欢迎来到《四川大学学报(医学版)》

丁苯酞联合骨髓间充质干细胞治疗小鼠实验性自身免疫性脑脊髓炎的疗效及机制研究

耿嘉 刘国懿 马舒 李姗姗 周红雨

耿嘉, 刘国懿, 马舒, 等. 丁苯酞联合骨髓间充质干细胞治疗小鼠实验性自身免疫性脑脊髓炎的疗效及机制研究[J]. 四川大学学报(医学版), 2021, 52(5): 759-766. doi: 10.12182/20210960206
引用本文: 耿嘉, 刘国懿, 马舒, 等. 丁苯酞联合骨髓间充质干细胞治疗小鼠实验性自身免疫性脑脊髓炎的疗效及机制研究[J]. 四川大学学报(医学版), 2021, 52(5): 759-766. doi: 10.12182/20210960206
GENG Jia, LIU Guo-yi, MA Shu, et al. Effect and Mechanism of Treating Experimental Autoimmune Encephalomyelitis in Mice with Butylphthalide Combined with Bone Marrow Mesenchymal Stem Cells[J]. JOURNAL OF SICHUAN UNIVERSITY (MEDICAL SCIENCE EDITION), 2021, 52(5): 759-766. doi: 10.12182/20210960206
Citation: GENG Jia, LIU Guo-yi, MA Shu, et al. Effect and Mechanism of Treating Experimental Autoimmune Encephalomyelitis in Mice with Butylphthalide Combined with Bone Marrow Mesenchymal Stem Cells[J]. JOURNAL OF SICHUAN UNIVERSITY (MEDICAL SCIENCE EDITION), 2021, 52(5): 759-766. doi: 10.12182/20210960206

丁苯酞联合骨髓间充质干细胞治疗小鼠实验性自身免疫性脑脊髓炎的疗效及机制研究

doi: 10.12182/20210960206
基金项目: 国家自然科学基金(No. 81760226)和云南省高层次卫生计生技术人才培养经费(No. D-2018029)资助
详细信息
    通讯作者:

    E-mail:zhouhy@scu.edu.cn

Effect and Mechanism of Treating Experimental Autoimmune Encephalomyelitis in Mice with Butylphthalide Combined with Bone Marrow Mesenchymal Stem Cells

More Information
  • 摘要:   目的  探索丁苯 探索丁苯酞(3-n-butylphthalide, NBP)和骨髓间充质干细胞(bone mesenchymal stem cells, BMSCs)联合治疗小鼠实验性自身免疫性脑脊髓炎(experimental autoimmune encephalomyelitis, EAE)的疗效和机制。  方法  用髓鞘少突胶质糖蛋白(myelin oligodendrocyte glycoprotein 35-55, MOG35-55)诱导C57BL/6小鼠建立EAE模型,将小鼠随机分为EAE组〔腹腔注射磷酸盐缓冲生理盐水 (phosphate-buffered saline, PBS)〕、NBP处理EAE组(NBP组,腹腔注射NBP)、BMSCs移植EAE组(BMSCs组,BMSCs注入侧脑室,腹腔注射PBS)、BMSCs和NBP联合处理EAE组(BMSCs+NBP组,BMSCs注入侧脑室,腹腔注射NBP),每组各10只。并设正常小鼠10只为空白对照组(腹腔注射PBS)。每日记录小鼠神经功能评分。诱导EAE 22 d后处死小鼠,劳克坚劳蓝(Luxol fast blue, LFB)脊髓髓鞘染色观察脱髓鞘情况;酶联免疫吸附法(enzyme-linked immunosorbent assay, ELISA)检测血清白介素(interleukin, IL)-6、IL-10、IL-17、IL-22和转化生长因子-β(transforming growth factor-β, TGF-β)水平;免疫荧光染色检测脑中胶质纤维酸性蛋白(glial fibrillary acidic protein, GFAP)、微管相关蛋白-2(microtubule-associated protein-2, MAP-2)、髓鞘碱性蛋白(myelin basic protein, MBP)的表达;Western blot法检测脊髓核因子κB〔nuclear factor (NF)-κB〕通路、磷脂酰肌醇-3激酶(phosphoinositide-3 kinase, PI3K)/蛋白激酶(protein kinase B, PKB or Akt)通路及IL-17、叉状头/翅膀状螺旋转录因子(forkhead box P3, Foxp3)的表达。  结果  各治疗组神经功能评分及平均分均较EAE组降低(P<0.05);BMSCs+NBP组评分下降较单独治疗组(NBP组和BMSCs组)更为明显(P<0.05);LFB染色与神经功能评分及平均分一致。与EAE组相比,各治疗组EAE小鼠血清促炎因子IL-6、IL-17和IL-22降低(P<0.05),抑炎因子IL-10和TGF-β增加(P<0.05);BMSCs+NBP组细胞因子表达的变化较BMSCs组更为显著(P<0.05)。BMSCs+NBP组的GFAP、MAP-2、MBP表达均较BMSCs组增加(P<0.05)。与EAE组相比,各治疗组p-NF-κB/NF-κB比值下降,而p-IκBα/IκBα比值上升,p-PI3K/PI3K及p-Akt/Akt比值均上升,IL-17/Foxp3比值降低,以BMSCs+NBP组最为显著(P<0.05)。  结论  NBP和BMSCs联合治疗可缓解EAE模型小鼠的症状,且疗效优于单独治疗。其机制与抑制NF-κB通路以调节Th17/Foxp3比例及激活PI3K/Akt通路以促进BMSCs成神经分化有关。
  • 图  1  人骨髓间充质干细胞的形态和表面标记物的表达

    Figure  1.  Human BMSCs morphology and surface marker expression

    BMSCs were attached to a polygonal form. After BMSCs grew to the third generation, their shape became similar to that of fibroblasts (×100) in light microscope (A) and fluorescence microscope (B); C-F: CD29, CD34, CD45 and CD90 surface marker expression was determined by flow cytometry. The result showed that cultured BMSCs expressed CD29 and CD90, while there was no expression of CD34 and CD45 on the surface of BMSCs. R1: Regin 1; RN1: Positive fluorescence regin.

    图  2  NBP联合BMSCs移植治疗对EAE小鼠神经功能评分的影响

    Figure  2.  Effect of NBP combined with BMSCs transplantation on the neurological function score of EAE mice

    n=10. * P<0.05, **P<0.01, ***P<0.001, ****P<0.000 1. Data compared at Day 22 in left figure.

    图  3  NBP联合BMSCs移植治疗对EAE小鼠脱髓鞘病变的影响。 LFB染色 ×400

    Figure  3.  Effect of NBP combined with BMSCs transplantation on demyelinating lesions in EAE mice. LFB staining ×400

    A: Control group, no demyelination; B: EAE group, large areas of demyelination (black arrows); C: NBP group, after NBP treatment, compared with the EAE group, there was significantly less demyelination (black arrows); D: BMSCs group, after BMSCs transplantation, the area of demyelination (black arrows) was significantly reduced compared with that of the EAE group; E: BMSCs+NBP group, after combined treatment with NBP and BMSCs, compared with EAE group, demyelination was greatly reduced; compared with NBP and BMSCs alone treatment group, demyelination was further reduced (black arrows). F: Pathological score, n=10, *P<0.05, **P<0.01, *** P<0.001, ****P<0.000 1.

    图  4  NBP对BMSCs移植后GFAP、MAP-2和MBP荧光表达量的影响

    Figure  4.  Effect of NBP on GFAP, MAP-2 and MBP fluorescence expression after BMSCs transplantation

    Compared with the group that had BMSCs transplantation alone, the expression of GFAP (A), MAP-2 (B), MBP (C) and the immunofluorescence double labeling(merged with GFP)in the brain of mice significantly increased after NBP was used in combination. White arrows: Immunofluorescence positive cells.* P<0.05; ****P<0.0001; n=10.

    图  5  各组小鼠脊髓组织NF-κB、PI3K/Akt信号通路及IL-17、Foxp3表达的变化

    Figure  5.  Changes of NF-κB and PI3K/Akt signaling pathways and IL-17 and Foxp3 expression in spinal cord of mice in each group

    A: Control group; B: EAE group; C: NBP group; D: BMSCs group; E: BMSCs+NBP group. * P<0.05,** P<0.01, *** P<0.001,****P<0.000 1, n=10.

    表  1  各组小鼠血清IL-6、IL-17、IL-22、IL-10和TGF-β水平的变化

    Table  1.   Changes of serum levels of IL-6, IL-17, IL-22, IL-10 and TGF-β in the mice of each group

    IndexGroup
    Control (n=10)EAE (n=10)NBP (n=10)BMSCs (n=10)BMSCs+NBP (n=10)
    IL-6/(pg/mL) 91.1±18.5 188.1±35.0# 145.1±17.9 132.6±19.6△△ 101.3±21.6*, △△
    IL-17/(pg/mL) 50.4±9.1 120.6±10.5# 84.0±6.2 74.4±7.7△△ 60.4±8.1*, △△
    IL-22/(pg/mL) 223.5±80.6 515.1±58.6# 402.0±77.9 339.8±89.0△△ 213.3±56.2*, △△
    IL-10/(pg/mL) 111.5±10.2 41.7±5.9# 58.9±6.8 77.1±10.3△△ 127.1±8.5*, △△
    TGF-β/(pg/mL) 12.5±1.3 5.1±0.5# 6.7±0.7 7.7±0.9△△ 11.5±1.7*, △△
     #P<0.01, vs. control group; △P<0.05, △△P<0.01, vs. EAE group; *P<0.05,vs. BMSCs group.
    下载: 导出CSV
  • [1] LEMUS H N, WARRINGTON A E, RODRIGUEZ M. Multiple Sclerosis: Mechanisms of disease and strategies for myelin and axonal repair. Neurol Clin, 2018, 36(1): 1−11.
    [2] KIPP M, NYAMOYA S, HOCHSTRASSER T, et al. Multiple sclerosis animal models: A clinical and histopathological perspective. Brain Pathol, 2017, 27(2): 123-137.
    [3] KIM E A, KIM S Y, YE B R, et al. Anti-inflammatory effect of Apo-9'-fucoxanthinone via inhibition of MAPKs and NF-kB signaling pathway in LPS-stimulated RAW 264.7 macrophages and zebrafish model. Int Immunopharmacol, 2018, 59: 339−346[2020-11-09]. https://doi.org/10.1016/j.intimp.2018.03.034.
    [4] HU R, LV W, ZHANG S, et al. Combining miR-23b exposure with mesenchymal stem cell transplantation enhances therapeutic effects on EAE. Immunol Lett, 2021, 229: 18−26[2020-11-09]. https://doi.org/10.1016/j.imlet.2020.11.007.
    [5] ZHANG J, BRODIE C, LI Y, et al. Bone marrow stromal cell therapy reduces proNGF and p75 expression in mice with experimental autoimmune encephalomyelitis. J Neurol Sci, 2009, 279(1/2): 30−38[2020-11-09]. https://doi.org/10.1016/j.jns.2008.12.033.
    [6] WANG W, WANG T, BAI S, et al. Dl-3-n-butylphthalide attenuates mouse behavioral deficits to chronic social defeat stress by regulating energy metabolism via AKT/CREB signaling pathway. Transl Psychiat, 2020, 10(1): 49[2020-11-09]. https://doi.org/10.1038/s41398-020-0731-z.
    [7] LUO R, WANGQIN R, ZHU L, et al. Neuroprotective mechanisms of 3-n-butylphthalide in neurodegenerative diseases. Biomed Rep, 2019, 11(6): 235−240.
    [8] CORREA J O, AARESTRUP B J, AARESTRUP F M. Effect of thalidomide and pentoxifylline on experimental autoimmune encephalomyelitis (EAE). Exp Neurol, 2010, 226(1): 15−23 .
    [9] CAI Y, SHEN H, QIN C, et al. The spatio-temporal expression profiles of CD4+ T cell differentiation and function-related genes during EAE pathogenesis. Inflammation, 2017, 40(1): 195−204.
    [10] BALASA R, BARCUTEAN L, BALASA A, et al. THE action of TH17 cells on blood brain barrier in multiple sclerosis and experimental autoimmune encephalomyelitis. Hum Immunol, 2020, 81(5): 237−243.
    [11] SUN D, LUO F, XING J C, et al. 1, 25(OH)2 D3 inhibited Th17 cells differentiation via regulating the NF-kappaB activity and expression of IL-17. Cell Prolif, 2018, 51(5): e12461[2020-11-09]. https://doi.org/10.1111/cpr.12461.
    [12] MOAAZ M, YOUSSRY S, ELFATATRY A, et al. Th17/Treg cells imbalance and their related cytokines (IL-17, IL-10 and TGF-beta) in children with autism spectrum disorder. J Neuroimmunol, 2019, 337: 577071[2020-11-09]. https://doi.org/10.1016/j.jneuroim.2019.577071.
    [13] WANG N, LIANG S, JIN J, et al. CD226 attenuates Treg suppressive capacity via CTLA-4 and TIGIT during EAE. Immunol Res, 2019, 67(6): 486−496.
    [14] CHAO C C, GUTIERREZ-VAZQUEZ C, ROTHHAMMER V, et al. Metabolic control of astrocyte pathogenic activity via cPLA2-MAVS. Cell, 2019, 179(7): 1483−1498 e1422[2020-11-09]. https://doi.org/10.1016/j.cell.2019.11.016.
    [15] HOU H, CAO R, QUAN M, et al. Rapamycin and fingolimod modulate Treg/Th17 cells in experimental autoimmune encephalomyelitis by regulating the Akt-mTOR and MAPK/ERK pathways. J Neuroimmunol, 2018, 324: 26−34[2020-11-09]. https://doi.org/10.1016/j.jneuroim.2018.08.012.
    [16] 毛国富, 欧阳玉龙, 房树华. 丁苯酞对老年急性脑梗死患者外周血Th17/Treg水平及相关细胞因子表达的影响. 湖北科技学院学报(医学版), 2019, 33(3): 212-215.
    [17] CHEN Q H, WU F, LIU L, et al. Mesenchymal stem cells regulate the Th17/Treg cell balance partly through hepatocyte growth factor in vitro. Stem Cell Res Ther, 2020, 11(1): 91[2020-11-09]. https://doi.org/10.1186/s13287-020-01612-y.
    [18] YANG M, DANG R, XU P, et al. Dl-3-n-Butylphthalide improves lipopolysaccharide-induced depressive-like behavior in rats: Involvement of Nrf2 and NF-kappaB pathways. Psychopharmacology (Berl), 2018, 235(9): 2573−2585.
    [19] LOTFY A, ALI N S, ABDELGAWAD M, et al. Mesenchymal stem cells as a treatment for multiple sclerosis: A focus on experimental animal studies. Rev Neurosci, 2020, 31(2): 161−179.
    [20] CHEN T, NOTO D, HOSHINO Y, et al. Butyrate suppresses demyelination and enhances remyelination. J Neuroinflamm, 2019, 16(1): 165[2020-11-09]. https://doi.org/10.1186/s12974-019-1552-y.
    [21] LI Q, HOUDAYER T, LIU S, et al. Induced neural activity promotes an oligodendroglia regenerative response in the injured spinal cord and improves motor function after spinal cord injury. J Neurotrauma, 2017, 34(24): 3351−3361.
    [22] FAN W, LI X, HUANG L, et al. S-oxiracetam ameliorates ischemic stroke induced neuronal apoptosis through up-regulating α7 nAChR and PI3K/Akt/GSK3β signal pathway in rats. NeurochemInt, 2018, 115: 50−60[2020-11-09]. https://doi.org/10.1016/j.neuint.2018.01.008.
    [23] CHOI N Y, KIM J Y, HWANG M, et al. Atorvastatin rejuvenates neural stem cells injured by oxygen-glucose deprivation and induces neuronal differentiation through activating the PI3K/Akt and ERK pathways. Molecul Neurobiol, 2019, 56(4): 2964−2977 .
    [24] ZHANG X, HE X, LI Q, et al. PI3K/AKT/mTOR signaling mediates valproic acid-induced neuronal differentiation of neural stem cells through epigenetic modifications. Stem Cell Rep, 2017, 8(5): 1256−1269 .
    [25] ZHOU Z, DUN L, WEI B, et al. Musk ketone induces neural stem cell proliferation and differentiation in cerebral ischemia via activation of the PI3K/Akt signaling pathway. Neuroscience, 2020, 435: 1−9[2020-11-09]. https://doi.org/10.1016/j.neuroscience.2020.02.031.
    [26] SUN B, FENG M, TIAN X, et al. DL-3-n-Butylphthalide protects rat bone marrow stem cells against hydrogen peroxide-induced cell death through antioxidation and activation of PI3K-Akt pathway. Neurosci Lett, 2012, 516(2): 247-252.
  • 加载中
图(5) / 表(1)
计量
  • 文章访问数:  75
  • HTML全文浏览量:  19
  • PDF下载量:  16
  • 被引次数: 0
出版历程
  • 收稿日期:  2020-11-29
  • 修回日期:  2021-05-21
  • 网络出版日期:  2021-12-06
  • 刊出日期:  2021-09-20

目录

    /

    返回文章
    返回