葛根素抑制小鼠实验性自身免疫性脑脊髓炎的作用

刘国懿; 赵清青; 武妍; 李姗姗; 钟莲梅; 耿嘉

doi:10.12259/j.issn.2095-610X.S20210401

葛根素抑制小鼠实验性自身免疫性脑脊髓炎的作用

doi: 10.12259/j.issn.2095-610X.S20210401

1.
昆明医科大学第一附属医院神经内科，云南昆明　650032
2.
昆明医科大学基础医学院人体解剖学与组织胚胎学系，云南昆明　650500

基金项目: 国家自然科学基金资助项目（81760226）；云南省高层次卫生计生技术人才培养基金资助项目（D-2018029）；云南省科技厅-昆明医科大学应用基础研究联合专项基金资助项目[2018FE001（-145）]

详细信息

作者简介:
刘国懿（1982～），女，四川乐山人，医学硕士，副主任医师，主要从事神经内科临床及研究工作

通讯作者:
耿嘉，E-mail：basler777@163.com

中图分类号: R392
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出版历程
- 收稿日期: 2020-12-26
- 刊出日期: 2021-04-01

Effect and Mechanism of Puerarin on Experimental Autoimmune Encephalomyelitis in Mice

1.
Dept. of Neurology，The 1st Affiliated Hospital of Kunming Medical University，Kunming Yunnan 650032
2.
Dept of Human Anatomy，Histology and Embryology，School of Basic Medicine，Kunming Medical University，Kunming Yunnan 650500，China

摘要

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。
- 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.
- Experimental autoimmune encephalomyelitis /
- Puerarin /
- Th17 /
- Treg /
- NF-κB pathway

HTML全文

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

下载: 全尺寸图片幻灯片

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

下载: 全尺寸图片幻灯片

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

下载: 全尺寸图片幻灯片

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

下载: 全尺寸图片幻灯片

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。

下载: 导出CSV

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。

下载: 导出CSV

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