Syringin targeted inhibition DLAT protein and application of syringin targeted inhibition DLAT protein in inhibition of mycobacterium tuberculosis

Abstract

The invention belongs to the technical field of medicines, and particularly relates to syringin targeted inhibition DLAT protein and application of syringin targeted inhibition DLAT protein in inhibition of mycobacterium tuberculosis. Research finds that natural micromolecule syringin can inhibit DLAT protein activity in a targeted manner, reduce acetylation of MYC protein and promote ubiquitination and degradation of MYC protein, so that the MYC protein level is reduced, M2 type polarization of macrophages is inhibited, the polarization state of the macrophages is adjusted, growth of mycobacterium tuberculosis in the macrophages is inhibited, and the MYC-MYC-MYC-MYC-MYC-MYC-MYC-MYC-MYC-MYC is inhibited. The anti-tuberculosis effect is effectively achieved. Compared with an existing anti-tuberculosis technology, the anti-tuberculosis scheme is clear in action mechanism and accurate in targeting effect, and blindness of a traditional therapy is avoided. According to the present invention, the tuberculosis condition can be effectively controlled, the treatment period is expected to be shortened, the physical, mental and economic burden of the patient is reduced, the untoward effect risk is substantially reduced due to the high biological safety of the natural small molecules, and the new reliable path is provided for the clinical anti-tuberculosis treatment.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and particularly relates to the targeted inhibition of DLAT protein by syringin and its application in inhibiting Mycobacterium tuberculosis. Background Art

[0002] Mycobacterium tuberculosis (M. tuberculosis) is a major global health threat, infecting nearly 2 billion people worldwide, with approximately 10% developing active tuberculosis. Currently, clinically available anti-tuberculosis drugs are very limited, and the growing problem of drug resistance necessitates the urgent development of new anti-tuberculosis drugs. During infection, M. tuberculosis is phagocytosed by alveolar macrophages, but some can resist the killing effects of macrophages, subsequently developing primary tuberculosis or entering a latent phase, where they can survive in the host for decades. Therefore, the ability of macrophages to kill M. tuberculosis is crucial for the treatment of tuberculosis.

[0003] Studies have shown that infection with Mycobacterium tuberculosis activates the defense response of macrophages, causing them to exhibit an M1-type polarization-like response in the early stages of infection (4-8 hours), producing highly toxic reactive oxygen species, nitrogen species, bactericidal peptides, and other substances, and transporting ingested pathogens to lysosomes for destruction. Individuals lacking the M1-type polarization response are highly susceptible to infection with intracellular mycobacteria. In the middle and late stages of infection (24-48 hours), macrophages initiate an M2-type polarization-like response. Although this response plays an important homeostatic regulatory role in immune regulation, tissue remodeling, and resistance to parasitic infection, it is beneficial to the survival and replication of Mycobacterium tuberculosis. For example, overexpression of IL-4 promotes the progression of tuberculosis and the formation of typical organized necrotizing granulomas, and parasitic infections such as helminths can also antagonize the host's protective response to mycobacteria. Therefore, the purpose of anti-tuberculosis can be achieved by regulating the polarization response of macrophages.

[0004] Macrophage polarization is a process of metabolic reprogramming. MYC is a major transcription factor regulating cellular metabolism, regulating the expression of at least 15% of human genes. It has been identified as a hallmark protein of M2-polarized macrophages and plays a role in promoting M2 polarization. Multiple studies have demonstrated that MYC directly regulates the expression of genes involved in mitochondrial biogenesis and maintains normal intracellular oxidative phosphorylation levels. Conversely, knockdown or deletion of MYC significantly decreases basal oxygen consumption, ATP production, and maximal mitochondrial oxygen consumption, thereby inhibiting macrophage M2 polarization. Therefore, modulating MYC activity is an important target for regulating macrophage polarization. However, the highly disordered structure of MYC, the lack of binding pockets, and the lack of enzymatic activity present significant challenges in the development of inhibitors targeting MYC. Notably, MYC's highly disordered structure results in a short intracellular half-life of 15-30 minutes and a high turnover rate. Furthermore, MYC undergoes extensive post-translational modifications that affect its degradation. It can be seen that by regulating the modification, degradation and level of MYC protein, macrophage polarization can be regulated. Therefore, the development of drugs that regulate MYC protein modification to control macrophage polarization is of great significance for the treatment or research of tuberculosis. Summary of the Invention

[0005] In order to overcome the deficiencies of the above-mentioned prior art, the present invention discovered a natural small molecule compound, syringin, which can directly bind to DLAT protein in macrophages and inhibit its activity, thereby reducing the acetylation of MYC protein, promoting the ubiquitination and degradation of MYC protein, thereby regulating the polarization state of macrophages and inhibiting the growth of Mycobacterium tuberculosis in macrophages.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] The first aspect of the present invention provides the use of syringin in the preparation of anti-Mycobacterium tuberculosis drugs.

[0008] The second aspect of the present invention provides the use of syringin in the preparation of a medicament for treating tuberculosis.

[0009] Preferably, the syringin inhibits the activity of DLAT protein, thereby reducing the level of MYC protein and inhibiting the M2 polarization of macrophages, thereby exerting an anti-Mycobacterium tuberculosis effect.

[0010] The present invention has found through research that the natural small molecule lilacin can target and inhibit the activity of DLAT protein, thereby reducing the level of MYC protein, inhibiting the M2 polarization of macrophages, and then inhibiting the growth of Mycobacterium tuberculosis in macrophages, effectively exerting an anti-tuberculosis effect.

[0011] Preferably, the Mycobacterium tuberculosis includes a clinical isolate of Mycobacterium tuberculosis, a standard strain of Mycobacterium tuberculosis or Mycobacterium tuberculosis carried by a Mycobacterium tuberculosis infected patient.

[0012] A third aspect of the present invention provides an anti-Mycobacterium tuberculosis drug, wherein the drug contains syringin as a main active ingredient.

[0013] Preferably, the effective concentration of syringin is 25-200 μM.

[0014] Preferably, the drug further comprises pharmaceutically acceptable excipients.

[0015] More preferably, the excipients include at least one of excipients, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrators, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, antioxidants, adsorbents, filter aids, and release retardants.

[0016] Preferably, the dosage form of the drug includes tablets, capsules, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents or suppositories.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The polarization state of macrophages determines its killing effect on Mycobacterium tuberculosis, and is of great significance in the pathogenesis and treatment of tuberculosis. The present invention has been found through research that the natural small molecule syringin can target and inhibit the activity of DLAT proteins, reduce the acetylation of MYC proteins, promote the ubiquitination and degradation of MYC proteins, thereby reducing the level of MYC protein, inhibiting the M2 polarization of macrophages, regulating the polarization state of macrophages, thereby inhibiting the growth of Mycobacterium tuberculosis in macrophages, and effectively exerting an anti-tuberculosis effect. This shows that syringin has a targeting characteristic and can specifically inhibit the activity of DLAT proteins. By inhibiting the acetylation of MYC proteins, promoting ubiquitination and degradation, it can achieve effective regulation of MYC protein levels. The reduction of MYC protein levels can accurately inhibit the M2 polarization of macrophages, reshape the polarization state of macrophages, and fundamentally destroy the microenvironment in which Mycobacterium tuberculosis grows in macrophages, significantly inhibiting its proliferation, and thus exerting an anti-tuberculosis effect. Compared to existing anti-TB technologies, the anti-TB regimen of the present invention has a clear mechanism of action and precise targeting, avoiding the blind spots of traditional therapies. It can effectively control TB symptoms, potentially shortening treatment cycles and alleviating the physical, mental, and financial burdens on patients. Furthermore, due to the high biosafety of natural small molecules, the risk of adverse reactions is significantly reduced, providing a new and reliable approach for clinical anti-TB treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the chemical structural formula of syringin.

[0020] Figure 2 Syringin inhibits IL-4-induced M2 polarization of macrophages; (A) Effect of syringin on IL-10 release; (B) Effect of syringin on ARG1 protein level; (C, D) Flow cytometry analysis of the effect of syringin on the level of CD206, an M2 polarization marker.

[0021] Figure 3 The effect of syringin on regulating macrophage M2 polarization depends on the regulation of MYC protein; (A) Effect of syringin on MYC protein level; (B) Effect of knockdown of MYC protein and its complement on syringin regulating ARG1 protein level; (C) Effect of knockdown of MYC protein and its complement on syringin regulating IL-10 release.

[0022] Figure 4Figure 3. Syringin regulates MYC protein levels by binding to DLAT proteins. (A) Effect of syringin on Myc mRNA levels. (B) Effect of syringin on MYC protein degradation after blocking protein degradation with CHX. (C) Effect of syringin on the thermal stability of MYC protein in an intracellular thermal shift assay. (D, E) Immunoprecipitation assay to detect the acetylation and ubiquitination levels of MYC protein. (F) Effect of syringin on the thermal stability of DLAT proteins in an intracellular thermal shift assay. (G) BLI assay to detect the binding of DLAT proteins to syringin.

[0023] Figure 5 Syringin blocks the binding of DLAT to the substrate acetyl-CoA; (A, B) Immunoprecipitation assay detects the interaction between DLAT protein and MYC protein in the presence or absence of syringin; (C) BLI assay detects the interaction between DLAT protein and the substrate acetyl-CoA in the presence or absence of syringin.

[0024] Figure 6 Figure 3. Syringin inhibits the growth of H37Rv in macrophages; (A) The knockdown effect of DLAT in BMDMs cells; (B) The effect of DLAT knockdown on the inhibition of H37Rv growth in macrophages by syringin. DETAILED DESCRIPTION

[0025] The following is a further description of specific embodiments of the present invention. It should be noted that the description of these embodiments is intended to facilitate understanding of the present invention and does not constitute a limitation of the present invention. In addition, the technical features involved in the various embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

[0026] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0027] Example: Study on the Targeted Inhibition of DLAT Protein by Syringin and Its Inhibition of Mycobacterium tuberculosis

[0028] 1. Experimental methods:

[0029] 1.1 Eight-week-old C57BL / 6 male mice (purchased from Ruisiyuan Biotechnology Co., Ltd.) were sacrificed by cervical dislocation and disinfected with 70% ethanol. The femur and tibia were cut open at both ends in a clean bench, and sterile PBS was aspirated with a syringe to flush the bone marrow from the bone cavity. The collected bone marrow cells were filtered through a 70 μm mesh to remove bone fragments and other impurities, then resuspended in PBS and centrifuged at 500 g for 5 minutes. The supernatant was removed. The resulting cell pellet was inoculated in DMEM high-glucose medium supplemented with 10% fetal bovine serum (FBS), 100 μg / mL streptomycin, 100 U / mL penicillin, and 100 ng / mL macrophage colony-stimulating factor (M-CSF). After 6 days of culture, bone marrow-derived macrophages (BMDMs) were obtained. The medium was then replaced with medium without M-CSF, and 20 ng / mL IL-4 was added for stimulation. M2-polarized macrophages were obtained after 24 hours. At the same time, when IL-4 was added, different concentrations (0, 25, 50, 100 μM) of syringin (Syringin, structural formula: Figure 1 Cells were collected for Western blot and flow cytometry to measure the expression of proteins associated with M2 polarization. In addition, the culture supernatant was collected and IL-10 levels were measured using an ELISA (kit purchased from Elabscience, E-HSEL-M0004). The specific assay procedure was followed according to the kit instructions. Among them, the experimental methods for BMDMs culture and M2 polarization were referred to the literature (PINEDA-TORRAI, GAGE ​​M, de JUANA, et al. Isolation, culture, and polarization of murine bone marrow-derived and peritoneal macrophages[J]. Methods Mol Biol, 2015, 1339: 101-109.), while Western blot, qPCR and flow cytometry all adopted conventional experimental protocols. The detailed operations were referred to the literature (MILYA, KALSUM S, LORETI MG, et al. Polarization of M1 and M2 human monocyte-derived cells and analysis with flow cytometry upon mycobacterium tuberculosis infection[J]. J Vis Exp, 2020, (163).), and the qPCR primers are shown in Table 1 below.

[0030] Table 1 qPCR primer sequences

[0031]

[0032] 1.2. To knock down MYC protein in BMDMs, a shRNA sequence (5'-GAGAACAGTTGAAACACAA-3') was used to target the MYC gene coding region and constructed in the pLKO.1 vector (purchased from U-Bio). The full-length mouse MYC protein corresponds to the CDS sequence NM_010849.4, and its encoding gene was cloned from the cDNA of BMDM cells. To restore MYC protein expression in cells, the MYC protein CDS sequence was amplified using KOD PLUS NEO (TOYOBO) DNA polymerase and constructed into the pCDH-CMV-MCS-EF1-BSD plasmid (the pCDH-CMV-MCS-EF1-PURO plasmid was modified by replacing the BSD sequence with the PURO sequence from the pLV-EF1α-MCS-IRES-BSD vector using conventional molecular biology methods. All plasmids were purchased from U-Bio). In this process, the coding sequence of two amino acids in the Myc gene (5'-GAGA A CAATTGAA G CACAA-3' (corresponding to nucleotide positions 1316–1334, the underlined bases indicate the mutation site) was mutated to render the gene resistant to shRNA degradation. The two plasmids were then packaged into lentivirus in 293T cells with the psPAX2 and pMD2.G plasmids, respectively. The specific method is described in the literature (Prochownik EV, Wang H. Lessons inaging from Myc knockout mouse models [J]. Frontiers in Cell and Developmental Biology, 2023, 11: 1244-321).

[0033] On the second day of BMDMs cell culture, slow virus was added and 10 μg / mL Polybrene was added to knock down MYC protein, or to restore the expression of MYC protein in the protein knocked down. On the 4th day, 5 μg / mL puromycin was used to screen the cells that MYC knocked down, or 20 μg / mL Blasticidin was used to screen the cells that MYC protein was replenished. The specific method is referenced to literature (Kofoed EM, Vance R E.Innate immune recognition of bacterial ligands by NAIPs determines inflammasome specificity[J]. Nature, 2011, 477(7366): 592-595.). Finally, on the 8th day, cells were treated with 20 ng / mL IL-4 and 100 μM syringin, and cells were collected and analyzed.

[0034] 1.3. In M2 polarized macrophages, 100 μM syringin was added to treat the cells for 30 minutes, and then 10 μM cycloheximide (CHX) was added to inhibit protein synthesis. The cells were then collected at 0 min, 15 min, 30 min, 1 h, 3 h, and 6 h. Western analysis was performed to detect the amount of MYC protein remaining in the cells and evaluate the effect of syringin on MYC protein. The intracellular protein degradation experiment was specifically referred to the literature (Xiao L, Yao J, Miao Y, et al. Tubuloside B, isolated from Cistanche tubulosa, a promising agent against M1 macrophage activation via synergistically targeting Mob1 and ERK1 / 2[J]. Biomedicine & Pharmacotherapy, 2022, 153: 113414.).

[0035] 1.4. After treating M2 polarized macrophages with 100 μM syringin for 60 minutes, the cells were collected, washed once with PBS, and resuspended in PBS containing protease inhibitors (Biyuntian, P1005) to adjust the cell density to 5×10 6 / mL. The cell suspension was immersed in liquid nitrogen for 30 seconds, then thawed at room temperature and repeatedly frozen and thawed three times to ensure complete cell lysis. Subsequently, the cell suspension was aliquoted into 50 μL tubes and treated at a temperature gradient (37°C, 39°C, 41°C, 43°C, 45°C, 47°C) for 3 minutes, followed by cooling on ice for 3 minutes. The sample was then centrifuged at 20,000g for 20 minutes at 4°C to separate soluble and aggregated proteins. The supernatant contained soluble proteins, and the degradation of the target protein was analyzed by Western blot. The intracellular thermal shift experiment was specifically referred to the literature (Xiao L, Yao J, Miao Y, et al. Tubuloside B, isolated from Cistanche tubulosa, a promising agent against M1 macrophage activation via synergistically targeting Mob1 and ERK1 / 2[J]. Biomedicine&Pharmacotherapy, 2022, 153: 113414.).

[0036] 1.5. BMDMs were treated with 20 ng / mL IL-4 and 100 μM syringin for 24 hours. If the MG132 proteasome inhibitor was added, 20 μM MG132 was added 2 hours before cell harvest to inhibit protein ubiquitination. After treatment, cells were washed once with PBS and treated with IP lysis buffer containing protease inhibitors (Biyuntian, P1005). Cells were scraped and lysed on ice for 30 minutes. The supernatant was then centrifuged at 20,000 g for 20 minutes. 100 μL of the supernatant was retained for analysis of the original input protein content. 1 μg of MYC antibody or DLAT antibody was added to the remaining supernatant and incubated at 4°C with shaking overnight to form antibody-protein complexes. Protein A / G magnetic beads were then added and incubated at 4°C with shaking for 1 hour to precipitate the antibody-protein complexes. The beads were then washed five times with IP lysis buffer to remove nonspecifically bound substances. Then, 1× SDS loading buffer was added to the magnetic beads and heated at 95°C for 10 min. Western blot analysis was performed. Acetylation and ubiquitination levels of MYC protein were detected using acetylated protein antibodies or polyprotein antibodies, or protein interactions were detected using DLAT antibodies or MYC antibodies. The specific procedures for protein co-immunoprecipitation experiments were referred to the literature (Liu Y, Xu P, Rivara S, et al. Clathrin-associated AP-1 controls termination of STING signaling [J]. Nature, 2022, 610(7933): 761-767.).

[0037] 1.6. The full-length mouse DLAT protein (CDS sequence of NM_145614.4) was amplified using KOD PLUS NEO (TOYOBO) DNA polymerase and subcloned into the pLVX-IRES-Puro-3Flag plasmid (purchased from U-Bio). The plasmid was then transfected into 293T cells to express the DLAT-3Flag protein. The recombinant protein was purified using conventional Flag purification agarose (Genscript, L00432-25). The specific method was referred to the literature (Liu Y, Xu P, Rivara S, et al. Clathrin-associated AP-1 controls termination of STING signaling [J]. Nature, 2022, 610(7933): 761-767.).

[0038] 50 μL of 1 mg / mL DLAT-3Flag was mixed with 0.4 μL of 1 mg / mL NHS-LC-Biotin and incubated at 37°C for 60 min to form DLAT-3Flag-biotin. The product was then desalted using a desalting column (Thermo). Biolayer interferometry experiments were performed at 30°C using a Gator Biolayer Interferometry System (SNGC00070, ProbeLife, Inc.). Kinetics buffer (PBS containing 0.05% Tween-20 and 0.1% BSA) was used to reduce nonspecific binding. SMAP biosensors (Gator Bio) were used for immobilization of the DLAT-3Flag-biotin protein. After hydration in kinetics buffer for 10 minutes, the biosensor was loaded with DLAT-3Flag-biotin protein at a concentration of 15 μg / mL for 800 seconds, and the sensor was equilibrated in kinetics buffer for 60 seconds. Subsequently, the sensor was exposed to 10, 20, and 40 μM syringin and the binding was monitored for 120 seconds, followed by a 120-second dissociation process in kinetic buffer. A negative control of the reference sensor was included in the experiment to eliminate the effect of nonspecific binding and retain only R 2 Data with a p-value > 0.95 were analyzed to evaluate the direct binding of DLAT proteins to syringin. The specific experimental method of biolayer interferometry (BLI) was referred to the literature (Wang Y, Sun Y, Deng S, et al. Galectin-8 is a major ligand of LILRB4 prompting MDSC functions in the tumor microenvironment [J]. Biorxiv, 2022: 2022.07.27.501694.).

[0039] 1.7. In the experiment to investigate the effect of syringin on the binding of DLAT-3Flag to acetyl-CoA, the SMAP biosensor was loaded with DLAT-3Flag-biotin as described in 1.6. The sensor was equilibrated in kinetic buffer for 60 seconds and then exposed to 40 μM syringin for 120 seconds of binding. The sensor was then exposed to a mixture of 40 μM syringin and acetyl-CoA (25, 50, and 100 μM concentrations, respectively) for 120 seconds of binding monitoring, followed by 120 seconds of dissociation in kinetic buffer. The analysis method used was the same as in 1.6 to analyze the effect of syringin on the binding of DLAT to acetyl-CoA.

[0040] 1.8. To knock down DLAT (dihydrolipoyl transacetylase) protein in BMDMs, a shRNA sequence for DLAT (5′-CCAACAGAAGTGACCAGCTTA-3′) was inserted into the lentiviral transfer vector pLKO.1 (purchased from Ubao Bio). This plasmid, along with psPAX2 and pMD2.G plasmids, was then packaged into lentivirus in 293T cells. The specific method was described in the literature (Xiao L, Yao J, Miao Y, et al. Tubuloside B, isolated from Cistanche tubulosa, apromoting agent against M1 macrophage activation via synergistically targeting Mob1 and ERK1 / 2[J]. Biomedicine & Pharmacotherapy, 2022, 153:113414.). On the second day of BMDM cell culture, the lentivirus was added, along with 10 μg / mL Polybrene, to knock down DLAT protein. On the 4th day, DLAT knockdown cells were screened using 5 μg / mL puromycin. The specific operation method was referred to the literature (Kofoed EM, Vance RE. Innate immune recognition of bacterial ligands by NAIPs determines inflammasome specificity [J]. Nature, 2011, 477(7366): 592-595.; Wang J, Yang Y, Shao F, et al. Acetate reprogrammes tumour metabolism and promotes PD-L1 expression and immune evasion by upregulating c-Myc [J]. Nat Metab. 2024; 6(5): 914-932.).

[0041] Mycobacterium tuberculosis H37Rv strain (ATCC 27294) was grown at 37°C in Middlebrook 7H9 broth containing 0.2% glycerol, 0.5% bovine serum albumin, 0.05% Tween 80, 0.2% glucose, and 0.085% sodium chloride. Or it was grown in Middlebrook 7H11 agar containing 10% oleic acid-albumin-dextrose-catalase (7H11-OADC). On day 7 of BMDM differentiation, macrophages were stimulated with 20 ng / mL IFNγ. After 24 hours of treatment, macrophages were infected with a logarithmic phase culture of the H37Rv strain at an MOI of 5. After 4 hours of infection, the cells were washed twice with PBS to remove excess extracellular bacteria. Fresh culture medium was then added, along with 100 μM syringin and 20 ng / mL IFNγ. 120 h after infection, the cells were lysed with 0.5% Triton X-100, and the lysate was inoculated onto Middlebrook 7H11 agar plates using a serial dilution method. The colony count was evaluated after being incubated at 37°C for 2-3 weeks to assess the effect of syringin on the survival rate of Mycobacterium tuberculosis in macrophages. For specific operation methods, please refer to the literature (Shi S, Ehrt S. Dihydrolipoamide acyltransferase is critical for Mycobacterium tuberculosispathogenesis [J]. Infection and immunity, 2006, 74 (1): 56-63.; Ehrt S, Schnappinger D, Bekiranov S, et al. Reprogramming of the macrophage transcriptome in response to interferon-γ and Mycobacterium tuberculosis: signaling roles of nitricoxide synthase-2and phagocyte oxidase[J]. The Journal of experimental medicine, 2001, 194(8):1123-1140.).

[0042] 2. Experimental results:

[0043] In mouse BMDMs cells, 20 ng / mL IL-4 was used to induce M2 polarization, and it was found that syringin could reduce the protein expression level of ARG1 (arginase 1) in a concentration-dependent manner ( Figure 2 A), reduce the release of inflammatory factor IL-10 ( Figure 2 B), and reduced the expression level of CD206, a cell surface M2 polarization marker ( Figure 2 CD), indicating that syringin can significantly inhibit the M2 polarization of macrophages induced by IL-4.

[0044] In BMDMs cells, 20ng / mL IL-4 can promote the increase of MYC protein level ( Figure 3 A). After knocking out MYC protein, the inhibitory effect of syringin on macrophage M2 polarization disappeared, including the regulation of ARG1 protein levels and IL10 release ( Figure 3 BC), indicating that MYC protein plays an important role in the process of syringin regulating macrophage polarization.

[0045] Syringin can reduce the concentration of MYC protein in a concentration-dependent manner, but has no significant effect on its mRNA level ( Figure 4 A), indicating that syringin's regulation of MYC protein levels is not achieved by regulating its mRNA level. By inhibiting protein degradation through actinomycin, syringin was given to M2 polarized macrophages and found that syringin can promote the degradation of MYC protein, increase the turnover rate of MYC protein in cells, and promote its degradation ( Figure 4 B). Subsequently, an intracellular thermal shift assay was used to investigate whether syringin affects MYC protein degradation by directly binding to it. Direct binding of small molecules to proteins can affect the thermal stability of proteins. Syringin treatment in M2-polarized macrophages revealed no significant effect on the thermal stability of MYC protein. Figure 4 C), which also indicates that syringin cannot directly bind to MYC protein, but regulates the degradation of MYC protein through other mechanisms.

[0046] The level of MYC protein is regulated by its post-translational modification, among which DLAT protein can acetylate and modify MYC protein, inhibiting its ubiquitination and degradation. Studies have found that the presence of syringin can inhibit the acetylation and ubiquitination of MYC protein ( Figure 4 DE), suggesting that the mechanism of action of syringin is related to DLAT protein. In the intracellular thermal migration experiment, syringin reduced the denaturation rate of DLAT protein ( Figure 4 F), indicating that syringin has a direct effect on DLAT protein. Further BLI was used to detect the binding of syringin and DLAT protein, and it was found that the two showed a fast binding and slow dissociation morphology curve ( Figure 4 G), indicating that syringin directly binds to DLAT protein. Protein immunoprecipitation experiments were used to detect the interaction between DLAT protein and MYC protein, and it was found that in the presence of syringin, there was no significant change in the binding between the two ( Figure 5AB), indicating that syringin is not achieved by directly inhibiting the interaction between DLAT protein and MYC protein. Subsequently, BLI was used to detect the binding of DLAT to its substrate acetyl-CoA in the presence or absence of syringin. It was found that in the presence of syringin, the direct binding of DLAT to acetyl-CoA was significantly inhibited ( Figure 5 C), there is no obvious rise in the binding curve, suggesting that the protein structure changes significantly after syringin binds to DLAT protein, and loses the binding with the substrate. Therefore, it is speculated that syringin inhibits the acetyltransferase activity of DLAT protein, thereby inhibiting the acetylation of MYC protein and promoting its ubiquitination and degradation.

[0047] The M1 / M2 polarization state of macrophages can affect their killing effect on Mycobacterium tuberculosis. Syringin can inhibit the M2 polarization of macrophages and prolong the inflammatory response of macrophages. Studies have found that in IFNγ-stimulated macrophages, syringin can reduce the number of H37Rv Mycobacterium tuberculosis remaining in macrophages, indicating that syringin can play an anti-tuberculosis effect. However, in DLAT-knockdown cell lines, syringin cannot reduce the number of Mycobacterium tuberculosis ( Figure 6 ), indicating that the effect of syringin depends on DLAT protein, further proving the mechanism of action of syringin in inhibiting Mycobacterium tuberculosis by targeting DLAT protein.

[0048] The embodiments of the present invention are described in detail above, but the present invention is not limited to the described embodiments. It is apparent to those skilled in the art that various changes, modifications, substitutions, and variations of these embodiments may be made without departing from the principles and spirit of the present invention, and the changes still fall within the scope of protection of the present invention.

Claims

1. Use of syringin in the preparation of a drug against Mycobacterium tuberculosis.

2. Use of syringin in the preparation of a drug for treating tuberculosis.

3. The application according to claim 1, characterized in that The syringin exerts an anti-Mycobacterium tuberculosis effect by inhibiting the activity of DLAT protein, thereby reducing the level of MYC protein and inhibiting the M2 polarization of macrophages.

4. The application according to claim 1, characterized in that The Mycobacterium tuberculosis includes clinical isolates of Mycobacterium tuberculosis, standard strains of Mycobacterium tuberculosis, or Mycobacterium tuberculosis carried by patients infected with Mycobacterium tuberculosis.

5. A drug against Mycobacterium tuberculosis, characterized in that, The drug uses syringin as the main active ingredient.

6. The anti-Mycobacterium tuberculosis drug according to claim 5, characterized in that, The effective concentration of the syringin is 25 - 200 μM.

7. A drug against Mycobacterium tuberculosis according to claim 5, characterized in that, The drug further includes pharmaceutically acceptable excipients.

8. A drug against Mycobacterium tuberculosis according to claim 7, characterized in that, The excipients include at least one of excipients, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, antiadhesives, chelating agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickeners, clathrates, humectants, absorbents, diluents, flocculants and deflocculants, antioxidants, adsorbents, filter aids, release retardants.

9. A drug against Mycobacterium tuberculosis according to claim 5, characterized in that, The dosage form of the drug includes tablets, capsules, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, or suppositories.

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