Research Article The Extract of Fructus Psoraleae Promotes...

Research ArticleThe Extract of Fructus Psoraleae Promotes Viability andCartilaginous Formation of Rat Chondrocytes In Vitro

Xin Pan1 Kang Xu23 Xuefeng Qiu34 Wen Zhao5 Dong Wang3 and Li Yang2

1College of Pharmacy South-Central University for Nationalities Wuhan China2National Innovation and Attracting Talents ldquo111rdquo Base Key Laboratory of Biorheological Science and TechnologyMinistry of Education College of Bioengineering Chongqing University Chongqing China3Department of Bioengineering University of California at Los Angeles 410 Westwood Plaza 5121 Engineering VLos Angeles CA 90095 USA4Department of Cardiovascular Surgery Union Hospital Tongji Medical School Huazhong University of Science and TechnologyWuhan Hubei China5School of Life Sciences Northwestern Polytechnical University Xirsquoan Shaanxi China

Correspondence should be addressed to Xin Pan xinpanmailscueceducn and Li Yang yanglibmecqueducn

Received 9 August 2016 Accepted 20 October 2016

Academic Editor Antonella Fioravanti

Copyright copy 2016 Xin Pan et al This is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

This study aimed to investigate the extract components of FP on rat chondrocyte function and cartilaginous formation in vitroPetroleum ether extract (P-e) of FP extract components was selected to treat Sprague-Dawley rat chondrocytes Cell viability wastested with different concentrations (01 1 10 and 100 120583gmL) of P-e treatment Concentrations of 01 and 1 120583gmL P-e conditionedculturemediumswere used for treating chondrocytes in experiments Cell proliferationwasmeasured viaDNA incorporation assayType II collagen aggrecan and Sox-9 genes expression levels were measured with RT-PCR Additionally cartilaginous formationwas analyzed with type II collagen immunofluorescence HampE and alcian blue staining Concentrations of 01 and 1 120583gmL P-eshowed low cytotoxicity and demonstrated stimulatory effects on chondrocyte proliferation in early stages Following 6 days ofP-e culture aggrecan and Sox-9 gene expression levels of the 1 120583gmL P-e group were upregulated by 182- (119901 lt 005) and 206-fold (119901 lt 005) respectively versus controls Moreover 1120583gmL P-e significantly stimulated cell aggregation and type II collagendeposits after 1 week of treatment Noteworthy tight cartilaginous structures formed in the 10-day 1 120583gmL P-e conditioned cultureThese findings suggest that P-e has the potential to treat cartilage degeneration induced by chondrocyte failure

1 Introduction

Chondrocytes and cartilage matrix are important compo-nents of cartilage structure Chondrocytes are highly spe-cialized cells involved in synthesizing collagen and proteo-glycans and the maintenance of cartilage homeostasis Well-recognized cartilage diseases attributable to extracellularmatrix (ECM) degeneration which affect normal chondro-cyte function include osteoarthritis achondroplasia andarticular cartilage injury [1 2] Importantly chondrocytedeath suppresses synthesis of the cartilage matrix resultingin delayed tissue remodeling and poor recovery function [3ndash5] Further exacerbating these diseases affecting chondro-cytes is the finding that chondrocyte proliferation is slow

[6] Therefore effective pharmacologic therapy promotingchondrocyte proliferation and function may be an importanttarget in treating cartilage disease

In Traditional Chinese Medicine (TCM) theory FructusPsoraleae is used to treat a range of conditions includingbone fractures and joint diseases Phytochemical studiessuggest that coumarins flavonoids and meroterpenoids arethe primary active chemical components in FP [7 8] Sev-eral studies have observed that extract components of FPdemonstrate a stimulatory effect on osteogenesis leading tonew bone formation [9] In particular it has been reportedthat prenyl compounds relating to FP stimulate osteoblastproliferation and differentiation [10] Additionally our grouppreviously demonstrated that psoralen the main coumarins

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2016 Article ID 2057631 8 pageshttpdxdoiorg10115520162057631

2 Evidence-Based Complementary and Alternative Medicine

of FP activates chondrocyte cartilaginous cellular functions[11] Yang et al also showed that psoralen regulates chondro-cyte gene expression while also attenuating degeneration ofintervertebral lumbar discs [12]

In this study we identified and assessed extract com-ponents involved in stimulating chondrocyte proliferationand cartilaginous formation After experiment screening P-e of FP extract components was selected and it showed lowcytotoxicity and exhibited promotive effects on chondrocytesproliferation in the early stage Moreover P-e stimulatedcell ECM gene expression aggregation depositing type IIcollagen and forming a tight cartilaginous structure Thesepositive promotive effects on chondrocytes provide the basisfor rechondrogenesis of injured cartilage The P-e of FPcould be further studied and developed some promisingactive medications for treating cartilage degeneration whichis compromised by chondrocytes dysfunction

2 Materials and Methods

21 Chondrocytes Isolation Six healthy Sprague-Dawley ratswere euthanized in order to identify and isolate chondrocytesfor this study as described previously [11] In brief after cellisolation chondrocytes were identified using type II collagenimmunofluorescence (see Supplementary Figure 1 in Supple-mentaryMaterial available online at httpdxdoiorg10115520162057631) while being cultured via culture mediumconsisting of low glucose Dulbecco modified Eagle medium(DMEM) supplemented with 10 FBS and 100UmL peni-cillin and 100 120583gmL streptomycin The third passage of cellswas used for experiments

22 Preparation and HPLC Analysis of P-e Fructus Psoraleaewas purchased from Sichuan Province Traditional ChineseMedicineHospital and authenticated by ProfessorXian-MingLu (Chengdu University of Traditional Chinese Medicine)Fructus Psoraleaewaswashed and decocted by refluxing twicewith 75 ethyl alcohol (1 8 wv) followed by water (1 10wv) Filtrates were combined and condensed to form a rawextract Following this raw extract was further extractedwith petroleum ether using Soxhlet extraction and driedunder reduced pressure to form the petroleum ether extract(yield 528 plusmn 041 119899 = 6 and percentage value representsextractFP rawmaterials)The quality of FP extract was mea-sured via HPLC Briefly 001 g P-e extract was dissolved inmethanol and filtered using 045120583m filters in preparation forHPLC analysis Chromatographic conditions were ShimadzuLC-20AT-DADHPLC system (ShimadzuCorp) C18 column(46 times 250mm 5 120583m) injected volume of 10120583L columntemperature of 30∘C flow velocity at 1mLmin UV wave-length at 230 nm and gradient elution of acetonitrilewater(Supplementary Table 2)

23 Preparation of the Conditioned Culture Medium P-eof FP was dissolved with DMSO to form stock solution(extracts concentration 1 gmL expressed in the weight ofrawmaterials permL) sterilized using a 022120583maseptic filterand stored at 4∘C for later use In preparing the conditionedculture medium stock solution was added in low glucose

DMEM supplemented with 10 FBS to form the conditionalculture medium with the final concentration of 100 120583gmL10 120583gmL 1 120583gmL and 01 120583gmL (DMSO volume was 01in the conditional culture medium) The blank containeduntreated cells with low glucose DMEM supplemented with10 FBS whereas the control group contained the sameproportion of DMSO compared to the test samples

24 Cell Viability andMorphology Analysis Cell viability wasdetermined with the MTS assay A standard curve was firstestablished to test the linear relationship between cell numberand optical density value Following this approximately 5 times103 chondrocytes were seeded in each well of a 96-well plateand then treated with a different conditional culture medium(P-e 100 10 1 and 01 120583gmL)After a culture period of 3 daysMTS colorimetric testing was performed each day to checkODvalues at 490 nm in eachwell Additionally cells were alsomicroscopic observation and recording occurred to assess theeffect of P-e on cell morphology on day 3

25 EdU Incorporation Cell Proliferation Assay PreviousMTS viability assays showed cell viability and proliferationwere optimal at the final concentration of 01 and 1120583gmLTherefore we also studied chondrocyte DNA synthesis usinga treatment of 1 and 01 120583gmL P-e extract for 24 hours viaa Cell-Light EdU Apollo 488 in vitro Imaging Kit In briefcells were seeded in 96-well plates at 5 times 103 cellswell andthen treated with 1 and 01 120583gmL P-e extract conditionedculture medium for 24 hours Following this culture periodcells were incubated with EdU-labeling medium (50 120583M) for2 hThereafter cells were detected using anApollo fluorescentdye counterstained with DAPI and observed using a fluo-rescent microscopeThe percentage of EdU-positive cells wascalculated from five fields randomly selected in each well

26 Cell Cycle Analysis After 1 day of treatment at the finalconcentration of 01 and 1120583gmL cells were trypsinized andthen resuspended in PBS at 5 times 105mL followed by fixationwith 70precooled ethanol overnight at 4∘C Fixed cells werecentrifuged washed and stained with PIRNase stainingbuffer for 1 h in a dark room at 4∘C Cell counts at differentphases of cell cycle were analyzed by flow cytometry

27 qRT-PCR Assay Chondrocytes were seeded at a densityof 1 times 105 per well into 6-well plates Cells were thencultured in normal culture medium or different conditionedmedium Gene expression was evaluated by qRT-PCR after3 and 6 days Total RNA was extracted using the total RNAextraction kit according to manufacturerrsquos guidelines Firststrand cDNA synthesis was followed with the RT-PCR pro-tocol SD rat specific primers were designed using the NCBIPrimer-BLAST and synthesized by Invitrogen (Table 2) TheSsoAdvanced Universal SYBR Green Supermix and CFX96Real-Time PCRDetection Systemwere used to perform qRT-PCR The real-time PCR condition was as follows 95∘C for30 sec followed by 39 cycles of a two-temperature program

Evidence-Based Complementary and Alternative Medicine 3

Table 1 The molecular structure of compounds

Compound Molecular structure Content ()

(1) PsoralenOO O

385

(2) IsopsoralenOOO

269

(3) BavachinHO

O

OH

O002

(4) IsobavachalconeHO

OOH

OH

101

(5) Bavachalcone

OHO

O

H3CO

029

(6) Bakuchiol

OH

845

for five sec at 95∘C and 30 sec at 60∘C The gene expressionlevels were evaluated by the 2minusΔΔct method

28 Immunofluorescent Assay Chondrocytes were seeded ata density of 1times 104 per well into 96-well plates Cells were thencultured in normal culturemediumor the final concentrationof 01 and 1 120583gmL conditionedmedium for 7 days Followingthis cells were fixed with 4 PFA for 30min blocked with1 bovine serum albumin for 3min at room temperatureand stained with rabbit anti-type II collagen overnight at 4∘CAfter washing the cells with PBS Alexa Fluor 488 dye-labeledgoat anti-rabbit antibody was used as a secondary identifyingantibody Finally cells were counterstained with DAPI

29 Cartilaginous Formation Assay Approximately 2 times 106chondrocyteswere centrifuged in the bottomof each tube andthen cultured at a final concentration of 1 120583gmL conditionedculture medium and normal medium for 10 days to formthe cartilaginous pellets Thereafter pellets were collectedand fixed with liquid nitrogen followed by frozen section

processing Section samples were stained with HampE andalcian blue prior to examination under the microscope

3 Results

31 Identification ofMain Components in FP P-e When com-paredwith FP total extract FP P-e contained less compoundsSix known main components in the FP P-e were found andidentified by comparing the retention time with that of anaccepted standard (Figure 1) The percentage content of (1)psoralen (2) isopsoralen (3) bavachin (4) isobavachalcone(5) bavachalcone and (6) bakuchiol in the extract was 385269 002 101 029 and 845 respectively (Table 1)

32 Effect of FP P-e Extract on Chondrocytes Morphology andViability After a 3-day culture period chondrocytes under1 120583gmL and 01 120583gmL P-e conditioned culture mediumsgrew well and were polygonal elliptical and spindle-shaped(Figure 2(a)) However the concentration of 100 120583gmLand 10 120583gmL showed increased cytotoxicity towards thechondrocytes leading to death (Figure 2 100 120583gmL and


Table 2 List of primer sequences for real-time PCR

Genename Primers (51015840-31015840) Production

size (bp)

CollagenII

FAATTTGGTGTGGACATAGGG

94RAAGTATTTGGGTCCTTTGGG

Aggrecan

FGGTGGACCTGTAACAATCTT

509RTCTTCTCCTGAGTATGAGGG

SOX-9

FAAGTGAAGGTAACGATTGCT

153RCTCACTAACTCTGAAGGAGC

GADPH

FCAAGGTCATCCATGACAACT

253RCAGATCCACAACGGATACAT

Total extract

P-e

(1) (2)(3)

(4)(5)

(6)

350100 150 200 250 300 400 4505000(min)

Figure 1 HPLC chromatogram for petroleum ether and totalextract of Fructus Psoraleae (1) Psoralen (2) isopsoralen (3)bavachin (4) isobavachalcone (5) bavachalcone and (6) bakuchiol

10 120583gmL) The OD value from MTS testing illustrates cellviability while indirectly reflecting cell proliferation Follow-ing a culture conditioning period of 3 days P-e showed anincreasing influence on cell proliferation at P-e concentra-tions of 1 120583gmL and 01 120583gmL (Figure 2(b)) In contrast P-econcentrations of 100120583gmL and 10 120583gmL led to profoundcell death (Figure 2(b)) On day 1 1120583gmL and 01 120583gmL P-esignificantly increased cell viability by 049- (119901 lt 005) and035-fold (119901 lt 005) versus the control group (01 DMSO)respectively On day 2 compared with the control group cellviability of the 1 120583gmL and 01 120583gmL P-e extract increasedby 029- and 025-fold respectively On day 3 the favorableeffects of the P-e extract on cell viability plateaued HowevertheODvalue of 1120583gmL and 01120583gmLP-e extract treatmentsshowedmodest increases compared to the control group (119901 gt005)

33 Effect of FP P-e Extract on Chondrocytes ProliferationThe EdU incorporation assay illustrated cell DNA synthesisshowing the percentage of EdU+ nuclei was approximately184 159 and 113 for 1 120583gmL 01 120583gmL and control

group respectively following 24 h culture periods (Figures3(a) and 3(b)) Compared to the control group the percentageof EdU+ nuclei at 1 120583gmL and 01 120583gmL P-e treatmentsincreased by 063- and 041-fold respectively (Figure 3(b))Additionally cell cycle detection showed that after 24 hculture periods with P-e 296 252 and 221 of cellswere at the SG2 phase for 1 120583gmL 01 120583gmL concentrationsand control group respectively (Figure 3(c)) The percentageof SG2 cells at 1 120583gmL P-e treatments showed significantdifference versus control group (Figure 3(d)) Lastly therewas no proliferation stimulating effect of 3 days P-e treatment(Supplementary Figure 3) Thus in the early cell cycle stagesP-e promoted cell proliferation via enhanced DNA synthesisand an increased cell cycle SG2 phase

34 Effect of FP P-e Extract on Chondrocytes Gene Expressionand Cartilaginous Formation The gene transcript levels ofcollagen II aggrecan and SOX-9 of chondrocytes wereanalyzed via qRT-PCR for 3- and 6-day culture with normalculture medium and the conditioned culture medium withthe final concentration of 01 120583gmL and 1 120583gmL P-e (Fig-ure 4(a)) On day 3 compared with control group the P-econcentration at 1120583gmL decreased type II collagen aggre-can and SOX-9 gene expression by 069- (119901 lt 005) 056-(119901 lt 005) and 067-fold (119901 lt 005) respectively Comparedto the control the P-e concentration at 01 120583gmL decreasedtype II collagen aggrecan and SOX-9 gene expression by011- 017- and 011-fold respectively (all 119875 gt 005) Incontrast on day 6 compared to the control group aggrecanand SOX-9 gene expression in the 1 120583gmL P-e group wereupregulated by 182-fold (119901 lt 005) and 206-fold (119901 lt 005)respectively Type II collagen gene expression demonstratedmodest upregulation of 019-fold compared to controls (119901 gt005) Additionally although not significant 01 120583gmL P-eleads to upregulation of type II collagen aggrecan and SOX-9gene expression by 016- 027- and 051-fold respectively

Type II collagen immunofluorescent staining showed that1 120583gmL P-e significantly stimulated cell aggregation and typeII collagen deposits (Figure 4(b)) compared to the controlgroup after 1 week of conditioned culture

Based on previous qRT-PCR and immunofluorescenceanalyses 1 120583gmL P-e was suggested to demonstrate thestrongest effects on cartilaginous ECM gene expression Cellpellets were cultured with 1 120583gmL P-e conditioned culturemedium for 10 days in vitro and the cartilaginous con-structions were evaluated by the frozen sections which werestainedwithHampE and alcian blue (Figure 4(c)) HampE stainingshowed that compared to the control group the majority ofchondrocytes aggregated towards the outer edge forming acartilaginous rim in the P-e group (Figure 4(c) indicated byarrow) moreover the structure of the pellet of the P-e groupwas more dense and complete in shape (Figure 4(c)) Addi-tionally according to the alcian blue staining stain densityof the outer edge of the pellet was higher in the P-e extractgroup versus controls (Figure 4(c) indicated by arrow) Thisstaining indicated that there were more glycosaminoglycandeposits in the P-e group Lastly we detected type II collagenwith immunofluorescence on the frozen sections while


100120583gmL 10120583gmL 1120583gmL

01 120583gmL DMSO Blank

(a)

1 day 2 days 3 days000

025

050

075

OD

val

ue

100120583gmL10120583gmL1120583gmL

01 120583gmL01 DMSOBlank

lowastlowast

lowastlowast

(b)

Figure 2 Effects of P-e treatments with different concentration on chondrocytes morphology and viability for 3 days (a) Cell morphologyunder the phase contrast microscope (b) Cell viability was tested via MTS assay and data are presented as mean plusmn sd (119899 = 6) lowast119901 lt 005versus DMSO group

observing more type II collagen deposits in the P-e groupcompared to controls (Supplementary Figure 4)

4 Discussion

A lack of active blood supply within cartilage compro-mises the recovery of the cartilage degenerative diseasesTherefore it is important to identify and develop targetedpharmacologic therapy that acts to inhibit cartilage failurewhile improving chondrocyte function In previous studiesfrom our group we observed that psoralen promotes carti-laginous ECM synthesis as well as increased cartilaginousgene expression However we found that psoralen does notsimulate cell proliferation In this study we first in orderprepared the solvent extracts of FP with petroleum etherethyl acetate and n-butyl alcohol according to the chemicalpolarity (Supplementary Figure 2) Then these differentsolvent extracts were screened with MTS assay as a pilotstudy to determinate the active extract for cell viability(data not shown) The findings of this pilot study showedthat petroleum ether extract demonstrates a stimulatingeffect on cell viability while exhibiting low cytotoxicity atconcentrations of 01 and 1 120583gmLThus the petroleum etherextract was selected for ongoing experiments

We acknowledge that extracts from herbal medicineswhich are used in pharmacological studies are limited byunstable quality control and undefined material basis of thechemical components Therefore in this study we calculatedthe P-e extraction yield to confirm a stable and repeatablemethodology for sample preparation After HPLC analysisand identification we identified the main components ofP-e We found 6 known compounds psoralen isopsoralen

bavachin isobavachalcone bavachalcone and bakuchiolThus quality control and HPLC analysis ensure a reliable P-ein future experiments

It is worth noting that in the extract the percentagecontent of bakuchiol reached to 845 Previous reportssuggest that bakuchiol which is a meroterpenoid may playimportant roles in antimicrobial [13] antioxidant [14] anti-tumor [15] and inflammatory suppression activities [16] Limet al compared different compounds from Psoralea corylifoliaL and demonstrated that bakuchiol is a key compound withestrogenic activity due to a high estrogen receptor-bindingaffinity [17] Several studies have confirmed that estrogenand its similar active compounds regulate cell cycle forproliferation [18ndash21] Therefore we suggest that bakuchiolas an active compound demonstrating estrogenic activitymay be used as a therapy for cartilage degenerative diseaseFuture studies are needed to investigate the potential role ofbakuchiol as a therapy for cartilage degenerative disease

Additionally we observed that with P-e treatment car-tilaginous gene (ie type II collagen aggrecan and Sox-9) expression levels were modestly downregulated in earlyphases which was contrasted by upregulation in later phasesAs such we speculate that because cell proliferation is anenergy intensive process that depends on cyclins and otherregulated peptides cells remaining in the S phase are notable to synthesize an extracellular matrix Thus in this studyexpression levels of cartilaginous genes (ie type II collagenaggrecan and Sox-9 expression) were suppressed in earlystages likely due to the effects of prior cell proliferationWhereas another potential explanation is that some activecompounds in P-e show demonstrate an inhibitory effect onECM gene expression via high affinity receptors others show


1120583

gm

L01

120583g

mL

EdU EdUDAPI

Merge

Con

trol

(a)

1120583gmL01 120583gmLControl

1 day

0

5

10

15

20

25

Pro

lifer

ativ

e cel

ls pe

rcen

tage

()

lowast

(b)

200 400 600200 400 600

Control

SG2 296SG2 252SG2 221

01 120583gmL 1120583gmL

1k800600400200

0

1k800600400200

0

1k800600400200

0

0100200300400500

0100200300400500

0100200300400500

400 600200

(c)

1120583gmL01 120583gmLControl20

25

30

SG

2 ph

ase (

)

lowast

(d)

Figure 3 The effects of P-e on cell proliferation (a and b) EdU incorporative DNA synthesis of rat chondrocytes with P-e treatment (a)EdU staining (red) and DAPI (blue) in chondrocytes with P-e treatment for 24 h (b) The histogram showed the percentage of EdU+ nucleiEach bar represents the mean plusmn sd (119899 = 5) lowast119901 lt 005 versus DMSO group (control) was accepted as statistically significant (c) Cell cycleby flow cytometry indicating that 221 252 and 296 of the cells were at the SG2 phase for DMSO (control) 01 120583gmL P-e 1 120583gmLP-e respectively (d) Statistical analyses for flow cytometry (119899 = 3) lowast119901 lt 005 versus DMSO group (control) was accepted as statisticallysignificant


Collagen II Aggrecan Sox-9

Control01 120583gmL1120583gmL


3 days 6 days 3 days 6 days

Nor

mal

ized

fold

expr

essio

n

00

05

10

15

20

(targ

etG

APD

H)

Nor

mal

ized

fold

expr

essio

n

00

05

10

15

20

25

(targ

etG

APD

H)

Nor

mal

ized

fold

expr

essio

n

00

05

10

15

20

25

(targ

etG

APD

H)

3 days 6 days

lowastlowast

lowast

lowast

lowast


(a)

Con

trol

01

120583g

mL

1120583

gm

L

(b)

P-e

Con

trol

Phase contrast HampE Alcian blue

(c)

Figure 4 The effect of P-e on chondrocytes ECM gene expression and cartilaginous formation (a) Gene expression level of differentcartilaginous-related genes collagen II aggrecan and Sox-9 at days 3 and 6 with P-e treatmentsThe data were analyzed by the 2minusΔΔct relativequantification method (mean plusmn sd 119899 = 3) lowast119901 lt 005 versus DMSO group (control) was accepted as statistically significant (b) Collagen IIimmunofluorescent staining of chondrocytes with P-e treatment for 1-week culture (c) HampE and alcian blue staining of cartilaginous tissuesections of the pellet with P-e conditioned culturing for 10 days

stimulating effects due to low affinity receptors Neverthelessin supporting this hypothesis future studies need to inves-tigate different cell receptors signal transduction pathwaysinvolving each chemical compound associated with P-e

In addition to the P-e stimulating effect on cell prolifera-tion and gene expression we also observed tight cartilaginousstructures (ie pellets) in 1 120583gmL P-e conditioned culturesthat lasted 10 days Thus these data further support the sug-gestion that P-e treatment is able to promote cell proliferationas well as synthesize ECM for cartilaginousmatrix formationAlthough we did not study human chondrocytes the presentanimal model observations provide a promising approach indeveloping new therapies for cartilage degenerative diseasetreatment Future mechanism studies targeting human chon-drocytes using the techniques in this study are warranted toimprove the clinical translation of these data

5 Conclusions

The results suggest that P-e may promote chondrocytesviability and proliferation regulate cartilaginous extracellular

matrix gene expression and stimulate cartilaginous for-mation These positive promotive effects on chondrocytesprovide the basis for rechondrogenesis of injured cartilageThe P-e of FP could be further studied and developed somepromising active medications for treating cartilage degenera-tion which is compromised by chondrocytes dysfunction

Competing Interests

The authors declare that there are no competing interests

Authorsrsquo Contributions

Xin Pan and Kang Xu designed the project performed theexperiments collected the data and wrote the manuscriptthese two authors contributed equally to this study and sharethe first authorship Xuefeng Qiu and Dong Wang analyzedthe data and wrote and revised the manuscript Xin Pan LiYang and Wen Zhao gave financial support and wrote andrevised the manuscript All authors read and approved thefinal manuscript


Acknowledgments

This work was supported by South-Central University forNationalities National Innovation and Attracting TalentsProject (ldquo111rdquo Project) (B06023) College of Biomedical Engi-neering Chongqing University National Natural ScienceFoundation of China (11532004 31270990 and 81170110) andNatural Science Basic Research Plan in Shaanxi Province ofChina (Program no 2015JM5177) Kang Xuwas sponsored byldquoChina Scholarship Council 201406050084rdquo

References

[1] A D Pearle R F Warren and S A Rodeo ldquoBasic science ofarticular cartilage and osteoarthritisrdquoClinics in SportsMedicinevol 24 no 1 pp 1ndash12 2005

[2] MMaldonado and J Nam ldquoThe role of changes in extracellularmatrix of cartilage in the presence of inflammation on thepathology of osteoarthritisrdquo BioMed Research International vol2013 Article ID 284873 10 pages 2013

[3] M B Goldring ldquoChondrogenesis chondrocyte differentiationand articular cartilage metabolism in health and osteoarthritisrdquoTherapeutic Advances in Musculoskeletal Disease vol 4 no 4pp 269ndash285 2012

[4] P K Sacitharan and T L Vincent ldquoCellular ageingmechanismsin osteoarthritisrdquoMammalian Genome vol 27 no 7-8 pp 421ndash429 2016

[5] J J Vaca-Gonzalez M L Gutierrez J M Guevara and DA Garzon-Alvarado ldquoCellular automata model for humanarticular chondrocytes migration proliferation and cell deathan in vitro validationrdquo In Silico Biology 2015

[6] T Aigner and H A Kim ldquoApoptosis and cellular vitalityissues in osteoarthritic cartilage degenerationrdquo Arthritis andRheumatism vol 46 no 8 pp 1986ndash1996 2002

[7] L Zhao C Huang Z Shan B Xiang and L Mei ldquoFingerprintanalysis of Psoralea corylifolia L by HPLC and LC-MSrdquo Journalof Chromatography B Analytical Technologies in the Biomedicaland Life Sciences vol 821 no 1 pp 67ndash74 2005

[8] B Ruan L-Y Kong Y Takaya and M Niwa ldquoStudies on thechemical constituents of Psoralea corylifolia Lrdquo Journal of AsianNatural Products Research vol 9 no 1 pp 41ndash44 2007

[9] D-Z Tang F Yang Z Yang et al ldquoPsoralen stimulatesosteoblast differentiation through activation of BMP signalingrdquoBiochemical andBiophysical ResearchCommunications vol 405no 2 pp 256ndash261 2011

[10] W D Li C P Yan Y Wu et al ldquoOsteoblasts proliferation anddifferentiation stimulating activities of the main components ofFructus Psoraleae corylifoliaerdquo Phytomedicine vol 21 no 4 pp400ndash405 2014

[11] K Xu X Pan Y Sun W Xu L Njunge and L Yang ldquoPsoralenactivates cartilaginous cellular functions of rat chondrocytes invitrordquo Pharmaceutical Biology vol 53 no 7 pp 1010ndash1015 2015

[12] L Yang X Sun and X Geng ldquoEffects of psoralen on chondro-cyte degeneration in lumbar intervertebral disc of ratsrdquoPakistanJournal of Pharmaceutical Sciences vol 28 no 2 supplementpp 667ndash670 2015

[13] H Katsura R-I Tsukiyama A Suzuki and M KobayashildquoIn vitro antimicrobial activities of bakuchiol against oralmicroorganismsrdquo Antimicrobial Agents and Chemotherapy vol45 no 11 pp 3009ndash3013 2001

[14] S Adhikari R Joshi B S Patro et al ldquoAntioxidant activity ofbakuchiol experimental evidences and theoretical treatmentson the possible involvement of the terpenoid chainrdquo ChemicalResearch in Toxicology vol 16 no 9 pp 1062ndash1069 2003

[15] Z ChenK Jin LGao et al ldquoAnti-tumor effects of bakuchiol ananalogue of resveratrol on human lung adenocarcinoma A549cell linerdquo European Journal of Pharmacology vol 643 no 2-3pp 170ndash179 2010

[16] M L Ferrandiz B Gil M J Sanz et al ldquoEffect of bakuchiol onleukocyte functions and some inflammatory responses inmicerdquoJournal of Pharmacy and Pharmacology vol 48 no 9 pp 975ndash980 1996

[17] S-H Lim T-Y Ha J Ahn and S Kim ldquoEstrogenic activitiesof Psoralea corylifolia L seed extracts and main constituentsrdquoPhytomedicine vol 18 no 5 pp 425ndash430 2011

[18] M Maras C Vanparys F Muylle et al ldquoEstrogen-like prop-erties of fluorotelomer alcohols as revealed by MCF-7 breastcancer cell proliferationrdquo Environmental Health Perspectivesvol 114 no 1 pp 100ndash105 2006

[19] M Dalvai and K Bystricky ldquoCell cycle and anti-estrogen effectssynergize to regulate cell proliferation and ER target geneexpressionrdquo PLoS ONE vol 5 no 6 Article ID e11011 2010

[20] T J Spady R D McComb and J D Shull ldquoEstrogen action inthe regulation of cell proliferation cell survival and tumorige-nesis in the rat anterior pituitary glandrdquo Endocrine vol 11 no3 pp 217ndash233 1999

[21] C D Fowler F Johnson and Z Wang ldquoEstrogen regulationof cell proliferation and distribution of estrogen receptor-120572 inthe brains of adult female prairie and meadow volesrdquo Journal ofComparative Neurology vol 489 no 2 pp 166ndash179 2005

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Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


of FP activates chondrocyte cartilaginous cellular functions[11] Yang et al also showed that psoralen regulates chondro-cyte gene expression while also attenuating degeneration ofintervertebral lumbar discs [12]

In this study we identified and assessed extract com-ponents involved in stimulating chondrocyte proliferationand cartilaginous formation After experiment screening P-e of FP extract components was selected and it showed lowcytotoxicity and exhibited promotive effects on chondrocytesproliferation in the early stage Moreover P-e stimulatedcell ECM gene expression aggregation depositing type IIcollagen and forming a tight cartilaginous structure Thesepositive promotive effects on chondrocytes provide the basisfor rechondrogenesis of injured cartilage The P-e of FPcould be further studied and developed some promisingactive medications for treating cartilage degeneration whichis compromised by chondrocytes dysfunction

2 Materials and Methods

21 Chondrocytes Isolation Six healthy Sprague-Dawley ratswere euthanized in order to identify and isolate chondrocytesfor this study as described previously [11] In brief after cellisolation chondrocytes were identified using type II collagenimmunofluorescence (see Supplementary Figure 1 in Supple-mentaryMaterial available online at httpdxdoiorg10115520162057631) while being cultured via culture mediumconsisting of low glucose Dulbecco modified Eagle medium(DMEM) supplemented with 10 FBS and 100UmL peni-cillin and 100 120583gmL streptomycin The third passage of cellswas used for experiments

22 Preparation and HPLC Analysis of P-e Fructus Psoraleaewas purchased from Sichuan Province Traditional ChineseMedicineHospital and authenticated by ProfessorXian-MingLu (Chengdu University of Traditional Chinese Medicine)Fructus Psoraleaewaswashed and decocted by refluxing twicewith 75 ethyl alcohol (1 8 wv) followed by water (1 10wv) Filtrates were combined and condensed to form a rawextract Following this raw extract was further extractedwith petroleum ether using Soxhlet extraction and driedunder reduced pressure to form the petroleum ether extract(yield 528 plusmn 041 119899 = 6 and percentage value representsextractFP rawmaterials)The quality of FP extract was mea-sured via HPLC Briefly 001 g P-e extract was dissolved inmethanol and filtered using 045120583m filters in preparation forHPLC analysis Chromatographic conditions were ShimadzuLC-20AT-DADHPLC system (ShimadzuCorp) C18 column(46 times 250mm 5 120583m) injected volume of 10120583L columntemperature of 30∘C flow velocity at 1mLmin UV wave-length at 230 nm and gradient elution of acetonitrilewater(Supplementary Table 2)

23 Preparation of the Conditioned Culture Medium P-eof FP was dissolved with DMSO to form stock solution(extracts concentration 1 gmL expressed in the weight ofrawmaterials permL) sterilized using a 022120583maseptic filterand stored at 4∘C for later use In preparing the conditionedculture medium stock solution was added in low glucose

DMEM supplemented with 10 FBS to form the conditionalculture medium with the final concentration of 100 120583gmL10 120583gmL 1 120583gmL and 01 120583gmL (DMSO volume was 01in the conditional culture medium) The blank containeduntreated cells with low glucose DMEM supplemented with10 FBS whereas the control group contained the sameproportion of DMSO compared to the test samples

24 Cell Viability andMorphology Analysis Cell viability wasdetermined with the MTS assay A standard curve was firstestablished to test the linear relationship between cell numberand optical density value Following this approximately 5 times103 chondrocytes were seeded in each well of a 96-well plateand then treated with a different conditional culture medium(P-e 100 10 1 and 01 120583gmL)After a culture period of 3 daysMTS colorimetric testing was performed each day to checkODvalues at 490 nm in eachwell Additionally cells were alsomicroscopic observation and recording occurred to assess theeffect of P-e on cell morphology on day 3

25 EdU Incorporation Cell Proliferation Assay PreviousMTS viability assays showed cell viability and proliferationwere optimal at the final concentration of 01 and 1120583gmLTherefore we also studied chondrocyte DNA synthesis usinga treatment of 1 and 01 120583gmL P-e extract for 24 hours viaa Cell-Light EdU Apollo 488 in vitro Imaging Kit In briefcells were seeded in 96-well plates at 5 times 103 cellswell andthen treated with 1 and 01 120583gmL P-e extract conditionedculture medium for 24 hours Following this culture periodcells were incubated with EdU-labeling medium (50 120583M) for2 hThereafter cells were detected using anApollo fluorescentdye counterstained with DAPI and observed using a fluo-rescent microscopeThe percentage of EdU-positive cells wascalculated from five fields randomly selected in each well

26 Cell Cycle Analysis After 1 day of treatment at the finalconcentration of 01 and 1120583gmL cells were trypsinized andthen resuspended in PBS at 5 times 105mL followed by fixationwith 70precooled ethanol overnight at 4∘C Fixed cells werecentrifuged washed and stained with PIRNase stainingbuffer for 1 h in a dark room at 4∘C Cell counts at differentphases of cell cycle were analyzed by flow cytometry

27 qRT-PCR Assay Chondrocytes were seeded at a densityof 1 times 105 per well into 6-well plates Cells were thencultured in normal culture medium or different conditionedmedium Gene expression was evaluated by qRT-PCR after3 and 6 days Total RNA was extracted using the total RNAextraction kit according to manufacturerrsquos guidelines Firststrand cDNA synthesis was followed with the RT-PCR pro-tocol SD rat specific primers were designed using the NCBIPrimer-BLAST and synthesized by Invitrogen (Table 2) TheSsoAdvanced Universal SYBR Green Supermix and CFX96Real-Time PCRDetection Systemwere used to perform qRT-PCR The real-time PCR condition was as follows 95∘C for30 sec followed by 39 cycles of a two-temperature program




(1) PsoralenOO O

385

(2) IsopsoralenOOO

269

(3) BavachinHO

O

OH

O002


OOH

OH

101

(5) Bavachalcone

OHO

O

H3CO

029

(6) Bakuchiol

OH

845





3 Results






size (bp)

CollagenII



Aggrecan



SOX-9



GADPH



Total extract

P-e

(1) (2)(3)

(4)(5)

(6)

350100 150 200 250 300 400 4505000(min)









100120583gmL 10120583gmL 1120583gmL


(a)


025

050

075

OD

val

ue

100120583gmL10120583gmL1120583gmL


lowastlowast

lowastlowast

(b)



4 Discussion







1120583

gm

L01

120583g

mL

EdU EdUDAPI

Merge

Con

trol

(a)


1 day

0

5

10

15

20

25

Pro

lifer

ativ

e cel

ls pe

rcen

tage

()

lowast

(b)

200 400 600200 400 600

Control

SG2 296SG2 252SG2 221

01 120583gmL 1120583gmL

1k800600400200

0

1k800600400200

0

1k800600400200

0

0100200300400500

0100200300400500

0100200300400500

400 600200

(c)


25

30

SG

2 ph

ase (

)

lowast

(d)







Nor

mal

ized

fold

expr

essio

n

00

05

10

15

20

(targ

etG

APD

H)

Nor

mal

ized

fold

expr

essio

n

00

05

10

15

20

25

(targ

etG

APD

H)

Nor

mal

ized

fold

expr

essio

n

00

05

10

15

20

25

(targ

etG

APD

H)

3 days 6 days

lowastlowast

lowast

lowast

lowast


(a)

Con

trol

01

120583g

mL

1120583

gm

L

(b)

P-e

Con

trol


(c)




5 Conclusions



Competing Interests





Acknowledgments


References



























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















(1) PsoralenOO O

385

(2) IsopsoralenOOO

269

(3) BavachinHO

O

OH

O002


OOH

OH

101

(5) Bavachalcone

OHO

O

H3CO

029

(6) Bakuchiol

OH

845





3 Results






size (bp)

CollagenII



Aggrecan



SOX-9



GADPH



Total extract

P-e

(1) (2)(3)

(4)(5)

(6)

350100 150 200 250 300 400 4505000(min)









100120583gmL 10120583gmL 1120583gmL


(a)


025

050

075

OD

val

ue

100120583gmL10120583gmL1120583gmL


lowastlowast

lowastlowast

(b)



4 Discussion







1120583

gm

L01

120583g

mL

EdU EdUDAPI

Merge

Con

trol

(a)


1 day

0

5

10

15

20

25

Pro

lifer

ativ

e cel

ls pe

rcen

tage

()

lowast

(b)

200 400 600200 400 600

Control

SG2 296SG2 252SG2 221

01 120583gmL 1120583gmL

1k800600400200

0

1k800600400200

0

1k800600400200

0

0100200300400500

0100200300400500

0100200300400500

400 600200

(c)


25

30

SG

2 ph

ase (

)

lowast

(d)







Nor

mal

ized

fold

expr

essio

n

00

05

10

15

20

(targ

etG

APD

H)

Nor

mal

ized

fold

expr

essio

n

00

05

10

15

20

25

(targ

etG

APD

H)

Nor

mal

ized

fold

expr

essio

n

00

05

10

15

20

25

(targ

etG

APD

H)

3 days 6 days

lowastlowast

lowast

lowast

lowast


(a)

Con

trol

01

120583g

mL

1120583

gm

L

(b)

P-e

Con

trol


(c)




5 Conclusions



Competing Interests





Acknowledgments


References



























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















size (bp)

CollagenII



Aggrecan



SOX-9



GADPH



Total extract

P-e

(1) (2)(3)

(4)(5)

(6)

350100 150 200 250 300 400 4505000(min)









100120583gmL 10120583gmL 1120583gmL


(a)


025

050

075

OD

val

ue

100120583gmL10120583gmL1120583gmL


lowastlowast

lowastlowast

(b)



4 Discussion







1120583

gm

L01

120583g

mL

EdU EdUDAPI

Merge

Con

trol

(a)


1 day

0

5

10

15

20

25

Pro

lifer

ativ

e cel

ls pe

rcen

tage

()

lowast

(b)

200 400 600200 400 600

Control

SG2 296SG2 252SG2 221

01 120583gmL 1120583gmL

1k800600400200

0

1k800600400200

0

1k800600400200

0

0100200300400500

0100200300400500

0100200300400500

400 600200

(c)


25

30

SG

2 ph

ase (

)

lowast

(d)







Nor

mal

ized

fold

expr

essio

n

00

05

10

15

20

(targ

etG

APD

H)

Nor

mal

ized

fold

expr

essio

n

00

05

10

15

20

25

(targ

etG

APD

H)

Nor

mal

ized

fold

expr

essio

n

00

05

10

15

20

25

(targ

etG

APD

H)

3 days 6 days

lowastlowast

lowast

lowast

lowast


(a)

Con

trol

01

120583g

mL

1120583

gm

L

(b)

P-e

Con

trol


(c)




5 Conclusions



Competing Interests





Acknowledgments


References



























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















100120583gmL 10120583gmL 1120583gmL


(a)


025

050

075

OD

val

ue

100120583gmL10120583gmL1120583gmL


lowastlowast

lowastlowast

(b)



4 Discussion







1120583

gm

L01

120583g

mL

EdU EdUDAPI

Merge

Con

trol

(a)


1 day

0

5

10

15

20

25

Pro

lifer

ativ

e cel

ls pe

rcen

tage

()

lowast

(b)

200 400 600200 400 600

Control

SG2 296SG2 252SG2 221

01 120583gmL 1120583gmL

1k800600400200

0

1k800600400200

0

1k800600400200

0

0100200300400500

0100200300400500

0100200300400500

400 600200

(c)


25

30

SG

2 ph

ase (

)

lowast

(d)







Nor

mal

ized

fold

expr

essio

n

00

05

10

15

20

(targ

etG

APD

H)

Nor

mal

ized

fold

expr

essio

n

00

05

10

15

20

25

(targ

etG

APD

H)

Nor

mal

ized

fold

expr

essio

n

00

05

10

15

20

25

(targ

etG

APD

H)

3 days 6 days

lowastlowast

lowast

lowast

lowast


(a)

Con

trol

01

120583g

mL

1120583

gm

L

(b)

P-e

Con

trol


(c)




5 Conclusions



Competing Interests





Acknowledgments


References



























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















1120583

gm

L01

120583g

mL

EdU EdUDAPI

Merge

Con

trol

(a)


1 day

0

5

10

15

20

25

Pro

lifer

ativ

e cel

ls pe

rcen

tage

()

lowast

(b)

200 400 600200 400 600

Control

SG2 296SG2 252SG2 221

01 120583gmL 1120583gmL

1k800600400200

0

1k800600400200

0

1k800600400200

0

0100200300400500

0100200300400500

0100200300400500

400 600200

(c)


25

30

SG

2 ph

ase (

)

lowast

(d)







Nor

mal

ized

fold

expr

essio

n

00

05

10

15

20

(targ

etG

APD

H)

Nor

mal

ized

fold

expr

essio

n

00

05

10

15

20

25

(targ

etG

APD

H)

Nor

mal

ized

fold

expr

essio

n

00

05

10

15

20

25

(targ

etG

APD

H)

3 days 6 days

lowastlowast

lowast

lowast

lowast


(a)

Con

trol

01

120583g

mL

1120583

gm

L

(b)

P-e

Con

trol


(c)




5 Conclusions



Competing Interests





Acknowledgments


References



























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















Nor

mal

ized

fold

expr

essio

n

00

05

10

15

20

(targ

etG

APD

H)

Nor

mal

ized

fold

expr

essio

n

00

05

10

15

20

25

(targ

etG

APD

H)

Nor

mal

ized

fold

expr

essio

n

00

05

10

15

20

25

(targ

etG

APD

H)

3 days 6 days

lowastlowast

lowast

lowast

lowast


(a)

Con

trol

01

120583g

mL

1120583

gm

L

(b)

P-e

Con

trol


(c)




5 Conclusions



Competing Interests





Acknowledgments


References



























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Acknowledgments


References



























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Research Article The Extract of Fructus Psoraleae Promotes...

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