补肾益髓方及其拆方调控实验性自身免疫性脑脊髓炎小鼠轴突再生抑制信号通路相关分子的实验研究

doi:10.3969/j.issn.1006-7795.2020.02.010

首都医科大学学报 ›› 2020, Vol. 41 ›› Issue (2): 200-211.doi: 10.3969/j.issn.1006-7795.2020.02.010

补肾益髓方及其拆方调控实验性自身免疫性脑脊髓炎小鼠轴突再生抑制信号通路相关分子的实验研究

王蕾¹, 安辰¹, 赵晖¹, 薛冰², 齐放¹, 李君玲¹, 金良韵², 张楠¹, 樊永平³

1. 首都医科大学中医药学院中医临床方药学系中医络病研究北京市重点实验室, 北京 100069;
2. 首都医科大学中心实验室, 北京 100069;
3. 首都医科大学附属北京天坛医院中医科, 北京 100070

收稿日期:2020-01-15 出版日期:2020-04-21 发布日期:2020-04-16
通讯作者: 王蕾 E-mail:tmwangl@ccmu.edu.cn
基金资助:
国家自然科学基金（81873252，81573898），北京市自然科学基金（7182020）。

Experimental study on the regulation of Bushen Yisui formula and its disassembled formulas on related molecules of inhibitory signal pathway of axon regeneration in mice with experimental autoimmune encephalomyelitis

Wang Lei¹, An Chen¹, Zhao Hui¹, Xue Bing², Qi Fang¹, Li Junling¹, Jin Liangyun², Zhang Nan¹, Fan Yongping³

1. Department of Medicinal Formula and Pharmacy of TCM, School of Traditional Chinese Medicine, Beijing Key Lab of TCM Collateral Disease Theory Research, Capital Medical University, Beijing 100069, China;
2. Core Facility Center, Capital Medical University, Beijing 100069, China;
3. Department of Traditional Chinese Medicine, Beijing Tiantan Hospital, Capital Medical University, Beijing 100070, China

Received:2020-01-15 Online:2020-04-21 Published:2020-04-16
Supported by:
This study was supported by National Natural Science Foundation of China （81873252，81573898），Natural Science Foundation of Beijing （7182020）.

摘要/Abstract

摘要： 目的观察补肾益髓方（Bushen Yisui formula，BSYSF）及其拆方补肾方（Bushen formula，BSF）和化痰活血方（Huatan Huoxue formula，HTHXF）对实验性自身免疫性脑脊髓炎（experimental autoimmune encephalomyelitis，EAE）小鼠轴突再生抑制信号通路NogoA/NgR/RhoA/ROCK的调控作用。方法将雌性C57BL/6小鼠采用数字表法随机分为正常对照组（normal control，NC）、模型组（model，MO）、醋酸泼尼松组（prednisone acetate，PA，6 mg/kg）、梓醇组（catapol，CA，40 mg/kg）、BSYSF组（生药3.02 g/kg）、BSF组（生药1.44 g/kg）、HTHXF组（生药1.57 g/kg），每组16只。用髓鞘少突胶质细胞糖蛋白_35-55（myelin oligodendrocyte glycoprotein_35-55，MOG_35-55）免疫小鼠制备EAE模型。NC和MO组小鼠灌服蒸馏水，各治疗组予相应药物灌胃，每日1次，共40 d。免疫第25天和40天，分别取小鼠脑和脊髓。应用实时荧光定量-PCR（quantitative real time-PCR，qRT-PCR）检测NogoA、NgR、RhoA和ROCKⅡmRNA表达；免疫组织化学（immunohistochemistry，IHC）和蛋白质印迹（Western blotting，WB）法测定上述指标蛋白的表达。结果 MO组小鼠脑和脊髓NogoA、NgR、RhoA和 ROCKⅡmRNA和蛋白表达较NC组明显上调（P<0.05，P<0.01，P<0.001）。经BSYSF及其拆方BSF和HTHXF治疗后，NogoA、NgR、RhoA和ROCKⅡmRNA和蛋白的表达均有不同程度的降低，BSYSF组的上述指标差异有统计学意义（P<0.05，P<0.01，P<0.001）。结论 BSYSF及其拆方BSF和HTHXF均具有调节EAE小鼠轴突抑制因子NogoA/NgR及其信号通路RhoA/ROCK作用，并且BSYSF作用明显。上述结果为阐释BSYSF促进轴突再生的作用机制提供了实验依据。

关键词: 补肾益髓方及其拆方, 实验性自身免疫性脑脊髓炎, 轴突再生, NogoA/NgR/RhoA/ROCK

Abstract: Objectives To observe the regulation of Bushen Yisui formula (BSYSF) and its disassembled formulas, Bushen formula (BSF) and Huatan Huoxue formula (HTHXF) on related inhibition molecules in signal pathway of axon regeneration in mice with experimental autoimmune encephalomyelitis (EAE). Methods Female C57BL/6 mice were randomly divided into normal control (NC), model (MO), prednisone acetate (PA, 6mg/kg), catapol (CA, 40 mg/kg), BSYSF (raw drug 3.02 g/kg), BSF (raw drug 1.44 g/kg) and HTHXF(raw drug 1.57 g/kg), with 16 mice in each group. EAE model in mice was made by immunizing with myelin oligodendrocyte glycoprotein _35-55 (MOG_35-55). The mice in NC and MO groups were given distilled water by means of intragastric administration, the mice in treatment groups were given corresponding drugs once a day for 40 d. The brain and spinal cord of mice were taken on day 25 and 40 of immunization. The gene expression of NogoA, NgR, RhoA and ROCKⅡmRNA were determined by quantitative real time-PCR, the protein expression of the above indexes were detected by the immunohistochemistry and Western blotting technique. Results The mRNA and protein expressions of NogoA, NgR, RhoA and ROCKⅡin the brain and spinal cord in MO mice were increased significantly in EAE mice compared to NC mice (P<0.05, P<0.01, P<0.001). After the treatment of BSYSF and its disassembled formulas BSF和HTHXF, the mRNA and protein expressions of NogoA, NgR, RhoA and ROCKⅡwere reduced to some extent, the above indexes had statistically significant in BSYSF group compared with MO group (P<0.05, P<0.01, P<0.001). Conclusions BSYSF and its disassembled formulas BSF and HTHXF had the regulation on inhibitory factors of axon regeneration NogoA/NgR and its signal pathway RhoA/ROCK in EAE mice, and BSYSF had obvious effect. These findings provide an experimental basis for explaining the mechanism of BSYSF promoting axon regeneration.

Key words: Bushen Yisui formula and its disassembled formulas, experimental autoimmune encephalomyelitis, axon regeneration, NogoA/NgR/RhoA/ROCK

中图分类号:

王蕾, 安辰, 赵晖, 薛冰, 齐放, 李君玲, 金良韵, 张楠, 樊永平. 补肾益髓方及其拆方调控实验性自身免疫性脑脊髓炎小鼠轴突再生抑制信号通路相关分子的实验研究[J]. 首都医科大学学报, 2020, 41(2): 200-211.

Wang Lei, An Chen, Zhao Hui, Xue Bing, Qi Fang, Li Junling, Jin Liangyun, Zhang Nan, Fan Yongping. Experimental study on the regulation of Bushen Yisui formula and its disassembled formulas on related molecules of inhibitory signal pathway of axon regeneration in mice with experimental autoimmune encephalomyelitis[J]. Journal of Capital Medical University, 2020, 41(2): 200-211.

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补肾益髓方及其拆方调控实验性自身免疫性脑脊髓炎小鼠轴突再生抑制信号通路相关分子的实验研究

Experimental study on the regulation of Bushen Yisui formula and its disassembled formulas on related molecules of inhibitory signal pathway of axon regeneration in mice with experimental autoimmune encephalomyelitis

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