AU2020101537A4 - A kind of probiotic and application thereof in gastric injury - Google Patents

Abstract

The invention discloses a probiotic and its application in gastric injury, belonging to the technical field of microorganisms. The Lactobacillus plantarum HFY09 belongs to the lactic acid bacteria (LAB), and the preservation number is CGMCC No. 16633; This invention adopts a hydrochloric acid/ethanol acute gastric injury model to evaluate the preventive effect of Lactobacillus plantarum HFY09 on gastric injury; The results showed that HFY09 could inhibit gastric juice secretion, maintain the normal pH value of gastric acid, reduce the damage area of gastric mucosa, inhibit apoptosis of gastric mucosal epithelial cells, and play a biological role, which is positively correlated with concentration. HFY09 can be used in the production of probiotics or functional foods, which provides theoretical basis for its further development and utilization. Norml Modecl LB Rariitidjinc EFY09-H HFY09-I, Figure1I

Description

Norml Modecl LB

Rariitidjinc EFY09-H HFY09-I,

Figure1I

AUSTRALIA

PATENTS ACT 1990

PATENT SPECIFICATION FOR THE INVENTION ENTITLED:

A kind of probiotic and application thereof in gastric injury

The invention is described in the following statement:-

A KIND OF PROBIOTIC AND APPLICATION THEREOF IN GASTRIC INJURY TECHNICAL FIELD

[0001] The invention relates to the technical field of microorganisms, in particular to a

probiotic and its application in gastric injury.

BACKGROUND

[0002] Gastric injury is an infectious or chemical attack factor that damages mucosal

defence system and can cause gastric epithelial injury or ulcer. Current research shows

that bacteria, drugs, smoking and drinking can cause gastric mucosal damage. Ethanol is

the main component used in liquid alcohol and beverages because it is fat-soluble and

produces acetaldehyde molecules with strong carcinogenic effect, starting from entering

the oral cavity and continuing when entering the blood circulation. Although gastric

mucosa can metabolize ethanol and acetaldehyde through ethanol dehydrogenase and

acetaldehyde dehydrogenase, the carcinogenic effect of acetaldehyde cannot be

underestimated. Therefore, alcoholism can cause acute erosive haemorrhagic gastritis,

and long-term drinking can cause gastropathy, chronic atrophic gastritis, cancer, alcoholic

hepatitis, fatty liver, liver cirrhosis and other alcoholic diseases. Hydrochloric acid may

also cause gastric mucosal damage, and ethanol may reach mucosa and induce vascular

parietal cell rupture.

[0003] Probiotics and some new biological drugs have been widely studied as new

therapies for gastric related diseases. More and more evidences show that probiotics are

beneficial to host health. A large number of studies have confirmed that yeast and lactic

acid bacteria are the main resident microorganisms beneficial to human body in stomach.

Among them, yeast colonized in the secretory region of the stomach, while lactic acid

bacteria colonized in the non-secretory region, which is considered to be the main force

to maintain the microecological balance in the stomach.

Some researchers have studied ethanol-induced gastritis and liver injury in mouse models

and found that Lactobacillus plantarum LC27 and Bifidobacterium longum LC67 play an

important role in repairing gastritis by stimulating immune system and fibroblasts. Some

researchers' pretreated rats with probiotic mixture showed significant reduction of acute

gastric mucosal damage caused by aspirin or ethanol. Animal experiments have also

confirmed that Lactobacillus lute DSM17938 can prevent gastric injury caused by

ethanol.

[0004] Therefore, it is of great economic and social value to develop probiotics with the

function of preventing and treating gastric injury and to master the mechanism of

probiotics' influence on human health.

SUMMARY

[0005] The object of the invention is to provide a probiotic and its application in gastric

injury, so as to solve the problems existing in the above prior art.

[0006] In order to achieve the above object, the invention provides the following scheme:

[0007] The invention provides a Lactobacillus plantarum HFY09, which belongs to the

genus Lactic acid bacteria (LAB) and has been deposited in the China General

Microbiological Culture Collection Center, with the preservation date of October 25,

2018 and the preservation number CGMCC No. 16633.

[0008] The invention may also provide for an application of the Lactobacillus plantarum

HFY09 in preparing a medicine for preventing or treating gastric injury.

[0009] The invention discloses the following technical effects:

[0010] Lactobacillus plantarum HFY09 is a lactobacillus isolated from natural fermented

yak yogurt. The invention adopts a hydrochloric acid/ethanol acute gastric injury model

to evaluate the preventive effect of Lactobacillus plantarum HFY09 on gastric injury. The

results showed that HFY09 could inhibit gastric juice secretion, maintain the normal pH

value of gastric acid and reduce the damage area of gastric mucosa. Compared with the

model group, the levels of SP, ET-1, IL-6, IL-12, TNF-a and IFN-y in HFY09-H group

decreased, while the levels of SS and VIP increased. Further experiments showed that

HFY09 increased the expression of eNOS, nNOS, Cu/Zn SOD, Mn-SOD, EGF and

EGFR, and decreased the expression of iNOS and COX-2 in gastric tissue, indicating that

HFY09 can inhibit the apoptosis of gastric mucosal epithelial cells and play a biological

role, which is positively correlated with the concentration. The results show that HFY09

can be used in the production of probiotics or functional foods, providing theoretical

basis for its further development and utilization.

BRIEF DESCRIPTION OF THE FIGURES

[0011] In order to more clearly illustrate that embodiments of the present invention or the

technical solution in the prior art, a brief description will be given to the drawing which

are required to be used in the embodiments, and it is obvious that the drawings in the

following description are merely some embodiments of the present invention, and other

drawings may be obtained from these drawings without any creative effort by those

ordinary technical personnel in the art.

FIG. 1 is the appearance observation of gastric tissue in experimental group;

FIG. 2 is a pathological observation (100x) of H & E in mouse stomach;

FIG. 3 shows 3mRNA expression levels of Cu / Zn-SOD, Mn-SOD and CAT in mouse

stomach;

FIG. 4 shows 4mRNA expression levels of nNOS, eNOS, iNOS and COX-2 in mouse

stomach;

FIG. 5 shows 5 mRNA expression levels of EGF and EGFR in the mouse stomach.

DESCRIPTION OF THE INVENTION

[0012] Various exemplary embodiments of the invention are now described in detail,

which is not to be taken as a limitation of the invention, but is to be understood as a more

detailed description of certain aspects, features and embodiments of the invention.

[0013] It is to be understood that the terms used in the invention are only intended to

describe particular embodiments and are not intended to limit the invention. In addition,

with regard to the numerical range in the present invention, it should be understood that

each intermediate value between the upper and lower limits of the range is also

specifically disclosed. Each smaller range between any stated value or median value

within the stated range and any other stated value or median value within the range is also

included in the present invention. The upper and lower limits of these smaller ranges can

be independently included or excluded from the range.

[0014] Unless otherwise specified, all technical and scientific terms used herein have the

same meaning as would normally be understood by those ordinary technical personnel in

the art. Although this invention only describes preferred methods and materials, any

methods and materials similar or equivalent to those described herein may be used in the

practice or testing of the present invention. All documents referred to in this specification are incorporated by reference to disclose and describe methods and / or materials related to the documents. In the event of a conflict with any incorporated document, the contents of this specification shall prevail.

[0015] It will be apparent to those ordinary technical personnel in the art that various

modifications and changes may be made to the specific embodiments of the present

specification without departing from the scope or spirit of the invention. Other

embodiments from the description of the present invention will be readily apparent to

those ordinary technical personnel in the art. The specification and embodiment of the

present application are exemplary only.

[0016] As used herein, "comprising", "including", "having", "containing" and the like are

all open terms, meaning to include but not limited to.

[0017] Example 1

1. Materials and methods

1.1 Laboratory strains

Lactobacillus plantarum HFY09 was isolated from natural fermented yak yoghourt and

identified as Lactobacteria by 16S rDNA sequence analysis. The strain has been

deposited in the China General Microbiological Culture Collection Center (CGMCC,

address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen

West Road, Chaoyang District, Beijing) with the preservation date of October 25, 2018

and the preservation number CGMCC No. 16633.

[0018] Lactobacillus plantarum HFY09 was isolated and purified from yak yoghurt

samples, and all of them were positive by Gram staining; the colonies colour were mostly

white or milky white, round in shape, with neat edges, and smooth and moist surfaces.

Under 100 times oil microscope, the cells have long stem, short stem, globular shape, and

no budding reproduction.

[0019] The 16S rDNA base sequence of the strain HFY09 of the present invention is

shown as SEQ ID No. 1; the sequencing results were aligned on the NCBI website

(http://blast.ncbi.nlm.nih.gov/Blast.cgi). The homology was 100% with Lactobacillus

plantarum and MN368282.1.

[0020] Lactobacillus delbrueckii subsp. bulgaricus (LB; CGMCC No. 1.16075) was used

as a control strain for HFY09.

1.2 Mouse model of gastric injury

[0021] Randomly divide sixty Kunming mice (6 weeks old, Male rats)into 6 groups

(n=10): normal group, model group, ranitidine taken by intragastric administration group

(50mg/kg), Lactobacillus delbrueckii subsp. bulgaricus LB taken by intragastric

administration group (1.Ox1O 9 CFU/Kg), Lactobacillus plantarum HFY09 low-dose

intragastric administration group (1.Ox1O8CFU/ kg) and HFY09-H high-dose intragastric

administration group (1.0 x 109). In that first 14 day, the normal group and the model

group were given free diet and water drinking, the ranitidine group was given ranitidine

solution, the Lactobacillus plantarum HFY09 group received low dose and high dose of

bacterial suspension, and the LB group received LB suspension.

[0022] After the 15th day, the mice were fasted for 24 hours and then treated by

intragastric administration with induction solution hydrochloric acid/ethanol solution

(60% ethanol and 40% 150 mmol/L hydrochloric acid mixture). The induction dose was

ml/kg. Except for the normal group, other groups were given the same dose of

hydrochloric acid/ethanol solution, and the normal group was given the same volume of normal saline. After half an hour, the blood of the mice was collected and centrifuged at

4000 rpm at 4 °C for 10 minutes. Measure the secretion of gastric juice and pH value of

gastric juice. And use ImageJ software to analyse the gastric injury area and calculate the

gastric injury inhibition rate: gastric injury inhibition rate (%)= gastric injury area/gastric

tissue area x 100. See Table 1 for specific administration methods and doses.

Table 1 Experimental groups of animals Group 1st-14th days 15th day Normal Physiological saline (10 ml / kg) Model Physiological saline (10 ml / kg) Hydrochloric acid / ethanol (10 ml / kg) Ranitidine (50 mg / kg). Hydrochloric acid / ethanol (10 Ranitidine ml / kg) LB LB suspension Hydrochloric acid / ethanol (10 (1.Ox1O 9 CFU/kg,10*;K/&FM ) ml / kg) No. 09-h HFY09(1.0x10 9 CFU/kg,10*h/>Y) Hydrochloric acid / ethanol (10 ml / kg) HFY09-1 HFY09(1.0 x 10 8CFU/kg,10%h/>Y) Hydrochloric acid / ethanol (10 ml / kg) 1.3 Determination of ET-1, SP, SS and VIP levels in mice serum

[0023] Collect serum from mice and store it at -80°C. Measure serum endothelin-1 (ET

1), substance P (SP), somatostatin (SS) and vasoactive intestinal peptide (VIP) levels

according to the kit instructions (Shanghai Enzyme-Linked Biotechnology Co., Ltd.).

1.4 Determination of IL-6, IL-12, TNF and IFN-y levels in mice

[0024] The levels of cytokines interleukin-6 (IL-6), interleukin-12 (IL-12), tumour

necrosis factor-a and interferon-y(IFN-) in serum were determined according to the

corresponding kit instructions (Shanghai Enzyme-linked Biotechnology Co., Ltd.).

1.5 Pathological Observation of Mouse Gastric Tissue

[0025] One third of the gastric tissues of mice were taken and placed in 10% formalin

neutral fixation for 48 hours for histopathological analysis. After hematoxylin-eosin (H

E) staining, histopathological morphology was observed.

1.6 RNA expression assay (Q-PCR method)

[0026] Real-time fluorescence quantitative PCR (Q-PCR) is a method that uses

fluorescent chemicals to detect the total amount of products after each PCR cycle in DNA

amplification reaction. The cycle threshold (CT) of both the target gene and the

housekeeping gene was obtained by fluorescence quantitative PCR reaction, and the

relative expression of mRNA in the sample was calculated by relative comparison and

quantitative method (2 -ACt method). Q-PCR was used to detect the relative expressions

of antioxidant enzymes (Cu/Zn-SOD), Mn-SOD, CAT, inflammatory regulatory factors

(endothelial nitric oxide synthase (eNOS), neuronal nitric oxide synthase (nNOS),

inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2)), epidermal

growth factor (EGF) and receptor (eGFR) in different tissue RNA groups. The

corresponding gene primer sequences are shown in Table 2.

Table 2 Primer sequence for Q-PCR detection Gene name order

Forward : 5'-AACCAGTTGTT GT CAGAC-3' Cu/Zn-SOD Reverse: 5'-CCACCATGTT TCTTA GAGTGAGG-3'

Transmit : 5'-CAGACCT GCCTTA CCGACTATGG-3' Manganese-SOD Reverse: 5 "-CTCGGT GGCGTT GAATTGTT-3."

Forward: 5'-GGAGGCG GGAACCCAATA G-3' CAT Reverse : 5'-GTGTG CCATCTCGT CAGTGA-3'

Transmit : 5'-TCAGCCACAGTT CCC-3' eNOS Reverse: 5 "-ATAGCCCGCA TAGCGTA AG-3"

NOS Transmit : 5'-ACGGCAAACT GCACAAGC-3'

Reverse 5'-CGTTC TGAATACGGT TGTTG-3'

Transmit 5'-GT TCAGCCAACAATA CAGA-3' iNOS Reverse: 5'-GTGACGGGTC GATGTAC-3'

Forward : 5'-GTGCCTGGTGATGATG-3' COX -2 Reverse: 5 "-TGCT GTGT GGAATAGTT GCT-3"

Transmit: 5 "-GCCAA GCTC AGAAGGCTA C-3." egr Reverse : 5'-CAGCCAGCCTCGTCCTCT-3'.

Forward : 5'-CAGAA GCCACTCTGACT CCC-3' egrf Reverse : 5'-GTCCATGTCAACAGTG-3'.

Transmit: 5 "GGCT GTATT CCCC TCCATCG-3" 3 -actin Reverse : 5'CCAGTT GGTA ACATGCCATGT-3'

1.7 Statistical analysis

[0027] The data are expressed as mean standard deviation. The difference between the

average values of the groups was evaluated by single factor analysis. Analysis of variance

(ANOVA) by Duncan's multiple range test showed significant differences (P < 0.05). All

figures were plotted using Origin8.0 software.

2. Results

2.1 Determination of gastric mucosal damage

[0028] As shown in FIG. 1 and Table 3, compared with the normal group, the gastric

mucosa of the model group showed obvious bleeding and erosion, and the gastric

mucosal lesion area was the largest. After treatment with HFY09, ranitidine and LB, the

area of injury decreased significantly (P < 0.05), and the inhibitory rate of ranitidin on the

site of injury was the highest. The inhibitory rate of HFY09-H was between ranitidine and HFY09-L and significantly higher than that of LB (P < 0.05). Therefore, HFY09 can effectively reduce the effect of gastric injury on gastric mucosa.

Table 3 Gastric injury area and gastric injury inhibition rate (n=10) Group Gastric injury area (mm) Inhibition rate of gastric injury(%) 2) Normal 0.00 0. 00a 100 0. 00° Model 21.85 2. 38 Ranitidine 3.33 0. 81l 84.46 5. 37d LB 17.24 0. 65° 20.80 5. 64a No. 09-h 7.87 0. 31c 63.70 5. 38c HFY09-1 11.58 1. 00 d 46.95 ±1. 19

[0029] The value given is the standard deviation. In the same column, there was a

significant difference (p < 0.05) in the values of the different letters according to the

Duncan multiple range test.

[0030] Normal = normal mice; Model = mice treated with hydrochloric acid / ethanol (10

ml / kg); Ranitidine = 50 mg / kg / day, by gavage; HFY09 = mice treated with

hydrochloric acid / ethanol (day 15th) and Lactobacillus plantarum HFY09 (L,H)(108 , 109

CFU / kg / day); LB = mice treated with hydrochloric acid / ethanol (the 15th day) and

subspecies Lactobacillus delbrueckii; Lactobacillus bulgaricus (109 CFU per kg per day).

2.2 Determination of mice gastric juice volume and pH

[0031] See Table 4 for the volume and pH value of gastric juice in each group. The

gastric juice pH value was the highest in the model group, while the gastric fluid pH was

the lowest in the control group. HFY09-H group and ranitidine group could effectively

improve the adverse changes of gastric juice volume and pH induced by HCl / ethanol,

which showed that the gastric juice pH increased and the gastric fluid volume decreased.

Table 4 Gastric juice volume and pH of experimental mice (n=10) Group Gastric juice volume (mL). H value of gastric acid Normal 0.143±0.005a 3.700±0.483c Model 0.342±0.061d 1.400±0.882a Ranitidine 0. 17 5 ±0.0 4 3b 2.600±0.843b

LB 0.310±0.085d 1.444±0.699a No. 09-h 0.217±0.059BC 2.200g0.541ab HFY09-1 0.2690.079CD 1.500±0.707a

[0032] The value given is the standard deviation. In the same column, there was a

significant difference (p < 0.05) in the values of the different letters according to the

Duncan multiple range test.

[0033] Normal = normal mice; Model = mice treated with hydrochloric acid / ethanol (10

ml / kg); Ranitidine = 50 mg / kg / day, by gavage; HFY09 = mice treated with

hydrochloric acid / ethanol (day 15th) and Lactobacillus plantarum HFY09 (L,H)(108 , 109

CFU / kg / day); LB = mice treated with hydrochloric acid / ethanol (the 15th day) and

subspecies Lactobacillus delbrueckii; Lactobacillus bulgaricus (109 CFU per kg per day).

2.3 Pathological observation of mouse stomach tissue

[0034] A pathological section of gastric tissue of mice with acute gastric injury caused by

HCl / ethanol is shown in FIG. 2. In normal group, the structure of gastric mucosa was

complete, and no inflammatory cell infiltration was found, while in model group,

epithelial cells were decreased and the cell gap was enlarged, and the gastric lesion was

the most serious. After treatment, the gastric mucosa of HFY09 high-dose group and

ranitidine group was similar to that of normal group, with clear structure and no obvious

damage. Compared with the normal group, the gastric mucosal cells in the HFY09 low

dose group were still partially exfoliated, but also improved. Therefore, HFY09 has a

protective effect on gastric injury to a certain extent, and has a stronger effect at high

concentration.

2.4 Determination of ET-1, SP, SS and VIP levels in mice serum

[0035] The normal group had the highest levels of SS and VIP, and the lowest levels of

ET-1 and SP, as shown in Table 5. However, the four indexes of gastric ulcer in the

model group are opposite to those in the normal group. After treatment, the levels of SS

and VIP in serum increased significantly (P < 0.05), while the levels of SP and ET-1

decreased significantly (P < 0.05). The ability of HFY09-H in serum to regulate the levels

of S S, SP, VIP and ET-1 was slightly lower than that of ranitidine, but the effect was

stronger than that of LB control group.

Table 5 Levels of serum ET-1, SP, SS and VIP in mice (n=10) Group ET-1 (ng/L) SP (ng/L) SS (ng/L) VIP (ng/L) Normal 70.91±5.56a 65.82±4.14a 211.97±8.47° 431.27±28.55° Model . ± 12 2 6 1 12 5 1 . d 152.51±17.67f 112.55±2.59a 212.92±38.12a Ranitidine 8 5 . 2 3 ±8. 89 b 7 5 . 8 7 ±6. 04b 18 9 . 6 1 ± 15 . 15 d 423.18±20.43° LB 108.36±14.93c 129.35±16.59° 144.74±13.55c 312.87±26.92c No. 09-h 92.80±9.44 92.91±5.87c 152.51±12.04c 3 5 1 .0 4 ±34 . 5 6 d HFY09-1 109.29±12.51c . 10 3 2 9 ±3. 73 d . 12 2 6 0 ±4. 93 b 2 6 8 .5 9 ±34 . 3 3b

[0036] The value given is the standard deviation. In the same column, there was a

significant difference (p < 0.05) in the values of the different letters according to the

Duncan multiple range test.

[0037] Normal = normal mice; Model = mice treated with hydrochloric acid / ethanol (10

ml / kg); Ranitidine = 50 mg / kg / day, by gavage; HFY09 = mice treated with

hydrochloric acid / ethanol (day 15th) and Lactobacillus plantarum HFY09 (L,H)(108 , 109

CFU / kg / day); LB = mice treated with hydrochloric acid / ethanol (the 15th day) and

subspecies Lactobacillus delbrueckii; Lactobacillus bulgaricus (109 CFU per kg per day).

2.5 Determination of IL-6, IL-12, TNF-a and IFN-y levels in mice serum

[0038] The levels of IL-6, IL-12, TNF-a and IFN-y in healthy mice were the lowest.

However, the levels of inflammatory factors in HCl / ethanol-induced gastric injury mice

increased and decreased after treatment, as shown in Table 6. The greatest decrease was in the ranitidine group, followed by the HFY09-H treatment group. In the HFY09-H group, the effect of reducing inflammatory factors was stronger than that in the HFY09-L group.

Table 6 Levels of serum IL-6, IL-12, TNF and IFN-in mice (n=10) Group IL-6 (ng/L) IL-12 (ng/L) Tumor necrosis IFN-(ng/L) factor-(ng/L) Normal 102.74±10.01a 6.89±0.92a 675.64±63.24a 487.99+20.17a Model 183.82±15.21f 14.16±1.22° 1340.68±163.49° 821.75±36.09° Ranitidine 1 1 3 .4 9 ± 1 2 . 6 5b 8. 1 9 ±0. 8 5b 881. 8 4 ± 9 7 .4 2b 615.99+79.5 1 b LB 154.97±14.62° . 12 5 1 ±1. 23 d 997.38±73.06c 764.75±8.59d No. 09-h 127.51±7.64c 8 . 8 7 ±1. 2 4 b 9 3 8 .4 0 ± 1 2 3 .4 5 ab 683.67±44.81c HFY09-1 . ± 13 4 19 12 4 8 . d 11.21±0.96c . ± 10 9 5 18 84 2 1 . d 853.74±49.54°

[0039] The value given is the standard deviation. In the same column, there was a

significant difference (p < 0.05) in the values of the different letters according to the

Duncan multiple range test.

[0040] Normal = normal mice; Model = mice treated with hydrochloric acid / ethanol (10

ml / kg); Ranitidine = 50 mg / kg / day, by gavage; HFY09 = mice treated with

hydrochloric acid / ethanol (day 15th) and Lactobacillus plantarum HFY09 (L,H)(108 , 109

CFU / kg / day); LB = mice treated with hydrochloric acid / ethanol (the 15th day) and

subspecies Lactobacillus delbrueckii; Lactobacillus bulgaricus (109 CFU per kg per day).

2.6 Gene Expression of Cu/Zn-SOD, Mn-SOD and CAT in Mouse Gastric Tissue

[0041] The expression of Mn-SOD, Cu / Zn SOD and CAT in mouse stomach was

examined by Q-PCR method, as shown in FIG. 3. The results of FIG. 3 show that the

expression levels of Mn-SOD, Cu/Zn SOD and CATm RNA in gastric tissue of healthy

mice are the highest, while the expression levels of model group are the lowest. The

levels of Mn-SOD, Cu/Zn-SOD and CAT in Ranitidine group and HFY09-H group were

higher than those in model group, and the level of expression in Ranitidine group was higher than that in HFY09-H group. The expression level of LB was similar to that of

HFY09-L.

2.7 Gene Expression of eNOS, iNOS, nNOS and COX-2 in Mouse Gastric Tissue

[0042] The expression of COX-2, iNOS, eNOS and nNOS in mouse stomach tissue is

shown in FIG. 4. The expression of eNOS and nNOS was the highest in normal group,

but COX-2 and iNOS were the lowest in model group. The relative expression ofiNOS

and COX-2 in HFY09 group was significantly lower than that in model group, while that

in high-dose group (HFF09-H) was lower than those in low-dose (HFO9-L) and LB

treated groups. The difference between HFY09-H group and ranitidine group was small.

These data show that HFY09 can inhibit inflammation and protect gastric tissue of

HCl/ethanol-induced gastric injury model mice, and the effect is stronger at high

concentration.

2.8. Gene Expression of EGF and EGFR in Mouse Gastric Tissue

[0043] FIG. 5 shows gene expression of EGF and EGFR in mouse stomachs. The

expression level of EGF and EGFR in model group was significantly lower than that in

normal group (P < 0.05). The effects of ranitidine, LB and HFY09 on the expression of

EGF and EGFR genes were significantly different from those in the model group (P <

0.05). In the HFY09 high dose group, the gene expression of EGF and EGFR was lower

than that of ranitidine treatment group, but significantly higher than those of the LB

group and HFFY09 low dose group. At the same dose, the effect of HFY09H was more

obvious than that of HFFY09L.

3. Conclusion

[0044] Acute gastric injury is usually caused by acute alcohol abuse, with the main

symptoms being damage to the plasma membrane of epithelial cells, resulting in cell

shedding, multiple gastric erosions and ulcers. If blood vessels are involved, the damage

can lead to massive bleeding and inflammation of the stomach lining. Animal

experiments have shown that the extent of gastric injury can be determined by measuring

the area of ulceration through direct observation of gastric mucosal tissue ulceration.

[0045] Lactic acid bacteria (LAB) is widely distributed in the human gastrointestinal tract

and plays an important role in maintaining the microecological balance of the

gastrointestinal tract. A large number of studies have proved that beneficial bacteria have

certain therapeutic effect on various intestinal diseases, but there are few studies on the

effects of probiotics such as lactobacilli, bifidobacteria and yeasts on stomach-related

diseases.

[0046] The present invention determines that the bacterial concentration of the lactic acid

bacteria beverage must be higher than 107 CFU / mL by referring to related studies and

international standards, which is also why 109 CFU / ml is frequently used in animal

experiments. Therefore, in the present invention, 109 CFU / kg and 108 CFU / Kg were

selected as the study concentrations to investigate the preventive effect of Lactobacillus

plantarum HFY09 on acute gastric injury.

[0047] Gastric juice of healthy mice did not affect gastric mucosa. However, when the

gastric mucosa is damaged, the secretion of gastric juice increases, and thus the pH of the

gastric juice decreases. The increase of gastric juice volume and the decrease of pH value aggravate the damage of gastric mucosa and aggravate the injury of stomach, leading to the vicious circle. On the one hand, in the present study, the gastric juice pH of the model group was significantly lower than that of the normal group. HFY09 treatment can effectively reduce the amount of gastric juice, increase the pH value of the gastric juice so as to protect the gastric tissue and reduce the gastric injury caused by alcohol. On the other hand, by analysing the area of gastric injury and using ImageJ software to calculate the inhibition rate of gastric injury, the degree of gastric injury in each group can be directly compared. As shown in Table 3, the gastric mucosa of the model group showed significant erosion as compared with the normal group. Based on the area of gastric injury in model group, the gastric injury area in low dose group and high dose group were significantly decreased (P < 0.05), and the inhibition rates of stomach injury were 46.95% and 63.70%, respectively, but the inhibition rate of HFY09-H was lower than that in the positive control ranitidine group (84.46%). The results showed that HFY09 had a certain preventive effect on gastric injury.

[0048] In addition, histopathological observation is also an important clinical standard for

diagnosing gastric injury. In order to study and evaluate the protective effect of HFY09

on gastric injury induced by HCl/ethanol, the invention analyses gastric tissue sections of

mice, and HFY09 has certain preventive effect on gastric injury, indicating that gastric

mucosal epithelial cells fall off and intercellular gaps decrease.

[0049] Gastrointestinal hormones, such as SP, ET, SS and VIP, play an important role in

the regulation of gastrointestinal motility. Endothelin (ET) is an endothelium-derived

vasoconstrictor consisting of 21 amino acids. It is the most effective vasoconstrictor

peptide found so far and plays an important role in gastric mucosal injury. Previous studies have shown that ET causes injury primarily by reducing internal blood flow and weakening the protective mechanisms of the gastric mucosa, which leads to severe ulceration and plays a key role in the pathogenesis of ulcerative colitis. SS and VIP are gastrointestinal hormones that inhibit secretion of gastric juice. SS can protect gastric mucosa and promote the healing of gastric lesions by inhibiting the release of MTL and pepsin. When the stomach is stimulated, the decrease of SS will aggravate the secretion of gastrointestinal fluid and the inflammation of the gastrointestinal tract.

[0050] VIP level is closely related to immune regulation and inflammation caused by

gastric injury. At the same time, VIP can inhibit the transcription of iNOS in vivo and

prevent iNOS from being converted into excessive NO to cause damage to gastric

mucosa. SP is an excitatory gastrointestinal hormone. Under stress, its production will

increase, which will lead to a large amount of gastric juice secretion and strong gastric

acidity, leading to gastric mucosal injury. Table 5 shows that HCl / ethanol induced

gastrointestinal hormone secretion imbalance, serum ET-1 and SP significantly increased

(P < 0.05), and SS and VIP significantly decreased (P > 0.05) in mice. HFY09 can inhibit

gastric acid secretion, alleviate gastric mucosal damage, increase serum SS and VIP

levels, and decrease ET-1 and SP levels. Its effect is similar to ranitidine.

[0051] Another effective method is to control the number of pro-inflammatory factors.

Animal experiments show that the release of a large number of oxygen free radicals

caused by ethanol destroys the oxidation-reduction balance in the body, causes various

immune reactions, makes the immune system to release excessive pro-inflammatory

factors, and triggers inflammatory reactions. Therefore, the levels of IL-6, IL-12, IFN-,

TNF and other cytokines in the serum of patients with inflammatory diseases are higher than those of healthy people. Reducing these inflammatory cytokines may be an effective way to prevent gastric injury. Previous studies have also shown that probiotics can reduce ethanol-induced stomach damage through their own anti-inflammatory properties. Studies have found that Lactobacillus lutei DSM17938 supplement can protect mucosal adhesion caused by ethanol-induced gastric injury in mice. Lactobacillus fermentum can reduce the levels of serum inflammatory factors IL-6, IL-12, TNF and IFN-in HC/ethanol-induced gastric injury mice. In the current study, ranitidine, LB and HFY09 reduced the levels of

IL-6, IL-12, IFN-, TNF and other inflammatory related factors in serum. Among them,

ranitidine has the most obvious effect, followed by high-dose HFY09, which indicates

that high-dose HFY09 is effective in preventing gastric injury.

[0052] Alcohol-induced gastric injury is associated with the expression of antioxidant

genes. Studies have shown that the level of xanthine oxidase in the gastrointestinal tract is

much higher than in other tissues. Due to gastrointestinal metabolism, ethanol will be

converted to acetaldehyde. Acetaldehyde will further produce oxygen free radicals under

the action of xanthine oxidase, thus leading to oxidative damage of gastric mucosa.

Therefore, oxygen free radicals are one of the important mechanisms of ethanol - induced

gastric injury. Cu / Zn SOD and Mn-SOD can eliminate superoxide dismutase, hydrogen

peroxide, hydroxyl and lipid peroxide, regulate gastric injury, and even restore it to

normal levels. Therefore, the antioxidant effect of HFY09 can be determined by

measuring the levels of these important antioxidant-related genes.

[0053] According to the results of this experiment, high dose HFY09 increased the

expression of Cu / Zn SOD, Mn-SOD and CATm RNA compared to the model group,

indicating that free radicals of gastric oxidative metabolism induced by HCl / ethanol can be eliminated. HFY09 has certain antioxidation effect on gastric injury and can inhibit gastric injury. No is a highly unstable biological free radical which is lipophilic, can freely pass through the cell membrane and be highly oxidized with a half-life of only 3 to seconds. Its production is dependent on nitric oxide synthase (NOS), including nNOS, eNOS and iNOS. NOS and eNOS produce low levels of NO and protect gastric mucosa to some extent. The occurrence of gastric ulcer is related to the excessive inhibition of eNOS and nNOS levels. At the same time, the present invention also measures the expression of relevant inflammatory factors in gastric tissues, such as iNOS and COX-2, which can be used to inhibit the production of inflammatory cytokines.

[0054] COX-2 is the isozyme of COX. Cytokines and endotoxin can induce the

production of COX-2, participate in inflammatory reaction and promote the development

of inflammation. Therefore, when the cells are stimulated by inflammation, COX-2 is

highly expressed, and lycopene can down-regulate the expression of COX-2, so as to

alleviate the inflammatory reaction and thus alleviate the gastric injury. In addition, iNOS

interact with COX-2 to produce a synergistic effect, which can further reduce that

inflammatory response. In the present study, Lactobacillus plantarum HFY09 also

effectively controlled the expression of iNOS and COX-2 in mouse stomach tissue,

increased the expressions of nNOS and eNOS, and alleviated the stomach damage.

[0055] Epidermal growth factor can promote epithelial proliferation, tissue repair and cell

protection. It plays an important role in protecting gastric mucosa from injury factors and

maintaining the integrity of gastrointestinal mucosa. EGF exerts its biological effect

through its receptor EGFR. The results of the present invention show that the expression

of EGF and EGFR in gastric tissue of HCl / ethanol-induced gastric injury mice is significantly lower than that of normal mice. Epidermal growth factor, as a protective factor, binds to its receptor EGFR to accelerate ulcer healing, improve the ability of tissue repair, and maintain the quality of ulcer healing. In the present study, the treatment of

HFY09 significantly increased the expression level of EGF and EGFR genes in gastric

tissue of mice with gastric injury, suggesting that HFFY09 has a protective effect on HCl

/ ethanol-induced gastric injury in mice, and the effect is stronger at higher doses.

[0056] In conclusion, the present invention adopts the hydrochloric acid / ethanol acute

gastric injury model to evaluate the preventive effect of Lactobacillus plantarum HFY09

on gastric injury. The results showed that HFY09 could inhibit the secretion of gastric

juice, maintain the normal pH of gastric acid and reduce the area of gastric mucosal

lesion. Compared with the model group, the levels of SP, ET-1, IL-6, IL- 12, TNFa and

IFN- y in HFY09-H group were decreased, while those of SS and VIP were increased.

Further experiments showed that HFY09 increased the expression of eNOS, nNOS, Cu/

Zn SOD, Mn-SOD, EGF and EGFR, and decreased the expressions ofiNOS and COX-2

in gastric tissue. indicating that HFY09 can inhibit the apoptosis of gastric mucosal

epithelial cells and play a biological role, which is positively correlated with the

concentration. The results show that HFY09 can be used in the production of probiotics

or functional foods, providing theoretical basis for its further development and utilization.

[0057] The above-described embodiments are merely a description of the preferred mode

of the invention and are not intended to limit the scope of the invention. Without

departing from the spirit of the invention, various variations and modifications made by

those ordinary technical personnel in the art to the technical scheme of the invention

should fall within the scope of protection as determined in the claims of the invention.

Claims (2)

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. Lactobacillus plantarum (HFY09), is characterized in that the Lactobacillus

Plantarum HFY09 above belongs to the lactic acid bacteria (LAB), and has been

deposited in the China General Microbiological Culture Collection Center, with

the preservation date of October 25, 2018 and the preservation number CGMCC

No. 16633.

2. Use of a Lactobacillus plantarum HFY09 according to claim 1 in the

preparation of a medicament for preventing or treating gastric injury.

-1/5-

Figure 1

-2/5-

Figure 2

-3/5-

Figure 3

-4/5-

Figure 4

-5/5-

Figure 5