Application of LGALS1 and LGALS3 as targets in preparation of medicine for treating aplastic anemia

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of LGALS1 and LGALS3 as targets in preparation of medicines for treating aplastic anemia. The LGALS1 gene and the LGALS3 gene are remarkably and highly expressed in the abnormal activation process of the CD8 + T lymphocyte, and the result of the embodiment shows that the combined inhibitor Thiododigalacside of the Galectin-1 and the Galectin-3 can be competitively combined with a substrate of GAL to inhibit the activity of the Gal-1 and the Gal-3, so that the bone marrow infiltration capacity of the CD8 + T lymphocyte is slowed down, and the progress of AA diseases of mice is delayed. The invention provides a new application of the Galectin-1 and Galectin-3 combined inhibitor Thiododigalactoside, and provides a new drug treatment strategy for the treatment of AA (Acetic Acid).

Description

Technical Field

[0001] The present invention belongs to the technical field of medicine, and particularly relates to the application of LGALS1 and LGALS3 as targets in the preparation of drugs for treating aplastic anemia. Background Art

[0002] Aplastic anemia (AA) is a type of anemia characterized by pancytopenia caused by the decline or loss of bone marrow hematopoietic function. Pathologically, it is characterized by an "empty" bone marrow stroma (extremely few hematopoietic stem cells), and clinically, it is mainly manifested as anemia, bleeding, and infection. Currently, there are mainly two treatment methods for AA. For patients under 50 years old with a human leukocyte antigen (HLA)-matched sibling donor, hematopoietic stem cell transplantation (HSCT) is the first choice, and its cure rate is about 90%. However, age, organ function, matching donors, immune rejection reactions, and economic factors greatly limit its application. The second treatment option is immunosuppressive therapy (IST). The first-line IST treatment regimen of antithymocyte globulin (ATG) combined with cyclosporin A (CsA) can relieve the condition of 70% of patients. However, according to the follow-up survey results after treatment, 30% - 40% of patients experience recurrence, and the response rate of using IST again after recurrence is only about 25%; 15% - 20% of patients develop clonal hematological abnormalities. Although the application of HSCT and IST has greatly improved the survival rate of AA patients, the overall treatment effect of AA is still not ideal, the complete cure rate is less than half, and the problems of drug resistance and long-term suffering of patients are prominent. Therefore, there is an urgent need to develop more drugs or treatment regimens for AA. Summary of the Invention

[0003] The purpose of the present invention is to provide the application of LGALS1 and LGALS3 as targets in the preparation of drugs for treating aplastic anemia. By jointly inhibiting the functions of Galectin-1 and Galectin-3, the bone marrow infiltration ability of CD8 + T lymphocytes can be slowed down, and the progression of AA disease can be delayed.

[0004] The present invention provides the application of LGALS1 and LGALS3 as targets in the preparation of drugs for treating aplastic anemia.

[0005] As a preferred embodiment, the drug comprises inhibitors of Galectin-1 and Galectin-3; the inhibitor comprises a combined inhibitor of Galectin-1 and Galectin-3; alternatively, the inhibitor comprises an inhibitor of Galectin-1 and an inhibitor of Galectin-3.

[0006] As a preferred embodiment, the combined inhibitor comprises D-galactopyranosyl-β-D-thiogalactopyranoside.

[0007] As a preferred embodiment, the only active ingredient of the drug is D-galactopyranosyl-β-D-thiogalactopyranoside.

[0008] As a preferred embodiment, the drug is a unit dosage form, and based on body weight, the unit dosage form is formulated into a dosage form for administering D-galactopyranosyl-β-D-thiogalactopyranoside ≥ 10 mg / kg.

[0009] As a preferred embodiment, the effects of the drug include at least one of the following:

[0010] (1) Restoration of blood routine indexes;

[0011] (2) Inhibition of bone marrow infiltration of T lymphocytes;

[0012] (3) Increase in the number of nucleated bone marrow cells.

[0013] As a preferred embodiment, the blood routine indexes include at least one of white blood cells, platelets, red blood cells, and hemoglobin.

[0014] As a preferred embodiment, the T lymphocytes include CD8 + T lymphocytes.

[0015] As a preferred embodiment, the dosage form of the drug includes an injection.

[0016] As a preferred embodiment, the aplastic anemia includes immune-mediated aplastic anemia.

[0017] Beneficial effects: The present invention provides the use of LGALS1 and LGALS3 as targets in the preparation of drugs for treating aplastic anemia. The LGALS1 and LGALS3 genes are in CD8 +It is significantly highly expressed during the abnormal activation process of T lymphocytes. The LGALS1 and LGALS3 genes encode galactose-binding protein 1 (Galectin-1, abbreviated as Gal-1) and galactose-binding protein 3 (Galectin-3, abbreviated as Gal-3), respectively. The results of the examples show that using the combined inhibitor of Galectin-1 and Galectin-3, D-galactopyranosyl-β-D-thiogalactopyranoside (Thiodigalactoside, TDG), can slow down the + bone marrow infiltration ability of CD8 T lymphocytes and delay the progression of AA disease in mice. The present invention provides a new application of the combined inhibitor Thiodigalactoside of Galectin-1 and Galectin-3, providing a new drug treatment strategy for the treatment of AA. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments.

[0019] Figure 1 It is a single-cell sequencing and LGALS1 and LGALS3 expression result diagram; where A is the gene with significant changes in single-cell sequencing; B is the real-time quantitative PCR detection result of LGALS1 and LGALS3;

[0020] Figure 2 It is a diagram of the indexes of mice in each group; where A is the body weight of mice in each group; B is the survival time of mice in each group; C is the white blood cell count; D is the platelet count; E is the red blood cell count; F is the hemoglobin amount; G is the spleen weight of mice; H is the spleen cell count; I is the bone marrow cell count; * indicates significant difference in data, p < 0.05; ** indicates significant difference in data, p < 0.01; *** indicates extremely significant difference in data, p < 0.001;

[0021] Figure 3 It is a diagram of the proportion of different T cell subtypes in mice in each group; where A is the proportion of CD3 T lymphocytes in bone marrow cells; + the proportion of CD8 T lymphocytes; B is the proportion of CD8 T lymphocytes in bone marrow cells; + the proportion of CD4 T lymphocytes; C is the proportion of CD4 T lymphocytes in bone marrow cells; + the proportion of CD8 T lymphocytes; D is the proportion of CD8 T lymphocytes in bone marrow cells; + and CD4 T lymphocyte ratio; E is the proportion of CD3 T lymphocytes in spleen cells; + the proportion of CD8 T lymphocytes; F is the proportion of CD8 T lymphocytes in spleen cells; + the proportion of CD4 T lymphocytes; G is the proportion of CD4 T lymphocytes in spleen cells; + the proportion of CD8 T lymphocytes; H is the proportion of CD8 T lymphocytes in spleen cells; + the proportion of CD4 T lymphocytes; I is the proportion of CD8 T lymphocytes in spleen cells; + and CD4 T lymphocyte ratio;+ Ratio of T lymphocytes; * indicates significant difference in data, p < 0.05; ** indicates significant difference in data, p < 0.01; *** indicates extremely significant difference in data, p < 0.001;

[0022] Figure 4 These are HE staining images of the bone marrow of mice in each group. Detailed implementation manners

[0023] The present invention provides the use of LGALS1 and LGALS3 as targets in the preparation of drugs for treating aplastic anemia. As an implementation manner, the aplastic anemia described in the present invention includes immune-mediated aplastic anemia. Currently, there are mainly four mechanisms for the pathogenesis of aplastic anemia: abnormal hematopoietic stem cells, disordered bone marrow microenvironment, abnormal immune cells / molecules, and genetic predisposition. Among them, immune-mediated aplastic anemia is considered the main cause of the disease, mainly manifested as a significant inversion of the ratio of cytotoxic T lymphocytes CD4 + / CD8 + The ratio of which is significantly inverted, and on the one hand, activated CD8 + T lymphocytes can promote their own proliferation, and on the other hand, induce apoptosis of hematopoietic stem cells through the synergistic action of highly expressed IFN-γ, TNF and Fas / FasL. The LGALS1 and LGALS3 genes encode galactose-binding protein 1 (Galectin-1, abbreviated as Gal-1) and galactose-binding protein 3 (Galectin-3, abbreviated as Gal-3) respectively. These two proteins, Gal-1 and Gal-3, both belong to the galactose-binding protein family and play key roles in regulating immune responses, cell signal transduction, tumor development, apoptosis, and metabolic processes. The present invention uses single-cell sequencing technology to track the activation process of CD8 + T cells in the AA mouse model and finds that the LGALS1 and LGALS3 genes are significantly highly expressed during the abnormal activation process of CD8 + T cells, and LGALS1 and LGALS3 can be used as targets to prepare drugs for treating aplastic anemia.

[0024] As an embodiment, the drug of the present invention comprises inhibitors of Galectin-1 and Galectin-3; the inhibitor comprises a combined inhibitor of Galectin-1 and Galectin-3; or, the inhibitor comprises an inhibitor of Galectin-1 and an inhibitor of Galectin-3. As an embodiment, the combined inhibitor comprises D-galactopyranosyl-β-D-thiogalactopyranoside. D-galactopyranosyl-β-D-thiogalactopyranoside (Thiodigalactoside, TDG) is an orally active and effective galectin (GAL) inhibitor, which can inhibit the binding of GAL to β-galactoside through competitive binding. The Kd values for Gal-1 and Gal-3 are 24 μM and 49 μM respectively, and it can effectively inhibit the activities of Gal-1 and Gal-3.

[0025] As an embodiment, the only active ingredient of the drug of the present invention is D-galactopyranosyl-β-D-thiogalactopyranoside. As an embodiment, the drug is a unit dosage form, and calculated by body weight, the unit dosage form is formulated into a dosage form for administering D-galactopyranosyl-β-D-thiogalactopyranoside ≥ 10 mg / kg. The inventors found in previous studies that when the injection amount of Thiodigalactoside was 5 mg / kg calculated by mouse body weight, it had no effect on the recovery of various indicators of AA model mice. In a specific embodiment of the present invention, the injection amount of Thiodigalactoside is 10 mg / kg calculated by mouse body weight.

[0026] As an embodiment, the effects of the drug include at least one of the following: (1) recovery of blood routine indicators; (2) inhibition of bone marrow infiltration of T lymphocytes; (3) increase in the number of nucleated bone marrow cells. As an embodiment, the blood routine indicators include at least one of white blood cells, platelets, red blood cells and hemoglobin. In a specific embodiment of the present invention, after AA model mice are administered TDG, white blood cells (WBC), platelets (PLT), red blood cells (RBC), and hemoglobin (HGB) in the blood routine are significantly recovered.

[0027] As an embodiment, the T lymphocytes include CD8 + T lymphocytes. The inhibitor Thiodigalactoside of Galectin-1 and Galectin-3 of the present invention can slow down the bone marrow infiltration ability of CD8 + T cells and delay the progression of AA disease.

[0028] As an implementation manner, the dosage form of the drug includes an injection. The inventors found in previous studies that oral Thiodigalactoside had no obvious effect on the recovery of various indicators in AA mice. Therefore, an injection was selected as the dosage form of the drug.

[0029] To further illustrate the present invention, the application of LGALS1 and LGALS3 as targets in the preparation of drugs for treating aplastic anemia will be described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the protection scope of the present invention.

[0030] Example 1

[0031] Construct an AA model

[0032] ① Hybridize C57BL / 6 male mice with Babl / c male and female mice to breed first-generation CB6F1 mice. Select CB6F1 mice over 2 months old to establish an AA model (the modeling method reference: Chen J, Lipovsky K, Ellison FM, Calado RT, Young NS. Bystander destruction of hematopoietic progenitor and stem cells in a mouse model of infusion-induced bone marrow failure. Blood. 2004 Sep 15;104(6):1671-8. doi:10.1182 / blood-2004-03-1115.). Irradiate the CB6F1 mice with a total dose of 5 Gy under 60 Co-γ rays (ensuring a total dose of 5 Gy is sufficient, and there will be differences in the height and irradiation time of different models of radiation instruments).

[0033] ② Isolate the lymph node cells of C57BL / 6 male mice over two months old: Anesthetize the mice with isoflurane and then decapitate them. Isolate the inguinal and mesenteric lymph nodes and place them in cold PBS. Then transfer the lymph nodes to a 100 μm cell filter membrane. The cell filter membrane is placed on a well of a 6-well plate, and 5 mL of cold PBS is added to the bottom. Grind the lymph nodes with a cell grinding rod until there is no obvious tissue, collect the bottom liquid in a 15 mL centrifuge tube, centrifuge at 1500 rpm for 5 min, discard the supernatant, resuspend the cells with 1 mL of pre-cooled physiological saline, centrifuge again, discard the supernatant, and then resuspend the cells with 1 mL of pre-cooled physiological saline. Pipette 9 μL of the cell suspension and mix it with 1 μL of 10× trypan blue, and then count the cells on a hemocytometer. Finally, adjust the cell concentration to 5×10 6 / 100 μL to obtain a lymph node cell suspension.

[0034] ③ At 6 h after irradiating CB6F1 mice in step ①, inject 100 μL of the lymph node cell suspension in step ② into the mice through the tail vein using an insulin injection needle to construct an AA model.

[0035] ④ Select the bone marrow cells of AA mice after the onset of the disease and the lymph node cells of male C57BL / 6 mice for single-cell sequencing, and compare the expression values of CD8 + effector T lymphocytes in AA mice with CD8 + naive cells in the lymph node cells of male C57BL / 6 mice. The results are as Figure 1 shown in Figure A and Table 1.

[0036] Table 1 Genes with specific changes in CD8 + T cells in AA revealed by single-cell sequencing

[0037]

[0038] According to Figure 1 Figure A and Table 1, among the genes with significant changes in CD8 + effector T lymphocytes in AA mice compared with CD8 + naive T cells in the lymph node cells of male C57BL / 6 mice, LGALS1 and LGALS3 are the two genes with the most significant increase, indicating that LGALS1 and LGALS3 are increased in the CD8 + effector T lymphocytes of AA mice.

[0039] ⑤ Using GAPDH as an internal reference gene, use real-time quantitative QPCR to detect the expression of LGALS1 and LGALS3 in the lymph node cells (LIN) of male C57BL / 6 mice, the spleen (AA-spleen) and bone marrow cells (AA-BM) of AA mice 14 d after modeling.

[0040] A. Obtaining lymph node cell precipitate: Centrifuge the lymph node cell suspension in step ② to collect the lymph node cell precipitate.

[0041] B. Spleen treatment: Clean the surrounding tissue structures attached to the spleen and transfer it to a 100-μm cell filter membrane. The cell filter membrane is placed on one hole of a 6-well plate, 5 mL of PBS is added at the bottom, and the lymph node is ground with a cell grinding rod until there is no obvious tissue. Collect the bottom liquid in a 15-mL centrifuge tube, centrifuge at 1500 rpm for 5 min, discard the supernatant, resuspend the cells with 1 mL of red blood cell lysate, lyse at room temperature for 15 min, then add 9 mL of PBS, centrifuge at 1500 rpm for 5 min, repeat the lysis once, discard the supernatant after centrifugation, and resuspend the cells with 1 mL of PBS to obtain a spleen cell suspension, and centrifuge to collect the spleen cell precipitate.

[0042] C, Femur and tibia treatment: Thoroughly remove the muscle tissue around the femur and tibia. Use scissors to cut open both ends of the bone to expose the bone marrow cavity. Then, use an insulin syringe to aspirate 1 mL of PBS and insert it deep into the bone marrow cavity to flush the bone marrow into the wells of a 6-well plate. Flush it multiple times until there is no obvious red tissue in the bone marrow cavity. Collect the liquid in a 15 mL centrifuge tube and centrifuge it at 1500 rpm for 5 minutes. Discard the supernatant, resuspend the cells with 1 mL of red blood cell lysate, and lyse them at room temperature for 15 minutes. Then, add 9 mL of PBS, centrifuge it at 1500 rpm for 5 minutes, resuspend the cells with 1 mL of PBS to obtain a bone marrow cell suspension, and centrifuge to collect the bone marrow cell pellet.

[0043] D, RNA extraction: Add 1 mL of Trizol to the lymph node cell pellet, spleen cell pellet, and bone marrow cell pellet respectively to resuspend the cells and lyse them thoroughly for 5 minutes. Add 200 μL of chloroform, shake vigorously for 15 seconds, and then let it stand for 10 minutes. Then, centrifuge it at 13000 rpm at 4°C for 15 minutes. After centrifugation, transfer the centrifuge tube to ice, aspirate the top layer into a new centrifuge tube, add an equal volume of isopropanol, invert and mix well, and then let it stand for 10 minutes. Then, centrifuge it at 13000 rpm at 4°C for 10 minutes, pour out the supernatant, add 1 mL of 75% ethanol, invert and mix well, and then centrifuge it at 13000 rpm at 4°C for 5 minutes. Pour out the supernatant and completely dry the liquid in the tube. Dissolve the RNA by adding 20 - 50 μL of RNase-free water according to the amount of the precipitate. Aspirate 1 μL of the RNA solution and measure its concentration with a nucleic acid concentration detector. Then, reverse 2 μg of the RNA into cDNA using a reverse transcription kit (themo, 2996493). Then, mix it with the cDNA using a real-time quantitative PCR kit (ComWin Biotech, CW3360) and detect the expression levels of the LGALS1 and LGALS3 genes on a real-time quantitative PCR instrument. The relative expression of the genes is calculated based on the ΔΔCt method, and the results are as Figure 1 shown in B and Table 2.

[0044] The QPCR reaction system and procedure are set according to the general system and procedure of the kit. The primer sequences for QPCR are as follows:

[0045] LGALS1-F (SEQ ID NO.1): 5′-GCCTACACTTCAATCCTCGCT-3′;

[0046] LGALS1-R (SEQ ID NO.2): 5′-GTTCCCGGTGTTCGGTTCC-3′;

[0047] LGALS3-F (SEQ ID NO.3): 5′-GGAGAGGGAATGATGTTGCCT-3′;

[0048] LGALS3-R (SEQ ID NO.4): 5′-TCCTGCTTCGTGTTACACACA-3′;

[0049] GAPDH-F (SEQ ID NO.5): 5′-GACTTCAACAGCAACTCCCAC-3′;

[0050] GAPDH-R (SEQ ID NO.6): 5′-TCCACCACCCTGTTGCTGTA-3′.

[0051] Table 2 QPCR detection results

[0052] Group LIN AA-BM AA-spleen LGALS1 1±0.035 1.34±0.027 4.86±0.212 LGALS3 1±0.046 6.06±0.466 5.70±0.240

[0053] According to Figure 1 B in [reference] and Table 2, compared with the lymph node cells (LIN) of C57BL / 6 male mice, the expressions of LGALS1 and LGALS3 in the bone marrow cells (AA-BM) and spleen (AA-spleen) of AA mice were significantly up-regulated 14 days after modeling.

[0054] Example 2

[0055] Study on the therapeutic effect of Thiodigalactoside on AA mouse model

[0056] The experiments were set up as normal control group (N), total body irradiation control group (TBI), AA model control group (AA-CON), and AA model drug treatment group (AA-TDG); there were 8 mice in each of the AA model control group and the AA model drug treatment group, and 5 mice in each of the normal control group and the total body irradiation control group.

[0057] ① In the AA model drug treatment group, AA mice were intraperitoneally injected with drugs every day starting from 1 hour after modeling for 13 consecutive days, and the concentration of Thiodigalactoside was 10 mg / kg; the AA model control group, the normal and total body irradiation control groups were intraperitoneally injected with the same dose of normal saline every day. The body weight and health status of the mice in each group were detected every 3 days, and the results are shown in Figure 2 A in [reference] and Table 3. After two weeks, the survival time of the AA treated mice was evaluated as shown in Figure 2 B in [reference] and Table 4.

[0058] Table 3 Body weight of mice in each group

[0059] Time (d) N (g) TBI (g) AA-CON (g) AA-TDG (g) 0 27.96±0.33 27.92±0.4 28.06±0.39 28.54±0.38 3 28.22±0.48 27.5±0.51 26.48±0.36 26.77±0.26 6 28.14±0.48 28.1±0.61 27.75±0.39 28.12±0.34 9 28.24±0.37 27.5±0.54 24.4±0.49 24.65±0.34 12 28.68±0.53 27.56±0.33 24.35±0.63 24.03±0.36 14 28.16±0.65 27.42±0.4 24.08±0.94 23.68±0.73 15 28.94±0.69 27.3±0.29 22.16±0.79 23.12±1.05 16 28.84±0.59 27.4±0.39 22.08±0.71 23.98±0.39

[0060] According toFigure 2 As can be seen from Figure A and Table 3, compared with normal mice N and control irradiated mice TBI, the body weight of AA mice gradually decreased after 6 days of modeling, and the body weight began to recover on the 15th day after using TDG.

[0061] Table 4 Survival time of various mice

[0062] Group 10d 12d 14d 16d 18d 20d Normal mice N (number) 5 5 5 5 5 5 Control irradiated mice TBI (number) 5 5 5 5 5 5 AA-CON (number) 8 7 5 1 0 0 AA-TDG (number) 8 8 6 4 3 0

[0063] According to Figure 2 As can be seen from Figure B and Table 4, the average death time of AA mice was 14 days, and TDG could significantly delay the death time of AA mice.

[0064] ② Blood routine analysis: Living blood was collected from the submandibular vein of the mice. After two drops of blood were collected, hemostasis was performed with a cotton swab. The blood was placed in an EDTA anticoagulant tube and detected with a special blood routine detector for small animals. Record the values of white blood cells (WBC), red blood cells (RBC), platelets (PLT), and hemoglobin (HGB). The results are as Figure 2 shown in Figure C - F and Table 5.

[0065] Table 5 Detection results of blood routine and spleen weight of mice in each group

[0066]

[0067]

[0068] According to Figure 2 As can be seen from Figure C - F and Table 5, the white blood cells WBC, platelets PLT, red blood cells RBC, and hemoglobin HGB in the blood routine of AA mice were all significantly down - regulated, and each index of the blood routine significantly recovered after using TDG.

[0069] ③ Tissue infiltration analysis: After anesthetizing the AA model mice with isoflurane, they were sacrificed by cervical dislocation. The spleens, femurs, and tibias of the mice were separated. One pair of the two femurs and tibias was placed in neutral formalin for bone marrow pathological examination, and the other pair was used to extract bone marrow cells.

[0070] Treatment of the spleen: After cleaning the surrounding tissue structures attached to the spleen, it was weighed. The results are as Figure 2 shown in Figure G and Table 5; The operation of preparing the spleen cell suspension was carried out according to step ⑤ of Example 1 to obtain the spleen cell suspension; 20 μL of the spleen cell suspension was aspirated and counted on a full - automatic cell counter. The results are as Figure 2 shown in Figure H and Table 5.

[0071] Treatment of the femur and tibia: The operation of preparing the bone marrow cell suspension was carried out according to step ⑤ of Example 1 to obtain the bone marrow cell suspension. 20 μL of the bone marrow cell suspension was aspirated and counted on a full - automatic cell counter. The results are as Figure 2as shown in I and Table 5.

[0072] According to Figure 2 as can be seen from G - I and Table 5, the spleen weight of AA mice, and the cell numbers of spleen and bone marrow were significantly decreased, and all increased after using TDG, indicating that TDG has an obvious therapeutic effect on AA mice.

[0073] ④ Respectively pipette 100 μL of the spleen cell suspension and 100 μL of the bone marrow cell suspension from step ③, and add 1 μL (0.5 μg) of each of the PE - labeled CD3 + , FITC - labeled CD8 + , BV421 - labeled CD4 + flow - cytometry antibodies, incubate at 4°C for 30 min, then add 1 mL of PBS, centrifuge at 1500 rpm for 5 min, discard the supernatant, resuspend the cells with 300 μL of PBS, and then analyze the proportions of CD3 + , CD4 + , CD8 + in the total cells, and calculate the cell ratio of CD8 + to CD4 + . The results are as shown in Figure 3 and Table 6.

[0074] Table 6 Proportions of CD3 + , CD4 + , CD8 + in cells of each group

[0075]

[0076]

[0077] According to Figure 3 and Table 6, compared with normal mice N and control irradiated mice TBI, the proportions of CD3 + T lymphocytes, CD8 + T lymphocytes, CD4 + T lymphocytes in the bone marrow and spleen of AA mice were significantly increased, and the ratio of CD8 + T lymphocytes to CD4 + T lymphocytes increased. More than 60% of the cells in the AA bone marrow were infiltrated T lymphocytes. After using TDG, the proportions of these 3 types of T lymphocytes could be significantly decreased, and the ratio of CD8 + T lymphocytes to CD4 + T lymphocytes also decreased, indicating that TDG can inhibit the bone marrow infiltration of T lymphocytes. In the spleen, although the index of CD3 + T lymphocytes decreased significantly after drug administration, but the secondary subdivision of CD3 + T lymphocytes, CD4 +T lymphocytes and CD8 + The changes in the two indicators of T lymphocytes after drug administration were not significant, indicating that TDG inhibited CD8 to a certain extent on the one hand + The proliferation and activation of T lymphocytes, and on the other hand mainly inhibited the infiltration from the spleen to the bone marrow.

[0078] ⑤ Bone marrow pathological examination: HE pathological examinations of the bone marrow and tibial bone marrow were performed by Wuhan Sevier Biotechnology Co., Ltd., and the results were as Figure 4 shown.

[0079] According to Figure 4 It can be seen that compared with normal mice N and control irradiated mice TBI, most of the nucleated cells in the bone marrow of AA mice died and the number of adipocytes increased (white circles), which was similar to the bone marrow image of human AA patients; after treatment with TDG, the nucleated cells in the bone marrow increased significantly and the number of adipocytes decreased, indicating that TDG could significantly improve the proliferative ability of bone marrow cells and delay the onset of AA.

[0080] In summary, the LGALS1 and LGALS3 genes were significantly highly expressed during the abnormal activation of CD8 + T lymphocytes. The combined inhibitor of Galectin-1 and Galectin-3, Thiodigalactoside, could competitively bind to the substrates of GAL, inhibit the activities of Gal-1 and Gal-3, and then slow down the bone marrow infiltration ability of CD8 + T lymphocytes and delay the progression of AA disease in mice. The present invention provides a new application of the combined inhibitor of Galectin-1 and Galectin-3, Thiodigalactoside, and provides a new drug treatment strategy for the treatment of AA.

[0081] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, not all embodiments. People can also obtain other embodiments according to these embodiments without creative efforts, and these embodiments all belong to the protection scope of the present invention.

Claims

1. Application of LGALS1 and LGALS3 as targets in the preparation of drugs for the treatment of aplastic anemia.

2. The use according to claim 1, characterized in that: The drug includes inhibitors of Galectin-1 and Galectin-3; the inhibitor includes a combined inhibitor of Galectin-1 and Galectin-3; or, the inhibitor includes an inhibitor of Galectin-1 and an inhibitor of Galectin-3.

3. The use according to claim 2, characterized in that: The co-inhibitor includes D-galactopyranosyl-β-D-thiogalactopyranoside.

4. The use according to claim 3, characterized in that: The only active ingredient of the drug is D-galactopyranosyl-β-D-thiogalactopyranoside.

5. The use according to claim 3 or 4, characterized in that: The drug is a unit dose preparation, and the unit dose preparation is formulated in a dosage form for administration of D-galactopyranosyl-β-D-thiogalactopyranoside of ≥10 mg / kg based on body weight.

6. The use according to claim 1, characterized in that: The effects of the drug include at least one of the following: (1) Recovery of routine blood test indicators; (2) Inhibit T lymphocyte bone marrow infiltration; (3) Increase the number of bone marrow nucleated cells.

7. The use according to claim 6, characterized in that: The routine blood test index includes at least one of white blood cells, platelets, red blood cells and hemoglobin.

8. The use according to claim 6, characterized in that: The T lymphocytes include CD8 + T lymphocytes.

9. The use according to claim 1, characterized in that: The dosage form of the drug includes injection.

10. The use according to claim 1, characterized in that: The aplastic anemia includes immune-mediated aplastic anemia.

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