Lactobacillus paracasei and application thereof in improvement of gastric ulcer

Abstract

The invention belongs to the technical field of biology, and particularly relates to lactobacillus paracasei and application thereof in improvement of gastric ulcer. The lactobacillus paracasei NCU-21 is preserved in China General Microbiological Culture Collection Center (CGMCC) on September 25, 2023, the address of the lactobacillus paracasei NCU-21 is No.3, No.1 yard, Beichen West Road, Chaoyang District, Beijing, and the preservation number of the lactobacillus paracasei NCU-21 is CGMCC NO.28538. The lactobacillus paracasei NCU-21 is named as Lactobacillus paracasei NCU-21. The invention provides a strain of lactobacillus paracasei NCU-21, and the data result of the embodiment shows that the lactobacillus paracasei NCU-21 can promote the healing of ethanol gastric ulcer and improve gastric mucosal lesion caused by oxidative stress. In addition, the strain can also regulate stomach microbiota, improve ethanol-induced gastric mucosal lesion, improve ethanol-induced intestinal inflammation, repair intestinal barriers and relieve ethanol-induced intestinal microbiota disorder.

Description

Technical Field

[0001] This application belongs to the field of biotechnology, and specifically relates to a strain of Lactobacillus paracasei and its application in improving gastric ulcer. Background Art

[0002] Gastric ulcer mostly occurs in the lesser curvature of the stomach and the gastric antrum, usually caused by the self-digestion of the gastrointestinal mucosa by gastric acid or pepsin. In recent years, the incidence of gastric ulcer in China has been gradually increasing, which has seriously affected the normal life of patients. The main clinical symptoms after the onset of the disease are upper abdominal pain, which can be dull pain, distending pain, dull ache, etc. Gastric ulcer has the characteristics of a long course and easy recurrence. If gastric ulcer is not treated in time, it is also easy to cause some complications, and its threat to health far exceeds that of gastric ulcer. It mainly includes ulcer bleeding, perforation, pyloric obstruction, and ulcer canceration.

[0003] At present, the main treatment for gastric ulcer is drug treatment, mainly including proton pump inhibitors, H2 receptor antagonists, antibacterial drugs, gastric mucosal protective drugs, etc. Among them, most adopt the triple therapy of proton pump inhibitors plus two antibiotics, and there is also a treatment plan of adding bismuth agents and other gastric mucosal protectants to the triple therapy. Clinical practice has confirmed that the combination of traditional Chinese and Western medicine in the treatment of gastric ulcer also has certain advantages. However, the side effects of drugs are large, resulting in a decrease in the treatment compliance of patients, an increase in antibiotic resistance, and the recurrence rate of ulcers. Therefore, there is an urgent need to develop a new method. Summary of the Invention

[0004] The purpose of the present invention is to solve the deficiencies of the prior art, and provide a strain of Lactobacillus paracasei and its application in improving gastric ulcer. The specific technical solutions are as follows: In the first aspect, the present invention provides a strain of Lactobacillus paracasei NCU-21, which is characterized in that the Lactobacillus paracasei ( Lactobacillus paracasei ) NCU-21 was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on September 25, 2023. The address is: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC NO. 28538.

[0005] As a further preferred embodiment, the gene sequence of the 16S rDNA of the Lactobacillus paracasei NCU-21 is as shown in SEQ ID No.1.

[0006] SEQ ID No.1:

[0007] In a second aspect, the present invention provides the use of the above-mentioned Lactobacillus paracasei NCU-21 in the preparation of a product for preventing and / or improving and / or treating gastric ulcers.

[0008] As a further preferred embodiment, the gastric ulcer includes a gastric ulcer caused by ethanol.

[0009] As a further preferred embodiment, the product includes a drug or a microbial inoculum.

[0010] In a third aspect, the present invention provides a microbial inoculum, which includes the above-mentioned Lactobacillus paracasei NCU-21 or its ferment or its metabolite.

[0011] In a fourth aspect, the present invention provides a drug for improving gastric ulcers, which includes the above-mentioned Lactobacillus paracasei NCU-21.

[0012] As a further preferred embodiment, the drug further includes a pharmaceutical excipient.

[0013] As a further preferred embodiment, the pharmaceutical excipient is at least one of water, lactose, sodium chloride, and glucose.

[0014] As a further preferred embodiment, the dosage form of the drug includes one or more of powder, tablet, capsule, aqueous solution, gel, ointment, dripping pill, pill, and granule.

[0015] The beneficial effects of the present invention are as follows: The present invention provides a strain of Lactobacillus paracasei NCU-21, which can improve or treat gastric ulcers; according to the data results of this embodiment, Lactobacillus paracasei NCU-21 can inhibit ethanol-induced gastric mucosal injury, can also promote the healing of ethanol-induced gastric ulcers and improve gastric mucosal injury caused by oxidative stress. In addition, this strain can also regulate the gastric microbiota to improve ethanol-induced gastric mucosal injury, improve ethanol-induced intestinal inflammation and repair the intestinal barrier, and alleviate ethanol-induced intestinal microbiota dysbiosis. Description of the Drawings

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0017] Figure 1The figure shows the in vitro probiotic property results of Lactobacillus paracasei NCU-21; A, growth curve; B, antioxidant capacity measurement result; C, antibacterial activity result; D, acid tolerance result; E, bile salt tolerance result; F, antibiotic resistance test result; Figure 2 The figure shows the therapeutic effect of Lactobacillus paracasei NCU-21 on ethanol-induced gastric mucosal injury in mice; A, schematic diagram of the establishment of an ethanol-induced gastric ulcer mouse model; B, macroscopic damage and hematoxylin and eosin staining of gastric mucosa; C, gastric ulcer area; D, gastric ulcer index; E, gastric ulcer inhibition index; F-H, levels of MDA (F), MPO (G) and SOD (H) in ethanol-induced gastric ulcer tissue; Figure 3 The figure shows the effects of L. paracasei NCU-21 on the oxidative stress signaling pathway and growth factor signaling pathway in ethanol-induced gastric ulcer mice; A, immunohistochemical staining of TGF-β, VEGF and EGFR in gastric tissues of ethanol-induced gastric ulcer mice; B, Western blot detection of the expression of proteins related to the oxidative stress signaling pathway in gastric tissues; C-F, quantitative analysis of proteins related to the oxidative stress signaling pathway in gastric tissues, including HO-1 (C), NQO1 (D), Keap1 (E) and Nrf2 (F); Figure 4 The figure shows the effects of L. paracasei NCU-21 on the inflammatory signaling pathway and inflammatory factor signaling pathway in ethanol-induced gastric ulcer mice; A, immunohistochemical staining of PCNA in gastric tissues of mice with ethanol-induced gastric mucosal injury; B, Western blot detection of the expression of proteins related to the NF-κb signaling pathway in gastric tissues; C-G, quantitative analysis of proteins related to the NF-κb signaling pathway in gastric tissues, including p-Iκb (C), p-p65 (D), determination of the levels of IL-1β (E), TNF-α (F) and IL-1β (G) in gastric tissues; Figure 5 The figure shows that L. paracasei NCU-21 changed the gastric flora composition and diversity in ethanol-induced gastric ulcer mice; A, Chao 1 index representing the α-diversity of gastric microbiota; B, Shannon index representing the α-diversity of gastric microbiota; C, Beta diversity was determined by principal coordinate analysis (PCoA); D, Venn diagram showing the overlap of OTUs in the gastric microbiota; E, phylum-level distribution of gastric microbiota, F-H; relative abundances of Firmicutes (F), Bacteroidetes (G) and Proteobacteria (H). (I) genus-level distribution of gastric microbiota; I-M, relative abundances of Lactobacillus (J), Enterococcus (K), Oscillosra (L) and Allobaculum (M); Figure 6 Shown is the therapeutic effect of L. paracasei NCU-21 on ethanol-induced intestinal barrier injury and inflammation in mice; (A) Hematoxylin and eosin staining of colon tissue; B, Western blot of NF-κb-related protein expression in colon tissue; C-E, Quantitative analysis of TLR4 (C), MyD88 (D) and p-p65 (E) proteins; (F) Western blot of barrier-related protein expression in colon tissue; G-I, Quantitative analysis of proteins Claudin-1 (G) and Occuldin (H); (I) Determination of L-lactic acid in serum; Figure 7 Shown is that L. paracasei NCU-21 changed the intestinal flora composition and diversity in ethanol-induced gastric ulcer mice; A, Chao1 index representing the α-diversity of intestinal microbiota; B, Pielou_e index representing the α-diversity of intestinal microbiota; C, Beta diversity was determined by principal coordinate analysis (PCoA); D, Venn diagram showing the overlap of OTUs in intestinal microbiota; E, Phylum-level distribution of intestinal microbiota; F-H, Relative abundances of Firmicutes (F), Bacteroidetes (G) and Proteobacteria (H); (I) Genus-level distribution of intestinal microbiota; J-M, Relative abundances of Lactobacillus (J), Enterococcus (K), Lactobacillus (L) and Prevotella (M); Figure 8 Shown is the microscopic morphology diagram of Lactobacillus paracasei NCU-21; Figure 9 Shown is the pathological section diagram of mouse modeling. Detailed implementation manners

[0018] Next, the technical solutions in the embodiments of the present application will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present application.

[0019] Example 1 Screening of Lactobacillus paracasei NCU-21 and related experiments The probiotic strain in the present invention L. paracasei NCU-21 (CGMCC No.28538) was isolated from the fecal samples of healthy women, and the preservation date was September 25, 2023. The specific isolation method is as follows: Mix the fecal sample with 5 mL of PBS buffer, homogenize it, and place it at 37°C for 2-3 hours to allow natural sedimentation. Then, take the supernatant for serial dilution, spread it on MRS solid medium, and culture it for 24-48 hours under aerobic or anaerobic conditions. Select the plate with 200-400 colonies, and pick 5-10 single colonies according to the colony morphological characteristics and growth rate. Inoculate these single colonies into 5 mL of MRS liquid medium and culture it for 24-28 hours under aerobic or anaerobic conditions. Through comprehensive analysis of cell morphology, physiological and biochemical characteristics, 16S rRNA gene sequence (as shown in SEQ ID No.1), and dnaK gene sequence, the target strain was finally identified as Lactobacillus paracasei ( Lactobacillus paracasei ) NCU-21, and its microscopic morphology diagram is as shown in Figure 8 .

[0020] Example 2 Determination of in vitro probiotic properties of Lactobacillus paracasei NCU-21 (1) Acid tolerance experiment Culture the strain in MRS medium in a 37°C constant temperature incubator until the OD value reaches 0.6, then terminate the culture and reserve the strain. Take out 100 μL and dilute it 10 1 , 10 3 , 10 5 times with PBS buffer, centrifuge at 6000 g for 3 min and discard the supernatant; add PBS buffer with pH = 1, 3, 5, 7, place it for 4 hours, mix well, take 10 μL and spread it on a plate, culture it in a 37°C constant temperature incubator for 12-48 hours, perform viable count, and record the experiment well.

[0021] (2) Bile salt tolerance experiment Culture the strain in MRS (de Man, Rogosa and Sharpe) medium in a 37°C constant temperature incubator until the OD value reaches 0.6, then terminate the culture and reserve the strain. Take out 100 μL and dilute it 10 1 , 10 3 , 10 5 times with PBS buffer, centrifuge at 6000 g for 3 min and discard the supernatant; add the medium containing 0.1%-0.3% (0, 0.1, 0.2, 0.3) bovine bile salt and culture it in a 37°C constant temperature incubator for 12-48 hours, mix well, take 10 μL and spread it on a plate, culture it in a 37°C constant temperature incubator for 12-48 hours, perform viable count, and record the experiment well.

[0022] (3) Cell adhesion experiment The strain was cultured in MRS medium in a 37 °C constant temperature incubator until the OD value reached 0.6, and then the culture was terminated. The strain was reserved for later use. The six-well cell culture plate was washed once with sterile PBS buffer, and a sterilized coverslip was placed in it. 1 mL of the above bacterial culture solution was mixed with 1 mL of the culture solution containing a small amount of 293T cells, and then added to the six-well plate. The cells were cultured in a 37 °C cell culture incubator. After 1 - 1.5 hours of culture, the six-well tissue cell culture plate was taken out, the culture solution was aspirated, and it was washed repeatedly 5 times with PBS buffer, fixed with methanol, stained with Gram stain, and observed and photographed under an oil microscope for recording.

[0023] (4)Antibacterial experiment Lactobacillus paracasei was inoculated into MRS liquid medium and cultured in a carbon dioxide incubator at 37 °C for 36 - 72 h, and then centrifuged at 6,000 g for 5 min. Shigella flexneri ATCC 12022, β-hemolytic streptococcus, Salmonella typhimurium ATCC 13311, Staphylococcus aureus Cowan1, Escherichia coli O157, Salmonella enteritidis ATCC 13076 were spread on LB solid medium. An Oxford cup was gently placed on the plate, and 250 μL of the probiotic culture supernatant was aspirated into the Oxford cup (two replicates were made for each group). The cells were cultured at a constant temperature of 37 °C. The size of the antibacterial zone was observed every 2 h, and the diameter of the antibacterial zone was measured after 8 h. The experimental records were made.

[0024] (5)Growth curve Lactobacillus paracasei was cultured in a shaker at 37 °C, and the optical density value of the bacterial solution was measured every 2 hours.

[0025] (6)Antioxidant experiment a. Determination of DPPH free radical scavenging ability: Add 2 mL of (0.2 mmol / L) DPPH methanol solution to 2 mL of the supernatant of the bacterial culture solution, and react at room temperature in the dark for 30 min. Take the supernatant and measure the OD at 517 nm. The control is 2 mL of deionized water plus DPPH methanol solution. DPPH free radical scavenging rate = [1 - A517 (sample) / A517 (blank)] × 100% b. Determination of hydroxyl free radical scavenging ability: Take 1 mL of 2 mmol / L FeSO4 solution, 1 mL of 6 mmol / L H2O2, 1 mL of 6 mmol / L salicylic acid, add 1 mL of the supernatant of the bacterial culture solution, and let it stand at room temperature for 30 min. Using deionized water as the blank control, measure the absorbance at 510 nm, and calculate the scavenging rate of hydroxyl free radicals: Hydroxyl free radical scavenging rate = [1 - A510 (sample) / A510 (blank)] × 100% c. Determination of superoxide radical scavenging ability: In 0.5 mL of bacterial culture solution, 2 mL of 150 mmol / L Tris-Hcl solution with pH = 8.0 and 1 mL of 1.2 mmol / L pyrogallol solution were added successively. The reaction was carried out at room temperature for 30 min, and the absorbance at 330 nm was measured: Superoxide radical scavenging rate = [1 - (A11 - A10) / (A01 - A00)] × 100% A00: Without sample and pyrogallol; A01: Without sample but with pyrogallol A10: With sample but without pyrogallol; A11: With sample and pyrogallol d. Determination of the chelating ability towards Fe 2+ : 0.1 mL of 0.4% ferrous sulfate solution was added to 0.5 mL of sample solution. After gently inverting up and down to mix evenly, 0.1 mL of 1% VC and 1 mL of 0.2 mol / L NaOH solution were added. The reaction was carried out at room temperature for 20 min, and then 1 mL of 10% trichloroacetic acid was added. The mixture was centrifuged at 6,000 rpm for 10 min at 4 °C to remove proteins. 0.4 mL of the above solution was taken and 4 mL of 0.1% o-phenanthroline was added. The reaction was carried out at room temperature for 10 min, and the absorbance at 536 nm was measured: Chelating ability of Fe 2+ = [A blank - A sample / A blank] × 100% e. Determination of total reducing power 1 mL of bacterial culture solution was taken, 1 mL of phosphate buffer (pH = 6.6) and 1 mL of 1% K3[Fe(CN)6] solution were added. After thoroughly inverting up and down to mix evenly, it was incubated at 50 °C in an incubator for 2 min. 1 mL of 10% trichloroacetic acid solution was added and shaken well. 1 mL of the mixed solution was taken, 4 mL of deionized water and 0.4 mL of 0.1% FeCl3 solution were added, and it was left standing for 10 min. Then, using deionized water as the blank, the absorbance at 700 nm was measured. The larger the absorbance value, the stronger the reducing power of the test substance.

[0026] The above detection results are as Figure 1 shown. From the results, it can be seen that under aerobic culture conditions, L. paracasei NCU-21 reached the logarithmic growth phase within 4 hours and the maximum colony abundance within 20 hours. This indicates that L. paracasei NCU-21 has good activity and is suitable for subsequent experiments ( Figure 1A). To further study the probiotic properties of L. paracasei NCU-21, antioxidant activity, acid and bile salt tolerance, antibacterial activity, and antibiotic resistance tests were conducted. The results of the antioxidant experiment showed that the superoxide anion (·O 2- ) scavenging rate of L. paracasei NCU-21 was 69.96%, the hydroxyl radical (·OH) scavenging rate was 65.82%, the DPPH radical scavenging rate was 84.42%, the Fe 2+ chelating rate was 47.01%, and the total reducing power OD value was 0.86 ( Figure 1 B). In the antibacterial experiment, L. paracasei NCU-21 showed obvious inhibition zones against Shigella flexneri ATCC 12022 (S. flexneri), β-hemolytic streptococcus (S. haemolytic-β), Salmonella typhimurium ATCC 13311 (S. Typhimurium), Staphylococcus aureus Cowan1 (S. aureus), Escherichia coli O157:H7 (E. coil O157:H7), and Salmonella enteritidis ATCC 13076 (S. enteritidis). The diameters of the inhibition zones were 2.083 cm, 1.793 cm, 2.053 cm, 2.020 cm, 1.900 cm, and 1.903 cm, respectively ( Figure 1 C). In the acid and bile salt tolerance experiment, the number of bacterial colonies of L. paracasei NCU-21 did not significantly decrease in solutions with high acidity or high bile salt concentration. At pH = 1.0, the average was 3.69×10 5 CFU / mL; at a bile salt concentration of 0.3%, there were still 1.21×10 5 CFU / mL ( Figure 1 D-E). In the antibiotic resistance experiment, the growth of L. paracasei NCU-21 was significantly inhibited by different antibiotics. The sizes of the inhibition zones were 2.53 cm for sulfamethoxazole-trimethoprim (SXT), 2.59 cm for minocycline (MY), 4.26 cm for penicillin (PEN), 3.25 cm for ampicillin (AMP), 3.06 cm for erythromycin (E), 3.07 cm for ciprofloxacin (CIP), and 4.41 cm for tetracycline (TET) ( Figure 1 F). The above results indicate that L. paracasei NCU-21 has good probiotic properties.

[0027] Example 3 Investigation on the improvement effect of Lactobacillus paracasei NCU-21 on ethanol-induced gastric ulcers Establishment and treatment of a gastric mucosal injury mouse model 1. Mouse modeling (the schematic diagram of which is shown as A in Figure 2 ). Thirty-four healthy C57BL / 6 mice, SPF grade, body weight: 20±2 g, male. The mice were adaptively fed under conventional conditions for 1 week. On the 7th day, after fasting for 24 h without water deprivation, each group except the blank control group of mice was intragastrically administered 75% ethanol at a dose of 0.15 mL / 20 g. After 24 h, 2 model mice and 2 normal mice were randomly selected for model evaluation, and pathological sections were observed. The results are shown as Figure 9 (where C1 and C2 are the normal groups, and M1 and M2 are the model groups).

[0028] 2. Mouse grouping Blank control group (Group C, N = 6): Fed and watered normally.

[0029] Gastric mucosa damage model group (Group M, N = 6): Fed and watered normally. M + proton pump inhibitor (Group O, N = 6): Omeprazole (100 mg / kg / day) was dissolved in distilled water and intragastrically administered twice a day for 14 days.

[0030] M + Lactobacillus paracasei low-dose group (Group L, N = 6): After modeling, 1 ml of 1×10 7 CFU / ml Lactobacillus was intragastrically administered once a day for 14 days.

[0031] M + Lactobacillus paracasei high-dose group (Group H, N = 6): After modeling, 1 ml of 1×10 9 CFU / ml Lactobacillus was intragastrically administered once a day for 14 days.

[0032] The body weight of the mice was observed and recorded daily, and their mental activity status, hair color, food intake, and feces (feces were collected on the 7th and 14th days) were evaluated daily.

[0033] 3. Explore the effects on the gastric part and intestinal part respectively for the above-mentioned modeling and treatment 3.1 Histopathological analysis of gastric mucosa 3.1.1 Determination of gastric mucosal ulcer area and ulcer healing rate After the animals were sacrificed, the stomach was taken, the abdominal wall was cut open along the midline of the abdomen, and the stomach and small intestine were separated by cutting at the pyloric part and the gastric juice was collected. Then, the stomach was separated by cutting at the cardiac part. The stomach was cut open along the greater curvature of the stomach, the gastric contents were removed, and after rinsing with normal saline, the ulcer index (UI) and treatment index (TI) were measured. The gastric mucosal surface was lined with white paper and flattened, the degree of lesion was observed macroscopically, and the ulcer size was measured with a vernier caliper to calculate the gastric mucosal ulcer index and treatment index. The ulcer area (mm 2) = π × major axis × minor axis / 4; Ulcer healing rate = (ulcer area in the model group - ulcer area in the treatment group) / ulcer area in the model group × 100%.

[0034] 3.1.2 Gastritis and barrier function detection (HE staining, hematoxylin & eosin staining) Take gastric mucosa tissue, fix it with 4% formaldehyde and then perform HE staining. Observe the morphological integrity of the gastric mucosa tissue and the infiltration of inflammatory cells (neutrophils, lymphocytes and monocytes).

[0035] The results are as Figure 2 shown. From the results, it can be seen that after sacrificing the mice, the gastric tissues were collected for macroscopic damage assessment and histological analysis. In group C, H&E staining showed that the morphology and structure of the gastric mucosa, submucosa, muscularis propria and cell membrane were good and intact. In group M, the gastric mucosa was severely damaged, showing obvious hemorrhagic necrosis, local ulcers, exfoliation and loss of gastric mucosa epithelial tissue ( Figure 2 B). However, the use of the positive drug omeprazole and L. paracasei NCU-21 significantly reduced ethanol-induced gastric mucosal damage, as shown by a significant reduction in the ulcer area of the gastric ulcer. The average value of group M was 17.52, while those of groups L, H and O were 6.290, 3.208 and 2.878 respectively ( Figure 2 C). Similarly, after L. paracasei treatment with NCU-21, the gastric ulcer index was significantly reduced (p < 0.01) ( Figure 2 D). However, the gastric inhibition rate of group H was significantly higher than that of group M ( Figure 2 E). It is worth noting that L. paracasei the protective effect of NCU-21 on the gastric mucosa of mice showed a dose-dependent manner. In addition, omeprazole, a drug commonly used for the conventional treatment of gastric ulcers, significantly reduced the damage of ethanol to the gastric mucosa ( Figure 2 C-E). In addition, the study showed that ethanol significantly increased the level of malondialdehyde (MDA) in the gastric tissues of mice. The results showed that the MDA level in the ethanol-treated group was significantly increased compared with that in the normal control group (p < 0.001). Administration of low-dose and high-dose L. paracasei NCU-21 and omeprazole significantly reduced the MDA level in the gastric tissues. The MDA levels in the three treatment groups (group O, L and H) were 0.9905, 1.699 and 1.063 respectively, compared with the MDA level in group M. It is worth noting that the MDA levels in group H, the omeprazole group and group C were comparable ( Figure 2 F). Interestingly, the change trend of MPO level in each group was similar to that of MDA, while the change trend of SOD level was opposite to that of MDA ( Figure 2G-H). Ethanol gavage caused a significant increase in the levels of MDA and MPO in the gastric tissues of mice, while the level of SOD decreased significantly. However, L. paracasei the administration of NCU-21 and omeprazole reversed these trends ( Figure 2 G-H). These results indicate that L. paracasei NCU-21 has a significant therapeutic effect on ethanol-induced gastric mucosal injury, and its effect is dose-dependent.

[0036] 3.1.3 TGF-β, VEGF, and EGFR were measured in the gastric mucosal tissues of mice by immunohistochemistry, and the key protein indicators of the Nrf2 / Keap1 / ARE signaling pathway (antioxidant stress) were measured by WB: Nrf2, Keap1, ARE, NQO1, and HO-1.

[0037] The results are as Figure 3 shown. It can be seen from the results that the healing of ethanol-induced gastric mucosal injury is a multi-level process involving multiple mechanisms such as angiogenesis, epithelial regeneration, and remodeling. The regulation of these processes is mediated by various growth factors, such as TGF-β, VEGF, and EGFR. Compared with the M group, the expression levels of TGF-β, VEGF, and EGFR in the gastric mucosa were significantly increased in the O group and the H group. At the same time, the L group also showed a good recovery effect. In addition, compared with the omeprazole treatment group, L. paracasei there was no significant difference in the expression levels of TGF-β, VEGF, and EGFR in the high-dose NCU-21 treatment group ( Figure 3 A). These findings verified that L. paracasei NCU-21 treatment promoted the healing of gastric epithelium and the reconstruction of gastric mucosa, especially in the case of gastric mucosal injury. Oxidative stress is caused by the imbalance of redox reactions in gastric mucosal epithelial cells and persists during the occurrence and progression of gastric mucosal injury. Studies have shown that ethanol inhibits the activation of the Keap-1 / Nrf2 antioxidant stress signaling pathway. However, L. paracasei NCU-21 can reverse this inhibitory effect, enhance its antioxidant activity, and eliminate the accumulated lipid peroxides, thereby protecting gastric mucosal cells from ethanol-induced damage. Therefore, the protein changes related to the Nrf2 / HO-1 signaling pathway in gastric tissues were detected by Western blot. The results showed that the expression of Nrf2 in the H group was significantly restored compared with the M group (p < 0.01). In addition, the protein expression levels of HO-1 and NQO1 in the H group were also restored compared with the M group (p < 0.001). At the same time, L. paracasei NCU-21 showed dose-dependent activation of the Nrf2 / HO-1 signaling pathway. Interestingly, there was no significant difference in the expression levels of the above-mentioned proteins between the O group and the H group ( Figure 3 B-F). In summary,L. paracasei Administration of NCU-21 contributed to the healing process of ethanol-induced gastric mucosal injury and was achieved by stimulating the production of three growth factors and activating the antioxidant stress signaling pathway.

[0038] 3.1.4 PCNA was measured in mouse gastric mucosal tissues by immunohistochemistry, and key protein indicators of the NF-κb signaling pathway (antioxidant stress) were measured by WB: p-Iκb, Iκb, p-p65, and p65. IL-1β, IL6, and TNF in gastric tissues were detected by ELISA. The results are as Figure 4 shown. From the results, it can be seen that PCNA is closely related to cell DNA synthesis and plays an important role in cell proliferation. In addition, PCNA can effectively prevent the excessive production of MDA and SOD induced by ethanol. Existing studies have revealed that ethanol treatment leads to a significant decrease in the level of PCNA in mouse gastric cells, thus hindering the self-repair ability of gastric cells. Immunohistochemical analysis showed that L. paracasei NCU-21 significantly increased the level of PCNA in gastric tissues and promoted ulcer healing in a dose-dependent manner ( Figure 4 A). Multiple studies have shown that drinking ethanol significantly increases the production of proteins related to the NF-κB signaling pathway and the levels of pro-inflammatory cytokines in gastric tissues. Studies have shown that ethanol may trigger an inflammatory response by activating the NF-κB signaling pathway, leading to acute gastric mucosal injury. To clarify L. paracasei the potential mechanism of the gastric protective effect of NCU-21, we comprehensively studied the expression of multiple target proteins and cytokines in these signaling pathways. Western Blotting results showed that ethanol treatment increased the phosphorylation of IκBα protein and its degradation by ubiquitination, and at the same time led to the phosphorylation of p65 in gastric mucosal cells. These research results indicate that ethanol activates the NF-κB signaling pathway in ulcer tissues. However, the H group significantly decreased the p-p65 / p65 ratio (p < 0.05) and the p-IκBα / IκBα ratio (p < 0.001), while low-dose L. paracasei NCU-21 treatment also reversed the ethanol-induced p-IκBα / IκBα ratio (p < 0.05). In addition, there was no significant difference between the high-dose L. paracasei NCU-21 treatment group (H group) and the omeprazole treatment group (O group) ( Figure 4 B-D). Enzyme-linked immunosorbent assay (ELISA) showed that ethanol treatment significantly increased the levels of pro-inflammatory markers TNF-α (p < 0.001), IL-1β (p < 0.001), and IL-6 (p < 0.01). Low-dose and high-dose L. paracaseiAfter treatment with NCU-21, the expression of these pro-inflammatory factors was significantly inhibited, and at high doses L. paracasei there was no significant difference in the inhibitory effect between the NCU-21 treatment group and the normal control group ( Figure 4 E-G). These results indicate that L. paracasei NCU-21 can inhibit the NF-κB inflammatory signaling pathway, thereby reducing the production and release of pro-inflammatory substances in gastric tissues. In summary, L. paracasei NCU-21 promotes regeneration by increasing PCNA expression and reduces gastric inflammation during gastric injury healing by inhibiting the NF-κB inflammatory signaling pathway and the release of pro-inflammatory factors.

[0039] 3.1.5 Gastric high-throughput sequencing Collect mouse gastric contents and send them for high-throughput sequencing to observe the changes in the intestinal microbiota of each group of mice, including: observing the differences in α-diversity and β-diversity between groups, as well as the differences at the phylum level and genus level.

[0040] 3.1.6 Oxidative stress indicators (1) Determination of SOD (superoxide dismutase) SOD is an important antioxidant enzyme that reduces gastric injury by preventing oxidative damage. According to previous reports, gastric mucosal injury is mainly related to reactive oxygen species (ROS). SOD mainly converts harmful superoxides into hydrogen peroxide, which then decomposes into harmless water molecules, thereby protecting the stomach from oxidative damage.

[0041] (2) Determination of MDA (malondialdehyde) MDA is used as an indicator to measure lipid peroxidation. The gastric mucosal lesion index is related to the content of malondialdehyde in the gastric mucosa.

[0042] (3) Determination of MPO (myeloperoxidase) MPO is an important peroxidase produced by neutrophils. It can not only quantitatively determine the number of neutrophils to reflect the infiltration of neutrophils, but also quantitatively reflect the degree of inflammatory damage.

[0043] The results are as Figure 5 shown. From the results, it can be seen that the imbalance of the gastric microbiota is closely related to the occurrence of gastric ulcer (GUI). To further explore this issue, we comprehensively analyzed the gastric contents by 16S rRNA high-throughput sequencing. The microbial α-diversity index reflects the richness and evenness of the gastric microbial population. Therefore, we used the Chao1 index and Shannon index to evaluate the α-diversity of the gastric microbiota. Compared with group C, ethanol treatment led to a significant decrease in the Shannon index of the gastric microbiota in group M (p<0.05). And at high doses L. paracaseiGavage treatment with NCU-21 effectively improved the abundance and uniformity of the gastric microbiota in mice (p<0.05) ( Figure 5 B). The Chao1 index showed a similar trend ( Figure 5 A). The β-diversity of the gastric microbiota was represented by the clustering patterns of different groups in the principal coordinate analysis (PCoA) score plot. The β-diversity of the gastric microbiota in the M group and the O group changed significantly compared with that in the C group. The symbols of the M group and the O group overlapped with each other and hardly overlapped with the C group, indicating that the gastric microbiota structures of the mice in the M group and the O group were significantly different from that in the C group. However, this change was significantly reversed after gavage treatment with high-dose and low-dose L. paracasei NCU-21 ( Figure 5 C). These results indicate that gavage administration of L. paracasei NCU-21 is an effective method to enhance the composition of the intestinal microbiota and reverse the ethanol-induced changes in the gastric microbiota. The Venn diagram is a useful tool for showing the degree of shared resources among groups, as it can visually display the degree of overlap among groups. The analysis results showed that a total of 18 identical operational taxonomic units (OTUs) were presented in the gastric microbiota of the mice in the C, M, O, L, and H groups, and each group also had 907, 117, 267, 169, and 439 unique OTUs, respectively. Notably, the C group and the H group were highly similar and shared 337 OTUs, while the C group and the M group only shared 36 OTUs. Therefore, L. paracasei the administration of NCU-21 led to a significant increase in the OTUs of the mice after ethanol treatment, and the levels of these OTUs showed a trend of recovering to the control group ( Figure 5 D). Subsequently, to further clarify the specific role of L. paracasei NCU-21 in regulating the changes in the gastric microbiota, we compared the relative abundances of the dominant phyla among different groups ( Figure 5 E). Ethanol treatment led to a decrease in the relative abundances of the Firmicutes phylum and the Bacteroidetes phylum ( Figure 5 F-G). At the same time, the relative abundance of the Proteobacteria phylum in the M group was significantly higher than that in the C group (p<0.005) ( Figure 5 H). In contrast, gavage with high-dose L. paracasei NCU-21 significantly upregulated the relative abundance of the Firmicutes phylum ( Figure 5 F). However, L. paracasei the effect of NCU-21 on the relative abundance of the Bacteroidetes phylum was not significant ( Figure 5 G). The relative abundance of the Proteobacteria phylum in the H group was significantly decreased compared with that in the M group (p<0.05) (Figure 5 Group (H). However, this phenomenon was not observed in the group intragastrically administered with omeprazole ( Figure 5 Groups E-H). Addition L. paracasei Strain NCU-21 reversed the downregulation of Lactobacillus , Oscillospira , Allobaculum and the upregulation of Enterococcus that occurred in 10 dominant genera ( Figure 5 Group C). Notably, at the genus level, Group O showed a trend similar to that of Group M. However, L. paracasei administration of NCU-21 led to partial reversal of the above changes, although this change was not observed in the presence of omeprazole ( Figure 5 Groups I-M).

[0044] 3.2 Intestinal tissue-related assays 3.2.1 Intestinal tissue morphology assay (1) Intestinal barrier function assay (HE staining) Colon tissue was taken, fixed with 4% formaldehyde, and then subjected to HE staining. The cell structure, size, nuclear-cytoplasmic ratio, atypia, morphological integrity of the intestinal tissue, and infiltration of inflammatory cells (neutrophils, lymphocytes, and monocytes) were observed.

[0045] Experimental procedures: ① Specimen collection and fixation: Mouse gastric tissue was taken and placed in the fixative, which was 4% paraformaldehyde (the role of fixation is to preserve the morphological structure of the tissue, prevent its morphological change and deterioration; in addition, fixation can denature proteins, facilitating staining); ② Dehydration: The fixed mouse gastric tissue was taken out, rinsed thoroughly with PBS, and then dehydrated with ethanol, successively dehydrated with 70% ethanol, 80% ethanol, 90% ethanol, 95% ethanol I, 95% ethanol II, 100% ethanol I, and 100% ethanol II for 1 h; ③ Embedding: The dehydrated tissue was treated with xylene for 30 min and then embedded in paraffin; ④ Sectioning and mounting: A smooth plane was cut out by a rotary microtome, the section thickness was set to 3 - 5 μM, the cut wax sections were carefully lifted and placed in the water bath of a spreading machine with a water temperature of 45 °C to flatten the wrinkled wax sections; the wax sections were placed on glass slides and baked on a 60 °C baking machine for 4 h; ⑤ Deparaffinization: The tissue sections to be tested were placed in xylene and soaked thoroughly for 10 min, and then the xylene was replaced and soaked for another 10 min. The purpose is to dissolve the paraffin in the sections with xylene, so that when the staining solution is used for staining, the staining solution can fully enter the tissue. At the same time, xylene can also play a role in making the sections transparent, making it easier to observe the sections; ⑥ Hydration: The tissue sample to be tested that has been soaked in xylene is first immersed in absolute ethanol for 5 minutes to wash out the xylene used during dewaxing and allow water to enter the tissue. Then, it is successively placed in 100%, 90%, 80%, and 70% ethanol for 5 minutes each to achieve sufficient hydration. ⑦ Hematoxylin staining, differentiation, and blueing of tissue sections: The sections of the hydrated tissue sample are soaked and washed with PBS solution, 5 minutes each time, for a total of 3 times. Then, use a pipette to aspirate the pre-prepared hematoxylin staining solution and add 100 μl to each tissue section, followed by thorough staining for 10 minutes. After staining, use distilled water to wash away the excess hematoxylin staining solution. Then, use 1% hydrochloric acid ethanol for differentiation to remove the excessive dye bound in the cell nucleus and the excess dye in the cytoplasm. After differentiation, rinse the tissue sections thoroughly with double-distilled water. To turn the hematoxylin blue, add a weakly alkaline blueing solution to the tissue sections to stain the cell nucleus blue. After blueing, first wash with clear water and then rinse the tissue sections thoroughly with double-distilled water. ⑧ Eosin staining and dehydration of tissue sections: Add eosin staining solution to the tissue sample sections from the previous step and allow the tissue to be fully stained for 3 minutes. After staining, dehydrate the tissue sections step by step using 80%, 95%, and absolute ethanol. Dehydrate with 80% ethanol for 5 seconds, 95% ethanol for 2 minutes, and absolute ethanol for 2 minutes. ⑨ Sealing the slides: Immerse the dehydrated tissue sample sections in xylene twice, 4 minutes each time, and then mount the tissue sample sections using neutral balsam. ⑩ Observation: Place the sealed slides under an optical microscope and take pictures and observe at 200x and 400x magnifications, and perform pathophysiological analysis.

[0046] (2) Observation of the morphology of the mouse colon and cecum Dissect the mouse and observe the length of the colon and the swelling of the cecum.

[0047] 3.2.2 High-throughput sequencing of mouse feces Collect mouse feces and gastric contents and send them for high-throughput sequencing to observe the changes in the intestinal microbiota of each group of mice, including observing the differences in α-diversity and β-diversity between groups, as well as the differences at the phylum level and genus level.

[0048] 3.2.3 Molecular biology detection Take colon tissue for molecular biology detection such as WB and ELISA. (1) WB (Western Blot) Detect the TLRs-MyD88-NF-κB inflammatory signaling pathway and measure TLR4, MyD88, P65, and P-P65.

[0049] The MAPK / ERK / P38 / TJ cascade activation signaling pathway was detected, and the levels of ZO-1 (zonula occludens-1), Occludin-1 (occludens-1) and claudin-5 (occludens-5) were measured.

[0050] Experimental steps: a. Preparation of protein samples: The gastrointestinal tissue taken out from the -80°C freezer was weighed, and then RIPA lysis buffer (a traditional cell tissue rapid lysis buffer, mainly used to extract soluble proteins from animal tissues and animal cells) was added at a ratio of 10µl / mg. The gastric tissue was lysed in RIPA lysis buffer, and the tissue sample was crushed on ice using an ultrasonic crusher (set power to 40%, ultrasonic time 3s, ultrasonic interval time 3s, and working times 4 times). After ultrasonic treatment, the sample was centrifuged at 12000rpm at 4°C for 15 minutes. After centrifugation, the sample was placed on ice. Collect the obtained supernatant for BCA assay. After the BCA assay, aspirate 200µl of the supernatant and add it to a new centrifuge tube. Then add protein loading buffer to the sample at a ratio of 4:1 and boil it at 100℃ for 8-15 minutes for thermal denaturation. After heating, take out the sample and cool it on ice. Centrifuge the cooled sample at 2000rpm for one minute at 4℃ (the centrifugal speed of the sample with loading buffer must be less than 3000rpm). The sample taken out after centrifugation can be stored at -80℃.

[0051] b. BCA assay: ① Divide the 96-well plate into areas; ② Prepare BSA protein standard sample: dilute 2mg / ml protein standard solution to 0.5mg / ml (if 200µl of 0.5mg / ml protein standard solution is prepared, 50µl of 2mg / ml protein standard solution and 150µl PBS solution are required); ③ Prepare BCA working solution: Mix BCA reagent A and B at a ratio of 50:1 (2450µl A + 49µl B) to make an appropriate amount of BCA working solution; ④ Dilute BSA protein standard: add 0, 1, 2, 4, 8, 12, 16, 20 µl of standard sample to the labeled area, then fill the wells with PBS to 20 µl, and add 200 µl BCA to each well; ⑤Dilute the protein sample to be measured: Dilute it to 20 µl with PBS (the sample needs to be diluted by a certain multiple for quantification, 2 µl of the sample + 18 µl of PBS, that is, diluted 10 times), and then add 200 µl of BCA to each well; ⑥Place the well - filled 96 - well plate in an incubator at 37 °C for 30 minutes; ⑦After incubation, place it on a microplate reader and shake gently for 3 - 10 s at medium speed, measure the absorbance at 595 nm for colorimetric determination. After recording the standard curve and the absorbance data of the samples, use the protein content (ml) as the abscissa and the absorbance as the ordinate to make a standard curve. (R 2 > 0.98); ⑧Calculate the protein content (note that the quantitative sample has been diluted 10 times) Table 1 Sample loading volume of the protein quantification standard curve c. Prepare the separating gel and stacking gel: (Separating gel: 10 ml, Stacking gel: 2 ml) The concentration of the separating gel depends on the size of the protein to be separated. Generally, a higher - concentration separating gel is used to separate small - molecule proteins, and a lower - concentration separating gel is used to separate large - molecule proteins; d. Make the polyacrylamide gel: After checking the device for leakage, add 7.5 ml of the separating gel to the gel - making plate, then add a certain volume of isopropanol (the purpose is to drive away air bubbles and flatten the separating gel). After the separating gel solidifies, pour off the isopropanol, wash it with deionized water, dry it with absorbent paper, and then add 2 ml of the stacking gel. Slowly place the comb from right to left into the stacking gel to prevent air bubbles from being generated, and let it stand for a period of time until the gel solidifies; e. Loading: After the gel solidifies, carefully remove the comb, add a certain amount of the sample to be measured to the sample wells, and add 2 µl of marker after loading; f. Electrophoresis: Run the gel in SDS running buffer at 80 V for 30 min, and then at 120 V for 1 h (the electrophoresis buffer can be recycled after electrophoresis); g. Blotting: Transfer the protein from the gel to a nitrocellulose membrane. Retain the gel with the desired bands for blotting. There are two specifications for NC membranes. 0.22 µm is suitable for proteins with a size of 15 - 20 bp, and 0.45 µm is suitable for proteins with a size of 70 - 100 bp. The membrane needs to be pre - soaked in methanol. Place the gel on the sandwich plate in a dish containing transfer buffer, ensure that the transfer buffer soaks the gel, then cover it with the pre - soaked NC membrane, taking care not to generate air bubbles. Then place the sandwich plate (sandwich) into the transfer tank for blotting (in an ice bath). The blotting time for small molecules is generally 90 mA for 90 min, and the blotting time for large molecules is generally 260 mA for 50 min; h. Blocking: After blotting, place the sandwich plate in a dish, take out the NC membrane, and block it in 5% skim milk for 1 h; i. Primary antibody incubation: Add the diluted primary antibody to the membrane and incubate it overnight on a shaker at 4°C; (recover the primary antibody) j. Washing: Wash the membrane three times with 0.2% PBST for 10 minutes each time; k. Secondary antibody incubation: Add the goat anti-mouse immunoglobulin-horseradish peroxidase (HRP)-labeled secondary antibody and incubate at room temperature for 1 h; (12) Washing: Wash the membrane three times with 0.2% PBST for 10 minutes each time; (13) Place the membrane in a sealed bag and add the freshly prepared chemiluminescent reagent for development.

[0052] (2) ELISA a. Collect blood from the femoral artery of mice and centrifuge it in a pre-cooled centrifuge (4°C, 8000 rpm, 10 minutes). After centrifugation, place the test tube on ice and immediately transfer the plasma portion to a new centrifuge tube. Determine the level of the factor to be measured in the serum sample by using an ELISA kit specific for the target protein; b. Take the kit out of the refrigerator 30 min in advance and equilibrate it to room temperature; c. Prepare the standards and washing solution, and set up the blank and standard wells in advance; d. Determine the number of plates required according to the number of test samples and standards, and add 1 well as a blank control well. Add the specimens and different concentrations of standards (100 µl / well) to the corresponding wells respectively (only add the standard / sample diluent to the zero well), seal the reaction wells with sealing tape, and incubate in a 37°C incubator for 90 minutes (except for the blank control well); e. Wash the plate 4 times: 1) Automatic plate washer: The required volume of washing solution to be injected is 350 µL, and the interval between injection and aspiration is 15 - 30 seconds. 2) Manual plate washing: Drain the liquid in the wells, add 350 µL of washing solution to each well, let it stand for 30 seconds and then drain the liquid, and pat dry on thick absorbent paper; f. Add the biotinylated antibody working solution (100 µl / well). Seal the reaction wells with sealing tape and incubate in a 37°C incubator for 60 minutes (except for the blank control well); g. Wash the plate 4 times; h. Add the enzyme conjugate working solution (100 µl / well). Seal the reaction wells with sealing tape and incubate in a 37°C incubator for 30 minutes (except for the blank control well); i. Wash the plate 4 times; j. Add 100 µl / well of the chromogenic reagent, protect from light, and incubate in a 37°C incubator for 10 - 20 minutes; k. Add 100 µl / well of the stop solution, mix well and immediately measure the OD450 value (within 5 minutes).

[0053] (3)qPCR a. Weigh 50 - 100 mg of mouse gastric tissue. After thoroughly cutting it into pieces, add 1 mL of Trizol solution and grind it using a homogenizer to lyse the cells. Note that the total volume of the sample should not exceed 10% of the volume of the Trizol used. Then, let it stand at room temperature (15 - 30 °C) for 5 min; b. Add chloroform at a ratio of 0.2 mL per 1 mL of Trizol solution. Tighten the centrifuge tube cap, and use a vortex oscillator to shake it vigorously for 15 s. Let it stand at room temperature for 2 - 3 min; c. Centrifuge at 12000 rpm at 4 °C for 15 min; d. Transfer the top aqueous phase to a new centrifuge tube (2 Ep tubes). Add isopropanol at a ratio of 0.5 mL per 1 mL of Trizol solution, and let it stand at room temperature for 10 min; e. Centrifuge at 1200 rpm at 4 °C for 15 min; f. Discard the supernatant. Wash it by adding 1 mL of 75% ethanol (prepared with DEPC water) per 1 mL of Trizol solution. Vortex to mix evenly, centrifuge at 12000 rpm at 4 °C for 15 min, and repeat twice; g. Discard the supernatant, and let the precipitated RNA air - dry at room temperature for 5 - 10 min; h. Dissolve the RNA precipitate with an appropriate amount of RNase - free water, and repeatedly pipette to mix evenly; i. Incubate in a 65 °C water bath for 15 min, and store at - 80 °C for later use; j. Measure the RNA concentration and purity by ultraviolet absorption method (NanoDrop 2000 spectrophotometer).

[0054] Reverse transcription to cDNA Use the TAKARA reverse transcription kit for RNA reverse transcription a. Genomic DNA removal reaction b. Reverse transcription reaction c. Real Time PCR: d. PCR conditions: Stage1: 95 °C, 30 s Stage2: (95 °C, 5 s; 60 °C, 34 s) for 40 cycles; Stage3: 95 °C, 15 s; 60 °C, 1 min; 95 °C, 15 s.

[0055] e. PCR results: Confirm the amplification curve and melting curve of Real Time PCR, and make a standard curve for PCR quantification.

[0056] The results are as Figures 6 - 7 shown. It can be seen from Figure 6 that ethanol-induced gut microbiota dysbiosis may lead to an increase in the level of lipopolysaccharide (LPS) in the gut, thereby disrupting the intestinal epithelial barrier. H&E staining showed that compared with the C group, the number of goblet cells in the intestine of mice in the M group decreased, the epithelium exfoliated, the crypts were deformed, there was infiltration of inflammatory cells, and the lamina propria became thinner. However, in the intestines of mice in the H group and the L group, the inflammatory cells were significantly reduced, and the anatomical structure of the colon returned to normal ( Figure 6 A). In addition, the improvement in the H group was particularly obvious. Lipopolysaccharide (LPS) mainly induces intestinal inflammation by activating the TLR4-Myd88-NF-κB signaling pathway. Therefore, we performed Western blot analysis on colon tissues, and the results showed that compared with the C group, the expression levels of TLR4, MYD88, and p-p65 proteins in the M group and the O group were significantly increased (p<0.05). However, L. paracasei the administration of NCU-21 successfully reversed the increase in intestinal inflammation level (p<0.05). The treatment with high-dose L. paracasei NCU-21 effectively and completely reversed the increase in intestinal inflammation level ( Figure 6 B-E). The research shows that the dysbiosis of the gut microbiota may lead to the damage and disruption of the intestinal epithelial barrier. The tight junctions of the intestinal epithelium play a crucial role in regulating the intestinal permeability and defense function. Therefore, we analyzed the expression levels of Claudin-1 and Occludin in the intestine by Western blotting. The results showed that compared with the C group, the expression level of Claudin-1 in the M group was significantly decreased (p<0.001), and the expression level of Occludin was also significantly reduced (p<0.001). In contrast, L. paracasei the administration of NCU-21 restored their levels to normal levels, and this restoration was dose-dependent. The intestinal Claudin-1 protein level in the H group was restored to the level of the M group (p<0.001) ( Figure 6 F-H). Related research shows that L. paracasei the key metabolite L-lactic acid of Figure 6 can effectively protect the gastric mucosa from ethanol-induced damage. The results showed that the serum L-lactic acid levels in the H group and the L group were significantly restored, which was statistically significant compared with the M group and the O group (p<0.01) ( L. paracasei I). Generally speaking,

[0057] By Figure 7It can be seen that the Shannon index and Simpson index were used to evaluate the α-diversity of the intestinal microbiota in mice. The results showed that ethanol treatment led to a significant decrease in the Simpson index of the intestinal microbiota in the M group compared with the C group (p < 0.05). In contrast, L. paracasei Gavage treatment with NCU-21 effectively enhanced the abundance and evenness of the intestinal microbiota in mice (p < 0.05). In addition, the results of the Shannon index also showed a similar trend ( Figure 7 A - B). Subsequently, the β-diversity of the intestinal microbiota in each group of mice was evaluated by non-metric multidimensional scaling analysis (NMDS), and the results showed that L. paracasei gavage treatment with NCU-21 significantly alleviated ethanol-induced intestinal microbiota dysbiosis, while omeprazole failed to show a similar effect ( Figure 7 C). The Venn diagram showed that 22 identical OTUs were shared by the gastrointestinal flora of mice in the C, M, B, I, and U groups, and the number of unique OTUs in each group was 251, 83, 67, 300, and 272, respectively. In addition, the total number of OTUs in the C group and the H group was significantly higher, reaching a total of 523, compared with other groups (Figure 7D). At the phylum level, we selected the genera related to ethanol-induced gastric ulcers for analysis. Ethanol treatment led to a significant decrease in the relative abundances of Firmicutes (p < 0.05) and Bacteroidetes, while the relative abundance of the Proteobacteria phylum increased significantly (p < 0.05). In contrast, the addition of L. paracasei NCU-21 effectively reversed this trend (p < 0.05) (Figure 7E - H). Among the 10 major genus levels, the application of L. paracasei NCU-21 also reversed some of the ethanol-induced changes in the intestinal flora composition. Compared with the control group, ethanol treatment led to Enterococcus and Bacteroides relative abundances to increase, while the relative abundances of Lactobacillus and Prevotella decreased. However, L. paracasei the administration of NCU-21 partially reversed these changes, and this phenomenon was not observed in the omeprazole group (Figure 7I - M).

[0058] The embodiments of the present application have been described above in conjunction with the accompanying drawings. Specific examples are used herein to elaborate on the principles and implementation manners of the present application. The description of the above embodiments is only used to help understand the core idea of the present application. However, the present application is not limited to the above specific implementation manners. The above specific implementation manners are merely illustrative and not restrictive. Under the inspiration of the present application, those of ordinary skill in the art can also make many forms without departing from the purpose of the present application and the scope protected by the claims, and all of them belong to the protection scope of the present application.

Claims

1. A Lactobacillus paracasei NCU-21, characterized in that, The Lactobacillus paracasei ( Lactobacillus paracasei ) NCU-21 was deposited at the China General Microbiological Culture Collection Center on September 25, 2023. The address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC NO. 28538.

2. The Lactobacillus paracasei NCU-21 according to claim 1, wherein The gene sequence of the 16S rDNA of the Lactobacillus paracasei NCU-21 is as shown in SEQ ID No.

1.

3. Use of the Lactobacillus paracasei NCU-21 according to any one of claims 1-2 in the preparation of a product for preventing and / or ameliorating and / or treating gastric ulcer.

4. The application according to claim 3, characterized in that, The gastric ulcer includes gastric ulcer caused by ethanol.

5. The application according to claim 3, characterized in that The product includes a drug or a microbial agent.

6. A microbial inoculant, characterized in that, The microbial agent includes the Lactobacillus paracasei NCU-21 according to any one of claims 1-2, or its ferment, or its metabolite.

7. A drug for improving gastric ulcer, characterized in that, The drug includes the Lactobacillus paracasei NCU-21 according to any one of claims 1-2.

8. The drug according to claim 7, wherein, The drug further includes a pharmaceutical excipient.

9. The drug according to claim 8, characterized in that, The pharmaceutical excipient is at least one of water, lactose, sodium chloride, and glucose.

10. The drug according to claim 9, characterized in that, The dosage form of the drug includes one or more of powder, tablet, capsule, aqueous solution, gel, ointment, dropping pill, pill, and granule.

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