Application of 11-MT in preparation of medicine for treating acute B lymphocytic leukemia

Abstract

The invention belongs to the field of medical treatment, and discloses an application of 11-MT in medicine preparation. The medicine refers to a medicine for treating acute B lymphocytic leukemia. Researches show that 11-MT shows the following advantages in treatment of acute B lymphocytic leukemia: in an in-vitro test, 11-MT significantly promotes apoptosis of BALL-1 cells and shows dose dependence, 11-MT significantly promotes generation of ROS in the BALL-1 cells and shows dose dependence, 11-MT can cause DNA damage of the BALL-1 cells and shows dose dependence, and in an in-vivo test, 11-MT can significantly promote apoptosis of the BALL-1 cells and shows dose dependence. The 11-MT has a regulating effect on specific organ lesions, shows good repairing and protecting effects, can recover multiple serum biochemical indexes abnormally increased due to tumor load to a normal level, and can well reduce tumor cell infiltration.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to application of 11-MT in preparing medicine for treating acute B lymphocytic leukemia. Background Art

[0002] The chemical structure of 11-MT is shown in Formula 1 below:

[0003]

[0004] Formula 1;

[0005] Regarding the anti-tumor application of 11-MT, we have observed in previous applications that it can inhibit human myeloid leukemia HL-60.

[0006] The applicant has conducted in-depth research on this drug to explore its effect on acute B-lymphocytic leukemia.

[0007] It should be noted that HL-60 cells and BALL-1 cells correspond to myeloid leukemia and lymphocytic leukemia, respectively. There are obvious differences in the treatment strategies and mechanisms of these two diseases.

[0008] Leukemia is a malignant tumor of the hematopoietic system originating from hematopoietic stem and progenitor cells. It is mainly manifested by the massive proliferation of abnormal leukemia cells in the bone marrow and infiltration into other tissues and organs, resulting in the suppression of normal hematopoietic function. According to the degree of differentiation and natural course of leukemia cells, it can be divided into acute leukemia and chronic leukemia; according to the affected cell lines, it can be divided into myeloid leukemia (ML) and lymphocytic leukemia (LL). Myeloid leukemia includes acute myeloid leukemia (AML) and chronic myeloid leukemia (CML). Lymphocytic leukemia includes acute lymphocytic leukemia (ALL) and chronic lymphocytic leukemia (CLL) [1] .

[0009] HL-60 cells are a type of human promyelocytic leukemia cell with typical myeloid leukemia cell characteristics. They morphologically exhibit the characteristics of promyelocytic cells, with larger cells, prominent nucleoli, and strong proliferation ability.

[0010] BALL-1 cells are acute B lymphoblastic leukemia cells that originate from B lymphocytes and express specific B lymphocyte-associated antigens on their surface. Under a microscope, the cells are small in morphology and have a large nuclear to cytoplasmic ratio.

[0011] For acute myeloid leukemia represented by HL-60 cells, traditional treatment drugs include cytarabine, daunorubicin, etc. Cytarabine mainly acts on the S phase of cells, inhibiting DNA polymerase and hindering DNA synthesis, thereby inhibiting cell proliferation. Taking cytarabine as an example, its action channel mainly involves the DNA synthesis pathway. After cytarabine enters the cell, it is phosphorylated by kinase to form cytarabine triphosphate, which in turn inhibits DNA polymerase and interferes with the DNA synthesis process. [2] . Cisplatin is a commonly used chemotherapy drug. In the treatment of HL-60 cells, cisplatin can bind to DNA, forming intra-chain and inter-chain crosslinks, destroying the structure and function of DNA, and thus inhibiting cell proliferation. Studies have shown that HL-60 cells are sensitive to cisplatin, but with the passage of time and the mutation of tumor cells, they may develop drug resistance. Related studies have shown that cisplatin treatment experiments on HL-60 cells have found that a certain concentration of cisplatin can induce HL-60 cell apoptosis and inhibit its proliferation, but the specific effect will vary depending on the experimental conditions and cell status. [3-4] Bortezomib is a proteasome inhibitor. In HL-60 cells, it can inhibit the activity of the proteasome, leading to the accumulation of misfolded proteins in the cells, activating the unfolded protein response (UPR), and ultimately inducing cell apoptosis. Studies have shown that bortezomib has a certain killing effect on HL-60 cells and can inhibit their proliferation. As the concentration of bortezomib increases, the survival rate of HL-60 cells decreases significantly and the apoptosis rate increases. [5] .

[0012] For acute B lymphoblastic leukemia represented by BALL-1 cells, commonly used chemotherapy drugs include vincristine and prednisone. Vincristine mainly acts on the pre-mitotic phase (M phase) of cells, and by binding to tubulin, it prevents microtubule polymerization, thereby inhibiting the formation of spindles and causing cell division to stagnate in the M phase. Vincristine acts on tubulin and interferes with spindle formation during cell mitosis. Its action channel is closely related to the microtubule dynamics associated with cell mitosis; prednisone is a glucocorticoid that can induce lymphocyte apoptosis, regulate immune response, and inhibit tumor cell growth; cyclophosphamide metabolizes in the body to generate cytotoxic substances, destroy DNA structure, and interfere with cell proliferation. [6]In BALL-1 cells, cisplatin also works by binding to DNA. However, due to the difference in biological characteristics between BALL-1 cells and HL-60 cells, the effect of cisplatin on BALL-1 cells is not exactly the same as that on HL-60 cells. BALL-1 cells are relatively less sensitive to cisplatin, and a higher concentration of cisplatin or a combination of other drugs may be needed to achieve better therapeutic effect. It is possible that BALL-1 cells have unique biological characteristics, signaling pathways, and protein metabolism, and compared to acute myeloid leukemia cells (such as HL-60 cells), BALL-1 cells are relatively less sensitive to proteasome inhibitors [7]. Chemotherapy is the basic means of treating BALL-1 related diseases. Commonly used chemotherapy drugs include vincristine, prednisone, cyclophosphamide, etc. Combination chemotherapy regimens are generally used, such as the classic VDLP regimen (vincristine, daunorubicin, asparaginase, prednisone), which improves the therapeutic effect through the synergistic effect of multiple drugs. In the early stage of treatment, induction remission therapy aims to quickly eliminate a large number of leukemia cells so that the patient can achieve complete remission; then, through consolidation and intensive treatment, residual leukemia cells are further killed to reduce the risk of relapse. Some acute B lymphoblastic leukemias have specific genetic abnormalities, such as BCR-ABL fusion gene positivity. For such patients, tyrosine kinase inhibitors (TKI) such as imatinib and dasatinib are important therapeutic drugs. TKI drugs can specifically inhibit the tyrosine kinase activity of BCR-ABL fusion protein, block downstream signal transduction pathways, thereby inhibiting the proliferation of leukemia cells and inducing their apoptosis. Rituximab is a monoclonal antibody against CD20, which is widely expressed on the surface of B lymphocytes. Rituximab binds to the CD20 antigen on BALL-1 cells, killing leukemia cells through mechanisms such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). It is often used in combination with chemotherapy drugs to enhance the effectiveness of treatment. CAR-T therapy is an emerging and effective immunotherapy. The patient's own T cells are collected and genetically engineered in vitro to express chimeric antigen receptors that specifically recognize BALL-1 cell surface antigens (such as CD19). Once the engineered CAR-T cells are infused back into the patient, they can accurately recognize and kill BALL-1 cells. CAR-T therapy often achieves excellent therapeutic results for patients with relapsed or refractory B-cell acute lymphoblastic leukemia. Some immune checkpoint molecules play a role in the immune escape of leukemia cells. By using immune checkpoint inhibitors, such as antibodies against programmed death protein 1 (PD-1) and its ligand (PD-L1), the immune checkpoint signaling pathway can be blocked, restoring the body's immune system's ability to recognize and kill BALL-1 cells. However, its application in acute B-lymphocytic leukemia is still in the research and exploration stage. For patients with high-risk or relapsed and refractory BALL-1-related acute B-lymphocytic leukemia, allogeneic hematopoietic stem cell transplantation is an important treatment. By transplanting hematopoietic stem cells from healthy donors, the patient's hematopoietic and immune systems are rebuilt, thereby achieving the goal of curing the disease. [8] .

[0013] The sources of the literature mentioned above are as follows:

[0014] [1]Arber DA,Orazi A,Hasserjian R,Thiele J,Borowitz MJ,Le Beau MM,Bloomfield CD,Cazzola M,Vardiman JW.The 2016revision to the World HealthOrganization classification of myeloid neoplasms and acuteleukemia.Blood.2016May 19;127(20):2391-405.doi:10.1182 / blood-2016-03-643544.Epub 2016Apr 11.PMID:27069254.

[0015] [2]Pan Q,Wang J,Jiang X,Yang E,Dong L,Gu K.Apatinib enhanceschemosensitivity of acute myeloid leukemia hl60 cells to cytarabine byinducing apoptosis.J BUON.2019Jan-Feb;24(1):374-381.PMID:30941994.

[0016] [3]Dasari S,Tchounwou PB.Cisplatin in cancer therapy:molecularmechanisms of action.Eur J Pharmacol.2014Oct 5;740:364-78.doi:10.1016 / j.ejphar.2014.07.025.Epub 2014Jul 21.PMID:25058905;PMCID:PMC4146684.

[0017] [4]Velma V,Dasari SR,Tchounwou PB.Low Doses of Cisplatin Induce GeneAlterations,Cell Cycle Arrest,and Apoptosis in Human Promyelocytic LeukemiaCells.Biomark Insights.2016Aug 24:11:113–21.doi:10.4137 / BMI.S39445.PMID:27594783;PMCID:PMC4998075.

[0018] [5]KlikováK, A,PilchovaI,Hatok J,Chudy P,Chudej J,Dobrota D, P.Differential impact of bortezomib on HL-60and K562 cells.Gen PhysiolBiophys.2015Jan34(1):33-42.doi:10.4149 / gpb_2014026.Epub 2014Nov 4.PMID:25367763.

[0019] [6]Garg N,Farhat J,Dhankhar S,Chauhan S,Bala R,Madaan R,Sharma H,Saini M,Singh TG,Aldahish AA,Almarhoon Z,Al-Omari B,Sharifi-Rad J.Vincristine Cancer Therapy:Mechanisms,Efficacy,and Future Prospects.Curr MedChem.2024Sep 23.doi:10.2174 / 0109298673319496240911060138.Epub ahead ofprint.PMID:39318002.

[0020] [7] Sin CF, Man PM. The Role of Proteasome Inhibitors in Treating Acute Lymphoblastic Leukaemia. Front Oncol. 2021 Dec 23; 11: 802832. doi: 10.3389 / fonc.2021.802832. PMID: 35004327; PMCID: PMC8733464.

[0021] [8] Aureli A, Marziani B, Venditti A, Sconocchia T, Sconocchia G. Acute Lymphoblastic Leukemia Immunotherapy Treatment: Now, Next, and Beyond. Cancers (Basel). 2023 Jun 26; 15(13): 3346. doi: 10.3390 / cancers15133346. PMID: 37444456; PMCID: PMC10340788.

[0022] From the above analysis, it can be seen that acute B lymphoblastic leukemia is the disease with the highest incidence in acute lymphoblastic leukemia, as high as 85%. At present, the clinical treatment is mainly chemotherapy. However, chemotherapy can cause damage to normal tissue cells, and the activation of a single apoptosis pathway (such as the mitochondrial pathway) is easy to be escaped by tumor cells through mechanisms such as Bcl-2 overexpression, which is easy to induce chemotherapy resistance and lead to leukemia recurrence.

[0023] Therefore, finding natural products that can induce multiple cell apoptosis and have low toxicity is a new perspective to overcome leukemia recurrence, which is expected to solve the treatment problem of B-ALL. SUMMARY

[0024] The purpose of the present application is to provide the use of 11-MT in the preparation of a drug for treating acute B lymphoblastic leukemia. Through research, we found that 11-MT has the following advantages in the treatment of acute B lymphoblastic leukemia: in vitro tests, 11-MT significantly promotes BALL-1 cell apoptosis and shows dose dependence, 11-MT significantly promotes the generation of ROS in BALL-1 cells and shows dose dependence, 11-MT can cause DNA damage in BALL-1 cells and shows dose dependence, in vivo tests, 11-MT has a regulatory effect on specific organ lesions, 11-MT shows good repair and protection effects, and can restore multiple serum biochemical indicators abnormally elevated due to tumor load to normal levels, and 11-MT can better reduce tumor cell infiltration.

[0025] To achieve the above object, the present invention provides the following technical solutions:

[0026] The invention relates to the use of 11-MT in the preparation of a medicine; the medicine is a medicine for treating acute B lymphocytic leukemia.

[0027] 11-methoxytabersonine (11-MT), molecular formula C 22 H 20 N2O3, with a molecular weight of 366. This compound is a quebracho-type monoterpene indole alkaloid and a derivative of tabersonine. The difference is that the carbon atom at the 11th position is replaced by a methoxy group. This compound is mainly found in plants of the genus Citrus in the Apocynaceae family. Its structure is also based on the indole ring, with a methoxy group introduced at the 11th position of the indole ring. The presence of this methoxy group greatly affects the electron cloud distribution and steric hindrance of the molecule. The electron-donating effect of the methoxy group changes the electron cloud density on the indole ring, affecting the binding mode and affinity of the molecule to the target. At the same time, other parts of the molecule, such as the side chain structure connected to the indole ring, contain specific carbon-carbon double bonds and other functional groups. These structural features together determine the chemical properties and biological activity of 11-MT, which may be the reason why it exerts anti-tumor activity in the BALL-1 cell line.

[0028] In the above application, the drug is used to reduce the level of CD45 in bone marrow blood. + Cell ratio to reduce the proliferation and infiltration of acute B-lymphoblastic leukemia cells in the bone marrow.

[0029] In the above application, the drug refers to a drug that reduces tumor burden and repairs and protects liver and kidney functions during the treatment of acute B lymphocytic leukemia.

[0030] The beneficial effects of the present invention are:

[0031] 1. In in vitro tests, 11-MT significantly promoted apoptosis of BALL-1 cells in a dose-dependent manner; 11-MT significantly promoted ROS generation in BALL-1 cells in a dose-dependent manner; and 11-MT caused DNA damage in BALL-1 cells in a dose-dependent manner.

[0032] 2. In in vivo tests, 11-MT has a regulatory effect on specific organ lesions; 11-MT exhibits good repair and protection effects, and can restore multiple serum biochemical indicators that are abnormally elevated due to tumor burden to normal levels; 11-MT can effectively reduce tumor cell infiltration. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a bar graph showing the effect of 11-MT on the survival rate of HUVEC cells in Example 1;

[0034] Figure 2 This is a graph showing the results of 11-MT-induced apoptosis of BALL-1 cells in Example 1;

[0035] Figure 3 This is a graph showing the results of 11-MT-induced ROS generation in BALL-1 cells in Example 1;

[0036] Figure 4 This is the DNA comet image of BALL-1 cells induced by 11-MT in Example 1;

[0037] Figure 5 For Example 1, the volcano plot of differentially expressed genes is shown in Figure 5 The meaning of each figure is as follows: (A) 11-MT low-dose group vs. blank group (B) 11-MT medium-dose group vs. blank group (C) 11-MT high-dose group vs. blank group, green dots indicate mRNA downregulation, red dots indicate mRNA upregulation, and gray dots indicate no significant difference in mRNA;

[0038] Figure 6 GO enrichment analysis diagram of differentially expressed genes in Example 1;

[0039] Figure 7 This is the KEGG enrichment analysis diagram of differentially expressed genes in Example 1;

[0040] Figure 8 This is a cluster analysis diagram of differentially expressed genes in the EBV infection signaling pathway in Example 1;

[0041] Figure 9 This is a graph showing the weight change of mice after intraperitoneal administration for 14 consecutive days in Example 2;

[0042] Figure 10 This is a bar graph of organ indexes in mice after intraperitoneal administration for 14 consecutive days in Example 2;

[0043] Figure 11 This is a diagram showing the effect of 11-MT on the pathological changes of the heart, liver, spleen, lung, kidney, and brain tissues of mice in Example 2;

[0044] Figure 12 This is an analysis chart of blood biochemical indicators in mice after intraperitoneal administration for 14 consecutive days in Example 2;

[0045] Figure 13 Figure 2 shows the status of mice in the control group (A) and mice in the model group (B).

[0046] Figure 14In Example 2, flow cytometry was used to detect CD45 in peripheral blood (A) and bone marrow blood (B). + Cell ratio result diagram;

[0047] Figure 15 Figure 2 shows the morphological changes of spleen and liver in normal and model group mice in Example 2;

[0048] Figure 16 This is a graph showing the analysis results of serum biochemical indicators in mice after intraperitoneal injection of the drug in Example 2;

[0049] Figure 17 This is a histogram of mouse organ indexes in Example 2;

[0050] Figure 18 These are the histopathological images of the liver (A) and spleen (B) in Example 2;

[0051] Figure 19 In Example 2, CD45 + Scale drawing. DETAILED DESCRIPTION

[0052] Below in conjunction with embodiments of the present invention, technical scheme of the present invention is clearly and completely described, it is obvious that described embodiment is only a part of embodiment of the present invention, rather than whole embodiment. Based on the embodiment in the present invention, every other embodiment obtained by those of ordinary skill in the art without making creative work premise all falls within the scope of protection of the present invention. Reagents used or instrument do not indicate manufacturer, all are conventional products that can be purchased by commercially available acquisition.

[0053] Example 1

[0054] In vitro experiments

[0055] cell lines

[0056] Human B lymphocytic acute leukemia cells (BALL-1) were purchased from the Cell Bank of Type Culture Collection Committee of the Chinese Academy of Sciences, and human umbilical vein endothelial cells (HUVEC) were purchased from the ATCC Cell Bank.

[0057] 1.1 Effects of compounds on normal cell survival

[0058] HUVEC cells were selected and cultured at 2.5×10 4 Cells were seeded at a density of 1000 cells / well in a 96-well plate. After 24 hours, different concentrations of 11-MT were added to treat the cells. The cells were cultured for another 20 hours. 10% CCK-8 was added to the wells and incubated in the dark for 3-4 hours. The absorbance at 450 nm was measured with a microplate reader. The experiment was repeated three times for valid results. Figure 1As shown in Table 1, 11-MT had no significant effect on the cell viability of HUVECs at a concentration below 2.5 μM.

[0059] These results indicate that 11-MT exerts tumor growth inhibitory activity without causing toxic effects on normal cells.

[0060] Table 1 IC of 11-MT against lymphoid system malignant tumor cells 50 (μM)

[0061] 11-MT (μM) CEM / C1 1.05±0.90 Jurkat 1.32±0.80 MOLT-4 1.34±0.80 Raji 1.34±0.80 BALL-1 0.89±0.16 Nalm-6 1.04±0.10 SR 1.58±0.31

[0062] 1.2 Effect of 11-MT on apoptosis of BALL-1 cells

[0063] Annexin V-FITC / PI staining combined with flow cytometry was used to quantitatively detect the apoptosis of BALL-1 cells induced by 11-MT.

[0064] Take cells in the logarithmic growth phase, centrifuge and discard the supernatant, then resuspend the cells in RPMI-1640 complete medium; count the cells using a cell counter and adjust the cell density to 1×10 6 Cells were plated at 1 mL / well in a 6-well plate. 1 mL of culture medium containing different concentrations of the sample was added to the 6-well plate to achieve a final concentration of 11-MT of 0.5 μM, 1 μM, and 2 μM. The cells were cultured in an incubator for 48 hours. After the culture, the cells were collected by centrifugation at 3000 rpm for 5 minutes and washed twice with pre-cooled PBS. The treated cells were operated according to the instructions of the Annexin V-FITC / PI apoptosis kit. Finally, the cells were detected by flow cytometry. The results are shown in the figure below. Figure 2 shown.

[0065] After 11-MT treatment, the apoptosis rate of BALL-1 cells was 1.55±0.04% in the blank control group, 25.29±0.12% in the CDDP-treated group, and 40.84±1.20% in the VCR-treated group. When treated with 0.5 μM 11-MT, the apoptosis rate was 10.22±3.88%; when treated with 1 μM 11-MT, the apoptosis rate was 30.19±0.62%; and when treated with 2 μM 11-MT, the apoptosis rate was 43.25±1.70%. The apoptosis rate of BALL-1 cells increased with increasing 11-MT concentration in a dose-dependent manner, indicating that 11-MT induces apoptosis in BALL-1 cells in a dose-dependent manner.

[0066] 1.3 Effect of 11-MT on ROS generation in BALL-1 cells

[0067] Take cells in the logarithmic growth phase, centrifuge and discard the supernatant, then resuspend the cells in complete culture medium; count the cells using a cell counter and adjust the cell density to 1×10 6 / mL, 1mL was inoculated into each well of a 6-well plate; 1mL of culture medium containing samples of different concentrations was added to the 6-well plate to make the final concentration of 11-MT reach 0.5μM, 1μM, and 2μM, and cultured in an incubator for 48h; after the culture, the cells were collected by centrifugation at 3000rpm for 5min, and the cells were washed twice with pre-cooled PBS; DCFH-DA was prepared into a 10mmol stock solution with DMSO, and then diluted to a concentration of 10μmol with pre-cooled PBS and added to the cells, and incubated in an incubator for 30min; the cells were washed twice with pre-cooled PBS to remove unbound DCFH-DA probes, and appropriate pre-cooled PBS was added according to the cell concentration to mix the cells, and the DCF fluorescence intensity of BALL-1 cells was analyzed by flow cytometry. The ROS level in BALL-1 cells was significantly increased. The results are as follows Figure 3 As shown in the results, compared with the normal control group, CDDP treatment increased ROS levels from 42.27±0.35% to 55.95±0.77%; VCR treatment increased ROS levels from 42.27±0.35% to 76.35±0.47%. 11-MT (0.5, 1, and 2 μM) treatment significantly increased ROS levels in BALL-1 cells from approximately 42.27±0.35% to 51.37±1.44%, 78.86±2.26%, and 88.50±1.67%, respectively. These results suggest that 11-MT may induce apoptosis in BALL-1 cells by increasing ROS levels (P<0.05).

[0068] refer to Figure 4 Single-cell gel electrophoresis results showed that the degree of DNA damage in BALL-1 cells treated with 11-MT was dose-dependent compared with the control group. Higher concentrations of 11-MT resulted in more significant DNA damage, with the percentage of tail DNA increasing to 20.69±2.11% at 0.5μM 11-MT, 43.71±3.04% at 1μM 11-MT, and 69.39±1.22% at 2μM 11-MT. Meanwhile, treatment of HUVEC cells with the same concentration of 11-MT did not cause DNA damage. These results further confirm that 11-MT induces cell apoptosis through a DNA damage mechanism and has no toxic effects on normal cells.

[0069] 1.4 RNA-Seq analysis of the effect of 11-MT on differentially expressed genes (DEGs) in BALL-1 cells

[0070] After BALL-1 cells were treated with different concentrations of 11-MT for 48 hours, the cells were collected, RNA was extracted, and DEGs were detected using RNA sequencing technology. DESeq2 software was used to perform differential expression analysis between sample groups. By setting Fold Change ≥ 1.0 and pvalue < 0.05 as conditions, 3500 DEGs were screened and further volcano plots were drawn to intuitively display the overall distribution of DEGs between different 11-MT dose groups and the blank control group. Figure 5 As can be seen, there were 1722 DEGs in the 11-MT low-dose group vs. the blank group, 773 DEGs were upregulated, and 949 DEGs were downregulated. There were 2558 DEGs in the 11-MT medium-dose group vs. the blank group, 1180 DEGs were upregulated, and 1378 DEGs were downregulated. There were 3398 DEGs in the 11-MT high-dose group vs. the blank group, 1437 DEGs were upregulated, and 1961 DEGs were downregulated.

[0071] The above DEGs were subjected to GO enrichment analysis, and the biological process, cellular component and molecular function were displayed in a bar graph. Figure 6 As shown, the 10 most significantly enriched items were selected for display.

[0072] The biological processes include immune response, adaptive immune response, peptide-tyrosine phosphorylation, interferon signaling pathway, positive regulation of T cell proliferation, antigen processing and presentation of exogenous peptide antigen via MHC class II, lipopolysaccharide (LPS)-mediated signaling pathway, immunoglobulin production involved in immunoglobulin-mediated immune response, B cell receptor signaling pathway, and positive regulation of T-helper 1 type immune response.

[0073] The cellular components mainly include the external side of plasma membrane signaling pathway, lamellipodium, clathrin-coated endocytic vesicle membrane, integral component of lumenal side of endoplasmic reticulum membrane, extrinsic component of cytoplasmic side of plasma membrane, MHC class II protein complex, proteasome complex signaling pathway, cytoplasmic side of plasma membrane, immunological synapse, and proteasome core complex (beta-subunit complex).

[0074] Molecular function mainly includes protein serine / threonine / tyrosine kinase activity, protein heterodimerization activity, GTPase activity, peptide antigen binding, protein tyrosine kinase activity, GTP binding, non-membrane spanning protein tyrosine kinase activity, MHC class II protein complex binding, MHC class II receptor activity, and protein tyrosine / threonine phosphatase activity.

[0075] KEGG pathway enrichment analysis of DEGs showed that Figure 7 As shown. The DEGs in the present invention were enriched in 330 signal pathways, and the 10 most significant KEGG pathways were selected to draw a scatter plot. Among them, the more significant pathways involved: EBV infection (Epstein-Barr virus infection), allogeneic transplant rejection (Leishmaniasis), leishmaniasis (Allograft rejection), viral myocarditis (Viral myocarditis) and intestinal immune network for IgA production and other signal pathways. EBV exists in the human body in the form of latent infection and lytic infection, and can cause the occurrence of a wide range of epithelial cell and lymphocyte-like malignancies through a variety of pathogenic mechanisms, indicating that EBV infection is related to the occurrence and development of acute lymphoblastic leukemia.

[0076] The above differentially expressed genes were clustered and analyzed by the FPKM values ​​of the genes using mainstream hierarchical clustering. We selected the EBV infection signaling pathway (Epstein-Barr virus infection) that is closely related to the occurrence and development of acute lymphoblastic leukemia for gene cluster analysis. The results are as follows Figure 8 As shown in Figure 2 . Among these genes, PSMD11, CDK4, and AKT2 were significantly downregulated, potentially serving as key targets for 11-MT's anti-BALL-1 cell proliferation. Studies have shown that PSMD11 promotes hepatocellular carcinoma proliferation by regulating the ubiquitination and degradation of CDK4. Treatment of acute leukemia cells, U937 and RS4;11, with CDK4 / 6 inhibitors reduced proliferation and promoted apoptosis. Studies have shown that aberrant activation of AKT2 in T-ALL cell lines and relapsed / refractory ALL patients leads to inhibition of the FoxO3a / Bim pathway, resulting in glucocorticoid resistance. Inhibition of AKT2 using the specific inhibitor CCT128930 significantly enhances the FoxO3a / Bim signaling pathway, increases the cytotoxicity of glucocorticoids against drug-resistant T-ALL cells, and reverses glucocorticoid resistance without significantly affecting the hematopoietic, cardiac, and renal systems.

[0077] Through the above in vitro cell experiments, the following conclusions can be drawn:

[0078] (1) The Annexin V-FITC / PI double staining method was used to detect the effect of 11-MT on the apoptosis of BALL-1 cells. The results showed that 11-MT significantly promoted the apoptosis of BALL-1 cells in a dose-dependent manner.

[0079] (2) The DCFH-DA probe was used to detect the effect of 11-MT on ROS generation in BALL-1 cells. The results showed that 11-MT significantly promoted ROS generation in BALL-1 cells in a dose-dependent manner.

[0080] (3) Single-cell gel electrophoresis experiments were used to evaluate the effect of 11-MT on DNA damage in BALL-1 cells. The results showed that 11-MT could cause DNA damage in BALL-1 cells in a dose-dependent manner.

[0081] (4) RNA-seq sequencing technology was used to study the effect of 11-MT on gene expression in BALL-1 cells. The results showed that a total of 3500 significantly differentially expressed genes (DEGs) were identified in BALL-1 cells treated with 11-MT. Among them, there were 1722 DEGs in the 11-MT low-dose group VS the blank group, 773 DEGs were upregulated, and 949 DEGs were downregulated. There were 2558 DEGs in the 11-MT medium-dose group VS the blank group, 1180 DEGs were upregulated, and 1378 DEGs were downregulated. There were 3398 DEGs in the 11-MT high-dose group VS the blank group, 1437 DEGs were upregulated, and 1961 DEGs were downregulated.

[0082] (5) GO and KEGG enrichment analysis of significantly differentially expressed genes revealed that 11-MT may inhibit BALL-1 cell proliferation mainly through the EBV infection pathway. Cluster analysis of DEGs showed that PSMD11, CDK4, and AKT2 may be key targets of 11-MT inhibiting BALL-1 cell proliferation.

[0083] Example 2

[0084] In vivo experiments

[0085] 1.1 Experimental Animals

[0086] Four-week-old female ICR mice were purchased from the Yunnan University Animal Experimental Center, and four-week-old NOD / SCID mice were purchased from Beijing Huafu Biotechnology Co., Ltd. (License No. SCXK(Jing)2024-0003). Mice were housed in a specific pathogen-free (SPF) environment at the Yunnan University Animal Experimental Center under a 12-h light and 12-h dark cycle. All experimental procedures followed the guidelines approved by the Yunnan University Laboratory Animal Care Committee. The Yunnan University Ethics Committee approved the animal use and experimental protocols (Ethics Review No. YNU20251171).

[0087] 1.2 Experimental Materials

[0088] The main consumables and reagents used in this example are shown in Table 1.

[0089] Table 1 Information of consumables and reagents

[0090]

[0091]

[0092] 1.3 Instruments and Equipment

[0093] The main instruments and equipment used in this chapter are shown in Table 2.

[0094] Table 2 Information of instruments required for experiment

[0095] Instrument name factory Ice Maker Suzhou Fuwang Company Fully automatic biochemical analyzer Shenzhen Leishe Life Science, China Tail vein syringe Kunming Tai'a Biotechnology Co., Ltd.

[0096] 1.3 Safety evaluation of 11-MT in vivo

[0097] Twelve ICR female mice were randomly divided into six groups (blank control group, 11-MT group, CDDP group, VCR group, 11-MT+CDDP group, 11-MT+VCR group), with 6 mice in each group. Each mouse was injected intraperitoneally with 0.2 mL of solution per day. During the experiment, the reaction of each animal before and after administration was observed. Body weight change was measured every two days. At the end of the experimental period, the heart, liver, spleen, lung, kidney, brain and other tissues of the mice were taken to evaluate the organ index of the mice.

[0098] The selected mice were randomly divided into groups, with 6 mice in each group. This grouping method aims to minimize experimental errors and ensure good comparability between experimental groups. The administration scheme of each experimental group was carefully designed and considered. The 11-MT group was administered with a dose of 5 mg / kg, which was injected intraperitoneally at regular intervals every day for 14 days. This dose and frequency were determined based on previous research results and pre-experimental results. The CDDP administration dose was determined by reference to the research of Chen YF et al. As a classic anti-tumor drug, CDDP has a significant effect on inhibiting tumor growth, but previous studies have shown that it often leads to significant weight loss in tumor mice and has high sensitivity to the kidney, easily causing kidney damage. Considering these factors, the CDDP group in this experiment was administered with a dose of 2 mg / kg, which was injected intraperitoneally every 3 days. This dose and interval can ensure the anti-tumor activity of CDDP while reducing its adverse effects on mouse weight and kidney to some extent. The VCR group was administered according to the research of Crazzolara R et al. VCR performs well in inhibiting leukemia cell proliferation and inducing leukemia cell apoptosis, but improper dose selection in clinical application can easily cause serious adverse reactions such as neurotoxicity and hematological toxicity. Based on this, the administration scheme of the VCR group was determined as 0.15 mg / kg, which was injected intraperitoneally once a week, in an attempt to maximize the therapeutic effect while minimizing the risk of adverse reactions. The 11-MT+CDDP group used a combined administration method, with 5 mg / kg of 11-MT administered at the same time as 2 mg / kg of CDDP injected intraperitoneally every 3 days. The 11-MT+VCR group was administered with 5 mg / kg of 11-MT at the same time as 0.15 mg / kg of VCR injected intraperitoneally every week, in order to further study the safety of this combination in mice.

[0099] Reference Figure 9During the 14-day dosing period, the mice's diet and activity were carefully observed regularly every day. The mice's weight was measured and recorded every other day. Throughout the dosing process, all mice had a normal diet, actively ate, moved freely, did not show any signs of lethargy or abnormal behavior, grew well, and no deaths occurred. Analysis showed that there was no significant difference in the weight of mice in each dosing group compared with the blank control group. The results showed that each treatment group had no obvious adverse effects on the weight gain of mice.

[0100] After the experiment, the mice were euthanized and immediately dissected. Under sterile conditions, the heart, liver, spleen, lung, kidney, and brain tissues of the mice were carefully removed, quickly weighed, and the organ coefficients were calculated. The results are as follows: Figure 10 As shown in the figure, compared with the blank control group, the organ coefficients of each drug-treated group did not change significantly (P>0.05). This result shows that the dosage selected in this experiment did not have adverse effects on the mouse organs.

[0101] To further comprehensively and deeply evaluate the effects of the administered dose on the organs of mice, histopathological analysis was performed on the heart, liver, spleen, lung, kidney and brain tissues of mice. Figure 11 As shown, the cell morphology and tissue structure of the mouse organs and tissues remained normal, and no obvious signs of damage such as cell degeneration, necrosis, and inflammatory cell infiltration were found.

[0102] In addition, a systematic analysis of the whole blood of each group of mice was performed (Table 3). The key indicators in the blood routine were detected, including red blood cell count, white blood cell count, platelet count, hemoglobin content, etc. After analysis, there was no significant difference in blood indicators between different groups, and all indicator values ​​were within the normal reference range of this strain of mice. At the same time, an in-depth analysis of the serum biochemical indicators of each group of mice was conducted. Focus was placed on indicators such as AST, ALT and ALP that are closely related to liver and kidney function damage. Compared with the blank group, there was no significant difference in the liver and kidney function-related indicators of each drug-treated group ( Figure 12 ).

[0103] The present invention has demonstrated the good safety of 11-MT in mice through a systematic safety evaluation. During the experiment, food intake was continuously observed, body weight changes were regularly recorded, and indices of major organs (heart, liver, spleen, lungs, kidneys, and brain) were measured. Histopathological analysis was performed in conjunction with H&E staining. Complete blood cell analysis and serum biochemical markers were also performed. The results showed that 11-MT did not cause any adverse reactions within the dosage range established by the present invention. These important findings not only provide a scientific basis for elucidating the safety characteristics of this drug, but also lay a solid foundation for subsequent in-depth pharmacodynamic studies and potential clinical applications.

[0104] Table 3 Hematological analysis results of samples injected intraperitoneally into mice 14 days later

[0105]

[0106]

[0107]

[0108] 1.4 Establishment of acute lymphoblastic leukemia mouse model

[0109] After the purchased NOD / SCID mice were acclimated in the feeding room for one week, they were labeled for subsequent experiments. Cells in the logarithmic growth phase were cultured to the required amount, centrifuged and the supernatant was discarded. The cells were washed twice with PBS and the cells were adjusted to the required concentration and a cell suspension was prepared. Leukemia group: BALL-1 cells (1×10 7Each mouse received a 100 μL injection of 100 μL of leukemia cell extract (cells / mouse) subcutaneously into the tail vein of NOD / SCID mice. For the normal control group, each mouse received a 100 μL injection of normal saline via the tail vein. Following cell inoculation, the mice were monitored daily for signs of hunched backs, changes in hair coat, hair loss, slowed movements, or paralysis of the lower limbs. Body weights were recorded every other day. The immunophenotype of the leukemia cells was also monitored regularly. Under the aseptic conditions of a clean bench, sterile forceps and scissors were quickly used to cut open the skin of the mouse's lower limbs. The muscle tissue was then carefully separated to expose the femur and tibia. The knee and hip joints were carefully severed and the femur and tibia were completely removed, taking care to avoid damaging the bone marrow attached to the bone surface. The removed femur and tibia were placed in a culture dish containing sterile PBS buffer and washed clean of any residual muscle tissue and blood. Sterile ophthalmic scissors were then used to remove the epiphyses at both ends of the femur and tibia to expose the bone marrow cavity. Under a sterile operating environment, use a sterile syringe to draw up an appropriate amount of PBS. Carefully insert the syringe needle into one end of the bone marrow cavity, and then slowly push the syringe piston to inject PBS into the bone marrow cavity for flushing. During the flushing process, bone marrow cells will slowly flow into the sterile centrifuge tube along with the buffer solution. Repeatedly flush the bone marrow cavity 3-4 times until the contents of the bone marrow cavity are drained and the bone marrow turns white, ensuring that as many bone marrow cells as possible are flushed out. Centrifuge the centrifuge tube containing the bone marrow cell suspension at 2500-3000rpm for 3-5 minutes to allow the cells to precipitate at the bottom of the tube. Discard the supernatant, add an appropriate amount of PBS, and gently blow the cell pellet with a pipette to fully disperse the cells and prevent cell aggregation. After preparing the mouse bone marrow single cell suspension, the immunophenotyping of leukemia cells was performed as follows: centrifuge at 4°C, 2500-3000 rpm for 3-5 minutes, discard the supernatant after centrifugation → transfer the cells to a 1.5 mL sterile centrifuge tube, add flow cytometry antibodies, incubate at 4°C in the dark for 30 minutes → centrifuge and discard the supernatant, wash with pre-cooled PBS to remove unbound antibodies → resuspend the cells in 300 μL PBS for flow cytometry antibody detection. Flow cytometry revealed that CD45 was detected in the peripheral blood of B-ALL model mice. + When the proportion of leukemia cells in the bone marrow blood to all non-erythroid hematopoietic cells is about 10% or more, this time point is set as the time point for starting treatment.

[0110] After successful modeling, the leukemia mice were randomly divided into a 11-MT low-dose group (n=6); intraperitoneal injection of 11-MT 1.25 mg / kg per day; a 11-MT medium-dose group (n=6): intraperitoneal injection of 11-MT 2.5 mg / kg per day; a 11-MT high-dose group (n=6): intraperitoneal injection of 11-MT 5 mg / kg per day; a CDDP group: intraperitoneal injection of CDDP 2 mg / kg once every three days; a 11-MT+CDDP group: intraperitoneal injection of CDDP 2 mg / kg + 11-MT 5 mg / kg once every three days; a VCR group: intraperitoneal injection of VCR 0.15 mg / kg once a week; and a 11-MT+VCR group: intraperitoneal injection of VCR 0.15 mg / kg + 11-MT 5 mg / kg once a week.

[0111] General observation indicators of mice in the model group:

[0112] From the perspective of behavior and appearance, when the model group received tail vein injection of cells for 8 weeks, due to the abnormal proliferation of white blood cells in the body, a large amount of nutrients in the body were consumed, which interfered with the normal metabolic process, causing the mice to lose physical strength and reduce activity, and then become listless and less active, and huddled together for warmth. This phenomenon is also consistent with previous studies. In addition, the abnormal metabolism of tumor cells will change the body's metabolic microenvironment, such as causing disorders in blood sugar, blood lipids and amino acid metabolism. These metabolic changes will interfere with the signal transduction pathways in hair follicle cells and affect the proliferation and differentiation ability of hair follicle stem cells. Therefore, the ears of mice in the model group turned red, which may be related to the infiltration of leukemia cells into blood vessels, causing local vasodilation and abnormal blood flow. The mice in the blank group did not show any of the above abnormal signs. Their activity status was normal, their fur was smooth and neat, and their ears were uniform in color, indicating that they were not invaded by leukemia cells and their bodies were in a healthy state ( Figure 13 ).

[0113] Flow cytometry was used to detect CD45 in peripheral blood and bone marrow + The proportion of cells:

[0114] Nine weeks after modeling, flow cytometry was used to detect CD45 + The percentage of cells.

[0115] The method for detecting the proportion of leukemia cells in the peripheral blood and bone marrow of mice is as follows:

[0116] Peripheral blood was collected from mice according to the above method and the blood in the blood collection tube was transferred to an EP tube. Under a sterile environment, an appropriate amount of red blood cell lysis buffer was added to the EP tube. After gentle mixing, the EP tube was placed on ice and incubated for 3 to 5 minutes to lyse the red blood cells. After incubation, the EP tube was placed in a centrifuge, the centrifuge temperature was set to 4°C, the speed was adjusted to 2000 rpm, and the centrifugation time was set to 6 minutes. After centrifugation, the supernatant was carefully discarded and the pellet in the tube was washed twice with PBS. Finally, the pellet was resuspended in 300 μL PBS and the flow cytometry antibody was added to prepare for flow cytometry detection to determine the proportion of leukemic cells in peripheral blood nucleated cells.

[0117] Mouse bone marrow single cell suspension was prepared according to the above method. After treatment, the pellet was resuspended in 300 μL PBS and flow cytometry antibodies were added to prepare for flow cytometry detection to determine the proportion of leukemia cells in the bone marrow cells.

[0118] The results are as follows Figure 14 As shown, no human CD45 was detected in the peripheral blood of control mice. + cells, while human CD45 + The cell ratio was >1%. At the same time, human CD45 was detected in the bone marrow blood. + The proportion of cells is 15%. According to previous studies, when CD45 + A ratio of ≥1% can be considered a successful model. The experimental results show that the acute B lymphoblastic leukemia mouse model was successfully established.

[0119] Routine blood analysis of mice:

[0120] During the experiment, the blood routine of the mice in the blank group and the model group was analyzed. The results are shown in Table 4. Compared with the blank group, the white blood cell count of the mice in the model group increased, the lymphocyte count increased, and anemia occurred. The red blood cell count decreased, the hemoglobin content decreased, and the platelet count decreased, which is very similar to the blood picture of human acute B lymphocytic leukemia.

[0121] Table 4 Changes in peripheral blood routine of mice after injection of BALL-1 cells

[0122]

[0123] Histomorphological observation of liver and spleen:

[0124] The mice were dissected and bone marrow blood was taken. The liver and spleen tissues were also taken for observation. Figure 15As shown, compared with the control group, the liver and spleen tissues of the model group mice were significantly enlarged. Macroscopically, diffuse tumor nodules or granular appearance were observed. The tumor masses had clear boundaries and irregular margins, consistent with literature descriptions. These results fully demonstrate that the acute B-lymphocytic leukemia mouse model was successfully established and ready for further drug administration.

[0125] 1.5 Effect of 11-MT on blood routine of mice with acute B lymphoblastic leukemia

[0126] The drugs were administered continuously for 14 days. After the end of the experimental period, the peripheral blood of mice in different drug administration groups was tested to analyze the changes in the concentrations of white blood cells, red blood cells, lymphocytes and mean corpuscular hemoglobin in the blood of mice in different drug administration groups.

[0127] The method for routine detection of mouse peripheral blood is as follows:

[0128] After leukemia cell injection in normal mice and after leukemia model mice have received treatment, peripheral blood routine tests were performed at designated time points. The specific method is as follows: Gently place the mouse in a restraining device, with the body in a naturally extended position and the head secured and slightly tilted upward, fully exposing the submandibular area. Wipe the submandibular area with a 75% alcohol-containing cotton ball, slightly larger than the blood collection site, starting from below the mandible and moving toward the neck. Disinfection aims to reduce microorganisms on the skin surface and minimize the risk of contamination during blood collection. After disinfection, allow the alcohol to air dry to prevent residual alcohol from diluting the blood and potentially affecting subsequent testing. Insert the needle with the bevel facing upward at a 15-30° angle to the submandibular skin. If bleeding is visible, adjust the needle position appropriately, but avoid rapid advancement or withdrawal to avoid damaging blood vessels. Collect blood using a sterile blood collection tube. After collection, apply pressure to the collection site with a dry cotton ball for 3-5 minutes until bleeding stops. If the pressing time is too short, it may cause bleeding or hematoma at the blood collection site, affecting the health of the mouse. Follow the detection steps of the fully automatic whole blood analyzer to detect the routine blood test of the mouse peripheral blood.

[0129] The results are shown in Table 5. Compared with the blank group, the number of white blood cells, the number of neutrophils, the percentage of neutrophils, the number of red blood cells, the hemoglobin content and the number of platelets in the peripheral blood of the model group mice all showed significant changes (P < 0.0001). Specifically, the number of white blood cells in the model group increased to 13.01 ± 1.45 (10 9 / L), and after treatment with different concentrations of drugs, the number of white blood cells in all groups decreased significantly (P < 0.0001). The number of neutrophils in the model group increased to 2.13 ± 0.02 (10 9 / L). After treatment with different concentrations of drugs, except for a slight decrease in the number of white blood cells in the CDDP group (P < 0.01), the number of white blood cells in all other groups decreased significantly (P < 0.0001). The percentage of neutrophils in the model group was significantly increased to 55.37 ± 0.15%, among which the percentage of neutrophils did not decrease after CDDP treatment. The percentage of neutrophils in the 11-MT + CDDP group treated with 11-MT and CDDP decreased slightly (P < 0.05), and the percentage of neutrophils in all other groups decreased significantly. The number of red blood cells in the model group decreased to 7.03 ± 1.03 (10 12 / L). After treatment with different concentrations of drugs, the number of red blood cells in the CDDP group increased slightly (P < 0.05), while the number of red blood cells in all other groups decreased significantly (P < 0.0001). The hemoglobin content in the model group decreased to 129 ± 11.53 (g / L). After treatment with different concentrations of drugs, the hemoglobin content in the 11-MT + CDDP group did not increase. The platelet count in the model group decreased to 985.33 ± 1.53 (10 9 / L), after treatment with different concentrations of drugs, the CDDP treatment group had no significant effect on the platelet count, the platelet count in the 11-MT+CDDP group was slightly increased (P < 0.01), and the platelet counts in all other groups were significantly increased (P < 0.0001). In the present invention, after 11-MT treatment, the blood routine indicators of mice with acute B lymphocytic leukemia were significantly improved, the number of white blood cells, the number and percentage of neutrophils decreased significantly, and the number of red blood cells, hemoglobin content and platelet count showed an upward trend in the drug-treated group. Studies have shown that some traditional chemotherapy drugs, such as vincristine, can inhibit the proliferation of leukemia cells, but will cause bone marrow suppression, leading to an excessive decrease in the number of white blood cells and platelets. In comparison, 11-MT can more effectively make various indicators tend to normal levels when regulating blood routine indicators, thereby reducing the inhibition of normal hematopoietic function. From a mechanism of action perspective, 11-MT may induce leukemia cell apoptosis and reduce the interference of abnormally proliferating cells on the bone marrow hematopoietic microenvironment, thereby promoting the proliferation and differentiation of normal hematopoietic stem cells and restoring routine blood test indicators. Based on the trends of routine blood test results, all groups except the CDDP group and the 11-MT+CDDP group demonstrated good in vivo anti-leukemia activity.

[0130] 1.6 Effects of 11-MT on serum biochemical parameters in mice with acute B-lymphocytic leukemia

[0131] After the experiment, the serum biochemical indicators of mice in different drug-treated groups were tested to analyze the effects of different drug treatments on the serum biochemical indicators of leukemia mice. ALT, AST, total protein TP, and albumin ALB are liver function test indicators. Among them, elevated ALT and AST indicate liver damage, and elevated TP indicates an association with chronic inflammation. Elevated ALB indicates dehydration. Elevated UREA indicates impaired renal function. Elevated TG and T-CHO indicate an association with inflammation and cardiovascular risk. Sodium, potassium, and chloride ions are indicators for assessing the body's electrolyte balance.

[0132] The results are as follows Figure 16 As shown in the results, the ALT of the model group increased significantly, and after treatment with different concentrations of the drug, the ALT values ​​showed a significant decrease (P < 0.01); the AST of the model group increased significantly, and after treatment with different concentrations of the drug, the AST values ​​showed a significant decrease (P < 0.05); the TP of the model group increased significantly, and after treatment with different concentrations of the drug, only the TP value of the 11-MT high-dose group decreased significantly (P < 0.001); the ALB of the model group increased significantly, and after treatment with different concentrations of the drug, only the 11- ALB levels significantly decreased in the high-dose MT, medium-dose 11-MT, VCR, and 11-MT+VCR groups (P < 0.005). UREA and TG levels significantly increased in the model group, and after treatment with different concentrations, UREA levels were significantly decreased in all groups (P < 0.0001). T-CHO levels significantly increased in the model group, and after treatment with different concentrations, UREA levels were significantly decreased in all groups (P < 0.01). There were no differences in sodium, potassium, and chloride ion levels among all groups. 11-MT significantly reduced elevated serum biochemical parameters in mice with acute B-cell lymphoblastic leukemia, including ALT, AST, TP, UREA, TG, and T-CHO. Previous studies have shown that some leukemia drugs, while capable of reducing tumor burden, may adversely affect liver or kidney function. For example, imatinib, when used to treat leukemia, can cause elevated liver enzymes, increasing the burden on the liver. In contrast, 11-MT has been shown to reduce tumor burden while also repairing and protecting liver and kidney function. This may be because 11-MT regulates the body's metabolic and immune functions, alleviating tumor damage to organs while also suppressing inflammation and reducing organ dysfunction caused by inflammation.

[0133] 1.7 Effects of 11-MT on organs of mice with acute B-lymphocytic leukemia

[0134] (1) Effects of 11-MT on organ indices in mice with acute B lymphoblastic leukemia

[0135] After the experimental period, the heart, liver, spleen, lung, kidney and brain of the mouse were quickly separated and weighed, and the organ coefficient was calculated. The results are as follows: Figure 17 As shown, there were no significant differences in the weights of the heart, liver, lung, kidney, and brain tissues between the model and drug-treated groups compared with the blank control group. However, the spleen organ coefficient increased significantly in the model group. After treatment with different concentrations of 11-MT, the organ coefficients of mice in the high-dose 11-MT and 11-MT+VCR groups approached normal levels. These results suggest that 11-MT and the combined administration of 11-MT and VCR have regulatory effects on diseased organs. Previous studies have shown that some drugs, while inhibiting tumor cells in leukemia treatment, may also be toxic to organs, leading to abnormal organ coefficients or tissue damage. For example, some chemotherapy drugs can cause cardiotoxicity, resulting in myocardial cell damage and altered cardiac coefficients. 11-MT exhibits advantages in this regard. It not only reduces leukemia cell infiltration in organs but also regulates diseased organs, maintaining their normal structure and function. This may be related to its ability to modulate immune responses and inhibit tumor cell migration and invasion.

[0136] (2) H&E staining of mouse liver and spleen tissue

[0137] H&E staining results of mouse liver and spleen tissues are shown in Figure 2. Figure 18 As shown, the hepatocytes of mice in the blank group were neatly arranged, with normal nuclear morphology. The red and white pulp of the spleen tissue showed clear demarcation, and the tissue structure was normal. In the model group, extensive tumor cell infiltration was observed in both the liver and spleen tissues. Compared with the model group, leukemia cell infiltration in the liver and spleen tissues of all treatment groups showed slight improvement, with the 11-MT treatment group showing a significant improvement compared with the other treatment groups.

[0138] Table 6.5 Changes in blood routine of mice after administration

[0139]

[0140]

[0141] Note: Data are presented as mean ± standard deviation. * P<0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 was considered statistically significant between groups.

[0142] 1.8 Effect of 1-MT on the growth of leukemia cells in mice with acute B-lymphoblastic leukemia

[0143] This example uses flow cytometry to analyze the effects of different drug treatments on CD45 + The effect of cell ratio ( Figure 19 ).

[0144] The results showed that CD45 + The proportion of cells was as high as 36.89%, indicating that the disease model successfully led to abnormal proliferation or infiltration of immune cells. After drug treatment, each treatment group showed different degrees of CD45 + The proportion of cells decreased, and the 11-MT low-dose group increased CD45 + The cell ratio dropped to 30.93% and the 11-MT medium dose group + The cell ratio dropped to 28.96% in the 11-MT high-dose group. + The cell ratio dropped to 27.08% in the CDDP group. + The cell ratio dropped to 33.36% in the VCR group. + The cell ratio dropped to 30.39% in the 11-MT+CDDP group. + The cell ratio dropped to 34.33% in the 11-MT+VCR group. + The cell ratio dropped to 28.66%.

[0145] The above results showed that among the treatment groups, the high-dose 11-MT group had a significant effect in inhibiting the growth of leukemia cells in acute B lymphocytic leukemia mice and reducing the expression of CD45 + The role of cell ratio is prominent, and the 11-MT+VCR combined administration group also showed a good synergistic inhibitory effect. Studies have shown that although many traditional therapeutic drugs can inhibit the growth of leukemia cells to a certain extent, they are often accompanied by serious side effects and drug resistance problems. For example, after long-term use of some chemotherapy drugs, leukemia cells will develop drug resistance mechanisms, resulting in a significant reduction in the treatment effect. 11-MT shows unique advantages in inhibiting the growth of leukemia cells. Its high-dose group can significantly reduce the level of CD45 in bone marrow blood. + This suggests that 11-MT can effectively reduce the proliferation and infiltration of leukemia cells in the bone marrow, fundamentally controlling the progression of the disease. In terms of its mechanism of action, 11-MT may inhibit leukemia cell growth by affecting the signaling pathways of leukemia cells, interfering with their proliferation, differentiation, and survival. Furthermore, 11-MT may enhance the body's own immune function, activating immune cells to recognize and kill leukemia cells.

[0146] Summarize:

[0147] (1) The safety of 11-MT, positive drugs, and combined administration in mice was evaluated using ICR mice. The results showed that all drugs were non-toxic in mice.

[0148] (2) Successfully constructed a mouse model of acute B lymphoblastic leukemia.

[0149] (3) The effect of 11-MT on mouse organs was explored. The results showed that compared with the normal group, the organ coefficients of liver and spleen in the model group increased significantly, and the organ coefficients of 11-MT treated mice were close to the normal level, indicating that 11-MT had a regulatory effect on specific organ lesions.

[0150] (4) The effect of 11-MT on serum biochemical indicators of acute B lymphoblastic leukemia mice was explored. The results showed that 11-MT showed a little repair and protection effect, and could restore multiple serum biochemical indicators abnormally elevated due to tumor load to normal level.

[0151] (5) Through H&E staining, blood routine, serum biochemical indicators and leukemia cell immunophenotyping detection, it was proved that 11-MT could better reduce tumor cell infiltration.

Claims

1. 11-MT is used in the preparation of a drug for the treatment of acute B-lymphocytic leukemia.

2. The use according to claim 1, characterized in that The drug is used to reduce the level of CD45 in bone marrow blood + Cell ratio to reduce the proliferation and infiltration of acute B-lymphoblastic leukemia cells in the bone marrow.

3. The use according to claim 1, characterized in that The drug is a drug that reduces tumor burden and repairs and protects liver and kidney functions during the treatment of acute B lymphocytic leukemia.

Images