中药二黄胶囊为何可治疗多发性硬化？

doi:10.3969/j.issn.1673-5374.2013.06.005

中国神经再生研究(英文版) ›› 2013, Vol. 8 ›› Issue (6): 523-531.doi: 10.3969/j.issn.1673-5374.2013.06.005

• 原著:退行性病与再生 • 上一篇下一篇

中药二黄胶囊为何可治疗多发性硬化？

收稿日期:2012-09-08 修回日期:2012-11-08 出版日期:2013-02-25 发布日期:2013-02-25

Mechanism of Erhuang capsule for treatment of multiple sclerosis

Kangning Li^{1, 2}, Yongping Fan¹, Tao Yang¹, Lei Wang³

1 Department of Traditional Chinese Medicine, Beijing Tiantan Hospital, Capital Medical University, Beijing 100050, China
2 Institute of Basic Research in Clinical Medicine, China Academy of Chinese Medical Sciences, Beijing 100700, China
3 Department of Prescription, School of Traditional Chinese Medicine, Capital Medical University, Beijing 100069, China

Received:2012-09-08 Revised:2012-11-08 Online:2013-02-25 Published:2013-02-25
Contact: Yongping Fan, M.D., Professor, Chief physician, Doctoral supervisor, Department of Traditional Chinese Medicine, Beijing Tiantan Hospital, Capital Medical University, Beijing 100050, China, yongpingf@ hotmail.com
About author:Kangning Li☆, M.D., Research assistant.
Supported by:
This study was supported by the Key Combination Program of Capital Medical Development Foundation, No. 2005-SF-I-001; the Natural Science Foundation of Beijing, No. 7102051, 7092014; the Beijing Science and Technology Development Foundation of Traditional Chinese Medicine, No. JJ2009-27; and the National Natural Science Foundation of China, No. 81072765, 30873230.

摘要/Abstract

摘要：

二黄胶囊可减轻多发性硬化症状、减轻炎症反应和糖皮质激素的副作用。少突胶质细胞前体细胞对髓鞘的修复与再生有重要作用。补肾化痰活血复方中药二黄胶囊可以有效改善实验性自身免疫性脑脊髓炎小鼠临床症状和神经功能评分，降低死亡率，促进神经功能恢复。其作用机制体现在明显促进小鼠脑和脊髓特定部位少突胶质细胞前体细胞的增殖，显著增加Olig2基因和组织的表达，促进少突胶质细胞前体细胞的分化。

关键词: 神经再生, 中医药, 少突胶质细胞前体细胞, 二黄胶囊, 中药复方, 补肾化痰活血法, 少突胶质细胞转录因子2, 实验性自身免疫性脑脊髓炎, 多发性硬化

Abstract:

Erhuang capsule, a typical formula based on traditional Chinese medicine theory, is widely used to ameliorate multiple sclerosis, inflammation and side effects of glucocorticoid treatment. Oligodendrocyte precursor cells are neural stem cells that are important for myelin repair and regeneration. In the present study, Erhuang capsule effectively improved clinical symptoms and neurological function scores, reduced mortality and promoted recovery of neurological functions of mice with experimental autoimmune encephalomyelitis. The mechanism of action involved significant increases in oligodendrocyte precursor cell proliferation in specific regions of the brain and spinal cord, increased oligodendrocyte lineage gene 2 expression and enhanced oligodendrocyte precursor cell differentiation.

Key words: neural regeneration, traditional Chinese medicine, neurodegenerative diseases, oligodendrocyte precursor cells, Erhuang capsule, Chinese compound recipe, methods of tonifying the kidney, resolving phlegm and activating blood, oligodendrocyte lineage gene 2, experimental autoimmune encephalomyelitis, multiple sclerosis, central nervous system, grants-supported paper, photographs-containing paper, neuroregeneration

. 中药二黄胶囊为何可治疗多发性硬化？[J]. 中国神经再生研究(英文版), 2013, 8(6): 523-531.

Kangning Li, Yongping Fan, Tao Yang, Lei Wang. Mechanism of Erhuang capsule for treatment of multiple sclerosis[J]. Neural Regeneration Research, 2013, 8(6): 523-531.

参考文献

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引言

材料方法

Design

A randomized, controlled animal study.

Time and setting

The experiment was performed at the School of Traditional Chinese Medicine of Capital Medical University, China from December 2009 to January 2011.

Materials

Animals

A total of 125 healthy, female, specific pathogen-free, C57BL/6 mice, weighing 16–20 g, aged 7–8 weeks, were provided by Beijing Vital River Laboratories, Beijing, China (SCXK (Jing) 2007-0001). Experimental procedures were performed in accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals, formulated by the Ministry of Science and Technology of China^[41].

Drugs

Erhuang capsule was composed of Radix Rehmanniae15 g, prepared Radix Rehmanniae15 g, Rhubarb(stir-fried with wine) 3 g, Radix Polygoni Multiflori Preparata 12 g, Chekiang Fritillary Bulb 9 g, Hirudo 3 g, Scorpion 3 g, Rhizoma Gastrodiae5 g, Weeping Forsythia Fruit9 g and Common Motherwort Herb 15 g, and was provided by Beijing Institute of Pharmaceutical Analysis, China (No. 071206). Prednisone acetate tablets (an effective drug generally accepted in multiple sclerosis clinical trials) were provided by Tianjin Pacific Pharmaceutical Co., Ltd., China (No. 061203). Erhuang capsule was dissolved in deionized water to prepare a suspension at a ratio of 41.6 g Erhuang capsule in 80 mL deionized water for Erhuang capsule high-dose group and 10.7 g Erhuang capsule in 80 mL deionized water for the Erhuang capsule low-dose group. A prednisone acetate tablet was dissolved in deionized water to prepare a suspension at a ratio of 41 mg Prednisone acetate tablets in 80 mL deionized water for the hormone group.

Methods

Induction of experimental autoimmune encephalomyelitis model and drug intervention

Experimental autoimmune encephalomyelitis was induced in mice by subcutaneous injection in the back with 0.2 mL emulsion, containing 50 μg myelin oligodendrocyte glycoprotein 35–55 peptide (No.C213406; Beijing Scilight Biotechnology Co., Ltd., Beijing, China) in 100 μL 0.9% physiological saline and 100 μL complete Freund’s adjuvant (No. 049K8700; Sigma-Aldrich, St. Louis, MO, USA; to which 4 mg/mL Mycobacterium tuberculosis was added), followed by two intraperitoneal injections with 500 ng pertussis toxin (No.029K1314; Sigma-Aldrich) on days 0 (the first day of immunization was regarded as day 0) and 2 post- immunization. The mice from the normal group were given physiological saline. On day 1, the two Erhuang capsule groups were intragastrically administrated with Erhuang capsule 7.8 g/kg per day for the high-dose group and 2.0 g/kg per day for the low-dose group, once daily, for 42 consecutive days. The normal group and model group were intragastrically administrated with the same volume of saline. The dose of crude drug for humans was 23.47 g per day. The body weight of each person is regarded as 60 kg. Thus, the mean dose administered to humans was 23.47 g crude drug/60 kg body mass (that is, 0.39 g crude drug/kg body mass). The conversion ratio of body weight between mouse and human is 0.11. For the high-dose group, mice were given 7.8 g powdered extract/kg body mass, that is, 19.42 g crude drug/kg body mass. Taking the dose into consideration for humans, that would be equivalent to 19.42 g crude drug/kg body mass × 0.11 = 2.14 g crude drug/kg body mass, which is 5.5 times the mean dose given to humans. For the low-dose group, the mice were given 2.0 g powdered extract/kg body mass, that is, 4.98 g crude drug/kg body mass. Taking the dose into consideration for humans, that would be 4.98 g crude drug/kg body mass × 0.11 = 0.55 g crude drug/kg body mass, which is 1.4 times the mean dose for humans. On day 12, the hormone group was intragastrically administrated with prednisone acetate tablets at 7.714 mg/kg, once a day, for 31 consecutive days. Body mass, incidence, mortality rate and neurological scores were observed. Neurological scores were graded on a scale of 0 to 5 using previously established standard criteria^[36-37]: 0: normal; 1: flaccid tail; 2: moderate hind or front leg weakness; 3: severe hind or front leg weakness; 4: complete paralysis of limb(s); 5: death.

5-bromodeoxyuridine administration

5-bromodeoxyuridine (No. B5002; Sigma-Aldrich) was dissolved in nonpyrogenic 0.9% physiological saline at a concentration of 5 mg/mL. Intraperitoneal injections of 5-bromodeoxyuridine (50 mg/kg) were given once daily for 4 consecutive days preceding tissue harvest. Twenty-four hours after the last injection, mice were sacrificed (on days 23 or 43 post-immunization, respectively).

Sample preparation

On day 23 (period I) and day 43 (period II) post- immunization, mice were anesthetized with an intraperitoneal injection of 10% chloral hydrate (5 mL/kg). The brain and spinal cord of mice were harvested, fixed, and paraffin embedded. Sections, 5 μm in thickness, were cut for hematoxylin-eosin staining and immunohistochemical analysis. Three mice from each group were prepared for harvest of brain tissues that were stored at –80°C for real-time PCR analysis. The paraffin blocks for immunohistochemistry and the tissues for real-time PCR used the same region (the middle-third parts of mouse brain).

Hematoxylin-eosin staining

Sections were dewaxed using xylene I and II, dehydrated with gradient alcohol for 5 minutes each, and stained with Harris hematoxylin (No. HHS16; Sigma-Aldrich) for 1 minute, followed by eosin (No. E4009; Sigma-Aldrich) for 10 minutes. The sections were dehydrated in gradient alcohol, permeabilized with xylene, mounted on neutral gum, and observed by light microscopy (Nikon Eclipse 80i, Tokyo, Japan).

Immunohistochemistry detection for oligodendrocyte lineage gene 2

The sections were dewaxed using xylene I and II for 5 minutes each and dehydrated with gradient alcohol for 3 minutes each. The sections were treated with 3% hydrogen peroxide for 5 minutes at room temperature, and washed three times with PBS (pH 7.4) for 2 minutes each. The sections were pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6) for 20 minutes and then washed three times with PBS. The sections were then incubated with anti-oligodendrocyte lineage gene 2 antibody (1:150; rabbit polyclonal antibody to oligodendrocyte lineage gene 2, ab33427, No.735325; Abcam, Cambridge, UK) at 4°C overnight and washed three times with PBS. Secondary antibody (1 drop; two-step IHC Detection Reagent, PV-6001; Zhongshan Goldenbridge Biotechnology Co., Ltd., Beijing, China) was added, incubated at 37°C for 30 minutes, and then washed three times with PBS. DAB was used as the chromogen. The sections were then counterstained with hematoxylin. Five sections from each group were observed by light microscopy (Nikon Eclipse 80i, Tokyo, Japan). Six random high power fields (× 400) were selected for each section of the brain and spinal cord. The results were presented by integral optical density using NIS-Elements BR 3.0 software.

Immunohistochemistry detection for 5-bromodeoxyuridine

Sections were dewaxed using xylene I and II for 5 minutes each and dehydrated with gradient alcohol for 3 minutes each. The sections were treated with 3% hydrogen peroxide for 5 minutes at room temperature and washed three times with PBS. The sections were digested with pancreatin (diluted at a ratio of 3:1) at 37°C for 30 minutes. The sections were then treated with 2 N HCl at 37°C for 30 minutes and washed under running water for 15 minutes. Anti-BrdU antibody (1:100; sheep polyclonal to 5-bromodeoxyuridine, No.661267; Abcam) was added, incubated at 4°C overnight and then washed three times with PBS. Secondary antibody (one drop; two-step IHC Detection Reagent, PV-6001; Zhongshan Goldenbridge Biotechnology Co., Ltd.) was added, incubated at 37°C for 30 minutes and then washed three times with PBS. Diaminobenzidine was used as the chromogen. The sections were then counterstained with hematoxylin. Five sections from each group were observed by light microscopy (Nikon Eclipse 80i). Six random high power fields (× 400) were selected for each section in the brain and spinal cord. The results were presented by integral optical density using NIS-Elements BR 3.0 software.

Oligodendrocyte lineage gene 2 gene expression detected by real-time PCR

RNA from brain tissues was extracted using Trizol (CW0580, CWbio Co., Ltd., Beijing, China). Reverse transcription and real-time quantitative PCR were performed. The oligodendrocyte lineage gene 2 forward primer sequence was 5’-GGGGATTATGGGGAGTCGA-3’ and reverse primer 5’-GGAGTGTTCAGCCAAAGAGTCA-3’. β-actin served as the internal reference using the forward primer 5’-GCCTTCCTTCTTGGGTAT-3’ and reverse primer 5’- GGCATAGAGGTCTTTACGG-3’. The primers were synthesized by CWbio Co., Ltd.

Changes in gene expression were determined by the quantitative method (2^－^△△CT). Following reverse transcription, quantitative PCR reaction was prepared as follows: 10 μL REAL SYBR Mixture (2 × ; CW0767, CWbio Co., Ltd), 0.5 μL upstream primer (10 μM), 0.5 μL downstream primer (10 μM), 2 μL cDNA template and dH₂O to a final volume of 20 μL. Sample loading was done on ice. The reaction solution was placed in 96-well plates, centrifuged at 5 000 × g for 15 seconds, followed by real-time PCR reaction in a quantitative PCR apparatus (ABI-7500, Carlsbad, CA, USA). PCR conditions were as follows: 95°C for 5 minutes; 40 cycles at 95°C for 10 seconds and 60°C for 31 seconds. β-actin and oligodendrocyte lineage gene 2 were quantified using PCR, and the relative expression of oligodendrocyte lineage gene 2 mRNA was calculated with the following formula: fold change = 2^－^ΔΔCt= 2^－^{(ΔCt(treated samples)}^－^{ΔCt(control samples))}.

Statistical analysis

The data were analyzed using SPSS 13.0 (SPSS, Chicago, IL, USA), and were expressed as mean ± SD. Intergroup differences were compared using one-way analysis of variance. A value of P < 0.05 was considered statistically significant.

讨论

文章快阅

延伸阅读

1 实验设计构思介绍
实验着重探讨二黄胶囊对实验性自身免疫性脑脊髓炎模型小鼠少突胶质细胞前体细胞增殖和分化的作用，为探究以二黄胶囊为代表的补肾中药和方剂对实验性自身免疫性脑脊髓炎和多发性硬化的作用机制提供证据。研究使用国际上公认的实验性自身免疫性脑脊髓炎小鼠模型为研究对象，观察二黄胶囊对实验性自身免疫性脑脊髓炎小鼠发病情况、神经组织病理变化及脑、脊髓不同部位Olig2基因和组织表达的影响。由实验结果可推测二黄胶囊可能是通过促进少突胶质细胞前体细胞增殖和分化，促进髓鞘修复，而对实验性自身免疫性脑脊髓炎小鼠及多发性硬化患者起到防治作用。

2 国内外同类研究水平的介绍
Neurochemistry International. 2009 ;54:192-198.
局灶或者迁移的Olig2前体细胞的增殖和分化促进脱髓鞘区域的髓鞘再生
Nature. 2003;422(6933): 688-694.
通过鞘内或者静脉注射成体神经前体细胞可以促进慢性多发性硬化模型多病灶髓鞘再生和功能恢复
Journal of Neuroscience Methods. 2006;157: 187-194.
少突胶质细胞前体细胞的分化伴随Olig2基因表达上调，Olig2对少突胶质细胞的形成是必须的
Neuron 2011;69;906-917.
Olig2磷酸化可以调节神经前体细胞增殖
Mol Cell Neurosci. 2006;33:371-380.
诱导Olig2表达是少突胶质细胞形成的充分条件，但并不是运动神经元和星形胶质细胞形成的充分条件
Journal of the Neurological Sciences. 2005;233:179-181.
神经干细胞是少突胶质细胞髓鞘修复的潜在来源

3 先进性、创新性及其验证说明特色内容
（1）提供证据：检索Pubmed数据库，检索时间为2011年10月，检索关键词为Olig2，EAE，OPCs，MS。
（2）创新性说明：本研究采用神经病理、IHC、Real-time PCR技术检测与少突胶质细胞前体细胞增殖分化相关的Olig2基因和组织表达，从组织和分子层面探讨补肾化痰活血中药二黄胶囊对实验性自身免疫性脑脊髓炎小鼠少突胶质细胞前体细胞增殖和分化的影响，为中医药治疗多发性硬化提供理论依据。

4 专家外审意见与作者答疑
问题1：说明免疫组化研究与RT-PCR之间的关系，取材部分是否存在交叉与重叠。
回复：用免疫组化法标记Olig2可以对其蛋白表达进行定位与半定量研究，RealTime-PCR 可以对Olig2的DNA水平进行定量研究，从而在基因和蛋白两个方面对Olig2进行综合分析。取材部位尽量保持一致，免疫组化包埋的蜡块和 RealTime-PCR 的取材组织均固定于端脑（不包括小脑与脑干、延髓）冠状面中间三分之一的组织块。
问题2：研究的时间点在多个研究中的联系。
回复：基因的检测分为两个时点进行，免疫第23天基本处于发病高峰期，免疫第43天处于逐渐恢复的平台期，RealTime-PCR 显示发病高峰期时二黄胶囊大、小剂量组Olig2基因的增加较平台期更为明显。免疫组化检测Olig2和BrdU的统计结果是两个时间点的综合，重点强调不同部位的表达。
问题3：为何PA组从12天时开始给药，而EH组从1天时开始？
回复：根据临床用药规范，PA从发病期开始用药，小鼠发病大多从12天开始；而中药可以作为预防用药，故使用时间可以提前。

中药二黄胶囊为何可治疗多发性硬化？

Mechanism of Erhuang capsule for treatment of multiple sclerosis

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