and the change of the wind degree of helicobacter pylori can be seen, and the lactic acid bacteria have certain prevention effect and can play a role in protecting the stomach micro-ecological environment, reducing the content of pathogenic bacteria in the stomach and reducing the planting rate of the helicobacter pylori in the stomach. Wherein the Lactobacillus plantarum ZJ316 has a better effect than Lactobacillus rhamnosus LGG.Analysis of glutathione content in liver tissue

Numerous studies have reported that vacuolating toxin (VacA) in h.pylori causes Glutathione (GSH) metabolic damage to gastric epithelial cells. Glutathione is the most important small molecular active oligopeptide in an antioxidant defense system in an organism and has the function of maintaining an immune system. Oxidative stress and redox reactions in gastrointestinal tissues are strongly linked to helicobacter pylori infection. Glutathione content in mouse liver tissue is shown in Table 6, and GSH content in liver of H.pylori-infected model building group (YG group) is low, and the content is 1.43 + -0.23 μmol/gprot. The content of Lactobacillus plantarum ZJ316 prophylactic treatment group (YT group) and Lactobacillus rhamnosus LGG prophylactic treatment group (YC group) was increased compared to YG group, and was 1.65. + -. 0.07. mu. mol/gprot and 1.54. + -. 0.16. mu. mol/gprot, respectively. The YT group was more elevated than the YC group. The results of this experiment suggest that Lactobacillus plantarum ZJ316 may protect the body from oxidative damage caused by H.pylori infection through antioxidant action.

TABLE 3.6 glutathione content in mouse liver tissue

Tab.3 .6 The levels of GSH in liver of mice

Second, treatment effect of Lactobacillus plantarum ZJ316 on mice infected by helicobacter pyloriExperimental materials and methods

Experimental strains

Laboratory animal

40 mice were 6-week-old female SPF (Specific pathogen free) grade C57BL/6J mice, 40 were purchased from Shanghai Seiparl-Bikay laboratory animals Co., Ltd, and were housed in the laboratory animal house of the public health clinic center in Shanghai. The temperature of the animal room is 24 +/-2 ℃, and padding, feed and drinking water need to be replaced every week under the environment of 12 hours of day and night alternation. Wherein the drinking water and the padding need to be sterilized at high temperature and high pressure. The feed was purchased from shanghai shilin biotechnology limited.

Main reagent and consumable

TABLE 7 Experimental reagents and consumables

Tab.7 List of main reagents and consumables

Laboratory apparatus and device

The instruments and equipment used in the experiments are described above.

Media and related solution formulations

Preparation of culture Medium

Brain Heart Infusion (BHI) broth culture: 25g of the medium was weighed, dissolved in 500mL of ultrapure water under heating, and autoclaved at 121 ℃ for 15 minutes for use.

Other solution formulation

Preparing combined antibiotics: the dosage of the antibiotics is converted into the dosage of the mice according to the instruction of the antibiotics. Dissolving 12.5g of amoxicillin, 82.5mg of omeprazole and 2.1g of clarithromycin in 1L of sterile water, fully and uniformly mixing, and placing in a 4-degree refrigerator for later use.

Experimental methods and contents

Activation and culture of bacterial species

(1) Activation and culture of lactic acid bacteria

(2) Activation and culture of helicobacter pylori

Lactobacillus treatment on pyloric screwEstablishment of bacterial mouse gastritis model

40C 57BL/6J mice, after 1 week of acclimatization, were divided into 5 groups of 8 mice each, and the experimental procedures were carried out under normal diet conditions, as follows:

(1) ZH group (n ═ 8): the culture medium BHI is perfused with 400 mu L of gastric lavage BHI per day once every other day, and the perfusion of normal saline is perfused with 400 mu L of gastric lavage BHI per day for four weeks after three weeks.

(2) ZT group (n ═ 8): the H.pyrori bacterial suspension is perfused at 400 mu L/one per stomach, the perfusion is carried out once every other day for three weeks, and the normal saline is perfused at 400 mu L/one per day for four weeks after the three weeks.

(3) ZC group (n ═ 8): h.pyrori bacterial suspension of 400 mu L/one, gavage once every other day for three weeks, and after three weeks, gavage lactobacillus plantarum ZJ 316400 mu L/one/day for four weeks.

(4) ZG group (n ═ 8): h.pyrori bacterial suspension is irrigated at intervals of 400 mu L/one per stomach for three weeks, and after three weeks, the rhamnose lactobacillus LGG is irrigated at intervals of 400 mu L/one per day.

(5) Group ZI (n ═ 8): h.pyrori bacterial suspension of 400 mu L/one, gavage once every other day for three weeks, gavage combined with antibiotics for 10 days after three weeks, and continuously gavage normal saline of 400 mu L/one/day for 18 days after 10 days.

Mice were weighed once a week during the animal experiments and observed for active growth status. At the end of the molding, the vertebroplasty was sacrificed and the mice were dissected in a biosafety cabinet. Aseptically excising gastric fundus, washing the remaining gastric mucosa tissue with sterile normal saline, dividing into three parts, placing in a freezing tube, treating with liquid nitrogen, refrigerating at-80 deg.C, and measuring the experimental index. Meanwhile, taking out the liver to prepare tissue homogenate for measuring the indexes of glutathione and malondialdehyde. Wherein, the gastric mucosa of the helicobacter pylori model building group (ZT group) needs to be immediately put into a urease indicator to confirm whether the helicobacter pylori infection mouse model is successfully established. Treatment time points for the treatment groups can be seen with reference to fig. 12.

Gastric mucosal tissue section, H&E staining and tissue damage evaluation

Specific experimental methods refer to the description above.

Determination of inflammatory factors in gastric mucosal tissue

Specific experimental methods refer to the description above.

And (3) performing liquid nitrogen treatment on the gastric mucosa tissue in the cryopreserved tube in the established helicobacter pylori mouse gastritis model, performing DNA extraction by using Huada gene, and sequencing.

Detection of glutathione in liver tissue

Specific experimental methods refer to the description above.

Data processing and analyzing method

Analysis and discussion of results

Dynamic change of mouse body weight and growth activity condition

After the molding is finished, the gastric mucosa of the helicobacter pylori molding group (ZT group) is immediately put into a urease indicator, and the color changes instantly, which indicates that a helicobacter pylori infection mouse model is successfully established. In an experiment in which the therapeutic effect of Lactobacillus plantarum ZJ316 on H.pylori-infected mice was investigated for day 56, the body weight change of each group was as shown in FIG. 13. After one week of acclimatization, the weight of the mice was weighed, at which time the weight average of the bodies reached 17.72. + -. 0.88 g. After 56 days of culture and molding, the weight of the product finally reaches 22.63 +/-1.27 g. Among them, the weight decreased slightly at week 4, which may be that continuous gavage had some effect on the growth of mice, and also included the weight fluctuation within the normal range. In the experiment process of 56 days, the diet drinking water condition of each group of mice has no obvious difference during the molding period, so that the H.pyrori infected mice have no influence on the weight, normal diet drinking water and activity condition of the H.pyrori infected mice. Pylori infection is a slow disease process and has no major impact on the body weight and diet of the animal. Consistent with the experimental results of this study.

Histopathological staining observation and tissue damage evaluation are currently effective means for diagnosing the severity of gastritis, and are widely used in clinical examination. The results of gastric mucosal staining of mice in different treatment groups are shown in FIG. 14. The ZH group has no gavage any strain as a blank control, and the staining result shows that the structure of the tissue mucosa is clear, the epithelium is complete, the arrangement of the mucosa glands is compact, the shape is normal, and no obvious inflammatory reaction is seen. The ZT group is perfused with helicobacter pylori only, and the staining result shows that the tissue mucous membrane layer has clear structure, the mucous membrane layer glands are arranged closely, the submucosa layer and the serosal layer can be infiltrated by more inflammatory cells, and the serosal layer can be seen to have lymphocytes, granulocytes and the like (black arrows), and the serosal layer can be seen to bleed (red arrows). The ZC group treats mice infected by helicobacter pylori with lactobacillus plantarum 316 for intragastric administration, and the staining result shows that the tissue structure is complete, the mucosal layer glands are arranged closely and have normal shapes, a small amount of inflammatory cells infiltrate the submucosa, and mast cells, granulocytes, lymphocytes and the like (black arrows) exist, and no other obvious abnormalities are seen. The ZG group is used for treating mice infected by helicobacter pylori by virtue of the Lactobacillus rhamnosus LGG, and the staining result shows that the tissue structure is complete, the arrangement of mucosa glands is compact, the shape is normal, a small amount of inflammatory cell infiltration can be seen in submucosa, and mast cells, lymphocytes and the like are arranged (red arrows). ZI was a mouse treated with mixed antibiotics for H.pylori infection, and the staining was identical to that of the above-mentioned blank control group (ZH group). The results show that the antibiotic treatment effect is most obvious, the inflammation can be basically eradicated, the lactobacillus plantarum ZJ316 and the lactobacillus rhamnosus LGG can play a role in treating the inflammatory reaction caused by helicobacter pylori infection, and the infiltration of inflammatory cells is reduced.

Changes in gastric mucositis factor in mice

The variation of gastric mucositis factors in mice from different treatment groups is shown in figure 15. Compared with ZH, after modeling, the obvious increase of IFN-gamma and IL-6 and the obvious decrease of IL-10 appear under the condition that ZT group is infected by helicobacter pylori without any prevention and treatment measures, which shows that the helicobacter pylori after gastric lavage can arouse the inflammatory reaction of the organism, cause the increase of inflammatory factors and reduce the inflammatory factors. Compared with the ZT group, the ZC group and the ZG group which treat mice infected by helicobacter pylori through the lactobacillus plantarum ZJ316 and the Lactobacillus rhamnosus LGG respectively have obvious reduction of IFN-gamma and IL-6, and IL-10 has no obvious change, which shows that the lactobacillus plantarum ZJ316 and the Lactobacillus rhamnosus LGG which are intragastric have certain treatment effect on inflammatory reaction caused by the helicobacter pylori infection and inhibit the production of proinflammatory factors. However, there is still a gap between lactobacillus treatment and antibiotic treatment, as shown in FIG. 15, the inflammation factors (IFN-. gamma., IL-6) in group ZI are significantly less than those in group ZC and group ZG, indicating that antibiotic treatment is more effective than lactobacillus for treating inflammation in mice caused by helicobacter pylori infection. Above and mouse gastric mucosa H&The pathological staining observation results are similar, and further prove that the plant lactobacillus ZJ316 has obvious treatment effect on the inflammatory reaction caused by the helicobacter pylori infection of the mice.Analysis of mouse stomach microecological change condition by 16SrRNA gene high-throughput sequencing detection

16SrRNA gene high-throughput sequencing result

The Illumina MiSeq sequencing platform performs 16s rDNA (V3, V4 hypervariable region) sequencing on microbial communities in 40 stomach samples of 5 groups of mice, and the obtained data are spliced to obtain the final data shown in table 8. As can be seen, the total number of effective Tags sequences obtained from the sequencing data of 40 stomach samples was 2091105, with 52219. + -. 70 for the ZC group, 52276. + -. 93 for the ZG group, 52078. + -. 74 for the ZH group, 52408. + -. 165 for the ZI group and 52405. + -. 212 for the ZT group. The splicing rate of Tags obtained by a sequencing result reaches more than 97 percent, which indicates that the sequencing result is relatively ideal. Fig. 16 shows that the ascending trend of the curve end is gradually gentle, which indicates that the sampling amount is sufficient, i.e. the sequencing depth meets the experimental requirements. As can be seen from fig. 17, as the abundance of the sample OUT increases, the curve becomes wider in the horizontal direction and tends to be smooth in the vertical direction, indicating that the species are abundant and the distribution is more uniform.

TABLE 8 high throughput sequencing sequence basis information

Tab.8 Information of Illumina sequencing

Effect of Lactobacillus plantarum ZJ316 on mouse gastric mucosa flora structural composition through therapeutic action

According to the results of the species annotation of OTU, the flora in the gastric mucosa samples of 5 groups of mice were analyzed separately on the basis of the bacterial "phylum" level and plotted as a bar graph as shown in fig. 18. The dominant flora in the gastric mucosa of each group of mice were Firmicutes, Bacteroidetes and Proteobacteria, respectively, wherein Firmicutes and Bacteroidetes are the highest flora and account for an average of about 60% of the total OUT of each sample. Proteobacteria (Proteobacteria) is the largest of bacteria, including pathogenic bacteria such as Escherichia coli, Salmonella, helicobacter pylori, and the like, and is the beginning of main gastrointestinal diseases. As can be seen from FIG. 18, the ZT group had no preventive measures because of infection with helicobacter pylori, in which Proteobacteria (Proteobacteria) ratio was significantly maximized as compared with the other groups. The ratio of Proteobacteria (Proteobacteria) in the treatment group with Lactobacillus plantarum ZJ316 (ZC group) was significantly lower than that of ZT, and the ratio of Firmicutes (Firmicutes) to Bacteroides (Bacteroides) was also higher. The structural change of the mouse gastric mucosa flora on the 'portal' level shows that the lactobacillus plantarum ZJ316 can play a certain role in inhibiting the propagation of pathogenic bacteria through the treatment effect.

A histogram is plotted at the "genus" level for the flora at the first 18 relative abundances in the gastric mucosa samples of 5 groups of mice as shown in fig. 19 and the 5 groups of samples are analyzed for the flora at the genus level, which mainly comprises the genera Lactobacillus, Helicobacter, Staphylococcus, and Veillonella, wherein the Lactobacillus content is the highest, and it can be reflected from the side that Lactobacillus is the main flora in the gastrointestinal tract and is responsible for maintaining the microecological balance of the gastrointestinal tract of the host. As is clear from FIG. 19, the ZT group had no preventive measures against infection with Helicobacter pylori, and the proportion of Helicobacter was the largest as compared with the other groups. The ratio of Helicobacter pylori ZJ316 treated group (ZC group) and the antibiotic treated group (ZI group) was significantly decreased compared with ZT, and although the decrease of the antibiotic treated group (ZI group) was more significant, the Lactobacillus plantarum ZJ316 treated group (ZC group) also had a significant effect of inhibiting Helicobacter. The structural change of mouse gastric mucosa flora on the genus level shows that the lactobacillus plantarum ZJ316 can play a certain role in inhibiting the growth and reproduction of helicobacter pylori through the treatment effect.

Beta diversity analysis of the flora in the gastric mucosa of the treated mice

As can be seen from Beta diversity heat map 20A and FIG. 20B, the difference between the heat indexes of the blank control group (ZH group) and the helicobacter pylori infection model group (ZT group) is large, and the heat colors have obvious difference, which indicates that the two groups of mouse gastric mucosa microbial flora have great difference on the whole. Meanwhile, a certain heat index difference exists between a lactobacillus plantarum 316 treatment group (ZC group) and a helicobacter pylori artificial module (ZT group), so that the change of the helicobacter pylori on the gastric microbial flora can be improved to a certain extent by carrying out treatment on the lactobacillus plantarum ZJ316 for intragastric administration of mice.

Analysis of species significant differences in gastric mucosa of mice in each treatment group

In order to comparatively analyze the remarkable difference of the related floras of the helicobacter pylori infection modeling group and the lactobacillus plantarum treatment group, LEfSe is used for comparatively analyzing the relative abundance of the floras of the five groups of ZI, ZT, ZG, ZC and ZH respectively so as to find out the floras with remarkable change. As shown in FIG. 4.10, the groups in which a significant increase occurs in the gastric microflora of the Helicobacter pylori colonization module (ZT) are Proteobacteria at the "phylum" level, Deferribacteriaceae, Tenericus, "Epsilon protobacteria at the" class "level," Camphylobacteria at the "order" level, Deferribacteriales, Anaeroplasmates, Mollicutes, Sphingobacterials, "Helicobacter eraceae at the" order "level, Ruminococcus, Prevollaceae, Anaeroplaticaceae, Clostridiaceae, Destriobacteriaceae, Dehalocerae, Sphingobacteriaceae, Odoboribacteriaceae," Heliciella, Oscilaria, Preostriniaceae, Bugularia, Sphingobacterium, Odooribacteriaceae, "Kluyveromyces, Brucescens, Brucescenella, Kluyveromyces, Sphingobacteria, Brucella, and Brucella at the" level. The most remarkable increase of the pathogenic bacteria is Proteobacteria which is the largest of bacteria, including pathogenic bacteria such as escherichia coli, salmonella, helicobacter pylori and the like and is a warrior of primary gastrointestinal diseases. The most remarkable increase of the genus is that Helicobacter can cause various digestive tract diseases such as chronic active gastritis, peptic ulcer, gastric cancer and the like, and belongs to one of pathogenic bacteria. Prevotella interferes with the immune system, causes the autoimmune system to attack joints, induces rheumatoid arthritis, and also belongs to pathogenic bacteria. The main fermentation metabolites of Ruminococcus are acetic acid and formic acid, and bacteria in the intestinal type mainly including Ruminococcus mainly acquire energy by absorbing monosaccharides and degrading mucin.

The groups in which a significant increase in the Lactobacillus plantarum ZJ316 therapeutic treatment group (ZC) gastric microflora occurred were Firmicutes at the "phylum" level and Bacilli at the "class" level, lactobacillals at the "order" level, clostridium at the "genus" level, Roseburia. The lactobacillus on the level of the eye is increased most obviously, and the lactobacillus plantarum ZJ316 for intragastric administration reduces the number of pathogenic bacteria in the gastric microecological flora and increases the number of lactic acid bacteria. Meanwhile, the content of flora in the Firmicutes is greatly increased, and most of the Firmicutes are beneficial bacteria for maintaining gastrointestinal health. For example, the order Lactobacillales (Lactobacillus) in the class Bacillales (Bacillus) has probiotic functions of promoting gastrointestinal digestion, maintaining the health of the gastrointestinal tract, and the like. Roseburia can reduce inflammation and atherosclerosis in vivo. From the above, the Lactobacillus plantarum ZJ316 improves the abundance of probiotics such as Lactobacillus and Roseburia in mouse gastric mucosa and reduces the abundance of pathogenic bacteria such as Prevotella and Helicobacter through treatment. The flora in which the lactobacillus rhamnosus LGG treatment group (ZG) gastric microbial flora is remarkably increased is Veillonella and Staphylococcus on the 'genus' level, and the two or more bacteria also have certain pathogenicity. However, no significant increase in Helicobacter was detected, indicating that lactobacillus rhamnosus LGG also has some therapeutic effect.

As can be seen from fig. 20A and 20B, in ZT group, the average relative abundance of Helicobacter pylori (Helicobacter) was 0.236, while in ZC group, ZG group, and ZI group were 0.017, and mice were treated with lactobacillus after being infected with Helicobacter pylori, and using antibiotics as control, the significant difference in gastric microecological flora and the change in the anemometry of Helicobacter pylori showed that the treatment effect of antibiotics was the best, but lactobacillus also had a certain treatment effect, and had the effects of protecting gastric microecological environment, reducing the colonization content in stomach, and reducing the Helicobacter pylori rate in stomach. Wherein the Lactobacillus plantarum ZJ316 has a better treatment effect relative to Lactobacillus rhamnosus LGG.

Analysis of glutathione content in liver tissue

The glutathione content in mouse liver tissue is shown in Table 4.3, and the GSH content in liver of helicobacter pylori infection model group (ZT group) is low, and the content is 0.87 +/-0.36 mu mol/gprot. The content of the lactobacillus plantarum ZJ316 treatment group (ZC group) and the lactobacillus rhamnosus LGG treatment group (ZG group) is increased compared with the ZT group, and is respectively 1.79 +/-0.15 mu mol/gprot and 1.90 +/-0.12 mu mol/gprot. The content of GSH in the group treated by mixed antibiotic treatment (ZI group) is obviously increased to 2.66 +/-0.03 mu mol/gprot. The antibiotic has the strongest treatment capacity, but the lactobacillus also has the capacity of resisting oxidation and removing free radicals and improves the content of GSH.

TABLE 9 glutathione content in mouse liver tissue

Tab.9 The levels of GSH in liver of mice

It will be appreciated by persons skilled in the art that the embodiments of the invention described above and shown in the drawings are given by way of example only and are not limiting of the invention. The objects of the invention have been fully and effectively accomplished. The functional and structural principles of the present invention have been shown and described in the examples, and any variations or modifications of the embodiments of the present invention may be made without departing from the principles.

Claims

1. The lactobacillus for preventing and treating gastritis caused by helicobacter pylori is lactobacillus plantarum ZJ316 with the deposit number of CCTCC No: m208077.

2. The lactic acid bacterium according to claim 1, wherein the lactic acid bacterium is capable of reducing infiltration of inflammatory cells.

3. The lactic acid bacterium according to claim 1, wherein the lactic acid bacterium is capable of increasing the abundance of beneficial bacterial flora in the treatment of gastritis induced by helicobacter pylori.

4. The lactic acid bacterium according to claim 1, wherein the lactic acid bacterium is capable of increasing the abundance of beneficial bacterial flora in the prevention of helicobacter pylori-induced gastritis.

5. The lactic acid bacterium according to claim 1, wherein the lactic acid bacterium is capable of reducing the abundance of pathogenic bacteria in the prevention of helicobacter pylori-induced gastritis.

6. The lactic acid bacterium according to claim 1, wherein the lactic acid bacterium is capable of reducing the abundance of the pathogenic flora in the treatment of gastritis caused by helicobacter pylori.

7. The lactic acid bacterium according to claim 1, wherein the lactic acid bacterium is capable of increasing glutathione content in the liver in the prevention of helicobacter pylori-induced gastritis.

8. The lactic acid bacterium according to claim 1, wherein the lactic acid bacterium is capable of increasing glutathione content in the liver in the treatment of helicobacter pylori-induced gastritis.

9. The lactic acid bacterium according to claim 1, wherein the lactic acid bacterium is capable of up-regulating the expression levels of proinflammatory cytokines IL-6 and IFN- γ in the gastric mucosa in the prevention of helicobacter pylori-induced gastritis.

10. The lactic acid bacterium according to claim 1, wherein said lactic acid bacterium is capable of up-regulating the expression levels of the pro-inflammatory cytokines IL-6 and IFN- γ in the gastric mucosa in the treatment of helicobacter pylori-induced gastritis.

11. The lactic acid bacterium according to claim 1, wherein the lactic acid bacterium is a culture solution containing one or a combination of both of a cell surface substance and a cell secretion substance.

12. The application of lactobacillus is characterized in that lactobacillus plantarum ZJ316 is applied to prevent and treat gastritis caused by helicobacter pylori, CCTCC No: m208077.

13. The use according to claim 12, wherein the lactic acid bacteria are used to increase the abundance of beneficial bacterial flora.

14. The use of claim 12, wherein the lactic acid bacteria use up-regulates the expression of the pro-inflammatory cytokines IL-6 and IFN- γ in the gastric mucosa.

15. The use according to claim 12, wherein the lactic acid bacteria are used to increase glutathione content in the liver.

16. The use according to claim 12, wherein the lactic acid bacteria is a culture solution containing one or a combination of both of a bacterial surface substance and a bacterial secretion substance.

17. A food product comprising lactic acid bacteria and an edible material, wherein the lactic acid bacteria is lactobacillus plantarum ZJ316, CCTCC No: m208077.

18. The food product of claim 17, wherein the edible material is selected from one or more of a group consisting of water, fluid milk, condensed milk, yogurt, frozen yogurt, lactobacillus fermented beverages, milk powder, ice cream, cheese, soy milk, fermented soy milk, vegetable juice, fruit juice, sports drinks, desserts, jellies, candies, baby food, health food, animal feed, herbal compositions, and dietary supplements.

19. The food product of claim 17, wherein the lactic acid bacteria is a culture fluid comprising a combination of one or both of a bacterial surface material or a bacterial secretion material.

20. The food product of claim 17, wherein the lactic acid bacteria are live bacteria or dead bacteria.