Introduction

Cerebral ischemia is a common neurological condition and leads to high rates of disability and mortality. At present, the only approved therapy is human tissue-type plasminogen activator (Lees et al., 2010). However, only 5% of patients can receive this treatment because it has to be administered within 4.5 hours of an ischemic event (Fonarow et al., 2011).Moreover, even with effective thrombolysis, most patients will have varying degrees of neurological de fi cits (Yamamoto et al., 2009). Stroke elicits disruption of myelin and impairs axonal conductivity, which exacerbates functional outcome(Benowitz and Carmichael, 2010). Axonal remodeling plays a crucial role in endogenous brain repair (Zhang et al., 2010).Some inhibitory factors of neurological regeneration, such as neurite outgrowth inhibitor protein A (Nogo-A), play an important role in restraining axon regeneration and lateral shoot formation (Schwab, 2004). Downregulation of RhoA/Rho-associated kinase, a downstream switch of Nogo-A, can promote axonal regeneration (Yang et al., 2013). Also, low expression levels of axonal growth factors may be another difficulty for neural regeneration. Netrin-1 is an important member of the netrin axonal guidance family, and is expressed in the mature central nervous system but not during development (Izzi and Charron, 2011). The Netrin family proteins can promote the recovery of neuronal function after cerebral ischemia (Bayat et al., 2012), while Netrin-1 can promote axon growth by activating Rho GTPases, including Rac1 and Cdc42, which are crucial for transmitting extracellular signals into axonal remodeling within the growth cone(Antoine-Bertrand et al., 2011). Furthermore, both promotive and inhibitory factors of axonal growth exert their functions by binding with their receptors and activating downstream molecular switches (Sakumura et al., 2005). Based on these fi ndings, exploring alternative therapies that boost axonal repair is a matter of urgency for stroke treatment.

Houshiheisan (HSHS) was the fi rst prescription for stroke created by Zhang Zhongjing and has been clinically used in China to promote safe and effective repair of neurological function for over 2000 years. According to basic theory of traditional Chinese medicine, HSHS is composed of a wind-dispelling drug (WDD) and a deficiency-nourishing drug (DND). Flos Chrysanthemi and Radix et Rhizoma Ginseng are considered as representative WDD and DND drugs, respectively. Our previous analysis of HSHS by high performance liquid chromatography showed that the main components are chlorogenic acid, apigenin-7-O-glucoside,and luteolin-7-O-glucoside (Chang et al., 2016). Importantly,chlorogenic acid ameliorates brain damage and edema by inhibiting matrix metalloproteinase-2 and 9 (Lee et al., 2012),while luteolin-7-O-glucoside inhibits inflammation after focal cerebral ischemia in vivo via conversion to luteolin(Zhang et al., 2012). Furthermore, HSHS can inhibit amyloid precursor protein (APP) deposition, reduce the release of in fl ammatory response factors, and up-regulate the expression of brain-derived neurotrophic factor, thereby improving neurological function (Bayat et al., 2012; Zhang et al., 2012;Wang et al., 2014). Our previous fi ndings demonstrated that HSHS has a potent effect on neural regeneration. However,the mechanism of this effect has not been fully investigated.This study focused on the axonal signaling pathways of Nogo-A/RhoA/Rock2 and Netrin-1/DCC/Rac1, and explored the mechanisms by which HSHS promotes neural regeneration after cerebral ischemia via axon recovery.

明乎此，让我们来审视当今文学、绘画、音乐、舞蹈、书法、建筑等学科门类的审美诉求。当前，这些学科门类中一些学者和艺术家往往误把基于道德、政治等社会批评的“尚拙”“斥巧”与基于技巧、技艺等的艺术批评混同为一，以至于出现了以丑为美、以怪为美、以俗为美的不良艺术追求。这种不良风尚的生成并且成为气候，在一定程度上是由于受到近古时期以来的宋明理学影响而形成的，有其历史文化的背景。但“尚拙”“斥巧”的审美选择，并非中国文化的全部传统，我们应该检讨自宋代之后若干文化范畴的被误读和误用问题，还原其本来面貌。

Materials and Methods

Establishment of focal cerebral ischemia models

Seventy-two healthy, specific-pathogen-free, 3-month-old male Sprague-Dawley rats weighing 300—350 g, were provided by Beijing Vital River Laboratory Animal Technology Co.,Ltd., China [Animal license No. SCXK (Jing) 2012-0001]. The ethical license number from the Institution Animal Care and Use Committee was AEEI-2016-054. Rats were housed in the laboratory animal center of the Capital Medical University,China [Animal license No. SYXK (Jing) 2010-0020]. Protocols conform to the Guidance Suggestions for the Care and Use of Laboratory Animals, formulated by the Ministry of Science and Technology of China. The rats were randomly divided into sham group (n = 12), permanent middle cerebral artery occlusion (pMCAO) group (n = 15), HSHS group (n = 15),WDD group (n = 15) and DND group (n = 15). The pMCAO model was based on our previously published method (Bayat et al., 2012). Rats were anesthetized via face mask inhalation of 1.5% to 2.5% isoflurane in a 2:1 N2O:O2 atmosphere and fi xed in the supine position. The origins of the right common carotid artery, the internal carotid artery and the external carotid artery were exposed and isolated by blunt separation. A 4-0 mono fi lament nylon suture (Beijing Sunbio Biotech Co.,Ltd., Beijing, China) was inserted into the lumen of the internal carotid artery to block the right middle cerebral artery.Rats in the sham group were subjected to the same operation without insertion of the nylon suture. Five rats died within 7 days of pMCAO (two in the pMCAO group, two in the WDD group and one in the HSHS group).

HSHS preparation

HSHS contains 13 Chinese herbs, including Flos Chrysanthemi, Radix Saposhnikoviae, Ramulus Cinnamomi, Rhizoma Chuanxiong, Radix et Rhizoma Asari, Radix Platycodonis,Rhizoma Atractylodis macrocephalae, Poria, Rhizoma Zingiberis, Radix Angelicae sinensis, Radix et Rhizoma Ginseng,Radix Scutellariae and Concha Ostreae. According to the theory of traditional Chinese medicine, HSHS can be divided into two main components, WDD and DND. WDD consists of six Chinese herbs, including Flos Chrysanthemi, Radix Saposhnikoviae, Ramulus Cinnamomi, Rhizoma Chuanxiong, Radix et Rhizoma Asari and Radix Platycodonis. DND is composed of five Chinese herbs: Rhizoma Atractylodis macrocephalae, Poria, Rhizoma Zingiberis, Radix Angelicae sinensis and Radix et Rhizoma Ginseng. All the above Chinese herbs were purchased from Tong-ren-tang Chinese Medicine Co., Ltd. (Beijing, China) and authenticated by Associate Professor Jia Li (Capital Medical University, Beijing,China) according to the Chinese Pharmacopoeia (2015 edition). Their voucher specimens were deposited at the Beijing Key Laboratory of Traditional Chinese Medicine Collateral Disease Theory Research (Beijing, China). Quality control of these Chinese herbs was previously performed by high performance liquid chromatography as published in our previous study (Bayat et al., 2012).

HSHS, WDD and DND were prepared separately with our previously published methods (Bayat et al., 2012). Briefly,the air-dried herbs were mixed according to the prescription and then re fl uxed twice with 30% ethanol (1:10, w/v) for 2 hours. After filtration, the solution was evaporated under vacuum at 50°C to remove ethanol to give liquid extracts of HSHS, WDD and DND. One milliliter of HSHS, WDD and DND extract was produced from 1.7 g, 1.25 g and 0.42 g of crude herbs, respectively.

Drug administration

According to a previous study (Bayat et al., 2012), the rats in the HSHS, WDD and DND groups were given 10.5 g/kg HSHS, 7.7 g/kg WDD and 2.59 g/kg DND, respectively. The rats in the sham and pMCAO groups were given the same volume of normal saline (1 mL/100 g). The doses of HSHS,WDD and DND were calculated using the body surface area normalization method and normal clinical usage of crude drugs. Drugs were intragastrically administrated for the fi rst time at 6 hours after the operation, and then every 24 hours for 7 consecutive days. Brain tissues were removed at 7 days after pMCAO for analysis (Figure 1).

李建军[1]认为，农业研究与开发体系大致包括政府所属的研究机构，私人公司和其他非政府性的研究机构以及各个国际化农业中心等，而我国已经建立起了以农业科研院所为主体，以农业大学为主干的农业研究与开发体系和独特的农业自助体系，这些体系的职责是实现农业技术创新。农业技术创新是一个系统工程，涵盖了农业技术推广在内的多个环节。本研究的农业科研人员指的是农业科研院所、农业大学内从事农业技术创新的人员，其工作职责包括农业技术研究与开发、农业推广、农业科技成果转化等。

Neurofunctional scoring

Neurological deficit was assessed using the improved Zea Longa fi ve-grade scoring method (Longa et al., 1989): 0 = no apparent neurological symptoms; 1 = failure to fully stretch the left forelimb; 2 = rotating to the left; 3 = falling to the leftside during walking; 4 = loss of consciousness or walking only by stimulation.

学生讲坛活动已经走过了五个春秋。从低年级讲绘本故事，到中年级介绍自己喜欢的书籍，再到高年级融入自己思想的学生讲坛，孩子们的思维在发展，思想在成熟，生命在成长。学生每次演讲少则半个小时，多则一个多小时，演讲的主题都是学生们自定的，涉及历史、文学、科学、军事、戏剧、体育等诸多方面，比如，“匈奴的传奇”“帝国的兴起—大秦统一之路”“曹家谋士贾诩”“第二次世界大战中的欧洲战场”“解读二战”，等等。演讲台上，孩子们旁征博引，谈古论今，一个个俨然是所讲主题的专家。讲座中，全年级同学认真听讲做笔记，台上台下不时的互动让所有人都参与其中。

Beam walking test

The beam walking test was performed at 3 and 7 days after pMCAO (Roof et al., 2001). Rats were trained from 3 days before the operation to ensure they could successfully cross a bar 100 cm long, 3 cm wide and located 60 cm above the fl oor. The assessment was evaluated with the following grading criteria: 0 = unable to stay on the crossbar; 1 = able to stay on the crossbar without movement; 2 = trying to traverse the beam but falling off; 3 = traversing the beam with more than 50% left hindlimb slips; 4 = traversing the beam with less than 50% left hindlimb slips; 5 = walking across the beam with only one left hindlimb slip; 6 = crossing the beam successfully without any hindlimb slips.

Neuropathological assessments

Three rats were randomly selected from each of the five experimental groups at 7 days after modeling for hematoxylin-eosin staining. After deep anesthesia, rats were transcardially perfused with 500 mL 0.9% saline, followed by 500 mL of 4% paraformaldehyde in 0.1% M phosphate buffered solution (PBS, pH 7.4). Brains were then carefully dissected,fi xed in 4% paraformaldehyde, embedded in paraffin, and 3 μm-thick sections prepared for hematoxylin-eosin staining.Stained sections were observed by optical microscopy (E100,Nikon, Tokyo, Japan) at 400× magni fi cation.

股骨髁复杂骨折往往存在多向移位情况，仅仅依靠常规复位技术或皮牵引床很难达到满意复位并有效维持[12]。骨折牵引器为我们在股骨髁骨折复位及维持复位中提供了很好的帮助，但其为单侧牵引，其在纠正骨折端旋转及成角移位上空间不大，如骨折端移位较大，术中在安置牵引器前仍需要助手在尽可能纠正成角及旋转移位并维持正常力线下手法牵引维持直至安置牵引器完成。单侧骨折牵引因其术中操作简便，术中牵引有效，并且价格相对低廉，值得在复杂股骨髁骨折中辅助复位及临时固定，为微创内固定提供了确实可行的帮助，使MIPPO技术变得相对简化，值得应用。

GAP-43 is an indicator of axonal recovery. The expression of GAP-43 was up-regulated after pMCAO, and was further elevated after HSHS or WDD treatment (GAP-43, F(4,20) =19.394, P ＜ 0.001; Figure 4A). Notably, the expression of GAP-43 was higher after HSHS treatment than after WDD or DND treatment (P ＜ 0.05; Figure 4A).

为研究铷和铌钽等在不同粒级中的分布情况，我们将样品碎磨至-1 mm后进行粒度分级，并进行了筛析样的化学分析研究，分析结果见表4。

Immuno fl uorescence for microtubule-associated protein-2 (MAP-2)

MAP-2 plays a critical role in axon growth (Wang et al.,2017). Sections for immunofluorescence staining of MAP-2 were heated with 0.01 M citrate buffer, and incubated with primary antibody (MAP-2, mouse monoclonal, 1:3000; Abcam, Cambridge, MA, USA) at 4°C for 40 hours and then at 37°C for 1 hour. Sections were then washed with PBS and incubated with 50 μL secondary antibody (goat anti-mouse IgG ＜H+L＞-FITC, 1:200; SouthernBiotech, Birmingham,AL, USA) at 37°C for 2 hours. Sections were mounted using Dapi-Fluoromount-G (SouthernBiotech, Birmingham, AL,USA) and preserved at 4°C. Sections were observed by fl uorescence microscopy (Olympus, Tokyo, Japan), and images were collected by digital photomicroscopy (Leica, Wetzlar,Germany). Five fi elds of view were randomly selected from each section for quantitative analysis, and the average value of integral optical density calculated using the NIS-Elements Basic Research image acquisition system (Nikon).

Western blot assay

Total protein was extracted from ischemic hippocampi and quanti fi ed using a bicinchoninic acid protein assay kit. Proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then transferred onto polyvinylidene fl uoride membranes (Neobioscience Biotech Co., Ltd., Beijing, China). The membranes were blocked with 5% nonfat milk in Tris-buffered saline containing 0.1%Tween-20 for 1 hour, and incubated with primary antibodies, including APP (1:100,000; rabbit monoclonal; Epitomics,Burlingame, CA, USA), growth associated protein-43 (GAP-43) (1:100,000; rabbit monoclonal; Epitomics), Nogo-A(1:5000; rabbit polyclonal; Abcam), NgR (1:5000; rabbit monoclonal; Abcam), Rock2 (1:10,000; rabbit polyclonal;Abcam), RhoA (1:15,000; rabbit monoclonal; Cell Signaling Technology, Danvers, MA, USA), Netrin-1 (1:2000; rabbit monoclonal; Abcam), deleted in colorectal cancer (DCC)(1:5000; rabbit polyclonal; Abcam), Rac1 (1:15,000; mouse monoclonal; Abcam), Cdc-42 (1:5000; rabbit polyclonal; Abcam), and GAPDH (1:10,000; mouse monoclonal; GeneTex,Irvine, CA, USA) at 4°C overnight. After several washes with Tris-buffered saline containing 0.1% Tween-20, the membranes were incubated in horseradish peroxidase-linked goat-anti-rabbit antibody (IgG, 1:20,000; Neobioscience Biotech Co., Ltd.) for 1 hour at 37°C and washed. Blots were visualized using the ECL chemiluminescent system (Millipore,Burlington, MA, USA) and quantified using Image Quant TL software (Amersham Biosciences, Piscataway, NJ, USA).

Figure 1 Schedule of experimental manipulations.

HSHS: Houshiheisan; WDD: wind-dispelling drug; DND: deficiency-nourishing drug; h:hours; i.g.: intragastrically; HE: hematoxylin-eosin.

Figure 2 Neurological function and pathological changes in each group after ischemia.

(A) Neurological function scores from 1 to 7 days after pMCAO; (B) Beam walking scores at 3 and 7 days after pMCAO. Higher scores in (A) &(B) represent better neurological function, while low scores mean more serious neurological damage. (C) Hematoxylin-eosin staining of surviving neurons (arrows) in peri-infarct cortex (upper) and hippocampi (lower) at 7 days after cerebral ischemia (original magni fi cation: 400×; Scale bars:50 μm). (a—e) Sham, pMCAO, WDD, DND, and HSHS groups, respectively. (D) Quanti fi cation of the number of surviving neurons. (A, B, D)Data are expressed as the mean ± SD, and were analyzed by analysis of variance followed by the least signi fi cant difference post hoc test. *P ＜ 0.05,**P ＜ 0.01, vs. pMCAO group; ΔP ＜ 0.05, ΔΔP ＜ 0.01, vs. HSHS group. (E) Transmission electron microscopy images showing neurons with an irregular nuclear membrane (arrow) in peri-infarct hippocampi at 7 days (original magni fi cation: 1500×, scale bars: 2 μm). (a—e) Sham, pMCAO,WDD, DND, and HSHS group, respectively. pMCAO: Permanent middle cerebral artery occlusion; WDD: wind-dispelling drug; DND: de fi ciency-nourishing drug; HSHS: Houshiheisan.

Statistical analysis

Figure 3 Myelin sheath and axonal changes 7 days after cerebral ischemia.

(A) Transmission electron microscopy images: Myelinated nerve fibers were disorganized; layers of the myelin sheath were wrinkled and axoplasms of axons were degenerated in the peri-infarct hippocampus (arrow) of the pMCAO group (original magnification: 4000×;scale bar: 1 μm); (a—e) Sham, pMCAO,WDD, DND, and HSHS groups, respectively. (B) g-ratio of rats in each group.(C) APP protein expression in peri-infarct hippocampi detected by western blot assays. Data are expressed as the mean ± SD, and were analyzed by analysis of variance followed by the least significant difference post hoc test. *P＜ 0.05, **P ＜ 0.01, vs. pMCAO group;ΔΔP ＜ 0.01, vs. HSHS group. pMCAO:Permanent middle cerebral artery occlusion; WDD: wind-dispelling drug;DND: deficiency-nourishing drug;HSHS: Houshiheisan.

Figure 4 MAP-2 and GAP-43 expression in the peri-infarct hippocampus of rats in each group at 7 days after cerebral ischemia.

(A) GAP-43 protein expression in peri-infarct hippocampi detected by western blotting. (B) Immunofluorescence staining of peri-infarct hippocampi showing MAP-2 positive neurons(arrow). Scale bar: 50 μm; original magni fi cation: 400×. (a—e) Sham, pMCAO,WDD, DND, and HSHS group, respectively. (C) Quantification of MAP-2 immuno fl uorescence staining. Data are expressed as the mean ± SD, and were analyzed by analysis of variance followed by the least signi fi cant difference post hoc test. *P ＜ 0.05, **P ＜ 0.01, vs.pMCAO group; ΔP ＜ 0.05, ΔΔP ＜ 0.01,vs. HSHS group. pMCAO: Permanent middle cerebral artery occlusion; WDD:wind-dispelling drug; DND: deficiency-nourishing drug; HSHS: Houshiheisan; MAP-2: microtubule-associated protein-2; GAP-43: growth associated protein-43.

Figure 5 Nogo-A/RhoA/Rock2 expression in the peri-infarct hippocampus of rats in each group at 7 days after cerebral ischemia.

(A) Nogo-A and NgR protein expression in peri-infarct hippocampi detected by western blotting. (B) RhoA and Rock2 expression in peri-infarct hippocampi detected by western blotting. Data are expressed as the mean ± SD, and were analyzed by analysis of variance followed by the least significant difference post hoc test. *P ＜ 0.05, **P ＜ 0.01, vs. pMCAO group; ΔP ＜ 0.05, vs. HSHS group.pMCAO: Permanent middle cerebral artery occlusion; WDD: wind-dispelling drug; DND: de fi ciency-nourishing drug;HSHS: Houshiheisan.

Figure 6 Netrin-1/DCC/Rac1/Cdc42 expression in the peri-infarct hippocampus of rats in each group at 7 days after cerebral ischemia.

(A) Netrin-1 and DCC protein expression in peri-infarct hippocampi detected by western blot assay. (B) Rac1 and Cdc42 expression in peri-infarct hippocampi detected by western blot assay. Data are expressed as the mean± SD, and were analyzed by analysis of variance followed by the least significant difference post hoc test. *P ＜ 0.05,**P ＜ 0.01, vs. pMCAO group; ΔP ＜0.05, ΔΔP ＜ 0.01, vs. HSHS group. pMCAO: Permanent middle cerebral artery occlusion; WDD: wind-dispelling drug;DND: deficiency-nourishing drug;HSHS: Houshiheisan.

The inhibitory effect of Nogo-A/NgR depends on the activity of downstream molecules, RhoA and Rock. The expression of RhoA and Rock2 [a subtype of Rock with cardio-cerebral vascular distribution (Iizuka et al., 2012)] was detected by western blot assay. Both RhoA and Rock2 levels were signi fi cantly increased in the pMCAO group at 7 days after cerebral ischemia, and HSHS, WDD and DND treatments down-regulated the expression of RhoA and Rock2 compared with the pMCAO group (one-way analysis of variance: RhoA, F(4,20) = 14.025, P ＜ 0.01; Rock2, F(4,20) = 10.834,P ＜ 0.01). Interestingly, the expression of RhoA after HSHS treatment was signi fi cantly lower than that after DND treatment (post-hoc, P ＜ 0.05; Figure 5B).

本次采用的问卷调查主要包括两大部分的内容。一是词汇学习观念。观念分为三种：学习词汇应该死记硬背、词汇应该在上下文中学习和词汇应该通过运用来学习。二是词汇学习策略：认知策略和元认知策略。前者分为死记硬背策略、构词法策略、分类策略、联想策略、查字典策略和搭配策略等，后者分为制定计划、自我评估、自我检查和选择性分配注意力四种。问卷的内容以选择的形式，要求调查对象根据自己的实际学习情况进行选择。总计发放问卷100份，收回问卷100份，问卷回收率100%。

HSHS promotes neuronal recovery after cerebral ischemia by up-regulating certain neurotrophic factors (Bayat et al., 2012). Axonal remodeling is the main manifestation of neuronal recovery after cerebral ischemia (Liu et al., 2017).GAP-43, a growth-related protein promoting neuronal growth, development, nerve regeneration and synaptic remodeling, is a marker of axonal recovery after cerebral ischemia (Li et al., 2017). MAP-2 is another important molecule in regulating axonal reorganization (Roof et al., 2001). Our results show that HSHS and WDD up-regulate the expression of GAP-43 and MAP-2, which means that HSHS and WDD had positive effects on axonal recovery. Furthermore,the effect of HSHS was significantly stronger than that of WDD. However, DND did not produce a signi fi cant effect.These results illustrate that HSHS promotes neural regeneration by promoting axonal recovery and that the effect may derive from the complementarity of WDD and DND.

Results

Effects of HSHS on improving neurological function and relieving neuronal damage in cerebral ischemia model rats

Neurological function was evaluated by the Zea Longa five-level neurological deficit score every 24 hours for 7 days after pMCAO. Compared with the pMCAO group,the neurological de fi cit score was signi fi cantly decreased in HSHS-treated rats from 3—7 days after pMCAO (neurological deficit score: Day 3, F(3,16) = 2.024, P ＜ 0.01; Day 4, F(3,16) =4.515, P ＜ 0.01; Day 5, F(3,16) = 4.515, P ＜ 0.01; Day 6, F(3,16) =2.260, P ＜ 0.01; Day 7, F(3,16) = 2.384, P ＜ 0.01) (Figure 2A),while WDD and DND treatment reduced the neurological de fi cit score from 4—5 days (P ＜ 0.05) (Figure 2A).

The beam walking test was performed to observe fi ne motor control. Compared with the pMCAO group, the scores were higher in the HSHS and WDD groups at 3 and 7 days (Day 3,F(3,16) = 6.713, P ＜ 0.01; Day 7, F(3,16) = 7.515, P ＜ 0.01) (Figure 2B). Scores in the DND group showed a prominent improvement at 7 days (P ＜ 0.05; Figure 2B). It was noteworthy that scores in DND group were markedly lower at 3 and 7 days compared with the HSHS group (P ＜ 0.05; Figure 2B), which indicated that HSHS had a greater effect than DND.

解得：Q=n（e）×NA×1.6×10-19=9.6/64×2×6.02×10-23×1.6×10-19=2.89×104C

观察组的肠鸣音恢复时间、肛门自主排气时间以及护理满意度 为（16.56±8.20）h、（22.12±7.85）h、（92.14±2.15） 分；对照组的上述指标为（38.15±14.33）h、（47.96±18.98）h、（81.23±3.65）分。

Transmission electronic microscopy showed structural abnormalities of neurons following pMCAO. HSHS, WDD and DND treatments alleviated neuronal deformation compared with the pMCAO group (Figure 2E).

由图5表明，随着溶液矿浆浓度升高，溶液中金的浸出率逐渐升高。当矿浆浓度为25%时，浸出曲线趋向于平缓，综合考虑金的浸出率和试验效率，选择最佳矿浆浓度为25%，此时金的浸出率为98.23%。

Effect of HSHS on reducing myelin sheath and axonal damage after cerebral ischemia

Pathological changes to myelin sheaths and axons were observed by transmission electron microscopy at 7 days after pMCAO (Figure 3A). Myelin sheath damage was severe in pMCAO rats but was was markedly relieved after administration of HSHS, WDD or DND (Figure 3).

The g-ratio was increased after focal cerebral ischemia and was decreased after HSHS, WDD or DND treatments compared with the pMCAO group (one-way analysis of variance:g-ratio, F(4,45) = 47.247, P ＜ 0.01) (Figure 3). More importantly, HSHS-treated rats had a lower g-ratio compared with WDD- and DND-treated rats (post hoc, P ＜ 0.01; Figure 3).The degree of axon damage was detected by western blot assay for APP. APP levels were signi fi cantly increased after pMCAO, but HSHS, WDD and DND treatments reduced APP expression compared with the pMCAO group (one-way analysis of variance: APP, F(4,20) = 14.000, P ＜ 0.01; Figure 3).

HSHS promoted axon recovery in the ischemic hippocampus by up-regulating the expression of GAP-43 and MAP-2

The ultrastructure of ischemic peri-infarcts in the hippocampus was observed by transmission electron microscopy(JEM-1230, Jeol, Japan). One rat was randomly selected from each of the fi ve groups, and perfused transcardially with 2%paraformaldehyde mixed with 2% glutaraldehyde for 1 hour,followed by 3% glutaraldehyde for 2 hours. Brains were then removed and ultrathin sections prepared. The ratio of axon diameter to total fiber diameter is called the g-ratio, and data were collected from 10 fi elds of view from each section and averaged using Image-Pro Plus 6.0 (Media Cybernetics,Rockville, MD, USA) (Zheng et al., 2015).

MAP-2 is another useful marker for evaluation of axonal recovery (Roof et al., 2001). MAP-2 staining was significantly decreased in the pMCAO group compared with the sham group and was up-regulated to different degrees after HSHS,WDD and DND treatments (one-way analysis of variance: APP,F(4,20) = 32.638, P ＜ 0.01; Figure 4B, C). Meanwhile, HSHS had a signi fi cant effect on the expression of MAP-2 compared with WDD or DND alone (post-hoc, P ＜ 0.05; Figure 4C).

HSHS promoted axon recovery by down-regulating the Nogo-A/NgR/RhoA/Rock2 signaling pathway in the pMCAO rat hippocampus

The limitation of our study was that we only explored the mechanism of HSHS on axonal recovery at 7 days after cerebral ischemia; more time points should be observed in future research.

Data are shown as the mean ± SD, and were analyzed with SPSS 19.0 software (IBM, Armonk, NY, USA). One-way analysis of variance followed by the least significant difference post hoc test was utilized to compare mean difference among multiple groups. A value of P ＜ 0.05 was considered statistically signi fi cant.

HSHS promoted axon growth by up-regulating the Netrin-1/DCC/Rac1/Cdc42 signaling pathway in the pMCAO rat hippocampus

Netrin-1 promotes axonal remodeling by binding with its receptor, DCC (Blasiak et al., 2016). Netrin-1 and DCC levels were increased in the pMCAO group compared with the sham group, and HSHS, WDD, and DND treatment further elevated netrin-1 and DCC levels compared with the pMCAO group (Netrin-1, F(4,20) = 48.947, P ＜ 0.01; DCC, F(4,20) =41.473, P ＜ 0.01; Figure 6A). Moreover, HSHS up-regulated the expression of Netrin-1 and DCC compared with WDD or DND alone (P ＜ 0.01; Figure 6A).

Rac1 and Cdc42 are downstream molecular switches of Netrin-1 and DCC (Ji et al., 2016). Ischemic injury signi ficantly reduced the expression of Rac1 and Cdc42, while HSHS, WDD and DND up-regulated Rac1 and Cdc42 expression to different degrees (Rac1, F(4,20) = 21.108, P ＜0.01; Cdc42, F(4,20) = 41.471, P ＜ 0.01). Further comparison revealed that HSHS had a greater effect on up-regulating the expression of Rac1 and Cdc42 compared with WDD or DND alone (P ＜ 0.01) (Figure 6B).

Discussion

Regeneration of neurons after cerebral ischemia, has been extensively investigated (He et al., 2016a; Yin et al., 2016).This study investigated the effect and molecular mechanisms of HSHS and its components WDD and DND in neural regeneration and axon remodeling in the pMCAO rat model.

周冬梅 女 1973年生，成都理工大学副教授,西南交通大学在读博士，研究方向：微波技术、通信技术、交通安全工程.

在森林资源管理过程当中，相关林业管理部门要发挥其根本职能，要做到依法治林的原则。森林资源管理要充分发挥法律的功效，为森林资源管理提供重要的法律保障，更好的发挥森林管理部门的职能。通常情况下，森林资源管理者要加强对林地管理，做好林地管理各个环节的工作，严格履行申报审批程序。严格执行林业部门相关的规章管理制度，加强对毁林开荒的惩处管理，做好执法必严、违法必究，对森林资源造成破坏的行为给予严厉的打击。

Hematoxylin-eosin staining revealed severe necrosis, in fi ltration of in fl ammatory cells and vascular edema in the cortex and hippocampus in the non-treatment pMCAO group.There were different degrees of improvement in morphology after HSHS, WDD and DND treatment at 7 days after cerebral ischemia (Figure 2C). Quantitative light microscopy analysis showed that survival of neurons in the cortex and hippocampus of HSHS, WDD and DND groups was signi ficantly increased compared with the pMCAO group (survival neurons: cortex, F(4,20) = 174.610, P ＜ 0.001; hippocampus,F(4,20) = 40.263, P ＜ 0.001; Figure 2D). Many more neurons survived in the HSHS group than in the WDD and DND groups (P ＜ 0.01; Figure 2D).

Neuroprotection plays an important role in neural regeneration after ischemic stroke (Shi et al., 2016). HSHS and its components signi fi cantly alleviated ischemic injury in both neurons and axons. Importantly, HSHS and its components decreased the expression of APP, a neuronal transmembrane glycoprotein transported by rapid axonal transport, which rapidly accumulates after cerebral ischemia (Coleman,2005). Decreased levels of APP indicate less axon damage.Our results indicate that HSHS and its components have protective effects on axons, and that HSHS was more potent than WDD or DND alone in protecting the myelin sheath.

（3）合理分流。通常来说，路基施工不会对公路的正常运营产生严重的影响，但桥梁拼接施工和桥梁路面施工过程都会对桥梁的正常运行产生较为严重的影响，所以需要封闭施工，在施工期间，为了保证通常的交通，可以通过分流的方式分散交通压力。

Axon recovery is regulated by guidance and inhibitory molecules (Schmidt and Minnerup, 2016; Dun and Parkinson, 2017). However, axonal growth in adult neurons after cerebral ischemia is limited because of a lack of intrinsic capacity. More importantly, some inhibitory neurite outgrowth factors can induce axonal dysplasia in central neurons after ischemic lesion (Papadopoulos et al., 2002; Pernet and Schwab, 2012). Nogo-A, a myelin-derived transmembrane protein, is a strong inhibitor on axonal growth by inducing axonal dysplasia (Bandtlow, 2003; Schweigreiter et al., 2004).Under physiological conditions, Nogo-A is highly expressed in immature neurons, which reduces when the neurons become mature. However, ischemic stimulation can evoke an increase of Nogo-A in mature neurons (Buss et al., 2005; Rolando et al., 2012; Feng et al., 2016). Nogo-A can lead to collapse of the growth cone (GrandPré et al., 2000; Lindau et al.,2014). In an NgR-knockdown ischemic animal model, axon growth was more active and was accompanied by recovery of neurological function (Wang et al., 2010). The present study showed an obvious increase in Nogo-A/NgR levels at 7 days after pMCAO, while HSHS, WDD and DND treatments had an inhibitory effect on Nogo-A and NgR expression. RhoA/Rock2, the Rock subtype mainly expressed in brain tissue, is a downstream molecule in the Nogo-A/NgR pathway (Liu et al., 2011a). HSHS, WDD and DND had bene fi cial effects on down-regulating the expression of RhoA/Rock2, while HSHS had a greater effect than DND on the expression of RhoA.Overall, HSHS, WDD and DND contributed to axon growth by down-regulating the expression of the inhibitory Nogo-A/NgR/RhoA/Rock2 signaling pathway, and the effect of HSHS was more powerful than that of DND in inhibiting RhoA.

Netrin-1, the vertebrate homolog of Unc6, is the strongest known chemoattractant for the promotion of axon extension and is a regulator of axonal guidance and angiogenesis (Lai Wing Sun et al., 2011). The combination of Netrin-1 and its receptor, DCC, can precisely regulate the growth direction of the axonal growth cone (Shoja-Taheri et al., 2015). In this study, the expression of Netrin-1 and DCC was spontaneously elevated after cerebral ischemia, which is consistent with previous results (Liu et al., 2011b; Bayat et al., 2012).HSHS, WDD and DND treatments upregulated the expression of Netrin-1/DCC to varying degrees to promote recovery after cerebral ischemia. HSHS promoted the release of axon guidance factors more effectively than WDD and DND after focal cerebral ischemia. Furthermore, the Netrin-1/DCC-mediated axonal recovery needed a downstream effector to exert its function. Rac1/Cdc42 is a molecular switch in axon regeneration (Sit and Manser, 2011). In our study,the expression of Rac1/Cdc42 markedly decreased after pMCAO, while HSHS, WDD and DND treatments up-regulated Rac1/Cdc42 to promote axonal recovery after cerebral ischemia. Importantly, HSHS had a greater effect on the molecular switches than WDD or DND.

Based on these results, we confirmed that HSHS, WDD and DND can reduce axon damage, promote axon recovery,and facilitate axonal regeneration by regulating the expression of Nogo-A/RhoA/Rock2 and Netrin-1/Rac1/Cdc42 after cerebral ischemia. HSHS had better therapeutic effects than WDD or DND alone after focal cerebral ischemia in the following ways: (1) protecting neurons, reducing axonal damage; (2) promoting axonal recovery; (3) up-regulating guidance pathways to encourage axon growth. The potent effect of HSHS on promoting neural regeneration was probably derived from the complementarity of WDD and DND,with WDD participating in all the therapeutic effects by promoting axonal recovery and performing better than DND.SHS has been used in China to treat stroke for approximately 2000 years, or since the Han Dynasty. Recently, doctors of traditional Chinese medicine have expanded the clinical application of HSHS to treat neurodegenerative diseases,such as cerebral ischemia (He et al., 2016b) and Parkinson’s disease (Pang, 2015). Our fi ndings indicate that WDD and DND combined can achieve better therapeutic effects on ischemic cerebral stroke than when used separately.

Nogo-A is a potent axonal inhibitory factor (Schwab, 2004).The expression of Nogo-A and its receptor, NgR, were clearly increased in ischemic rats without treatment, but this expression decreased after administration of HSHS, WDD or DND (one-way analysis of variance: Nogo-A, F(4,20) = 12.763,P ＜ 0.01; NgR, F(4,20) = 9.476, P ＜ 0.01; Figure 5A).

Acknowledgments: Thanks for the assistance of Pei-Yuan Zhao, Man-Zhong Li and Yu Zhan from School of Traditional Chinese Medicine,Capital Medical University (Beijing, China) in manipulating the fi gures.

Author contributions: YL conducted animal behavior experiments and molecular biology experiments. FH conducted paper writing. JHC conducted molecular biology experiments and revised the paper. XQY conducted molecular biology experiments. HZ designed the study and revised the paper. HYZ revised the preparation method of Chinese herbs and the composition of Houshiheisan. QXZ and LW revised the paper. All authors approved the fi nal version of the paper.

Con fl icts of interest: The authors declare no competing fi nancial interests.

Financial support: This study was supported by the National Natural Science Foundation of China, No. 81373526. The funding body played no role in the study design, in the collection, analysis and interpretation of data, in the writing of the paper, or in the decision to submit the paper for publication.

Institutional review board statement: The study protocol was approved by the Institution Animal Care and Use Committee (Approval number:AEEI-2016-054). The experimental procedure followed the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 8023, revised 1985).

Copyright license agreement: The Copyright License Agreement has been signed by all authors before publication.

Data sharing statement: Datasets analyzed during the current study are available from the corresponding author on reasonable request.

Plagiarism check: Checked twice by iThenticate.

Peer review: Externally peer reviewed.

Open access statement: This is an open access journal, and articles are distributed under the terms of the Creative Commons Attribution-Non-Commercial-ShareAlike 4.0 License, which allows others to remix, tweak,and build upon the work non-commercially, as long as appropriate credit is given and the new creations are licensed under the identical terms.

Open peer reviewer: Na fi sa M Jadavji, Carleton University, Canada.

Additional fi le: Open peer review report 1.

References

Antoine-Bertrand J, Villemure JF, Lamarche-Vane N (2011) Implication of rho GTPases in neurodegenerative diseases. Curr Drug Targets 12:1202-1215.

Bandtlow CE (2003) Regeneration in the central nervous system. Exp Gerontol 38:79-86.

Bayat M, Baluchnejadmojarad T, Roghani M, Goshadrou F, Ronaghi A, Mehdizadeh M (2012) Netrin-1 improves spatial memory and synaptic plasticity impairment following global ischemia in the rat.Brain Res 1452:185-194.

Benowitz LI, Carmichael ST (2010) Promoting axonal rewiring to improve outcome after stroke. Neurobiol Dis 37:259-266.

Blasiak A, Kilinc D, Lee GU (2016) Neuronal cell bodies remotely regulate axonal growth response to localized netrin-1 treatment via second messenger and DCC dynamics. Front Cell Neurosci 10:298.

Buss A, Sellhaus B, Wolmsley A, Noth J, Schwab ME, Brook GA (2005)Expression pattern of NOGO-A protein in the human nervous system. Acta Neuropathol 110:113-119.

Chang J, Yao X, Zou H, Wang L, Lu Y, Zhang Q, Zhao H (2016) BDNF/PI3K/Akt and Nogo-A/RhoA/ROCK signaling pathways contribute to neurorestorative effect of Houshiheisan against cerebral ischemia injury in rats. J Ethnopharmacol 194:1032-1042.

Coleman M (2005) Axon degeneration mechanisms: commonality amid diversity. Nat Rev Neurosci 6:889-898.

Dun XP, Parkinson DB (2017) Role of netrin-1 signaling in nerve regeneration. Int J Mol Sci 18:E491.

Feng N, Hao G, Yang F, Qu F, Zheng H, Liang S, Jin Y (2016) Transplantation of mesenchymal stem cells promotes the functional recovery of the central nervous system following cerebral ischemia by inhibiting myelin-associated inhibitor expression and neural apoptosis.Exp Ther Med 11:1595-1600.

Fonarow GC, Smith EE, Saver JL, Reeves MJ, Bhatt DL, Grau-Sepulveda MV, Olson DM, Hernandez AF, Peterson ED, Schwamm LH(2011) Timeliness of tissue-type plasminogen activator therapy in acute ischemic stroke: patient characteristics, hospital factors, and outcomes associated with door-to-needle times within 60 minutes.Circulation 123:750-758.

GrandPré T, Nakamura F, Vartanian T, Strittmatter SM (2000) Identi fi cation of the Nogo inhibitor of axon regeneration as a Reticulon protein. Nature 403:439-444.

He G, Xu W, Tong L, Li S, Su S, Tan X, Li C (2016a) Gadd45b prevents autophagy and apoptosis against rat cerebral neuron oxygen-glucose deprivation/reperfusion injury. Apoptosis 21:390-403.

He TT, Chen WM, Qian YL (2016b) Houshiheisan intervention of Ischemic stroke in different stages of treatment application. Nei Mongol Zhongyiyao 35:87-88.

Iizuka M, Kimura K, Wang S, Kato K, Amano M, Kaibuchi K, Mizoguchi A (2012) Distinct distribution and localization of Rho-kinase in mouse epithelial, muscle and neural tissues. Cell Struct Funct 37:155-175.

Izzi L, Charron F (2011) Midline axon guidance and human genetic disorders. Clin Genet 80:226-234.

Ji X, Liu H, An C, Wang Y, Zhao H, Zhang Q, Li M, Qi F, Chen Z, Wang X, Wang L (2016) You-Gui pills promote nerve regeneration by regulating netrin1, DCC and Rho family GTPases RhoA, Racl, Cdc42 in C57BL/6 mice with experimental autoimmune encephalomyelitis. J Ethnopharmacol 187:123-133.

Lai Wing Sun K, Correia JP, Kennedy TE (2011) Netrins: versatile extracellular cues with diverse functions. Development 138:2153-2169.

Lee K, Lee JS, Jang HJ, Kim SM, Chang MS, Park SH, Kim KS, Bae J,Park JW, Lee B, Choi HY, Jeong CH, Bu Y (2012) Chlorogenic acid ameliorates brain damage and edema by inhibiting matrix metalloproteinase-2 and 9 in a rat model of focal cerebral ischemia. Eur J Pharmacol 689:89-95.

Lees KR, Bluhmki E, von Kummer R, Brott TG, Toni D, Grotta JC,Albers GW, Kaste M, Marler JR, Hamilton SA, Tilley BC, Davis SM,Donnan GA, Hacke W; ECASS, ATLANTIS, NINDS and EPITHET rt-PA Study Group, Allen K, Mau J, Meier D, del Zoppo G, De Silva DA, et al. (2010) Time to treatment with intravenous alteplase and outcome in stroke: an updated pooled analysis of ECASS, ATLANTIS, NINDS, and EPITHET trials. Lancet 375:1695-1703.

Li Y, Gao X, Wang Q, Yang Y, Liu H, Zhang B, Li L (2017) Retinoic acid protects from experimental cerebral infarction by upregulating GAP-43 expression. Braz J Med Biol Res 50:e5561.

Lindau NT, Banninger BJ, Gullo M, Good NA, Bachmann LC, Starkey ML, Schwab ME (2014) Rewiring of the corticospinal tract in the adult rat after unilateral stroke and anti-Nogo-A therapy. Brain 137:739-756.

Liu K, Li Z, Wu T, Ding S (2011a) Role of rho kinase in microvascular damage following cerebral ischemia reperfusion in rats. Int J Mol Sci 12:1222-1231.

Liu N, Huang H, Lin F, Chen A, Zhang Y, Chen R, Du H (2011b) Effects of treadmill exercise on the expression of netrin-1 and its receptors in rat brain after cerebral ischemia. Neuroscience 194:349-358.

Liu Y, Li C, Wang J, Fang Y, Sun H, Tao X, Zhou XF, Liao H (2017) Nafamostat Mesilate improves neurological outcome and axonal regeneration after stroke in rats. Mol Neurobiol 54:4217-4231.

Longa EZ, Weinstein PR, Carlson S, Cummins R (1989) Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke 20:84-91.

Pang YY (2015) The clinical observation on Parkinson’s disease from the treatment of warming yang and extinguishing wind. Jinan, Shandong province, China: Shandong University of Traditional Chinese Medicine.

Papadopoulos CM, Tsai SY, Alsbiei T, O’Brien TE, Schwab ME, Kartje GL (2002) Functional recovery and neuroanatomical plasticity following middle cerebral artery occlusion and IN-1 antibody treatment in the adult rat. Ann Neurol 51:433-441.

Pernet V, Schwab ME (2012) The role of Nogo-A in axonal plasticity,regrowth and repair. Cell Tissue Res 349:97-104.

Rolando C, Parolisi R, Boda E, Schwab ME, Rossi F, Buffo A (2012)Distinct roles of Nogo-a and Nogo receptor 1 in the homeostatic regulation of adult neural stem cell function and neuroblast migration. J Neurosci 32:17788-17799.

Roof RL, Schielke GP, Ren X, Hall ED (2001) A comparison of longterm functional outcome after 2 middle cerebral artery occlusion models in rats. Stroke 32:2648-2657.

Sakumura Y, Tsukada Y, Yamamoto N, Ishii S (2005) A molecular model for axon guidance based on cross talk between rho GTPases.Biophys J 89:812-822.

Schmidt A, Minnerup J (2016) Promoting recovery from ischemic stroke. Expert Rev Neurother 16:173-186.

Schwab ME (2004) Nogo and axon regeneration. Curr Opin Neurobiol 14:118-124.

Schweigreiter R, Walmsley AR, Niederost B, Zimmermann DR, Oertle T, Casademunt E, Frentzel S, Dechant G, Mir A, Bandtlow CE (2004)Versican V2 and the central inhibitory domain of Nogo-A inhibit neurite growth via p75NTR/NgR-independent pathways that converge at RhoA. Mol Cell Neurosci 27:163-174.

Shi Z, Ren H, Luo C, Yao X, Li P, He C, Kang JX, Wan JB, Yuan TF, Su H (2016) Enriched endogenous omega-3 polyunsaturated fatty acids protect cortical neurons from experimental ischemic injury. Mol Neurobiol 53:6482-6488.

Shoja-Taheri F, DeMarco A, Mastick GS (2015) Netrin1-DCC-mediated attraction guides post-crossing commissural axons in the hindbrain.J Neurosci 35:11707-11718.

Sit ST, Manser E (2011) Rho GTPases and their role in organizing the actin cytoskeleton. J Cell Sci 124:679-683.

Wang C, Sun C, Hu Z, Huo X, Yang Y, Liu X, Botchway BOA, Davies H, Fang M (2017) Improved neural regeneration with olfactory ensheathing cell inoculated PLGA scaffolds in spinal cord injury adult rats. Neurosignals 25:1-14.

Wang H, Wang L, Zhang N, Zhang Q, Zhao H, Zhang Q (2014)Houshiheisan compound prescription protects neurovascular units after cerebral ischemia. Neural Regen Res 9:741-748.

Wang T, Wang J, Yin C, Liu R, Zhang JH, Qin X (2010) Down-regulation of Nogo receptor promotes functional recovery by enhancing axonal connectivity after experimental stroke in rats. Brain Res 1360:147-158.

Yamamoto Y, Suzuki M, Kawada Y, Kamogawa K, Okamoto K, Okuda B (2009) Pulseless arrest in an elderly patient treated with intravenous tissue plasminogen activator for cardioembolic ischemic stroke.Nihon Ronen Igakkai zasshi Japanese journal of geriatrics 46:352-357.

Yang Z, Wang J, Liu X, Cheng Y, Deng L, Zhong Y (2013) Y-39983 downregulates RhoA/Rho-associated kinase expression during its promotion of axonal regeneration. Oncol Rep 29:1140-1146.

Yin XH, Yan JZ, Yang G, Chen L, Xu XF, Hong XP, Wu SL, Hou XY,Zhang G (2016) PDZ1 inhibitor peptide protects neurons against ischemia via inhibiting GluK2-PSD-95-module-mediated Fas signaling pathway. Brain Res 1637:64-70.

Zhang Q, Zhao H, Wang L, Zhang Q, Wang H (2012) Effects of wind-dispelling drugs and de fi ciency-nourishing drugs of Houshiheisan compound prescription on astrocyte activation and in fl ammatory factor expression in the corpus striatum of cerebral ischemia rats.Neural Regen Res 7:1851-1857.

Zhang QM, Peng Y, Zhuang WH, Lan J, Li CY (2010) Effects of bone marrow mesenchymal stem cell transplantation on growth-associated protein-43 expression and infarction volume following permanent middle cerebral artery occlusion in rats. Zhongguo Zuzhi Gongcheng Yanjiu 14:6738-6743.

Zheng Q, Yang T, Fang L, Liu L, Liu H, Zhao H, Zhao Y, Guo H, Fan Y, Wang L (2015) Effects of Bu Shen Yi Sui Capsule on Th17/Treg cytokines in C57BL/6 mice with experimental autoimmune encephalomyelitis. BMC Complement Altern Med 15:60.