A method for enhancing the immunoregulatory ability of regulatory b cells and its use

Abstract

The application discloses application of mitochondrial acid 5 in preparation of a product for enhancing the immunoregulation ability of regulatory B cells, and provides a method for enhancing the immunoregulation ability of regulatory B cells; the regulatory B cells are pretreated by mitochondrial acid 5, the expression of interleukin-10 in the regulatory B cells is promoted, and the treatment effect of the regulatory B cells on autoimmune diseases, chronic inflammatory diseases and / or organ transplant rejection is enhanced, so that the application further requests to protect the application of mitochondrial acid 5 in preparation of a synergist for treating autoimmune diseases, chronic inflammatory diseases and / or organ transplant rejection. The application develops a safe, efficient and specific method for promoting differentiation of B lymphocytes into functional Bregs and secreting interleukin-10, and the method can effectively inhibit an excessively active immune response and avoid self-tissue damage.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, and in particular to a method and application for enhancing the immunomodulatory capacity of regulatory B cells. Background Technology

[0002] Regulatory B cells (Bregs) are a functionally defined subset of B lymphocytes that primarily regulate immune responses by secreting the inhibitory cytokine interleukin-10 (IL-10). Impairment in the number or function of Bregs is associated with various pathological conditions, including autoimmune diseases (such as multiple sclerosis, systemic lupus erythematosus, and rheumatoid arthritis), chronic inflammatory diseases (such as inflammatory bowel disease), and organ transplant rejection. Therefore, therapeutic strategies that selectively induce functional Bregs have significant clinical value.

[0003] Currently, some molecules have been reported to have the ability to induce Bregs, such as certain Toll-like receptor agonists and CD40 ligands. However, existing inducers generally have the following drawbacks: (1) they are not very specific and may activate other pro-inflammatory immune cells while inducing Bregs; (2) their induction efficiency is limited; and (3) their in vivo application may be accompanied by significant side effects or toxicity.

[0004] Mitochonic acid 5 (MA-5), or 4-(2,4-difluorophenyl)-2-(1H-indole-3-yl)-4-oxobutyric acid, is a compound derived from the plant growth hormone indole-3-acetic acid. MA-5 improves mitochondrial function by targeting the mitochondrial protein mitofilin, regulating energy metabolism and reducing mitochondrial oxidative stress. It also upregulates mitophagy to reduce mitochondrial apoptosis.

[0005] Therefore, exploring technologies to enhance the immunomodulatory capacity of Bregs is helpful for drug development for diseases that rely on the immunomodulatory capacity of Bregs. Developing a novel inducer that can safely, efficiently, and specifically promote the differentiation of B lymphocytes into functional Bregs and the secretion of IL-10 has important scientific significance and clinical application value. Summary of the Invention

[0006] The purpose of this invention is to overcome the above-mentioned shortcomings of the prior art and to provide a method and application for enhancing the immunomodulatory capacity of regulatory B cells.

[0007] The primary objective of this invention is to provide the use of mitochondrial acid 5 in the preparation of products for enhancing the immunomodulatory capacity of regulatory B cells.

[0008] A second objective of this invention is to provide a product that enhances the immunomodulatory capacity of regulatory B cells.

[0009] A third objective of this invention is to provide a method for enhancing the immunomodulatory capacity of regulatory B cells.

[0010] A fourth objective of this invention is to provide the use of mitochondrial acid 5 as an enhancer in the preparation of medicaments for the treatment of autoimmune diseases, chronic inflammatory diseases and / or organ transplant rejection.

[0011] A fifth objective of the present invention is to provide a composition for treating autoimmune diseases, chronic inflammatory diseases and / or organ transplant rejection.

[0012] To achieve the above objectives, the present invention is implemented through the following solution: This invention seeks protection for the use of mitochondrial acid 5 (MA-5) in the preparation of products for enhancing the immunomodulatory capacity of regulatory B cells (Bregs).

[0013] Preferably, the enhancement of the immunomodulatory capacity of regulatory B cells specifically refers to promoting the expression of interleukin-10 (IL-10) on regulatory B cells.

[0014] Regulatory B cells (Bregs) actively suppress excessive immune responses by secreting inhibitory cytokines (mainly IL-10), thereby maintaining immune tolerance and preventing autoimmune damage.

[0015] Therefore, the present invention also claims protection for a product that enhances the immunomodulatory capacity of regulatory B cells, said product using mitochondrial acid 5 as an active ingredient.

[0016] Preferably, the concentration of mitochondrial acid 5 in the product is 5–100 μM.

[0017] More preferably, the concentration of mitochondrial acid 5 in the product is 50 μM.

[0018] The present invention also claims protection for a method for enhancing the immunomodulatory capacity of regulatory B cells by mixing the product described above with regulatory B cells.

[0019] Preferably, when any of the above-described products is mixed with regulatory B cells, the concentration of the regulatory B cells after mixing is 1×10⁻⁶. 6 The concentration of mitochondrial acid 5 after mixing is 50 μM.

[0020] The present invention also claims protection for the use of mitochondrial acid 5 as an enhancer in the preparation of medicaments for the treatment of autoimmune diseases, chronic inflammatory diseases and / or organ transplant rejection.

[0021] Preferably, the autoimmune disease is multiple sclerosis, systemic lupus erythematosus, and / or rheumatoid arthritis; The chronic inflammatory disease mentioned is inflammatory bowel disease.

[0022] More preferably, the inflammatory bowel disease is colitis.

[0023] More preferably, the colitis is ulcerative colitis.

[0024] Preferably, the drug is a regulatory B cell.

[0025] This invention involves culturing regulatory B cells (Bregs) in a medium containing mitochondrial acid 5 (MA-5), or B lymphocytes in a medium containing LPS and mitochondrial acid 5 (MA-5). The results showed that MA-5 pretreatment significantly enhances the ability of regulatory B cells (Bregs) to secrete IL-10, thereby enhancing the therapeutic effect of regulatory B cells on autoimmune diseases, chronic inflammatory diseases, and / or organ transplant rejection. The concentration of mitochondrial acid 5 in the medium was 50 μM, the concentration of LPS was 3 μg / mL, and the concentration of regulatory B cells or B lymphocytes was 1 × 10⁻⁶. 6 LPS can induce B lymphocytes to transform into regulatory B cells, which then come into contact with mitochondrial acid 5 in the culture medium, thereby enhancing the immunomodulatory capacity of regulatory B cells.

[0026] The present invention also claims protection for a composition for treating autoimmune diseases, chronic inflammatory diseases and / or organ transplant rejection, said composition using mitochondrial acid 5 pretreated regulatory B cells as the active ingredient.

[0027] Preferably, the pretreatment specifically involves: thoroughly mixing mitochondrial acid 5 and regulatory B cells in a culture medium, wherein the concentration of mitochondrial acid 5 in the culture medium is 50 μM, and the concentration of regulatory B cells in the culture medium is 1 × 10⁻⁶. 6 per mL.

[0028] Compared with the prior art, the present invention has the following beneficial effects: This invention provides the use of mitochondrial acid 5 in the preparation of products for enhancing the immunomodulatory capacity of regulatory B cells, and provides a method for enhancing the immunomodulatory capacity of regulatory B cells. By pretreating regulatory B cells with mitochondrial acid 5, the expression of interleukin-10 in regulatory B cells can be effectively promoted, thereby enhancing the therapeutic effect of regulatory B cells against autoimmune diseases, chronic inflammatory diseases, and / or organ transplant rejection. Therefore, this invention also claims protection for the use of mitochondrial acid 5 as an adjuvant in the preparation of drugs for treating autoimmune diseases, chronic inflammatory diseases, and / or organ transplant rejection. This invention develops a method that can safely, efficiently, and specifically promote the differentiation of B lymphocytes into functional Bregs and the secretion of interleukin-10, effectively inhibiting overactive immune responses, avoiding damage to self-tissues, and providing an efficient treatment for autoimmune diseases, chronic inflammatory diseases, and / or organ transplant rejection. Attached Figure Description

[0029] Figure 1 Figure A shows the effect of mitochondrial acid 5 on IL-10 expression in regulatory B cells in Example 1; A represents the results of ELISA detection in the Activated B group and the Activated B group. MA-5 The image shows the IL-10 detection results for group B; B represents group Activated B and group Activated B. MA-5 Immunoblotting and quantitative expression levels of IL-10 protein in group B; C represents the expression levels of group Activated B and Activated B in flow cytometry. MA-5 The results of B cell detection for IL-10 secretion regulation in the group are shown in Figure D; D represents the Activated B group and Activated B group in the ELISA detection. MA-5 The graph shows the TNF-α detection results for group B; E represents the results of ELISA detection for group B and group B. MA-5 The results of IL-10 detection in the group; Figure 2 This is a graph showing the effect of mitochondrial acid 5 on the immunophenotype of regulatory B cells in Example 2. Figure 2 For flow cytometry detection of Activated B group and Activated B MA-5 Figure showing the detection results of B cell-related surface molecular markers regulating expression in the group; Figure 3 The figure shows the effect of mitochondrial acid 5 on the level of mitophagy in regulatory B cells in Example 3; A represents the Activated B group and the Activated B group. MA-5Immunoblotting and quantitative expression levels of LC3Ⅰ, LC3Ⅱ, and P62 proteins in group B; B represents group Activated B. MA-5 Group and Activated B MA-5 +Laser confocal plot and Pearson coefficient results for mitochondrial autophagy colocalization in group Mdivi-1; C represents the results of flow cytometry analysis of Activated B group and Activated B... MA-5 Group and Activated B MA-5 Figure 1; Detection results of B cells regulating IL-10 secretion in the +Mdivi-1 group; Figure 4 Figure A shows the therapeutic effect of mitochondrial acid 5 on ulcerative colitis in Example 4; Figure B shows the experimental process of DSS-induced colitis mouse models in each group; Figure C shows the weight change of mice in each group; Figure D shows the disease activity index curve of mice in each group; Figure D shows the comparative representative diagram of colon tissue of mice in each group and the colon length results of mice in each group; Figure E shows the HE staining observation of colon tissue of mice in each group; Figure F shows the histopathological score of colitis in mice in each group. Detailed Implementation

[0030] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0031] The mitochondrial acid 5 (MA-5) used in this embodiment of the invention has the CAS number 1354707-41-7 and the structural formula is as follows: The manufacturer is Selleck, and the product number is S0881-25mg.

[0032] Example 1: Effect of mitochondrial acid 5 on IL-10 expression in regulatory B cells I. Experimental Methods 1. Group settings Activated B MA-5 Group: Using MojoSort TM Mouse Pan B Cell Isolation Kit II was used to isolate B lymphocytes from the spleen of C57BL / 6 mice. The isolated B lymphocytes were then processed at a concentration of 3 × 10⁻⁶ cells / mL. 6 pcs / cm 2The cells were seeded in complete culture dishes containing LPS (3 μg / mL) and MA-5 (50 μM) and cultured in a saturated humidity incubator at 37℃ and 5% (v / v) CO2 for 48 h to obtain MA-5 pretreated regulatory B cells. The complete culture medium was RPMI 1640 medium containing 10% (v / v) fetal bovine serum, 1% (v / v) glutamine (100×) and 0.1% (v / v) 2-mercaptoethanol (1000×).

[0033] B MA-5 Group and Activated B MA-5 The difference between the groups is that the complete culture medium does not contain LPS, but the other culture methods are exactly the same, resulting in MA-5 pretreated B lymphocytes.

[0034] Activated B group and Activated B MA-5 The difference between the groups is that the complete culture medium does not contain MA-5, but the other culture methods are exactly the same, resulting in regulatory B cells.

[0035] Control group (Group B) and Activated Group B MA-5 The difference between the groups is that the complete culture medium does not contain LPS and MA-5, but the other culture methods are exactly the same, and B lymphocytes are obtained.

[0036] 2. ELISA testing Collect Activated B group, Activated B MA-5 Group B, Group B and B MA-5 The supernatant in the group will contain Activated B group and Activated B group. MA-5 Group B, Group B and B MA-5 The supernatant from the groups was analyzed for IL-10 using an IL-10 enzyme-linked immunosorbent assay kit (BD, 555252) according to the kit instructions; and the IL-10 levels in the Activated B group and Activated B group were also analyzed. MA-5 The supernatant in the group was used to detect TNF-α using the TNF-α enzyme-linked immunosorbent assay kit (BDI, 558534) according to the kit instructions.

[0037] 3. Immunoblotting assay Activated B MA-5 The MA-5 pretreated regulatory B cells obtained from each group were washed twice with PBS, and then... 6Add 150 μL of lysis buffer (1×RIPA lysis buffer + 100× protease inhibitor + 100× phosphatase inhibitor, v:v), incubate on ice for 5 min, then transfer to a 1.5 mL centrifuge tube, incubate on ice for 30 min, centrifuge at 12000 rpm for 15 min at 4 °C, and collect the supernatant as Activated B. MA-5 The group obtained MA-5 pretreated regulatory B cell proteins.

[0038] Next, activate B MA-5 The MA-5 pretreated regulatory B cell proteins obtained from the group were mixed with an equal volume of 5×SDS loading buffer and boiled in a water bath for 5 min. Then, an equal volume of total protein extract was separated using a 12% (w / v) polyacrylamide electrophoresis gel (ACE, ET15012LGel) and transferred to a PVDF membrane. The PVDF membrane was blocked with 5% (w / v) skim milk powder at 37°C for 2 h, followed by incubation at 4°C with 3 mL of primary antibody (IL-10 antibody, Santa Cruz, sc-365858) for 12 h overnight. After incubation, the membrane was washed three times with 1×TBST, and then incubated with 3 mL of secondary antibody (Anti-mouse lgG, CST, 7076S) at room temperature with shaking for 1 h. After washing three times with 1×TBST, the membrane was then contrasted with enhanced chemiluminescence imaging solution (Merck). The chemiluminescence immunoassay (MIL-10, WBKLS0500) was dropped onto the target band (the location on the PVDF membrane where the IL-10 antibody was incubated), and the image was captured in a chemiluminescence analyzer. The luminescent band was recorded, and the gray value of the protein band was quantitatively analyzed using ImageJ software. Three sets of results were measured in parallel.

[0039] The regulatory B cells obtained from the activated B group were subjected to Western blotting experiments according to the above method, and the results were recorded.

[0040] 4. Flow cytometry detection: Activated B... MA-5 After culturing the cells for 42 h, the culture medium was supplemented with phorbol ester (PMA, Sigma-Aldrich, P8139-1MG) at a final concentration of 50 ng / mL, ionomycin (Ionomycin, Sigma-Aldrich, I0634-1MG) at 1 μg / mL, and brevidin A (BFA, Sigma-Aldrich, B7651-5MG) at 10 μg / mL. After culturing for another 6 h, the cells were collected and centrifuged at 1800 rpm for 5 min. The cell pellet was collected, resuspended in 1×PBS solution, and then centrifuged at 1×10⁻⁶ ppm. 5Cells / samples were aliquoted into flow cytometry tubes and washed once with PBS containing 2% (v / v) FBS. Then, fixative (4% paraformaldehyde, w / v, eagene, DF0135) was added to each tube at 500 μL / tube. The tubes were incubated at 37°C in the dark for 30 min, centrifuged at 1800 rpm for 5 min, and the supernatant was discarded. Permeabilization buffer (Perm / Wash Buffer, BD, 554723) was added to each tube at 2 mL / tube. The tubes were incubated at 37°C in the dark for 10 min, centrifuged at 1800 rpm for 5 min, and the supernatant was discarded. 2 μL of PE-IL-10 (Biolegend, 505008) and 98 μL of permeabilization buffer were added to each tube. The mixture was vortexed, incubated at 37°C in the dark for 30 min, centrifuged at 1800 rpm for 5 min, and the supernatant was discarded. The cells were washed once with 1×PBS and resuspended in PBS. The cells were then analyzed using a flow cytometer.

[0041] The regulatory B cells obtained from the Activated B group were treated as described above, and the results were recorded.

[0042] II. Experimental Results The effect of mitochondrial acid 5 on IL-10 expression in regulatory B cells is shown in the figure below. Figure 1 As shown, Figure 1 In the diagram, A represents the Activated B group and Activated B in the ELISA test. MA-5 The results of IL-10 detection in the group; Figure 1 In the text, B represents the Activated B group and Activated B group in the immunoblotting experiment. MA-5 The luminescent band pattern and grayscale results of IL-10 in the group; Figure 1 In the diagram, C represents the Activated B group and Activated B in the flow cytometry detection. MA-5 Figure showing the results of IL-10-positive regulatory B cells in the group; Figure 1 In the diagram, D represents the Activated B group and Activated B in the ELISA test. MA-5 The detection results of TNF-α in the group are shown in the figure; Figure 1 In the figure, E represents group B and B in the ELISA test. MA-5 The image shows the results of IL-10 testing in the group.

[0043] The results showed that after LPS treatment, B lymphocytes differentiated into regulatory B cells (Bregs), and MA-5 treatment effectively promoted the secretion of the anti-inflammatory cytokine IL-10 by regulatory B cells without increasing the secretion of the pro-inflammatory factor TNF-α; while mitochondrial acid 5 had no effect on the secretion of IL-10 in resting B lymphocytes (differentiated into Bregs without LPS treatment).

[0044] The results indicate that mitochondrial acid 5 can effectively promote the expression of interleukin-10 in regulatory B cells (Bregs), thereby enhancing the immunomodulatory capacity of regulatory B cells.

[0045] Example 2: Effects of mitochondrial acid 5 on immunophenotype in regulatory B cells I. Experimental Methods As shown in Example 1, control group (Group B), Activated Group B, and Activated Group B were set up respectively. MA-5 Group.

[0046] Immunophenotyping: Activated B MA-5 After culturing the cells for 42 h, the culture medium was supplemented with phorbol ester (PMA, Sigma-Aldrich, P8139-1MG) at a final concentration of 50 ng / mL, ionomycin (Ionomycin, Sigma-Aldrich, I0634-1MG) at 1 μg / mL, and brevidin A (BFA, Sigma-Aldrich, B7651-5MG) at 10 μg / mL. After culturing for another 6 h, the cells were collected and centrifuged at 1800 rpm for 5 min. The cell pellet was collected, resuspended in 1×PBS solution, and then centrifuged at 1×10⁻⁶ ppm. 5Cells / samples were aliquoted into different flow cytometry tubes, and APC-CD1d (Biolegend, 123521), PEcy7-CD5 (Biolegend, 100622), APC-CD21 (Biolegend, 123411), BV421-CD23 (Biolegend, 101621), FITC-CD24 (Biolegend, 101805), and PEcy7-CD43 (Biolegend, 143209) were added to the different flow cytometry tubes respectively (each antibody was added at a concentration of 2...). Add 100 μL of antibody-containing PBS solution to 98 μL of the antibody-containing PBS solution. Vortex to mix, then incubate at 37°C in the dark for 30 min. After incubation, centrifuge at 1800 rpm for 5 min, wash once with PBS solution containing 2% (v / v) FBS, then add 500 μL of fixative (4% paraformaldehyde, w / v, eagene, DF0135) per tube. Incubate at room temperature in the dark for 30 min, centrifuge at 1800 rpm for 5 min, discard the supernatant, and add 2 mL of permeabilization buffer (Perm / Wash) per tube. Buffer (BD, 554723), incubate at room temperature in the dark for 10 min, centrifuge at 1800 rpm for 5 min, discard the supernatant, add 2 μL of PE-IL-10 and 98 μL of membrane rupture buffer to each tube, vortex to mix, incubate at room temperature in the dark for 30 min, centrifuge at 1800 rpm for 5 min, discard the supernatant, wash once with 1×PBS solution, resuspend in PBS, and then use a flow cytometer for detection.

[0047] The activated B group and the control group (B group) were tested according to the above treatment method, and the flow cytometry results were obtained.

[0048] II. Experimental Results The effect of mitochondrial acid 5 on immunophenotype in regulatory B cells is shown in the figure below. Figure 2 As shown, Figure 2 For flow cytometry detection of Activated B group and Activated B MA-5 The detection results of Bregs-related surface molecular markers in the group are shown in the figure.

[0049] The results showed that the expression levels of relevant surface molecular markers of regulatory B cells, CD1d, CD5, CD21, CD23, CD24 and CD43, did not change significantly after mitochondrial acid 5 pretreatment.

[0050] Note: Treatment of regulatory B cells with mitochondrial acid 5 does not affect the phenotypic characteristics of regulatory B cells, indicating that mitochondrial acid 5 can enhance the immunomodulatory function of regulatory B cells without altering their phenotypic characteristics.

[0051] Example 3: Effect of mitochondrial acid 5 on the level of mitophagy in regulatory B cells I. Experimental Methods As shown in Example 1, Activated B group and Activated B are set up respectively. MA-5 Group.

[0052] Activated B MA-5 +Mdivi-1 group same as Activated B MA-5 The difference between the groups is that the complete culture medium also contains 5 μM of Mdivi-1 (Selleck, S7162, CAS: 338964-87-6), while the rest of the culture methods are exactly the same, resulting in Mdivi-1-treated regulatory B cells.

[0053] Immunoblot assay: Activated B MA-5 The MA-5 pretreated regulatory B cells obtained from the group were washed twice with PBS, then lysed with lysis buffer (RIPA lysis buffer + protease inhibitor + phosphatase inhibitor) for 5 min on ice. The cells were then transferred to 1.5 mL centrifuge tubes, incubated on ice for 30 min, and centrifuged at 12000 rpm for 15 min at 4°C. The supernatant was collected as activated B cells. MA-5 The group obtained MA-5 pretreated regulatory B cell proteins.

[0054] Next, Activated B MA-5The MA-5 pretreated regulatory B cell proteins obtained from the group were mixed in equal volumes with 5×SDS loading buffer and boiled in a water bath for 5 min. Then, equal volumes of total protein extract were separated using a 12% (w / v) polyacrylamide electrophoresis gel (ACE, ET15012LGel) and transferred to a PVDF membrane. The PVDF membrane was blocked with 5% (w / v) skim milk powder at 37°C for 2 h. Then, 3 mL of primary antibody [including LC3 antibody (Proteintech, 14600) and P62 antibody (Proteintech, 18420), independently incubated at different locations on the PVDF membrane, with the antibodies diluted 1:1000 using primary antibody buffer (Beyotime, P0256)] was incubated at 4°C for 12 h. After incubation, the membrane was washed three times with 1×TBST and incubated with secondary antibody (Anti-rabbit) at room temperature with shaking. After washing three times with 1×TBST for 1 hour, enhanced chemiluminescence developing solution (Merck Millipore, WBKLS0500) was added to the target band (the position on the PVDF membrane where P62 antibody and LC3 antibody were incubated), and the image was formed in a chemiluminescence analyzer. The luminescent band was recorded and the gray value of the protein band was quantitatively analyzed using ImageJ software. Three sets of results were measured in parallel.

[0055] The regulatory B cells obtained from the Activated B group were treated as described above, and the results were recorded.

[0056] Immunoconfocal assay: Activated B group, Activated B... MA-5 Group and Activated B MA-5After culturing the +Mdivi-1 group cells for 24 h, the cells from each group were collected and centrifuged at 1800 rpm for 5 min. The cell pellets were collected and resuspended in 300 μL of complete culture medium. After reconstitution, the pellets were added to confocal microplates coated with Cell Tak solution (Corning, 354240) [285 μL of NaHCO3 (0.1 M), 5 μL of NaOH (1 N), and 10 μL of Cell Tak mixed and treated for 30 min]. The plates were incubated for 1 h to allow the cells to settle. The culture medium in the confocal microplates was then discarded, and the cells were washed three times with 1×PBS. The cells were then stained with 300 μL of MitoTracker Red (Invitrogen, M7512, 400 nM) in the dark for 45 min. Finally, 1×PBS was added. Wash 3 times with PBS, fix with 4% (w / v) PFA in the dark for 20 min, wash 3 times with 1×PBS, add 500uL of 0.1% (v / v) Triton-X and incubate for 10 min, add 300uL of 3% (w / v) BSA and block for 1 h, then add 200uL of LC3 primary antibody (1:200) and incubate at 4℃ for 12 h. After incubation, wash 3 times with 1×PBS, incubate with 200uL of secondary antibody (1:400, invitroge, A21206) in the dark for 1 h, discard the secondary antibody, wash 2 times with 1×PBS, then add 200uL of DAPI (1:1000) and counterstain for 5 min in the dark, add 2mL of sterile PBS, and capture confocal images to detect the results.

[0057] Flow cytometry detection: Activated B group, Activated B MA-5 Group and Activated B MA-5 After culturing Mdivi-1 cells for 42 h, phorbol ester (PMA, Sigma-Aldrich, P8139-1MG), ionomycin (Ionomycin, Sigma-Aldrich, I0634-1MG), and brevidin A (BFA, Sigma-Aldrich, B7651-5MG) were added to each culture medium at final concentrations of 50 ng / mL, 1 μg / mL, and 10 μg / mL, respectively. After further culturing for 6 h, cells were collected and centrifuged at 1800 rpm for 5 min. The cell pellet was collected, resuspended in 1×PBS, and then centrifuged at 1×10⁻⁶ ppm. 5Cells / samples were aliquoted into flow cytometry tubes and washed once with PBS containing 2% (v / v) FBS. Then, fixative (4% paraformaldehyde, w / v, eagene, DF0135) was added to each tube at 500 μL. The tubes were incubated at room temperature in the dark for 30 min, centrifuged at 1800 rpm for 5 min, and the supernatant was discarded. Permeabilization buffer (Perm / Wash Buffer, BD, 554723) was added to each tube at 2 mL. The tubes were incubated at room temperature in the dark for 10 min, centrifuged at 1800 rpm for 5 min, and then 2 μL of PE-IL-10 (Biolegend, 505008) and 98 μL of permeabilization buffer were added to each tube. The mixture was vortexed and incubated at room temperature in the dark for 30 min, centrifuged at 1800 rpm for 5 min, and the supernatant was discarded. The cells were washed once with 1×PBS and resuspended in PBS. The cells were then analyzed using a flow cytometer.

[0058] II. Experimental Results The effect of mitochondrial acid 5 on the level of mitochondrial autophagy in regulatory B cells is shown in the figure below. Figure 3 As shown; Figure 3 In the diagram, A represents Activated B group and Activated B... MA-5 Immunoblotting experiments and quantitative analysis of LC3-I, LC3-II and P62 protein expression levels in the group; Figure 3 In this context, B stands for Activated B group, Activated B MA-5 Group and Activated B MA-5 Laser confocal plot and Pearson coefficient results for colocalization of mitochondrial autophagy in the +Mdivi-1 group; Figure 3 In the text, C represents the Activated B group and Activated B in the flow cytometry detection. MA-5 Group and Activated B MA-5 The results of B cell detection for IL-10 secretion regulation in the +Mdivi-1 group are shown in the figure.

[0059] The results showed that autophagic flux in regulatory B cells was activated after treatment with mitochondrial acid 5, and the colocalization of mitochondria and LC3 in regulatory B cells was significantly increased after treatment with mitochondrial acid 5, indicating that mitochondrial acid 5 can effectively enhance mitophagy in regulatory B cells. Furthermore, Mdivi-1 can significantly inhibit the enhancing effect of mitochondrial acid 5 on mitophagy in regulatory B cells. At the same time, the inhibition of mitophagy reduces the ability of mitochondrial acid 5 to promote the secretion of IL-10 by regulatory B cells.

[0060] Note: Mitochondrial acid 5 promotes the expression of interleukin-10 in regulatory B cells by upregulating mitophagy.

[0061] Example 4: The therapeutic effect of mitochondrial acid 5 on ulcerative colitis I. Experimental Methods Activated B was constructed according to the method shown in Example 1. MA-5 The cells were divided into group A and group B, and cells from both groups were collected. The cells were centrifuged at 1800 rpm for 5 min, and the cell pellet was collected and resuspended in PBS to obtain MA-5 pretreated regulatory B cells and regulatory B cells that could be used for intraperitoneal injection.

[0062] Mouse model establishment: Control group mice: C57BL / 6 mice, which drank water that did not contain sodium dextran sulfate (DSS) daily.

[0063] Model mice (IBD): C57BL / 6 mice were given water containing 3% (w / v) DSS daily to induce an inflammatory response in the colon, mimicking the pathological features of ulcerative colitis.

[0064] Activated B group mice: C57BL / 6 mice were given water containing 3% (w / v) DSS daily, and on the second day after drinking, they were injected intraperitoneally with 1×10 6 One regulatory B cell was given to the mice. They continued to drink water containing 3% (w / v) DSS. On day 7, the water containing 3% (w / v) DSS was replaced with water without DSS.

[0065] Activated B MA-5 Group mice: C57BL / 6 mice were given water containing 3% (w / v) DSS daily, and on the second day after drinking, they were injected intraperitoneally with 1×10 6 The mice were given MA-5 pretreated regulatory B cells and continued to drink water containing 3% (w / v) DSS. On day 7, the water containing 3% (w / v) DSS was replaced with DSS-free water.

[0066] After treating the mice according to the above methods, the mice in each group were weighed daily, and their condition, bloody stools, etc. were observed. The Disease Activity Index (DAI) of the mice was calculated. On the 8th day, the mice were sacrificed, and the colon length of each group was measured. At the same time, the colon tissue of each group of mice was taken, fixed with 4% paraformaldehyde (w / v, eagene, DF0135), and observed using hematoxylin-eosin staining (HE staining). The histopathological scores of colitis in each group of mice were recorded.

[0067] II. Experimental Results The therapeutic effect of mitochondrial acid 5 on ulcerative colitis is shown in the figure below. Figure 4 As shown, Figure 4A in the diagram represents the experimental process of DSS-induced colitis mouse models in each group; Figure 4 B in the graph represents the weight changes of mice in each group; Figure 4 C in the graph represents the disease activity index curve of each group of mice; Figure 4 In the diagram, D represents a comparative image of colon tissue from each group of mice and an image showing the colon length of each group of mice. Figure 4 E in the figure represents HE staining of colon tissue from mice in each group; Figure 4 F in the figure represents the histopathological score of colitis in each group of mice.

[0068] The results showed that compared with the control group, the IBD group mice (inducing colonic inflammation to simulate ulcerative colitis) had significantly lower body weight, significantly higher disease activity index (DAI), and significantly shorter colon length, with obvious pathological damage to the colonic tissue; after intraperitoneal injection of regulatory B cells (Activated B group), the mice showed varying degrees of relief of disease symptoms; compared with intraperitoneal injection of regulatory B cells pretreated with MA-5, the intraperitoneal injection of MA-5 pretreated regulatory B cells (Activated B group) showed greater improvement. MA-5 In the control group, the mice's body weight, disease activity index, colon length, and colon tissue were significantly closer to those of the control group.

[0069] The results indicate that regulatory B cells can effectively treat ulcerative colitis, and MA-5 pretreated regulatory B cells have stronger immunomodulatory capabilities. MA-5 pretreatment can significantly enhance the therapeutic effect of regulatory B cells on ulcerative colitis, and MA-5 can serve as a potentiator for drugs used to treat ulcerative colitis.

[0070] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description and ideas, and it is neither necessary nor possible to exhaustively describe all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. The application of mitochondrial acid 5 in the preparation of potentiators for drugs used to treat ulcerative colitis; The drug is for regulatory B cells.

2. A composition for treating ulcerative colitis, characterized in that, The composition uses mitochondrial acid 5-pretreated regulatory B cells as the active ingredient. The pretreatment specifically involves thoroughly mixing mitochondrial acid 5 and regulatory B cells in a culture medium, wherein the concentration of mitochondrial acid 5 in the culture medium is 50 μM, and the concentration of regulatory B cells in the culture medium is 1 × 10⁻⁶. 6 per mL.

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