Application of Galectin-3 inhibitor TD139 in treatment of tuberculous granuloma

Abstract

The invention provides an application of a Galectin-3 inhibitor TD139 in the treatment of tuberculous granuloma. The TD 139 is proved to be a specific inhibitor of the Galectin-3, and the TD 139 is a specific inhibitor of the Galectin-3. According to the invention, the dosage of intraperitoneal injection of mice is 200 [mu] g / mouse. D <-1 >. By adopting the technical scheme of the invention to treat a mycobacterium marinum induced mouse tuberculosis model through the TD139, the formation of tuberculous granuloma is reduced in vivo through the TD139. TD139 is subjected to intraperitoneal injection once every other day and 5 times continuously, the granuloma area, the intracellular bacterium carrying amount and the inflammatory factor expression of the tail skin of a tuberculosis model can be remarkably reduced, and mice do not die in the whole process; the intrinsic mechanism is that PDIM is a toxic structure of a mycobacterium marinum bacterial wall and causes skin ulceration and granuloma. The Galectin-3 gene can inhibit phagocytosis and elimination of Mycobacterium marinum by macrophages and activation of innate immune pathways of the Mycobacterium marinum, and TD139 promotes the macrophages to eliminate pathogenic bacteria and reduces MMP-9 expression, so that formation of granuloma is reduced. The new pharmacological efficacy and the tuberculosis treatment mechanism of the TD139 are found for the first time, and a foundation is laid for future clinical application.

Description

Technical Field

[0001] The present invention relates to a new therapeutic use of the TD139 compound. It is found that the chemical composition (intraperitoneal injection) has a strong inhibitory effect on the granuloma induced by Mycobacterium marinum, which helps to expand its indications and provide a new option for the treatment of human tuberculosis. Background Art

[0002] Mycobacterium tuberculosis ( Mycobacterium tuberculosis , Mtb) infection causes 10 million infections and 1.5 million deaths worldwide each year. Tuberculous granuloma is a highly variable pathological change unique to tuberculosis, and it is also one of the reasons why tuberculosis is difficult to treat. It is a dynamic spatial structure composed of macrophages, granulocytes, lymphocytes and fibroblasts. On the one hand, the infection site is isolated from the uninvolved lung tissue to limit the spread, allowing the bacteria to remain in the body for a long time; on the other hand, the upregulated tolerance pathways in the granuloma site will limit the clearance of Mycobacterium tuberculosis and block the entry of drugs into the granuloma. Therefore, inhibiting granuloma formation is an important treatment idea to reduce the prolongation of tuberculosis. If combined with anti-tuberculosis drugs, the host's clearance of tuberculosis bacteria can be improved as soon as possible. At present, the only way to remove granulomas is through surgical treatment.

[0003] Galectins (Gal) are sugar-binding proteins with highly conserved N-terminal amino acid sequences and specific recognition of β-galactosidic structures. They belong to the lectin superfamily. So far, 15 galectins have been identified in mammals, most of which are widely distributed in various tissues in the animal body, and a small number of galectins are specifically expressed in limited tissues. Galectin-3 (Gal3) is a chimeric oligomeric protein composed of 31kDa subunits, containing an N-amino terminal region and a CRD structure, which can form a hexamer. Gal-3 is a typical cytoplasmic protein, but its expression can also be found in the extracellular matrix, cell surface, and cell nucleus, and is highly expressed in macrophages, tumor cells, epithelial cells, fibroblasts and other immune cells. Existing studies have found that Gal3 can promote participation in pulmonary fibrosis and participate in the differentiation of macrophage M2. However, the role of Gal3 in tuberculosis is still unclear.

[0004] The present invention is the first to discover that the key pathogenic gene for granuloma formation is the Galectin-3 protein of macrophages (gene name LGALS3, Gal3 We injected Mycobacterium marinum ( Mycobacterium marinum , Mm ) simulated human Mycobacterium tuberculosis to produce skin granuloma formation, and compared the effect of wild-type Mycobacterium marinum infection on granuloma formation by inhibiting Gal3 MmGal3 overexpression (overexpression of Gal3 plasmid) can significantly inhibit macrophage phagocytosis and clearance Mm ability to inhibit the innate immune response. Knocking down or knocking out Gal3 expression (siRNA technology or Gal3 knockout mice) can alleviate the immune response and inhibit the growth of Mycobacterium marinum ( Mm ) induced granuloma formation.

[0005] This discovery is the first to use TD139, a novel synthetic galectin-3 inhibitor, to treat tuberculous granulomas. TD139 is also known as GB0139, 33DFTG, bis-(3-deoxy-3-(4-(3-fluorophenyl)-1H-1,2,3-triazol-1-yl)-β-D-galactopyranosyl, and bis-(3-deoxy-3-(4-(3-fluorophenyl)-1H-1,2,3-triazol-1-yl)-β-D-galactopyranosyl. TD139 has a high affinity for galectin-3 with a Kd (dissociation constant) of 14 nM; its Kd for galectin-1 is 10 nM, but its affinity for Galectins-2, Galectins-4N, Galectins-4C, Galectins-7, Galectins-8N or Galectins-9N is lower. In vivo experiments have confirmed that TD139 can significantly inhibit the formation of tuberculous granulomas. Summary of the invention

[0006] The present invention discloses that TD139 can alleviate Mycobacterium marinum ( Mm )-induced granuloma formation. This model first uses tail vein injection of Mycobacterium marinum ( Mm 10 days later, TD139 (200 μg / mouse / day) was intraperitoneally injected -1 , once every other day, for 5 consecutive times) to make a tuberculosis model.

[0007] The present invention first discovered the important role of Galectin-3 (Gal3) in promoting tuberculous granulomas. Through in vivo and in vitro experiments, it was found that downregulation or upregulation of Gal3 can change the formation of granulomas in the mouse tail skin induced by Mycobacterium marinum. Inhibition of Gal3 in in vivo and in vitro infection models can significantly improve granuloma formation and tissue damage caused by mycobacterium infection. PDIM, a component of the bacterial wall of Mycobacterium marinum, is the main component that promotes granulomas and skin ulcers.

[0008] A Galectin-3 inhibitor, wherein the inhibitor is TD139.

[0009] Another technical solution of the present invention is to provide a granuloma inhibitor, wherein the inhibitor is TD139.

[0010] Another technical solution of the present invention is to provide a use of a Galectin-3 inhibitor in preparing a drug for treating tuberculous granuloma, wherein the inhibitor is TD139.

[0011] The inhibitor TD139 is used in drugs for reducing tuberculosis induced by Mycobacterium marinum.

[0012] The inhibitor TD139 is used in the preparation of a drug for reducing tuberculosis induced by Mycobacterium marinum or Mycobacterium tuberculosis.

[0013] The inhibitor TD139 is used in the preparation of a drug for reducing one or more of the granuloma area, intracellular bacterial load, and inflammatory factor expression of tuberculosis tail skin ulcers.

[0014] The TD139 is used in the preparation of a drug for promoting macrophages to clear pathogens and reduce the expression of MMP-9.

[0015] The inhibitor TD139 is used in the preparation of a drug for treating tuberculous granuloma by injection.

[0016] In the preparation of the drug for treating tuberculous granuloma, the subjects suffering from tuberculous granuloma disease include mammals and fish; In some preferred cases, the mammals include mice, rabbits, monkeys, humans, horses, dogs, cats, sheep, and pigs; and the fish include zebrafish.

[0017] (1) Confirming the important role of Gal3 in tuberculosis: Mm In vitro infection of macrophages combined with proteomics technology first discovered the inducible high expression gene Gal3; by infecting mice Mm Induced granuloma formation, confirmed that granulomas highly expressed Gal3. In animal models, other animals (mice, rabbits, marmosets, humans) infected with human tuberculosis mycobacterium (MTB) and other species (zebrafish) infected with marine mycobacterium also obtained similar conclusions (all formed granulomas and highly expressed Gal3). These all confirm that Gal3 is highly positively correlated with the occurrence of tuberculosis and may be an important factor promoting tuberculosis. See Examples 2, 3, and 4.

[0018] (2) Confirming the feasibility of Gal3 downregulation in improving tuberculous granulomas: In vivo experiments, Gal3 knockout mice were infected Mm It was found that the area of ​​tuberculous granulomas was significantly reduced, the amount of bacteria in the tissues was reduced, and the morphology of the granulomas was significantly alleviated; in vitro experiments: it was found that Gal3 knockout primary cultured macrophages were infected MmAfter that, the number of intracellular bacteria can be effectively reduced. The same conclusion can be obtained by knocking down Gal3 using siRNA technology (in mouse macrophage Raw264.7 or primary cultured macrophages). Knocking down Gal3 in vitro may also promote the lysosome and macrophage Mm The fusion of the above mentioned compounds can achieve the effect of eliminating pathogens. See Example 5.

[0019] (3) Confirmation of the mechanism by which Gal3 promotes granuloma formation: In vitro experiments revealed that: Mm Bacteria induce the production of Gal3, which can inhibit the IKKα / β→NF-κB→TNFα pathway; knocking out Gal3 can activate the IKKα / β→NF-κB→TNFα pathway. Gal3 knockdown can also promote inflammatory response, thereby accelerating the clearance of pathogens. Gal3 overexpression (plasmid) can reduce the production of pro-inflammatory factors (TNF-α, iNOS) and increase the production of anti-inflammatory factors (TGF-β), which will be detrimental to the clearance of pathogens. See Example 6.

[0020] (4) TD139 reduces the formation of tuberculous granulomas and the amount of bacteria carried by inhibiting Gal3: Based on the research foundation of the first three parts, Gal3 is an important target for the treatment of tuberculous granulomas, and inhibiting Gal3 is an important therapeutic idea for reducing tuberculous granulomas. The present invention uses the Gal3-specific inhibitor TD139 to verify its anti-granuloma effect in animals. Through in vivo experiments (wild bacteria Mm Ten days after infection, mice were injected intraperitoneally with TD139 five times, 200 μg each time, once every other day. It was found that TD139 could significantly reduce Mm The area of ​​skin granuloma induced by bacteria was reduced, and the amount of bacteria in tail granuloma was reduced (microscopic morphology, quantitative count), and its effect was close to that of low-virulence strain infection (△PDIM); TD139 could downregulate the expression of Gal3 and MMP-9 in granuloma tissue. See Examples 1 and 6. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 : Figure 1 A shows that Raw264.7 cells were infected with WT (wild type, virulent type) or mutant bacteria (PDIM, virulence-deficient type), and cytoplasmic and nuclear proteins were extracted respectively. A quantitative proteomics method based on amino acid stable isotope labeling (AACT / SILAC) in cell culture was used to find that the differential protein Galectin-3 was significantly increased in the WT infection group. Figure 1B is the western blot method to verify the high expression of Gal3 in the WT group (two repeated experiments). Group 1C is the laser confocal microscopy found that the WT group Gal3 was highly expressed in Raw264.7 cells (4 hours after inoculation), while the mock infection group (mock) and △PDIM group Gal3 expression was very low.

[0022] Figure 2 : Figure 2 A shows the intracellular Gal3 levels of Raw264.7 cells after infection with WT group and different concentrations of PDIM groups (ELISA method, cell lysate was collected). Figure 2 B shows the intracellular bacterial counts of Raw264.7 cells after infection with WT group and different concentrations of PDIM groups (the bacteria were labeled with tdTomato red fluorescent protein, directly infected with Raw264.7 cells, and directly counted under a laser confocal microscope). Figure 2 C shows the total Gal3 expression of Raw264.7 cells infected with WT group and different concentrations of PDIM groups (cell supernatant + cell lysate were collected, counted separately and then superimposed).

[0023] Figure 3 : Infection of different species (mice, rabbits, marmosets, humans), infection with human Mycobacterium tuberculosis (MTB), infection of zebrafish Mm HE staining, Gal3 staining (immunohistochemistry), and Ziehl-Neelsen (ZN) staining (red shows tuberculosis bacteria) of the tuberculosis granuloma.

[0024] Figure 4 : Figure 4 A is the gross view of the tail of wild-type (Gal3+ / +) and Gal3 systemic knockout mice (Gal3- / -) 21 days after WT bacteria were injected into the tail vein; Figure 4 B is the statistics of the skin ulcer area of ​​the mouse tail at different time points (3, 7, 14, and 21 days) after WT bacteria or △PDIM bacteria were injected into wild-type (Gal3+ / +) or Gal3 systemic knockout mice (Gal3- / -) through the tail vein.

[0025] Figure 5 : Figure 5 A shows the bacterial content in the granuloma after WT bacteria and △PDIM bacteria were injected into wild-type or Gal3 knockout mice, respectively (unit: CFU / g). Figure 5 B Figure 5 A is a section of a granuloma (HE staining).

[0026] Figure 6 : Figure 6A: Primary cultures of bone marrow-derived macrophages (BMDM) from wild-type or Gal3 knockout mice were infected with WT bacteria or PDIM bacteria, and the number of intracellular bacteria was calculated. Figure 6 B shows the effect of knocking down Gal3 in BMDM cells (siRNA technology) on the number of intracellular bacteria in BMDM after infection with WT bacteria or △PDIM bacteria.

[0027] Figure 7 : Figure 7 A is the effect of overexpression (transient transfection with Gal3 plasmid) or knockdown (siRNA technology) of Gal3 expression of Raw264.7 on the fusion between LAMP1 (lysosomal marker) and intracellular bacteria (WT or △PDIM bacteria). Figure 7 B is correct Figure 7 LAMP1 and Mm Statistical analysis of colocalization.

[0028] Figure 8 : Figure 8 A is the knockdown of Gal3 expression in Raw264.7 cells to observe the effect of WT bacteria and △PDIM bacteria on the mRNA expression of inflammatory factors in Raw264.7 cells (qRT-PCR method); Figure 8 B is the separation of bone marrow-derived macrophages (BMDM) from Gal3+ / + and Gal3- / - mice, which were infected with WT or △PDIM bacteria for 4 hours, and the mRNA expression of inflammatory factors in BMDM was observed (qRT-PCR method).

[0029] Fig. 9 : Fig. 9 A shows the expression of TNF-α and TGF-β (qRT-PCR method) in primary culture of bone marrow-derived macrophages (BMDM) from wild-type or Gal3 knockout mice after adding different strains. Fig. 9 B shows the MRNA expression of inflammatory factors in Raw264.7 cells after transient transfection of Gal3 plasmid (qRT-PCR method).

[0030] Fig.10 : Fig.10 A is the molecular structure of TD139. Fig.10 B shows the changes in tail skin ulcers after WT bacteria or △PDIM bacteria were injected into the tail vein of wild-type mice with or without TD139 treatment.

[0031] Fig.11 : Fig.11 A is the statistics of the skin ulcer area of ​​mice induced by wild-type bacteria or △PDIM mutant bacteria after TD139 treatment. Fig.11 B is the number of bacteria contained in the tail tissue of mice induced by wild-type or mutant bacteria after TD139 treatment.

[0032] Fig.12 : HE staining and ZN staining of skin ulcers in mice infected with wild-type bacteria (WT), mice infected with △PDIM mutant bacteria, and mice infected with wild-type bacteria after TD139 treatment.

[0033] Fig.13 : Gal3 staining (Figure A) and MMP-9 staining (Figure B) of skin ulcers in mice infected with wild-type bacteria (WT), △PDIM mutant bacteria, and wild-type bacteria-infected mice after TD139 treatment (immunohistochemistry, DAB-stained brown areas indicate gene expression). DETAILED DESCRIPTION

[0034] Example 1 Preparation of TD139 solution (for animal experiments) Take TD139 (MCE, product number HY-19940, MW: 648.64) 10 mg + DMSO 15.4169 mL (dimethyl sulfoxide, cell culture grade, purity> 99.5%, Sigma, product number D2650-100 mL, the same below), dissolve into 1 mM storage solution, store the storage solution at -20°C for 1 month, and at -80°C for 6 months; Example 2 Experiments on infection of macrophage cell lines with wild-type or mutant bacteria Phthiocerol dimycocerosate (PDIM) is one of the lipid components of the tuberculosis wall (including Mycobacterium marinum and Mycobacterium tuberculosis), which is closely related to pathogenicity. In this study, wild-type (WT, strong virulence) or PDIM mutant (i.e., unable to synthesize PDIM, so mild virulence) was used to infect mouse macrophage cell line (Raw264.7), and total protein, cytoplasmic protein, and nuclear protein were extracted 4 hours after infection.

[0035] 2.1 Figure 1 A shows the identification of wild-type by quantitative proteomics based on amino acid stable isotope labeling in cell culture (AACT / SILAC) Mm Can induce high expression of Gal3 in the cytoplasm.

[0036] 2.2 Figure 1 B shows the detection of Gal3 expression in the total protein of Raw264.7 by immunoblotting, verifying that wild bacteria can induce increased Gal3 expression in macrophage cell lines.

[0037] 2.3 Figure 1 C shows the results of immunofluorescence analysis, which showed that wild-type bacteria could induce high expression of Gal3 in the cytoplasm and nucleus of Raw264.7 cells compared with mutant bacteria and sham infection groups.

[0038] 2.4 Figure 2 A shows the Gal3 content in macrophages at different time points (0, 0.5, 1, 2, 3, 4h) after infection with wild-type bacteria or mutant bacteria (the supernatant was discarded, cell lysate was added, and the Gal3 content in the cells was detected by ELISA). It was found that the wild-type bacteria had significantly increased Gal3. Figure 2 B shows the intracellular bacterial content at different time points (0, 0.5, 1, 2, 4, 6, 8 h) after infection with wild-type bacteria or mutant bacteria (bacteria were fluorescently labeled with tdTomato and the number of bacteria was quantified by laser confocal microscopy). It was found that the wild-type bacteria had significantly increased Gal3. Figure 2 C shows the intracellular and supernatant Gal3 content at different time points (0, 0.5, 1, 2, 3, 4 h) after infection with wild-type or mutant bacteria. It was found that wild-type bacteria significantly increased total Gal3, and mutant bacteria also caused an increase in total Gal3. MOI is the dose of infected bacteria.

[0039] Example 3 Expression of Gal3 in different species of animals infected with Mycobacterium tuberculosis Different species infection models (mice, rabbits, marmosets, humans infected with Mycobacterium tuberculosis (MTB); zebrafish infected with Mm ) of tuberculosis granuloma, HE staining, Gal3 staining (immunohistochemistry, DAB shows brown-yellow granules), and Ziehl-Neelsen (ZN) staining (acid-fast staining, red shows tuberculosis bacteria).

[0040] Figure 3 The results showed that Gal3 was highly expressed in tuberculous granulomas of different species, and MTB or Mm bacteria.

[0041] Example 4 Study on the inoculation of Gal3 knockout mice with different types of tuberculosis WT bacteria and PDIM bacteria were microinjected into wild-type mice or Gal3 knockout mice, and tail tissues were taken at different time points (0, 7, 14, and 21 days) to measure the following indicators: 4.1 Figure 4 A shows the general view of the tail (day 21). The results show that the tail of wild-type mice is more ulcerated, while the tail damage of Gal3 knockout mice is relatively mild.

[0042] 4.2 Figure 4 B pair Figure 5 Statistical analysis of the ulcer area on the tail skin of A. The results showed that the ulcer area of ​​wild-type mice inoculated with WT bacteria was the largest, while the ulcer area of ​​Gal3 knockout mice was smaller; the ulcer area of ​​wild-type or knockout mice inoculated with △PDIM was smaller.

[0043] 4.3 Figure 5 A shows the tail sampling to measure the amount of Mycobacterium marinum in the tissues of different groups. The results showed that the ulcer area of ​​wild-type mice inoculated with WT bacteria increased with time, while the ulcer area of ​​WT knockout mice, or mutant bacteria in wild-type or knockout mice decreased with time.

[0044] 4.4 Figure 5 B Figure 5 HE staining of the tail skin ulcers of the four groups in A. The results showed that only when WT bacteria were inoculated into wild-type mice would thick-walled granulomas be formed, which contained a large number of Mycobacterium marinum, but inflammatory cells could not infiltrate into the granulomas.

[0045] Example 5 Experiment on the effect of down-regulating macrophage Gal3 expression on the clearance of Mycobacterium marinum This study used two methods: (1) Primary cultured bone marrow-derived macrophages (BMDM) (derived from wild-type mice or Gal3 knockout mice) were infected with WT bacteria or △PDIM bacteria, and the content of intracellular bacteria was observed; (2) siRNA technology was used to knock down Gal3 expression in BMDM of wild-type mice, and WT bacteria or △PDIM bacteria were infected to observe the content of intracellular bacteria, cell immunofluorescence was used to observe the co-localization of Mycobacterium marinum and lysosomes, cell RNA was extracted to detect the expression of inflammatory genes (mRNA), and total protein was extracted to detect protein expression (Western blotting).

[0046] 5.1 Figure 6 A shows Gal3 + / + WT bacteria were the most abundant in macrophages, while Gal3 - / - There are fewer WT bacteria in macrophages. There are fewer △PDIM in both cells. The results show that high expression of Gal3 reduces the clearance of pathogens by host macrophages, while Gal3 knockout is beneficial for host cells to clear pathogens.

[0047] 5.2 Figure 6 B shows that the WT bacteria in macrophages are the most, while the WT bacteria in Gal3 knockdown macrophages are less. Both cells have less △PDIM. The results show that high expression of macrophage Gal3 reduces the host macrophage's clearance of pathogens, while Gal3 knockdown is conducive to the clearance of pathogens by macrophages.

[0048] 5.3 Figure 7 A shows that Gal3 knockdown (siRNA) or Gal3 overexpression (transient Gal3 wild-type plasmid) was introduced into Raw264.7 cells, and then Mycobacterium marinum ( Mm ) After 4 hours, the co-localization of bacteria and lysosomes was observed. The results showed that when Gal3 was overexpressed or under normal conditions, Mm Bacteria can induce Gal3 expression and inhibit the fusion of lysosomes and tuberculosis bacteria to form phagolysosomes. Knockdown of Gal3 promotes the formation of phagolysosomes, which is beneficial for the clearance of Mm .

[0049] 5.4 Figure 7 B is correct Figure 8 Statistical analysis of A. Raw264.7 cells overexpressing Gal3 had no significant difference from the control group, but knocking down Gal3 in Raw264.7 cells increased the expression of lysosomal markers LAMP1 and Mm Co-localization of bacteria.

[0050] 5.5 Figure 8 A is the effect of knocking down the Gal3 expression of Raw264.7 by siRNA technology on the mRNA expression of inflammatory factors after WT bacteria and △PDIM bacteria infected Raw264.7 cells (qRT-PCR method). The results showed that in response to WT bacteria infection, macrophage Raw264.7 inhibited the expression of pro-inflammatory genes (TNF-α, iNOS) and increased the expression of anti-inflammatory genes (TGF-β, IL-10) (which will inhibit the clearance of pathogens and cause immune escape). However, in response to WT bacteria infection, RAW264.7 with low Gal3 expression increased the expression of pro-inflammatory genes (TNF-α, iNOS) and reduced the expression of anti-inflammatory genes (TGF-β, IL-10) (which will be beneficial to the clearance of pathogens). △PDIM is weaker than wild bacteria, and it increases the expression of pro-inflammatory genes in macrophages and reduces the expression of anti-inflammatory genes.

[0051] 5.6 Figure 8 B is the Gal3 expression of BMDM cells knocked down by siRNA technology and then infected Mm The expression of inflammatory factors mRNA in host cells was observed (qRT-PCR). Fig. 9 A is the same.

[0052] 5.7 Fig. 9 A is in Fig. 9 B, total proteins were extracted respectively, and the inflammatory pathway was detected by immunoblotting. The results showed that compared with wild-type host cells (BMDM macrophages), Gal3 knockout host cells could promote the activation of their inflammatory pathways (IKKα / β, NF-kB p65), which would be beneficial to the elimination of pathogens.

[0053] 5.8 Fig. 9 B is the detection of inflammatory gene mRNA (qRT-PCR method). Wild-type Raw264.7 cells were transfected with Gal3 plasmid or infected with △PDIM bacteria. The results showed that increasing Gal3 expression can inhibit the expression of pro-inflammatory genes (TNF-α, iNOS) and increase the expression of anti-inflammatory genes (TGF-β).

[0054] Example 6 TD139 Intervention Mm Evaluation of therapeutic efficacy in a mouse model of bacterial infection First, use Mycobacterium marinum (wild bacteria or △PDIM bacteria, inject 5×10 8 Bacteria, dissolved in 100 μL PBS) were injected once into the tail vein of C57BL / 6J mice (8-10 weeks old, female, SPF grade). TD139 (MCE, dissolved in 0.5% DMSO) was given 10 days after infection (200 μg per mouse, once every 2 days, for 5 times) (molecular structure see Fig.10 A). Then take pictures and remove the tail for morphological examination.

[0055] 6.1 Fig.10 B. General view of the tails of mice in each group (10th and 20th days). The results showed that the tail skin of mice infected with WT bacteria would have severe ulceration, which was most obvious on the 20th day. TD139 treatment could significantly improve the ulcer area. △ The tail skin of mice infected with PDIM bacteria would have milder ulceration, which was most obvious on the 20th day. No ulcer wounds were seen after TD139 treatment.

[0056] 6.2 Fig.11 A pair Fig.11 The skin ulcer area of ​​B was statistically analyzed and the same results were obtained.

[0057] 6.3 Fig.11 B: Samples were taken from the mouse tails in Figure 1 to measure the bacterial content per unit tissue. The results showed that TD139 can significantly reduce the amount of bacteria in the tissue after infection with wild bacteria.

[0058] 6.4 Fig.12 HE staining and ZN staining of mouse tails are shown. HE staining shows that WT bacteria can significantly induce granuloma formation, which is more severe than PDIM. TD139 can significantly reduce the formation of granulomas. ZN staining (acid-fast staining) shows tuberculosis bacteria in granulomas. TD139 can significantly reduce the number of bacteria in tissues after WT bacteria infection.

[0059] 6.5 Fig.13 A and 13B show the expression of Gal3 and MMP-9 in each group by immunohistochemistry. MMP-9 has been shown to recruit macrophages and promote granuloma formation. The results showed that the expression of Gal3 and MMP-9 in the WT bacterial group was significantly increased, significantly higher than that in the △PDIM group. TD139 can significantly reduce the expression of Gal3 and MMP-9 in tissues. TD139 can significantly reduce the expression of MMP-9 in granulation tissue. These prove that TD139 can inhibit granuloma formation.

Claims

1. A Galectin-3 inhibitor, characterized in that The inhibitor is TD139.

2. A granuloma inhibitor, characterized in that The inhibitor is TD139.

3. Use of a Galectin-3 inhibitor in the preparation of a drug for treating tuberculous granuloma, characterized in that: The inhibitor is TD139.

4. The use according to claim 3, characterized in that The inhibitor TD139 is used in drugs for reducing tuberculosis induced by Mycobacterium marinum.

5. The use according to claim 3, characterized in that: The inhibitor TD139 is used in the preparation of a drug for reducing tuberculosis induced by Mycobacterium marinum or Mycobacterium tuberculosis.

6. The use according to claim 5, characterized in that The inhibitor TD139 is used in the preparation of a drug for reducing one or more of the granuloma area, intracellular bacterial load, and inflammatory factor expression of tuberculosis tail skin ulcers.

7. The use according to claim 5, characterized in that The TD139 is used in the preparation of a drug for promoting macrophages to clear pathogens and reduce the expression of MMP-9.

8. The use according to any one of claims 3 to 7, characterized in that: The inhibitor TD139 is used in the preparation of a drug for treating tuberculous granuloma by injection.

9. The use according to claim 8, characterized in that In the preparation of the drug for treating tuberculous granuloma, the subjects suffering from tuberculous granuloma disease include mammals and fish; The mammals include mice, rabbits, monkeys, humans, horses, cats, sheep, and pigs; the fish include zebrafish.

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