Application of lactobacillus gasseri in preparation of medicine for relieving ulcerative colitis

Abstract

The invention belongs to the technical field of biology, and particularly relates to application of lactobacillus gasseri in preparation of a medicine for relieving ulcerative colitis. The invention discloses a mechanism for treating ulcerative colitis by regulating ferroptosis pathway through lactobacillus gasseri, and solves the defect of imperfect research on the mechanism for treating UC by probiotics in the prior art. And a theoretical basis and a practical basis are provided for developing a new strategy for targeted treatment of the ulcerative colitis based on the probiotics.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to application of Lactobacillus gasseri (L. gasseri) in preparing medicine for alleviating ulcerative colitis. Background Art

[0002] Ulcerative colitis (UC) is a chronic inflammatory bowel disease characterized by abdominal pain, diarrhea, and bloody stools. Long-term inflammation can increase the risk of colon cancer. Existing studies have shown that intestinal flora disturbances play a key role in the pathogenesis of UC. Lactobacilli, a key member of the Firmicutes phylum, have attracted considerable attention for their immunomodulatory properties. Lactobacillus rhamnosus is known to regulate the Th17 / Treg ratio through the JAK-STAT signaling pathway, while Lactobacillus reuteri can inhibit neutrophil recruitment and increase the proportion of Tregs.

[0003] Probiotics have been widely validated clinically and experimentally for restoring gut microbial diversity and protecting against inflammatory bowel disease (IBD). Current research focuses on the role of lactic acid bacteria in alleviating ulcerative colitis (UC) through immune regulation, such as regulating the Th17 / Treg balance and inhibiting neutrophil recruitment. However, their role in regulating cell death pathways remains understudied.

[0004] As a natural strain in traditional fermented foods (such as kimchi and yogurt), Lactobacillus gasseri has been consumed for over a century and has been granted GRAS (Generally Recognized as Safe) certification by the FDA, posing no risk of genetic modification. It exhibits excellent tolerance to salivary enzymes, low pH levels, and pancreatic juice, colonizes in intestinal epithelial cells, and contributes to the health and biological function of the host environment. Summary of the Invention

[0005] In view of this, the present invention aims to reveal the mechanism by which L. gasseri treats UC by regulating the ferroptosis pathway, thereby addressing the problem of incomplete research on the mechanism of probiotic treatment of UC in the prior art. By establishing an in vitro ferroptosis cell model (induced by RSL3) and an in vivo UC animal model (induced by DSS), combined with multi-omics analysis techniques, the present invention systematically studies the regulatory effects of Lactobacillus gasseri on key ferroptosis pathways, thereby clarifying its specific molecular mechanism for treating UC and providing a theoretical basis and practical foundation for the development of new strategies for the targeted treatment of UC based on probiotics.

[0006] The present invention provides application of Lactobacillus gasseri in preparing a medicine for alleviating ulcerative colitis.

[0007] The present invention also provides the use of Lactobacillus gasseri in preparing a medicine for reducing the mortality rate of colon epithelial cells.

[0008] The present invention also provides the use of Lactobacillus gasseri in preparing a medicine for inhibiting cell ferroptosis.

[0009] The present invention also provides the use of Lactobacillus gasseri in preparing a drug for reducing the expression of ferroptosis-related genes.

[0010] In the present invention, the ferroptosis-related genes include FTH1 and ASCL4.

[0011] The present invention also provides the use of Lactobacillus gasseri in preparing medicine for reducing cellular inflammatory factors.

[0012] In the present invention, the cellular inflammatory factors include IL-6, IL-1β and TNF-α.

[0013] The present invention also provides the use of Lactobacillus gasseri in preparing a medicine for reducing colon barrier damage.

[0014] The present invention also provides the use of Lactobacillus gasseri in preparing a medicine for maintaining the structural integrity of mitochondria.

[0015] The present invention also provides the use of Lactobacillus gasseri combined with mesalazine sustained-release granules and / or sulfasalazine enteric-coated tablets in the preparation of a medicament for alleviating ulcerative colitis.

[0016] The present study found that Lactobacillus gasseri can delay DSS-induced colitis in mice (mice weight loss is significantly reduced, DAI scores are lowered, colon length is restored; DSS-induced colon pathological damage and intestinal damage are weakened, etc.), and exerts a protective effect against ulcerative colitis by inhibiting ferroptosis (maintaining mitochondrial structural integrity; reducing the production of lipid peroxides and iron ion content in the colon of UC mice; at the same time, the expression of ferroptosis-related genes such as FTH1 and ASCL4 is reduced, and the expression of GPX4 is increased, etc.). When we interfered with genes in the ferroptosis-related pathway (such as GPX4), the results were the opposite, indicating that Lactobacillus gasseri can alleviate ulcerative colitis by inhibiting the ferroptosis pathway. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Figure 2 is the 24-hour growth curve of L. gasseri in MRS liquid medium; Figure (A) is the 24-hour growth curve of L. gasseri in MRS liquid medium; Figure (B) is the Gram-stained L. gasseri observed under a 63× field of view;

[0018] Figure 2L. gasseri alleviated DSS-induced colitis in mice; (A) Body weight change; (B) DAI score; (C) to (D) Colon gross photographs and length; (E) H&E staining of colon; (F) FD-4 intestinal permeability test; (G) Colon Alcian blue staining; Scale bar: 200 μm.

[0019] Figure 3 L. gasseri inhibited DSS-induced colonic ferroptosis in mice. (A) Transmission electron microscopy was used to observe the mitochondrial structure in mouse colonic epithelial cells. The yellow arrow indicates mitochondrial damage. (B) Immunohistochemical staining was used to detect the expression of 4-HNE. (C) Prussian blue staining was used to detect intracellular iron deposition. (D) Iron determination kit was used to detect the iron content in colonic tissue. (E) GSH concentration in colonic tissue. (F) MDA level in colonic tissue. Scale bars: 1.0-2.0 μm.

[0020] Figure 4 L. gasseri inhibits inflammation and RSL3-induced ferroptosis in NCM-460 cells; (A) to (C) mRNA levels of IL-6, IL-1β, and TNF-α; (D) to (F) RSL3 was used to induce ferroptosis in cells after pretreatment with L. gasseri, and the intracellular iron content, GSH, and MDA levels were detected; (G) Cell viability was analyzed by CCK-8 assay; (H) mRNA expression levels of ferroptosis-related genes; (I) Protein expression levels of ACSL4, FTH1, and GPX4 were detected by Western blotting. DETAILED DESCRIPTION

[0021] For reagents or instruments used below, if specific techniques or conditions are not specified, conventional experimental conditions were followed. If the reagent company's instructions are not clearly stated, the recommended conditions were followed. For reagents or instruments used without manufacturer information, all are commercially available, conventional products.

[0022] Example

[0023] 1. Anti-inflammatory and anti-ferroptosis effects in vivo

[0024] 1. Experimental Materials

[0025] MRS medium was purchased from Beina Biotechnology; L. gasseri strain (BNCC339385) was purchased from Beina Microbial and Cell Resource Collection Center (BNCC); C57BL / 6J mice (male, 6-8 weeks old) were provided by Jiangsu Jicui Yaokang Biotechnology Co., Ltd.

[0026] 2. Experimental Methods

[0027] 2.1 L. gasseri strain recovery and culture

[0028] (1) Prepare MRS liquid medium: accurately weigh 10.0 g peptone, 10.0 g beef extract, 4.0 g yeast extract, 20.0 g glucose, 0.2 g magnesium sulfate, 5.0 g sodium acetate, 2.0 g triammonium citrate, 2.0 g potassium dihydrogen phosphate, 0.04 g manganese sulfate, and 1.0 g Tween 80, mix well with 1 L ultrapure water, and sterilize by autoclaving at 121°C for 15 min.

[0029] (2) Activation of L. gasseri strains: Disinfect the surface of the ampoule tube containing the freeze-dried strain powder, burn the top with alcohol in a biosafety cabinet, and add sterile water to break the glass;

[0030] (3) Add 0.5 mL of MRS medium to fully dissolve the lyophilized powder and inoculate the entire bacterial solution into a 15 mL sterile centrifuge tube filled with MRS medium;

[0031] (4) Culture in an anaerobic incubator at 37°C and subculture every 24 to 48 hours.

[0032] 2.2 Plotting the growth curve of L. gasseri strain

[0033] Inoculate fresh L. gasseri bacterial suspension at a 2% (v / v) inoculum volume into 200 ml of MRS liquid medium and culture in an anaerobic incubator at 37°C. Take the bacterial suspension every 3 hours and measure the absorbance at 600 nm. Before each absorbance measurement, centrifuge the bacteria (4000 g, 10 min) and wash and resuspend them in sterile PBS. Draw a 24-hour growth curve of L. gasseri based on the incubation time and absorbance values. Take a smear of L. gasseri bacterial suspension in the logarithmic growth phase and perform Gram staining to observe bacterial morphology. For details, see Figure 1 .

[0034] 2.3 Animal modeling and drug administration methods

[0035] (1) The mice were adaptively raised for 7 days and randomly divided into three groups: control group (Ctrl), model group (DSS), and L. gasseri intervention group (DSS+L. gasseri), with 8 mice in each group.

[0036] (2) The control group had a normal diet and water intake throughout the whole process, while the model group and L. gasseri intervention group were fed with 3% DSS in water for 7 days to induce UC. During the modeling period, the L. gasseri intervention group was given 5×10 8 The mice in the DSS group were orally administered with fresh bacterial solution of CFU once, and the mice in the DSS group were orally administered with an equal volume of pure water as a control.

[0037] (3) After 7 days, feces were collected, and blood, colon tissue, and mesenteric lymph nodes of the mice were taken for subsequent testing.

[0038] Test results are shown in Figure 2 and Figure 3 .

[0039] Results: L. gasseri significantly improved colon inflammation. L. gasseri treatment significantly reduced weight loss, lowered DAI scores, restored colon length, and reduced colon barrier damage. It also maintained mitochondrial structural integrity and reduced lipid peroxide and iron ion levels.

[0040] 2. In vitro anti-inflammatory and anti-ferroptosis effects

[0041] 1. Experimental Materials

[0042] RSL3 (HY-100218A) was purchased from MCE; human normal colon epithelial cells NCM-460 were from the American Type Culture Collection (ATCC); L. gasseri strain (BNCC339385) was purchased from the Beina Microbial and Cell Collection (BNCC).

[0043] 2. Experimental Methods

[0044] 2.1 Establishment of cell inflammation model and L. gasseri intervention L. gasseri (10 5 -10 7 CFU / mL) pretreated NCM-460 cells (2×10 5 / well) for 12 hours, then 1 μg / mL LPS was used to induce cell inflammation, and total cell RNA was extracted to detect the expression levels of inflammatory factor mRNA.

[0045] 2.2 Establishment of cell ferroptosis model and L. gasseri intervention L. gasseri (10 5 -10 7 CFU / mL) pretreated NCM-460 cells (2×10 5 gasseri pretreatment, 3 μM RSL3 was used to induce cell ferroptosis, and total cellular RNA and total protein were extracted for subsequent detection.

[0046] Test results such as Figure 4 shown.

[0047] Results: L. gasseri pretreatment could reduce the expression of inflammatory factors in NCM-460 cells induced by LPS; in the RSL3-induced ferroptosis model of colon epithelial cells NCM-460, L. gasseri could significantly reduce the cell death rate and inhibit the characteristic indicators of ferroptosis (FTH1 and ASCL4).

[0048] Based on the above conclusions, it can be considered that Lactobacillus gasseri combined with mesalazine sustained-release granules and / or sulfasalazine enteric-coated tablets can effectively relieve ulcerative colitis.

[0049] The above description is only a preferred embodiment of the present invention and is not intended to limit the scope of the present invention. Any changes and improvements made to the present invention should be included in the scope of protection of the present invention.

Claims

1. Application of Lactobacillus gasseri in the preparation of a medicine for alleviating ulcerative colitis.

2. Application of Lactobacillus gasseri in the preparation of drugs for reducing the mortality rate of colon epithelial cells.

3. Application of Lactobacillus gasseri in the preparation of drugs for inhibiting cell ferroptosis.

4. Application of Lactobacillus gasseri in the preparation of drugs for reducing the expression of ferroptosis-related genes.

5. The use according to claim 4, characterized in that The ferroptosis-related genes include FTH1 and ASCL4.

6. Application of Lactobacillus gasseri in the preparation of drugs for reducing cellular inflammatory factors.

7. The use according to claim 6, characterized in that The cellular inflammatory factors include IL-6, IL-1β and TNF-α.

8. Use of Lactobacillus gasseri in the preparation of drugs for reducing colon barrier damage.

9. Use of Lactobacillus gasseri in the preparation of drugs for maintaining mitochondrial structural integrity.

10. Use of Lactobacillus gasseri combined with mesalazine sustained-release granules and / or sulfasalazine enteric-coated tablets in the preparation of a drug for relieving ulcerative colitis.

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