New use of lactobacillus debrueckii surface protein duf4430

Abstract

The application belongs to the technical field of biological medicine, and particularly discloses application of Lactobacillus delbrueckii surface protein DUF4430 in preparation of a medicine for improving symptoms of ulcerative colitis, and preventing and / or treating ulcerative colitis. The application provides a new use of the Lactobacillus delbrueckii surface protein DUF4430, including application in preparation of a medicine 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 ulcerative colitis in mice, improve weight loss, shorten colon length, and reduce colon mucosal injury, reduce the content of pro-inflammatory cytokines (such as IL-1β and 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 therefore can be used for preparing a medicine for improving symptoms of ulcerative colitis, and preventing and treating ulcerative colitis.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to application of DUF4430 on the surface of lactobacillus delbrueckii in preparation of a medicine for improving symptoms of ulcerative colitis, and preventing and / or treating ulcerative colitis. BACKGROUND

[0002] Ulcerative colitis (UC) is a chronic nonspecific inflammatory bowel disease mainly involving rectal and colonic mucosa and submucosa, and is clinically manifested as abdominal pain, diarrhea, hematochezia and the like, and affects the life quality of patients, and is even evolved into cancer in severe cases.

[0003] At present, the etiology and pathogenesis of ulcerative colitis are not clear, and may be related to heredity, immunity, nutrition, bacteria, viruses and other environmental factors. Clinically, ulcerative colitis is mainly treated by using antibiotic drugs, such as mesalazine enteric-coated tablets, aminosalicylic acid preparations and the like. However, the use of antibiotic drugs will change the balance of intestinal flora, and long-term taking will produce dependency and drug resistance.

[0004] Chinese patent CN112646744B (Jiangnan University) discloses a strain of Lactobacillus reuteri (accession number GDMCC No: 61102), which can reduce the disease activity index during ulcerative colitis, improve colon mucosa damage, reduce MPO activity, reduce the content of pro-inflammatory factors TNF-a, IL-6, IFN-g in colon, up-regulate the transcription level of colon tight junction related proteins Claudin-3, ZO-1, ZO-2 and Occludin, up-regulate the transcription level of colon antibacterial peptides Reg3g, Reg3b, improve intestinal flora diversity, and reduce the relative abundance of Acinetobacter in feces, which can be used to prepare products (such as probiotic preparations, drugs, etc.) for improving the symptoms of ulcerative colitis. In addition, other existing patents also use probiotics to intervene to relieve ulcerative colitis, for example, Chinese patent CN114657084B (Nanchang University) discloses a strain of Bifidobacterium longum NSP001 (accession number GDMCC No: 61439), which can improve the weight loss and DAI index of ulcerative colitis mice under mouse or human intestinal flora conditions. Chinese patents CN112111422B and CN114231470B (Jiangnan University) protect a strain of Bifidobacterium pseudocatenulatum MY40C (accession number GDMCC No: 60954) and Lactobacillus acidophilus CCFM1200 (accession number GDMCC No: 62128) respectively, which can relieve ulcerative colitis; Chinese patent application CN116747248A (Jinjiao Biotechnology) claims to protect a strain of Lactobacillus rhamnosus MP108 (accession number: DSM24229, German Microorganism Preservation Center), which can relieve weight loss and colon shortening, restore colon barrier, treat colon tissue damage, inhibit colonic inflammation, and regulate intestinal flora, thereby relieving ulcerative colitis. In addition, Chinese patent CN117562249A claims to protect a fermentation product for improving ulcerative colitis, using lactoprotein hydrolysate as the fermentation substrate and Lactobacillus plantarum BHR4 (accession number CGMCC No: 26686) as the fermentation strain. The obtained fermentation product has good antioxidant activity, can effectively inhibit intestinal inflammatory response, maintain intestinal epithelial barrier function, change intestinal microbial beta diversity, increase the relative abundance of Firmicutes phylum in intestinal microorganisms, and reduce the relative abundance of 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, but the application of probiotics has limitations: first, the quality of the bacterial agent is difficult to guarantee, and its effect depends on sufficient viable bacterial count; 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, and in some special groups (such as premature infants and immunocompromised individuals), serious adverse reactions also need to be vigilant. 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 the probiotics and determine the effect molecules in the probiotics that play a probiotic role.

[0006] Studies have shown that many active substances present on the surface of probiotics, such as extracellular polysaccharides, surface proteins and peptidoglycans, play an important role in probiotic function. Therefore, finding effective surface active substances has potential application value for developing new probiotic products and alleviating ulcerative colitis. SUMMARY

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

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

[0009] To solve the above technical problems, the present application adopts the following technical solutions:

[0010] The new use of Lactobacillus delbrueckii surface protein DUF4430 is the 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.

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

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

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

[0014] (3) a sequence comprising a truncated sequence of the amino acid sequence set forth 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 Accession No: 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). The sequence is the sequence after the signal peptide of the amino acid sequence set forth 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). The sequence comprises a truncated sequence of the amino acid sequence set forth in SEQ ID NO: 2.

[0021] As a preferred embodiment of the present application, the improvement of symptoms of ulcerative colitis, prevention and / or treatment of ulcerative colitis comprises at least one of the following aspects:

[0022] (1) improving body weight loss;

[0023] (2) improving colon length shortening;

[0024] (3) improving colon mucosa injury;

[0025] (4) reducing the content of pro-inflammatory cytokines in the colon;

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

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

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

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

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

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

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

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

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

[0035] A medicine for improving the symptoms of ulcerative colitis, preventing and / or treating ulcerative colitis, wherein the pharmacodynamic component of the medicine comprises Lactobacillus debrueckii surface protein DUF4430.

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

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

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

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

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

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

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

[0043] As a preferred embodiment of the present application, the medicine further comprises other pharmacodynamic components, including but not limited to pharmacodynamic components for assisting in improving the symptoms of ulcerative colitis, preventing and / or treating ulcerative colitis.

[0044] As a preferred embodiment of the present application, the medicine 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, etc.

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

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

[0047] The beneficial effects of the present application are as follows:

[0048] The present application provides the use of the Lactobacillus delbrueckii surface protein DUF4430 in the preparation of a medicine for improving the symptoms of ulcerative colitis, preventing and / or treating ulcerative colitis. Animal experiments show that the Lactobacillus delbrueckii surface protein DUF4430 can relieve DSS-induced ulcerative colitis in mice, improve weight loss, shorten the length of the colon, and reduce the damage to the colon mucosa, 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, which can be used for preparing a medicine for improving the symptoms of ulcerative colitis, preventing and / or treating ulcerative colitis.

[0049] The present application provides a medicine for improving the symptoms of ulcerative colitis, preventing and / or treating ulcerative colitis, which comprises an effective amount of the Lactobacillus delbrueckii surface protein DUF4430 and a pharmaceutically acceptable carrier or excipient for forming a dosage form that is convenient to carry, store or use. BRIEF DESCRIPTION OF DRAWINGS

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

[0051] Figure 2 Figure 7. SDS-PAGE analysis of supernatant purification of DUF4430 protein in experimental example.

[0052] Figure 3 Figure 8. DUF4430 protein detection results (left) and Western Blot identification results (right) in experimental example.

[0053] Figure 4 Figure 9. Effect of DUF4430 treatment on body weight of DSS-induced colitis mice in experimental example.

[0054] Figure 5 Figure 10. Effect of DUF4430 treatment on colon length of DSS-induced colitis mice in experimental example.

[0055] Figure 6 Figure 11. Effect of DUF4430 treatment on colon morphology of DSS-induced colitis mice in experimental example.

[0056] Figure 7 Figure 12. Effect of DUF4430 treatment on IL-1β, IL-6 and IL-10 content in mouse colon in experimental example.

[0057] Figure 8 Figure 13. Effect of DUF4430 treatment on mouse spleen macrophage typing in experimental example.

[0058] Figure 9 Figure 14. Effect of DUF4430 treatment on mouse colon macrophage typing in experimental example.

[0059] Figure 10 Figure 15. Effect of different concentrations of DUF4430 treatment on RAW264.7 cell viability in experimental example.

[0060] Figure 11 Figure 16. Effect of DUF4430 treatment on LPS-induced macrophage pro-inflammatory cytokine expression in experimental example.

[0061] Figure 12 Figure 17. Effect of DUF4430 treatment on macrophage gene expression profile in experimental example.

[0062] Figure 13 Figure 18. Effect of DUF4430 treatment on Mincle receptor pathway gene and protein expression in experimental example.

[0063] Figure 14 Figure 19. Map of recombinant expression plasmid containing DUF4430 gene in experimental example.

[0064] In order to more clearly illustrate the technical solutions of the present application, the above drawings in the examples or experimental examples are briefly introduced. It should be understood that the above drawings should not be regarded as any limitation on the protection scope of the present application. For those skilled in the art, other related drawings can also be obtained from these drawings without creative labor. DETAILED DESCRIPTION

[0065] The technical solutions of the present application will be clearly and completely explained in combination with specific examples and experimental examples.

[0066] Those skilled in the art should understand that the examples and experimental examples described below are only used to illustrate the technical solutions and technical effects of the present application, and should not be regarded as any limitation on the protection scope of the present application. Based on the examples described below, other technical solutions obtained by those skilled in the art without creative labor, such as modifications, deformations or simple replacements, all belong to the protection scope of the present application.

[0067] The raw materials, reagents, equipment and the like used in the examples or experimental examples are all commercially available goods, unless otherwise specified.

[0068] Example 1

[0069] The present example provides a new use of Lactobacillus delbrueckii surface protein DUF4430, specifically: the use of Lactobacillus delbrueckii surface protein DUF4430 (the amino acid sequence is shown as 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) improving weight loss;

[0071] (2) improving shortening of colon length;

[0072] (3) improving colon mucosal injury.

[0073] Example 2

[0074] The present example provides a new use of Lactobacillus delbrueckii surface protein DUF4430, specifically: the use of Lactobacillus delbrueckii surface protein DUF4430 (the amino acid sequence is shown as 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) reducing the content of pro-inflammatory cytokines in the colon;

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

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

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

[0079] (8) increasing 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 Mincle-related signaling pathway-related genes in macrophages, or reducing the expression of Mincle, Malt1 and / or P50 in macrophages.

[0081] In other embodiments of the present application, the use of Lactobacillus debrueckii surface protein DUF4430 (amino acid sequence as shown in SEQ ID NO: 2) in the preparation of a drug for preventing and / or treating ulcerative colitis, substantially the same as embodiment 2.

[0082] Embodiment 3

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

[0084] Embodiment 4

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

[0086] In other embodiments of the present application, the drug for preventing and / or treating ulcerative colitis, comprising a pharmacologically effective amount of Lactobacillus debrueckii surface protein DUF4430 (amino acid sequence as shown in SEQ ID NO: 2), and a pharmaceutically acceptable carrier and adjuvant for preparing the drug into a tablet.

[0087] Experimental Example

[0088] I. Expression and purification of DUF4430 protein

[0089] 1. DUF4430 protein sequence

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

[0091] 2. Plasmid construction

[0092] The latest developed codon optimization software MaxCodon of DeTai Bio was adopted TM The nucleotide sequence of the above-mentioned DUF4430 protein was optimized by using the Optimization Program (V13), the optimized synthetic sequence of the DUF4430 was inserted into the expression vector pET28a through the restriction enzyme sites Ndel and Xhol by using whole gene synthesis, the accuracy of the final expression vector was confirmed by enzyme digestion and sequencing, and a recombinant expression plasmid containing the DUF4430 gene was obtained (the plasmid map is shown in Figure 14 ).

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

[0094] CAGAGCATTCAGGCAGCGAAAACCACCAAAACCGCGAAAAAGACCACCAAGAAGGCGAAAGCGAAACAGATTGGCG TTACCTATACCCTGAAGGACACCACCAAAGCGAAGAACAAGCAGACCCTGGCGAAGAAAACCTTCAAGGTCAAGAA GGGCACCAGCGTTTTTACCGTCCTGAAAAAGGCCTGGAAGGTCAACTACACCAAAAGCAGCAAGTACGGCGTCTTC ATCACCAAGATCAAAGGCCTGGGCGACGAAAAGAAGAAACTGTACTGGACCTACACCGTCGACGGTAAAATGAGCA AAGTTGCGGCGGATAAACAGAAGCTGACCAAGAACAAGAGCAAGGTCGTCTTCACCCTGAAGCAGTAC TAATGA CACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGAT (SEQ ID NO: 4).

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

[0096] ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATG (italic).

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

[0098] CAGAGCATTCAGGCAGCGAAAACCACCAAAACCGCGAAAAAGACCACCAAGAAGGCGAAAGCGAAACAGATTGGCGTTACCTATACCCTGAAGGACACCACCAAAGCGAAGAACAAGCAGACCCTGGCGAAGAAAACCTTCAAGGTCAAGAAGGGCACCAGCGTTTTTACCGTCCTGAAAAAGGCCTGGAAGGTCAACTACACCAAAAGCAGCAAGTACGGCGTCTTCATCACCAAGATCAAAGGCCTGGGCGACGAAAAGAAGAAACTGTACTGGACCTACACCGTCGACGGTAAAATGAGCAAAGTTGCGGCGGATAAACAGAAGCTGACCAAGAACAAGAGCAAGGTCGTCTTCACCCTGAAGCAGTAC (underlined).

[0099] It should be noted that the DUF4430 protein is added with His tag and contains part of the sequence on the vector when expressed.

[0100] 3. Transformation of expression vector and induction of expression

[0101] The recombinant expression plasmid containing the DUF4430 gene was transformed into BL21(DE3) competent cells and uniformly spread on LB plates (containing 50 μg / mL kanamycin sulfate) and then inverted in a 37°C incubator overnight. Single colonies were selected from the transformed plates and inoculated into 4 mL of LB medium (containing 50 μg / mL kanamycin sulfate) for expansion culture. When the culture reached OD600 of 0.8, 0.2 mM IPTG was added to the test tube culture solution, and the bacteria were collected after 16 h of induction at 15°C. If purification is not performed on the same day, the bacteria are frozen at -20°C.

[0102] SDS-PAGE identification: the induced culture solution was centrifuged at 12000 rpm for 5 min, the supernatant was removed, PBS buffer was added to resuspend the precipitate, and finally SDS-PAGE sample buffer was added to heat the sample at 100°C for 10 min, and the supernatant was electrophoresed. The whole bacteria were ultrasonically lysed with 20 mM Tris (pH 8.0), 300 mM NaCl, 20 mM Imidazole (containing 1% Triton X-100, v / v), 1 mM DTT, 1 mM PMSF, and the supernatant and precipitate were subjected to SDS-PAGE analysis, respectively. The analysis results are shown in Figure 1 .

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

[0104] 4. Purification of DUF4430 protein

[0105] Purification of DUF4430 protein in supernatant by affinity chromatography (the entire purification process was operated at low temperature): the whole bacteria were ultrasonically lysed with 50 mM Tris (pH 8.0), 300 mM NaCl, 20 mM Imidazole (containing 1% Triton X-100, v / v), 1 mM DTT, 1 mM PMSF, and the Ni-IDA affinity chromatography column was equilibrated with 50 mM Tris (pH 8.0), 300 mM NaCl, 20 mM Imidazole buffer. Then the target protein was eluted with different concentrations of imidazole equilibration buffer, 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 were collected, dialyzed into 1xPBS (pH 7.4), 5% glycerol, filtered with a 0.22 μm filter after dialysis, and aliquoted and frozen at -80°C. 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] II. DUF4430 relieves dextran sodium sulfate (DSS)-induced ulcerative colitis in mice

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

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

[0110] The degree of body weight change was evaluated by the percentage change of body weight of each mouse relative to its initial body weight during the experiment Figure 4 . At the end of the experiment, the body weight percentage of the CON group mice increased compared to the initial body weight, indicating that they had normal food intake and drinking water, good growth and development, and natural growth trend. The body weight percentage of the DSS group was significantly lower than that of the CON group (P<0.001), while each concentration of DUF4430 treatment could alleviate the weight loss induced by DSS to varying degrees, among which the body weight percentage of the L4430 + DSS group mice was significantly higher than that of the DSS group mice (P<0.05), indicating that low concentration of DUF4430 treatment was more effective.

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

[0112] From Figure 5As can be seen, the colon length of CON group mice was 9-10 cm, the feces in the intestinal canal was shaped, and the intestinal mucosa morphology was normal without hyperemia and swelling. Compared with the CON group mice, the colon length of DSS group mice was extremely significantly shortened (P < 0.0001), only about 7 cm. After treatment with low concentration of DUF4430, the colon length of mice was significantly increased compared with the DSS group (P < 0.001), while the colon length was increased after treatment with medium and high concentrations of DUF4430, but there was no significant difference compared with the DSS group.

[0113] 3. Histopathological examination of colon tissue

[0114] The severity of colonic inflammation was evaluated by observing the tissue with H&E staining method. Figure 6 Under light microscope, the histopathological changes of H&E staining were observed: the colon specimen of the control group showed the normal histological morphology of the mouse colon, the colonic tissue mucosal epithelium was complete and continuous, the glands were arranged in order, the crypt structure was normal, and there was little inflammatory cell infiltration. The colon section of the DSS group showed typical inflammatory changes, such as loss of crypts and surface epithelial cells, infiltration of inflammatory cells, and spread of inflammation to the mucosa, mucosal muscle layer and submucosa. Compared with the DSS group, the degree of colon injury in the L4430+DSS group was significantly reduced, although the crypt morphology was still changed and the mucosal epithelium was locally discontinuous, but the inflammatory cell infiltration was reduced and the mucosal structure was more complete.

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

[0116] The contents of IL-1β, IL-6 and IL-10 in the colon tissue of mice were detected using ELISA kit. Figure 7 The results showed that compared with the CON group, the contents of pro-inflammatory cytokines IL-1β and IL-6 in the colon of DSS group mice were significantly increased (P < 0.05), while the content of anti-inflammatory cytokine IL-10 was extremely significantly reduced (P < 0.001). Low concentration of DUF4430 treatment reduced the contents of IL-1β and IL-6, and increased the content of IL-10.

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

[0118] The mononuclear cells in the spleen were obtained using the spleen mononuclear cell separation kit, the white blood cells were labeled with CD45, the macrophages were labeled with F4 / 80 and CD11b, and the pro-inflammatory macrophages were labeled with CD16 / 32, and flow cytometry analysis was performed. Figure 8 The results showed that compared with the CON group, the proportion of pro-inflammatory macrophages in the spleen of DSS group mice was significantly increased (P < 0.05), and DUF4430 treatment significantly reduced the proportion of pro-inflammatory macrophages (P < 0.05).

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

[0120] Pro-inflammatory macrophages in the mouse colon were labeled with CD68, and anti-inflammatory macrophages were labeled with CD206, followed by immunofluorescence staining. Results showed ( Figure 9 Compared to the CON group, the DSS group mice showed an increase in pro-inflammatory macrophages and a decrease in anti-inflammatory macrophages in the colon. DUF4430 treatment reversed this phenomenon, reducing pro-inflammatory macrophages and increasing anti-inflammatory macrophages.

[0121] III. Effects of DUF4430 on cell viability

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

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

[0124] IV. Effects of DUF4430 on the pro-inflammatory phenotype of macrophages

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

[0126] from Figure 11 It can be seen that DUF4430 inhibited the pro-inflammatory factors of RAW264.7 cells in a dose-dependent manner, with the 0.25 μg / mL DUF4430 treatment showing the best inhibitory effect, significantly inhibiting the expression levels 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.Figure 12 RT-qPCR and protein identification confirmed that DUF4430 pretreatment significantly reduced the expression of Mincle, Malt1, and IL-1β compared to the LPS group ( Figure 13 ). These results indicate that DUF4430 inhibits LPS-induced macrophage proinflammatory phenotype by regulating Mincle receptor-associated signaling pathways.

[0127] Although the technical solutions and technical effects of the present application have been described in detail above with general descriptions, specific embodiments and experimental examples, modifications, replacements or improvements made by those skilled in the art without departing from the spirit and scope of the present application shall fall within the protection scope of the present application.

Claims

1. A novel application of Lactobacillus delbrueckii surface protein DUF4430, characterized by: The use of Lactobacillus delbrueckii surface protein DUF4430 in the preparation of drugs for improving symptoms of ulcerative colitis, preventing and / or treating ulcerative colitis; The amino acid sequence of the Lactobacillus delbrueckii surface protein DUF4430 is shown in SEQ ID NO: 1.

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