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

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

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CN116509873A
CN116509873A CN202310449937.6A CN202310449937A CN116509873A CN 116509873 A CN116509873 A CN 116509873A CN 202310449937 A CN202310449937 A CN 202310449937A CN 116509873 A CN116509873 A CN 116509873A
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carboplatin
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eae
multiple sclerosis
apoptosis
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CN116509873B (en
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杜昌升
吕婕
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Tongji University
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Abstract

本发明属于生物医药技术领域,具体而言,提供了一种卡铂在制备预防或治疗多发性硬化症药物中的应用。试验结果表明,口服卡铂可以减轻实验性自身免疫性脑脊髓炎的总体临床症状,减少脊髓炎症和脱髓鞘现象;体外细胞实验数据表明卡铂抑制活化T细胞的增殖并诱导其凋亡;从而表明卡铂可安全有效地应用于多发性硬化症治疗药物中。

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

卡铂在制备预防或治疗多发性硬化症药物中的应用Application of carboplatin in the preparation of drugs for preventing or treating multiple sclerosis

技术领域technical field

本发明属于生物医药技术领域,尤其涉及卡铂在制备预防或治疗多发性硬化症药物中的应用。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.

背景技术Background technique

多发性硬化(Multiple sclerosis,MS)是一种中枢神经系统(Central NervousSystem,CNS)的自身免疫性疾病。其特征是局部淋巴细胞浸润、髓鞘破裂、星形胶质细胞增殖、小胶质细胞活化和神经变性。MS的分子机制很复杂,人们普遍认为自身反应性T细胞在疾病的发展中起着关键作用。目前,美国至少批准了19种疾病修饰疗法(DMT)用于治疗多发性硬化症,所有这些疗法都可以通过发挥免疫抑制或免疫调节作用在一定程度上降低其复发率。然而,目前的药物仅对一些MS患者有效,对继发性进展性MS没有明显治疗作用。此外,这些治疗的长期效果与并发症相关,也会引起一些严重的副反应。因此,迫切需要开发一种新的、有效的治疗药物。实验性自身免疫性脑脊髓炎(experimental autoimmuneencephalomyelitis,EAE)是研究MS发病机制和开发新疗法的最佳动物模型。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.

卡铂(carboplatin,CA)是一种静脉给药的铂类配合物和烷基化剂,用作治疗各种癌症的化疗剂,主要是卵巢癌、头颈癌和肺癌。卡铂和顺铂作为烷基化剂,导致DNA链之间和链内的交联,导致DNA、RNA和蛋白质合成受到抑制,并触发程序性细胞死亡,主要发生在快速分裂的细胞中。1989年,卡铂在美国被批准用于癌症化疗。然而,关于卡铂在免疫系统中的调节功能的报道很少。因此,卡铂是否对MS有治疗作用尚不清楚。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.

发明内容Contents of the invention

本发明所要解决的技术问题是提供一种卡铂在制备预防或治疗多发性硬化症药物中的应用,为多发性硬化症的治疗和预防提供方向。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.

本发明为解决上述技术问题而采用的技术方案是提供一种卡铂在制备预防或治疗多发性硬化症药物中的应用。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.

进一步地,所述多发性硬化症包括:局部淋巴细胞浸润、髓鞘破裂、星形胶质细胞增殖、小胶质细胞活化和神经变性。Further, the multiple sclerosis includes: local lymphocyte infiltration, myelin sheath rupture, astrocyte proliferation, microglial activation and neurodegeneration.

进一步地,所述药物抑制T细胞增殖,并促进活化的T细胞和髓鞘少突胶质细胞糖蛋白特异性T细胞凋亡。Further, the drug inhibits the proliferation of T cells and promotes the apoptosis of activated T cells and myelin oligodendrocyte glycoprotein-specific T cells.

进一步地,所述药物包括所述卡铂及药学上可接受的载体。Further, the drug includes the carboplatin and a pharmaceutically acceptable carrier.

进一步地,所述药物在预防多发性硬化症时,每隔两天口服一次,并控制卡铂的用量为30mg/kg-100mg/kg。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.

本发明对比现有技术有如下的有益效果:本发明研究了卡铂对EAE神经病理学的影响。研究发现,口服卡铂可以减轻EAE的总体临床症状,减少脊髓炎症和脱髓鞘现象。此外,卡铂处理组EAE小鼠外周组织中T细胞的比例和数量明显降低。同时,质谱数据分析也表明对照组和卡铂处理组EAE小鼠的脾细胞中的差异表达蛋白主要与凋亡调控途径有关。体外细胞实验数据表明卡铂抑制活化T细胞的增殖并诱导其凋亡。本发明的数据表明卡铂可安全有效地应用于MS治疗药物中。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.

附图说明Description of drawings

图1表明卡铂治疗减轻EAE小鼠的临床症状;Figure 1 shows that carboplatin treatment alleviates the clinical symptoms of EAE mice;

图2表明卡铂治疗减少了CNS中致病性T细胞的浸润;Figure 2 demonstrates that carboplatin treatment reduces the infiltration of pathogenic T cells in the CNS;

图3表明卡铂治疗减少了EAE小鼠外周T细胞数量;Figure 3 shows that carboplatin treatment reduces the number of peripheral T cells in EAE mice;

图4表明卡铂治疗增强了EAE期间的凋亡信号;Figure 4 demonstrates that carboplatin treatment enhanced apoptotic signaling during EAE;

图5表明卡铂介导活化T细胞凋亡;Figure 5 shows that carboplatin mediates the apoptosis of activated T cells;

图6表明卡铂增强EAE小鼠中MOG特异性T细胞凋亡。Figure 6 demonstrates that carboplatin enhances MOG-specific T cell apoptosis in EAE mice.

具体实施方式Detailed ways

下面结合附图和实施例对本发明作进一步的描述。The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

本发明试图评估口服卡铂是否可以用于改善EAE的病情。卡铂治疗降低了EAE小鼠的脊髓炎症、脱髓鞘和疾病评分。此外,卡铂治疗组的EAE小鼠的脾脏和引流淋巴结中的致病性T细胞(尤其是Th-1和Th-17)的数量和比例明显减少。蛋白质组差异富集分析表明,卡铂处理组脾细胞所表达的与凋亡信号相关蛋白发生显著变化。CFSE实验结果显示卡铂显著抑制T细胞增殖。最后,体外实验证明卡铂可以促进活化的T细胞和髓鞘少突胶质细胞糖蛋白(简称MOG)特异性T细胞凋亡。研究结果表明卡铂在EAE的发生和发展中起着保护作用,并且为治疗MS的新药设计成为可能。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.

以下详细公开实验、检测、理论分析。The experiment, detection, and theoretical analysis are disclosed in detail below.

一、实验中涉及的材料(动物模型)与本领域通用的实验和检测方法1. The materials (animal model) involved in the experiment and the common experiments and detection methods in this field

1.动物模型1. Animal model

雄性C57BL/6小鼠购自江苏集萃药康生物科技股份有限公司(南京,中国)。所用C57BL/6小鼠为8-10周龄,饲养于同济大学实验动物中心SPF级实验室,自由食用水和饲料。所有实验均获得批准,并按照同济大学的动物保护委员会的指导进行。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.方法:建立小鼠EAE模型及药物治疗2. Methods: establishment of mouse EAE model and drug treatment

C57BL/6小鼠(8-10周)皮下注射200μg完全溶于完全弗氏佐剂并包含有热灭活结核杆菌(H37Ra,5mg/ml,BD Diagnostics)的MOG35-55(MEVGWYRSPFSRVVHLYRNGK)。免疫第0天及第2天每只小鼠腹腔注射200ng/只百日咳毒素(Calbiochem)。每天根据临床症状为小鼠评分,评分标准如下:0分,无临床症状;1分,尾部瘫痪;2分,轻度瘫痪(单侧或双侧后肢无力,不完全瘫痪);3分,截瘫(双侧后肢完全瘫痪);4分,截瘫并前肢无力或瘫痪;5分,濒死状态或死亡。药物治疗,卡铂溶于无菌水中并按照(10mg/kg,30mg/kg,100mg/kg)剂量在免疫第3天灌胃给药直至实验结束,相同体积的200μl无菌水作为对照。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.方法:组织病理和免疫组织化学分析3. Methods: Histopathological and Immunohistochemical Analysis

每只小鼠深度麻醉后0.9%NaCl心脏灌流去除各器官中的外周血,4%(w/v)多聚甲醛灌流进行固定。取腰髓组织样品置于4%多聚甲醛溶液,4℃固定过夜。石蜡包埋后H&E染色分析炎症浸润,快蓝染色分析脊髓脱髓鞘,Image-Pro软件进行统计分析。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.方法:中枢神经系统浸润细胞分离和分析4. Methods: CNS Infiltrating Cell Isolation and Analysis

脑和脊髓用预冷的组织匀浆器进行匀浆,70μm细胞滤网过滤收集至15ml离心管中,500g 4℃离心10min获得细胞,然后将细胞重悬在8ml 37%Percoll中。在15ml离心管中先轻轻加入4ml 70%Percoll,瞬时离心去除离心管壁上残留的70%Percoll,避免与后面加入的37%Percoll互溶;沿着管壁缓缓加入37%Percoll细胞悬液,避免破坏界面层。780g25℃密度梯度离心25min,收集位于37/70%Percoll界面处的细胞,进行分析。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.方法:细胞染色和流式分析5. Methods: Cell Staining and Flow Cytometry

脾脏细胞和引流淋巴结细胞进行表面染色,FITC-CD4、APC-CD8、APC-B220和FITC-CD11b在4℃避光染色30分钟,洗涤,然后用1×PBS重悬。脾脏细胞,引流淋巴结细胞,或CNS浸润细胞用PMA(50ng/ml;Sigma-Aldrich)、ionomycin(750ng/ml;Sigma-Aldrich)和brefeldin A(5μg/ml;Sigma-Aldrich)37℃刺激5小时。细胞表面标记CD4用抗体4℃避光染色30min,然后细胞用固定通透液重悬,分别进行胞内IL-17A(BioLegend,506904,1:100)和IFN-γ(BioLegend,505810,1:100)染色。流式分析为BD FACS Verse系统,并用Flow JoV10软件进行分析。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.方法:ELISA检测外周血中炎症因子表达6. Method: ELISA to detect the expression of inflammatory factors in peripheral blood

卡铂(100mg/kg体重)或溶媒(无菌水)处理小鼠构建EAE模型。在建模第10天时,从眼眶静脉血中收集全血约150μl。眼眶静脉血在室温静置30分钟。30分钟后,4℃下以4000rpm离心10分钟,仔细收集上清,尽可能不要吸到下方的沉淀,获得血清。根据制造商的说明,使用特定的ELISA试剂盒(eBioscience)测量血清中IL-17A和IFN-γ的浓度。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.方法:T细胞增殖实验7. Method: T cell proliferation assay

用10mM羧基荧光素二乙酸琥珀酰亚胺酯(CFSE)标记脾细胞,然后接种至96孔板中,加入200μL包含10%FBS、2mML-谷氨酰胺和50μMβ-巯基乙醇的RPMI1640完全培养基进行培养,加入anti-CD3(2μg/ml;BD Pharmingen)和anti-CD28(2μg/ml;BD Pharmingen)抗体激活细胞。各种浓度的卡铂同时加入,培养48小时。收集细胞,流式细胞术检测CFSE荧光。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.方法:T细胞凋亡实验8. Method: T cell apoptosis experiment

在含有2μg/ml anti-CD28抗体和2μg/ml anti-CD3抗体的RPMI1640完全培养基中以5×105/孔的密度在96孔板中培养幼稚脾细胞。在不同浓度的卡铂存在下培养24、48、72小时。在免疫后第10天,分离EAE小鼠的脾细胞,用MOG35-55(20μg/ml)刺激24、48、72小时。然后用7-AAD和Annexin-V结合抗CD4或抗CD8抗体对细胞进行染色。流式细胞术检测活细胞或凋亡细胞(7-AAD和Annexin-V双阳)的百分比。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.方法:LC-MS/MS分析9. Method: LC-MS/MS Analysis

用400μl尿素裂解缓冲液(8M尿素,100mM Tris-HCl pH8.0)、4μl蛋白酶抑制剂(PierceTM,Thermo Fisher Scientific)以保护蛋白质免于降解,并使用Bradford方法(Eppendorf Biospectrometer)测量蛋白质浓度。样品在Orbitrap Fusion、OrbitrabFusion Lumos和Q Exactive Plus质谱仪上进行分析,质谱仪与连接到UltiMate3000RSLCnano系统的Easy nLC 1000纳米流LC系统相连。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.方法:统计学分析10. Methods: Statistical Analysis

EAE小鼠处理组间统计学差异用two-way ANOVA test分析,其他数据统计用Student’s t-test分析,数据表示为mean±SEM,P<0.05被认为有统计学意义。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.

实验1对应方法:建立小鼠EAE诱导模型及药物治疗;组织病理和免疫组织化学分析;Corresponding methods of experiment 1: establishment of mouse EAE induction model and drug treatment; histopathological and immunohistochemical analysis;

实验2对应方法:中枢神经系统浸润细胞分离和分析;Corresponding method of experiment 2: isolation and analysis of central nervous system infiltrating cells;

实验3对应方法:细胞染色和流式分析;ELISA检测外周血中炎症因子表达;Corresponding methods of experiment 3: cell staining and flow cytometry analysis; ELISA detection of inflammatory factor expression in peripheral blood;

实验4对应方法:LC-MS/MS分析;Experiment 4 corresponding method: LC-MS/MS analysis;

实验5对应方法:T细胞增殖实验;T细胞凋亡实验。Corresponding methods of experiment 5: T cell proliferation experiment; T cell apoptosis experiment.

二、实验及结果2. Experiment and results

实验1卡铂改善EAE小鼠的临床症状Experiment 1 Carboplatin improves the clinical symptoms of EAE mice

为了评估卡铂是否对多发性硬化症有治疗作用,本发明通过MOG35-55肽免疫在C57BL/6小鼠中诱导EAE模型,从免疫后第3天到实验结束,每天或每两天口服卡铂一次(图1A-B),而给予水作为溶剂对照。卡铂在一系列实验中显示了对EAE的严重程度有剂量依赖性的抑制作用。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.

低剂量卡铂口服给药不能改善EAE的严重程度。在30mg/kg时,卡铂延迟了EAE的发作,并降低了EAE缓解期间的临床评分(图1A)。100mg/kg卡铂的治疗也延迟了EAE的发作,并显著降低了其严重程度和累积临床评分(图1A)。本发明发现,每隔两天口服卡铂也可以延缓EAE的发作,缓解EAE的症状,且药效更好(图1B)。有趣的是,在发病后(第12天)给予卡铂(100mg/kg,口服)仍能降低EAE的严重程度,这表明CA除了预防作用之外,还有治疗作用。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.

图1中:(A-C)通过MOG35-55免疫在雄性C57BL/6小鼠中诱导EAE。从免疫后第3天开始每天(A)或隔天(B)给予卡铂一次,或从第12天开始隔天口服一次(C)直到实验结束,每天记录临床评分。对照组口服同体积无菌水。数据表示平均值±SEM(n=5-7),*p<0.05,**p<0.01,***p<0.001与对照组相比(two-way ANOVA test)。(D-E)免疫后第17天,对未建模组、溶剂对照组或卡铂(100mg/kg,口服,从第3天开始)处理的EAE小鼠分离的脊髓石蜡切片进行H&E染色和快蓝染色。顶部行中的方框区域在底部放大显示,标尺,200μM。(F-G)定量H&E染色切片中CNS浸润的细胞数量和快蓝染色切片中脱髓鞘的面积。每组三只动物,并分析每只动物的15个脊髓切片,***p<0.001与未建模组相比,###p<0.001与溶剂对照组相比(Student's t test)。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).

实验2卡铂减少EAE小鼠中枢神经系统中白细胞浸润和神经病变Experiment 2 Carboplatin reduces leukocyte infiltration and neuropathy in the central nervous system of EAE mice

EAE小鼠发病过程中伴随着CNS(脑和脊髓)的炎性细胞浸润及髓鞘损伤,在发病高峰期现象最明显,其中腰髓的白质部分现象最为突出。因此,本发明选取卡铂给药组和无菌水对照组EAE小鼠,在发病最高峰(第17天)时取脊髓切片进行病理组织检测,确定卡铂给药是否能减少EAE小鼠CNS病灶部位炎性细胞的浸润和减轻髓鞘损伤。同时,本发明选取健康的C57BL/6小鼠(naive小鼠)作为没有炎性细胞浸润和髓鞘损伤的阴性对照。与对照组相比,卡铂导致脊髓白细胞浸润显著减少(图1D,1F)。与对照组相比,快蓝染色还显示卡铂处理的EAE小鼠的脱髓鞘较少(图1E,1G)。免疫后第17天,通过流式细胞术分析对CNS白细胞浸润进行定量。结果证实了卡铂治疗后EAE小鼠CNS中CD4+T细胞积聚减少(图2A-D)。产生IL-17A的Th17细胞和产生IFN-γ的Th1细胞是EAE的主要致病性T效应细胞。因此,本发明量化了CNS浸润中Th1和Th17细胞的数量。卡铂治疗后,IFN-γ+CD4+T细胞(Th1细胞)和IL-17A+CD4+T细胞(Th17细胞)的百分比和数量显著降低(图2E-J)。上述这些数据表明卡铂治疗可显著降低EAE的严重程度,减少CNS炎症和脱髓鞘。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.

图2中:(A)在免疫后第17天,用37/70%Percoll从对照组或卡铂组(100mg/kg,从第3天开始)EAE小鼠的大脑和脊髓中分离总CNS浸润,并通过流式细胞术定量总浸润的数量。(B,E,H)用卡铂(100mg/kg,从第3天开始)或无菌水处理的EAE小鼠的CNS中CD4+T细胞、Th-1细胞和Th-17细胞的代表性FACS图。(C,F,I)通过GraphPad Prism 8软件对CNS中IFN-γ和IL-17A的细胞内染色进行FACS分析的统计数据。(D,G,J)CD4+T细胞、Th-1细胞和Th-17细胞的细胞数统计。数据为平均值±SEM(每组n=7-8),统计测试采用Student's t test,与对照组相比,*p<0.05,**p<0.01,***p<0.001。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.

实验3卡铂降低EAE小鼠外周CD4+T和CD8+T细胞Experiment 3 Carboplatin reduces peripheral CD4+T and CD8+T cells in EAE mice

MS是由外周免疫系统的免疫细胞引起的自身免疫性疾病;T细胞、B细胞和单核细胞参与了MS的发病机制。为了检测卡铂是否影响基础免疫应答,从对照组和卡铂处理组的EAE小鼠脾脏中收集白细胞,并通过流式细胞术分析脾白细胞中CD4+T细胞、CD8+T细胞、B细胞和CD11b+细胞的比例。表面染色显示,卡铂处理显著降低了脾脏中CD4+T细胞和CD8+T细胞的比例,而不影响B细胞和CD11b+细胞群体(图3A顶部两行,C和G)。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).

接下来,本发明用细胞内细胞因子染色检测脾脏中Th-1和Th-17细胞的数量。研究发现,在卡铂处理的EAE小鼠中,CD4+群体中Th-1和Th-17细胞的比例数量显著减少(图3A底行,D和H)。对引流淋巴结中的免疫细胞进行相同分析,本发明也获得了类似的结果(图3B、E、F、I和J)。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).

最后,在卡铂治疗的EAE小鼠中,血清中的细胞因子IFN-γ和IL-17A的分泌显著减少(图3K和L)。Finally, the secretion of cytokines IFN-γ and IL-17A in serum was significantly reduced in carboplatin-treated EAE mice (Fig. 3K and L).

图3中:(A-B)免疫后第10天,从卡铂(100mg/kg,口服,从第3天开始)或无菌水处理的EAE动物的脾脏(A)和引流淋巴结(dLN)(B)分离白细胞,并用表面染色和流式细胞术分析CD4+T细胞、CD8+T细胞、B细胞(B220+)和CD11b+细胞的百分比。Th-1和Th-17细胞通过胞内染色流式分析CD4阳性细胞中IFN-γ和IL-17A的比例。(C-J)A和B的统计分析。数据表示平均值±SEM(n=9)。与对照组相比,*p<0.05,**p<0.01,***p<0.01(Student's t test)。(K-L)从用100mg/kg卡铂或相同体积的水处理的EAE小鼠收集血清,分析其中的细胞因子IFN-γ和IL-17A的分泌量。数据为平均值±SEM(n=9-13)。与对照组相比,*p<0.05(Student'sttest)。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.

实验4卡铂治疗增强EAE期间的凋亡通路信号Experiment 4 Carboplatin treatment enhances apoptotic pathway signaling during EAE

为了探讨卡铂在EAE发病机制中的作用,本发明对分别用无菌水和卡比处理的EAE小鼠(免疫第10天)的脾细胞进行了质谱分析。卡铂处理后,表达差异蛋白质主要影响凋亡信号通路调节、蛋白质稳定性调节、细胞杀伤和细胞周期负调节的生物学功能(图4A和B)。KEGG分析显示卡铂主要通过凋亡、抗原处理和呈递等影响EAE进展。其中,与抗原处理和呈现相关的蛋白质(例如,Hsp90ab1、Canx、Hsp90aa1、Pdia3和Hspa8)和凋亡(例如,Ybx3、Runx3、Rps7和Hsph1)在卡铂处理后显著下调(图4C和D)。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) .

图4中:(A-B)通过GO富集分析(A)和网络分析(B)按功能对EAE小鼠的对照组和卡铂组的比较蛋白质组分析进行分类。(C-D)KEGG富集分析(C)和KEGG热图(D)显示免疫后第10天EAE小鼠的对照组和卡铂(100mg/kg,从第3天开始)组脾细胞的总蛋白显著富集的信号通路。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.

实验5卡铂抑制T细胞增殖并促进T细胞凋亡Experiment 5 Carboplatin inhibits T cell proliferation and promotes T cell apoptosis

实验3的脾细胞和淋巴细胞的表面染色结果表明,卡铂治疗后,外周免疫组织中T细胞的频率显著降低。同时,质谱数据分析也表明差异表达蛋白主要与凋亡调控有关。因此推测卡铂可能影响T细胞的增殖或凋亡。为了验证这一假设,本发明利用小鼠脾细胞进行了体外细胞增殖或凋亡实验。在卡铂存在下,用抗小鼠CD3和抗小鼠CD28的抗体活化从野生型小鼠分离得到的脾细胞48小时。如图5A所示,卡铂剂量依赖性抑制T细胞抗原刺激的细胞增殖。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.

接下来,在卡铂(3μM和10μM)处理不同持续时间(24-72小时)后,用annexin V和7-AAD染色检测凋亡细胞比例。体外实验表明卡铂增强了CD4+和CD8+T细胞群的凋亡(图5B-G)。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).

本发明同时测试了卡铂对MOG特异性T细胞的影响。在免疫后第10天从EAE小鼠中分离脾细胞,并在卡铂的存在下用MOG(20μg/ml)在体外重刺激,用FACS测量CD4+和CD8+T亚群中的凋亡(图6)。卡铂处理显著促进MOG特异性CD4+和CD8+T细胞的凋亡。上述这些数据都表明了卡铂诱导了T细胞凋亡(图6A-F)。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).

图5中:(A)分离野生型小鼠的脾细胞,用2μg/ml抗小鼠CD3和2μg/ml抗小鼠CD28抗体活化细胞;用CFSE染色评估细胞增殖。(B-C)经卡铂处理的CD4+(B)或CD8+(C)T细胞凋亡的代表性FACS图。(D-E)B和C中的凋亡统计分析。数据来自三个独立实验(平均值±SEM)。与对照组相比,*p<0.05,***p<0.001(two-way ANOVA)。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).

图6中:(A-B)免疫后第10天从EAE小鼠分离脾细胞,并用MOG35-55(20μg/ml)再刺激。用卡铂处理细胞24、48或72小时,通过流式细胞术检测CD4+(A)或CD8+(B)T细胞的凋亡情况。(C-D)A和B的统计分析。数据来自三次独立的实验(平均值±SEM)。与对照组相比,*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).

三、实验后的讨论和小结3. Discussion and conclusion after the experiment

多发性硬化(Multiple sclerosis,MS)是一种慢性神经系统疾病,具有潜在的破坏性和长期并发症。实验性自身免疫性脑脊髓炎(experimental autoimmuneencephalomyelitis,EAE)是一种CD4+T细胞介导的中枢神经系统脱髓鞘疾病,常被用作人类MS的动物模型。激活的CD4+T细胞从外周迁移到中枢神经系统,在那里通过分泌细胞因子和趋化因子启动炎症反应级联。CD8+T细胞也参与了EAE的发病机制。在MS病变中检测到的CD8+T淋巴细胞表现出活化和克隆扩增细胞的特征,支持了这些细胞积极参与观察到的损伤的概念。已有研究表明,髓磷脂反应性CD8+T细胞直接引发轴突损失,而神经元损伤已被发现与CD8+T细胞浸润的程度相关。因此,诱导T细胞凋亡可能有助于改善EAE。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.

凋亡是大多数多细胞动物生长和存活的先决条件,并确保组织稳态的维持。它在免疫学中的矛盾作用可以通过以下事实来说明:这个过程不仅有助于平衡功能,而且会产生有害影响,包括组织损伤。使用EAE模型的实验表明,细胞死亡影响浸润淋巴细胞和CNS驻留细胞,并有助于轴突损伤和炎症的消退。研究报告称,醋酸格拉替雷和干扰素β等药物通过诱导外周T细胞凋亡来治疗MS。同样,本发明的数据表明卡铂降低了脾脏和引流淋巴结中CD4+T细胞的比例。可能是由于外周T细胞凋亡的增加,因此浸润CNS的CD4+T细胞的数量也减少。此外,本发明的体外实验数据也表明卡铂诱导了脾细胞中CD4+T细胞和CD8+T细胞凋亡。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.

细胞凋亡可能由线粒体途径、内质网应激途径或死亡受体介导的外源性途径触发。在这些途径中,线粒体途径可被线粒体中的caspase级联和Bcl-2家族成员激活。Bcl-2家族包含多种促凋亡和抗凋亡蛋白,它们是“内在”通路的关键组成部分。有文献报道卡铂处理显著降低Bcl-2蛋白的表达,并以时间依赖性方式诱导Raji细胞凋亡。本发明的质谱数据显示,对照组和卡铂治疗组之间脾细胞中的差异表达的蛋白主要与凋亡信号的调控有关。因此,本发明推测卡铂促进T细胞凋亡也可能是受到Bcl-2家族调控。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.

总之,本发明证明卡铂通过抑制EAE小鼠外周组织中致病性T细胞的数量来减轻EAE的临床和组织病理学症状。此外,本发明的结果表明卡铂抑制T细胞增殖并促进T细胞凋亡。本发明的研究表明卡铂是治疗MS的潜在治疗剂。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.

虽然本发明已以较佳实施例揭示如上,然其并非用以限定本发明,任何本领域技术人员,在不脱离本发明的精神和范围内,当可作些许的修改和完善,因此本发明的保护范围当以权利要求书所界定的为准。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.卡铂在制备预防或治疗多发性硬化症药物中的应用。1. Application of carboplatin in the preparation of drugs for preventing or treating multiple sclerosis. 2.根据权利要求1所述的应用,其特征在于,所述多发性硬化症包括:局部淋巴细胞浸润、髓鞘破裂、星形胶质细胞增殖、小胶质细胞活化和神经变性。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.根据权利要求1所述的应用,其特征在于,所述药物抑制T细胞增殖,并促进活化的T细胞和髓鞘少突胶质细胞糖蛋白特异性T细胞凋亡。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.根据权利要求1所述的应用,其特征在于,所述药物包括所述卡铂及药学上可接受的载体。4. The application according to claim 1, characterized in that the drug comprises the carboplatin and a pharmaceutically acceptable carrier. 5.根据权利要求1所述的应用,其特征在于,所述药物在预防多发性硬化症时,每隔两天口服一次,并控制卡铂的用量为30mg/kg-100mg/kg。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.
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