Effect and Mechanism of Treating Experimental Autoimmune Encephalomyelitis in Mice with Butylphthalide Combined with Bone Marrow Mesenchymal Stem Cells
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。Abstract: Objective To explore the efficacy and mechanism of using 3-n-butylphthalide (NBP) in combination with bone marrow mesenchymal stem cells (BMSCs) in the treatment of experimental autoimmune encephalomyelitis (EAE) in mice. Methods Myelin oligodendrocyte glycoprotein (MOG35-55) was used for the induction and establishment of the EAE model in C57BL/6 mice. The mice were randomly assigned to the EAE group, which received intraperitoneal injection of phosphate-buffered saline (PBS), the NBP-treated EAE group, or the NBP group, which received intraperitoneal injection of NBP, the BMSCs transplantion EAE group, or the BMSCs group, which received BMSCs injected into the lateral ventricle and intraperitoneal injection of PBS, and the BMSCs and NBP combination treatment EAE group, or the BMSCs+NBP group, which received BMSCs injected into the lateral ventricle and intraperitoneal injection of NBP. Each group had 10 mice, while ten normal mice were used as the blank control group receiving intraperitoneal injection of PBS. The neurological function scores were documented daily. The mice were sacrificed 22 days after EAE induction, and the demyelination state of of the spinal cords was observed through Luxol fast blue (LFB) staining. In addition, the levels of serum interleukin-6 (IL-6), IL-10, IL-17, IL-22 and transforming growth factor-β (TGF-β) were examined with ELISA. The levels of glial fibrillary acidic protein (GFAP), microtubule associated protein-2 (MAP-2) and myelin basic protein (MBP) in the brain were examined with immunofluorescence staining. Western blot was used to check the expressions of nuclear factor (NF)-κB pathway, phosphoinositide-3 kinase (PI3K)/protein kinase B (PKB or Akt) pathway, IL-17 and forkhead box P3 (Foxp3) in the spinal cords. Results The neurological function scores and average scores of each treatment group were significantly lower than those of the EAE group (P<0.05). The scores of the BMSCs+NBP group decreased more significantly than those of the single treatment groups (the NBP group and the BMSCs group) (P<0.05). LFB staining results of the spinal cords were consistent with the neurological function scores and the average scores. Compared with the EAE group, the levels of pro-inflammatory cytokines, including IL-6, IL-17 and IL-22, significantly decreased (P<0.05), and the levels of anti-inflammatory cytokines IL-10 and TGF-β significantly increased (P<0.05). The change in cytokine expression was more significant in the BMSCs+NBP group (P<0.05). The expressions of GFAP, MAP-2 and MBP in the BMSCs+NBP group were significantly higher than those of the BMSCs group (P<0.05). Compared with the EAE group, the p-NF-κB/NF-κB ratio and the IL-17/Foxp3 ratio in NBP group, BMSCs group and BMSCs+NBP group decreased, while P-IκBα/IκBα, p-pI3k/PI3K and P-Akt/Akt ratios increased, especially in the BMSCs+NBP group(P<0.05). Conclusion The combined treatment of NBP and BMSCs can help alleviate the symptoms of EAE model mice, showing better efficacy than treatment with NBP or BMSCs alone. The mechanism is related to the inhibition of the NF-κB pathway to regulate Th17/Foxp3 ratio and the activation of the PI3K/Akt pathway to promote the neurogenic differentiation of BMSCs.
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 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
Index Group 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.
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