Effect and Mechanism of Puerarin on Experimental Autoimmune Encephalomyelitis in Mice
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摘要:
目的 探索葛根素（Puerarin）治疗小鼠实验性自身免疫性脑脊髓炎（experimental autoimmune encephalomyelitis, EAE）的作用和机制。 方法 将C57BL/6小鼠随机分为对照组、EAE组、Puerarin处理EAE组（Puerarin组）。从诱导EAE当日开始，Puerarin组用葛根素（80 mg/kg）灌胃，其余2组用同体积生理盐水灌胃，每日1次。每天记录神经功能评分及体重变化；劳克坚劳蓝（luxol fast bBlue，LFB）髓鞘染色检测脊髓脱髓鞘情况；ELISA法检测血清中IL-6、IL-10、IL-17、IL-22和TGF-β的水平；Western 印迹法检测脊髓组织NF-κB、IκBα、IL-17、Foxp3等蛋白表达。 结果 Puerarin组小鼠神经功能评分及平均分均较EAE组明显降低（P < 0.01），而体重明显增加（P < 0.01）。应用葛根素后，EAE小鼠脱髓鞘面积（P < 0.01）及病理评分（P < 0.05）均明显下降。Puerarin组小鼠促炎因子IL-6、IL-17和IL-22水平显著降低（P < 0.01），抑炎因子IL-10和TGF-β显著增加（P < 0.05）。应用葛根素后：NF-κB及IL-17表达明显减少（P < 0.05）；IκBα及Foxp3表达显著增加（P < 0.01）。 结论 葛根素可缓解EAE小鼠症状，减少脱髓鞘。作用机制是抑制NF-κB通路，调节IL17/Tregs平衡，实现对免疫功能的调节。 -
关键词:
- 实验性自身免疫性脑脊髓炎 /
- 葛根素 /
- 辅助性T细胞17 /
- 调节性T细胞 /
- NF-κB通路
Abstract:Objective To explore the effect and mechanism of Puerarin on experimental autoimmune encephalomyelitis （EAE） in mice. Methods C57BL / 6 mice were randomly divided into control group, EAE group and purearin treated EAE group. From the first day of EAE induction, puerarin group was gavaged with Puerarin （80 mg/kg）, and the other two groups were given the same volume of normal saline once a day. The neurological function score and weight changes were recorded every day; the demyelination of spinal cord was detected by LFB myelination staining; the levels of IL-6, IL-10, IL-17, IL-22 and TGF-β in serum were detected by ELISA; the protein expression of NF-κB, IκBα, IL-17, Foxp3 in spinal cord tissue were detected by Western blot. Results Compared with EAE group, the neurological function score and average score of purearin group were significantly decreased （P < 0.01）, while the body weight was significantly increased （P < 0.01）. The demyelinating area （P < 0.01） and pathological score （P < 0.05） of EAE mice were significantly decreased after puerarin treatment. The levels of proinflammatory cytokines IL-6, IL-17 and IL-22 were significantly decreased in puerarin group （P < 0.01）, while IL-10 and TGF-β were significantly increased （P < 0.05）. After puerarin treatment, the expressions of NF-κB and IL-17 significantly decreased （P < 0.05）, and the expressions of IκBα and Foxp3 significantly increased （P < 0.01）. Conclusion Puerarin can alleviate the symptoms of EAE mice and reduce demyelination. The mechanism of action is to inhibit NF-κB pathway, to balance of IL-17/Tregs, and to regulate of immune function. -
Key words:
- Experimental autoimmune encephalomyelitis /
- Puerarin /
- Th17 /
- Treg /
- NF-κB pathway
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图 1 葛根素对EAE小鼠神经功能及体重的影响
A：小鼠神经功能评分；B：小鼠神经功能平均评分；C：小鼠体重。与Puerarin group比较，*P < 0.05。
Figure 1. Effects of Puerarin on neural function and body weight of EAE mice
图 2 对照组小鼠、EAE小鼠、葛根素组EAE小鼠的LFB染色
A：对照组；B：EAE组；C：葛根素组；D：葛根组病理学评分。与Puerarin group比较，*P < 0.05。
Figure 2. LFB staining of control group mice，EAE mice and puerarin-treated EAE mice
图 3 各组小鼠血清IL-6，IL-10，IL-17，IL-22 和TGF-β水平的变化
与对照组比较，##P < 0.01，####P < 0.000 1；与EAE组比较，#P < 0.05，##P < 0.01，####P < 0.0001。
Figure 3. Changes of serum levels of IL-6，IL-10，IL-17，IL-22 and TGF - β in mice
图 4 各组小鼠脊髓组织NF-κB信号通路及IL-17、Foxp3表达的变化
A：各组小鼠Western印迹蛋白表达；B：核转录因子κB相对表达水平；C：核转录因子κB抑制蛋白α相对表达水平；D：核转录因子κB与核转录因子κB抑制蛋白α表达比值；E：转录因子叉头蛋白3相对表达水平；F：白介素-17相对表达水平；G：转录因子叉头蛋白3与白介素-17表达比值。与对照组比较，#P < 0.05，##P < 0.01，###P < 0.001，####P < 0.0001；与EAE组比较，#P < 0.05，##P < 0.01，###P < 0.001，####P < 0.0001。
Figure 4. Changes of NF - κ B signaling pathway and expression of IL-17 and Foxp3 in spinal cord of mice in each group
表 1 各组小鼠血清IL-6、IL-17、IL-22、IL-10和TGF-β水平的变化（
$ \bar x \pm s$ ）（pg/mL）Table 1. Changes of serum levels of IL-6，IL-17，IL-22，IL-10 and TGF-β in mice （
$\bar x \pm s $ ）（pg/mL）项目 对照组 EAE 葛根素 IL-6 87.4 ± 15.4 179.3 ± 17.4# 151.7 ± 14.5##△ IL-17 42.7 ± 8.4 110.0 ± 15.5# 60.1 ± 9.9##△ IL-22 209.2 ± 54.8 507.4 ± 57.3# 429.4 ± 64.2##△ IL-10 110.4 ± 12.5 50.5 ± 7.5# 68.7 ± 9.5##△ TGF-β 12.02 ± 1.71 5.10 ± 0.56# 6.39 ± 0.73##△ 与对照组相比较，#P < 0.05，##P < 0.01；与EAE组比较，△P < 0.05。 
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表 2 各组小鼠脊髓组织NF-κB信号通路及IL-17、Foxp3表达的变化（
$\bar x \pm s $ ）（蛋白相对表达水平）Table 2. Changes of NF-κB signaling pathway and expression of IL-17 and Foxp3 in spinal cord of mice （
$\bar x \pm s $ ）（Relative protein expression level）项目 对照组 EAE 葛根素 NF-κB 1.15 ± 0.06 2.27 ± 0.36# 1.64 ± 0.13△ IκBα 1.09 ± 0.09 1.43 ± 0.07# 2.08 ± 0.10##△ NF-κB/IκBα 1.08 ± 0.13 1.98 ± 0.20# 0.58 ± 0.09##△ IL-17 1.03 ± 0.03 2.19 ± 0.09# 1.62±0.21##△ Foxp3 1.20 ± 0.08 0.49 ± 0.10# 0.41±0.09##△ IL-17/Foxp3 0.97 ± 0.11 3.21 ± 0.34# 与对照组比较，#P < 0.05，##P < 0.01；与EAE组比较，△P < 0.05。
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