Traditional Mongolian medicine Eerdun Wurile improves stroke recovery through regulation of gene expression in rat brain

Journal of Ethnopharmacology 222 (2018) 249–260

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Traditional Mongolian medicine Eerdun Wurile improves stroke recovery through regulation of gene expression in rat brain

T

Saren Gaowaa,b,c, Narisi Baoa,b, Man Dac, Qiburi Qiburid, Tsogzolmaa Ganboldd, Lu Chend, ⁎ ⁎⁎ Altanzul Altangereld, Temuqile Temuqileb,c, , Huricha Baiguded, a

School of Basic Medical Science, Beijing University of Chinese Medicine, Chaoyang District, Beijing 100029, PR China Inner Mongolia Medical University, Hohhot, Inner Mongolia 010020, PR China c International Hospital of Mongolian Medicine, Hohhot, Inner Mongolia 010021, PR China d Institute of Mongolian Medicinal Chemistry, School of Chemistry & Chemical Engineering, Inner Mongolia University, Hohhot, Inner Mongolia 010020, PR China b

A R T I C LE I N FO

A B S T R A C T

Chemical compounds studied in this article: povidone-iodine (PubChem CID: 410087) chloral hydrate (PubChem CID: 2707) geniposide (PubChem CID: 107848) (+)-(7 S,8 R,8′R)-lyoniresinol 9-O-β-D-(6″-Otrans-sinapoyl) glucopyranoside (PubChem CID: 10395492) 3,5-di-O-caffeoylquinic acid (PubChem CID: 6474310) kaempferol-3-O-rutinoside (PubChem CID: 24211973) myristicin (PubChem CID: 4276) costunolide (PubChem CID: 6436243) isoliquiritigenin (PubChem CID: 638278) toosendanin (PubChem CID: 115060) 4-dihydro-2-(4’ - hydroxyphenylmethyl)−6 [(3″,4″ -dihydroxy-5″- methoxyphenyl) methylene]-pyran-3, 5-dione 2,3- dihydro-2-(4′- hydroxy-phenylethyl)-6[(3″,4″ -dihydroxy-5″-methoxy) phenyl]-5pyrone

Ethnopharmacological relevance: Eerdun Wurile (EW) is one of the key Mongolian medicines for treatment of neurological and cardiological disorders. EW is ranked most regularly used Mongolian medicine in clinic. Components of EW which mainly originate from natural products are well defined and are unique to Mongolian medicine. Aim of the study: Although the recipe of EW contains known neuroactive chemicals originated from plants, its mechanism of action has never been elucidated at molecular level. The objective of the present study is to explore the mechanism of neuroregenerative activity of EW by focusing on the regulation of gene expression in the brain of rat model of stroke. Materials and methods: Rat middle cerebral artery occlusion (MCAO) models were treated with EW for 15 days. Then, total RNAs from the cerebral cortex of rat MCAO models treated with either EW or control (saline) were extracted and analyzed by transcriptome sequencing. Differentially expressed genes were analyzed for their functions during the recovery of ischemic stroke. The expression level of significantly differentially expressed genes such as growth factors, microglia markers and secretive enzymes in the lesion was further validated by RTqPCR and immunohistochemistry. Results: Previously identified neuroactive compounds, such as geniposide (Yu et al., 2009), myristicin (Shin et al., 1988), costunolide (Okugawa et al., 1996), toosendanin (Shi and Chen, 1999) were detected in EW formulation. Bederson scale indicated that the treatment of rat MCAO models with EW showed significantly lowered neurological deficits (p < 0.01). The regional cerebral blood circulation was also remarkably higher in rat MCAO models treated with EW compared to the control group. A total of 186 genes were upregulated in the lesion of rat MCAO models treated with EW compared to control group. Among them, growth factors such as Igf1 (p < 0.05), Igf2 (p < 0.01), Grn (p < 0.01) were significantly upregulated in brain after treatment of rat MCAO models with EW. Meanwhile, greatly enhanced expression of microglia markers, as well as complementary components and secretive proteases were also detected. Conclusion: Our data collectively indicated that EW enhances expression of growth factors including Igf1 and Igf2 in neurons and microglia, and may stimulate microglia polarization in the brain. The consequences of such activity include stimulation of neuron growth, hydrolysis and clearance of cell debris at the lesion, as well as the angiogenesis.

Keywords: Ischemic stroke Mongolian medicine Eerdun Wurile RNA-seq Igf2 Microglia

Abbreviations: tPA, tissue plasminogen; MCAO/R, middle cerebral artery occlusion/reperfusion; EW, Eerdun Wurile; NGF, nerve growth factor; BDNF, brain-derived neurotrophic factor; DEGs, differentially expressed genes; Igf2, insulin-like growth factor 2; IGFBP2, insulin-like growth factor binding protein 2; Tgf-b1, transforming growth factor beta 1; Vim, vimentin; Grn, granulin; ApoD, apolipoprotein D; Aif1, allograft inflammatory factor 1; Csf1r, colony stimulating factor 1 receptor; CX3CR1, C-X3-C motif chemokine receptor 1; C3, complementary component 3; C1qa, complementary component 1, q subcomponent, A chain ⁎ Corresponding author at: International Mongolian Hospital, Hohhot 010021, PR China. ⁎⁎ Corresponding author. E-mail address: [email protected] (H. Baigude). https://doi.org/10.1016/j.jep.2018.05.011 Received 29 November 2017; Received in revised form 28 April 2018; Accepted 10 May 2018 0378-8741/ © 2018 Elsevier B.V. All rights reserved.

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1. Introduction

2. Materials and methods

Stroke happens when blood supply to brain is insufficient, or due to bleeding in the brain, leading to the damage of brain tissue and subsequent dysfunction in central nervous system. Ischemic stroke, which is a predominant type of stroke, may happen due to blockage of blood vessel caused by thrombosis, embolism, or systemic hypoperfusion (Donnan et al., 2008; Shuaib and Hachinski, 1991). Cryptogenic stroke, which occurs without obvious reasons listed above, constitutes about 35% of all ischemic stroke (Guercini et al., 2008). Ranked after cardiovascular disease and before cancer, stroke is the second major cause of death worldwide (Donnan et al., 2008). It is a tremendous threat to the health of world population. Although seriousness of stroke hit varies depending on the size and location of the damaged brain area, considerably high disability rate has been reported that can lead to numbness, incontinence, speech and vision loss. The main principle for stroke therapy is quick restoration of blood supply by removing the blockage (Saver, 2006). Early thrombolysis using tissue plasminogen (tPA) can remarkable decrease the rate of disability (Emberson et al., 2014). Stroke rehabilitation is crucial to the reduction of brain injury as well as promotion of recovery. Constraint-induced movement therapy (Siebers et al., 2010), brain repair by electrical stimulation (Brown, 2006), and Bobath (Brock et al., 2011) are among the popular stroke rehabilitations. Natural products such as isoliquiritigenin (root of Glycyrrhiza glabra) has protective effects in rat middle cerebral artery occlusion (MCAO)-induced ischemic stroke model (Zhan and Yang, 2006). Diphenylheptanes (fruits of Amomum tsaoko) protects H2O2-induced nerve injury (Zhang et al., 2016). Some traditional Chinese folk medicines have significant therapeutic efficacy for post-stroke recovery (Young et al., 2010). Eerdun Wurile (EW) is a well established Mongolian medicine with a long history of clinical application for treatment of CNS diseases. Effective compounds groups are molecular base for the various biological activities of Mongolian medicine such as neuroprotective effect (Liu et al., 2011). The main components originated from plants in EW are Terminalia chebula Retz (fruits), Carthamus tinctorius (flowers), Gardenia jasminoides Ellis (fruits), Amomum tsaoko (fruits), Glycyrrhiza uralensis Fis (roots and rhizomes), Myristica fragrans (seeds), Abutilon theophrasti (seeds), Melia toosendan Sieb (fruits), Cassia obtusifolia (seeds), Saussurea costus (roots), and Cinnamomum cassia (bark) (Table S1). The key components in EW contain a group of neuroactive natural products. For example, Gardenia jasminoides Ellis contains geniposide, (+)-(7S,8R,8′R)-lyoniresinol 9-O-β-D-(6″-O-trans-sinapoyl) glucopyranoside and 3,5-di-O-caffeoylquinic acid; Amomum tsaoko contains 4dihydro-2-(4’ - hydroxyphenylmethyl) -6-[(3″,4″ -dihydroxy-5″- methoxyphenyl) methylene]-pyran-3, 5-dione and 2,3- dihydro-2-(4′- hydroxy-phenylethyl)-6-[(3″,4″ -dihydroxy-5″-methoxy) phenyl]-5pyrone. The therapeutic effect of EW in the treatment of limb numbness and other nerve related diseases has been clinically proved. It is one of the key remedies used in post-stroke recovery in Mongolian medicine (Hua et al., 2014; Tian, 2011). Recent study on MCAO/R injury rat model has shown that EW may improve nerve growth by up-regulating expression of neurotrophins such as nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) (Hua et al., 2016). However, full spectrum of gene regulation by EW is not understood, and more detailed neuroprotective and possible neuroregenerative mechanism of EW is not clear. In this report, we first confirmed the therapeutic efficiency of EW on MCAO rat model by Bederson scaling, brain microcirculation measurement, TTC staining and H&E staining of infarction area. Then, we analyzed full transcription patterns in the cerebral infarction of MCAO model animals treated with EW using RNA-seq technology. Significant differential expression was observed in genes functioning in cell recovery and growth, as well as immune activity, which may provide the neurorecovery mechanism of EW.

2.1. Chemicals and instruments Eerdun Wurile (internal medicine number M14010080, batch number 20150301) was provided by National Mongolian Pharmaceutical Preparation Center, International Mongolian Hospital, Inner Mongolia, China. Voucher specimens were deposited in the Virtual Herbarium of Inner Mongolia Medical University (Hohhot, China). Chloral hydrate was purchased from Tianjin Zhiyuan Chemical Reagent Co., Ltd. (Tianjin, China). Monofilament nylon suture was purchased from Beijing Cinontech Co. Ltd. (Beijing, China). Waters Acquity UPLC/qTOF (Waters, USA) includes quaternary solvent manager, online degasser, sample manager, column manager, Acquity PDA detector, ESI source, Lockspray source, Xevo G2-XS QTof four-pole flight time tandem mass spectrometer and Masslynx V4.1 Workstation. Acetonitrile is purchased from Fisher Scientific, USA. Formic acid solution is purchased from Sigma-Aldrich, China. Leucine enkephalin is purchased from Waters, USA. Ethanol absolute, petroleum, ethyl acetate and n-butanol were purchased from Tianjin Fengchuan Chemical Reagent Technologies Co., Ltd (Tianjin, China). 2.2. Sample preparation for UPLC-qTOF mass measurement First, EW powder was weighed into 5 round bottom flasks (0.2 g per flask), and added 20 mL of distilled water (Ⅰ), anhydrous ethanol (Ⅱ), nbutanol (Ⅲ), ethyl acetate (Ⅳ) and petroleum ether (Ⅴ), respectively. After immersing for 16 h, the flasks were heated in oil bath to reflux for 8 h at 35 °C while stirring at 700 r/min, followed by filtration through a sand funnel with diatomite. Then, the solvents were removed under nitrogen flow and the samples were stored at 4 °C. 2.3. Analysis of UPLC-QTof-MS For analysis, the following chromatographic conditions were applied: the column (Waters ACQUIY UPLC® BEH Shield RP18, 2.1 × 100 mm Column, 1.7 µm) was connected to a Vanguard HSS T3 guard column. Column temperature was 40 °C; the mobile phase was as following: A: water (containing 0.1% formic acid), B: acetonitrile (containing 0.1% formic acid); the mobile phase gradient elution was: 50% → 90% B, 10 → 15 min: 100% B, 15 → 20 min: 50% B; the flow rate was 0.4 mL/min; the injection volume was: 2 μL. The mass spectrometry conditions were as following: Electrospray Ionization (ESI) positive ion mode was used for detection. Mass detection range was 100–1200 Da; capillary voltage was 3 kV; sample cone was 40 V; extraction cone was 4 V; source temperature was 100 ℃; desolvation temperature was 400 °C; desolvation gas was 800 L/h, lockmass was 556.2771 (positive ion mode). The accuracy error threshold was fixed at 5 mDa. Data acquisition is controlled by MassLynx 4.1 software. According to the report of the chemical composition of the EW medicinal materials which were associated with nerves system, the information on the chemical constituents were collected and that chemical compositions structure were drawn through the "Chemdraw" software, building the molecular formula and theoretical relative molecular mass database. 2.4. Animals A total of 55 eight-week-old male Wistar rats weighing 200–240 g (purchased from Experimental Animal Center of Inner Mongolia University, Hohhot, Inner Mongolia, China) were used for this experiment. The rats were acclimated for 7 days before the start of any experiments. They were housed in a controlled environment (4 animals per cages, 55 ± 5% relative humidity, 22 °C, 12 h:12 h light/dark cycle) and provided with free access to food and water. All experimental procedures involving animals were approved by the Animal 250

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Fig. 1. UPLC-QTOF-MS BPI chromatogram of EW extracted in water (A), ethanol (B), butyl alcohol (C), ethyl acetate (D), and petroleum ether (E).

Care and Use Committee of Inner Mongolia University (approval number: 2016004). We made all efforts to minimize the number of animals used and their suffering.

2.5. Model The rat model of middle cerebral artery occlusion/reperfusion (MCAO/R) was established according to a previously published method 251

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Fig. 2. Plot of the identified neuroactive compounds extracted from EW in water (A), ethanol (B), butyl alcohol (C), ethyl acetate (D), and petroleum ether (E) phase using UPLC-QTOF-MS and UNIFI data processing.

used to assess the effects of occlusion.

(Longa et al., 1989). Operation procedures: (1) Before operation, rats were fasted for 12 h, anesthetized with 10% chloral hydrate (3.5 mL/ 100 g body weight, i.p. injection); then cut off fur around the neck and sterilized the skin using povidone-iodine. The rectal temperature was recorded and maintained at 37 ± 0.5 °C throughout the surgical procedure; (2) Isolated and ligated common carotid artery, external carotid artery, internal carotid artery, vagus nerve, wing palate artery, occipital artery, vascular nerve and surrounding tissue; (3) In the external carotid artery on a small thorn, insert the bolt, fixed bolt, cut off the external carotid artery, and the internal carotid artery into a line, the bolt line to the internal carotid artery into about (18.0 ± 2.0 mm), at the bifurcation ligation, loose artery clip ischemia for 90 min; (4) After 90 min of ischemia, the torsion was removed from the common carotid artery bifurcation, confirmed after the non-active bleeding iodine disinfection, coated with penicillin, suture the skin, the construction of MCAO/R model. The neurologic status of each rat was evaluated carefully 24 h after surgery by an observer. A grading scale of 0–3 was

2.6. Treatment groups The 55 rats were randomly divided into five groups and were continuously injected (intragastric administration) saline, positive control (nimodipine) and EW by intragastric administration (once a day) for 2 weeks. The doses for each group were as following: untreated group (NT, n = 10, saline), MCAO/R (IS, n = 12, saline), MCAO/R + Nimodipine (NI, n = 6, nimodipine, 4 mg/100 g body weight), MCAO/R + EW (EWlow, n = 13, EW 61.7 mg /100 g body weight), and MCAO/R + EW (EWhigh, n = 14, EW 123.4 mg /100 g body weight). Drug doses were determined according to the dose of EW in clinic: the dose of EWlow is 7 times of dose for human; the dose of EWhigh is double amount of EWlow.

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Table 2 Bederson scale scores of MCAO/R rat models before and after treatment. Each value represents the mean ± SEM. The symbol “*” and “**” indicate significant difference p < 0.05 and p < 0.01, respectively.

(Yu et al., 2009) (Yu et al., 2013) (Ablat et al., 2016) (Ablat et al., 2016) (Zhu et al., 2003) (Yu et al., 2009) (Shin et al., 1988) (Okugawa et al., 1996) (Okugawa et al., 1996) (Yu et al., 2012) (Zhan and Yang, 2006) (Zhang et al., 2016) (Yu et al., 2012) (Shi and Chen, 1999) (Zhang et al., 2016) Gardenia jasminoides Ellis Carthamus tinctorius Carthamus tinctorius Carthamus tinctorius Carthamus tinctorius Gardenia jasminoides Ellis Myristica fragrans Saussurea costus Saussurea costus Gardenia jasminoides Ellis Glycyrrhiza uralensis Fis Amomum tsaoko Gardenia jasminoides Ellis Melia toosendan Sieb Amomum tsaoko 411.1245a 449.1081b 1045.2835b 595.1632b 613.1765b 453.1407a 193.0838b 231.1363b 255.1324a 789.2979b 279.0633a 371.1145b 539.1119a 569.2380a 357.1325b 388.1369 448.1006 1044.2747 594.1585 612.1690 430.1475 192.0786 230.1307 232.1463 788.2892 256.0736 370.1053 516.1268 546.2465 356.1260 C19H26O11 C21H20O11 C48H52O26 C27H30O15 C27H32O16 C25H24O12 C15H12O4 C29H38O10 C15H18O2 C39H48O17 C20H18O7 C20H20O6 C39H48O17 C11H12O3 C17H24O10

Source Calculated m/z Chemical formula

Obtained m/z

Reference

S. Gaowa et al.

Groups

Non-treated (NT) MCAO/R model (IS) MCAO/R model + nimodipine (NI) MCAO/R model + EW (low) (EWlow) MCAO/R model + EW (high) (EWhigh)

No. of animals

Bederson scalea Before treatment

After treatment

10 12 6

0±0 2.16 ± 0.38* 2.16 ± 0.40

0±0 1.58 ± 0.66 0.33 ± 0.51*

13

2.07 ± 0.27

0.38 ± 0.50**

14

2.07 ± 0.26

0.21 ± 0.42**

a

Average scale of animals in each group. * P < 0.05. ** P < 0.01.

2.7. TTC and H&E staining Triphenyl tetrazolium chloride (TTC) staining was performed according to the previous report (Saraf et al., 2010). Hematoxylin and eosin (H&E) staining was conducted in a similar method that has been published previously (Lillie et al., 1976). Additionally, to analyze the toxicity effect of EW in liver, the liver enzyme ALT and AST was analyzed by biochemistry analyzer (Pronto Evolution, Italy). Alanine Aminotransferase Activity Assay Kit (ALT, lot number: 172611) and Aspartate Aminotransferase Activity Assay Kit (AST, lot number: 171331) were purchased from Biosino Bio-technology and Science Inc., Beijing, China. Automated biochemistry analyzer (PRONTO Evolution BIOCHEMISTRY ANALYZER, Italy)

The brain tissue (hippocampus and cerebral cortex area) was collected and immediately froze in liquid nitrogen. RNA isolation and subsequent RNA-seq were performed by BGI (Shenzhen, China). To determine mRNA levels in collected tissue, total RNA was extracted with TRIZOL (Invitrogen, Carlsbad, CA, USA). The expression of mRNA was measured using iScriptTM Reverse Transcription Supermix for RTqPCR and iTaqTM Universal SYBR Green Supermix (Bio-Rad, Hercules, CA, USA) for quantitative PCR. The primer sequences used for this study are listed in Table S2. 2.9. Immunohistochemistry analysis Igf2 expression in neuron and microglia was examined using immunohistochemistry (IHC) according to previously published methods. Fixed slides were probed with primary antibodies for neuron marker, NeuN (1:1000, ab177487, Abcam, Cambridge, MA), or microglia marker Iba1 (1:1000, ab178847, Abcam, Cambridge, MA). For visualization, the secondary antibodies, goat anti-rabbit IgG-FITC (1:1000, ab6717, Abcam, Cambridge, MA) and goat anti-rabbit Alexa Fluor ® 594 conjugate (1:1000,﹟8889, Cell Signaling Technology, Inc.) were used along with the nuclear marker DAPI (1:1000, Life Technologies Corp.). Images were acquired by sequential scanning of the immunostained tissues with an Olympus Fluorescence confocal microscope (Olympus, Fluoview FV 1000).

[M+Na]+. [M+H]+.

2.10. Statistical analysis Statistical significance was determined using unpaired t- test or oneway analysis of variance (ANOVA) with a Dunnett's multiple comparisons test. P values of < 0.05 were considered statistical significance. All the results were expressed as mean ± SEM. The analysis was

b

a

Geniposide Kaempferol-3-O-glucoside Anhydrosafflor yellow B Kaempferol-3-O-rutinoside Hydroxysafflor yellow A 6′-O-acetylgeniposide Myristicin Dehydrocostus lactone Costunolide (+)-(7S,8R,8′R)-lyoniresinol 9-O-β-D-(6″-O-trans-sinapoyl) glucopyranoside Isoliquiritigenin 4-dihydro-2-(4’ - hydroxyphenylmethyl) -6 - [(3″,4″ -dihydroxy-5″- methoxyphenyl) methylene]-pyran-3, 5-dione 3,5-di-O-caffeoylquinic acid Toosendanin 2,3- dihydro-2-(4′- hydroxy-phenylethyl)-6-[(3″,4″ -dihydroxy-5″-methoxy) phenyl]-5-pyrone

Compound

Table 1 Identification of neuroactive compounds in EW by UPLC-qTOF analysis.

2.8. Sample collection, RNA-seq and RT-qPCR

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Fig. 3. Regional cortical blood flow recovery after treatment of MCAO rat model with EWhigh. The cerebral blood flow perfusion image (color) and detected position image (grey) for MCAO/R model and MCAO/R + EWhigh were shown.

acetate and petroleum ether, respectively. Using ChemDraw software, we built a structural library of 24 compounds that exist in the plants used in the EW prescription and were previously report to be neuroactive. By using UPLC-qTOF mass analysis, we were able to identify a total of 15 compounds in the overall extraction (Fig. 1, Fig. 2, Table 1), confirming that the formulation process of EW maintained most of the active compounds intact.

Table 3 The rate of rational cortical blood flow.a Treatment

Before MCAO

After MCAO/R (0 d)

After MCAO/R (15 d)

Saline treated MCAO/R EWhigh treated MCAO/R

377.4 ± 37.2 PU

206.9 ± 10.8 PU

221.6 ± 55.9 PU

369.6 ± 55.4 PU

200.6 ± 17.1 PU

310.7 ± 16.1 PU

3.2. Functional assessment of rat MCAO/R model after treatment The therapeutic effect of EW on rat MCAO/R model after 15 days of treatment was first assessed by Bederson scaling. The Bederson scale is widely used to assess neurological impairments after ischemic stroke (Bederson et al., 1986). Significantly low score was observed in the rats treated with EWlow and EWhigh for 14 days (Table 2). The IS group scored 2.16 immediately after the operation, indicating that the establishment of MCAO/R model was successful. After 15 days, the IS group showed some extent of self-recovery, scaling 1.58. Remarkably, MCAO/R model treated with EWlow as well as EWhigh showed significant recovery after 15 days of treatment: the scale dropped from 2.16 before treatment to 0.38 (p < 0.01) and 0.21 (p < 0.01) after treatment, respectively.

a

The values were average of 6 animals in each group. PU: perfusion unit, which equals concentration of moving red blood cells (CMBC) multiplied by average moving speed of blood cells (V).

performed using GraphPad Prism 7.0. 3. Results 3.1. Analysis of neuroactive components in Eerdun Wurile The EW recipe contains several natural products that have been reported to have neuroactive properties. Because the neuroprotective activity of EW may relay on the combined effects of the multiple compounds, we first identified these compounds in the extracts of EW. By using solvents with different polarity, we extracted and analyzed components of EW that is soluble in water, ethanol, butyl alcohol, ethyl

3.3. Regional cortical blood perfusion Blood perfusion and microcirculation in the infarction area of MCAO rat brain was measured by PeriCam PSI system. Based on laser 254

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Fig. 4. TTC staining of rat brain slices for non-treated and all treated groups after 15 days of drug administration. Sever infarction was observed in non-treated MCAO model IS group; significantly reduced infarct areas were observed in EW treated groups.

speckle contrast imaging, PeriCam PSI system can visualize brain blood perfusion in real-time. Ischemia-induced brain injury induces reduction in regional cortical blood flow. To evaluate the effect of EW on microcirculation, the regional cortical blood flow was measured before and after MCAO modeling for non-treated control (NT) and EWhigh treated group, respectively. Significant recovery of cerebral blood flow was detected in EWhigh treated MCAO model animals (Fig. 3, Table 3), indicating that EW may improve microcirculation in cerebral infarction.

EW treatment reduces the infarct volume. To do this, we conducted TTC staining after 15 days of continuous treatment. Infarction was clearly observed in IS group, indicating that the MCAO modeling was successful. All drug treated groups showed reduction to some extent. Significantly, the EWhigh group showed most reduced infarct area (Fig. 4). H&E staining of affected rat brain tissues revealed that, in the infarction region of MCAO model cells shrunk with karyopyknosis, showing apparent encephalomalacia in the extracellular matrix. The intensity of cells in the region also decreased, with irregular arrangement, apparent edema formation and cellular swelling (Fig. 5 A, B). Treatment by Nimodipine reduced karyopyknosis and encephalomalacia to some extent, although apparently decreased number

3.4. Morphological assessment of infarction Next we examined the infarction in all groups to test whether or not

Fig. 5. H&E staining. Brain sections from normal (A), MCAO model (B) and MCAO treated with nimodipine (C), EWlow (D) and EWhigh (E) rats were subjected to H&E staining. Original magnification: x 400. 255

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Fig. 6. Representative liver histopathology in different group of rat by hematoxylin-eosin staining (H&E). A, non-treated; B, MCAO model; C, Nimodipine treated; D, EW treated (low dose); E, EW treated (high dose). Table 4 Liver enzyme analysis a Each value represents the mean ± SEM (n = 6). The symbol “*” indicate significant difference p < 0.05. Groups

No. of animals

AST (U/L)

ALT (U/L)

Non-treated MCAO/R model MCAO/R model + nimodipine MCAO/R model + EWlow MCAO/R model + EWhigh

6 6 6 6 6

120.8 118.5 115.7 111.0 114.1

55.1 57.5 77.5 53.8 54.8

± ± ± ± ±

19.6 21.4 24.7 14.3 9.80

± ± ± ± ±

7.80 15.0 10.7* 12.1 20.0

* P < 0.05. a The doses used for liver toxicity test: nimodipine was 4.0 mg per 100 g body weight; EWlow: 61.7 mg per 100 g body weight; EWhigh: 123.4 mg per 100 g body weight. AST, aspartate aminotransferase; ALT, alanine aminotransferase.

Fig. 7. Representative genes differentially expressed in EW treated groups identified by RNA-seq were validated by RT-qPCR analysis. Each value represents the mean ± SEM (n = 6). The symbol “*” indicates significant difference (p < 0.05).

groups showed normal cell morphology and tissue arrangement (Fig. 6). Moreover, marker enzymes (AST, ALT) in serum (of 6 animals randomly chosen for analysis) were not elevated in EW treated animals, indicating the structural integrity of the hepatocellular membrane (Table 4). Therefore, the cytotoxicity of EW in the liver is negligible.

of neurons was also observed (Fig. 5 C). In both EWlow and EWhigh treated groups, edema formation, karyopyknosis and encephalomalacia were reduced compared to MCAO model group, while increased infiltration of microglia and astrocytes was also observed (Fig. 5 D, E). 3.5. Liver toxicity assessment

3.6. Differential expression of growth factors after EW treatment Herbal medicines may induce liver toxicity. To investigate whether EW has liver toxicity, we examined the liver histopathology as well as the serum level of liver enzymes in rats treated with different doses of EW, and compared to the NT, IS and NI groups. Liver histology of all

Total RNAs was extracted from the cerebral cortex and hippocampus of untreated group (NI, 3 biological replicates), MCAO rat model (IS, 3 biological replicates) and MCAO treated with EW (EWhigh,

Table 5 Significantly upregulated genes in the brain of EW treated MCAO model rats compared to untreated MCAO model rats. Gene ID

Description

Log2 ratioa

Function

24483 25662 59086 81818 29143

Insulin-like growth factor 2 (Igf2) Insulin-like growth factor binding protein 2 (IGFBP2) Transforming growth factor beta 1 (Tgf-β1) Vimentin (Vim) Granulin (Grn)

5.33 2.04 2.02 2.84 2.51

25239 287435 29427 307403

Apolipoprotein D (ApoD) Cd68 molecule (CD68) Allograft inflammatory factor 1 (Aif1) Colony stimulating factor 1 receptor (Csf1r)

2.19 2.93 2.38 2.08

Growth, neuro protection (Ye et al., 2015) Important in Igf1action in activated microglia (Chesik et al., 2004) Suppress apoptosis in stroke (Zhu et al., 2017); promote angiogenesis (Meng et al., 2016) Interacts with Igf1 receptor to promote axonal growth (Shigyo et al., 2015) Attenuate neuronal injury (Egashira et al., 2013); promote activation of microglia (Zhu et al., 2013) Involve in regenerative and scar formation (Rickhag et al., 2008) Microglia marker (Ramprasad et al., 1996) Microglia marker (Deininger et al., 2000) Microglia marker (Walter and Crews, 2017)

a

Log2 ration between EW treated MCAO rats versus untreated MCAO rats. 256

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Fig. 8. EW enhances expression of Igf2 in both neurons and microglia cells. (A) EW treatment of rats dramatically increases Igf2 protein level in NeuN+ cells in cerebral infarction; (B) EW also enhances Igf2 expression in Iba1+ cells in infarction area.

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formation in the infarct. Treatment of rat MCAO model with EW also reduced edema formation, karyopyknosis and encephalomalacia and increased infiltration of microglia and astrocytes in the lesion (Fig. 5) without showing any apparent toxicity to liver (Table 4, Fig. 6). The mechanism of EW has not been systemically studied. The possible mechanism for neuro-recovery includes neurogenesis, angiogenesis, axonal sprouting and oligodendrogenesis (Lo, 2010). To explore the possible mechanism of EW, we chose to study the changes in gene expression pattern in MCAO/R model rats with or without EW treatment. We took RNA-seq approach and focused on the gene regulation of EW at the level of protein coding genes. RNA-seq is a powerful tool to study alternatively spliced transcripts, mutations/single-nucleotide polymorphism, post-transcriptional modifications and differences in gene expression in different treatments (Wang et al., 2009). Treatment of MCAO model of rat with EW significantly up-regulated several growth factors (Table 5). Insulin-like growth factor 2 (Igf2) is a major fetal growth factor that has an elevated expression in adult brain compared to most other tissues (Stylianopoulou et al., 1988). Treatment of MCAO rats with EW significantly up-regulated the expression of Igf2, which can promote cell survival (Lehtinen et al., 2011) and is required for memory consolidation (Lehtinen et al., 2011). Insulin-like growth factor binding protein 2 (Igfbp2) plays a dominant role in regulation of Igf1, a mitogen for microglia, in the central nerve system, although the exact function of Igfbp2 in Igf therapeutic strategy is not clear (Chesik et al., 2007). Granulin is a growth factor. Treatment of MCAO rat model with recombinant granulin suppressed neutrophil recruitment to ischemic-reperfusion (I/R) brain, leading to the reduction of NF-κB and MMP-9 activation (Egashira et al., 2013). Granulin is highly expressed in activated microglia but not in astrocytes and oligodendrocytes (Baker et al., 2006). Granulin has neurotrophic properties. While most nerve growth factors poorly have effect on PC12 cell line, progranulin as well as Igf1 and Igf2 promote growth of PC12 cells (Ahmed et al., 2007). Microglia sustain regular neuronal function and in injured CNS, play a role of neuroprotection and proregeneration (Hanisch and Kettenmann, 2007; Streit, 2002). Classically activated microglia (M1) produce destructive proinflammatory mediators, while alternatively activated microglia (M2) which express signature proteins such as Tgfβ1, go through phagocytosis and clear cell debris (David and Kroner, 2011; Hu et al., 2015). In the early stage of ischemic injury, M2 phenotype of microglia is predominant, but transition to M1 phenotype happens in the later stage of injury (Hu et al., 2012), suggesting that adjusting the balance of M1 and M2 phenotype may be beneficial for stroke therapies (Xia et al., 2015). Three distinct markers for microglia, i.e. CD68, Aif1, Csf1r and CX3CR1, were significantly upregulated upon treatment of MCAO rats model with ER, suggesting that EW treatment may induce the proliferation, activation and accumulation of microglia at stroke site. The expression of secreted enzymes such as cathepsins and complement components was further stimulated by EW in the MCAO rats, which may facilitate hydrolysis of extracellular macromolecules, and clearance of cell debris. Moreover, significantly upregulated Tgf-β1 after EW treatment of MCAO rats suggest that EW may specifically polarize microglia to M2 phenotype, which contribute to axonal remodeling, neural repair, neurogenesis, angiogenesis and neovascularization (Fig. 9).

Fig. 9. Neuroregenerative mechanism of Eerdun Wurile. EW upregulates the expression of microglia markers, growth factors and hydrolytic proteases in the lesion, leading to the activation, proliferation or polarization of microglia, which facilitates neuroregeneration, angiogenesis and phagocytosis in the lesion.

4 biological replicates) after 15 days. RNA-seq analysis revealed that the gene expression level in hippocampus of untreated group, MCAO group and MCAO + EW group did not show significant difference (data not shown). However, in the cerebral cortex gene expression differed dramatically between different groups of animals: 60 genes were differentially expressed in the MCAO group compared to untreated group, and a total of 186 genes were upregulated in the EW treated group compared to MCAO model group (Supplementary content Fig. S1, Fig. S2). Most significantly upregulated genes (log2ratio ≥2.0) include growth factors such as Igf2, Igfbp2, Tgf-β1, vimentin, and granulin, as well as microglia markers such as CD68, Aif1, Csf1r and complementary components such as C3 and C1qa (Table 5). The RNA-seq results were validated by RT-qPCR. The expression level of most upregulated genes in RNA-seq (ApoD, Grn, Igf2, Tgf-β1, Vim) was confirmed by qPCR. Remarkably, expression level of Igf2 was confirmed to be upregulated 12 fold in the cerebral cortex in EW treated MCAO rats (Fig. 7). To confirm the gene expression regulation of EW at protein level, we performed immunohistochemistry analysis of the infarction area of rat brain in all groups. To do this, we double stained the tissues with neuron marker (anti-NeuN antibody) and microglia marker (anti-Iba1 antibody), respectively, followed by staining with anti-Igf2 to determine the cell types that express Igf2. In neurons of NT group, IS group and NI group, basic level of Igf2 was detected. Significantly higher cell density was observed in EWhigh treated rats. Remarkably, significantly increased Igf2 expression in neurons was observed in the brain of rats treated with EW (Fig. 8A). Similar Igf2 expression pattern was also observed in microglia (Fig. 8B), suggesting that EW can significantly upregulate Igf2 expression in both neurons and microglia cells. 4. Discussion The clinically established neuro-recovery effect of EW possibly depends on the combined activity of the compounds that contained in the individual plant components, which have been report previously. Administration of EW to rat MCAO model effectively lowered neurological impairment, as assessed by Bederson scaling (Table 2). After 15 days of treatment, the overall activities of MCAO/R model treated with EW significant recovered. Bederson scaling showed remarkably dropped scale (2.16 before treatment and 0.21 after treatment, p < 0.01). Meanwhile, the microcirculation of MCAO rats treated with EW was enhanced in the affected area (221 PU to 310 PU) (Table 3, Fig. 3), suggesting that EW may contribute to the new blood vessel

5. Conclusions Our data collectively showed that the effective group of compounds in the traditional Mongolian medicine Eerdun Wurile significantly improves recovery from ischemic stroke by upregulating the expression of a group of genes including some growth factors in microglia cells in the lesion, potentially prompting a M1 to M2 polarization of microglia. Further studies on the identification of active compounds groups are underway in our laboratory.

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Acknowledgements

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This research was kindly supported by Inner Mongolia Plan of Science and Technology (201502109), Internal Funding from Research Institute of Mongolian Medicine of Inner Mongolia Autonomous Region (2016YJS31), Natural Science Foundation of Inner Mongolia Autonomous Region (2017MS0821), and funding from Department of Finance of Inner Mongolia Autonomous Region, China (CZT_201701). Author's contributions Saren Gaowa ([email protected]) and Lu Chen ([email protected]) performed animal experiments. Qiburi Qiburi ([email protected]) analyzed the chemical components. Tsogzolmaa Ganbold ([email protected]) performed IHC analysis. Altanzul Altangerel ([email protected]) contributed to RT-PCR analysis. Narisi Bao (narsu1988 @163.com) and Man Da ([email protected]) were involved in the data analysis. Temuqile Temuqile (tmqyx01@ 163.com) and Huricha Baigude ([email protected]) designed and analyzed the research. Huricha Baigude wrote the manuscript. All authors reviewed the results and approved the final version of the manuscript. Conflict of interest All authors report no conflicts of interest and no competing financial interests exist. Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.jep.2018.05.011. References Ablat, N., Lv, D., Ren, R., Xiaokaiti, Y., Ma, X., Zhao, X., Sun, Y., Lei, H., Xu, J., Ma, Y., Qi, X., Ye, M., Xu, F., Han, H., Pu, X., 2016. Neuroprotective effects of a standardized flavonoid extract from safflower against a rotenone-induced rat model of parkinson's disease. Molecules 21 (9). Ahmed, Z., Mackenzie, I.R., Hutton, M.L., Dickson, D.W., 2007. Progranulin in frontotemporal lobar degeneration and neuroinflammation. J. Neuroinflamm. 4, 7. Baker, M., Mackenzie, I.R., Pickering-Brown, S.M., Gass, J., Rademakers, R., Lindholm, C., Snowden, J., Adamson, J., Sadovnick, A.D., Rollinson, S., Cannon, A., Dwosh, E., Neary, D., Melquist, S., Richardson, A., Dickson, D., Berger, Z., Eriksen, J., Robinson, T., Zehr, C., Dickey, C.A., Crook, R., McGowan, E., Mann, D., Boeve, B., Feldman, H., Hutton, M., 2006. Mutations in progranulin cause tau-negative frontotemporal dementia linked to chromosome 17. Nature 442 (7105), 916–919. Bederson, J.B., Pitts, L.H., Tsuji, M., Nishimura, M.C., Davis, R.L., Bartkowski, H., 1986. Rat middle cerebral artery occlusion: evaluation of the model and development of a neurologic examination. Stroke 17 (3), 472–476. Brock, K., Haase, G., Rothacher, G., Cotton, S., 2011. Does physiotherapy based on the Bobath concept, in conjunction with a task practice, achieve greater improvement in walking ability in people with stroke compared to physiotherapy focused on structured task practice alone? A pilot randomized controlled trial. Clin. Rehabil. 25 (10), 903–912. Brown, J.A., 2006. Recovery of motor function after stroke. Prog. Brain Res. 157, 223–228. Chesik, D., De Keyser, J., Wilczak, N., 2007. Insulin-like growth factor binding protein-2 as a regulator of IGF actions in CNS: implications in multiple sclerosis. Cytokine Growth Factor Rev. 18 (3–4), 267–278. Chesik, D., Glazenburg, K., Wilczak, N., Geeraedts, F., De Keyser, J., 2004. Insulin-like growth factor binding protein-1-6 expression in activated microglia. Neuroreport 15 (6), 1033–1037. David, S., Kroner, A., 2011. Repertoire of microglial and macrophage responses after spinal cord injury. Nat. Rev. Neurosci. 12 (7), 388–399. Deininger, M.H., Seid, K., Engel, S., Meyermann, R., Schluesener, H.J., 2000. Allograft inflammatory factor-1 defines a distinct subset of infiltrating macrophages/microglial cells in rat and human gliomas. Acta Neuropathol. 100 (6), 673–680. Donnan, G.A., Fisher, M., Macleod, M., Davis, S.M., 2008. Stroke. Lancet 371 (9624), 1612–1623. Egashira, Y., Suzuki, Y., Azuma, Y., Takagi, T., Mishiro, K., Sugitani, S., Tsuruma, K., Shimazawa, M., Yoshimura, S., Kashimata, M., Iwama, T., Hara, H., 2013. The growth factor progranulin attenuates neuronal injury induced by cerebral ischemiareperfusion through the suppression of neutrophil recruitment. J. Neuroinflamm. 10, 105.

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