CN116509873A

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

Info

Publication number: CN116509873A
Application number: CN202310449937.6A
Authority: CN
Inventors: 杜昌升; 吕婕
Original assignee: Tongji University
Current assignee: Tongji University
Priority date: 2023-04-25
Filing date: 2023-04-25
Publication date: 2023-08-01
Anticipated expiration: 2043-04-25
Also published as: CN116509873B

Abstract

The invention belongs to the technical field of biological medicines, and particularly provides application of carboplatin in preparation of a medicament for preventing or treating multiple sclerosis. The test result shows that oral carboplatin can reduce the overall clinical symptoms of experimental autoimmune encephalomyelitis and reduce spinal cord inflammation and demyelination; in vitro cell experimental data show that carboplatin inhibits proliferation and induces apoptosis of activated T cells; therefore, the carboplatin can be safely and effectively applied to the medicines for treating the multiple sclerosis.

Description

Application of carboplatin in preparation of medicine for preventing or treating multiple sclerosis

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to application of carboplatin in preparation of a medicament for preventing or treating multiple sclerosis.

Background

Multiple sclerosis (Multiple sclerosis, MS) is an autoimmune disease of the central nervous system (Central Nervous System, CNS). It is characterized by local lymphocyte infiltration, myelin sheath rupture, astrocyte proliferation, microglial activation, and neurodegeneration. The molecular mechanism of MS is complex and it is widely believed that autoreactive T cells play a critical role in the progression of the disease. Currently, at least 19 Disease Modifying Therapies (DMT) are approved in the united states for the treatment of multiple sclerosis, all of which can reduce their recurrence rate to some extent by exerting immunosuppressive or immunomodulatory effects. However, current drugs are only effective in 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 serious side effects. Therefore, there is an urgent need to develop a new and effective therapeutic agent. Experimental autoimmune encephalomyelitis (experimental autoimmune encephalomyelitis, EAE) is the best animal model to study MS pathogenesis and develop new therapies.

Carboplatin (CA) is a platinum complex and alkylating agent administered intravenously and is 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, resulting in cross-linking between and within DNA strands, leading to inhibition of DNA, RNA and protein synthesis and triggering programmed cell death, mainly in rapidly dividing cells. Carboplatin was approved in the united states for cancer chemotherapy in 1989. However, there are few reports on the regulatory function of carboplatin in the immune system. Thus, it is unclear whether carboplatin has a therapeutic effect on MS.

Disclosure of Invention

The technical problem to be solved by the invention is to provide an application of carboplatin in preparing a medicament for preventing or treating multiple sclerosis, and provide a direction for treating and preventing multiple sclerosis.

The technical scheme adopted by the invention for solving the technical problems is to provide application of carboplatin in preparing a medicament for preventing or treating multiple sclerosis.

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

Further, the agents inhibit T cell proliferation and promote activated T cell and myelin oligodendrocyte glycoprotein specific T cell apoptosis.

Further, the medicament comprises the carboplatin and a pharmaceutically acceptable carrier.

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

Compared with the prior art, the invention has the following beneficial effects: the present invention investigated the effect of carboplatin on EAE neuropathology. Oral administration of carboplatin was found to alleviate the overall clinical symptoms of EAE, reducing spinal inflammation and demyelination. In addition, the proportion and number of T cells in peripheral tissues of carboplatin treated group EAE mice were significantly reduced. At the same time, mass spectrometry data analysis also shows that the differentially expressed proteins in spleen cells of EAE mice in the control group and the carboplatin treated group are mainly related to apoptosis regulation pathways. In vitro cell experimental data indicate that carboplatin inhibits proliferation and induces apoptosis of activated T cells. The data of the present invention indicate that carboplatin can be safely and effectively used in MS therapeutic drugs.

Drawings

FIG. 1 shows that carboplatin treatment alleviates clinical symptoms in EAE mice;

figure 2 shows that carboplatin treatment reduces infiltration of pathogenic T cells in the CNS;

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

figure 4 shows that carboplatin treatment enhances apoptotic signals during EAE;

FIG. 5 shows carboplatin mediated activation of T cell apoptosis;

figure 6 shows carboplatin enhances MOG-specific T cell apoptosis in EAE mice.

Detailed Description

The invention is further described below with reference to the drawings and examples.

The present invention seeks to assess whether oral carboplatin can be used to improve the condition of EAE. Carboplatin treatment reduced myelitis, demyelination and disease scores in EAE mice. In addition, the number and proportion of pathogenic T cells (especially Th-1 and Th-17) in the spleen and draining lymph nodes of EAE mice of the carboplatin treated group were significantly reduced. Proteome differential enrichment analysis shows that the protein related to apoptosis signals expressed by spleen cells of the carboplatin treatment group is significantly changed. CFSE experimental results show that carboplatin significantly inhibited T cell proliferation. Finally, in vitro experiments prove that carboplatin can promote apoptosis of activated T cells and myelin oligodendrocyte glycoprotein (MOG for short) specific T cells. The results of the study indicate that carboplatin plays a protective role in the development and progression of EAE and that new drug designs for the treatment of MS are possible.

Experiments, assays, theoretical analysis are disclosed in detail below.

1. Materials involved in experiments (animal models) and methods of experimentation and detection common in the art

1. Animal model

Male C57BL/6 mice were purchased from Jiangsu Jiuyaokang biotechnology Co., ltd (Nanjing, china). The C57BL/6 mice are 8-10 weeks old and are fed to SPF laboratory at the center of experimental animals of the university of ataxia, free water and feed. All experiments were approved and conducted as directed by the animal protection committee of the university of homotaxis.

2. The method comprises the following steps: establishing an EAE model and drug treatment for mice

C57BL/6 mice (8-10 weeks) were subcutaneously injected with 200. Mu.g of MOG completely dissolved in complete Freund's adjuvant and containing heat-inactivated tubercle bacillus (H37 Ra,5mg/ml, BD Diagnostics) _35-55 (MEVGWYRSPFSRVVVVHLYRNGK). Each mouse was intraperitoneally injected with 200 ng/pertussis toxin (Calbiochem) on day 0 and day 2 of immunization. Mice were scored daily according to clinical symptoms, the scoring criteria were as follows: 0 minutes, no clinical symptoms; 1, tail paralysis; 2 minutes, mild paralysis (weakness of unilateral or bilateral hind limbs, incomplete paralysis); 3 minutes, paraplegia (bilateral hind limb complete paralysis); 4 minutes, paraplegia with weakness or paralysis of the forelimbs; 5 minutes, dying state or death. For drug treatment, carboplatin was dissolved in sterile water and administered by gavage at a dose of (10 mg/kg,30mg/kg,100 mg/kg) on day 3 of immunization until the end of the experiment, 200 μl of sterile water of the same volume was used as a control.

3. The method comprises the following steps: histopathological and immunohistochemical analysis

Peripheral blood was removed from each organ by cardiac perfusion with 0.9% NaCl after deep anesthesia in each mouse, and fixation was performed by 4% (w/v) paraformaldehyde perfusion. Samples of lumbar spinal tissue were taken and placed in 4% paraformaldehyde solution and fixed overnight at 4 ℃. H & E staining after paraffin embedding analyzed inflammatory infiltrates, fast blue staining analyzed spinal cord demyelination, image-Pro software for statistical analysis.

4. The method comprises the following steps: central nervous system infiltrating cell isolation and analysis

Brain and spinal cord were homogenized with a pre-chilled tissue homogenizer, collected by filtration on a 70 μm cell strainer into a 15ml centrifuge tube, centrifuged at 500g at 4℃for 10min to obtain cells, and then resuspended in 8ml of 37% Percoll. 4ml of 70% Percoll is firstly added into a 15ml centrifuge tube, and the residual 70% Percoll on the wall of the centrifuge tube is removed by instantaneous centrifugation, so that the mutual dissolution with the added 37% Percoll is avoided; the 37% percoll cell suspension was slowly added along the tube wall to avoid disruption of the interfacial layer. 780g of cells at the 37/70% Percoll interface were collected for analysis by density gradient centrifugation at 25℃for 25 min.

5. The method comprises the following steps: cell staining and flow analysis

Spleen cells and draining lymph node cells were surface stained, and FITC-CD4, APC-CD8, APC-B220 and FITC-CD11B were stained at 4℃for 30 minutes in the absence of light, washed, and then resuspended in 1 XPBS. Spleen cells, draining lymph node cells, or CNS infiltrating cells were stimulated with PMA (50 ng/ml; sigma-Aldrich), ionomycin (750 ng/ml; sigma-Aldrich) and brefeldin A (5 μg/ml; sigma-Aldrich) for 5 hours at 37 ℃. Cell surface marker CD4 was stained with antibody at 4deg.C for 30min in the dark, and then cells were resuspended in a fixative permeate for intracellular IL-17A (BioLegend, 506904, 1:100) and IFN-gamma (BioLegend, 505110, 1:100) staining, respectively. The Flow analysis was a BD FACS Verse system and was performed using Flow Jo V10 software.

6. The method comprises the following steps: ELISA detection of inflammatory factor expression in peripheral blood

Carboplatin (100 mg/kg body weight) or vehicle (sterile water) treated mice constructed the EAE model. On day 10 of modeling, approximately 150 μl of whole blood was collected from orbital venous blood. Orbital venous blood was allowed to stand at room temperature for 30 minutes. After 30 minutes, the supernatant was collected carefully by centrifugation at 4000rpm for 10 minutes at 4℃and the sediment was collected as little as possible without being sucked down to obtain serum. The concentration of IL-17A and IFN-gamma in serum was measured using a specific ELISA kit (eBioscience) according to the manufacturer's instructions.

7. The method comprises the following steps: t cell proliferation assay

Spleen cells were labeled with 10mM carboxyfluorescein diacetate succinimidyl ester (CFSE), then inoculated into 96-well plates, cultured with 200. Mu.L of RPMI1640 complete medium containing 10% FBS, 2mM L-glutamine and 50. Mu.M beta. -mercaptoethanol, and activated with anti-CD3 (2. Mu.g/ml; BD Pharmingen) and anti-CD28 (2. Mu.g/ml; BD Pharmingen) antibodies. Carboplatin at various concentrations was added simultaneously and incubated for 48 hours. Cells were collected and CFSE fluorescence was detected by flow cytometry.

8. The method comprises the following steps: t cell apoptosis assay

In RPMI1640 complete medium containing 2. Mu.g/ml anti-CD28 antibody and 2. Mu.g/ml anti-CD3 antibody at 5X 10 ⁵ Density of wells naive spleen cells were cultured in 96-well plates. Culturing for 24, 48 and 72 hours in the presence of carboplatin at different concentrations. On day 10 post immunization, splenocytes from EAE mice were isolated and stimulated with MOG35-55 (20. Mu.g/ml) for 24, 48, 72 hours. Cells were then stained with 7-AAD and Annexin-V binding anti-CD 4 or anti-CD 8 antibodies. Flow cytometry detects the percentage of living or apoptotic cells (7-AAD and Annexin-V double positive).

9. The method comprises the following steps: LC-MS/MS analysis

Mu.l of urea lysis buffer (8M urea, 100mM Tris-HCl pH 8.0), 4. Mu.l of protease inhibitor (Pierce ^TM Thermo Fisher Scientific) to protect the protein from degradation, and protein concentration was measured using Bradford method (Eppendorf Biospectrometer). Samples were analyzed on an Orbitrap Fusion, orbitrab Fusion Lumos and Q exact Plus mass spectrometer connected to an Easy nLC 1000 nanofluid LC system connected to a UltiMate 3000RSLCnano system.

10. The method comprises the following steps: statistical analysis

Statistical differences between EAE mice treated groups were analyzed by two-way ANOVA test, other data statistics were analyzed by Student's t-test, data expressed as mean±sem, P <0.05 were considered statistically significant.

Experiment 1 corresponds to the method: establishing a mouse EAE induction model and drug treatment; histopathological and immunohistochemical analysis;

experiment 2 corresponds to the method: isolation and analysis of central nervous system infiltrating cells;

experiment 3 corresponds to the method: cell staining and flow analysis; ELISA (enzyme Linked immunosorbent assay) for detecting inflammatory factor expression in peripheral blood;

experiment 4 corresponds to the method: LC-MS/MS analysis;

experiment 5 corresponds to the method: t cell proliferation experiments; t cell apoptosis experiments.

2. Experiment and results

Experiment 1 carboplatin improves clinical symptoms in EAE mice

To evaluate whether carboplatin has therapeutic effect on multiple sclerosis, the present invention is directed to a method of treating multiple sclerosis by MOG _35-55 Peptide immunization induced EAE models in C57BL/6 mice, carboplatin was orally administered daily or every two days from day 3 post immunization to the end of the experiment (fig. 1A-B), while water was administered as a solvent control. Carboplatin showed a dose-dependent inhibition of EAE severity in a series of experiments.

Oral administration of low doses of carboplatin did not improve the severity of EAE. Carboplatin delayed the onset of EAE at 30mg/kg and reduced clinical scores during EAE remission (fig. 1A). Treatment with 100mg/kg carboplatin also delayed the onset of EAE and significantly reduced its severity and cumulative clinical score (fig. 1A). The invention discovers that oral administration of carboplatin every two days can also delay the onset of EAE, relieve the symptoms of EAE and have better efficacy (figure 1B). Interestingly, administration of carboplatin (100 mg/kg, orally) after onset (day 12) still reduced EAE severity, suggesting that CA has therapeutic effects in addition to prophylactic effects.

In fig. 1: (A-C) passage through MOG _35-55 Immunization induced EAE in male C57BL/6 mice. Carboplatin was administered once daily (a) or every other day (B) starting on day 3 after immunization, or orally once every other day (C) starting on day 12 until the end of the experiment, with daily clinical scores recorded. The control group was orally administered the same volume of sterile water. Data represent mean ± SEM (n=5-7), p<0.05，**p<0.01，***p<0.001 compared to the control group (two-way ANOVA test). (D-E) on day 17 post immunization, H was performed on spinal paraffin sections isolated from EAE mice treated with either non-modular, solvent control or carboplatin (100 mg/kg, oral administration, starting on day 3)&E staining and fast blue staining. The box area in the top row is shown enlarged at the bottom, scale, 200 μm. (F-G) quantification of H&Fine CNS infiltration in E-stained sectionsCell number and area of demyelination in fast blue stained sections. Three animals per group and 15 spinal cord sections of each animal were analyzed<0.001 compared to the unmodeled group, # # p<0.001 compared to the solvent control group (Student's t test).

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

The inflammatory cell infiltration and myelin damage of CNS (brain and spinal cord) are accompanied by the onset of EAE mice, the most pronounced phenomenon being seen in peak onset, with the white matter portion of lumbar marrow being the most prominent. Therefore, the invention selects EAE mice of a carboplatin administration group and a sterile water control group, and takes spinal cord slices for pathological tissue detection at the highest incidence (day 17) to determine whether the carboplatin administration can reduce inflammatory cell infiltration at CNS focus parts of the EAE mice and reduce myelin damage. Meanwhile, healthy C57BL/6 mice (naive mice) were selected as negative controls without inflammatory cell infiltration and myelin damage. Carboplatin resulted in a significant reduction in spinal cord leukocyte infiltration compared to the control group (fig. 1d,1 f). Fast blue staining also showed less demyelination in carboplatin treated EAE mice compared to control (fig. 1e,1 g). CNS leukocyte infiltration was quantified by flow cytometry analysis on day 17 post immunization. The results demonstrate reduced cd4+ T cell accumulation in the CNS of EAE mice following carboplatin treatment (fig. 2A-D). Th17 cells producing IL-17A and Th1 cells producing IFN-gamma are the main pathogenic T effector cells of EAE. Thus, the present invention quantifies the number of Th1 and Th17 cells in CNS infiltration. After carboplatin treatment, the percentage and number of IFN-. Gamma. + CD4+ T cells (Th 1 cells) and IL-17A+ CD4+ T cells (Th 17 cells) were significantly reduced (FIGS. 2E-J). These data indicate that carboplatin treatment can significantly reduce EAE severity, reduce CNS inflammation and demyelination.

In FIG. 2, (A) total CNS infiltration was isolated from the brain and spinal cord of EAE mice on day 17 post immunization with 37/70% Percoll from the control group or carboplatin group (100 mg/kg, starting on day 3) and the amount of total infiltration was quantified by flow cytometry. (B, E, H) representative FACS plots of CD4+ T cells, th-1 cells and Th-17 cells in the CNS of EAE mice treated with carboplatin (100 mg/kg, starting on day 3) or sterile water. (C, F, I) statistical data from FACS analysis of intracellular staining of IFN-gamma and IL-17A in the CNS by GraphPad Prism 8 software. Cell count statistics of (D, G, J) CD4+ T cells, th-1 cells and Th-17 cells. Data are mean ± SEM (n=7-8 per group), statistical test using Student's t test, p <0.05, p <0.01, p <0.001 compared to control group.

Experiment 3 carboplatin reduced EAE mice peripheral CD4+ T and CD8+ T cells

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. To examine whether carboplatin affected the basal immune response, leukocytes were collected from the spleens of EAE mice in the control group and carboplatin-treated group, and the proportion of cd4+ T cells, cd8+ T cells, B cells, and cd11b+ cells in the spleen leukocytes was analyzed by flow cytometry. Surface staining showed that carboplatin treatment significantly reduced the proportion of cd4+ T cells and cd8+ T cells in the spleen without affecting the B cell and cd11b+ cell populations (top two rows, C and G of fig. 3A).

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

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

In fig. 3: (A-B) on day 10 post immunization, leukocytes were isolated from spleen (A) and draining lymph nodes (dLN) (B) of carboplatin (100 mg/kg, orally, starting on day 3) or sterile water-treated EAE animals and analyzed for percentage of CD4+ T cells, CD8+ T cells, B cells (B220+) and CD11b+ cells by surface staining and flow cytometry. Th-1 and Th-17 cells were analyzed by intracellular staining flow-through the ratio of IFN-gamma and IL-17A in CD4 positive cells. Statistical analysis of (C-J) A and B. Data represent mean ± SEM (n=9). P <0.05, < p <0.01 (Student's t test) compared to the control group. (K-L) serum was collected from EAE mice treated with 100mg/kg carboplatin or the same volume of water and assayed for secretion of cytokines IFN-gamma and IL-17A. Data are mean ± SEM (n=9-13). P <0.05 (Student's ttest) compared to control group.

Experiment 4 carboplatin treatment enhances apoptotic pathway signaling during EAE

To investigate the role of carboplatin in EAE pathogenesis, the present invention performed mass spectrometry analysis of splenocytes from EAE mice treated with sterile water and with calix, respectively (day 10 of immunization). Following carboplatin treatment, the expression of differential proteins primarily affected the biological functions of apoptosis signaling pathway regulation, protein stability regulation, cell killing, and cell cycle down regulation (fig. 4A and B). KEGG analysis showed that carboplatin affects EAE progression primarily through apoptosis, antigen processing, and presentation, etc. Among them, proteins associated with antigen processing and presentation (e.g., hsp90ab1, canx, hsp90aa1, pdia3, and Hspa 8) and apoptosis (e.g., ybx3, runx3, rps7, and Hsph 1) were significantly down-regulated after carboplatin treatment (fig. 4C and D).

In fig. 4: (A-B) the comparative proteome analysis of the control and carboplatin groups of EAE mice was functionally categorized by GO enrichment analysis (A) and network analysis (B). (C-D) KEGG enrichment analysis (C) and KEGG heat map (D) show signaling pathways that significantly enrich total protein of splenocytes in the control group and carboplatin (100 mg/kg, starting on day 3) group of EAE mice on day 10 post immunization.

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

The surface staining results of splenocytes and lymphocytes from experiment 3 showed a significant decrease in the frequency of T cells in peripheral immune tissue following carboplatin treatment. At the same time, mass spectrometry data analysis also shows that the differentially expressed proteins are mainly related to apoptosis regulation. It is therefore speculated that carboplatin may affect proliferation or apoptosis of T cells. To verify this hypothesis, the present invention performed in vitro cell proliferation or apoptosis experiments using mouse spleen cells. Spleen cells isolated from wild-type mice were activated with antibodies against mouse CD3 and mouse CD28 in the presence of carboplatin for 48 hours. As shown in fig. 5A, carboplatin dose-dependent inhibited T cell antigen-stimulated cell proliferation.

Next, apoptotic cell fractions were detected with annexin V and 7-AAD staining after carboplatin (3. Mu.M and 10. Mu.M) treatment for various durations (24-72 hours). In vitro experiments showed that carboplatin enhanced apoptosis in cd4+ and cd8+ T cell populations (fig. 5B-G).

The invention also tests the effect of carboplatin on MOG-specific T cells. Splenocytes were isolated from EAE mice on day 10 post immunization and re-stimulated in vitro with MOG (20 μg/ml) in the presence of carboplatin and apoptosis in cd4+ and cd8+ T subpopulations were measured by FACS (fig. 6). Carboplatin treatment significantly promoted apoptosis of MOG-specific cd4+ and cd8+ T cells. These data indicate that carboplatin induces T cell apoptosis (FIGS. 6A-F).

In fig. 5: (A) Spleen cells from wild-type mice were isolated and cells were 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. Statistical analysis of apoptosis in (D-E) B and C. Data from three independent experiments (mean ± SEM). P <0.05, < p <0.001 (two-way ANOVA) compared to the control group.

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

3. Discussion and summary after experiments

Multiple sclerosis (Multiple sclerosis, MS) is a chronic neurological disease with potentially damaging and long-term complications. Experimental autoimmune encephalomyelitis (experimental autoimmune encephalomyelitis, EAE) is a cd4+ T cell mediated demyelinating disease of the central nervous system, 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 exhibit the characteristics of activating and clonally expanding cells, supporting the notion that these cells are actively involved in the observed lesions. Studies have shown that myelin-reactive CD8+ T cells directly trigger axonal loss, while neuronal damage has been found to correlate with the extent of CD8+ T cell infiltration. Thus, induction of T cell apoptosis may help to improve EAE.

Apoptosis is a prerequisite for the growth and survival of most multicellular animals and ensures maintenance of tissue homeostasis. Its contradictory role in immunology can be illustrated by the following facts: this process not only aids in balancing function, but can also have deleterious effects, including tissue damage. Experiments using the EAE model have shown that cell death affects infiltrating lymphocytes and CNS resident cells and contributes to the regression of axonal injury and inflammation. Research reports that glatiramer acetate and interferon beta drugs treat MS by inducing apoptosis of peripheral T cells. Likewise, the data of the present invention demonstrate that carboplatin reduces the proportion of cd4+ T cells in the spleen and draining lymph nodes. Probably due to the increased apoptosis of peripheral T cells, the number of cd4+ T cells infiltrating the CNS is also reduced. In addition, the in vitro experimental data of the present invention also demonstrate that carboplatin induces apoptosis of cd4+ T cells and cd8+ T cells in spleen cells.

Apoptosis may be triggered by a mitochondrial pathway, an endoplasmic reticulum stress pathway, or an exogenous pathway 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 pro-and anti-apoptotic proteins that are key components of the "intrinsic" pathway. Carboplatin treatment has been reported to significantly reduce Bcl-2 protein expression and induce Raji apoptosis in a time-dependent manner. Mass spectrometry data of the present invention show that differentially expressed proteins in spleen cells between control and carboplatin treated groups are primarily involved in the regulation of apoptosis signals. Thus, the present invention speculates that carboplatin promotion of T cell apoptosis may also be regulated by the Bcl-2 family.

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

While the invention has been described with reference to the preferred embodiments, it is not intended to limit the invention thereto, and it is to be understood that other modifications and improvements may be made by those skilled in the art without departing from the spirit and scope of the invention, which is therefore defined by the appended claims.

Claims

1. Application of carboplatin in preparing medicine for preventing or treating multiple sclerosis is provided.

2. The use of claim 1, wherein the multiple sclerosis comprises: local lymphocyte infiltration, myelin sheath rupture, astrocyte proliferation, microglial activation, and neurodegeneration.

3. The use of claim 1, wherein the medicament inhibits T cell proliferation and promotes activated T cell and myelin oligodendrocyte glycoprotein specific T cell apoptosis.

4. The use according to claim 1, wherein the medicament comprises the carboplatin and a pharmaceutically acceptable carrier.

5. The use according to claim 1, wherein the medicament is administered orally once every two days for preventing multiple sclerosis and the amount of carboplatin is controlled to be 30mg/kg-100mg/kg.