Use of methylene blue for the preparation of a medicament for the treatment of lymphoma

Abstract

This invention discloses the application of methylene blue in the preparation of drugs for treating lymphoma, belonging to the field of therapeutic drug technology. This invention creatively discovers that methylene blue can be used as a drug for treating lymphoma, exhibiting cytotoxic effects against human diffuse large B-cell lymphoma cell lines including U2932, TMD8, SUDHL-4, and OCI-LY19, as well as human peripheral T-cell lymphoma cell lines including KARPAS299, SMZ-1, and KHYG-1, and its in vitro activity is superior to first-line chemotherapy drugs. This invention provides an effective option for researching lymphoma treatment strategies and preparing new drugs for treating lymphoma.

Description

Technical Field

[0001] This invention belongs to the field of therapeutic drug technology, specifically relating to the application of methylene blue in the preparation of drugs for treating lymphoma. Background Technology

[0002] The statements herein provide only background information in relation to this invention and do not necessarily constitute prior art.

[0003] Lymphoma is a malignant tumor originating from the lymphohematopoietic system. Based on pathological characteristics, it can be divided into Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (NHL). The latter is further subdivided into dozens of subtypes derived from B cells, T cells, and NK cells, exhibiting extremely high biological heterogeneity. Currently, first-line clinical strategies still primarily rely on traditional combination chemotherapy regimens such as CHOP (cyclophosphamide + doxorubicin + vincristine + prednisone) or ABVD (doxorubicin + bleomycin + vincristine + dacarbazine). While these regimens can achieve remission in 60%-80% of treatment-naïve patients, the associated risks of myelosuppression, cardiotoxicity, gastrointestinal reactions, and secondary infections significantly reduce tolerability in elderly patients or those with underlying diseases. Targeted therapies, such as the BTK inhibitor ibrutinib, the Bcl-2 inhibitor venetoclax, and the CD20 monoclonal antibody rituximab, improve treatment precision by blocking specific signaling pathways. However, with long-term use, tumor cells can escape through mechanisms such as MDR1 overexpression, Bcl-2 upregulation, and BTK C481S mutation, leading to a year-on-year increase in the proportion of relapsed / refractory cases. Immunotherapy, such as PD-1 / PD-L1 antibodies and CAR-T cell therapy, has achieved deep remission in some patients, but its long preparation cycle, high degree of personalization, and cost of hundreds of thousands of yuan per course of treatment, coupled with serious adverse reactions such as cytokine storms and neurotoxicity, limit its accessibility. In addition, the complex structures of multi-drug combinations or biologics make it difficult to control quality consistency, further increasing the difficulty of regulation and clinical application.

[0004] Therefore, there is an urgent clinical need for single small molecule drugs with well-defined structures, low prices, controllable toxicity, and effectiveness against drug-resistant cells to compensate for the shortcomings of existing treatment systems.

[0005] Methylene blue (C 16 H 18ClN3S (CAS No. 61-73-4) is a synthetic thiazide small molecule that has been on the market for over a century. It was first used as a dye and was later included in the WHO Essential Medicines List. Its pharmacological effects mainly include: ① Redox cycle: Methylene blue can accept electrons from reduced coenzyme I (NADH) in cells to be reduced to colorless methylene blue, and then directly transfer electrons to cytochrome c (bypassing complex I / III), promoting ATP production; under certain conditions, it can also use NADPH to transfer electrons to methemoglobin, forming a redox cycle and reversing methemoglobinemia; ② Nitric oxide synthase (NOS) inhibition: By inhibiting NOS, it reduces NO production and improves sepsis-related hypotension; ③ Monoamine oxidase (MAO) inhibition: It increases the level of monoamines in the central nervous system and is used as an adjunct treatment for Alzheimer's disease and mental illness; ④ Photosensitization: It generates reactive oxygen species (ROS) in photodynamic therapy (PDT) to kill Propionibacterium acnes or tumor cells; ⑤ Autophagy-apoptosis regulation: Recent studies have shown that methylene blue can induce autophagic death in glioma and breast cancer cells by inhibiting mTOR and activating the Beclin-1 pathway.

[0006] Although the anti-tumor potential of methylene blue has been preliminarily validated in solid tumors such as melanoma, glioma, and prostate cancer, no in vitro experimental, animal model, or clinical studies on methylene blue as a monotherapy or in combination with other drugs for the treatment of lymphoma have been found in the publicly available literature to date. Summary of the Invention

[0007] To address the shortcomings of existing technologies and overcome the drawbacks of current lymphoma treatment drugs, such as high toxicity, high drug resistance rates, high preparation and administration costs, and complex structures, this invention provides the application of methylene blue in the preparation of drugs for treating lymphoma. This application relates to a novel therapeutic approach for lymphoma using the small molecule compound methylene blue as the active ingredient. As a new pharmaceutical use of methylene blue, it can effectively kill multiple subtypes of lymphoma cell lines even at micromolar concentrations, thereby providing a safe, economical, convenient, and scalable treatment option for patients with relapsed / refractory lymphoma.

[0008] To achieve the above objectives, the present invention provides the following technical solution: The use of methylene blue in the preparation of medicaments for the treatment of lymphoma, wherein methylene blue is used as a single active ingredient or in combination with other active ingredients.

[0009] Preferably, the lymphoma includes B-cell lymphoma and T-cell lymphoma.

[0010] This invention creatively discovers that methylene blue can be used as a drug to treat lymphoma. Through in-depth research, this invention has found that the core mechanism by which methylene blue kills lymphoma cells involves the activation of the endoplasmic reticulum stress (ER stress) pathway.

[0011] The endoplasmic reticulum (ER) is a vital organelle for protein synthesis, folding, and calcium storage within cells. When the intracellular environment is disrupted (e.g., by oxidative stress or calcium homeostasis), unfolded or misfolded proteins accumulate in the ER lumen, triggering the unfolded protein response (UPR). A moderate UPR can restore ER function, but persistent or severe ER stress induces cell death by activating apoptotic signals.

[0012] Methylene blue, as a redox reactive molecule, interferes with the mitochondrial electron transport chain after entering lymphoma cells, generating oxidative stress and leading to endoplasmic reticulum protein folding dysfunction, thereby activating the PERK-eIF2α-ATF4-CHOP signaling axis. Experiments have verified its specific manifestations as follows: (1) CHOP (C / EBP homolog) expression is significantly upregulated: CHOP is a key transcription factor for ER stress-induced apoptosis. Its overexpression can inhibit the anti-apoptotic protein Bcl-2, disrupt the balance of mitochondrial apoptosis regulation, and ultimately activate the Caspase cascade reaction to induce apoptosis in lymphoma cells.

[0013] (2) Significant downregulation of Bcl-2 expression: Bcl-2 is a common anti-apoptotic protein in diffuse large B-cell lymphoma, and its high expression is closely related to chemotherapy resistance. Methylene blue inhibits Bcl-2 transcription through the endoplasmic reticulum stress-CHOP pathway, thereby relieving the inhibition of apoptosis and restoring the sensitivity of lymphoma cells to apoptotic signals.

[0014] This mechanism differs from the direct DNA damage mechanism of existing chemotherapy drugs (such as alkylating agents and antimetabolites), providing a new approach to overcome the problem of lymphoma drug resistance.

[0015] Preferably, the drug is capable of killing lymphoma cell lines.

[0016] This invention found that, under in vitro culture conditions, co-incubation of methylene blue with lymphoma cell lines for 48 h resulted in a dose-dependent decrease in cell viability; CCK-8 assays showed a half-maximal inhibitory concentration (IC50) after 48 h. 50 The concentration is 1-5 μM.

[0017] Based on in vitro IC 50Based on mouse equivalent dose conversion, the clinical dosage of methylene blue is 10-25 mg / kg body weight, which can be administered via intravenous infusion, oral administration, intratumoral injection, or after loading into a pharmaceutically acceptable delivery system.

[0018] Preferably, the lymphoma cell line includes a human diffuse large B-cell lymphoma cell line and a human peripheral T-cell lymphoma cell line.

[0019] Preferably, the human diffuse large B-cell lymphoma cell lines include U2932, TMD8, SUDHL-4, and OCI-LY19.

[0020] Preferably, the human peripheral T-cell lymphoma cell lines include KARPAS299, SMZ-1, and KHYG-1.

[0021] As a marketed small molecule, methylene blue has complete chemical structure, metabolic pathway and toxicological data, which can avoid the interactions and unknown risks caused by the combined use of multiple drugs.

[0022] The experimental results of this invention show that methylene blue has a 48-h IC50 effect on various lymphoma cell lines, including U2932, TMD8, SUDHL-4, and SMZ-1. 50 With a concentration of only 1-5 μM, its activity is superior to or equivalent to some first-line chemotherapy drugs (such as cyclophosphamide in vitro IC50). 50 Typically >10 μM).

[0023] Toxicity data for methylene blue have been documented in numerous published papers. For example, the paper "Toxicology and carcinogenesis studies of formamide (Cas No. 75-12-7) in F344 / N rats and B6C3F1 mice (gavage studies)" discloses the acute LD50 of methylene blue in mice. 50 >200 mg / kg; while the equivalent clinical dose is only 1-2 mg / kg, which is far below the known safety limit (7 mg / kg for human use), and the expected bone marrow suppression and cardiotoxicity are significantly lower than those of traditional chemotherapy.

[0024] In the treatment regimen using methylene blue to treat lymphoma, the market price of the active pharmaceutical ingredient is $2-3 / g, and the cost of the active pharmaceutical ingredient per person per course of treatment (2mg / kg×7 days) is less than $1, which is far lower than that of existing targeted or immunotherapies.

[0025] Preferably, the drug further includes pharmaceutically acceptable excipients.

[0026] Preferably, in the drug, methylene blue exists in anhydrous form or in the form of a pharmaceutically acceptable salt, solvate, or hydrate; the drug formulation may be an injection, lyophilized powder, tablet, capsule, liposome, or nanoparticle.

[0027] In the application described in this invention, methylene blue is the sole active ingredient in the drug used to prepare a drug that kills lymphoma cell lines. It is administered as a monotherapy for patients requiring treatment, or in combination with conventional supportive care or radiotherapy, but methylene blue always remains the sole active antitumor ingredient.

[0028] Routine supportive care includes standard palliative care measures such as hematopoietic growth factor support, anti-infection prevention and treatment, prevention of tumor lysis syndrome, symptomatic management of adverse reactions to chemotherapy, and nutritional and psychological support.

[0029] The hematopoietic growth factor support is bone marrow hematopoietic function support, and the hematopoietic growth factors include granulocyte colony-stimulating factor (G-CSF), erythropoietin (EPO), and thrombopoietin receptor agonist (TPO-RA).

[0030] The infection prevention and treatment include antimicrobial prophylaxis with fluoroquinolones, antiviral prophylaxis with acyclovir / valacyclovir, and antifungal prophylaxis with voriconazole and posaconazole.

[0031] The symptomatic treatment for adverse reactions to chemotherapy includes antiemetic therapy, treatment of peripheral neuropathy (CIPN), and treatment of oral mucositis.

[0032] The antiemetic drugs include 5-HT3 receptor antagonists, NK1 receptor antagonists, dexamethasone, and olanzapine; the drugs for treating peripheral neuropathy (CIPN) include duloxetine, venlafaxine, calcium and magnesium infusion, and glutathione; the treatment for oral mucositis includes oral care, local analgesia, and administration of growth factors.

[0033] Preferably, the drugs contain only methylene blue as the active ingredient and do not contain other antitumor drugs.

[0034] Furthermore, the drug can be used as a single agent for treating lymphoma patients or animal models, or as a drug used in combination with conventional supportive care or radiotherapy.

[0035] The beneficial effects achieved by one or more embodiments of the present invention described above are as follows: 1. This invention creatively discovers that methylene blue can be used as a drug agent for treating lymphoma. It exhibits killing effects against human diffuse large B-cell lymphomas, including U2932, TMD8, SUDHL-4, and OCI-LY19, as well as human peripheral T-cell lymphomas, including KARPAS299, SMZ-1, and KHYG-1, and its in vitro activity is superior to first-line chemotherapy drugs. This invention provides an effective option for researching lymphoma treatment strategies and preparing new drugs for treating lymphoma.

[0036] 2. Considering that methylene blue already possesses the following characteristics: ① it has been on the market for a century and has complete pharmacokinetic / toxicological data; ② the raw material is inexpensive (US$2-3 / gram); ③ it can be administered via multiple routes, including intravenous, oral, and intratumoral administration; ④ its mechanism of action is complementary to existing chemotherapy, targeted therapy, and immunotherapy, it suggests that its "repurposing of old drugs" in lymphoma has great development value and clinical translation potential. Attached Figure Description

[0037] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0038] Figure 1 This is a schematic diagram illustrating the killing of lymphoma cells by methylene blue.

[0039] Figure 2 This is a schematic diagram illustrating the experimental principle of the killing effect of methylene blue on lymphoma cells.

[0040] Figure 3 The image shows the cytotoxic effect of methylene blue on human diffuse large B-cell lymphoma after 48 h; where a is the U2932 cell line, b is the OCI-LY19 cell line, c is the TMD8 cell line, and d is the SUDHL-4 cell line; the concentrations are 0 μM (DMSO), 0.078125 μM, 0.15625 μM, 0.3125 μM, 0.625 μM, 1.25 μM, 2.5 μM, 5 μM, and 10 μM, respectively.

[0041] Figure 4 The image shows the cytotoxic effect of methylene blue on human peripheral T-cell lymphoma over 48 hours; where a is the KARPASS299 cell line, b is the KHYG-1 cell line, and c is the SMZ1 cell line; the concentrations are 0 μM (DMSO), 0.078125 μM, 0.15625 μM, 0.3125 μM, 0.625 μM, 1.25 μM, 2.5 μM, 5 μM, and 10 μM, respectively.

[0042] Figure 5The graph shows the half-maximal inhibitory concentration (IC50) of methylene blue against human diffuse large B-cell lymphoma; where a is the U2932 cell line, b is the OCI-LY19 cell line, c is the TMD8 cell line, and d is the SUDHL-4 cell line.

[0043] Figure 6 The graph shows the half-maximal inhibitory concentration (IC50) of methylene blue against human peripheral T-cell lymphoma; where a is the KARPASS299 cell line, b is the KHYG-1 cell line, and c is the SMZ-1 cell line.

[0044] Figure 7 A diagram illustrating the expression of endoplasmic reticulum stress and apoptosis-related proteins in U2932 cells under time-dependent activation by methylene blue. Detailed Implementation

[0045] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0046] Methylene blue (MB), also known as methyl blue, is widely used as an electron acceptor in biological oxidation processes. Its oxidized form is blue, while its reduced form is colorless. At low doses, methylene blue can reduce methemoglobin back to hemoglobin, thus treating methemoglobinemia caused by poisoning from nitrites, chlorates, quinones, aniline, and nitrobenzene. At high doses, it oxidizes hemoglobin back to methemoglobin, an effect that can be used to treat cyanide poisoning. Methylene blue has also been used as a urinary tract disinfectant and an antiseptic dye.

[0047] Recent studies have found that methylene blue has a high affinity for cancer cells, especially melanin, and can kill cancer cells, with its killing effect showing a significant time-dependent effect. Significant progress has been made in the diagnosis and treatment of metastatic melanoma using radiolabeled methylene blue, as well as in the treatment of various tumors. Current research has also revealed that methylene blue-mediated photodynamic therapy and its combination with doxorubicin have anti-tumor effects on breast cancer.

[0048] In some embodiments of the present invention, the use of methylene blue in the preparation of a medicament for treating lymphoma is provided, wherein methylene blue is a single active ingredient or in combination with other active ingredients.

[0049] Preferably, the lymphoma includes B-cell lymphoma and T-cell lymphoma.

[0050] Preferably, the lymphoma cell line includes a human diffuse large B-cell lymphoma cell line and a human peripheral T-cell lymphoma cell line.

[0051] Preferably, the human diffuse large B-cell lymphoma cell lines include U2932, TMD8, SUDHL-4, and OCI-LY19.

[0052] Preferably, the human peripheral T-cell lymphoma cell lines include KARPAS299, SMZ-1, and KHYG-1.

[0053] Preferably, the drug further includes pharmaceutically acceptable excipients.

[0054] Preferably, in the drug, methylene blue exists in anhydrous form or in the form of a pharmaceutically acceptable salt, solvate, or hydrate; the drug formulation may be an injection, lyophilized powder, tablet, capsule, liposome, or nanoparticle.

[0055] In the embodiments of this invention, human diffuse large B-cell lymphoma U2932, TMD8, SUDHL-4, OCI-LY19 and human peripheral T-cell lymphoma KARPAS299, SMZ-1, KHYG-1 cell lines were used. These cell lines are key in vitro models for studying the drug activity assays in human diffuse large B-cell lymphoma (DLBCL) and human peripheral T-cell lymphoma (PTCL). By simulating the state of tumor cells under different subtypes and genetic backgrounds, they can be used to evaluate the killing effect and mechanism of action of drugs.

[0056] Among them, U2932 belongs to the ABC type (activated B cell-like), representing a subtype with poor response to existing therapies (such as ibrutinib), and is used to evaluate the activity of drugs in drug-resistant or refractory models; TMD8 belongs to the ABC type (activated B cell-like) and is used to evaluate novel inhibitors targeting signaling pathways such as BCR, NF-κB, and STAT3; SUDHL-4 belongs to the GCB type (germinal center B cell-like) and is used to compare the selectivity of drugs to different DLBCL subtypes. It is used in combination with ABC type cell lines to evaluate the subtype specificity of drugs. OCI-LY19 belongs to the GCB type (germinal center B cell-like) and is used to verify the activity of drugs on GCB type cells or to study the role of specific genes (such as STAT3) in this subtype.

[0057] KARPAS-299 belongs to ALCL (anaplastic large cell lymphoma) and carries the NPM-ALK fusion gene, making it a key model for studying ALK inhibitors (such as crizotinib) and the downstream STAT3 signaling pathway. SMZ-1 belongs to PTCL-NOS (non-specific type), representing the most common PTCL subtype, and is used to evaluate the broad-spectrum antitumor activity of drugs in non-ALK-dependent PTCL. KHYG-1 belongs to the NK / T-cell lymphoma-derived lineage, derived from NK / T-cell lymphoma patients, and is an important tool for studying the drug sensitivity of this highly aggressive subtype.

[0058] Preferably, the drug further includes pharmaceutically acceptable excipients.

[0059] Preferably, in the drug, methylene blue is present in anhydrous form or in the form of a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[0060] Preferably, the formulation of the drug may be an injection, lyophilized powder, tablet, capsule, liposome, or nanoparticle.

[0061] Preferably, methylene blue is the sole active ingredient in the drug, which is used as a single drug for treating lymphoma patients or animal models, or as a drug used in combination with conventional supportive care or radiotherapy. The conventional supportive care includes hematopoietic growth factor support, anti-infection prevention and treatment, prevention of tumor lysis syndrome, symptomatic treatment of chemotherapy adverse reactions, and nutritional and psychological support.

[0062] The present invention will be further described below with reference to the embodiments.

[0063] Example 1 In vitro antitumor effect experimental study In this embodiment, the effect of methylene blue on the in vitro proliferation activity of lymphoma cells was investigated.

[0064] 1. Experimental Materials 1.1 Cell lines: Human diffuse large B-cell lymphomas U2932, TMD8, SUDHL-4, OCI-LY19 and human peripheral T-cell lymphomas KARPAS299, SMZ-1, KHYG-1.

[0065] 1.2 Reagents and Instruments: Reagents: CCK8 assay kit, 1640 medium, double antibody, FBS, PBS, DMSO. Main instruments: benchtop centrifuge, cell counting chamber, CO2 incubator, inverted microscope, microplate reader (450 nm filter), pipettes.

[0066] 2. Experimental Methods: The experimental procedure is as follows Figure 1 and Figure 2As shown, cells in the logarithmic growth phase were used for experiments. Cells were digested, counted, and prepared into a cell suspension. 10,000 cells / well (100 μL) were seeded into 96-well plates. Cell growth and density were observed under a microscope. Wells with good growth and uniform cell distribution and density were selected for experiments. Sample solutions of different concentration gradients were prepared, including 10 μM, 5 μM, 2.5 μM, 1.25 μM, 0.625 μM, 0.3125 μM, 0.15625 μM, 0.078125 μM, and 0 μM. Each concentration (1 μL) was added in triplicate to 96-well plates and cultured at 37℃, 5% CO2, and 90% humidity for 48 h.

[0067] Thaw CCK-8 at 4°C before use. Add 20 μL of CCK-8 solution to each well. Incubate at 37°C, 5% CO2, and 90% humidity for 1 h. Measure absorbance at 450 nm using a microplate reader. Process and analyze the results using Excel and Graphpad Prism. Calculate cell viability and inhibition rate using the following formulas by substituting the absorbance values: Cell viability = [(experimental wells - blank wells) / (negative control wells - blank wells)] × 100%.

[0068] Inhibition rate = [(Negative control wells - experimental wells) / (Negative control wells - blank wells)] × 100%.

[0069] The experimental wells contain cell culture medium, CCK-8, and the test substance; the negative control wells contain cell culture medium, CCK-8, and no test substance; and the blank wells contain neither cell culture medium nor test substance, nor CCK-8.

[0070] 3. Experimental Results It is evident that, under in vitro culture conditions, co-incubation of the lymphoma cell line with methylene blue at a single concentration gradient of 0 μM-10 μM for 48 h resulted in a dose-dependent decrease in cell viability. Figure 1 As shown, methylene blue can kill human diffuse large B-cell lymphomas including U2932, TMD8, SUDHL-4, and OCI-LY19, and human peripheral T-cell lymphomas including KARPAS299, SMZ-1, and KHYG-1. Figure 3 and Figure 4The cytotoxic effects of methylene blue on human diffuse large B-cell lymphoma U2932, TMD8, SUDHL-4, and OCI-LY19 cell lines and on human peripheral T-cell lymphoma KARPAS299 and KHYG-1 cell lines over 48 h were demonstrated at concentrations of 0 μM, 0.078125 μM, 0.15625 μM, 0.3125 μM, 0.625 μM, 1.25 μM, 2.5 μM, 5 μM, and 10 μM, respectively. Figure 3 The killing effects of cell lines a) on U2932, b) on OCI-LY19, c) on TMD8, and d) on SUDHL-4 were studied. Figure 4 The results of methylene blue (0.5 μM to 10 μM) on the KARPASS299 cell line (a) and KHYG-1 cell line (b) showed that, at 48 h, methylene blue at concentrations of 0.5 μM to 10 μM had a significant killing effect on human diffuse large B cells and T-cell lymphoma cell lines.

[0071] like Figure 5 The results of the half-maximal inhibitory concentrations (IC50) of methylene blue against the U2932 cell line, OCI-LY19 cell line, TMD8 cell line, and SUDHL-4 cell line (a) and (d) respectively show the half-maximal inhibitory concentrations (IC50) of methylene blue against diffuse large B-cell lymphoma U2932, TMD8, SUDHL-4, and OCI-LY19 cell lines after 48 h. 50 The concentrations were 0.6076 μM, 1.421 μM, 3.873 μM, and 0.5343 μM, respectively. Figure 6 The results of the half-maximal inhibitory concentrations (IC50) of methylene blue against the KARPAS299, KHYG-1, and SMZ-1 cell lines (a) and (b) respectively show the half-maximal inhibitory concentrations (IC50) of methylene blue against human peripheral T-cell lymphoma KARPAS299, KHYG-1, and SMZ-1 cell lines after 48 h. 50 The concentrations were 1.236 μM, 1.379 μM, and 2.934 μM, respectively.

[0072] Example 2 Mechanism study of methylene blue inducing lymphoma cell apoptosis by activating the PERK-eIF2α-ATF4-CHOP endoplasmic reticulum stress pathway 1. Experimental Materials 1.1 Cell line: Human diffuse large B-cell lymphoma U2932 cell line.

[0073] 1.2 Main Reagents: Methylene Blue trihydrate (CAS 7220-79-3, MedChemExpress, purity ≥98%); Rabbit anti-human phosphorylated PERK (p-PERK, Thr980) monoclonal antibody (CellSignaling Technology, #3179); Rabbit anti-human ATF4 polyclonal antibody (Cell Signaling Technology, #11815); Rabbit anti-human eIF2α monoclonal antibody (Cell Signaling Technology, #9722); Rabbit anti-human phosphorylated eIF2α (p-eIF2α, Ser51) monoclonal antibody (Cell Signaling Technology, #3398); Mouse anti-human CHOP monoclonal antibody (Cell Signaling Technology, #2895); Rabbit anti-human Caspase-3 monoclonal antibody (CellSignaling Technology, #9662); Mouse anti-human Bcl-2 monoclonal antibody (Cell Signaling Technology, #9662); Technology, #15071); Rabbit anti-human β-Tubulin monoclonal antibody (ABclonal, A12289); HRP-labeled goat anti-rabbit IgG secondary antibody (ABclonal, AS014); HRP-labeled goat anti-mouse IgG secondary antibody (ABclonal, AS003); RIPA lysis buffer (containing PMSF protease inhibitor and phosphatase inhibitor); BCA protein quantification kit, SDS-PAGE gel preparation kit, ECL ultrasensitive chemiluminescence kit.

[0074] 1.3 Main instruments: CO2 constant temperature incubator, biosafety cabinet, benchtop low temperature high speed centrifuge, vertical electrophoresis system, wet membrane transfer system, chemiluminescence imaging system.

[0075] 2. Experimental Methods 2.1 Cell Culture and Drug Treatment: U2932 cells in logarithmic growth phase were fed a 2×10⁻⁶ dose. 6 Cells were seeded at a density of 1 / well in 6-well plates and cultured in a CO2 incubator at 37°C, 5% CO2, and saturated humidity. After the cells stabilized, 5 μM methylene blue was added at different time points (0 h (control group), 12 h, 24 h, 36 h, and 48 h).

[0076] 2.2 Protein Sample Preparation Cells were collected at the same time point and washed twice with pre-chilled PBS. 150 μL of RIPA lysis buffer containing 1% PMSF and 1% phosphatase inhibitor was added to each well, and the cells were lysed on ice for 30 min, gently shaking to mix every 10 min. The cells were centrifuged at 12000 rpm for 10 min at 4 °C, and the supernatant was collected. Protein concentration was determined using the BCA method. 5×SDS loading buffer was added, and the cells were denatured at 95 °C for 5 min and stored at -80 °C for later use.

[0077] 2.3 Western Blot Detection Take 30 μg of protein sample and perform 12.5% ​​SDS-PAGE electrophoresis (stacking gel 80 V, separating gel 120 V). Wet transfer to a 0.22 μm PVDF membrane (250 mA constant current, 120 min). Block with 5% skim milk powder at room temperature for 1 h, wash 3 times with TBST, and incubate overnight at 4°C with the following primary antibodies: p-PERK (1:1000), ATF4 (1:1000), eIF2α (1:1000), p-eIF2α (1:1000), CHOP (1:1000), Caspase-3 (1:1000), Bcl-2 (1:1000), β-Tubulin (1:5000, internal control).

[0078] Wash three times with TBST, add HRP-labeled goat anti-rabbit / mouse IgG secondary antibody (1:5000) and incubate at room temperature for 1 h. Wash three times with TBST, develop color with ECL chemiluminescence reagent, and acquire images using a chemiluminescence imaging system.

[0079] 3. Experimental Results 3.1 Methylene blue time-dependent activation of the PERK-eIF2α-ATF4 signal axis After treating U2932 cells with 5 μM methylene blue for 0 h, 12 h, 24 h, 36 h, and 48 h, Western blotting was used to detect changes in the expression of endoplasmic reticulum stress-related proteins (p-PERK, ATF4, p-eIF2α, eIF2α, CHOP), apoptosis-related proteins (full-length Caspase-3, Bcl-2), and the internal control (β-Tubulin). Figure 7 As shown, methylene blue time-dependently activates the expression of endoplasmic reticulum stress and apoptosis-related proteins in U2932 cells. The phosphorylation level of upstream endoplasmic reticulum stress kinase PERK (p-PERK) begins to increase at 12 h, peaks at 24 h, and then gradually declines; the expression of downstream transcription factor ATF4 begins to be upregulated at 12 h, remains at a high level from 24 to 36 h, and slightly decreases at 48 h; at the same time, the phosphorylation trend of eIF2α (p-eIF2α) is consistent with that of p-PERK, and the total eIF2α expression shows no significant change.

[0080] The results confirm that methylene blue can time-dependently activate the classical endoplasmic reticulum stress signaling pathway PERK-eIF2α-ATF4.

[0081] 3.2 Methylene blue time-dependently induces upregulation of CHOP expression As a downstream effector molecule of the PERK-eIF2α-ATF4 pathway, the expression of the pro-apoptotic transcription factor CHOP began to increase significantly at 24 h, peaked at 36 h, and remained at a high level at 48 h. This result suggests that endoplasmic reticulum stress signals have been transmitted to the apoptosis execution phase.

[0082] 3.3 Methylene blue time-dependently upregulates total Caspase-3 protein expression Apoptosis-related protein assays showed that the expression of total Caspase-3 protein (full-length Caspase-3, molecular weight approximately 32 kDa) progressively increased with increasing methylene blue treatment time: an upward trend began at 12 h, significant upregulation occurred at 24 h, and high levels were maintained from 36 to 48 h. These results suggest that methylene blue may promote Caspase-3 expression through a CHOP-mediated transcriptional regulation mechanism, providing a sufficient reserve of effector molecules for apoptosis, or indicating that cells have entered the pre-apoptotic preparation stage.

[0083] 3.4 Methylene blue time-dependent inhibition of the anti-apoptotic protein Bcl-2 The expression of the anti-apoptotic protein Bcl-2 progressively decreased with increasing methylene blue treatment time: a decreasing trend began at 12 h, with a significant downregulation at 24 h, and reaching its lowest level between 36 and 48 h. This change was significantly negatively correlated with the upregulation of CHOP and Caspase-3, consistent with the molecular mechanism by which CHOP transcriptionally inhibits Bcl-2 and relieves its inhibitory effect on apoptosis.

[0084] The results show that methylene blue can time-dependently activate the PERK-eIF2α-ATF4-CHOP pathway, upregulate total Caspase-3 protein expression, and downregulate Bcl-2 expression.

[0085] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The use of methylene blue in the preparation of drugs for treating lymphoma, characterized in that, Methylene blue can be used as a single active ingredient or in combination with other active ingredients.

2. The use of methylene blue according to claim 1 in the preparation of a drug for treating lymphoma, characterized in that, The lymphomas include B-cell lymphomas and T-cell lymphomas.

3. The use of methylene blue according to claim 1 in the preparation of a drug for treating lymphoma, characterized in that, The drug can kill lymphoma cell lines.

4. The use of methylene blue according to claim 3 in the preparation of a drug for treating lymphoma, characterized in that, The lymphoma cell lines include human diffuse large B-cell lymphoma cell lines and human peripheral T-cell lymphoma cell lines.

5. The use of methylene blue according to claim 4 in the preparation of a drug for treating lymphoma, characterized in that, The human diffuse large B-cell lymphoma cell lines include U2932, TMD8, SUDHL-4, and OCI-LY19.

6. The use of methylene blue according to claim 4 in the preparation of a medicament for treating lymphoma, characterized in that, The human peripheral T-cell lymphoma cell lines include KARPAS299, SMZ-1, and KHYG-1.

7. The use of methylene blue according to claim 1 in the preparation of a drug for treating lymphoma, characterized in that, The drug also includes pharmaceutically acceptable excipients.

8. The use of methylene blue according to claim 1 in the preparation of a medicament for treating lymphoma, characterized in that, In the aforementioned drug, methylene blue exists in its anhydrous form or in the form of its pharmaceutically acceptable salt, solvate, or hydrate.

9. The use of methylene blue according to claim 1 in the preparation of a medicament for treating lymphoma, characterized in that, The formulation of the drug may be an injection, lyophilized powder, tablet, capsule, liposome, or nanoparticle.

10. The use of methylene blue according to claim 1 in the preparation of a medicament for treating lymphoma, characterized in that, Methylene blue is the sole active ingredient in the drug, which is used as a single agent to treat lymphoma patients or animal models, or as a drug used in combination with conventional supportive care or radiotherapy. The conventional supportive care includes hematopoietic growth factor support, anti-infection prevention and treatment, prevention of tumor lysis syndrome, symptomatic treatment of chemotherapy adverse reactions, and nutritional and psychological support.

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