Effects of Angelica Extract on Schwann Cell Proliferation ...

Research ArticleEffects of Angelica Extract on Schwann Cell Proliferation andExpressions of Related Proteins

Xiaowen Jiang1 Lin Liu1 Binqing Zhang1 Ziyin Lu1 Lu Qiao1

Xinxin Feng1 andWenhui Yu12

1College of Veterinary Medicine Northeast Agricultural University Harbin 150030 China2Key Laboratory of the Provincial Education Department of Heilongjiang for Common Animal Disease Prevention and TreatmentNortheast Agricultural University Harbin 150030 China

Correspondence should be addressed to Wenhui Yu yuwenhuineaueducn

Received 12 March 2017 Accepted 25 May 2017 Published 18 July 2017

Academic Editor Raffaele Capasso

Copyright copy 2017 Xiaowen Jiang et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The present study investigated the effects ofAngelica extract (AE) on Schwann cell proliferation and expressions of related proteinsincluding brain derived neurotrophic factor (BDNF) neural cell adhesionmolecule (NCAM) and proliferating cell nuclear antigen(PCNA) Proliferation activity and cell cycles of SCs were evaluated by MTT assay and flow cytometry methods respectively after12 h treatment of AE at different concentrations (625 125 250 1000 2000 4000 and 8000mgL) SCs were treated by 500 1000and 2000mgL AE for 24 h or 48 h the related genes mRNA and proteins expressions in SCs were detected by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) kit At theconcentration range of 125ndash2000mgL the SC proliferation was induced by AE in a dose-dependent manner especially 1000 and2000mgL cells in drug-treated groups showed the most increase Cells counts were ascended significantly in (G2M + S) phasecompared to control group BDNF NCAM and PCNA protein expressions significantly increased at drug-treated groups Relativegenes mRNA expressions levels were also significantly higher compared to control group The results indicated that AE facilitatedSC proliferation and related genes and proteins expressions which provided a basic guideline for nerve injury repair in clinic

1 Introduction

Schwann cells (SCs) are unique glial cells in the peripheralnervous system and they play vital roles in generationdevelopment morphology maintaining function and otheraspects of peripheral nerves [1] SCs are important for theregeneration and repairment of peripheral nerve injury [2]When distal site of injured nerve witnessed Waller degen-eration SCs began to proliferate and then participated inswallowing degeneration of axons and myelin debris form-ing a longitudinal continuous cell cord (Bngnerrsquos band)which guided the growth of regenerating axons SCs areessential for regeneration of microenvironment [3] whichis the important condition for neuronal survival and neu-rite growth SCs have nutrition effects on neuron due tosynthetic neurotrophic factors and they also contribute togenerating neurite growth factors [4] SCs secrete a varietyof neurotrophic factors such as nerve growth factor (NGF)

fibroblast growth factor (FGF) BDNF NCAM PCNA andciliary neurotrophic factor (CNTF) These factors play majorroles in peripheral nerve cell growth development regener-ation and maintaining normal nerve cells alive and they arebeneficial to axons regeneration and myelination [5 6] Lackof neurotrophic factors may cause neurological diseases andfailure of nerve regeneration [7]

Angelica Umbelliferae Angelica sinensis (Oliv) Dielsdried roots is regarded as a traditional natural medicinefor invigorating the circulation of blood Currently manyresearches showed that Angelica had a variety of pharma-cological effects including anti-inflammatory [8] anticancer[9] wound healing [10] and nerve regeneration [11] effectsNerve injury is a common clinical disease as the researchon the treatment of the disease gradually increases moreand more natural plants were applied in peripheral nerverepair It was reported that natural plants showed fewerside effects and minimize drug resistance [12] Some studies

HindawiEvidence-Based Complementary and Alternative MedicineVolume 2017 Article ID 6358392 9 pageshttpsdoiorg10115520176358392

2 Evidence-Based Complementary and Alternative Medicine

have demonstrated the effects of Angelica on nerve repairIt was also reported that AE attenuated neuropathic painwhich is associated with proinflammatory cytokines suchas tumor necrosis factor-120572 (TNF-120572) interleukin-1120573 (IL-1120573)and interleukin-6 (IL-6) TRPV1 and p-ERK in peripheralnervous pain systems [13] Some findings suggested thatAngelica injection improved the sciatic nerve crush injuryand the mechanism might be through the increase of BDNFand NGF protein expressions [14] Angelica dahuricae radixdecreased the levels of TNF-120572 IL-1120573 IL-6 inducible nitricoxide synthase (iNOS) and cyclooxygenase-2 (COX-2) ina lipopolysaccharide- (LPS-) activated microglial cell lineand provided neuroprotection by alleviating inflammationand oxidative stress [15] Another research elucidated thatAE promoted PC12 cell proliferation in vitro assay [16]The AE contains volatile oil organic acids polysaccharidesflavonoids and other ingredients [17]These findings showedthat Angelica exerted a positive-effect in nerve injury repairHowever the studies of AE in vitro assays are not commonand particularly effects of AE on SCs (important peripheralnerve cells) have not been reported In the present studywe investigated the effects of AE on SC proliferation andcycle protein and mRNA expressions of BDNF NCAM andPCNA which are related to peripheral nerve repair [18ndash20]The detection of these neural factors in AE-treated SCs willprovide a mechanistic framework for further studies of theuse of AE as an effective treatment for peripheral nerve injury

2 Materials and Methods

21 Equipment and Reagents HB050 type inverted phasecontrast microscope was manufactured by Zeiss companyK330 refrigerated centrifuge was manufactured by SigmaCorporation iMark550 microplate reader was manufacturedby Bio-Rad company BD flow cytometer was manufacturedby American companies GeneQuant 1300 spectrophotome-ter was manufactured by General Electric Company Light-Cycler 96 was produced by Roche in Switzerland

AE was purchased from Baoji FangSheng BiologicalDevelopment Co Ltd Rat SCs (RSC96) were obtainedfrom China Cell Line Repository Dulbecco Modified EagleMedium (DMEM) was purchased from HyClone companyFetal Bovine Serum (FBS) was purchased from Sijiqingcompany in ChinaMTT and Propidium Iodide (PI) reagentswere purchased from American Sigma company Rat BDNFNCAM and PCNA-ELISA kits were produced by BeyotimeInstitute of Biotechnology in JiangSu First-strand cDNA syn-thesis kit was purchased from Thermo Scientific CompanyAll primers were designed and synthesized by InvitrogenBiotechnology Co Ltd (Shanghai China)

22 Cell Toxic Assays The fifth passage of SCs was usedfor experiments and the trypsinized cells were diluted withDulbecco Modified Eagle Medium (DMEM) containing 10Fetal Bovine Serum (FBS) to cell suspension at a concen-tration of 50 times 104 cellsmL SCs were seeded in 96-wellculture plates and 200 120583L cell suspension was added to everywell Cells were incubated at 37∘C under 5 CO2 for 24 hthen the medium was removed and DMEM without FBS

was added per well and cells were hungered for 12 h andwere randomly divided into control group and drug-treatedgroups The control group and drug-treated groups werecultured dividedly by DMEM containing 10 FBS without orwith the medium containing indicated concentrations (625125 250 1000 2000 4000 and 8000mgL) of AE for 12 h at37∘C Cell viability was measured by MTT assay Five parallelwells were set up for a group and theMTT assay was repeated3 times

23 The Effects of AE on Proliferation of SCs SCs growth wasinterfered with by AE with the indicated concentration thatincreased SC proliferation positively SCs treated by DMEMcontaining 10 FBS acted as control and all groups are setup with 7 parallel wells The experiments were performed forduplicating at least three times Cells were cultured for 12 h24 h 36 h and 48 h The effects of AE on cell proliferationwere evaluated by MTT assay as previously described

24 The Effects of AE on the Cell Cycles of SCs SCs inlogarithmic phase were trypsinized and centrifuged SCswere diluted with DMEM containing 10 FBS and seededin 25mm3 glass screw-cap cell bottle at a density of 20 lowast105 cellsmL After 24 h of incubation at 37∘C the mediumwas removed and then cells were treated with DMEM for12 h In the drug-treated groups AE mixed with DMEMcontaining FBS was added to every well at a concentrationthat promoted cell proliferation and the cells in control groupwere only treated byDMEMcontaining FBSThe supernatantwas discarded after incubating for 24 h and 48 h The cellcycle of each group was detected by flow cytometry ModfitLT software was used for analyzing cell cycle of each groupThe experiments were performed for repeating at least threetimes

25 Effects of AE on Expressions of BDNF NCAM andPCNA in SCs SCs growths were interfered with by AEwith the concentrations which promoted SC proliferationand then cells were cultured for 24 h and 48 h respectivelyThen the number of SCs was counted and the culturesupernatants were collected to determine the amount ofBDNF NCAM and PCNA secreted by the cultured SCs Cellculture supernatants were centrifuged and assayed by usingan ELISA kit following the manufacturerrsquos instructions cellswere trypsinized and cell supernatantswere treated accordingto manufacturerrsquos instruction of ELISA The protein sampleswere stored at minus80∘C until being assayed The samples wereused to measure BDNF NCAM and PCNAThe experimentwas repeated three times

26 Effects of AE on Gene Expression of BDNF NCAM andPCNA SCs were treated by AE for 48 h at concentrationsof 500 1000 and 2000mgL respectively Total RNA wasisolated from SCs using TRIzol reagent according to themanufacturerrsquos instructions The RNA concentration wasdetermined using a GeneQuant 1300 spectrophotometer andthe purity of RNA was determined using the 260280 nmabsorbance ratio The 119860260119860280 ratio of the RNA samples

Evidence-Based Complementary and Alternative Medicine 3

Table 1 Primers used for RT-PCR

Gene Serial Primer (51015840rarr31015840) Product size

BDNF NM 0012706381 Forward CGGTATCAAAAGGCCAACTGReverse GTAGTTCGGCATTGCGAGTT 121 bp

NCAM NM 0315211 Forward AACGGACTCCAAACCATGACReverse TGGCTTTGCTTCTGACTCCT 122 bp

PCNA NM 0223813 Forward TTGGAATCCCAGAACAGGAGReverse TTTGCACAGGAGATCACCAC 115 bp

120573-Actin NM 0311443 Forward TGTCACCAACTGGGACGATAReverse GGGGTGTTGAAGGTCTCAA 165 bp

was 18ndash20 First-strand cDNA was synthesized from 5 120583gof total RNA according to the manufacturerrsquos instructionsAll primers were designed and synthesized by InvitrogenBiotechnology Co Ltd The primers used are shown inTable 1

RT-PCR was performed using a LightCycler 96 Theannealing temperatures and the thermocycling conditions forthe target genes were as follows BDNF (579∘C 35 cycles)NCAM (579∘C 35 cycles) PCNA (579∘C 35 cycles) and 120573-actin (59∘C 35 cycles) The standard PCR conditions wereas follows 94∘C for 30 s and 45 cycles of 94∘C for 5 s avariable annealing temperature for 15 s and 72∘C for 10 sThe experiments were performed in triplicate and repeatedat least three times Mean Ct values were used to calculatethe relative expression levels of the target genes for theexperimental groups relative to those in the negative controlgroup The relative expression of target genes was obtainedusing the 2minusΔΔCt formula using 120573-actin as a housekeepinggene

27 Statistical Analyses All data were presented as meanvalues plusmn standard deviation and statistical analysis wasperformed using a SPSS 130 statistical package The multiplecomparisons of data were performed by one-way ANOVAfollowed by Dunnettrsquos test Significant differences weredefined at values of119875 lt 005 and extreme significant differen-ces were defined at values of 119875 lt 001

3 Results

31 AE Stimulated SC Proliferation at Indicated ConcentrationSC proliferation is important for the healing of nerve injuryCell toxic assays were preformed to explore effects of AE onSC proliferation According to Figure 1 result of cell toxicassay showed that after treatment with different concentra-tions of AE the concentration range of AE which was con-ducive to the growths of SCs was 125ndash2000mgL and showeddose-dependent manner and when concentration of AE waslower than 125mgL the SCs growths in drug-treated groupsand control group had no significant differences Howeverwhen AE concentration was higher than 4000mgL SC pro-liferation was inhibited significantly compared to the controlgroup (119875 lt 005) Therefore these findings indicated thatAE stimulated SC proliferation at appropriate concentrationrange and the optimal concentration was 1000mgL

6250

0

1

2

3

4

125 250 500Groups (mgL)

1000 2000 4000 8000

Abso

rban

ce (D

490n

m)

lowastlowast

lowastlowast

lowastlowastlowastlowast


Figure 1 Cell toxic effects of AE on SCs (119899 = 6 plusmns) Thisphotograph showed SC viability after 12 h treatment with differentconcentrations of AE DMEM with 10 FBS was used as controlNotes lowast119875 lt 005 lowastlowast119875 lt 001 versus the control group (Studentrsquos119905-test)

32 AE Promoted SC Proliferation at Different Time PointsThrough the cytotoxicity test the effective concentrationsof AE were determined To further study the effects on SCproliferation at different time points with treatment of AEcell proliferation test was performed As shown in Figure 2when AE concentration was in the 250ndash2000mgL range AEpromoted SC proliferation after treating for 24 h 48 h and72 h at a concentration of 500ndash2000mgL cell proliferationin different groups was significantly increased by comparisonwith control group (119875 lt 005) when the concentrationswere 1000 and 2000mgL drug-treated groups showed theextremely significant difference compared to control group(119875 lt 001)

33 The Effects of AE on the Cell Cycles of SCs We examinedthe cell cycle in AE-treated SCsTheG2 phase is the late stageof DNA synthesis and the M phase is the stage of mitosisand they all reflect the state of cell proliferation to someextent As shown in Figures 3 and 4 SCs were treated by500 1000 and 2000mgL concentrations of AE for 24 h or48 h compared with control group and the results showedthat cells percentage composition had differences in G0G1S and G2M phase respectively Moreover we found that


00

1

2

3

4

12Time after AE (h)

24

Control

36 48

Abso

rban

ce (D

490n

m) lowastlowast

lowastlowast

lowastlowast

lowastlowast∙∙

∙∙

∙∙∙∙

250mgL500mgL

1000 mgL2000 mgL

Figure 2 Drug concentration and application time effects onSC proliferation (119899 = 12 plusmns) The viability of SC at 0 h 12 h24 h 36 h and 48 h was assessed after being treated with differentconcentrations (250sim2000mgmL) of AE Notes lowastlowast∙∙119875 lt 001versus control group at the same time (Studentrsquos 119905-test)

the proliferation index (PI = S + G2M) was significantlyincreased The percentage of drug-treated cells in (G2M +S) phase was higher than control group (119875 lt 001) and thisfinding showed that AE activated synthesis of DNA whichleads to cell proliferation (Figure 5)

34 AE Increased BDNF NCAM and PCNA Protein Expres-sions in SCs Expressions of BDNF NCAM and PCNAprotein are important for regeneration of nerve TheELISA assays were performed When AE concentration was1000mgL BDNF and PCNA contents in cells were checkedafter incubating for 24 h and the results showed significantdifference compared with control group (119875 lt 005) theexpression of PCNA in cells cultured for 48 h were detectedand these results revealed that drug-treated cells expressedmore PCNA protein compared with cells in control group(119875 lt 001) 2000mgL of AE-treated cells for 24 h and expres-sions of BDNF and PCNA were extremely significant higherthan control group (119875 lt 001) NCAM was upregulatedsignificantly compared with the control group (119875 lt 005)After incubation of 48 h expressions of BDNF NCAM andPCNA increased obviously compared with the control group(119875 lt 001) and all were shown in Figure 6

35 AE Promoted Relative Genes Expressions in SCs To fur-ther examine the effects of AE on BDNF NCAM and PCNAgene expressions were checked 500 1000 and 2000mgLof AE interfered with cells growths for 48 h RNA puritywas detected and generally the extracted RNA are availablewhen the detection results belong to 18ndash20 As shown inFigure 7 AE at concentrations of 500 1000 and 2000mgL alleffectively promoted the gene expressions of BDNF NCAMand PCNA compared with control group (119875 lt 001) and

expressions of these genes had the most significant increasewhen concentration of AE was 1000mgL (119875 lt 001)

4 Discussion

For the regeneration of injured peripheral nervous system(PNS) SCs play critical roles during this period via thesynergetic effects with macrophages and neurons (repairinginjured peripheral nerves bridging the gap) Researchesshowed that in mammals after peripheral nerve injury SCsgreatly reduce the expression of myelin [21ndash24] At the firstday after peripheral nervous damage SCs digest intracellularmyelin debris in the cavity to make the extracellular myelindebris exposure [25ndash27] which facilitate the macrophagephagocytosis and finally promote the axon regeneration [28]Therefore the proliferation of SC is crucial for the repairof peripheral nerve injury in our experiments When theAE concentration was at 250ndash2000mgmL AE significantlyenhanced the proliferation of SCs However DP inhibitedcell proliferation obviously when the drug concentrationexceeded 4000mgL Many researches had demonstrated theextracts of natural plants can accelerate cell proliferationNormally they play positive roles when they treat cells withinan optimal concentration range However negative effects ofthe same extracts are often observed when the drug-treatedconcentration is too high [29 30] In order to further test theeffects of AE on SCs cycles SCs were treated with differentconcentrations of AE in vitro AE at 500 1000 and 2000mgLpromoted the proliferation of SCs significantly respectivelyand elevated the cells counts in S phase and G2M phaseIn SCs cells in G0G1 phase were decreased at the sametime cells in S phase and G2M phase increased comparedwith control group The mechanism of how AE promotedSC proliferation can be inferred AE shortened the growthretardation stimulate the cell to enter the active S phase fromthe stationary G0 phase and increased the number in G2Mcells which will lead to the proliferation of cells [31]

Neural factors such as BDNF NCAM and PCNA playvery important roles for the peripheral nerve cell growthdevelopment and regeneration and maintain the survival ofthe nerve cells under normal condition BDNF can promoteregeneration of peripheral nerve protect the damaged neu-rons and nerve cells andmaintain sensitivity to neurons [32]BDNF also regulate and promote synthesis of adhesion inSCs [33 34] and together BDNF and neurotrophic tyrosinereceptor kinase type 2 (Ntrk2) are capable of activating theadhesion angiogenesis apoptosis and proliferation path-ways [35] BDNF and NGF play essential roles in centralnervous system [36] NCAM is a kind of immunoglobulinsecreted by SCs and it also has a close relation to the axonregeneration for example combining L1 to form L1-NCAMcomplexes which enhanced biological functions such asidentification and adhesion [37 38] NCAM is a signal of glialcell-derived neurotrophic factor (GDNF) family receptorsGDNF activate NCAM and intracellular Fyn and FAK andpromote the growth of nerve cells [28] PCNA protein isessential for cell DNA synthesis which is closely related tocell proliferation and can effectively reflect the activity of cellproliferation [39] PCNA is a unique intranuclear protein


800

200

400

600

00 50 250100 150 200

Dip G0-G1Dip G2-MDip S

Num

ber

Channels (PI-A)

(a) Control

800

200

400

600

0

Num

ber

2500 50 100 150 200Channels (PI-A)


(b) 500mgL

200

400

600

0

Num

ber

0 50 250100 150 200Channels (PI-A)


(c) 1000mgL

Num

ber

800

200

400

600

00 50 250100 150 200

1000

Channels (PI-A)


(d) 2000mgL

Figure 3 Different concentrations of AE exerted an influence on the SCs cycles (24 h) SCs were gathered at 24 h after being treated with 0500 1000 and 2000mgmL AE and then SCs cycles were detected using flow cytometry in sequence The results were shown respectivelyin (a) (b) (c) and (d)

which plays a key role in DNA synthesis damage healingand regulation of cell cycle and usually is regarded as cellproliferation index [40] Expressions of BDNF NCAM andPCNA in SCs treated by AE significantly increased comparedto control group and increased mRNA expressions of thosewere detected by RT-PCR BDNF plays an essential rolein promoting axonal regeneration and remyelination whenSCs were transplanted into nerve injury lesions [41] The

overexpression of NCAM promotes neurite outgrowth and isimplicated in myelination [42] The results of PCNA proteinupregulation were consistent with SC proliferation assayThese results showed that AE played an underlying role inperipheral nerve repair through promoting SC proliferationand stimulating SC to secrete neurotrophic factors Howeverthe active ingredients of Angelica such as polysaccharidessodium ferulate and volatile oil need to be further studied


Num

ber

200

400

600

00 50 100 150 200 250

Channels (PI-A)


(a) ControlN

umbe

r

60

120

180

00 50 100 150 200 250

240

Channels (PI-A)


(b) 500mgL

Num

ber

00 50 100 150 200 250

100

200

300

400

500

600

Channels (PI-A)


(c) 1000mgL

0 50 100 150 200 250

Num

ber

0

100

200

300

400

500

600

Channels (PI-A)


(d) 2000mgL

Figure 4 Different concentrations of AE exerted an influence on the SCs cycles (48 h)SCs were gathered at 48 h after being treated with 0500 1000 and 2000mgmL AE and then SCs cycles were detected using flow cytometry in sequence The results were shown respectivelyin (a) (b) (c) and (d)

5 Conclusion

In this work we demonstrated that AE promoted SC prolifer-ation furthermore the results of cell cycle detection showedAE increased significantlyDNApercentage in (G2 + S) phaseSimultaneously BDNF NCAM and PCNA protein expres-sions in SC significantly enhanced with the treatment of AEfor 24 h or 48 h Gene expressions of BDNF NCAM and

PCNA also were upregulated markedly after drug treatmentAlthough the effects of AE on nerve repair have been alreadyreported inmany researches we first studied the effects of AEon SC proliferation and cycle moreover many neurologicalfactors which are related to nerve regeneration increasedwithAE treatment Taken together all of these results suggestedpotential application of AE in the clinical therapy of nerveinjury


Control 5000

20

40

60

80

Perc

enta

ge o

f cel

ls (

)

1000Groups (mgL)

2000

24 h G0G1 phase24 h S phase24 h G2M phase


lowastlowast

lowastlowastlowast


lowastlowast

Figure 5 Effects of different concentrations ofAEon SCs cycles (119899 =5plusmns )Modfit LT softwarewas used for analyzing cell cycle of eachgroup after 24 or 48 h treatment with 500 1000 and 2000mgmL ofAE Notes lowast119875 lt 005 and lowastlowast119875 lt 001 as compared with the controlgroup (Studentrsquos 119905-test)

Control 50000

05

10

Prot

ein

cont

ent

15

20

25

1000 2000Groups (mgL)

휕휕 휕휕

lowastlowast lowastlowastlowast

∙∙ ∙∙ ∙∙

radicradic

BDNF 24 hBDNF 48 h

NCAM 48 h

NCAM 24 hPCNA 24 hPCNA 48 h

㶋㶋

Figure 6 Effects of different concentrations of AE on expressions ofthe relative secretion cytokine of SCs (119899 = 5plusmns) BDNFNCAM andPCNA expressions were detected by ELISA kits at 24 and 48 h afterstimulation without or with indicated concentrations of AE Notessingle symbol such as ldquolowastrdquo means 119875 lt 005 compared with controldouble symbols such as ldquolowastlowastrdquomean119875 lt 001 compared with control

Conflicts of Interest

The authors declare that they have no financial and personalrelationships with other people or organizations that caninappropriately influence their work and there is no profes-sional or other personal interest of any nature or kind in anyproduct service or company

0

BDNFNCAMPCNA

Control 500 1000

Groups (mgL)2000

2

4

6

8

Relat

ive m

RNA

expr

essio

n le

vels

lowastlowast

lowastlowast

lowastlowast

∙∙

∙∙

∙∙

Figure 7 Effects of different concentrations of AE on mRNAexpressions of BDNF NCAM and PCNA in SCs (119899 = 4 plusmns) AEinterfered with SCs growths for 48 h at concentrations of 500 1000and 2000mgL respectively gene expressions of BDNF NCAMand PCNA were checked by RT-PCR Results were presented as thefolds change compared with untreated cells Notes lowast∙119875 lt 005comparedwith control group lowastlowast∙∙119875 lt 001 comparedwith controlgroup (Studentrsquos 119905-test) ldquolowastrdquo and ldquo∙rdquo refer to differences in each geneexpression between control group and drug-treated groups

Authorsrsquo Contributions

Xiaowen Jiang and Lin Liu contributed equally to this workWenhui Yu and Xiaowen Jiang designed experiments Lin Liuand Xiaowen Jiang carried out experiments mainly BinqingZhang and Lu Qiao analyzed experimental results Ziyin LuLin Liu and Xinxin Feng wrote the manuscript together

Acknowledgments

The authors thank Professor Li for her equipment andinstrument support They also express heartfelt thanks toDr Shi for assistance with the experiments and valuablediscussion

References

[1] U Namgung ldquoThe role of schwann cell-axon interaction inperipheral nerve regenerationrdquo Cells Tissues Organs vol 200no 1 pp 6ndash12 2014

[2] A Cattin ldquoMacrophage-induced blood vessels guide schwanncell-mediated regeneration of peripheral nervesrdquo Cell vol 162no 5 pp 1127ndash1139 2015

[3] F Liu H W Zhang K M Zhang X Y Wang S P Li and Y XYin ldquoRapamycin promotes Schwann cell migration and nervegrowth factor secretionrdquo Neural Regeneration Research vol 9no 6 pp 602ndash609 2014

[4] A M Avellino D Hart A T Dailey M Mackinnon DEllegala and M Kliot ldquoDifferential macrophage responses inthe peripheral and central nervous system during wallerian


degeneration of axonsrdquo Experimental Neurology vol 136 no 2pp 183ndash198 1995

[5] D J Carey and R P Bunge ldquoFactors influencing the release ofproteins by cultured Schwann cellsrdquo Journal of Cell Biology vol91 no 3 part 1 pp 666ndash672 1981

[6] H Fansa T Dodic G Wolf W Schneider and G KeilhoffldquoTissue engineering of peripheral nerves epineurial grafts withapplication of cultured Schwann cellsrdquoMicrosurgery vol 23 no1 pp 72ndash77 2003

[7] Y Y Choi M H Kim J Hong K Kim andWM Yang ldquoEffectofDangguibohyul-Tang a Mixed Extract ofAstragalus mem-branaceusandAngelica sinensis on allergic and inflammatoryskin reaction compared with single extracts of astragalus mem-branaceusor angelica sinensisrdquo Evidence-Based Complementaryand Alternative Medicine vol 2016 pp 1ndash9 2016

[8] J Xu E Xiao-Qiang H-Y Liu J Tian and J-L Yan ldquoAngelicasinensis attenuates inflammatory reaction in experimental ratmodels having spinal cord injuryrdquo International Journal ofClinical and Experimental Pathology vol 8 no 6 pp 6779ndash6785 2015

[9] S Zheng W Ren and L Huang ldquoGeoherbalism evaluation ofRadix Angelica sinensis based on electronic noserdquo Journal ofPharmaceutical and Biomedical Analysis vol 105 pp 101ndash1062015

[10] C-Y Hsiao C-Y Hung T-H Tsai and K-F Chak ldquoA studyof the wound healing mechanism of a traditional chinesemedicine Angelica sinensis using a proteomic approachrdquoEvidence-Based Complementary and Alternative Medicine vol2012 Article ID 467531 14 pages 2012

[11] R Li J Zhang L Zhang Q Cui andH Liu ldquoAngelica injectionpromotes peripheral nerve structure and function recoverywithincreased expressions of nerve growth factor and brain derivedneurotrophic factor in diabetic ratsrdquo Current NeurovascularResearch vol 7 no 3 pp 213ndash222 2010

[12] J Wang X-Y Qiao and F Lin ldquoPresent situation and devel-opment strategies of Chinese medicine preparation in medicalinstitutionsrdquo China Journal of Chinese Materia Medica vol 40no 21 pp 4117ndash4121 2015

[13] R Li C Zhao M Yao Y Song Y Wu and A Wen ldquoAnal-gesic effect of coumarins from Radix angelicae pubescentis ismediated by inflammatory factors and TRPV1 in a spared nerveinjury model of neuropathic painrdquo Journal of Ethnopharmacol-ogy vol 195 pp 81ndash88 2017

[14] Q Cui J Zhang L Zhang R Li andH Liu ldquoAngelica injectionimproves functional recovery and motoneuron maintenancewith increased expression of brain derived neurotrophic factorand nerve growth factorrdquo Current Neurovascular Research vol6 no 2 pp 117ndash123 2009

[15] Y J Moon J Y Lee M S Oh et al ldquoInhibition of inflamma-tion and oxidative stress by Angelica dahuricae radix extractdecreases apoptotic cell death and improves functional recoveryafter spinal cord injuryrdquo Journal of Neuroscience Research vol90 no 1 pp 243ndash256 2012

[16] MH Jeong J S Kim Y Zou et al ldquoEnhancement of pheochro-mocytoma nerve cell growth by consecutive fractionization ofAngelica gigas Nakai extractsrdquoCytotechnology vol 62 no 5 pp461ndash472 2010

[17] S Gnavi B Elena Fornasari C Tonda-Turo et al ldquoThe effect ofelectrospun gelatin fibers alignment on schwann cell and axonbehavior and organization in the perspective of artificial nervedesignrdquo International Journal of Molecular Sciences vol 16 no6 pp 12925ndash12942 2015

[18] S Geuna S Raimondo S Nicolino et al ldquoSchwann-cellproliferatin in muscle-vein combined conduits for bridging ratsciatic nerve defectsrdquo Journal of Reconstructive Microsurgeryvol 19 no 2 pp 119ndash123 2003

[19] S Han B Wang W Jin et al ldquoThe collagen scaffold with colla-gen binding BDNF enhances functional recovery by facilitatingperipheral nerve infiltrating and ingrowth in canine completespinal cord transectionrdquo Spinal Cord vol 52 no 12 pp 867ndash873 2014

[20] F Klaiber B Schonfeld and G Kostorz ldquoBrief transvertebralelectrical stimulation of the spinal cord improves the specificityof femoral nerve reinnervationrdquoNeurorehabilitation andNeuralRepair vol 27 no 3 pp 260ndash268 2013

[21] C De Felipe and S P Hunt ldquoThe differential control of c-junexpression in regenerating sensory neurons and their associatedglial cellsrdquo Journal of Neuroscience vol 14 no 5 part 1 pp 2911ndash2923 1994

[22] B D Trapp P Hauer and G Lemke ldquoAxonal regulation ofmyelin protein mRNA levels in actively myelinating Schwanncellsrdquo Journal of Neuroscience vol 8 no 9 pp 3515ndash3521 1988

[23] P J Arthur-Farraj M Latouche D K Wilton et al ldquoc-Junreprograms Schwann cells of injured nerves to generate a repaircell essential for regenerationrdquo Neuron vol 75 no 4 pp 633ndash647 2012

[24] K R Jessen and RMirsky ldquoNegative regulation of myelinationrelevance for development injury and demyelinating diseaserdquoGlia vol 56 no 14 pp 1552ndash1565 2008

[25] M H Han Y J Piao D W Guo and K Ogawa ldquoThe role ofSchwann cells and macrophages in the removal of myelin dur-ingWallerian degenerationrdquo Acta Histochemica Et CytochemicaOfficial Journal of the Japan Society of Histochemistry ampampCytochemistry vol 22 no 2 pp 161ndash172 1989

[26] K Hirata and M Kawabuchi ldquoMyelin phagocytosis bymacrophages and nonmacrophages duringWallerian degenera-tionrdquoMicroscopy Research and Technique vol 57 no 6 pp 541ndash547 2002

[27] H Mei Liu L H Yang and Y J Yang ldquoSchwann cell prop-erties 3 U-fos expression bFGF production phagocytosisand proliferation during wallerian degenerationrdquo Journal ofNeuropathology and Experimental Neurology vol 54 no 4 pp487ndash496 1995

[28] G Paratcha F Ledda and C F Ibanez ldquoThe neural celladhesion molecule NCAM is an alternative signaling receptorfor GDNF family ligandsrdquoCell vol 113 no 7 pp 867ndash879 2003

[29] J W Ren K M Chan P K K Lai et al ldquoExtracts from RadixAstragali and Radix Rehmanniae promote keratinocyte prolif-eration by regulating expression of growth factor receptorsrdquoPhytotherapy Research vol 26 no 10 pp 1547ndash1554 2012

[30] B H I Ruszymah S R Chowdhury N A B A Manan OS Fong M I Adenan and A B Saim ldquoAqueous extract ofCentella asiatica promotes corneal epithelium wound healingin vitrordquo Journal of Ethnopharmacology vol 140 no 2 pp 333ndash338 2012

[31] CWebber and D Zochodne ldquoThe nerve regenerative microen-vironment early behavior and partnership of axons andSchwann cellsrdquo Experimental Neurology vol 223 no 1 pp 51ndash59 2010

[32] Y Takemura S Imai H Kojima et al ldquoBrain-derived neu-rotrophic factor from bone marrow-derived cells promotespost-injury repair of peripheral nerverdquo PLoS ONE vol 7 no9 Article ID e44592 2012


[33] MMeyer I Matsuoka CWetmore L Olson and HThoenenldquoEnhanced synthesis of brain-derived neurotrophic factor inthe lesioned peripheral nerve differentmechanisms are respon-sible for the regulation of BDNF and NGF mRNArdquoThe Journalof Cell Biology vol 119 no 1 pp 45ndash54 1992

[34] C Geral A Angelova and S Lesieur ldquoFrommolecular to nan-otechnology strategies for delivery of neurotrophins emphasison brain-derived neurotrophic factor (BDNF)rdquo Pharmaceuticsvol 5 no 1 pp 127ndash167 2013

[35] J MWessels L Wu N A Leyland HWang andW G FosterldquoThe brain-uterus connection brain Derived NeurotrophicFactor (BDNF) and its receptor (Ntrk2) are conserved in themammalian uterusrdquo PLoS ONE vol 9 no 4 Article ID e940362014

[36] S Coskun S Varol H H Ozdemir E Agacayak B Aydın OKapan et al ldquoAssociation of brain-derived neurotrophic factorand nerve growth factor gene polymorphisms with susceptibil-ity tomigrainerdquoNeuropsychiatric Disease and Treatment vol 12no 1 pp 1779ndash1785 2016

[37] M Dezawa K Kawana and E Adachi-Usami ldquoThe role ofSchwann cells during retinal ganglion cell regeneration inducedby peripheral nerve transplantationrdquo Investigative Ophthalmol-ogy amp Visual Science vol 38 no 7 pp 1401ndash1410 1997

[38] R Kleene C Cassens R Bahring et al ldquoFunctional conse-quences of the interactions among the neural cell adhesionmolecule NCAM the receptor tyrosine kinase TrkB and theinwardly rectifying K+ channel KIR33rdquo Journal of BiologicalChemistry vol 285 no 37 pp 28968ndash28979 2010

[39] S Bhattacharya N Muhammad R Steele G Peng and RB Ray ldquoImmunomodulatory role of bitter melon extract ininhibition of head and neck squamous cell carcinoma growthrdquoOncotarget vol 7 no 22 pp 33202ndash33209 2016

[40] X Xu C Yan B R Kossmann and I Ivanov ldquoSecondary inter-action interfaces with PCNA control conformational switchingof DNA polymerase PolB from polymerization to editingrdquoJournal of Physical Chemistry B vol 120 no 33 pp 8379ndash83882016

[41] C Tep M L Kim L I Opincariu et al ldquoBrain-derivedneurotrophic factor (BDNF) induces polarized signaling ofsmall GTPase (Rac1) protein at the onset of schwann cellmyelination through partitioning-defective 3 (Par3) proteinrdquoThe Journal of Biological Chemistry vol 287 no 2 pp 1600ndash1608 2012

[42] A A Lavdas J Chen F Papastefanaki et al ldquoSchwann cellsengineered to express the cell adhesion molecule L1 acceleratemyelination and motor recovery after spinal cord injuryrdquoExperimental Neurology vol 221 no 1 pp 206ndash216 2010

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


have demonstrated the effects of Angelica on nerve repairIt was also reported that AE attenuated neuropathic painwhich is associated with proinflammatory cytokines suchas tumor necrosis factor-120572 (TNF-120572) interleukin-1120573 (IL-1120573)and interleukin-6 (IL-6) TRPV1 and p-ERK in peripheralnervous pain systems [13] Some findings suggested thatAngelica injection improved the sciatic nerve crush injuryand the mechanism might be through the increase of BDNFand NGF protein expressions [14] Angelica dahuricae radixdecreased the levels of TNF-120572 IL-1120573 IL-6 inducible nitricoxide synthase (iNOS) and cyclooxygenase-2 (COX-2) ina lipopolysaccharide- (LPS-) activated microglial cell lineand provided neuroprotection by alleviating inflammationand oxidative stress [15] Another research elucidated thatAE promoted PC12 cell proliferation in vitro assay [16]The AE contains volatile oil organic acids polysaccharidesflavonoids and other ingredients [17]These findings showedthat Angelica exerted a positive-effect in nerve injury repairHowever the studies of AE in vitro assays are not commonand particularly effects of AE on SCs (important peripheralnerve cells) have not been reported In the present studywe investigated the effects of AE on SC proliferation andcycle protein and mRNA expressions of BDNF NCAM andPCNA which are related to peripheral nerve repair [18ndash20]The detection of these neural factors in AE-treated SCs willprovide a mechanistic framework for further studies of theuse of AE as an effective treatment for peripheral nerve injury

2 Materials and Methods

21 Equipment and Reagents HB050 type inverted phasecontrast microscope was manufactured by Zeiss companyK330 refrigerated centrifuge was manufactured by SigmaCorporation iMark550 microplate reader was manufacturedby Bio-Rad company BD flow cytometer was manufacturedby American companies GeneQuant 1300 spectrophotome-ter was manufactured by General Electric Company Light-Cycler 96 was produced by Roche in Switzerland

AE was purchased from Baoji FangSheng BiologicalDevelopment Co Ltd Rat SCs (RSC96) were obtainedfrom China Cell Line Repository Dulbecco Modified EagleMedium (DMEM) was purchased from HyClone companyFetal Bovine Serum (FBS) was purchased from Sijiqingcompany in ChinaMTT and Propidium Iodide (PI) reagentswere purchased from American Sigma company Rat BDNFNCAM and PCNA-ELISA kits were produced by BeyotimeInstitute of Biotechnology in JiangSu First-strand cDNA syn-thesis kit was purchased from Thermo Scientific CompanyAll primers were designed and synthesized by InvitrogenBiotechnology Co Ltd (Shanghai China)

22 Cell Toxic Assays The fifth passage of SCs was usedfor experiments and the trypsinized cells were diluted withDulbecco Modified Eagle Medium (DMEM) containing 10Fetal Bovine Serum (FBS) to cell suspension at a concen-tration of 50 times 104 cellsmL SCs were seeded in 96-wellculture plates and 200 120583L cell suspension was added to everywell Cells were incubated at 37∘C under 5 CO2 for 24 hthen the medium was removed and DMEM without FBS

was added per well and cells were hungered for 12 h andwere randomly divided into control group and drug-treatedgroups The control group and drug-treated groups werecultured dividedly by DMEM containing 10 FBS without orwith the medium containing indicated concentrations (625125 250 1000 2000 4000 and 8000mgL) of AE for 12 h at37∘C Cell viability was measured by MTT assay Five parallelwells were set up for a group and theMTT assay was repeated3 times

23 The Effects of AE on Proliferation of SCs SCs growth wasinterfered with by AE with the indicated concentration thatincreased SC proliferation positively SCs treated by DMEMcontaining 10 FBS acted as control and all groups are setup with 7 parallel wells The experiments were performed forduplicating at least three times Cells were cultured for 12 h24 h 36 h and 48 h The effects of AE on cell proliferationwere evaluated by MTT assay as previously described

24 The Effects of AE on the Cell Cycles of SCs SCs inlogarithmic phase were trypsinized and centrifuged SCswere diluted with DMEM containing 10 FBS and seededin 25mm3 glass screw-cap cell bottle at a density of 20 lowast105 cellsmL After 24 h of incubation at 37∘C the mediumwas removed and then cells were treated with DMEM for12 h In the drug-treated groups AE mixed with DMEMcontaining FBS was added to every well at a concentrationthat promoted cell proliferation and the cells in control groupwere only treated byDMEMcontaining FBSThe supernatantwas discarded after incubating for 24 h and 48 h The cellcycle of each group was detected by flow cytometry ModfitLT software was used for analyzing cell cycle of each groupThe experiments were performed for repeating at least threetimes

25 Effects of AE on Expressions of BDNF NCAM andPCNA in SCs SCs growths were interfered with by AEwith the concentrations which promoted SC proliferationand then cells were cultured for 24 h and 48 h respectivelyThen the number of SCs was counted and the culturesupernatants were collected to determine the amount ofBDNF NCAM and PCNA secreted by the cultured SCs Cellculture supernatants were centrifuged and assayed by usingan ELISA kit following the manufacturerrsquos instructions cellswere trypsinized and cell supernatantswere treated accordingto manufacturerrsquos instruction of ELISA The protein sampleswere stored at minus80∘C until being assayed The samples wereused to measure BDNF NCAM and PCNAThe experimentwas repeated three times

26 Effects of AE on Gene Expression of BDNF NCAM andPCNA SCs were treated by AE for 48 h at concentrationsof 500 1000 and 2000mgL respectively Total RNA wasisolated from SCs using TRIzol reagent according to themanufacturerrsquos instructions The RNA concentration wasdetermined using a GeneQuant 1300 spectrophotometer andthe purity of RNA was determined using the 260280 nmabsorbance ratio The 119860260119860280 ratio of the RNA samples











3 Results


6250

0

1

2

3

4

125 250 500Groups (mgL)

1000 2000 4000 8000

Abso

rban

ce (D

490n

m)

lowastlowast

lowastlowast







00

1

2

3

4

12Time after AE (h)

24

Control

36 48

Abso

rban

ce (D

490n

m) lowastlowast

lowastlowast

lowastlowast

lowastlowast∙∙

∙∙

∙∙∙∙

250mgL500mgL

1000 mgL2000 mgL






4 Discussion




800

200

400

600

00 50 250100 150 200


Num

ber

Channels (PI-A)

(a) Control

800

200

400

600

0

Num

ber

2500 50 100 150 200Channels (PI-A)


(b) 500mgL

200

400

600

0

Num

ber

0 50 250100 150 200Channels (PI-A)


(c) 1000mgL

Num

ber

800

200

400

600

00 50 250100 150 200

1000

Channels (PI-A)


(d) 2000mgL





Num

ber

200

400

600

00 50 100 150 200 250

Channels (PI-A)


(a) ControlN

umbe

r

60

120

180

00 50 100 150 200 250

240

Channels (PI-A)


(b) 500mgL

Num

ber

00 50 100 150 200 250

100

200

300

400

500

600

Channels (PI-A)


(c) 1000mgL

0 50 100 150 200 250

Num

ber

0

100

200

300

400

500

600

Channels (PI-A)


(d) 2000mgL


5 Conclusion




Control 5000

20

40

60

80

Perc

enta

ge o

f cel

ls (

)

1000Groups (mgL)

2000



lowastlowast

lowastlowastlowast


lowastlowast


Control 50000

05

10

Prot

ein

cont

ent

15

20

25


휕휕 휕휕


∙∙ ∙∙ ∙∙

radicradic

BDNF 24 hBDNF 48 h

NCAM 48 h


㶋㶋




0

BDNFNCAMPCNA

Control 500 1000

Groups (mgL)2000

2

4

6

8

Relat

ive m

RNA

expr

essio

n le

vels

lowastlowast

lowastlowast

lowastlowast

∙∙

∙∙

∙∙




Acknowledgments


References



















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


























3 Results


6250

0

1

2

3

4

125 250 500Groups (mgL)

1000 2000 4000 8000

Abso

rban

ce (D

490n

m)

lowastlowast

lowastlowast







00

1

2

3

4

12Time after AE (h)

24

Control

36 48

Abso

rban

ce (D

490n

m) lowastlowast

lowastlowast

lowastlowast

lowastlowast∙∙

∙∙

∙∙∙∙

250mgL500mgL

1000 mgL2000 mgL






4 Discussion




800

200

400

600

00 50 250100 150 200


Num

ber

Channels (PI-A)

(a) Control

800

200

400

600

0

Num

ber

2500 50 100 150 200Channels (PI-A)


(b) 500mgL

200

400

600

0

Num

ber

0 50 250100 150 200Channels (PI-A)


(c) 1000mgL

Num

ber

800

200

400

600

00 50 250100 150 200

1000

Channels (PI-A)


(d) 2000mgL





Num

ber

200

400

600

00 50 100 150 200 250

Channels (PI-A)


(a) ControlN

umbe

r

60

120

180

00 50 100 150 200 250

240

Channels (PI-A)


(b) 500mgL

Num

ber

00 50 100 150 200 250

100

200

300

400

500

600

Channels (PI-A)


(c) 1000mgL

0 50 100 150 200 250

Num

ber

0

100

200

300

400

500

600

Channels (PI-A)


(d) 2000mgL


5 Conclusion




Control 5000

20

40

60

80

Perc

enta

ge o

f cel

ls (

)

1000Groups (mgL)

2000



lowastlowast

lowastlowastlowast


lowastlowast


Control 50000

05

10

Prot

ein

cont

ent

15

20

25


휕휕 휕휕


∙∙ ∙∙ ∙∙

radicradic

BDNF 24 hBDNF 48 h

NCAM 48 h


㶋㶋




0

BDNFNCAMPCNA

Control 500 1000

Groups (mgL)2000

2

4

6

8

Relat

ive m

RNA

expr

essio

n le

vels

lowastlowast

lowastlowast

lowastlowast

∙∙

∙∙

∙∙




Acknowledgments


References



















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















00

1

2

3

4

12Time after AE (h)

24

Control

36 48

Abso

rban

ce (D

490n

m) lowastlowast

lowastlowast

lowastlowast

lowastlowast∙∙

∙∙

∙∙∙∙

250mgL500mgL

1000 mgL2000 mgL






4 Discussion




800

200

400

600

00 50 250100 150 200


Num

ber

Channels (PI-A)

(a) Control

800

200

400

600

0

Num

ber

2500 50 100 150 200Channels (PI-A)


(b) 500mgL

200

400

600

0

Num

ber

0 50 250100 150 200Channels (PI-A)


(c) 1000mgL

Num

ber

800

200

400

600

00 50 250100 150 200

1000

Channels (PI-A)


(d) 2000mgL





Num

ber

200

400

600

00 50 100 150 200 250

Channels (PI-A)


(a) ControlN

umbe

r

60

120

180

00 50 100 150 200 250

240

Channels (PI-A)


(b) 500mgL

Num

ber

00 50 100 150 200 250

100

200

300

400

500

600

Channels (PI-A)


(c) 1000mgL

0 50 100 150 200 250

Num

ber

0

100

200

300

400

500

600

Channels (PI-A)


(d) 2000mgL


5 Conclusion




Control 5000

20

40

60

80

Perc

enta

ge o

f cel

ls (

)

1000Groups (mgL)

2000



lowastlowast

lowastlowastlowast


lowastlowast


Control 50000

05

10

Prot

ein

cont

ent

15

20

25


휕휕 휕휕


∙∙ ∙∙ ∙∙

radicradic

BDNF 24 hBDNF 48 h

NCAM 48 h


㶋㶋




0

BDNFNCAMPCNA

Control 500 1000

Groups (mgL)2000

2

4

6

8

Relat

ive m

RNA

expr

essio

n le

vels

lowastlowast

lowastlowast

lowastlowast

∙∙

∙∙

∙∙




Acknowledgments


References



















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















800

200

400

600

00 50 250100 150 200


Num

ber

Channels (PI-A)

(a) Control

800

200

400

600

0

Num

ber

2500 50 100 150 200Channels (PI-A)


(b) 500mgL

200

400

600

0

Num

ber

0 50 250100 150 200Channels (PI-A)


(c) 1000mgL

Num

ber

800

200

400

600

00 50 250100 150 200

1000

Channels (PI-A)


(d) 2000mgL





Num

ber

200

400

600

00 50 100 150 200 250

Channels (PI-A)


(a) ControlN

umbe

r

60

120

180

00 50 100 150 200 250

240

Channels (PI-A)


(b) 500mgL

Num

ber

00 50 100 150 200 250

100

200

300

400

500

600

Channels (PI-A)


(c) 1000mgL

0 50 100 150 200 250

Num

ber

0

100

200

300

400

500

600

Channels (PI-A)


(d) 2000mgL


5 Conclusion




Control 5000

20

40

60

80

Perc

enta

ge o

f cel

ls (

)

1000Groups (mgL)

2000



lowastlowast

lowastlowastlowast


lowastlowast


Control 50000

05

10

Prot

ein

cont

ent

15

20

25


휕휕 휕휕


∙∙ ∙∙ ∙∙

radicradic

BDNF 24 hBDNF 48 h

NCAM 48 h


㶋㶋




0

BDNFNCAMPCNA

Control 500 1000

Groups (mgL)2000

2

4

6

8

Relat

ive m

RNA

expr

essio

n le

vels

lowastlowast

lowastlowast

lowastlowast

∙∙

∙∙

∙∙




Acknowledgments


References



















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Num

ber

200

400

600

00 50 100 150 200 250

Channels (PI-A)


(a) ControlN

umbe

r

60

120

180

00 50 100 150 200 250

240

Channels (PI-A)


(b) 500mgL

Num

ber

00 50 100 150 200 250

100

200

300

400

500

600

Channels (PI-A)


(c) 1000mgL

0 50 100 150 200 250

Num

ber

0

100

200

300

400

500

600

Channels (PI-A)


(d) 2000mgL


5 Conclusion




Control 5000

20

40

60

80

Perc

enta

ge o

f cel

ls (

)

1000Groups (mgL)

2000



lowastlowast

lowastlowastlowast


lowastlowast


Control 50000

05

10

Prot

ein

cont

ent

15

20

25


휕휕 휕휕


∙∙ ∙∙ ∙∙

radicradic

BDNF 24 hBDNF 48 h

NCAM 48 h


㶋㶋




0

BDNFNCAMPCNA

Control 500 1000

Groups (mgL)2000

2

4

6

8

Relat

ive m

RNA

expr

essio

n le

vels

lowastlowast

lowastlowast

lowastlowast

∙∙

∙∙

∙∙




Acknowledgments


References



















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Control 5000

20

40

60

80

Perc

enta

ge o

f cel

ls (

)

1000Groups (mgL)

2000



lowastlowast

lowastlowastlowast


lowastlowast


Control 50000

05

10

Prot

ein

cont

ent

15

20

25


휕휕 휕휕


∙∙ ∙∙ ∙∙

radicradic

BDNF 24 hBDNF 48 h

NCAM 48 h


㶋㶋




0

BDNFNCAMPCNA

Control 500 1000

Groups (mgL)2000

2

4

6

8

Relat

ive m

RNA

expr

essio

n le

vels

lowastlowast

lowastlowast

lowastlowast

∙∙

∙∙

∙∙




Acknowledgments


References



















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Effects of Angelica Extract on Schwann Cell Proliferation ...

Documents

Transcript of Effects of Angelica Extract on Schwann Cell Proliferation ...