Use of mannan peptide in preparation of alcoholic gastric ulcer drugs

Abstract

The application belongs to the technical field of biological medicine, and relates to application of a mannose peptide in preparation of an alcoholic gastric ulcer medicine. The application discloses that the mannose peptide can significantly relieve bleeding lesions and ulcer conditions of an alcoholic gastric ulcer, and the mechanism comprises the following aspects: reducing active oxygen generated by alcohol toxicity, improving body oxidation stress by improving the activity of antioxidant enzymes of gastric tissue; regulating the inflammation response of the body by regulating the Akt / NF-kappa B pathway; inhibiting the Bax-related apoptosis pathway to protect gastric mucosal cells from alcohol-induced apoptosis, so as to protect the integrity of the gastric mucosa; regulating the intestinal flora disorder caused by alcohol intake, and preventing acute alcoholic gastric ulcer by improving the intestinal flora composition of the alcoholic gastric ulcer. The application widens the application of the mannose peptide, and provides more solutions for the prevention or treatment of acute alcoholic gastric ulcer.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to the application of mannan peptide in the preparation of drugs for alcoholic gastric ulcers. Background Technology

[0002] Mannatide, formerly known as Polyactin A (PAA), is an immunostimulant. It is isolated from healthy human oral and pharyngeal α-hemolytic streptococcal strains through deep fermentation. It consists of sugar and peptide chains of varying lengths (20-100 kDa). The mannan is linked by β-(1-4) bonds to form the sugar backbone, while the peptide chains are mainly composed of aspartic acid, threonine, serine, glutamic acid, alanine, and leucine. Due to the large variation in the number of oligosaccharides and amino acid residues and the complex residue linkages, it possesses unique biological activities. Currently, its functional research mainly focuses on immunomodulation. As an immunomodulator, it has been shown to rapidly enhance the body's immunity and eliminate immunosuppression, with applications in cancer vaccine development, regulation of human monocyte function, tumor suppression, and relief of colitis. However, research on the protective effect of mannatide against alcoholic gastric ulcers and its related mechanisms is lacking.

[0003] Gastric ulcer is a common peptic ulcer disease caused by damage to the gastric mucosa. It is characterized by easy infection, long course, difficulty in treatment, and high recurrence rate. The pathogenesis of gastric ulcer is complex and not fully understood. Existing research suggests that it is mainly caused by an imbalance between aggressive factors and defense mechanisms of the gastric mucosa. In alcoholic gastric ulcers, alcohol acts as an attacking factor on the gastric mucosa. Compared with bacterial infection in Helicobacter pylori-related ulcers and COX-1 inhibition induced by NSAIDs, the pathogenesis of alcoholic gastric ulcers has the following unique mechanisms: (1) After alcohol enters the body, it is first digested and absorbed into the blood by the digestive system, including the gastrointestinal tract, thus promoting vasoconstriction and reduced blood flow, causing microcirculatory disorders and ischemia; (2) The direct toxicity of high concentrations of ethanol can dissolve the lipid layer of the gastric mucosa, destroy the mucus-bicarbonate barrier, and cause gastric acid and proteases to come into contact with epithelial cells, destroying the protective layer of the gastric mucosa and causing irreversible damage to the gastric mucosa; (3) Ethanol metabolites (such as acetaldehyde) can induce oxidative stress, which, through the production of reactive oxygen species, further causes inflammation and abnormal immune regulation, leading to the release of pro-inflammatory factors, thereby inducing acute gastric ulcers. Its typical pathological manifestation is superficial erosion of the gastric antrum. Reports on the adjuvant treatment of gastric ulcers with mannan peptides do not include research on their application in acute alcoholic gastric ulcers, and there is a lack of research on the mechanism by which mannan peptides target alcohol-induced ulcers. Summary of the Invention

[0004] The purpose of this invention is to broaden the application of mannan peptides and provide more solutions for the prevention or treatment of alcoholic gastric ulcers or acute alcoholic gastric ulcers.

[0005] On one hand, the present invention relates to the use of mannan peptides in the preparation of medicaments for the prevention and / or treatment of alcoholic gastric ulcers.

[0006] Furthermore, in the applications provided by this invention, the alcoholic gastric ulcer drug is used for the prevention and / or treatment of acute alcoholic gastric ulcers.

[0007] Furthermore, in the application provided by the present invention, the prevention and / or treatment are achieved by alleviating the bleeding lesions and ulcer symptoms in subjects with alcoholic gastric ulcers.

[0008] Furthermore, in the application provided by the present invention, the prevention and / or treatment are achieved by alleviating at least one of gastric tissue bleeding, diffuse necrosis and shedding of mucosal cells, atrophy of mucosal cells, glandular structural disorder, or inflammatory cell infiltration in subjects with alcoholic gastric ulcers.

[0009] Furthermore, in the application provided by the present invention, the prevention and / or treatment are achieved by increasing the activity of antioxidant enzymes in the gastric tissue of subjects with alcoholic gastric ulcers and / or improving the oxidative stress in the bodies of subjects with alcoholic gastric ulcers.

[0010] Furthermore, in the application provided by the present invention, the prevention and / or treatment are achieved by reducing the levels of pro-inflammatory factors in subjects with alcoholic gastric ulcers and / or regulating the body's inflammatory response in subjects with alcoholic gastric ulcers.

[0011] Furthermore, in the application provided by the present invention, the prevention and / or treatment are achieved by protecting the integrity of the gastric mucosa by regulating the inflammatory pathways and apoptosis-related pathways in subjects with alcoholic gastric ulcers.

[0012] Furthermore, in the application provided by the present invention, the prevention and / or treatment are achieved by improving the gut microbiota structure of subjects with alcoholic gastric ulcers.

[0013] Furthermore, in the application provided by this invention, the subject is a mammal.

[0014] Furthermore, in the application provided by this invention, the subject is a mammal, either human or mouse.

[0015] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages: This invention reveals for the first time the multi-pathway synergistic mechanism of mannan peptides in the prevention and treatment of acute alcoholic gastric ulcers: by enhancing the activity of antioxidant enzymes in gastric tissue and improving oxidative stress, reducing the content of pro-inflammatory factors and regulating the Akt / NF-κB pathway to inhibit inflammatory responses, while inhibiting the Bax apoptosis pathway to protect the integrity of gastric mucosal cells, and improving the function of the gut-gastric axis by regulating the gut microbiota structure, it significantly alleviates hemorrhagic lesions and ulcer symptoms in gastric tissue, blocking the development of alcoholic gastric ulcers at multiple targets, and providing more solutions for the prevention and treatment of alcoholic gastric ulcers. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 The figure shows the experimental results of the effect of mannan peptide on gastric tissue lesions and ulcer index in mice with acute alcoholic gastric ulcers.

[0018] Figure 2 This is a pathological image of gastric tissue from mice with acute alcoholic gastric ulcers, stained with H&E. Red arrows indicate regional hemorrhage; black arrows indicate diffuse necrosis and sloughing of mucosal cells; green arrows indicate mucosal cell atrophy; blue arrows indicate glandular structural disorder; and yellow arrows indicate inflammatory cell infiltration.

[0019] Figure 3 The figure shows the effect of mannan peptide on oxidative stress-related indicators in gastric tissue of mice with acute alcoholic gastric ulcers. In the figure, A represents SOD activity; B represents GSH-PX activity; and C represents MDA content.

[0020] Figure 4 The figure shows the effect of mannan peptide on pro-inflammatory factors in the serum of mice with acute alcoholic gastric ulcers. In the figure, A represents the level of the pro-inflammatory factor TNF-α; and B represents the level of the pro-inflammatory factor IL-1β.

[0021] Figure 5 The figure shows the effect of mannan peptide on gastric mucosal protection-related factors in the serum of mice with acute alcoholic gastric ulcers. In the figure, A represents PGE2 content; B represents EGF content.

[0022] Figure 6 The figure shows the effect of mannan peptide on the mRNA levels of genes related to the Akt / NF-κB pathway and Bax apoptosis pathway in mice with acute alcoholic gastric ulcers.

[0023] Figure 7 This is a graph showing the alpha diversity analysis of the mouse gut microbiota. The horizontal axis represents the flattening depth, and the vertical axis represents the median value of the alpha diversity index calculated over 10 iterations.

[0024] Figure 8 The graph shows the α-diversity index results of the mouse gut microbiota composition.

[0025] Figure 9 The figure shows the hierarchical clustering results at the genus level for β-diversity analysis of the mouse gut microbiota.

[0026] Figure 10 A heatmap of species composition was generated for species difference analysis of mouse gut microbiota.

[0027] Figure labeling: Ctrl represents the normal control group, Model represents the model group, P represents the positive drug group, LD represents the low-dose mannan peptide group, and HD represents the high-dose mannan peptide group. Detailed Implementation

[0028] The technical solution of the present invention will be described below with reference to embodiments. However, the present invention is not limited to the following embodiments. Unless otherwise specified, the experimental and detection methods described in each embodiment are conventional methods; the reagents and materials described are commercially available unless otherwise specified. Unless otherwise specified, all percentages in the following embodiments refer to mass percentages. Unless otherwise specified, all proportions in the following embodiments refer to mass ratios.

[0029] Animal grouping and materials: C56BL / 6 mice, weighing 18-20 g (BW), were provided by the Experimental Animal Center of the School of Medicine, Xi'an Jiaotong University, Animal Production License No.: SCXK (Shaanxi) 2023-002. After 7 days of acclimatization feeding, the mice were randomly divided into four groups: normal control group (Ctrl), model group (Model), positive control group (P: cimetidine 100 mg / kg BW, purchased from Xi'an Lijun Pharmaceutical), low-dose mannan peptide group (LD: 5 mg / kg BW), and high-dose mannan peptide group (HD: 100 mg / kg BW), with 8 mice in each group. Mannan peptide was obtained by fermentation by the Shaanxi Provincial Institute of Microbiology. The mannan peptide group was administered the drug once daily by gavage, while the normal control group and model group were given an equal volume of distilled water. Mice were administered the drug via gavage for 7 consecutive days. Two hours after the last administration, except for the normal control group, all treated mice were administered 10 mL / kg BW of 65% ethanol solution via gavage to establish an alcoholic gastric ulcer mouse model. One hour later, blood was collected, mice were euthanized by cervical dislocation, and gastric tissue was dissected.

[0030] Example 1 This embodiment provides an experiment on gastric tissue collection and ulcer index scoring.

[0031] Mice were euthanized by cervical dislocation, and their stomachs were dissected, photographed, and rinsed with 4°C saline to remove surface blood. The stomach tissue was then dissected parallel to the pylorus along the longest axis of the lesser curvature. The stomach was inverted, and any remaining contents were washed away with 4°C saline. The stomach was laid flat to observe hemorrhagic ulcer lesions on the inner wall. The ulceration status was evaluated using the ulcer index. The ulcer index scoring criteria were as follows: microulcers (1–2 mm) 1 point; medium ulcers (3–4 mm) 2 points; large ulcers (5–6 mm) 4 points; and extra-large ulcers (>6 mm) 8 points. The ulcer index for each treatment group was expressed as the ratio of the total ulcer score per mouse in the group to the number of mice in the group.

[0032] The gastric tissue of mice with gastric ulcers, pre-administered with positive drugs and mannan peptides, was observed, photographed, and the ulcer index was evaluated. The results are as follows: Figure 1 As shown, the normal group mice had normal macroscopic morphology of gastric tissue without obvious hemorrhagic lesions, while the ulcer model group mice had obvious hemorrhagic lesions and severe ulcers in their gastric tissue, with an ulcer index of 11.3±2.1. After treatment with mannan peptide (LD and HD groups), the hemorrhagic lesions and ulcers were significantly alleviated, and the ulcer index in the HD group decreased significantly to 3.9±1.4. p <0.05), showing no significant difference from the positive control drug, indicating that 100 mg / kg BW mannan peptide can significantly alleviate hemorrhagic lesions and ulceration in the gastric tissue of mice with alcoholic gastric ulcers.

[0033] Example 2 This embodiment provides a histological evaluation experiment.

[0034] After the ulcer index assessment, the gastric tissue was divided into multiple parts, one of which was used for histological evaluation. Fresh gastric tissue was soaked overnight in 10% neutral buffered formalin, then embedded in paraffin and sectioned (5 μm). The sections were stained with hematoxylin-eosin (H&E) and observed under a microscope at 100x and 400x magnification to observe changes in tissue morphology and structure.

[0035] H&E staining was performed on the gastric tissues of mice in each group, and the tissues were observed under a microscope at 100x and 400x magnification. Representative images are shown below. Figure 2As shown, in the normal group of mice, the gastric glands were neatly arranged, and the mucosa, submucosa, and muscle layer were structurally intact and clearly layered, with no obvious edema or inflammatory cell infiltration. Gastric tissue sections from the gastric ulcer model mice showed obvious regional hemorrhage (red arrow), diffuse necrosis and sloughing of mucosal cells (black arrow), disordered glandular structure (blue arrow), and inflammatory cell infiltration (yellow arrow). Mice pretreated with 5 mg / kg BW and 100 mg / kg BW showed significant relief of gastric damage; the HD group showed only mild inflammatory cell infiltration (yellow arrow), mucosal cell sloughing (black arrow), and atrophy (green).

[0036] Example 3 This embodiment provides an experiment for measuring biochemical indicators in serum and gastric tissue.

[0037] Example 2: The remaining portion of gastric tissue was used to analyze oxidative stress and inflammation-related biochemical indicators, including superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) activity, and malondialdehyde (MDA) content. Protein content was determined, and results were quantified as milligrams of protein (prot) per milliliter of gastric tissue. Serum indicators, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), prostaglandin E2 (PGE2), and epidermal growth factor (EGF), were detected using ELISA, and results were expressed as per milliliter of serum. All the above indicators were analyzed using commercially available kits purchased from Nanjing Jiancheng Biotechnology Research Institute.

[0038] The pathogenesis of acute alcoholic gastric ulcers is closely related to oxidative stress induced by ethanol metabolites, the release of pro-inflammatory factors, and their further oxidative stress. Superoxide dismutase (SOD) and gastrointestinal oxidative stress-related substances (GSH-PX), as substances associated with the body's oxidative stress levels, are important defenses against oxidative damage by regulating reactive oxygen species metabolism balance. Figure 3 As shown, the activities of antioxidant enzymes SOD and GSH-PX were significantly increased to 3644.9 U / mg prot and 45.0 U / mg prot, respectively, after treatment with 100 mg / kg BW mannan peptide, while the content of MDA, the end product of lipid peroxidation caused by free radicals, was significantly reduced. p <0.05, with no significant difference from the normal group ( p>0.05). This indicates that 100 mg / kg BW mannan peptide can alleviate alcoholic gastric ulcers in mice by enhancing the activity of antioxidant enzymes in gastric tissue and improving oxidative stress.

[0039] During the development of alcoholic gastric ulcers, excessive ethanol intake can stimulate the microcirculatory blood vessels on the gastric mucosa, producing a large number of inflammatory mediators and cytokines, exacerbating oxidative stress, and damaging the normal structure and physiological function of gastric tissue. Figure 4 As can be seen, the levels of pro-inflammatory cytokines TNF-α and IL-1β in the serum of mice with alcoholic gastric ulcer model were significantly increased, reaching 19.4 pg / mL and 156.8 pg / mL, respectively, while in the HD group they were significantly decreased to 14.3 pg / mL and 11.7 pg / mL (P>0.05), with no significant difference compared to the normal group and the positive drug group. Therefore, 100 mg / kg BW mannan peptide can reduce the levels of pro-inflammatory cytokines, regulate the body's inflammatory response, and alleviate alcoholic gastric ulcers in mice.

[0040] Example 4 This embodiment provides an experimental study on the protective effect of mannan peptide on the gastric mucosa of mice with acute alcoholic gastric ulcers.

[0041] RNA was extracted from the gastric tissue of mice in each group using the TaKaRa Mini-BEST and PrimeScipt™ RT Master Mix kits, and cDNA was synthesized. The primer sequences used for amplification are shown in Table 1. After amplification in triplicate, cDNA samples were amplified using TB Green™ Premix Ex Taq™ II chimeric fluorescence qPCR on a qPCR instrument under the following conditions: 95℃ for 30 s; 95℃ for 5 s; 60℃ for 30 s, for 40 cycles. The purity of the PCR products was analyzed by melting curve analysis using Equation 2. -△△Ct The relative expression level of mRNA was calculated and normalized to the mRNA level of GAPDH.

[0042] Table 1: qPCR primer sequence information

[0043] Intact gastric mucosa is the primary barrier protecting gastric tissue from direct damage by ethanol. PGE2 is an important regulator of gastric mucosal integrity, gastric pH environment, and gastric mucus secretion levels, and is of great significance for the prevention and treatment of gastric ulcers. Results are as follows... Figure 5In the HD group, the content of PGE2 was increased to nearly the positive control level (24.8 pg / mL), reaching 21.4 pg / mL, while the serum level in the model group was only 4.5 pg / mL. This experiment shows that mannan peptide treatment can effectively alleviate the inhibition of PGE2 production by ethanol intake. Furthermore, EGF can play a key role in gastric mucosal protection, repair, and regeneration by binding to the epidermal growth factor receptor (EGFR). Figure 4 As can be seen, compared with normal healthy mice (458.9 pg / mL), the serum EGF content in the gastric ulcer model group (274.6 pg / mL) was significantly decreased, while the HD group (454.4 pg / mL) restored it to the level of the normal group. EGF can also upregulate antioxidant enzymes (such as SOD and GSH) through the Akt / NF-κB pathway, reducing alcohol-induced reactive oxygen species damage; and protect gastric mucosal cells from alcohol-induced apoptosis by inhibiting the Bax-related apoptosis pathway. The mRNA levels of related genes were detected by qPCR. Figure 6 It is evident that 100 mg / kg BW mannan peptide can increase the content of gastric mucosal protection-related factors and alleviate alcoholic gastric ulcers in mice by regulating inflammatory and apoptosis-related pathways to protect the integrity of the gastric mucosa.

[0044] Example 5 This embodiment provides an experiment on the effect of mannan peptide on the intestinal flora structure of mice with acute alcoholic gastric ulcers.

[0045] Total genomic DNA was extracted from intestinal contents samples (n=5) according to the Fast SPIN extraction kit instructions. Genomic DNA concentration (spectrophotometry) and integrity (electrophoresis) were measured using primers 338F 5'-ACTCCTACGGGAGGCAGCA-3' and 806R 5'-GGACTACHVGGGTWTCTAAT-3'. The V3-V4 hypervariable region of the 16S rRNA gene was amplified. Amplicon purification and quantification were performed by Pasenuo Biotechnology Co., Ltd., and paired-end sequencing was performed on the Illumina MiSeq platform. Representative sequences and operational taxonomic units (OTUs) were then output using DADA2 (QIIME2 2019.4) and the Vsearch method (Vsearch (v2.13.4_linux_x86_64), cutadapt (v2.3)). Functional genes were corrected and sequence lengths were calculated using FrameBot software. The final representative sequences were selected and compared with the Greengenes database (QIIME2) to complete species annotation. Based on this, the number of taxonomic units included in each taxonomic level (from phylum to species) in the sample species annotation results was counted, and the impact of mannan peptides on the composition of the gut microbiota was evaluated through diversity and species difference analysis.

[0046] Gut microbiota can regulate gastric mucosal health through the gut-gastric axis. This example further analyzes the effect of mannan peptides on the gut microbiota composition of mice with acute alcoholic gastric ulcers. α-diversity analysis... Figure 7 The gradual flattening of the sparse curve indicates that the sequencing results are sufficient to reflect the diversity contained in the current sample. Figure 8 The α-diversity index results showed that the dose-dependent enhancement of mannan peptides on gut microbiota diversity in mice with acute alcoholic gastric ulcers increased the richness represented by the Chao1 and Observedspecies indices, the diversity represented by the Simpson index, and the evolutionary diversity represented by the Faith's PD index. β-diversity analysis was used to hierarchically cluster the gut microbiota at the genus level. Figure 9 ), and a species composition heatmap was drawn based on species difference analysis. Figure 10 As can be seen, 100 mg / kg BW mannan peptide can enhance... Dubosiella and Ileibacterium When the abundance of gut bacteria is reduced, Allobaculum and Desulfovibrio_R The abundance of [unspecified substance] allows gut microbiota to play a role in alleviating alcoholic gastric ulcers in the gut-gastric axis through competitive exclusion of Helicobacter pylori, promotion of short-chain fatty acid stimulation of mucus secretion to enhance gastric mucosal protection, and enhanced antioxidant and repair mechanisms. Therefore, 100 mg / kg BW mannan peptide can alleviate acute alcoholic gastric ulcers by improving the gut microbiota composition in mice with alcoholic gastric ulcers.

[0047] In summary, 100 mg / kg BW mannan peptide significantly alleviated hemorrhagic lesions and ulceration in the gastric tissue of mice with alcoholic gastric ulcers. The mechanisms include: (1) reducing reactive oxygen species produced by alcohol toxicity and improving oxidative stress by enhancing the activity of antioxidant enzymes in gastric tissue; (2) regulating the body's inflammatory response by modulating the Akt / NF-κB pathway; (3) protecting gastric mucosal cells from alcohol-induced apoptosis by inhibiting the Bax-related apoptosis pathway, thereby protecting the integrity of the gastric mucosa; and (4) regulating intestinal flora disorder caused by alcohol intake and preventing acute alcoholic gastric ulcers by improving the composition of the intestinal flora in mice with alcoholic gastric ulcers.

[0048] As described above, the basic principles, main features, and advantages of the present invention have been well described. The above embodiments and specifications are merely descriptions of preferred embodiments of the present invention, and the present invention is not limited to the above embodiments. Various changes and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit and scope of the present invention should fall within the protection scope defined by the present invention.

Claims

1. Use of a mannan peptide for the preparation of a medicament for acute alcoholic gastric ulcer, characterized in that, The acute alcoholic gastric ulcer medicine is used for preventing acute alcoholic gastric ulcer. The acute alcoholic gastric ulcer medicine is used for preventing acute alcoholic gastric ulcer.

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