CN116509873A - Application of carboplatin in preparation of medicine for preventing or treating multiple sclerosis - Google Patents

Abstract

The invention belongs to the technical field of biomedicine, and specifically provides an application of carboplatin in the preparation of drugs for preventing or treating multiple sclerosis. The test results showed that oral administration of carboplatin could alleviate the overall clinical symptoms of experimental autoimmune encephalomyelitis, reduce spinal cord inflammation and demyelination; in vitro cell experiment data showed that carboplatin inhibited the proliferation of activated T cells and induced their apoptosis; Thus showing that carboplatin can be safely and effectively used in the treatment of multiple sclerosis.

Description

Application of carboplatin in the preparation of drugs for preventing or treating multiple sclerosis

technical field

The invention belongs to the technical field of biomedicine, and in particular relates to the application of carboplatin in the preparation of drugs for preventing or treating multiple sclerosis.

Background technique

Multiple sclerosis (Multiple sclerosis, MS) is an autoimmune disease of the central nervous system (Central Nervous System, CNS). It is characterized by focal lymphocytic infiltration, myelin disruption, astrocyte proliferation, microglial activation, and neurodegeneration. The molecular mechanisms of MS are complex, and it is widely believed that autoreactive T cells play a key role in the development of the disease. Currently, at least 19 disease-modifying therapies (DMTs) are approved in the United States for the treatment of MS, all of which can reduce the relapse rate to some extent by exerting immunosuppressive or immunomodulatory effects. However, current drugs are only effective for some MS patients and have no significant therapeutic effect on secondary progressive MS. In addition, the long-term effects of these treatments are associated with complications, which can also cause some serious side effects. Therefore, there is an urgent need to develop a new and effective therapeutic drug. Experimental autoimmune encephalomyelitis (EAE) is the best animal model for studying the pathogenesis of MS and developing new treatments.

Carboplatin (CA) is an intravenously administered platinum complex and alkylating agent used as a chemotherapeutic agent in the treatment of various cancers, mainly ovarian, head and neck, and lung cancers. Carboplatin and cisplatin act as alkylating agents, causing cross-links between and within DNA strands, leading to inhibition of DNA, RNA, and protein synthesis and triggering programmed cell death, mainly in rapidly dividing cells. In 1989, carboplatin was approved for cancer chemotherapy in the United States. However, there are few reports on the regulatory function of carboplatin in the immune system. Therefore, whether carboplatin has a therapeutic effect on MS is unknown.

Contents of the invention

The technical problem to be solved by the present invention is to provide an application of carboplatin in the preparation of a drug for preventing or treating multiple sclerosis, so as to provide directions for the treatment and prevention of multiple sclerosis.

The technical scheme adopted by the present invention to solve the above-mentioned technical problems is to provide an application of carboplatin in the preparation of drugs for preventing or treating multiple sclerosis.

Further, the multiple sclerosis includes: local lymphocyte infiltration, myelin sheath rupture, astrocyte proliferation, microglial activation and neurodegeneration.

Further, the drug inhibits the proliferation of T cells and promotes the apoptosis of activated T cells and myelin oligodendrocyte glycoprotein-specific T cells.

Further, the drug includes the carboplatin and a pharmaceutically acceptable carrier.

Further, when preventing multiple sclerosis, the drug is taken orally once every two days, and the dosage of carboplatin is controlled at 30 mg/kg-100 mg/kg.

Compared with the prior art, the present invention has the following beneficial effects: the present invention studies the influence of carboplatin on EAE neuropathology. The study found that oral carboplatin can reduce the overall clinical symptoms of EAE, reduce spinal cord inflammation and demyelination. In addition, the proportion and number of T cells in the peripheral tissues of EAE mice in the carboplatin-treated group were significantly decreased. At the same time, mass spectrometry data analysis also showed that the differentially expressed proteins in splenocytes of EAE mice in control group and carboplatin-treated group were mainly related to apoptosis regulation pathways. In vitro cell experiment data showed that carboplatin inhibited the proliferation of activated T cells and induced their apoptosis. The data of the present invention demonstrate that carboplatin can be safely and effectively used in the treatment of MS.

Description of drawings

Figure 1 shows that carboplatin treatment alleviates the clinical symptoms of EAE mice;

Figure 2 demonstrates that carboplatin treatment reduces the infiltration of pathogenic T cells in the CNS;

Figure 3 shows that carboplatin treatment reduces the number of peripheral T cells in EAE mice;

Figure 4 demonstrates that carboplatin treatment enhanced apoptotic signaling during EAE;

Figure 5 shows that carboplatin mediates the apoptosis of activated T cells;

Figure 6 demonstrates that carboplatin enhances MOG-specific T cell apoptosis in EAE mice.

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

The present invention seeks to assess whether oral carboplatin can be used to improve the condition of EAE. Carboplatin treatment reduced spinal cord inflammation, demyelination, and disease scores in EAE mice. In addition, the number and proportion of pathogenic T cells (especially Th-1 and Th-17) were significantly reduced in the spleen and draining lymph nodes of EAE mice in the carboplatin-treated group. The differential enrichment analysis of proteome showed that the proteins related to apoptosis signal expressed in splenocytes of carboplatin treatment group changed significantly. The results of CFSE experiment showed that carboplatin significantly inhibited the proliferation of T cells. Finally, in vitro experiments proved that carboplatin can promote the apoptosis of activated T cells and myelin oligodendrocyte glycoprotein (abbreviated as MOG)-specific T cells. The results of the study showed that carboplatin played a protective role in the occurrence and development of EAE, and it was possible to design new drugs for the treatment of MS.

The experiment, detection, and theoretical analysis are disclosed in detail below.

1. The materials (animal model) involved in the experiment and the common experiments and detection methods in this field

1. Animal model

Male C57BL/6 mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. (Nanjing, China). The C57BL/6 mice used were 8-10 weeks old and were raised in the SPF laboratory of the Experimental Animal Center of Tongji University, with free access to water and feed. All experiments were approved and performed in accordance with the guidelines of the Animal Care Committee of Tongji University.

2. Methods: establishment of mouse EAE model and drug treatment

C57BL/6 mice (8-10 weeks) were subcutaneously injected with 200 μg of MOG _35-55 (MEVGWYRSPFSRVVHLYRNGK) completely dissolved in complete Freund's adjuvant and containing heat-killed Mycobacterium tuberculosis (H37Ra, 5 mg/ml, BD Diagnostics). On day 0 and day 2 of immunization, 200 ng/pertussis toxin (Calbiochem) was intraperitoneally injected into each mouse. The mice were scored according to clinical symptoms every day, and the scoring criteria were as follows: 0 points, no clinical symptoms; 1 point, tail paralysis; 2 points, mild paralysis (unilateral or bilateral hindlimb weakness, incomplete paralysis); 3 points, paraplegia (complete paralysis of bilateral hind limbs); 4 points, paraplegia with weakness or paralysis of the forelimbs; 5 points, dying state or death. For drug treatment, carboplatin was dissolved in sterile water and dosed (10mg/kg, 30mg/kg, 100mg/kg) by intragastric administration on the third day of immunization until the end of the experiment. The same volume of 200μl sterile water was used as a control.

3. Methods: Histopathological and Immunohistochemical Analysis

After each mouse was deeply anesthetized, the heart was perfused with 0.9% NaCl to remove the peripheral blood in each organ, and perfused with 4% (w/v) paraformaldehyde for fixation. The lumbar cord tissue samples were placed in 4% paraformaldehyde solution and fixed overnight at 4°C. After paraffin embedding, H&E staining was used to analyze inflammatory infiltration, fast blue staining was used to analyze spinal cord demyelination, and Image-Pro software was used for statistical analysis.

4. Methods: CNS Infiltrating Cell Isolation and Analysis

The brain and spinal cord were homogenized with a pre-cooled tissue homogenizer, filtered through a 70 μm cell strainer and collected into a 15ml centrifuge tube, centrifuged at 500g at 4°C for 10min to obtain the cells, and then the cells were resuspended in 8ml of 37% Percoll. Gently add 4ml 70% Percoll to the 15ml centrifuge tube first, then centrifuge briefly to remove the remaining 70% Percoll on the wall of the centrifuge tube to avoid miscibility with the 37% Percoll added later; slowly add 37% Percoll cell suspension along the tube wall , to avoid damage to the interface layer. The cells located at the interface of 37/70% Percoll were collected by density gradient centrifugation at 25°C for 25 minutes at 780g for analysis.

5. Methods: Cell Staining and Flow Cytometry

Splenocytes and draining lymph node cells were stained on the surface, FITC-CD4, APC-CD8, APC-B220 and FITC-CD11b were stained at 4°C in the dark for 30 minutes, washed, and then resuspended in 1×PBS. Spleen cells, draining lymph node cells, or CNS infiltrating cells were stimulated with PMA (50ng/ml; Sigma-Aldrich), ionomycin (750ng/ml; Sigma-Aldrich) and brefeldin A (5μg/ml; Sigma-Aldrich) for 5 hours at 37°C . The cell surface marker CD4 was stained with an antibody at 4°C in the dark for 30 minutes, and then the cells were resuspended in a fixed permeabilization solution, and the intracellular IL-17A (BioLegend, 506904, 1:100) and IFN-γ (BioLegend, 505810, 1:100) were analyzed respectively. 100) Dyeing. BD FACS Verse system was used for flow analysis, and Flow JoV10 software was used for analysis.

6. Method: ELISA to detect the expression of inflammatory factors in peripheral blood

Carboplatin (100mg/kg body weight) or vehicle (sterile water) treated mice to construct EAE model. On day 10 of modeling, approximately 150 μl of whole blood was collected from orbital venous blood. Orbital venous blood was left at room temperature for 30 minutes. After 30 minutes, centrifuge at 4000 rpm for 10 minutes at 4°C, carefully collect the supernatant, and try not to suck the sediment below to obtain serum. The concentrations of IL-17A and IFN-γ in serum were measured using specific ELISA kits (eBioscience) according to the manufacturer's instructions.

7. Method: T cell proliferation assay

Splenocytes were labeled with 10 mM carboxyfluorescein diacetate succinimidyl ester (CFSE), then inoculated into 96-well plates, and added to 200 μL RPMI1640 complete medium containing 10% FBS, 2 mM L-glutamine and 50 μM β-mercaptoethanol After culturing, the cells were activated by adding anti-CD3 (2 μg/ml; BD Pharmingen) and anti-CD28 (2 μg/ml; BD Pharmingen) antibodies. Various concentrations of carboplatin were added at the same time, and cultured for 48 hours. Cells were collected and CFSE fluorescence was detected by flow cytometry.

8. Method: T cell apoptosis experiment

Naive splenocytes were cultured in 96-well plates at a density of 5×10 ⁵ /well in RPMI1640 complete medium containing 2 μg/ml anti-CD28 antibody and 2 μg/ml anti-CD3 antibody. Incubate for 24, 48, 72 hours in the presence of different concentrations of carboplatin. On day 10 after immunization, splenocytes from EAE mice were isolated and stimulated with MOG35-55 (20 μg/ml) for 24, 48, and 72 hours. Cells were then stained with 7-AAD and Annexin-V combined with anti-CD4 or anti-CD8 antibodies. The percentage of living cells or apoptotic cells (7-AAD and Annexin-V double positive) was detected by flow cytometry.

9. Method: LC-MS/MS Analysis

Proteins were protected from degradation with 400 μl of urea lysis buffer (8M urea, 100 mM Tris-HCl pH 8.0), 4 μl of protease inhibitors (Pierce ^™ , Thermo Fisher Scientific), and protein concentrations were measured using the Bradford method (Eppendorf Biospectrometer). Samples were analyzed on Orbitrap Fusion, OrbitrabFusion Lumos, and Q Exactive Plus mass spectrometers connected to an Easy nLC 1000 nanoflow LC system connected to an UltiMate3000RSLCnano system.

10. Methods: Statistical Analysis

Statistical differences between EAE mouse treatment groups were analyzed by two-way ANOVA test, and other data statistics were analyzed by Student’s t-test, and the data were expressed as mean±SEM, and P<0.05 was considered statistically significant.

Corresponding methods of experiment 1: establishment of mouse EAE induction model and drug treatment; histopathological and immunohistochemical analysis;

Corresponding method of experiment 2: isolation and analysis of central nervous system infiltrating cells;

Corresponding methods of experiment 3: cell staining and flow cytometry analysis; ELISA detection of inflammatory factor expression in peripheral blood;

Experiment 4 corresponding method: LC-MS/MS analysis;

Corresponding methods of experiment 5: T cell proliferation experiment; T cell apoptosis experiment.

2. Experiment and results

Experiment 1 Carboplatin improves the clinical symptoms of EAE mice

In order to evaluate whether carboplatin has a therapeutic effect on multiple sclerosis, the present invention induces EAE model in C57BL/6 mice by MOG _35-55 peptide immunization, orally every day or every two days from the 3rd day after immunization to the end of the experiment Carboplatin was given once (Fig. 1A-B), while water was given as a solvent control. Carboplatin showed a dose-dependent inhibitory effect on the severity of EAE in a series of experiments.

Oral administration of low-dose carboplatin did not improve the severity of EAE. At 30 mg/kg, carboplatin delayed the onset of EAE and reduced clinical scores during EAE remission (Fig. 1A). Treatment with 100 mg/kg carboplatin also delayed the onset of EAE and significantly reduced its severity and cumulative clinical score (Fig. 1A). The present invention found that oral administration of carboplatin every two days can also delay the onset of EAE, alleviate the symptoms of EAE, and have better drug efficacy ( FIG. 1B ). Interestingly, administration of carboplatin (100 mg/kg, po) after onset (day 12) still reduced the severity of EAE, suggesting that CA has a therapeutic role in addition to its preventive role.

In Figure 1: (AC) EAE was induced in male C57BL/6 mice by MOG _35-55 immunization. Carboplatin was given once a day (A) or every other day (B) from the 3rd day after immunization, or administered orally every other day (C) from the 12th day until the end of the experiment, and the clinical scores were recorded every day. The control group took the same volume of sterile water orally. Data represent mean±SEM (n=5-7), *p<0.05, **p<0.01, ***p<0.001 compared with control group (two-way ANOVA test). (DE) On day 17 after immunization, H&E staining and fast blue were performed on paraffin sections of spinal cords isolated from EAE mice treated with unmodeled group, vehicle control group or carboplatin (100 mg/kg, orally, starting from day 3) dyeing. Boxed areas in the top row are shown enlarged at the bottom, scale bar, 200 μM. (FG) Quantification of the number of CNS-infiltrated cells in H&E-stained sections and the area of demyelination in Fast Blue-stained sections. Three animals per group, and 15 spinal cord sections per animal analyzed, ***p<0.001 vs. unmodeled group, ###p<0.001 vs. vehicle control group (Student's t test).

Experiment 2 Carboplatin reduces leukocyte infiltration and neuropathy in the central nervous system of EAE mice

The onset of EAE mice is accompanied by inflammatory cell infiltration and myelin sheath damage in the CNS (brain and spinal cord). Therefore, the present invention selects the carboplatin administration group and the EAE mice of the sterile water control group, and takes spinal cord sections at the peak of the onset (the 17th day) for pathological tissue detection to determine whether carboplatin administration can reduce the CNS of EAE mice. The infiltration of inflammatory cells in the lesion site and the reduction of myelin damage. At the same time, the present invention selects healthy C57BL/6 mice (naive mice) as a negative control without inflammatory cell infiltration and myelin sheath damage. Carboplatin resulted in a significant reduction in spinal cord leukocyte infiltration compared with controls (Fig. 1D, 1F). Fast blue staining also revealed less demyelination in carboplatin-treated EAE mice compared to controls (Fig. 1E, 1G). On day 17 after immunization, CNS leukocyte infiltration was quantified by flow cytometry analysis. The results confirmed the reduction of CD4+ T cell accumulation in the CNS of EAE mice after carboplatin treatment (Fig. 2A-D). Th17 cells producing IL-17A and Th1 cells producing IFN-γ are the main pathogenic T effector cells in EAE. Thus, the present invention quantifies the number of Th1 and Th17 cells in CNS infiltration. The percentages and numbers of IFN-γ+CD4+ T cells (Th1 cells) and IL-17A+CD4+ T cells (Th17 cells) were significantly decreased after carboplatin treatment (Fig. 2E-J). These data above indicate that carboplatin treatment can significantly reduce the severity of EAE, reduce CNS inflammation and demyelination.

In Figure 2: (A) Total CNS infiltrates were isolated from the brain and spinal cord of EAE mice in the control or carboplatin group (100 mg/kg, starting from day 3) with 37/70% Percoll on day 17 after immunization , and the number of total infiltrates was quantified by flow cytometry. (B, E, H) Representative CD4+ T cells, Th-1 cells and Th-17 cells in the CNS of EAE mice treated with carboplatin (100 mg/kg, starting from day 3) or sterile water FACS plot. (C, F, I) Statistical data of FACS analysis of intracellular staining for IFN-γ and IL-17A in CNS by GraphPad Prism 8 software. (D, G, J) Cell number statistics of CD4+ T cells, Th-1 cells and Th-17 cells. The data are mean±SEM (n=7-8 in each group), and the statistical test adopts Student's t test, compared with the control group, *p<0.05, **p<0.01, ***p<0.001.

Experiment 3 Carboplatin reduces peripheral CD4+T and CD8+T cells in EAE mice

MS is an autoimmune disease caused by immune cells of the peripheral immune system; T cells, B cells and monocytes are involved in the pathogenesis of MS. In order to detect whether carboplatin affects the basic immune response, leukocytes were collected from the spleens of EAE mice in control and carboplatin-treated groups, and CD4+T cells, CD8+T cells, B cells and Proportion of CD11b+ cells. Surface staining revealed that carboplatin treatment significantly reduced the proportions of CD4+ T cells and CD8+ T cells in the spleen without affecting the B cell and CD11b+ cell populations (Fig. 3A top two rows, C and G).

Next, the present invention uses intracellular cytokine staining to detect the number of Th-1 and Th-17 cells in the spleen. It was found that the proportional numbers of Th-1 and Th-17 cells in the CD4+ population were significantly reduced in carboplatin-treated EAE mice (Fig. 3A bottom row, D and H). The same analysis was performed on immune cells in the draining lymph nodes, and the present invention also obtained similar results (Fig. 3B, E, F, I and J).

Finally, the secretion of cytokines IFN-γ and IL-17A in serum was significantly reduced in carboplatin-treated EAE mice (Fig. 3K and L).

In Figure 3: (A-B) Day 10 after immunization, spleen (A) and draining lymph nodes (dLN) (B) from EAE animals treated with carboplatin (100 mg/kg, orally, from day 3) or sterile water ) to isolate leukocytes and analyze the percentages of CD4+ T cells, CD8+ T cells, B cells (B220+) and CD11b+ cells by surface staining and flow cytometry. The ratio of IFN-γ and IL-17A in CD4 positive cells was analyzed by intracellular staining flow cytometry in Th-1 and Th-17 cells. (C-J) Statistical analysis of A and B. Data represent mean ± SEM (n=9). Compared with the control group, *p<0.05, **p<0.01, ***p<0.01 (Student's t test). (K-L) Serum was collected from EAE mice treated with 100 mg/kg carboplatin or the same volume of water, and the secretion of cytokines IFN-γ and IL-17A was analyzed. Data are mean ± SEM (n=9-13). *p<0.05 (Student's sttest) compared to the control group.

Experiment 4 Carboplatin treatment enhances apoptotic pathway signaling during EAE

In order to explore the role of carboplatin in the pathogenesis of EAE, the present invention carried out mass spectrometry analysis on splenocytes of EAE mice (immunization day 10) treated with sterile water and carbine respectively. After carboplatin treatment, the differentially expressed proteins mainly affected the biological functions of apoptosis signaling pathway regulation, protein stability regulation, cell killing and cell cycle negative regulation (Fig. 4A and B). KEGG analysis showed that carboplatin mainly affected the progression of EAE through apoptosis, antigen processing and presentation. Among them, proteins related to antigen processing and presentation (e.g., Hsp90ab1, Canx, Hsp90aa1, Pdia3, and Hspa8) and apoptosis (e.g., Ybx3, Runx3, Rps7, and Hsph1) were significantly downregulated after carboplatin treatment (Fig. 4C and D) .

In Figure 4: (A-B) Comparative proteome analysis of control and carboplatin groups of EAE mice classified by function by GO enrichment analysis (A) and network analysis (B). (C-D) KEGG enrichment analysis (C) and KEGG heat map (D) show that the total protein of splenocytes in the control group and carboplatin (100 mg/kg, starting from day 3) group of EAE mice on day 10 after immunization was significantly enriched signaling pathways.

Experiment 5 Carboplatin inhibits T cell proliferation and promotes T cell apoptosis

The results of surface staining of splenocytes and lymphocytes in Experiment 3 showed that the frequency of T cells in peripheral immune tissues was significantly reduced after carboplatin treatment. At the same time, mass spectrometry data analysis also showed that the differentially expressed proteins were mainly related to the regulation of apoptosis. Therefore, it is speculated that carboplatin may affect the proliferation or apoptosis of T cells. In order to verify this hypothesis, the present invention uses mouse splenocytes to carry out in vitro cell proliferation or apoptosis experiments. Splenocytes isolated from wild-type mice were activated with anti-mouse CD3 and anti-mouse CD28 antibodies for 48 hours in the presence of carboplatin. As shown in Figure 5A, carboplatin dose-dependently inhibited T cell antigen-stimulated cell proliferation.

Next, the proportion of apoptotic cells was detected by annexin V and 7-AAD staining after carboplatin (3 μM and 10 μM) treatment for different durations (24–72 hours). In vitro experiments showed that carboplatin enhanced the apoptosis of CD4+ and CD8+ T cell populations (Fig. 5B-G).

The present invention also tested the effect of carboplatin on MOG-specific T cells. Splenocytes were isolated from EAE mice on day 10 after immunization and restimulated in vitro with MOG (20 μg/ml) in the presence of carboplatin, and apoptosis in CD4+ and CD8+ T subsets was measured by FACS (Fig. 6). Carboplatin treatment significantly promoted the apoptosis of MOG-specific CD4+ and CD8+ T cells. These above data all indicate that carboplatin induces T cell apoptosis (Fig. 6A-F).

In Figure 5: (A) Splenocytes from wild-type mice were isolated and activated with 2 μg/ml anti-mouse CD3 and 2 μg/ml anti-mouse CD28 antibodies; cell proliferation was assessed by CFSE staining. (B-C) Representative FACS plots of carboplatin-treated CD4+ (B) or CD8+ (C) T cell apoptosis. (D-E) Statistical analysis of apoptosis in B and C. Data are from three independent experiments (mean ± SEM). Compared with the control group, *p<0.05, ***p<0.001 (two-way ANOVA).

In Figure 6: (A-B) Splenocytes were isolated from EAE mice on day 10 after immunization and restimulated with MOG35-55 (20 μg/ml). The cells were treated with carboplatin for 24, 48 or 72 hours, and the apoptosis of CD4+(A) or CD8+(B) T cells was detected by flow cytometry. (C-D) Statistical analysis of A and B. Data are from three independent experiments (mean ± SEM). Compared with the control group, *p<0.05, ***p<0.001 (two-way ANOVA).

3. Discussion and conclusion after the experiment

Multiple sclerosis (MS) is a chronic neurological disease with potentially devastating and long-term complications. Experimental autoimmune encephalomyelitis (EAE) is a CD4+ T cell-mediated central nervous system demyelinating disease, which is often used as an animal model of human MS. Activated CD4+ T cells migrate from the periphery to the central nervous system, where they initiate the inflammatory response cascade by secreting cytokines and chemokines. CD8+ T cells are also involved in the pathogenesis of EAE. CD8+ T lymphocytes detected in MS lesions exhibited characteristics of activated and clonally expanded cells, supporting the concept that these cells are actively involved in the observed damage. It has been shown that myelin-responsive CD8+ T cells directly trigger axonal loss, while neuronal damage has been found to correlate with the degree of CD8+ T cell infiltration. Therefore, induction of T cell apoptosis may help to improve EAE.

Apoptosis is a prerequisite for growth and survival in most multicellular animals and ensures maintenance of tissue homeostasis. Its paradoxical role in immunology is illustrated by the fact that this process not only contributes to balanced function, but also has deleterious effects, including tissue damage. Experiments using the EAE model showed that cell death affects infiltrating lymphocytes and CNS resident cells and contributes to resolution of axonal injury and inflammation. Studies have reported that drugs such as glatiramer acetate and interferon beta treat MS by inducing apoptosis in peripheral T cells. Likewise, the present data show that carboplatin reduces the proportion of CD4+ T cells in the spleen and draining lymph nodes. Possibly due to increased apoptosis of peripheral T cells, the number of CD4+ T cells infiltrating the CNS was also reduced. In addition, the in vitro experimental data of the present invention also shows that carboplatin induces the apoptosis of CD4+T cells and CD8+T cells in splenocytes.

Apoptosis may be triggered by mitochondrial pathways, endoplasmic reticulum stress pathways, or extrinsic pathways mediated by death receptors. Among these pathways, the mitochondrial pathway can be activated by caspase cascades and Bcl-2 family members in mitochondria. The Bcl-2 family contains a variety of proapoptotic and antiapoptotic proteins that are key components of the "intrinsic" pathway. It has been reported that carboplatin treatment significantly reduces the expression of Bcl-2 protein and induces Raji cell apoptosis in a time-dependent manner. The mass spectrometry data of the present invention show that the differentially expressed proteins in splenocytes between the control group and the carboplatin treatment group are mainly related to the regulation of apoptosis signals. Therefore, the present invention speculates that carboplatin's promotion of T cell apoptosis may also be regulated by the Bcl-2 family.

In summary, the present invention demonstrates that carboplatin attenuates the clinical and histopathological symptoms of EAE by suppressing the number of pathogenic T cells in the peripheral tissues of EAE mice. Furthermore, the results of the present invention show that carboplatin inhibits T cell proliferation and promotes T cell apoptosis. The present study demonstrates that carboplatin is a potential therapeutic agent for the treatment of MS.

Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art may make some modifications and improvements without departing from the spirit and scope of the present invention. Therefore, the present invention The scope of protection should be defined by the claims.

Claims (5)

1. Application of carboplatin in the preparation of drugs for preventing or treating multiple sclerosis. 2 . The application according to claim 1 , wherein the multiple sclerosis includes: local lymphocyte infiltration, myelin sheath rupture, astrocyte proliferation, microglia activation and neurodegeneration. 3. The application according to claim 1, characterized in that the drug inhibits the proliferation of T cells and promotes the apoptosis of activated T cells and myelin oligodendrocyte glycoprotein-specific T cells. 4. The application according to claim 1, characterized in that the drug comprises the carboplatin and a pharmaceutically acceptable carrier. 5. The application according to claim 1, characterized in that, when preventing multiple sclerosis, the drug is taken orally once every two days, and the dosage of carboplatin is controlled to be 30 mg/kg-100 mg/kg.