New application of lactobacillus delbrueckii surface protein DUF4430

Abstract

The invention belongs to the technical field of biological medicines, and particularly discloses application of lactobacillus delbrueckii surface protein DUF4430 in preparation of medicines for improving symptoms of ulcerative colitis and preventing and / or treating ulcerative colitis. The novel application of the lactobacillus delbrueckii surface protein DUF4430, provided by the invention, comprises application in preparation of medicines for improving symptoms of ulcerative colitis and preventing and / or treating ulcerative colitis. Animal experiments show that the lactobacillus delbrueckii surface protein DUF4430 can relieve DSS-induced mouse ulcerative colitis, improve weight loss, colon length shortening and colonic mucosa injury, reduce the content of pro-inflammatory cytokines (such as IL-1beta and IL-6) in colon, increase the content of anti-inflammatory cytokines (such as IL-10), reduce the proportion of pro-inflammatory macrophages in spleen and colon, reduce the content of pro-inflammatory macrophages in spleen and colon, reduce the content of pro-inflammatory macrophages in spleen and colon, reduce the content of pro-inflammatory macrophages in spleen and colon and improve the The compound can inhibit the proinflammatory phenotype of macrophages, and can be used for preparing medicines for improving symptoms of ulcerative colitis and preventing and treating ulcerative colitis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the use of Lactobacillus delbrueckii surface protein DUF4430 in the preparation of a drug for improving the symptoms of ulcerative colitis, and preventing and / or treating ulcerative colitis. Background Art

[0002] Ulcerative colitis (UC) is a chronic nonspecific inflammatory bowel disease that mainly affects the rectum, colon mucosa and submucosa. Its clinical manifestations include abdominal pain, diarrhea, bloody stools, etc., which affect the patient's quality of life and may even develop into cancer in severe cases.

[0003] At present, the etiology and pathogenesis of ulcerative colitis are still unclear, and may be related to genetics, immunity, nutrition, bacteria, viruses and other environmental factors. Clinically, ulcerative colitis is mainly treated with antibiotics, such as mesalazine enteric-coated tablets, aminosalicylic acid preparations, etc. However, the use of antibiotics will change the balance of intestinal flora, and long-term use will produce dependence and drug resistance.

[0004] Chinese patent CN112646744B (Jiangnan University) discloses a strain of Lactobacillus reuteri (deposit number GDMCC No: 61102), which can reduce the disease activity index during ulcerative colitis, improve colonic mucosal damage, reduce MPO activity, reduce the content of pro-inflammatory factors TNF-α, IL-6, and IFN-γ in the colon, up-regulate the transcription levels of colon tight junction-related proteins Claudin-3, ZO-1, ZO-2, and Occludin, up-regulate the transcription levels of colon antimicrobial peptides Reg3g and Reg3b, increase the diversity of intestinal flora, and reduce the relative abundance of Acinetobacter in feces. It can be used to prepare products (such as probiotic preparations, drugs, etc.) that improve the symptoms of ulcerative colitis. In addition, other existing patents also use probiotic intervention to relieve ulcerative colitis. For example, Chinese patent CN114657084B (Nanchang University) discloses a strain of Bifidobacterium longum NSP001 (deposit number GDMCC No: 61439), which can improve the weight loss and DAI index of mice with ulcerative colitis under the conditions of mouse or human intestinal flora. Chinese patents CN112111422B and CN114231470B (Jiangnan University) respectively protect a strain of Bifidobacterium pseudoseudocatenulatum MY40C (deposit number GDMCC No: 60954) and Lactobacillus acidophilus CCFM1200 (deposit number GDMCC No: 62128) that can relieve ulcerative colitis; Chinese patent application CN116747248A (Jinqiao Biotechnology) requests protection of a strain of Lactobacillus rhamnosus MP108 (deposit number: DSM24229, German Microbiological Collection Center), which can relieve weight loss and colon shortening, restore colon barrier, treat colon tissue damage, inhibit colon inflammation and regulate intestinal flora, thereby relieving ulcerative colitis. In addition, Chinese patent CN117562249A seeks to protect a fermentation product that can be used to improve ulcerative colitis, using milk protein hydrolyzate as a fermentation substrate and Lactobacillus plantarum BHR4 (preservation number CGMCC No: 26686) as a fermentation strain. The resulting fermentation product has good antioxidant activity, can effectively inhibit intestinal inflammatory response, maintain intestinal epithelial barrier function, change intestinal microbial β diversity, increase the relative abundance of the Firmicutes phylum in intestinal microorganisms, and reduce the relative abundance of the Shigella genus, thereby maintaining intestinal health.

[0005] The probiotics and their preparations disclosed in the above patents can play a certain role in the prevention and treatment of ulcerative colitis. However, the application of probiotics has limitations: first, the quality of the bacterial preparation is difficult to guarantee, and its effect depends on a sufficient number of live bacteria; second, it is easy to cause adverse reactions in the body, such as bacterial and fungal sepsis, harmful metabolic activities, and excessive stimulation of immunity and gastrointestinal reactions. In certain special populations (such as premature infants and immunocompromised people), serious adverse reactions should be guarded against. The European Food Safety Authority (EFSA) believes that the health claims of most probiotics are insufficient in terms of strain characteristics (Scientific Opinion on the substantiation of health claims related to noncharacterised microorganisms pursuant to Article 13 (1) of Regulation (EC) No 1924 / 2006, 2009). In this case, it is particularly important to reveal probiotics and identify the effector molecules in probiotics that play a probiotic role.

[0006] Studies have shown that many active substances on the surface of probiotics, such as extracellular polysaccharides, surface proteins and peptidoglycan, play an important role in probiotic functions. Therefore, finding effective surfactants has potential application value in developing new probiotic products and relieving ulcerative colitis. Summary of the invention

[0007] The main technical problem solved by the present invention is to provide a new use of Lactobacillus delbrueckii surface protein DUF4430, specifically the use of surface protein DUF4430 in the preparation of drugs for improving the symptoms of ulcerative colitis, preventing and / or treating ulcerative colitis.

[0008] Secondly, the present invention provides a drug for improving the symptoms of ulcerative colitis, preventing and / or treating ulcerative colitis.

[0009] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0010] A new use of Lactobacillus delbrueckii surface protein DUF4430, which is: use of Lactobacillus delbrueckii surface protein DUF4430 in the preparation of a drug for improving the symptoms of ulcerative colitis, preventing and / or treating ulcerative colitis.

[0011] As a preferred embodiment of the present invention, the amino acid sequence of the Lactobacillus delbrueckii surface protein DUF4430 is selected from:

[0012] (1) the amino acid sequence shown in SEQ ID NO: 2;

[0013] (2) a truncated sequence of the amino acid sequence shown in SEQ ID NO: 2;

[0014] (3) A sequence comprising a truncated sequence of the amino acid sequence shown in SEQ ID NO:2.

[0015] Specifically, the amino acid sequence of the Lactobacillus delbrueckii surface protein DUF4430 is as follows:

[0016] MKKKILAVVVALAAFVGLFASQPVQKQSIQAAKTTKTAKKTTKKAKAKQIGVTYTLKDTTKAKNKQTLAKKTFKVKKGTSVFTVLKKAWKVNYTKSSKYGVFITKIKGLGDEKKKLYWTYTVDGKMSKVAADKQKLTKNKSKVVFTLKQY(SEQ ID NO: 2, NCBI registration number: WP_050889354.1).

[0017] Specifically, the amino acid sequence of the Lactobacillus delbrueckii surface protein DUF4430 is as follows:

[0018] QSIQAAKTTKTAKKTTKKAKAKQIGVTYTLKDTTKAKNKQTLAKKTFKVKKGTSVFTVLKKAWKVNYTKSSKYGVFITKIKGLGDEKKKLYWTYTVDGKMSKVAADKQKLTKNKSKVVFTLKQY (SEQ ID NO: 1). This sequence is the sequence obtained by truncating the signal peptide from the amino acid sequence shown in SEQ ID NO: 2.

[0019] Specifically, the amino acid sequence of the Lactobacillus delbrueckii surface protein DUF4430 is as follows:

[0020] MGSSHHHHHHSSGLVPRGSHMQSIQAAKTTKTAKKTTKKAKAKQIGVTYTLKDTTKAKNKQTLAKKTFKVKKGTSVFTVLKKAWKVNYTKSSKYGVFITKIKGLGDEKKKLYWTYTVDGKMSKVAADKQKLTKNKSKVVFTLKQY (SEQ ID NO: 3). This sequence comprises a truncated sequence of the amino acid sequence shown in SEQ ID NO: 2.

[0021] As a preferred embodiment of the present invention, the method for improving the symptoms of ulcerative colitis, preventing and / or treating ulcerative colitis comprises at least one of the following aspects:

[0022] (1) Improve weight loss;

[0023] (2) Improve the shortening of colon length;

[0024] (3) Improve colon mucosal damage;

[0025] (4) reduce the levels of pro-inflammatory cytokines in the colon;

[0026] (5) increase the content of anti-inflammatory cytokines in the colon;

[0027] (6) reduce the proportion of pro-inflammatory macrophages in the spleen;

[0028] (7) reduce the number of pro-inflammatory macrophages in the colon;

[0029] (8) Increase the number of anti-inflammatory macrophages in the colon;

[0030] (9) Inhibit the pro-inflammatory phenotype of macrophages.

[0031] Specifically, the inhibition of macrophage pro-inflammatory phenotype includes at least one of the following aspects:

[0032] (1) Downregulate the expression of pro-inflammatory cytokines in macrophages;

[0033] (2) inhibiting the expression of genes related to the Mincle-related signaling pathway in macrophages;

[0034] (3) Reduce the expression of Mincle, Malt1 and / or P50 in macrophages.

[0035] A drug for improving the symptoms of ulcerative colitis, preventing and / or treating ulcerative colitis, wherein the active ingredient of the drug comprises Lactobacillus delbrueckii surface protein DUF4430.

[0036] As a preferred embodiment of the present invention, the amino acid sequence of the Lactobacillus delbrueckii surface protein DUF4430 is selected from:

[0037] (1) the amino acid sequence shown in SEQ ID NO: 2;

[0038] (2) a truncated sequence of the amino acid sequence shown in SEQ ID NO: 2;

[0039] (3) A sequence comprising a truncated sequence of the amino acid sequence shown in SEQ ID NO:2.

[0040] Specifically, the amino acid sequence of the Lactobacillus delbrueckii surface protein DUF4430 is shown in SEQ ID NO:1.

[0041] Specifically, the amino acid sequence of the Lactobacillus delbrueckii surface protein DUF4430 is shown in SEQ ID NO:3.

[0042] As a preferred embodiment of the present invention, the content of Lactobacillus delbrueckii surface protein DUF4430 in the drug is an effective amount, which can be in the range of 0.01wt%-99.99wt%.

[0043] As a preferred embodiment of the present invention, the drug further comprises other medicinal ingredients, including but not limited to medicinal ingredients that assist in improving the symptoms of ulcerative colitis, preventing and / or treating ulcerative colitis.

[0044] As a preferred embodiment of the present invention, the drug further comprises a pharmaceutically acceptable carrier or excipient, including but not limited to excipients, preservatives, stabilizers, wetting agents, emulsifiers, salts for adjusting osmotic pressure, buffers, and the like.

[0045] As a preferred embodiment of the present invention, the dosage form of the drug is a pharmaceutically acceptable dosage form, including but not limited to powder injection, injection solution, tablet, pill, capsule, spray, dispersion and the like.

[0046] As a preferred embodiment of the present invention, the dosage of the drug is a pharmaceutically acceptable dosage.

[0047] Beneficial effects of the present invention:

[0048] The present invention provides an application of Lactobacillus delbrueckii surface protein DUF4430 in the preparation of a drug for improving the symptoms of ulcerative colitis, preventing and / or treating ulcerative colitis. Animal experiments show that Lactobacillus delbrueckii surface protein DUF4430 can alleviate DSS-induced ulcerative colitis in mice, improve weight loss, shortened colon length and colon mucosal damage, reduce the content of pro-inflammatory cytokines (such as IL-1β, IL-6) in the colon, increase the content of anti-inflammatory cytokines (such as IL-10), reduce the proportion of pro-inflammatory macrophages in the spleen and colon, and inhibit the pro-inflammatory phenotype of macrophages, and can be used to prepare a drug for improving the symptoms of ulcerative colitis, preventing and / or treating ulcerative colitis.

[0049] The present invention provides a drug for improving the symptoms of ulcerative colitis, preventing and / or treating ulcerative colitis. In addition to comprising an effective amount of Lactobacillus delbrueckii surface protein DUF4430, the drug also comprises a pharmaceutically acceptable carrier or excipient for preparing a dosage form that is convenient for carrying, storing or using. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 This is the SDS-PAGE analysis of the expression of DUF4430 protein in BL21 (DE3) in the experimental example.

[0051] Figure 2 This is the SDS-PAGE analysis of the DUF4430 protein supernatant purification results in the experimental example.

[0052] Figure 3 The results of DUF4430 protein detection (left) and Western Blot identification (right) in the experimental example.

[0053] Figure 4 This is the effect of DUF4430 treatment on the body weight of mice with DSS-induced colitis in the experimental example.

[0054] Figure 5 This is the effect of DUF4430 treatment on colon length in mice with DSS-induced colitis in the experimental example.

[0055] Figure 6 This is the effect of DUF4430 treatment on the colon morphology of mice with DSS-induced colitis in the experimental example.

[0056] Figure 7 This is the effect of DUF4430 treatment on the levels of IL-1β, IL-6 and IL-10 in the colon of mice in the experimental example.

[0057] Figure 8 This is the effect of DUF4430 treatment on the typing of mouse spleen macrophages in the experimental example.

[0058] Fig. 9 This is the effect of DUF4430 treatment on the typing of mouse colon macrophages in the experimental example.

[0059] Fig.10 This is the effect of different concentrations of DUF4430 on the viability of RAW264.7 cells in the experimental example.

[0060] Fig.11 This is the effect of DUF4430 treatment on the expression of pro-inflammatory cytokines in LPS-induced macrophages in the experimental example.

[0061] Fig.12 This is the effect of DUF4430 treatment on the gene expression profile of macrophages in the experimental example.

[0062] Fig.13 This is the effect of DUF4430 treatment on the expression of genes and proteins in the Mincle receptor pathway in the experimental example.

[0063] Fig.14 It is the map of the recombinant expression plasmid containing DUF4430 gene in the experimental example.

[0064] In order to more clearly illustrate the technical solution to be protected by the present invention, the drawings in the embodiments or experimental examples are briefly introduced above. It should be understood that the above drawings should not be regarded as any limitation on the protection scope of the present invention. For ordinary technicians in this field, other related drawings can also be obtained based on these drawings without paying creative work. DETAILED DESCRIPTION

[0065] The technical solution of the present invention will be clearly and completely explained below in conjunction with specific embodiments and experimental examples.

[0066] Those skilled in the art should understand that the following embodiments and experimental examples are only used to illustrate the technical solutions and technical effects of the present invention and should not be regarded as any limitation on the protection scope of the present invention. Based on the following embodiments, other technical solutions obtained by those skilled in the art without creative work, such as technical solutions obtained after modification, deformation or simple replacement, all belong to the protection scope of the present invention.

[0067] Unless otherwise specified, the raw materials, reagents, and equipment used in the examples or experimental examples are all commercially available products.

[0068] Example 1

[0069] This embodiment provides a new use of Lactobacillus delbrueckii surface protein DUF4430, specifically: use of Lactobacillus delbrueckii surface protein DUF4430 (amino acid sequence as shown in SEQ ID NO: 1) in the preparation of a drug for improving the symptoms of ulcerative colitis, including at least one of the following aspects:

[0070] (1) Improve weight loss;

[0071] (2) Improve the shortening of colon length;

[0072] (3) Improve colon mucosal damage.

[0073] Example 2

[0074] This embodiment provides a new use of Lactobacillus delbrueckii surface protein DUF4430, specifically: use of Lactobacillus delbrueckii surface protein DUF4430 (amino acid sequence as shown in SEQ ID NO: 3) in the preparation of a drug for preventing and / or treating ulcerative colitis, including at least one of the following aspects:

[0075] (4) reduce the levels of pro-inflammatory cytokines in the colon;

[0076] (5) increase the content of anti-inflammatory cytokines in the colon;

[0077] (6) reduce the proportion of pro-inflammatory macrophages in the spleen;

[0078] (7) reduce the number of pro-inflammatory macrophages in the colon;

[0079] (8) Increase the number of anti-inflammatory macrophages in the colon;

[0080] (9) Inhibiting the pro-inflammatory phenotype of macrophages, for example, down-regulating the expression of pro-inflammatory cytokines in macrophages, inhibiting the expression of genes related to the Mincle-related signaling pathway in macrophages, or reducing the expression of Mincle, Malt1 and / or P50 in macrophages.

[0081] In other embodiments of the present invention, the use of Lactobacillus delbrueckii surface protein DUF4430 (amino acid sequence as shown in SEQ ID NO: 2) in the preparation of a medicament for preventing and / or treating ulcerative colitis is basically the same as in Example 2.

[0082] Example 3

[0083] This embodiment provides a drug for improving the symptoms of ulcerative colitis, comprising a pharmaceutically effective amount of Lactobacillus delbrueckii surface protein DUF4430 (amino acid sequence as shown in SEQ ID NO: 1), and pharmaceutically acceptable carriers and excipients (such as excipients, preservatives and stabilizers), for preparing the drug into an injection.

[0084] Example 4

[0085] This embodiment provides a drug for preventing and / or treating ulcerative colitis, comprising a pharmaceutically effective amount of Lactobacillus delbrueckii surface protein DUF4430 (amino acid sequence as shown in SEQ ID NO: 3), and pharmaceutically acceptable carriers and excipients (such as preservatives and stabilizers), for preparing the drug into an injection.

[0086] In other embodiments of the present invention, a drug for preventing and / or treating ulcerative colitis comprises a pharmaceutically effective amount of Lactobacillus delbrueckii surface protein DUF4430 (amino acid sequence as shown in SEQ ID NO: 2), and pharmaceutically acceptable carriers and excipients for preparing the drug into tablets.

[0087] Experimental example

[0088] 1. Expression and purification of DUF4430 protein

[0089] 1. DUF4430 protein sequence

[0090] The amino acid sequence of Lactobacillus delbrueckii surface protein DUF4430 (NCBI accession number: WP_050889354.1) is shown in SEQ ID NO: 2, and the amino acid sequence after truncating the signal peptide is shown in SEQ ID NO: 1.

[0091] 2. Plasmid construction

[0092] Adopting MaxCodon, the latest codon optimization software developed by Detai Biotechnology TM The nucleotide sequence of the DUF4430 protein was optimized by the Optimization Program (V13). The optimized synthetic sequence of DUF4430 was inserted into the expression vector pET28a by whole gene synthesis and restriction enzyme cutting sites NdeI and Xho], and the accuracy of the final expression vector was confirmed by enzyme cutting and sequencing to obtain a recombinant expression plasmid containing the DUF4430 gene (see the plasmid map for details). Fig.14 ).

[0093] The nucleotide sequence of the recombinant expression plasmid is as follows:

[0094] CAGAGCATTCAGGCAGCGAAAACCACCAAAACCGCGAAAAAGACCACCAAGAAGGCGAAAGCGAAA CAGATTGGCGTTACCTATACCCTGAAGGACACCACCAAAGCGAAGAACAAGCAGACCCTGGCGAAGAAAACCTTCA AGGTCAAGAAGGGCACCAGCGTTTTACCGTCCTGAAAAAGGCCTGGAAGGTCAACTACACCAAAAGCAGCAAGTA CGGCGTCTTCATCACCAGATCAAAGGCCTGGGCGACGAAAAGAAGAAACTGTACTGGACCTACACCGTCGACGGT AAAATGAGCAAAGTTGCGGCGGATAAACAGAAGCTGACCAAGAACAAGAGCAAGGTCGTCTTCACCCTGAAGCAGT AC TAATGA CACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGAT (SEQ ID NO: 4).

[0095] In the above sequence, the NdeI restriction site is CATATG (italic bold), and the Xho I restriction site is CTCGAG (bold). The sequence on the vector carrying the HIS tag is as follows:

[0096] ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATG (italic).

[0097] The DUF4430 sequence connected to the vector is as follows:

[0098] CAGAGCATTCAGGCAGCGAAAACCACCAAAACCGCGAAAAAGACCACCAAGAAGGCGAAAGCGAAACAGATTGGCGTTACCTATACCCTGAAGGACACCACCAAAGCGAAGAACAAGCAGACCCTGGCGAAGAAAACCTTCAAGGTCAAGAAGGGCACCAGCGTTTTTTACCGTCCTGAAAAAGGCCTGG AAGGTCAACTACACCAAAAGCAGCAAGTACGGCGTCTTCATCACCAGATCAAAGGCCTGGGCGACGAAAAGAAGAAACTGTACTGGACCTACACCGTCGACGGTAAAATGAGCAAAGTTGCGGCGGATAAACAGAAGCTGACCAAGAACAAGAGCAAGGTCGTCTTCACCCTGAAGCAGTAC (underline).

[0099] It should be noted that the DUF4430 protein was added with a His tag during expression and contained part of the sequence on the vector.

[0100] 3. Expression vector transformation and induced expression

[0101] Transform the recombinant expression plasmid containing the DUF4430 gene into BL21 (DE3) competent cells and evenly spread on LB plates (containing 50 μg / mL kanamycin sulfate), then invert and place in a 37°C incubator overnight. Select a single clone from the transformed plate and inoculate it into 4 mL of LB medium (containing 50 μg / mL kanamycin sulfate) for expansion culture. When the OD600 is 0.8, add IPTG with a final concentration of 0.2 mM to the test tube culture solution, and collect the cells after induction at 15°C for 16 hours. If no purification operation is performed on the same day, freeze the cells at -20°C.

[0102] SDS-PAGE identification: Take the induced culture medium, centrifuge at 12000rpm for 5min, remove the supernatant, add PBS buffer to resuspend the precipitate, and finally add SDS-PAGE loading buffer to heat the sample at 100℃ for 10min, centrifuge and take the supernatant for electrophoresis. The whole bacteria are ultrasonically lysed with 20mM Tris (pH 8.0), 300mM NaCl, 20mM Imidazole (containing 1% Triton X-100, v / v), 1mM DTT, 1mM PMSF, and the supernatant and precipitate are respectively subjected to SDS-PAGE analysis and detection. The analysis results are shown in Figure 1 .

[0103] from Figure 1 It can be seen from the whole bacteria ultrasonic lysis analysis that the plasmid containing DUF4430 gene was transformed into BL21 (DE3) competent cells and expressed in the supernatant after induction.

[0104] 4. DUF4430 protein purification

[0105] Affinity chromatography purification of DUF4430 protein in the supernatant (the entire purification process was performed at low temperature): the whole bacteria were ultrasonically lysed with 50mMTris (pH 8.0), 300mMNaCl, 20mM Imidazole (containing 1% TritonX-100, v / v), 1mM DTT, and 1mM PMSF, and the Ni-IDA affinity chromatography column was equilibrated with 50mM Tris (pH 8.0), 300mM NaCl, and 20mM Imidazole buffer. The target protein was then eluted with equilibration buffers containing different concentrations of imidazole, and each elution fraction was collected for SDS-PAGE analysis. The analysis results are shown in Figure 2 .

[0106] After purification by Ni-IDA affinity chromatography, Lane 3-4 with relatively high purity and concentration was collected and dialyzed into 1×PBS (pH 7.4), 5% Glycerol. After dialysis, it was filtered with a 0.22μm filter and aliquoted and frozen at -80℃. The results of DUF4430 protein detection and Western Blot identification are shown in Figure 3 The amino acid sequence of the finally expressed DUF4430 protein is shown in SEQ ID NO:3.

[0107] DUF4430 alleviates dextran sulfate sodium (DSS)-induced ulcerative colitis in mice

[0108] Experimental methods: 35 SPF-grade ICR male mice were randomly divided into 5 groups, namely CON group, DSS group, L4430 (0.25μg / day) + DSS group, M4430 + DSS (1μg / day) and HDUF4430 (3μg / day) + DSS group. Different doses (low, medium and high) of DUF4430 protein were administered to mice by intraperitoneal injection every day. The CON group and DSS group were injected with equal volumes of normal saline every day. After 14 days of treatment, 3% DSS was added to the drinking water to induce ulcerative colitis. The CON group maintained normal drinking water, and body weight was recorded every day. Mice were killed after 23 days, and the spleen was collected for flow cytometry detection of macrophage typing, the colon was collected to measure the colon length, and a small section was taken for H&E staining and immunofluorescence staining, and another small section was used to extract protein for detection of IL-1β, IL-6 and IL-10. The results are shown in Figure 4-9 .

[0109] 1. Effect of DUF4430 treatment on body weight of mice with DSS-induced colitis

[0110] The degree of weight change was evaluated by the percentage change of the mice's weight during the experiment compared to their initial weight ( Figure 4 ). At the end of the experiment, the weight percentage of mice in the CON group increased compared with the initial weight, indicating that their food and water intake were normal, their growth and development were good, and they were in a natural growth trend. The weight percentage of the DSS group was significantly lower than that of the CON group (P<0.001), and the treatment with DUF4430 at various concentrations could alleviate the weight loss induced by DSS to varying degrees. The weight percentage of mice in the L4430+DSS group was significantly higher than that of mice in the DSS group (P<0.05), indicating that the treatment with low concentrations of DUF4430 was more effective.

[0111] 2. Effect of DUF4430 treatment on colon length in mice with DSS-induced colitis

[0112] from Figure 5It can be seen that the colon length of mice in the CON group was 9-10 cm, the feces in the intestine were formed, the intestinal mucosa was normal in morphology, and there was no congestion and swelling. Compared with the CON group, the colon length of mice in the DSS group was significantly shortened (P<0.0001), only about 7 cm. After treatment with low concentrations of DUF4430, the colon length of mice increased significantly compared with the DSS group (P<0.001), while the colon length increased after treatment with medium and high concentrations of DUF4430, but there was no significant difference compared with the DSS group.

[0113] 3. Colon histopathological examination

[0114] H&E staining was used to observe the tissue and evaluate the severity of colon inflammation. Figure 6 ). Observation of histopathological changes by H&E staining under light microscopy: The colon specimens of the control group showed normal histological morphology of the mouse colon, with intact and continuous colon tissue mucosal epithelium, neatly arranged glands, normal crypt structure, and little inflammatory cell infiltration. Colon sections of the DSS group showed typical inflammatory changes, such as loss of crypt and surface epithelial cells, infiltration of inflammatory cells, and spread of inflammation to the mucosa, muscularis mucosa, and submucosa. Compared with the DSS group, the degree of colon damage in the L4430+DSS group was significantly reduced. Although the crypt morphology was still changed and the mucosal epithelium was locally discontinuous, the inflammatory cell infiltration was reduced and the integrity of the mucosal structure was higher.

[0115] 4. Effects of DUF4430 treatment on the contents of IL-1β, IL-6 and IL-10 in colon tissue

[0116] ELISA kits were used to detect the levels of IL-1β, IL-6 and IL-10 in mouse colon tissues ( Figure 7 ). The results showed that compared with the CON group, the levels of pro-inflammatory cytokines IL-1β and IL-6 in the colon of mice in the DSS group were significantly increased (P<0.05), while the level of anti-inflammatory cytokine IL-10 was extremely significantly decreased (P<0.001). Low-concentration DUF4430 treatment reduced the levels of IL-1β and IL-6 and increased the level of IL-10.

[0117] 5. Effect of DUF4430 treatment on the phenotype of splenic macrophages

[0118] The spleen mononuclear cells were obtained using a spleen mononuclear cell isolation kit, and CD45 was used to mark leukocytes, F4 / 80 and CD11b were used to mark macrophages, and CD16 / 32 was used to mark pro-inflammatory macrophages for flow cytometry analysis. The results showed that ( Figure 8 ), compared with the CON group, the proportion of proinflammatory macrophages in the spleen of mice in the DSS group was significantly increased (P<0.05), while DUF4430 treatment significantly reduced the proportion of proinflammatory macrophages (P<0.05).

[0119] 6. Effects of DUF4430 treatment on the phenotype of colonic macrophages

[0120] Immunofluorescence staining was performed using CD68 to mark pro-inflammatory macrophages in the mouse colon and CD206 to mark anti-inflammatory macrophages. The results showed ( Fig. 9 ), compared with the CON group, the number of pro-inflammatory macrophages in the colon of mice in the DSS group increased, while the number of anti-inflammatory macrophages decreased. However, DUF4430 treatment reversed this phenomenon, reducing pro-inflammatory macrophages and increasing anti-inflammatory macrophages.

[0121] 3. Effect of DUF4430 on cell viability

[0122] RAW264.7 cells were seeded into 96-well plates, with 25,000 cells per well. The culture medium was high-glucose DMEM (10% fetal bovine serum). After overnight culture, the cells were treated with 0.01, 0.05, 0.1, 0.25, 0.5, 1, 5, 10 and 20 μg / mL of DUF4430. After 12 hours, 10 μL of CCK reagent was added to each well for incubation. After 2 hours, the absorbance was measured at a wavelength of 450 nm using an ELISA reader and the relative cell viability was calculated. The results are shown in Fig.10 .

[0123] from Fig.10 It can be seen that DUF4430 did not reduce macrophage viability in the concentration range of 0.01-1 μg / mL, but significantly inhibited cell viability at concentrations of 5, 10, and 20 μg / mL.

[0124] Effect of DUF4430 on the pro-inflammatory phenotype of macrophages

[0125] RAW264.7 cells were seeded into 12-well plates, with 250,000 cells per well, and the culture medium was high-glucose DMEM (10% fetal bovine serum). After overnight culture, the cells were treated with 0.01, 0.05, 0.1, and 0.25 μg / mL of DUF4430, respectively. After 12 hours, the culture medium was replaced and 100 ng / mL of lipopolysaccharide (LPS) was used to induce the pro-inflammatory phenotype of RAW264.7 cells. After 12 hours of induction, the culture medium was removed and the cells were collected for RNA and protein extraction. The results are shown in Figure 11-13 .

[0126] from Fig.11 It can be seen that DUF4430 inhibits the proinflammatory factors of RAW264.7 cells in a dose-dependent manner. The inhibitory effect of 0.25μg / mL DUF4430 treatment is the best, significantly inhibiting the expression of IL-1β, IL-6 and TNF-α genes. Transcriptome analysis showed that DUF4430 treatment of macrophages also inhibited the expression of genes related to the Mincle-related signaling pathway ( Fig.12 ). RT-qPCR and protein identification confirmed that DUF4430 pretreatment significantly reduced the expression of Mincle, Malt1, and IL-1β compared with the LPS group ( Fig.13 These results suggest that DUF4430 inhibits LPS-induced pro-inflammatory phenotype of macrophages by regulating Mincle receptor-related signaling pathways.

[0127] Although the technical scheme and technical effects of the present invention have been described in detail above with general descriptions, specific embodiments and experimental examples, for those skilled in the art, any modification, replacement or improvement made without departing from the spirit of the present invention shall fall within the scope of protection of the present invention.

Claims

1. A new use of Lactobacillus delbrueckii surface protein DUF4430, characterized in that: Application of Lactobacillus delbrueckii surface protein DUF4430 in the preparation of a drug for improving the symptoms of ulcerative colitis, and preventing and / or treating ulcerative colitis.

2. The novel use according to claim 1, characterized in that: The amino acid sequence of the Lactobacillus delbrueckii surface protein DUF4430 is selected from: (1) the amino acid sequence shown in SEQ ID NO: 2; (2) a truncated sequence of the amino acid sequence shown in SEQ ID NO: 2; (3) A sequence comprising a truncated sequence of the amino acid sequence shown in SEQ ID NO:

2.

3. The new use according to claim 1, characterized in that: The method of improving the symptoms of ulcerative colitis, preventing and / or treating ulcerative colitis comprises at least one of the following aspects: (1) Improve weight loss; (2) Improve the shortening of colon length; (3) Improve colon mucosal damage; (4) reduce the levels of pro-inflammatory cytokines in the colon; (5) increase the content of anti-inflammatory cytokines in the colon; (6) reduce the proportion of pro-inflammatory macrophages in the spleen; (7) reduce the number of pro-inflammatory macrophages in the colon; (8) Increase the number of anti-inflammatory macrophages in the colon; (9) Inhibit the pro-inflammatory phenotype of macrophages.

4. The new use according to claim 3, characterized in that: The inhibition of macrophage pro-inflammatory phenotype comprises at least one of the following aspects: (1) Downregulate the expression of pro-inflammatory cytokines in macrophages; (2) inhibiting the expression of genes related to the Mincle-related signaling pathway in macrophages; (3) Reduce the expression of Mincle, Malt1 and / or P50 in macrophages.

5. A drug for improving the symptoms of ulcerative colitis, preventing and / or treating ulcerative colitis, characterized in that: The active ingredient of the medicine comprises Lactobacillus delbrueckii surface protein DUF4430.

6. The drug according to claim 5, characterized in that: The amino acid sequence of the Lactobacillus delbrueckii surface protein DUF4430 is selected from: (1) the amino acid sequence shown in SEQ ID NO: 2; (2) a truncated sequence of the amino acid sequence shown in SEQ ID NO: 2; (3) A sequence comprising a truncated sequence of the amino acid sequence shown in SEQ ID NO:

2.

7. The drug according to claim 5, characterized in that: The content of Lactobacillus delbrueckii surface protein DUF4430 in the medicine is the effective amount, which is in the range of 0.01wt%-99.99wt%.

8. The drug according to claim 5, characterized in that: The drug also contains other medicinal ingredients, including but not limited to medicinal ingredients that assist in improving the symptoms of ulcerative colitis, preventing and / or treating ulcerative colitis.

9. The drug according to claim 5, characterized in that: The drug further comprises pharmaceutically acceptable carriers or excipients, including but not limited to excipients, preservatives, stabilizers, wetting agents, emulsifiers, salts for adjusting osmotic pressure, and buffers.

10. The drug according to claim 5, characterized in that: The dosage form of the drug is a pharmaceutically acceptable dosage form, including but not limited to powder injection, injection solution, tablet, pill, capsule, spray, and dispersion.

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