Application of brevibacitracin in preparation of medicine for treating systemic lupus erythematosus

Abstract

The invention provides application of brevibacitracin in preparation of a medicine for treating systemic lupus erythematosus. The treatment is realized by up-regulating expression of MTCH2. According to the application disclosed by the invention, the intervention value of the MTCH2 in the systemic lupus erythematosus is verified through experiments, and the fact that the brevibacterium peptide can inhibit the response of pathogenic B cells, recover the mitochondrial function and relieve the course of the systemic lupus erythematosus through pharmacologically up-regulating the MTCH2 is clear; therefore, the technical blank that in the prior art, functional research of the MTCH2 in autoimmune diseases, especially systemic lupus erythematosus is insufficient, and the application of the MTCH2-based target medicine is lacked is filled.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to a drug for treating systemic lupus erythematosus, specifically the use of bacitracin in the preparation of a drug for treating systemic lupus erythematosus. Background Technology

[0002] Systemic lupus erythematosus (SLE) is an autoimmune disease with a complex pathogenesis, characterized by the production of large amounts of autoantibodies and persistent chronic inflammation, which can lead to damage to multiple tissues and organs throughout the body. SLE is characterized by the formation of an immune response against nuclear self-antigens (such as nucleic acids and histones). Furthermore, antibodies can target mitochondria in autoimmune diseases, indicating an interaction between the adaptive immune system and mitochondria. Current research has reported multiple pathways by which mitochondrial dysfunction and its secondary oxidative stress contribute to the pathogenesis of SLE. Mitochondrial structural damage and functional alterations can lead to various pathological states that contribute to the development and progression of SLE, including changes in mitochondrial dynamics, abnormal mitochondrial biogenesis and energy metabolism, oxidative stress dysregulation, mitochondrial DNA (mtDNA) damage, and inflammatory responses. In addition, mitochondria participate in cell death pathways, including apoptosis, autophagy, necrosis, ferroptosis, and pyroptosis, which is another mechanism leading to SLE.

[0003] Mitochondrial carrier homolog 2 (MTCH2) is a key catalytic enzyme essential for controlling the insertion of various protein molecules, including biophysically diverse tail anchor proteins, signal anchoring proteins, and multichannel proteins, into the outer mitochondrial membrane.

[0004] The function of MTCH2 in autoimmune diseases, particularly systemic lupus erythematosus (SLE), has not been reported, and specific MTCH2 agonists have not been identified. How MTCH2 influences the development of SLE remains a crucial scientific question, and research in this field is currently lacking. Summary of the Invention

[0005] In view of the above-mentioned technical problems in the prior art, the present invention provides the use of bacitracin in the preparation of drugs for treating systemic lupus erythematosus, which solves the technical problem of poor efficacy of drugs for treating systemic lupus erythematosus in the prior art.

[0006] This invention provides the use of bacitracin in the preparation of medicaments for the treatment of systemic lupus erythematosus.

[0007] Furthermore, the treatment is achieved by upregulating the expression of MTCH2.

[0008] This invention utilizes a high-throughput screening platform based on a stable integration of the human MTCH2 promoter-EGFP reporter system in HEK293T cells. This platform directly reflects changes in MTCH2 transcriptional activity through fluorescence signal changes, enabling rapid, intuitive, and quantifiable screening of MTCH2 agonists. The invention screened a small molecule candidate, gramicidin, from an approved drug library that can stably activate MTCH2 transcription.

[0009] This invention experimentally verifies the intervention value of MTCH2 in systemic lupus erythematosus (SLE), clarifying that pharmacological upregulation of MTCH2 can inhibit pathogenic B cell responses, restore mitochondrial function, and alleviate the course of SLE. This fills the gap in existing technologies regarding insufficient research on the function of MTCH2 in autoimmune diseases, especially SLE, and the lack of technology for drug applications based on the MTCH2 target.

[0010] Compared with existing technologies, the technical effects of this invention are positive and significant. This invention discovers that gramicidin can significantly upregulate MTCH2 expression in primary B cells derived from SLE patients, improve mitochondrial membrane potential and mitochondrial functional status, and inhibit abnormal B cell activation and oxidative stress, indicating that it not only has molecular targeting effects but also plays a regulatory role at the level of cellular metabolism and immune function. Compared with existing treatment strategies that only target inflammatory responses or broad immunosuppression, this invention provides a novel technical approach based on mitochondrial metabolic checkpoint regulation, with the advantages of a clear mechanism of action and stronger targeting. Simultaneously, this invention provides new technical means and candidate drug sources for drug development targeting the mitochondrial intercalation enzyme MTCH2, thus possessing good social benefits and potential clinical application value. Attached Figure Description

[0011] Figure 1 A schematic diagram illustrating the process of constructing hMTCH2-EGFP reporter 293T cells is shown.

[0012] Figure 2 This is a plasmid map of pCDH-MTCH2-Pro 3kb-EGFP-puro.

[0013] Figure 3 The scatter plot shows the calculated average fluorescence intensity results after initial screening based on the HTS system.

[0014] Figure 4 The diagram shows the design of drug names, locations, and concentration gradients based on the HTS system for secondary screening.

[0015] Figure 5Shown is the qPCR verification of gramicidin specifically upregulating the expression of Mtch2 in wild-type HEK293T cells.

[0016] Figure 6 Shown is the qPCR verification of gramicidin specifically upregulating the expression of Mtch2 in B cells of lupus patients.

[0017] Figure 7 Shown is the verification that gramicidin reduces the proportion of activated B cells in lupus patients.

[0018] Figure 8 Shown is the verification that gramicidin reduces the mitochondrial function of B cells in lupus patients. Detailed implementation manners

[0019] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments. It should be understood that the following implementation manners are only used to illustrate the present invention and are not used to limit the protection scope of the present invention. Without departing from the spirit and essence of the present invention, equivalent substitutions or simple transformations made by those skilled in the art shall fall within the protection scope of the present invention.

[0020] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments. It should be understood that the following implementation manners are only used to illustrate the present invention and are not used to limit the protection scope of the present invention. Without departing from the spirit and essence of the present invention, various equivalent substitutions or deformations can be made to the present invention by those skilled in the art, and all shall fall within the protection scope of the present invention.

[0021] Through a high-throughput screening method based on a human MTCH2 promoter reporter system, the present invention discovers a small molecule agonist that can upregulate the expression of MTCH2. Through in vitro experiments on primary B cells derived from patients with systemic lupus erythematosus, the present invention proves that gramicidin can selectively upregulate the expression of MTCH2, restore the mitochondrial function of B cells, inhibit abnormal activation, and alleviate symptoms related to systemic lupus erythematosus.

[0022] The Chinese name of Gramicidin is gramicidin.

[0023] SLE is Systemic Lupus Erythematosus, and its common Chinese name is systemic lupus erythematosus.

[0024] MTCH2 is Mitochondrial Carrier Homolog 2, and its common Chinese name is mitochondrial carrier homolog 2.

[0025] The EGFP stands for Enhanced Green Fluorescent Protein.

[0026] The HEK293T is a commonly used adherent cell line derived from human embryonic kidney.

[0027] The FDA stands for Food and Drug Administration, commonly known as the U.S. Food and Drug Administration.

[0028] The TMRM is Tetramethylrhodamine Methyl Ester, commonly known as tetramethylrhodamine methyl ester.

[0029] The MitoTracker is a mitochondrial fluorescent probe, commonly known in Chinese as a mitochondrial tracer fluorescent probe.

[0030] The IncuCyte is a live-cell real-time imaging analysis system used to continuously acquire cell fluorescence, cell fusion degree, and cell growth status.

[0031] In this invention, the term "agonist" mainly refers to small molecule compounds that can enhance MTCH2 promoter activity, increase MTCH2 transcription levels, or promote MTCH2-related biological functions.

[0032] Example 1: Construction of hMTCH2-EGFP reporter cell line like Figure 1 As shown, an expression vector containing the human MTCH2 promoter sequence and the EGFP reporter gene was first constructed (e.g., Figure 2 (As shown in the diagram). In the vector, the human MTCH2 promoter (SEQ ID NO.1) is located upstream of the EGFP coding sequence, and the two are linked according to their transcriptional regulatory relationship, so that changes in the activity of the MTCH2 promoter can be expressed through changes in EGFP fluorescence intensity. The promoter sequence adopts a 3kb regulatory fragment upstream of the transcription start site of the human MTCH2 gene, containing the core promoter region and necessary proximal regulatory elements. The vector also includes a puromycin resistance gene, a selection marker gene, for subsequent screening of stable strains.

[0033] The following is the sequence of the human MTCH2 promoter: From: 47642558 to: 47645559 >NC_000011.10:c47644559-47641558 Homo sapiens chromosome 11,GRCh38.p14 Primary Assembly >NC_000011.10:c47645559-47642558 Homo sapiens chromosome 11,GRCh38.p14 Primary Assembly AGACGGGGTTTCACTATGTTGCCCAGGCTGGTCTCAAACTCCCGAGCTCAAAGCAATCCACCCAAGTGCT GGGATTACAGGCATGTGCCCTGCTCCCAAGAGGCTTTTAACTGGTAAATCCATTCAGCCTTCCCTGGGCT TCATTTTTTTCTGAGATGGAGTTTCACTCTTGTTGCCCAGGCTGGAGTGCAATGGCATGATCTCGGCTCA CTGCAACCTCCGCCTCCCGGGTTCAAGCGATTCTCCTGCCTCAGCCTCCCGAGTACCTGGGATTATAGGC ATGTGTCACCACGCCCGGCTAATTTTGTTTTTTAGTAGAGATGGGTTATCTCCATGTTGGTCAGGCTGGT CTTGAACTCCTGACCTCAGGTGATCCGCCCACCTCAGCCTCCCAAAGTGCTGGGATTACAGGCGTGAGCC ACCGCGCCCGGCTTCTGGGCTTCATTATTGACCTCTTGGAAGCTTTTGACATATTGACTATCCTTTCTTT TTCCAAACTTTTCTGTTGACTTCAGTGACATGCTGTTCTAGTTCTGTTTCCTTCCCAGTTAATTCTGCCC CTTTTGACTTCCGTTATTCCTCTCACCACTGCAATATGGGTCAGCAAATATAACCCATCTTAGGGTCATC ATACTGAACTGTATCAGGGAATGCCATTCAGATCATAGTCTTTGTGACTGGCCCCACTCTGAGTTATGCA GTGCACAGCCTAGGCAGCTGTACATAGCACCTCTTCCCTTATCTATACTGCTGACTTCGGTTACCTATTC TCTGCAGATTCTTCCCAAAACCTCATCAGCAGCTTCTGCCTCACACCATTGGAAAATATTGTGGAGCAGA CTGAATCCCCCAACCACCCACCCCTGGCAAGAGGCTCTGTGCCTCCCCATGCTCACTAGCATCAGTGGGA CAAGGCAATATTATTTGGCACCATCAATGCACAAATCCAAGAATAAATAATCCAACCAGGTGTGGTGACT CACACCTGTAATCCCAGCACTTTGGGAGGCAGGTGGATCACTTGAGTTCAAGAGTTTGAGACCAGCCTGG GGCAACATGGCGAGACCCCATCTCTACTAAAAATACAAAAAAAATAGCTGGGCATGGTGGTGCACACCTG TGGTCCCAGCTAATTGGGAGACTAAGGTAGGAGGATCGCTTGAGCCCAGGAGGTGGAGGTTGCAGTGAGC CGAGATTGCACCACTGCACTCCAGCCTGGGTGACAAAGAGAGACCCTGTCTCAAAAAAAAAAAGAAAAGA ATCCCCTGCTAAATAGTCTCTCTGGCTCAGCTTTATGTTTCTAGATTCAGTTAGAAGCAACTTGTTTCTT ACAACTTTATTTTTTATTTATTTATTCTTTTGAGACTGAGTCTCGCCCTGTCACCCAGGCTGGAGTGCAA TGGCGCAATCTTGGCTCACTGCAACCTCCAACTCCCGAGTTCAAGCGATTCTCCTGCCTCAGCCTCCCGA GTAGCTGGGATTACAGGCACCCGCCATCACACCCAACTAATTTTTGTACTTTTAGTACAGATGGGGTTTC ACCATGTCGGTCAGGCTGGTCTTGAACTCCCGACCTCAGGTGATCCACCCACCTCAGCCTTCTAAAGTGT TGGGATTACAGGTGTGAGCCACCGTGCCCAGCCAATATCTGTATTTCAATATAAAGTTTTTTAAAAAAAA CCCTTTCCTATGCATAATTTCACTTAATGCTCATAATAACCCTGAGAGATACACAGTAGTGTTAGATTTA GATACAAAAACTGGAAATTAAAGTAGCTAAAATGACAAATGGATGCAGTGGAAAAGCCAGGATCCAAATC CAAGTCTTTCCCCTACAGTACTTCACTCTGCTCCAAGCTCAGATTCACCAAATGTTGTTTGATAGAACAC GTGAGCTGGGGCTGAAGGAGCAGGGACAATGCCTTTTTCACTTTTGCCTCTGATAGCCTGTAGACTGGTA GGCACTGAAGTATTTGTTCACTTGTGCAATAAATATTTATTGAGCACCTCGTATGTGCTGGGCACTGAAA ACACAACCGTGAACAGAGGCTGTCACGGTTCCTGAACTCATGGAGCTTTCATTCCAGTGGAGGAGACATA ATTTCAGTGATAATGCTACGACGGGGCACAATGTGGGAGAAAATAAGAGGGAGGTGGCCAGGCGCGATGG CTCCTCACTCCTGTAATCCCAGCACTTTGGGAGGCCAAGGCGGGTGAATCACGAGGTCAGGAGTTTGAGA CCAGCCCGGCCAACATCGTGAAACCCCGTCTCTACTAAATAGACAAAAATTAGCCGGGCGTGGTGGCGTG TGCGTGTAATCCCAGCTACTAGGGAGGCTGAGGCAGGAGCATTGCTTGAACCTGGGAGGCGAAGGTTGCA GTGAGCTGAGATCGGGCCACTGCACTCCAGCCTGGGTGACAGAGCGAGACTCCGTCTCAAAAAAAAAAAA AAAAAGAAAGAAAAAGAAAAGGGGGAGGTAATTTTAGAATGGTCAGAGAAGGTTTTTTTGAAAAGATAAC ATTAATCAAACGAATAAATGAATGAAAAGCCACTCGCTAGCGGATTCATTATTTGCTATACTTATTTAGA GTTTGTCAGTCACGAGACTGTTACGGCTCTGATGCTGAGTATCTTTTGAATCCTATGTTGATGATGCCCA AAAGGCAAGTGCAGAGATGAAGCATAATGAAGAATCCTTTTCAATTTCTTTTCAGTCCTCTGACCTTGCC TCTGCCGTCACGGTCACACTCCCCACTGACACTCCCATTCTTCTGTTTCCGGACACTTGCGGCCTGGAA GCGCATGTGCAAGAGAAACCGTCATTCCTGGTGAAGGGCGAGCCCCCGGCAGAACGCATGCGCCCTGTC CCTTAAGCCCCGCCCGACCTCAGCGCCCCCTCGCGAGCGCCTGCCGTTTCTCGGGGCGGGACGGGGGGG CGGGGACTGGGCGGAGAGGCGCGTGCTGCTGCGTGCGTGCGCGCGCCGCGGGCGGGCCAG.

[0034] The constructed hMTCH2-EGFP reporter vector was introduced into HEK293T cells. In the reporter vector, the MTCH2 promoter is located upstream of the reporter gene and is used to drive the expression of the reporter gene; the reporter gene is enhanced green fluorescent protein (EGFP).

[0035] The purpose of this construction method is to enhance reporter gene expression when the test compound promotes MTCH2 promoter transcriptional activity, thereby indirectly reflecting the level of endogenous MTCH2 transcriptional upregulation through changes in the fluorescence intensity of green fluorescent protein (EGFP). Using stable integration rather than transient transfection reduces inter-well variability and transfection fluctuations, improving the consistency, reproducibility, and comparability of screening results.

[0036] The cell line was introduced via lentiviral infection. After introduction, the cells were cultured in a medium containing screening antibiotics to obtain a surviving cell population. Single-clonal cell lines were then obtained using limiting dilution or flow cytometry. The basal fluorescence intensity, fold change in induced response, passage stability, and growth status of the obtained clones were assessed. Cell lines with low background, large response window, and good genetic stability were selected for subsequent screening.

[0037] The cell culture medium can be DMEM complete medium containing 10% fetal bovine serum and 1% penicillin-dextrose antibody; the culture conditions are a 37°C, 5% CO2 incubator. These conditions are intended to maintain normal growth of HEK293T cells and ensure stable expression of the reporter system.

[0038] Example 2: Establishment of a high-throughput screening model The hMTCH2-EGFP reporter 293T cells obtained in Example 1 were seeded into 384-well multi-well plates. After seeding, the cells were cultured for 6 hours to allow them to adhere stably.

[0039] Subsequently, the compounds to be screened were added to each well. These compounds were sourced from the FDA-approved drug library (CiscoTech FDA Library). Compound addition was performed using an automated dispensing system to ensure consistency in dispensing volume, final concentration, and operation time across all wells. The solvent was DMSO (Dimethyl Sulfoxide). To minimize the solvent's impact on cells, the final DMSO concentration was controlled at 0.01% by mass.

[0040] like Figure 3 As shown, in this embodiment, over 2000 FDA-approved compounds were initially screened. An equal volume of DMSO was added to the negative control wells, while no compound was added to the blank control wells. These controls were set up to normalize the fluorescence signal and determine the dynamic range and stability of the screening system. Initially matched compounds were screened based on a comprehensive evaluation of factors such as EGFP signal enhancement fold, normalized calculated values, and cell viability retention. This step aims to rapidly identify candidates with potential MTCH2 activation capacity and acceptable toxicity from a large pool of compounds.

[0041] Example 3: Real-time fluorescence monitoring and determination of initial screening compounds After the compound was added, the cell plate was placed in the IncuCyte real-time live-cell imaging system, and bright-field and fluorescence images were automatically acquired at preset time points. Imaging data was collected within 24 hours of compound application. Image analysis software automatically calculated the EGFP fluorescence intensity, positive fluorescence area percentage, cell confluence, and morphological parameters for each well. The cell confluence was used to reflect cell activity and growth status, thus eliminating false signal changes caused by massive cell death or shedding.

[0042] In this embodiment, compounds whose EGFP fluorescence signal was significantly increased compared to the negative control group and whose cell viability remained within a preset range were defined as initial screening targets. Cell viability could be evaluated by cell confluence, cell counting, or an additional survival dye method. Preferably, the target compounds should enhance the fluorescence signal without causing significant cytotoxicity. Using the above methods, a total of 18 candidate compounds that could significantly enhance the EGFP signal were obtained, such as... Figure 4 As shown.

[0043] The technical role of this step is twofold: firstly, to utilize the MTCH2 promoter to drive EGFP expression and achieve visual detection of MTCH2 transcriptional activity; and secondly, to utilize real-time live-cell imaging to simultaneously monitor cell status, thereby improving the authenticity and reliability of the screening results.

[0044] Example 4: Secondary confirmatory screening and dose-response analysis like Figure 4 As shown, the 18 candidate compounds obtained in Example 3 were subjected to secondary screening for verification. Specifically, eight dose gradients were set for each candidate compound, and the doses could be obtained by serial dilution, such as 2-fold, 3-fold, or 10-fold serial dilutions. Preferably, two replicate wells were set for each concentration to improve statistical reliability.

[0045] Different concentrations of candidate compounds were added to hMTCH2-EGFP reporter 293T cells, and EGFP signaling and cell status were monitored using the IncuCyte system. Compounds that showed fluorescence enhancement only at high toxic concentrations or with no obvious dose dependence were discarded. Compounds that stably increased EGFP signaling at multiple dose points and maintained good cell viability were identified as preferred MTCH2 agonist candidates.

[0046] The specific process is as follows: Primary high-throughput screening: (1) On the morning of the same day, cells were inoculated into each 384-well test plate at a density of 2000 cells / well (initial inoculation density). Three parallel wells were set for each drug and 6 parallel wells were set for the DMSO negative control.

[0047] (2) Before screening, cells were seeded (50 μL / well) using an EL406 reagent dispenser and distributed into a 384-well detection plate (manufacturer code: Greiner 781280). The cells were incubated at 37 °C and 5% CO2 for 6 hours to allow them to adhere to the plate.

[0048] (3) After the cells adhered in the afternoon, a background EGFP fluorescence reading was performed. Then, each drug was added to each well using an Echo650 ultrasonic pipetting system to achieve a final concentration of 2.5 µM / well for the first high-throughput screening. EGFP detection readings and confluence measurements were performed every 6 hours after drug stimulation until 48 hours (using Incucyte to set up a long-term imaging program, and finally homogenization was performed based on the EGFP signal and cell number).

[0049] (4) The EGFP signal was detected using a fluorescence microplate reader (excitation light 485 nm, emission light 535 nm). Compounds with an MFI of more than 30% for EGFP in the initial drug screening were considered positive targets.

[0050] After initial high-throughput screening, appropriate cell concentrations, drug concentrations, and drug administration times were determined. Using the above methods, this invention preliminarily identified 18 candidate compounds from over 2000 compounds that could significantly enhance EGFP fluorescence signals.

[0051] 4. High-throughput secondary screening stage: Specifically, an eight-point dose gradient was set up for each candidate compound, and a concentration sequence was constructed using serial dilution. The reporter cells were then treated repeatedly. Details included seeding 2000 cells per well; setting up three parallel wells; and performing high-content imaging analysis after 48 hours of stimulation. The drug screening results were normalized and digitized by calculating the average fluorescence intensity of EGFP. The calculation method was: Intensity Cell Alexa 488 Mean / Mean per Well = Screening results. This step was used to evaluate the potency, maximum effect size, dose dependence, and specificity of candidate compounds in activating the MTCH2 promoter, and to eliminate compounds that only showed a transient increase in fluorescence within a narrow dose window or were accompanied by significant toxicity.

[0052] Through the above reaction experiments, this invention identifies gramicidin as a superior candidate small-molecule MTCH2 agonist. The candidate exhibits relatively stable MTCH2 transcriptional activation within a certain concentration range.

[0053] The molecular structure of gramidin is shown below. It is a commercially available product and can be purchased from Selleck, catalog number E2970.

[0054] .

[0055] Example 5: Validation of candidate compounds on endogenous MTCH2 expression like Figure 5 As shown, to verify the screening results and exclude non-specific factors that only affect EGFP fluorescence itself, the above-mentioned preferred candidates were further validated for endogenous MTCH2 expression. The candidate compounds were applied to HEK293T cells. After treatment, total RNA was extracted from the cells, and the MTCH2 mRNA level was detected by qPCR. qPCR stands for quantitative polymerase chain reaction.

[0056] Gramicidin was successfully validated as a positive drug, acting as an agonist that specifically upregulates the transcriptional expression of Mtch2.

[0057] Example 6: Detection of MTCH2 expression level in primary B cells of patients with systemic lupus erythematosus like Figure 6 As shown, this embodiment first verifies the regulatory effect of gramicidin on MTCH2 expression in primary B cells of SLE patients.

[0058] Specifically, peripheral blood samples were collected from SLE patients, peripheral blood mononuclear cells were isolated, and primary B cells were further purified. The B cells could be isolated using magnetic bead sorting, flow cytometry, or other conventional methods in the art. The isolated B cells were seeded in a suitable culture system and stimulated with gramicidin. The gramicidin concentration could be set at multiple gradients based on cell tolerance and pharmacological activity. The treatment time was 24 hours.

[0059] After treatment, total RNA was extracted from B cells to detect the mRNA expression level of MTCH2. Real-time quantitative polymerase chain reaction (qPCR) was the preferred method for detecting mRNA levels.

[0060] The results showed that bacitracin significantly upregulated MTCH2 expression in primary B cells of SLE patients. This step confirms that bacitracin upregulates MTCH2 expression in disease-associated cells and can reverse the abnormal immune activation of SLE-associated B cells, thus supporting its use as an MTCH2 agonist for alleviating SLE.

[0061] Example 7: The regulatory effect of gramicidin on the activation ratio of primary B cells in SLE patients like Figure 7 As shown, after confirming that gramicidin can upregulate MTCH2 expression, its effect on the activation status of primary B cells in SLE patients was further evaluated.

[0062] Specifically, primary B cells derived from SLE patients were cultured in vitro and stimulated with gramicidin, with a solvent control group included. After drug treatment, the activation rate of B cells was measured. This activation rate can be determined by detecting the expression levels of B cell activation markers, including but not limited to CD69, CD80, CD86, MHC-II, or other recognized B cell activation indicators. Detection methods may include flow cytometry, immunofluorescence, or other conventional analytical methods.

[0063] The results showed that treatment with gramicidin reduced the proportion of abnormally activated primary B cells (indicators of CD80+ B cells and CD86+ B cells) in SLE patients. This demonstrates that gramicidin not only upregulates MTCH2 but also reverses the abnormal immune activation of lupus-related B cells.

[0064] Example 8: The regulatory effect of gramicidin on mitochondrial function in primary B cells of SLE patients like Figure 8 As shown, after confirming that gramicidin can upregulate MTCH2 expression, we further evaluated its effect on mitochondrial function in primary B cells of SLE patients.

[0065] Simultaneously, mitochondrial function was assessed. MitoTracker Red staining and TMRM staining were used to evaluate mitochondrial functional status. Changes in MitoTracker Red fluorescence signal reflect mitochondrial status and activity, while TMRM fluorescence signal reflects the mitochondrial membrane potential difference.

[0066] The results showed that treatment with gramicidin improved mitochondrial function in primary B cells of SLE patients and alleviated abnormal oxidative stress, suggesting that it can alleviate abnormal oxidative stress and mitochondrial dysfunction in B cells.

[0067] This embodiment illustrates the mechanism of action of MTCH2 activation from the perspective of metabolism and mitochondrial function. It shows that gramicidin can not only upregulate MTCH2 expression, but also further inhibit the pathogenic activation of B cells from SLE patients and restore mitochondrial homeostasis, thereby alleviating the abnormality of pathogenic B cells and thus playing a role from both metabolic and immune function levels.

[0068] In summary, this invention provides a novel use of gramicidin in the preparation of drugs that upregulate MTCH2 to alleviate systemic lupus erythematosus (SLE), and establishes the important value of MTCH2 as a controllable metabolic checkpoint in autoimmune diseases. It offers new theoretical basis and technical pathways for the treatment of SLE and other autoimmune diseases, demonstrating significant social benefits and potential clinical application prospects. Those skilled in the art can implement this invention without inventive effort based on the disclosure of this specification.

Claims

1. Use of bacitracin in the preparation of drugs for the treatment of systemic lupus erythematosus.

2. The use according to claim 1, characterized in that, The treatment is achieved by upregulating the expression of MTCH2.

Images