calcium sensing receptor promotes cardiac fibroblast proliferation and extracellular matrix secretion

Department of Pathophysioloy, Harbin Medical University, Harbin, 150086 China Fax +8645186674548, E-Mail zhangwh116@126.com and E-Mail lufanghao1973@126.com Wei-hua Zhang and Fanghao L

Original Paper

Copyright © 2014 S Karger AG, Basel

NonCommercial 3.0 Unported license (CC BY-NC) (www.karger.com/OA-license), applicable to the online version of the article only Distribution permitted for non-commercial purposes only.

Department of Pathophysioloy, Harbin Medical University, Harbin, 150086 (China) Fax +8645186674548, E-Mail zhangwh116@126.com and E-Mail lufanghao1973@126.com

Wei-hua Zhang

and Fanghao Lu

Calcium Sensing Receptor Promotes

Cardiac Fibroblast Proliferation and

Extracellular Matrix Secretion

Xinying Zhanga,e Tiemin Zhangb,e Jichao Wua Xiangjing Yuc Dan Zhenga

Fan Yanga Ting Li a Lina Wanga Yajun Zhaoa Shiyun Donga Xin Zhonga

Songbin Fud Chang-qing Xua Fanghao Lua Wei-hua Zhanga

a Department of Pathophysioloy, d Department of Genetics, Harbin Medical University, b Department of

*HQHUDO6XUJHU\WKH)LUVW$IÀOLDWHG+RVSLWDORI+DUELQ0HGLFDO8QLYHUVLW\ c Clinical Laboratory, Harbin

Cancer Hospital of Harbin Medical University, Harbin, 150086, China; e represents equal contribution to

this paper

Key Words

&DOFLXPVHQVLQJUHFHSWRU‡&DUGLDFÀEUREODVW‡,QWUDFHOOXODUFDOFLXP‡&DUGLDFÀEURVLV

Abstract

Aims: Calcium-sensing receptor (CaR) acts as a G protein coupled receptor that mediates the

increase of the intracellular Ca2+FRQFHQWUDWLRQ7KHH[SUHVVLRQRI&D5KDVEHHQFRQÀUPHG

in various cell types, including cardiomyocytes, smooth muscle cells, neurons and vascular

HQGRWKHOLDOFHOOV+RZHYHUZKHWKHU&D5LVH[SUHVVHGDQGIXQFWLRQVLQFDUGLDFÀEUREODVWVKDV

remained unknown The present study investigated whether CaR played a role in cardiac

ÀEUREODVWSUROLIHUDWLRQDQGH[WUDFHOOXODUPDWUL[(&0VHFUHWLRQERWKLQFXOWXUHGUDWQHRQDWDO

FDUGLDF ÀEUREODVWV DQG LQ D PRGHO RI FDUGLDF K\SHUWURSK\ LQGXFHG E\ LVRSURWHUHQRO ,62

Methods and Results:,PPXQRÁXRUHVFHQFHLPPXQRKLVWRFKHPLVWU\DQG:HVWHUQEORWDQDO\VLV

UHYHDOHGWKHSUHVHQFHRI&D5LQFDUGLDFÀEUREODVWV&DOFLXPDQGFDOLQGRODVSHFLÀFDFWLYDWRU

RI&D5HOHYDWHGWKHLQWUDFHOOXODUFDOFLXPFRQFHQWUDWLRQLQFDUGLDFÀEUREODVWV3UHWUHDWPHQWRI

FDUGLDFÀEUREODVWVZLWKFDOKH[DVSHFLÀFLQKLELWRURI&D58DQG$3%DWWHQXDWHG

the calindol- and extracellular calcium-induced increase in intracellular calcium ([Ca2+]i)

&DUGLDFÀEUREODVWSUROLIHUDWLRQDQGPLJUDWLRQZHUHDVVHVVHGE\077'LPHWK\OWKLD]RO

2-yl)-2,5-diphenyltetrazolium bromide), cell count and the cell scratch assay ECM production

was detected by expression of matrix metalloproteinase-3 and -9 (MMP-3 and -9) Activation

RI &D5 SURPRWHG FDUGLDF ÀEUREODVW SUROLIHUDWLRQ DQG PLJUDWLRQ DQG (&0 VHFUHWLRQ 0RUH

LPSRUWDQWO\FDOKH[VXSSUHVVHGFDUGLDFÀEUREODVWSUROLIHUDWLRQDQGPLJUDWLRQDQG003

DQGH[SUHVVLRQ7RIXUWKHULQYHVWLJDWHWKHHIIHFWRI&D5RQFDUGLDFÀEURVLVDPRGHORI

,62LQGXFHG FDUGLDF K\SHUWURSK\ ZDV HVWDEOLVKHG 3UHWUHDWPHQW ZLWK FDOKH[ SUHYHQWHG

FDUGLDFÀEURVLVDQGGHFUHDVHGWKHH[SUHVVLRQRI003DQGH[SUHVVLRQConclusions: Our

UHVXOWVDUHWKHÀUVWUHSRUWWKDW&D5SOD\VDQLPSRUWDQWUROHLQ&D2+ signaling involved in cardiac

ÀEURVLVWKURXJKWKHSKRVSKROLSDVH&LQRVLWROSKRVSKDWH3/&,33) pathway

ƒ”†‹ƒ…ϐ‹„”‘„Žƒ•–•ƒ”‡–Š‡ƒ‹‘Ǧ›‘…›–‡…‡ŽŽ–›’‡‹–Š‡Š‡ƒ”–Ǥ†‡”’Š›•‹‘Ž‘‰‹…ƒŽ

…‹”…—•–ƒ…‡•ǡ …ƒ”†‹ƒ… ϐ‹„”‘„Žƒ•–• ’Žƒ› ƒ ‹’‘”–ƒ– ”‘Ž‡ ‹ ƒ‹–ƒ‹‹‰ Š‘‡‘•–ƒ•‹• ‘ˆ

–Š‡ ‡š–”ƒ…‡ŽŽ—Žƒ” ƒ–”‹šǡ ‹ ƒ††‹–‹‘ –‘ ’”‘†—…‹‰ ‰”‘™–Š ˆƒ…–‘”•ǡ …›–‘‹‡• ƒ† ƒ–”‹š

‡–ƒŽŽ‘’”‘–‡‹ƒ•‡•ȋ•ȌȏͳȐǤ‘™‡˜‡”ǡ…ƒ”†‹ƒ…ϐ‹„”‘„Žƒ•–•’”‘‘–‡›‘…ƒ”†‹ƒŽ•…ƒ””‹‰

ƒ†”‡‘†‡Ž‹‰‹‹Œ—”‡†Š‡ƒ”–•‹‹•…Š‡‹…ǡŠ›’‡”–‡•‹˜‡ƒ†Š›’‡”–”‘’Š‹…Š‡ƒ”–†‹•‡ƒ•‡•Ǥ

š…‡••‹˜‡’”‘Ž‹ˆ‡”ƒ–‹‘ƒ†‹‰”ƒ–‹‘‘ˆ…ƒ”†‹ƒ…ϐ‹„”‘„Žƒ•–•”‡•—Ž–‹ϐ‹„”‘•‹•ȏʹǡ͵ȐǤ›‘…ƒ”†‹ƒŽ

ϐ‹„”‘•‹• …ƒ ƒŽ–‡” –Š‡ ‡…Šƒ‹…ƒŽ ’”‘’‡”–‹‡• ‘ˆ –Š‡ Š‡ƒ”–•ǡ ‹’ƒ‹”‹‰ …ƒ”†‹ƒ… ˆ—…–‹‘

ƒ†’”‘‘–‹‰Š‡ƒ”–ˆƒ‹Ž—”‡ȏͶȐǤ‘‡‡˜‹†‡…‡Šƒ••Š‘™–Šƒ––Š‡…›–‘•‘Ž‹……ƒŽ…‹—‹‘

ȋȏƒʹΪȐiȌƒ…–•ƒ•ƒ•‡…‘†‡••‡‰‡”–‘”‡‰—Žƒ–‡˜‡”•ƒ–‹Ž‡…‡ŽŽˆ—…–‹‘•‹…Ž—†‹‰‡š…‹–ƒ–‹‘Ǧ

…‘†‹–‹‘…‘—’Ž‹‰ǡ‰‡‡–”ƒ•…”‹’–‹‘ǡ…‡ŽŽ‰”‘™–Šǡ†‹ˆˆ‡”‡–‹ƒ–‹‘ƒ†ƒ’‘’–‘•‹•Ǥ‘™‡˜‡”ǡ

Ž‹––Ž‡ ‹ˆ‘”ƒ–‹‘ ‹’Ž‹‡• –Šƒ– –Š‡ ƒʹΪ•‹‰ƒŽ‹‰ ’ƒ–Š™ƒ› ‹• ‹˜‘Ž˜‡† ‹ …ƒ”†‹ƒ… ϐ‹„”‘„Žƒ•–

’”‘Ž‹ˆ‡”ƒ–‹‘ƒ†‡š–”ƒ…‡ŽŽ—Žƒ”ƒ–”‹šȋȌ’”‘†—…–‹‘ȏͷȐǤ

ȋȌ•‹‰ƒŽ‹‰’ƒ–Š™ƒ›ǡ”‡•—Ž–‹‰‹ƒ‹…”‡ƒ•‡‹‹‘•‹–‘Ž’Š‘•’Šƒ–‡ȋȌƒ……——Žƒ–‹‘

ƒ†ƒʹڏ‘„‹Ž‹œƒ–‹‘ȏ͸ȐǤ—”’”‡˜‹‘—•™‘”†‡‘•–”ƒ–‡†–Šƒ–ƒ‹†—…‡†…ƒ”†‹‘›‘…›–‡

ƒ’‘’–‘•‹•–Š”‘—‰Š–Š‡‹–‘…Š‘†”‹ƒŽƒ†‡†‘ȋ•ƒ”…‘Ȍ’Žƒ•‹…”‡–‹…—Ž—ƒ’‘’–‘–‹…’ƒ–Š™ƒ›

‹ –Š‡ ˆƒ‹Ž‹‰ Š‡ƒ”–Ǥ ‘”‡‘˜‡”ǡ ‘—” ”‡•—Ž–• •Š‘™‡† ‘„˜‹‘—• ›‘…ƒ”†‹ƒŽ ϐ‹„”‘•‹• ˆ‘ŽŽ‘™‹‰

ƒ†‹‹•–”ƒ–‹‘‘ˆƒƒƒ…–‹˜ƒ–‘”ȏ͹ȐǤ‘™‡˜‡”ǡƒ–’”‡•‡–ǡ‘‡˜‹†‡…‡Šƒ†•Š‘™–Šƒ–ƒ

™ƒ•‡š’”‡••‡†‹…ƒ”†‹ƒ…ϐ‹„”‘„Žƒ•–•Ǥ

–Š‹••–—†›ǡ‘—””‡•—Ž–•†‡‘•–”ƒ–‡–Šƒ–ƒ‹•’”‡•‡–‹”ƒ–…ƒ”†‹ƒ…ϐ‹„”‘„Žƒ•–•Ǥ‡

‹†‡–‹ϐ‹‡†„‘–Š–Š‡’”‘–‡‹Ž‡˜‡Ž•ƒ†–Š‡ˆ—…–‹‘ƒŽ”‡‰—Žƒ–‹‘‘ˆƒ‹…ƒ”†‹ƒ…ϐ‹„”‘„Žƒ•–•Ǥ

Materials and Methods

Materials

•‘’”‘–‡”‡‘ŽȋȌǡ…ƒŽ‹†‘Žǡ…ƒŽŠ‡šʹ͵ͳǡ•‘†‹—–ƒ—”‘—”•‘†‡‘š›…Š‘Žƒ–‡™‡”‡’—”…Šƒ•‡†ˆ”‘‹‰ƒǦ

‡‘ƒ–ƒŽ”ƒ–…ƒ”†‹ƒ…ϔ‹„”‘„Žƒ•–•‹…—„ƒ–‹‘

‡‘ƒ–ƒŽ ”ƒ– …ƒ”†‹ƒ… ϐ‹„”‘„Žƒ•–• ™‡”‡ ’”‡’ƒ”‡† ˆ”‘ ʹǦ –‘ ͵Ǧ†ƒ›Ǧ‘Ž† ‡‘ƒ–ƒŽ ‹•–ƒ” ”ƒ–• ȋ‹ƒŽ

‡•‡ƒ”…Š •–‹–—–‡ ‘ˆ ƒ”„‹ ‡†‹…ƒŽ ‹˜‡”•‹–›ǡ Š‹ƒȌǤ Š‡ ”ƒ–• ™‡”‡ ƒ‡•–Š‡–‹œ‡† ƒ† •ƒ…”‹ϐ‹…‡† „›

‹‡”•‹‘ ‹–‘ ͹ͷΨ ȋ˜Ȁ˜Ȍ ƒŽ…‘Š‘ŽǤ Š‡ ˜‡–”‹…Ž‡• ™‡”‡ ”‡‘˜‡† ƒ† ™ƒ•Š‡† –Š”‡‡ –‹‡• ‹ Ǧƒ•

„ƒŽƒ…‡†•ƒŽ–•‘Ž—–‹‘ȋ‘ŽȀǣͲǤͶŽǡͲǤͲ͸ʹͶǡͺǤͲƒŽǡͲǤ͵ͷƒ͵ǡƒ†ͲǤͲ͸ƒʹͶ͹ʹǡ’

͹ǤʹȌƒ–Ͷιǡ–Š‡‹…‡†ƒ†‹…—„ƒ–‡†™‹–ŠͲǤʹͷΨȋ™Ȁ˜Ȍ–”›’•‹ˆ‘”ͳͲ‹ƒ–͵͹ιǤ‘–‡”‹ƒ–‡–Š‡

†‹‰‡•–‹‘ǡ ƒ ‡“—ƒŽ ˜‘Ž—‡ ‘ˆ …‘Ž† —Ž„‡……‘ǯ• ‘†‹ϐ‹‡†ƒ‰Ž‡ǯ• ‡†‹— ȋȌ …‘–ƒ‹‹‰ ͳͲ Ψ ȋ˜Ȁ˜Ȍ

‡™„‘”…ƒŽˆ•‡”—™ƒ•ƒ††‡†ǤŠ‡•—’‡”ƒ–ƒ–™ƒ•†‹•…ƒ”†‡†Ǥ‡ŽŽ•™‡”‡‹…—„ƒ–‡†™‹–Šˆ”‡•ŠͲǤʹͷΨ

–”›’•‹ˆ‘”ͳͷ‹ƒ–͵͹ιǡƒ†–Š‡•—’‡”ƒ–ƒ–™ƒ•…‘ŽŽ‡…–‡†ǤŠ‡Žƒ––‡”†‹‰‡•–‹‘•–‡’™ƒ•”‡’‡ƒ–‡†ˆ‘—”

–‹‡•Ǥ ‡ŽŽ• ‹ –Š‡ •—’‡”ƒ–ƒ– ™‡”‡ –Š‡ ‹•‘Žƒ–‡† „› …‡–”‹ˆ—‰ƒ–‹‘ ˆ‘” ͳ͵ ‹ ƒ– ʹͶͲͲ ‰Ȁ‹ ƒ– ”‘‘

–‡’‡”ƒ–—”‡Ǥ ‡ŽŽ• ™‡”‡ ”‡Ǧ•—•’‡†‡† ‹  …‘–ƒ‹‹‰ ʹͲ Ψ ȋ˜Ȁ˜Ȍ ‡™„‘” …ƒŽˆ •‡”—ǡ ͳͲͲ ȀŽ

’‡‹…‹ŽŽ‹ƒ†ͳͲͲ‰ȀŽ•–”‡’–‘›…‹ǡƒ†–Š‡™‡”‡…—Ž–—”‡†ƒ•‘‘Žƒ›‡”•ƒ–ƒ†‡•‹–›‘ˆͷέͳͲ Ͷ …‡ŽŽ•Ȁ

… ʹ ƒ–͵͹鋐ƒŠ—‹†‹ϐ‹‡†ƒ–‘•’Š‡”‡…‘–ƒ‹‹‰ͷΨȋ˜Ȁ˜ȌʹǤˆ–‡”ͳŠ‹…—„ƒ–‹‘‹ϐŽƒ••ƒ–͵͹ιǡ

‹ƒŠ—‹†‹ϐ‹‡†ƒ–‘•’Š‡”‡™‹–ŠͷΨʹǡ–Š‡—ƒ––ƒ…Š‡†…‡ŽŽ•™‡”‡†‹•…ƒ”†‡†ƒ†–Š‡ƒ––ƒ…Š‡†…‡ŽŽ•™‡”‡

…‘–‹—‘—•Ž›…—Ž–—”‡†ǤŠ‡‡†‹—™ƒ•…Šƒ‰‡†–™‘–‹‡•’‡”™‡‡Ǥ

Experimental protocol

Š”‡‡†ƒ›•ƒˆ–‡”„‡‹‰•‡‡†‡†ǡ–Š‡…ƒ”†‹ƒ…ϐ‹„”‘„Žƒ•–•™‡”‡†‹˜‹†‡†”ƒ†‘Ž›‹–‘ˆ‘—”‰”‘—’•ǣȋͳȌ

…‘–”‘Ž‰”‘—’ǣ‘–”‡ƒ–‡–ǢȋʹȌƒ‰‹‘–‡•‹ȋ‰Ȍ ‰”‘—’ǣ’”‡Ǧ–”‡ƒ–‡†™‹–ŠͳͲͲ‘ŽȀ‰ ˆ‘ŽŽ‘™‡†

„›–”‡ƒ–‡–™‹–ŠʹǤͷƒŽʹˆ‘”ʹͶŠǢȋ͵ȌƒŽ‹†‘ŽΪƒ‰‹‘–‡•‹ȋ‰Ȍ ‰”‘—’ǣ’”‡Ǧ‹…—„ƒ–‡†™‹–ŠͳͲͲ

’”‡Ǧ‹…—„ƒ–‡†™‹–ŠͳͲͲ‘ŽȀ‰ ˆ‘ŽŽ‘™‡†„›–”‡ƒ–‡–™‹–ŠʹɊ…ƒŽŠ‡šʹ͵ͳˆ‘”ʹͶŠǤ

—‘ϔŽ—‘”‡•…‡–ƒ†‹—‘Š‹•–‘…Š‡‹…ƒŽ•–ƒ‹‹‰

ƒ”ƒˆϐ‹Ǧ‡„‡††‡†–‹••—‡•‡…–‹‘•ȋͲǤʹɊȌ™‡”‡†‡’ƒ”ƒˆϐ‹‹œ‡†™‹–Šš›Ž‡‡•ƒ†”‡Š›†”ƒ–‡†™‹–Š

‰”ƒ†‡† ™ƒ•Š‡• ™‹–Š ‡–Šƒ‘Ž ˆ‘ŽŽ‘™‡† „› ’Š‘•’Šƒ–‡Ǧ„—ˆˆ‡”‡† •ƒŽ‹‡ ȋȌǤ Š‡ǡ ‡ƒ…Š •Ž‹…‡ ™ƒ• –”‡ƒ–‡†

™‹–Š͵ͲɊŽ͵Ψʹʹȋ”‡ƒ‰‡–Ȍǡ‹…—„ƒ–‡†ƒ–”‘‘–‡’‡”ƒ–—”‡ˆ‘”ʹͲ‹ƒ†™ƒ•Š‡†–™‹…‡™‹–ŠǤ

Š‡ǡ͵ͲɊŽ‰‘ƒ–•‡”—ȋ”‡ƒ‰‡–Ȍ™ƒ•ƒ††‡†ǡˆ‘ŽŽ‘™‡†„›‹…—„ƒ–‹‘ƒ–”‘‘–‡’‡”ƒ–—”‡ˆ‘”ʹͲ‹ƒ†

–™‘™ƒ•Š‡•™‹–ŠǤƒ…Š•Ž‹…‡™ƒ•‹…—„ƒ–‡†‹͵ͲɊŽ’”‹ƒ”›ƒ–‹„‘†›ȋ‘—•‡ƒ–‹Ǧ”ƒ–ƒ‘‘…Ž‘ƒŽ

ƒ–‹„‘†›ǡͳǣʹͲͲ†‹Ž—–‹‘Ȍƒ†’Žƒ…‡†‹ƒ™‡–„‘šƒ–Ͷι‘˜‡”‹‰Š–Ǥˆ–‡”™ƒ•Š‹‰™‹–Šǡ–Š‡•Ž‹…‡•™‡”‡

‹…—„ƒ–‡†‹͵ͲɊŽ„‹‘–‹›Žƒ–‡†’‘Ž›…Ž‘ƒŽ•‡…‘†ƒ”›ƒ–‹„‘†›ȋ”‡ƒ‰‡–Ȍƒ–”‘‘–‡’‡”ƒ–—”‡ˆ‘”͵Ͳ‹ǡ

ˆ‘ŽŽ‘™‡† „› ™ƒ•Š‹‰ ™‹–Š Ǥ Š‡ †‹ƒ‹‘„‡œ‹†‹‡ ȋȌ ‡–Š‘† ™ƒ• —•‡† ˆ‘” …‘Ž‘” †‡˜‡Ž‘’‡–ǡ

ˆ‘ŽŽ‘™‡† „› ™ƒ•Š‹‰ ™‹–Š –ƒ’ ™ƒ–‡”Ǥ Ž‹…‡• ™‡”‡ –Š‡ …‘—–‡”•–ƒ‹‡† ™‹–Š Š‡ƒ–‘š›Ž‹ǡ ‹…—„ƒ–‡† ‹

ƒ‘‹ƒǡ†‡Š›†”ƒ–‡†™‹–Š‰”ƒ†‹‡–‡–Šƒ‘Žǡ–”ƒ•’ƒ”‡–‹œ‡†™‹–Šš›Ž‡‡•ƒ†ϐ‹ƒŽŽ›•‡ƒŽ‡†™‹–Š‡—–”ƒŽ

‰—Ǥ‡ŽŽ•™‹–Š„”‘™•–ƒ‹‹‰’ƒ”–‹…Ž‡•‹–Š‡‹”…›–‘’Žƒ•ƒ†—…Ž‡—•™‡”‡†‡‘–‡†’‘•‹–‹˜‡—†‡”ƒŽ‹‰Š–

‹…”‘•…‘’‡Ǥ

›–‘’Žƒ•‹…’”‘–‡‹‹•‘Žƒ–‹‘ˆ”‘…ƒ”†‹ƒ…ϔ‹„”‘„Žƒ•–•

”‘–‡‹‹•‘Žƒ–‹‘ƒ†ƒƒŽ›•‹•™‡”‡’‡”ˆ‘”‡†ƒ•†‡•…”‹„‡†’”‡˜‹‘—•Ž›ȏͺȐǤ”‹‡ϐŽ›ǡ™Š‘Ž‡Ǧ…‡ŽŽŽ›•ƒ–‡•

™‡”‡…‘ŽŽ‡…–‡†‹Ž›•‹•„—ˆˆ‡”ȋ͸ʹǤͷ‘ŽȀ‘ˆ”‹•ǦŽǡʹ‘ŽȀ‘ˆǡʹǤ͵Ψƒ†ͳͲΨ‰Ž›…‡”‘Žȏ’

͸ǤͺȐȌǡ™‹–Šƒ’”‘–‡ƒ•‡‹Š‹„‹–‘”‹š–—”‡Ȍǡƒ†–‘–ƒŽ’”‘–‡‹…‘–‡–™ƒ•†‡–‡”‹‡†„›–Š‡„‹…‹…Š‘‹‹…

‹–”‘…‡ŽŽ—Ž‘•‡—•‹‰•–ƒ†ƒ”†–‡…Š‹“—‡•Ǥ

‡ƒ•—”‡‡–‘ˆ‹–”ƒ…‡ŽŽ—Žƒ”…ƒŽ…‹—‹…ƒ”†‹ƒ…ϔ‹„”‘„Žƒ•–•

ƒ”†‹ƒ…ϐ‹„”‘„Žƒ•–•™‡”‡Ž‘ƒ†‡†™‹–ŠͳɊ‘ŽȀ Ž‘—ǦͶȀƒ–͵͹ιˆ‘”͵Ͳ‹—–‡•‹„—ˆˆ‡”‡†

•ƒŽ‹‡ȋ‹‘ŽȀǣͳ͵ͲƒŽǡͷŽǡͳͲ‰Ž—…‘•‡ǡͳ‰ŽʹǡͳǤͲƒŽʹǡƒ†ʹͷȏ’͹ǤͶȐȌǡƒ†‰”‘—’•‘ˆͷ

–‘ͺ…‡ŽŽ•™‡”‡‘‹–‘”‡†—•‹‰ƒ‹˜‡”–‡†Ž›’—•Ǧ͹Ͳ‹…”‘•…‘’‡Ǥ’‡…–”‘ϐŽ—‘”‘‡–”‹…‡ƒ•—”‡‡–•

™‡”‡…‘ŽŽ‡…–‡†—•‹‰–Š‡‡Ž–ƒ…ƒ›•–‡•’‡…–”‘ϐŽ—‘”‘‡–‡”ȋŠ‘–‘‡…Š‘Ž‘‰›Ȍǡ™Š‡”‡–Š‡ϐ‹‡Ž†™ƒ•

‡š…‹–‡†ƒ–Ͷͺͺǡ™‹–Š‡‹••‹‘ƒ–ͷ͵ͲȏͻȐǤ

MTT Proliferation Assay

ƒ”†‹ƒ…ϐ‹„”‘„Žƒ•–•ȋͳšͳͲ ͷ …‡ŽŽ•’‡”™‡ŽŽȌ™‡”‡•‡‡†‡†‹͸Ǧ™‡ŽŽ’Žƒ–‡•ƒ†–”‡ƒ–‡†™‹–Š‘”™‹–Š‘—–

†‹ˆˆ‡”‡– ”‡ƒ‰‡–• ˆ‘” Ͷͺ Š‘—”•Ǥ Š‡”‡ƒˆ–‡”ǡ  ȋȋ͵ǦȋͶǡͷǦ‹‡–Š›Ž–Š‹ƒœ‘ŽǦʹǦ›ŽȌǦʹǡͷǦ†‹’Š‡›Ž–‡–”ƒœ‘Ž‹—

„”‘‹†‡Ȍȋͷ‰ȀȌ™ƒ•ƒ††‡†–‘‡ƒ…Š™‡ŽŽǡƒ†’Žƒ–‡•™‡”‡‹…—„ƒ–‡†ƒ–͵͹ιˆ‘”ͶȂ͸Š‘—”•ǤŠ‡‡†‹—

™ƒ•–Š‡”‡’Žƒ…‡†™‹–ŠͳͷͲɊ†‹‡–Š›Ž•—Žˆ‘š‹†‡ȋȌƒ†ƒ‰‹–ƒ–‡†ˆ‘”ͳͲ‹—–‡•Ǥ„•‘”„ƒ…‡™ƒ•

‡ƒ•—”‡†ƒ–ͶͻͲ—•‹‰ƒ‹…”‘’Žƒ–‡”‡ƒ†‡”Ǥ

‡ŽŽ‘—–‹‰

ƒ”†‹ƒ…ϐ‹„”‘„Žƒ•–•ȋͳšͳͲ ͷ ’‡”™‡ŽŽȌ™‡”‡•‡‡†‡†‹͸Ǧ™‡ŽŽ’Žƒ–‡•ƒ†–”‡ƒ–‡†™‹–Š‘”™‹–Š‘—–†‹ˆˆ‡”‡–

”‡ƒ‰‡–•ˆ‘”ƒ†‡•‹‰ƒ–‡†’‡”‹‘†ǤŠ‡…‡ŽŽ•™‡”‡–Š‡†‹‰‡•–‡†„›ͲǤʹͷΨ–”›’•‹ˆ‘”͸Ȃͺ‹—–‡•ƒ–͵͹ιǤ

Š‡…‡ŽŽ—„‡”™ƒ•…‘—–‡†—•‹‰ƒŠ‡‘…›–‘‡–‡”Ǥ

…”ƒ–…Š™‘—†”‡’ƒ‹”ƒ••ƒ›

ƒ–…ƒ”†‹ƒ…ϐ‹„”‘„Žƒ•–•™‡”‡–”‡ƒ–‡†™‹–Š…ƒŽ…‹—ǡ…ƒŽ‹†‘Žƒ†…ƒŽŠ‡šʹ͵ͳǡƒ†–Š‡•—„Œ‡…–‡†–‘–Š‡

in vitro •…”ƒ–…Šƒ••ƒ›ƒ•†‡•…”‹„‡†’”‡˜‹‘—•Ž›ȏͳͲǡͳͳȐǤƒ‰‡•™‡”‡…ƒ’–—”‡†ƒ–ͲǡʹͶƒ†ͶͺŠ‘—”•ƒˆ–‡”

–”‡ƒ–‡–—•‹‰’Šƒ•‡Ǧ…‘–”ƒ•–‹…”‘•…‘’›Ǥ

Animals

ƒŽ‡‹•–ƒ””ƒ–•ȋʹͲͲȂʹͷͲ‰Ȍ™‡”‡‘„–ƒ‹‡†ˆ”‘–Š‡š’‡”‹‡–ƒŽ‹ƒŽ‡–‡”‘ˆƒ”„‹‡†‹…ƒŽ

ˆ‘”–Š‡ƒ”‡ƒ†•‡‘ˆƒ„‘”ƒ–‘”›‹ƒŽ•ǯǯ’—„Ž‹•Š‡†„›–Š‡Š‹‡•‡ƒ–‹‘ƒŽ•–‹–—–‡•‘ˆ‡ƒŽ–ŠǤŠ‡

•–—†› ™ƒ• ƒ’’”‘˜‡† „› –Š‡ •–‹–—–‹‘ƒŽ ‹ƒŽ ‡•‡ƒ”…Š ‘‹––‡‡ ‘ˆ ƒ”„‹ ‡†‹…ƒŽ ‹˜‡”•‹–›Ǥ ŽŽ

ƒ‹ƒŽ•™‡”‡Š‘—•‡†ƒ––Š‡ƒ‹ƒŽ…ƒ”‡ˆƒ…‹Ž‹–›‘ˆƒ”„‹‡†‹…ƒŽ‹˜‡”•‹–›ƒ–ʹͷι™‹–ŠͳʹȀͳʹŠŽ‹‰Š–Ȁ

™‡”‡”ƒ†‘Ž›ƒ••‹‰‡†–‘†‹ˆˆ‡”‡––”‡ƒ–‡–‰”‘—’•Ǥ

š’‡”‹‡–ƒŽ‰”‘—’•

ȾǦ†”‡‡”‰‹…•–‹—Žƒ–‹‘…ƒ„‡‹†—…‡†„›ƒŠ‹‰Š†‘•‡‘ˆ‹•‘’”‘–‡”‡‘ŽȋȌǡ‹Œ‡…–‡†•—„…—–ƒ‡‘—•Ž›

ƒ•”‡’‘”–‡†„‡ˆ‘”‡ȏͳʹȐǤ‹š–›”ƒ–•™‡”‡”ƒ†‘Ž›†‹˜‹†‡†‹–‘ˆ‘—”‰”‘—’•Ǥ‰”‘—’ͳȋαͳͲȌǡ–Š‡”ƒ–•

™‡”‡•—„…—–ƒ‡‘—•Ž›‹Œ‡…–‡†™‹–Š•ƒŽ‹‡ˆ‘”Ͷ†ƒ›•ƒ•ƒ…‘–”‘ŽǤ‰”‘—’ʹȋǡαͳͷȌǡ–Š‡”ƒ–•™‡”‡

•—„…—–ƒ‡‘—•Ž›‹Œ‡…–‡†™‹–Šȋͳ͹Ͳ‰Ȁ‰Ȁ†‹•ƒŽ‹‡Ȍˆ‘”Ͷ†ƒ›•–‘‹†—…‡…ƒ”†‹ƒ…Š›’‡”–”‘’Š›Ǥ‰”‘—’

͵ȋǦǡαͳͺȌǡ–Š‡”ƒ–•™‡”‡•—„…—–ƒ‡‘—•Ž›‹Œ‡…–‡†™‹–Š…ƒŽ‹†‘ŽȋͳͲɊ‘ŽȀ‰Ȁ†‹•ƒŽ‹‡Ȍˆ‘”Ͷ™‡‡•

ƒˆ–‡”ƒ†‹‹•–”ƒ–‹‘‘ˆˆ‘”Ͷ†ƒ›•ǤƒŽ‹†‘Ž‹•ƒ•’‡…‹ϐ‹…ƒ…–‹˜ƒ–‘”‘ˆƒǤ‰”‘—’ͶȋǦǡαͳ͹Ȍǡ–Š‡”ƒ–•

™‡”‡•—„…—–ƒ‡‘—•Ž›‹Œ‡…–‡†™‹–Š…ƒŽŠ‡šʹ͵ͳȋͳͲɊ‘ŽȀ‰Ȁ†‹•ƒŽ‹‡Ȍˆ‘”Ͷ™‡‡•ƒˆ–‡”ƒ†‹‹•–”ƒ–‹‘‘ˆ

ˆ‘”Ͷ†ƒ›•ǤƒŽŠ‡šʹ͵ͳ‹•ƒ•’‡…‹ϐ‹…‹Š‹„‹–‘”‘ˆƒǤ

‡‘†›ƒ‹…‡ƒ•—”‡‡–

…Š‘…ƒ”†‹‘‰”ƒ•™‡”‡’‡”ˆ‘”‡†‘•‡ŽˆǦ„”‡ƒ–Š‹‰”ƒ–•—†‡”ƒ‡•–Š‡•‹ƒȋ‹–”ƒ’‡”‹–‘‡ƒŽ‹Œ‡…–‹‘‘ˆͳͲ

Ψ…ŠŽ‘”ƒŽŠ›†”ƒ–‡ƒ–ͲǤ͵ŽȀͳͲͲ‰„‘†›™‡‹‰Š–ȌǤ‡ˆ–˜‡–”‹…—Žƒ”ȋȌ’ƒ”ƒ‡–‡”•‡ƒ•—”‡†‹…Ž—†‡‹Ž‡–

˜‡–”‹…—Žƒ”•‡’–ƒŽ†‡ˆ‡…–•ȋ†Ȍƒ†Ž‡ˆ–˜‡–”‹…—Žƒ”‡Œ‡…–‹‘ˆ”ƒ…–‹‘ȋ ȌǤŽŽ’ƒ”ƒ‡–‡”•”‡’”‡•‡––Š‡

‡ƒ•‘ˆϐ‹˜‡…‘•‡…—–‹˜‡…ƒ”†‹ƒ……›…Ž‡•ȏͳ͵ȐǤ

ƒ••‘ǯ••–ƒ‹‹‰

‡ƒ”–•ˆ”‘†‹ˆˆ‡”‡–‰”‘—’•ǡϐ‹š‡†‹ͳͲΨˆ‘”ƒŽ‹ǡ™‡”‡’”‘…‡••‡†ƒ†’ƒ”ƒˆϐ‹‡„‡††‡†Ǥ‡ƒ”–

•‡…–‹‘• ȋͶ Ȍ ™‡”‡ –Š‡ •–ƒ‹‡† ™‹–Š ƒ••‘ǯ• –”‹…Š”‘‡ ”‡ƒ‰‡– ȏͳͶȐǤ Š‡ ’‡”…‡– ‘ˆ ϐ‹„”‘•‹• ȋ„Ž—‡

•–ƒ‹‹‰Ȍ–‘–‘–ƒŽ–‹••—‡™ƒ•ƒƒŽ›œ‡†ƒ†…ƒŽ…—Žƒ–‡†„›–Š‡Ǧ“—ƒ–‹–ƒ–‹˜‡ƒ—–‘ƒ–‹…’”‘‰”ƒ

„Ž‹†ˆƒ•Š‹‘Ǥ‡”‹ƒŽǡ–”ƒ•˜‡”•‡…”›‘•‡…–‹‘•ȋ͹–Š‹…Ȍ‘ˆŠ‡ƒ”–•™‡”‡…—–™‹–Š–Š‡ͳͻͷͲ ”‹‰‘…—–

Statistical analysis

ƒ–ƒƒ”‡‡š’”‡••‡†ƒ•–Š‡‡ƒάǤ‡Ǧ™ƒ›ƒƒŽ›•‹•‘ˆ˜ƒ”‹ƒ…‡ȋȌ™ƒ•—•‡†–‘†‡–‡”‹‡

•–ƒ–‹•–‹…ƒŽ•‹‰‹ϐ‹…ƒ…‡„‡–™‡‡‰”‘—’•ƒ†ƒŽ•‘–Š‡–™‘Ǧ–ƒ‹Ž‡†–—†‡–ǯ•–Ǧ–‡•–Ǥ’”‘„ƒ„‹Ž‹–›˜ƒŽ—‡‘ˆ’δͲǤͲͷ

™ƒ•–ƒ‡–‘‹†‹…ƒ–‡•–ƒ–‹•–‹…ƒŽ•‹‰‹ϐ‹…ƒ…‡Ǥ

Results

”‘–‡‹‡š’”‡••‹‘‘ˆƒ‹”ƒ–‡‘ƒ–ƒŽ…ƒ”†‹ƒ…ϔ‹„”‘„Žƒ•–•

‘ †‡–‡”‹‡ ™Š‡–Š‡” ƒ ‹• ‡š’”‡••‡† ‹ …ƒ”†‹ƒ… ϐ‹„”‘„Žƒ•–•ǡ ƒ ‡š’”‡••‹‘ ™ƒ•

†‡–‡…–‡† „› ‹—‘ϐŽ—‘”‡•…‡…‡ ƒ† ‹—‘Š‹•–‘…Š‡‹…ƒŽ •–ƒ‹‹‰Ǥ  ‡‘ƒ–ƒŽ …ƒ”†‹ƒ…

ϐ‹„”‘„Žƒ•–•ǡ †‡‡’ ›‡ŽŽ‘™ ‹—‘•–ƒ‹‹‰ ‘” ‰”‡‡ ϐŽ—‘”‡•…‡…‡ ™ƒ• ’”‡•‡– –Š”‘—‰Š‘—–

ƒŽŽ…ƒ”†‹ƒ…ϐ‹„”‘„Žƒ•–•‹†‹…ƒ–‹‰–Š‡‡š’”‡••‹‘‘ˆƒƒ––Š‡’”‘–‡‹Ž‡˜‡Ž‹–Š‡•‡…‡ŽŽ•Ǥ

Žƒ…‘ˆ•’‡…‹ϐ‹…•–ƒ‹‹‰™ƒ•†‡‘•–”ƒ–‡†‹–Š‡…‘–”‘Ž‰”‘—’ǡ‹–Š‡ƒ„•‡…‡‘ˆƒ–‹Ǧƒ

ƒ–‹„‘†›ȋ ‹‰Ǥͳƒƒ†„ȌǤ

Š‡ ‡š’”‡••‹‘ ‘ˆ ƒ ’”‘–‡‹ ™ƒ• ƒŽ•‘ ‡šƒ‹‡† ‹ ‡‘ƒ–ƒŽ …ƒ”†‹ƒ… ϐ‹„”‘„Žƒ•–• „›

™‡•–‡”„Ž‘––‹‰Ǥƒ’”‘–‡‹•™‹–Šƒ”‡Žƒ–‹˜‡‘Ž‡…—Žƒ”ƒ••„‡–™‡‡ͳʹͲǦͳͶͲƒ™‡”‡

†‡–‡…–‡†‹‡‘ƒ–ƒŽ…ƒ”†‹ƒ…ϐ‹„”‘„Žƒ•–•ȋ ‹‰Ǥͳ…ȌǤ —”–Š‡”‘”‡ǡ’”‡ƒ†•‘”’–‹‘‘ˆƒ–‹Ǧƒ

ƒ–‹„‘†›™‹–Š•–ƒ†ƒ”†ƒƒ–‹‰‡‡Ž‹‹ƒ–‡†–Š‡ͳ͵Ͳƒ„ƒ†Ǥ‘‰‡–Š‡”ǡ–Š‡•‡”‡•—Ž–•

‹†‹…ƒ–‡–Šƒ–ƒ‹•‡š’”‡••‡†‹‡‘ƒ–ƒŽ…ƒ”†‹ƒ…ϐ‹„”‘„Žƒ•–•Ǥ

…–‹˜ƒ–‹‘‘ˆƒ‡Ž‡˜ƒ–‡†–Š‡‹–”ƒ…‡ŽŽ—Žƒ”…ƒŽ…‹—…‘…‡–”ƒ–‹‘–Š”‘—‰Š–Š‡Ǧ͹

’ƒ–Š™ƒ›

‘ …‘ϐ‹” –Šƒ– ƒ ˆ—…–‹‘ƒŽŽ› ”‡‰—Žƒ–‡• ‹–”ƒ…‡ŽŽ—Žƒ” …ƒŽ…‹— …‘…‡–”ƒ–‹‘ ‹

‡‘ƒ–ƒŽ…ƒ”†‹ƒ…ϐ‹„”‘„Žƒ•–•ǡ…‡ŽŽ•™‡”‡–”‡ƒ–‡†™‹–ŠʹǤͷƒŽʹǡ‹ŽʹȋͳȌǡƒ‹Š‹„‹–‘”‘ˆ

–Š‡ƒΪǦƒʹΪ‡š…Šƒ‰‡”ǡƒ††ŽʹȋʹͲͲɊȌǡƒ‹Š‹„‹–‘”‘ˆ–Š‡Ǧ–›’‡…ƒŽ…‹—…Šƒ‡Žǡ‹–Š‡

‹‹Žƒ”Ž›ǡ ƒ†‹‹•–”ƒ–‹‘ ‘ˆ ͳͲ Ɋ …ƒŽ‹†‘Ž ‡˜‘‡† ƒ ‹…”‡ƒ•‡ ‹ ‹–”ƒ…‡ŽŽ—Žƒ” …ƒŽ…‹—

…‘…‡–”ƒ–‹‘ ‹ ‘”‡ –Šƒ ͻͷ Ψ ‘ˆ ‡‘ƒ–ƒŽ …ƒ”†‹ƒ… ϐ‹„”‘„Žƒ•–• ‹ ƒ ‰‹˜‡ ‘„•‡”˜ƒ–‹‘

ϐ‹‡Ž†Ǥˆ–‡”’”‡–”‡ƒ–‡–™‹–Š…ƒŽŠ‡šʹ͵ͳǡƒ•’‡…‹ϐ‹…‹Š‹„‹–‘”‘ˆƒǡ‹–”ƒ…‡ŽŽ—Žƒ”…ƒŽ…‹—

…‘…‡–”ƒ–‹‘™ƒ•‘–•‹‰‹ϐ‹…ƒ–Ž›‹…”‡ƒ•‡†…‘’ƒ”‡†–‘ƒ†‹‹•–”ƒ–‹‘‘ˆ…ƒŽ‹†‘ŽƒŽ‘‡Ǥ

™Š‹…Š ‹Š‹„‹–• ”‡ϐ‹ŽŽ‹‰ ‘ˆ –Š‡ ͵Ǧ•‡•‹–‹˜‡ …ƒŽ…‹— ”‡Ž‡ƒ•‡ ’‘‘Ž•Ǥ —„•‡“—‡–Ž›ǡ …‡ŽŽ•

Fig 1.ƒ‹•‡š’”‡••‡†‹‡‘ƒ–ƒŽ…ƒ”†‹ƒ…ϐ‹„”‘„Žƒ•–•ǤȋƒȌ—‘ϐŽ—‘”‡•…‡…‡•–ƒ‹‹‰ˆ‘”ƒȋ‰”‡‡Ȍ

ƒ†ȋ„Ž—‡ȌǤȋ„Ȍ—‘Š‹•–‘…Š‡‹•–”›ˆ‘”ƒȋ›‡ŽŽ‘™Ȍ’”‘–‡‹‡š’”‡••‹‘‹‡‘ƒ–ƒŽ…ƒ”†‹ƒ…ϐ‹„”‘Ǧ

„Žƒ•–•ǤŠ‡„ƒ”‰”ƒ’Š•Š‘™•–Š‡“—ƒ–‹ϐ‹…ƒ–‹‘‘ˆ–Š‡ƒ”‡ƒ•‘ˆ’‘•‹–‹˜‡…‡ŽŽ•ȋ”‹‰Š–’ƒ‡ŽǢαͺȌǤƒ–ƒƒ”‡’”‡Ǧ

•‡–‡†ƒ•–Š‡‡ƒάǤƒ‰‹ϐ‹…ƒ–‹‘ǣέͶͲͲǤȋ…Ȍ‡•–‡”„Ž‘––‹‰ƒƒŽ›•‹•‘ˆƒ‡š’”‡••‹‘‹‡‘ƒ–ƒŽ

…ƒ”†‹ƒ…ϐ‹„”‘„Žƒ•–•Ǥ

Fig 2. ƒ ƒ…–‹˜ƒ–‹‘ ‹†—…‡• ƒʹΪ

”‡Ž‡ƒ•‡ˆ”‘–Š‡‹‡‘ƒ–ƒŽ…ƒ”Ǧ

†‹ƒ… ϐ‹„”‘„Žƒ•–•Ǥ ȋƒȌ ƒ ƒ…–‹˜ƒ–‹‘

‹†—…‡† ƒ ʹΪ  ”‡Ž‡ƒ•‡ ˆ”‘ –Š‡ 

™‹–Š–Š‡–”‡ƒ–‡–‘ˆʹǤͷƒŽʹǤ

ȋ„Ȍ ƒŽ‹†‘Ž ‹†—…‡† ƒ ʹΪ  ”‡Ž‡ƒ•‡

ˆ”‘ –Š‡ Ǥ Š‡ …Šƒ‰‡ ‹ ϐŽ—‘”‡Ǧ

•…‡…‡ ‹–‡•‹–›ǡ …‘””‡•’‘†‹‰ –‘

‹–”ƒ…‡ŽŽ—Žƒ” …ƒŽ…‹— …‘…‡–”ƒ–‹‘ǡ

™ƒ• ‡ƒ•—”‡† „› Ž—‘ǦͶ  •–ƒ‹Ǧ

‹‰ǡȋαͶǦͷȌǤȗ’δͲǤͲͷ˜•Ǥ…‘–”‘ŽǤ

‹–”ƒ…‡ŽŽ—Žƒ” …ƒŽ…‹— …‘…‡–”ƒ–‹‘Ǥ ”‡‹…—„ƒ–‹‘ ‘ˆ ‡‘ƒ–ƒŽ …ƒ”†‹ƒ… ϐ‹„”‘„Žƒ•–• ˆ‘” ͳͲ

‹ ™‹–Š ͹͵ͳʹʹǡ ƒ ’Š‘•’Šƒ–‹†›Ž‹‘•‹–‘ŽǦ•’‡…‹ϐ‹…  „Ž‘…‡”ǡ ƒ„‘Ž‹•Š‡† –Š‡ ‹…”‡ƒ•‡ ‹

‹–”ƒ…‡ŽŽ—Žƒ”…ƒŽ…‹—‹†—…‡†„›ͳͲɊ…ƒŽ‹†‘Ž‘”ʹǤͷƒŽʹǤ”‡–”‡ƒ–‡–™‹–ŠʹǦǡ

ƒ͵”‡…‡’–‘”Ǧ•’‡…‹ϐ‹…‹Š‹„‹–‘”ǡˆ‘”ͳͲ‹‡Ž‹‹ƒ–‡†–Š‡‡ˆˆ‡…–‘ˆͳͲɊ…ƒŽ‹†‘Žƒ†ʹǤͷ

ƒŽʹ‘–Š‡‹†—…–‹‘‘ˆ‹–”ƒ…‡ŽŽ—Žƒ”…ƒŽ…‹—”‡Ž‡ƒ•‡ȋ ‹‰ǤʹȌǤŠ‡•‡”‡•—Ž–•†‡‘•–”ƒ–‡

–Šƒ– –Š‡ ƒ…–‹˜ƒ–‹‘ ‘ˆ ƒ ”‡•—Ž–‡† ‹ ‹–”ƒ…‡ŽŽ—Žƒ” …ƒŽ…‹— ‹…”‡ƒ•‡ ‹ ‡‘ƒ–ƒŽ …ƒ”†‹ƒ…

ϐ‹„”‘„Žƒ•–•–Š”‘—‰Š•–‹—Žƒ–‹‘‘ˆ–Š‡Ǧ͵’ƒ–Š™ƒ›Ǥ

ˆˆ‡…–•‘ˆƒ‘…‡ŽŽ’”‘Ž‹ˆ‡”ƒ–‹‘ƒ†‹‰”ƒ–‹‘‹‡‘ƒ–ƒŽ…ƒ”†‹ƒ…ϔ‹„”‘„Žƒ•–•

‘‹˜‡•–‹‰ƒ–‡™Š‡–Š‡”‡‘ƒ–ƒŽ…ƒ”†‹ƒ…ϐ‹„”‘„Žƒ•–’”‘Ž‹ˆ‡”ƒ–‹‘‹•”‡‰—Žƒ–‡†„›ƒǡ–Š‡

…‡ŽŽ•™‡”‡•–ƒ”˜‡†‘˜‡”‹‰Š–ƒ†’”‡–”‡ƒ–‡†™‹–Šƒƒ…–‹˜ƒ–‘”•ǡ‡‹–Š‡”ƒŽʹȋʹǤͷȌ‘”

…ƒŽ‹†‘ŽȋͳͲɊȌǡˆ‘”ͳŠ‘—”ƒ†–Š‡‹…—„ƒ–‡†™‹–Š‰ ˆ‘”ͶͺŠ‘—”•ǤŠ‡”‡•—Ž–•‹†‹…ƒ–‡†

–Šƒ– „‘–Š ƒŽʹ ƒ† …ƒŽ‹†‘Ž ‡Šƒ…‡† …‡ŽŽ †‡•‹–›ǡ ™Š‡”‡ƒ• –Š‡ ƒ „Ž‘…‡” …ƒŽŠ‡šʹ͵ͳ

ƒ„‘Ž‹•Š‡†–Š‡•‡‡ˆˆ‡…–•Ǥ••Š‘™‹ ‹‰—”‡•͵ƒƒ†„ǡƒŽʹƒ†…ƒŽ‹†‘Ž‹†—…‡†…ƒ”†‹ƒ…

ϐ‹„”‘„Žƒ•–’”‘Ž‹ˆ‡”ƒ–‹‘ǡƒ•‡˜‹†‡…‡†„›…‡ŽŽ˜‹ƒ„‹Ž‹–›ȋ ‹‰Ǥ͵ƒȌƒ†‹…”‡ƒ•‡†…‡ŽŽ—„‡”

ȋ ‹‰Ǥ͵„ȌǤ

‘ƒ††”‡••™Š‡–Š‡”–Š‡‹‰”ƒ–‹‘‘ˆ‡‘ƒ–ƒŽ…ƒ”†‹ƒ…ϐ‹„”‘„Žƒ•–‹•‹†—…‡†„›ƒǡ…‡ŽŽ

‹‰”ƒ–‹‘™ƒ••–—†‹‡†‹ƒ™‘—†ǦŠ‡ƒŽ‹‰ƒ••ƒ›Ǥ‡ŽŽ•‹…—Ž–—”‡™‡”‡•…”ƒ’‡†‘ˆˆ™‹–Šƒ

’‹’‡––‡–‹’ǡ’”‘†—…‹‰ƒ™‹†‡ƒ…‡ŽŽ—Žƒ”ƒ”‡ƒǤƒ”†‹ƒ…ϐ‹„”‘„Žƒ•–•‹‰”ƒ–‹‰‹–‘–Š‹•ƒ…‡ŽŽ—Ǧ

Žƒ”ƒ”‡ƒ™‡”‡…‘—–‡†ƒ†‡š’”‡••‡†ƒ•–Š‡—„‡”‘ˆ‹‰”ƒ–‡†…‡ŽŽ•Ǥ”‡ƒ–‡–™‹–ŠʹǤͷ

ƒŽʹƒ†ͳͲɊ…ƒŽ‹†‘Ž•‹‰‹ϐ‹…ƒ–Ž›‹…”‡ƒ•‡†–Š‡‹‰”ƒ–‹‘‘ˆ‡‘ƒ–ƒŽ…ƒ”†‹ƒ…ϐ‹„”‘Ǧ

„Žƒ•–•ƒˆ–‡”ʹͶŠ‹…—„ƒ–‹‘ǡ™Š‹Ž‡ƒ†‹‹•–”ƒ–‹‘‘ˆ…ƒŽŠ‡šʹ͵ͳ”‡†—…‡†–Š‹•‡ˆˆ‡…–ȋ ‹‰Ǥ͵…ȌǤ

Fig 3. ƒ

‹  † — … ‡ †

…ƒ”†‹ƒ… ϐ‹Ǧ

„ ” ‘ „ Ž ƒ • –

’”‘Ž‹ˆ‡”ƒǦ

–‹‘ ƒ†

‹‰”ƒ–‹‘Ǥ

‡ŽŽ ’”‘Ǧ

Ž ‹ ˆ‡ ”ƒ – ‹ ‘ 

‹†—…‡† „›

ƒ…–‹˜ƒ–‹‘

‘ˆ ƒǡ ƒ•

‡˜‹†‡…‡†

„› ȋƒȌ …‡ŽŽ

˜ ‹ ƒ „ ‹ Ž ‹ – ›

ƒ†ȋ„Ȍ…‡ŽŽ

 —  „ ‡ ” Ǥ

ȋ…Ȍ …”ƒ–…Š

™‘—† ”‡Ǧ

’ƒ‹” ƒ••ƒ›

ˆ‘” ‘„•‡”Ǧ

˜ƒ–‹‘ ‘ˆ

 ‡ ‘  ƒ – ƒ Ž

…ƒ”†‹ƒ… ϐ‹„Ǧ

”‘„Žƒ•– ‹Ǧ

‰”ƒ–‹‘Ǥ

Fig 4 Š‡‡ˆˆ‡…–‘ˆƒ‘‡š–”ƒ…‡ŽŽ—Žƒ”ƒ–”‹š’”‘†—…–‹‘Ǥš’”‡••‹‘‘ˆȋƒȌƒǡȋ„ȌǦʹǡȋ…ȌǦͻǡ

ȋ†ȌǦͶƒ†ȋ‡ȌƒǦ’”‘–‡‹•ǡƒ•‹†—…‡†„›–Š‡ƒ…–‹˜ƒ–‘”•…ƒŽ‹†‘Žƒ†ʹǤͷƒŽʹǤ”‘–‡‹Ž‡˜‡Ž•

ʹ ‰”‘—’Ǥ

ˆˆ‡…–•‘ˆƒ‘‡š–”ƒ…‡ŽŽ—Žƒ”ƒ–”‹šȋȌ’”‘†—…–‹‘‹‡‘ƒ–ƒŽ…ƒ”†‹ƒ…ϔ‹„”‘„Žƒ•–•

‘ ‹˜‡•–‹‰ƒ–‡ ƒ ”‡‰—Žƒ–‹‰  ’”‘†—…–‹‘ǡ ƒ ‡š’”‡••‹‘ ™ƒ• †‡–‡…–‡†Ǥ  –Š‡

…ƒŽ‹†‘Žƒ†ƒŽʹ‰”‘—’•ǡƒ‡š’”‡••‹‘™ƒ•—’”‡‰—Žƒ–‡†…‘’ƒ”‡†–‘…‘–”‘Žƒ†…ƒŽǦ

Š‡šʹ͵ͳǦ–”‡ƒ–‡†‰”‘—’ȋ ‹‰ǤͶƒȌǤ

–Š‡•‹• ”‡‰—Žƒ–‹‘Ǥ ‘ ”‡˜‡ƒŽ ™Š‡–Š‡” ƒ ‡ˆˆ‡…–‹‰ ‘  •‡…”‡–‹‘ǡ Ǧʹ ƒ† Ǧͻ

™‡”‡†‡–‡…–‡†„›‡•–‡”„Ž‘––‹‰Ǥ•‡š’‡…–‡†ǡͳͲɊ…ƒŽ‹†‘Žƒ†ʹǤͷƒŽʹ•‹‰‹ϐ‹Ǧ

…ƒ–Ž›‡Šƒ…‡†–Š‡‡š’”‡••‹‘‘ˆǦʹƒ†Ǧͻ…‘’ƒ”‡†–‘…‘–”‘Žƒ†…ƒŽŠ‡šʹ͵ͳǦ

–”‡ƒ–‡†‰”‘—’•ȋ ‹‰ǤͶ„ǡ…ȌǤǦͶ‡š’”‡••‹‘™ƒ••‹‰‹ϐ‹…ƒ–Ž›†‡…”‡ƒ•‡†‹…ƒŽ‹†‘Žƒ†

ƒŽʹ‰”‘—’•ǡ„—–™ƒ•‹…”‡ƒ•‡†‹…ƒŽŠ‡šʹ͵ͳǦ–”‡ƒ–‡†‰”‘—’ȋ ‹‰ǤͶ†ȌǤ

ȽǦȋƒŽ’ŠƒǦ•‘‘–Š—•…Ž‡ƒ…–‹Ȍ’‘•‹–‹˜‡ϐ‹„”‘„Žƒ•–•™‡”‡‹†‡–‹ϐ‹‡†ƒ•’”‹ƒ”›…‡ŽŽ

–›’‡”‡•’‘•‹„Ž‡ˆ‘”‹–‡”•–‹–‹ƒŽƒ–”‹šƒ……——Žƒ–‹‘‹ϐ‹„”‘–‹…†‹•‡ƒ•‡•Ǥ—””‡•—Ž–••Š‘™‡†

–Šƒ–ȽǦ‡š’”‡••‹‘™ƒ•‹…”‡ƒ•‡†‹–Š‡…ƒŽ‹†‘Žƒ†ƒŽʹ‰”‘—’•ȋ ‹‰ǤͶ‡ȌǤ

ˆˆ‡…–• ‘ˆ ƒ ƒ…–‹˜ƒ–‹‘ ‘ …ƒ”†‹ƒ… ϔ‹„”‘•‹• ƒ† ›‘…ƒ”†‹ƒŽ ˆ—…–‹‘ ‹ Ǧ‹†—…‡†

…ƒ”†‹ƒ…Š›’‡”–”‘’Š›

‘ˆ—”–Š‡”‹˜‡•–‹‰ƒ–‡–Š‡in vivo‡ˆˆ‡…–‘ˆƒ‘…ƒ”†‹ƒ…ϐ‹„”‘•‹•‹…ƒ”†‹ƒ…Š›’‡”–”‘’Š›ǡ

™‡ ‡•–ƒ„Ž‹•Š‡† ƒ ‘†‡Ž ‘ˆ …ƒ”†‹ƒ… Š›’‡”–”‘’Š› „› ‹Œ‡…–‹‰ ”ƒ–• ™‹–Š ƒ Š‹‰Š †‘•‡ ‘ˆ 

ȋͳ͹Ͳ‰Ȁ‰Ȍˆ‘”Ͷ†ƒ›•Ǥƒ••‘ǯ••–ƒ‹‹‰ƒƒŽ›•‡••Š‘™‡†–Šƒ–…‘ŽŽƒ‰‡†‡’‘•‹–‹‘™ƒ•

‹…”‡ƒ•‡†‹–Š‡ǦͶ™ƒ†ǦͶ™Ǧ…ƒŽ‹†‘Ž‰”‘—’•ǡ…‘’ƒ”‡†™‹–Š–Š‡ǦͶ™Ǧ…ƒŽŠ‡šʹ͵ͳ

‰”‘—’•ȋ ‹‰Ǥͷ…ȌǤ

Fig 5.˜‘Ž˜‡‡–‘ˆƒ‹–Š‡ˆ—…–‹‘ƒŽ…Šƒ‰‡•‘ˆǦ‹†—…‡†…ƒ”†‹ƒ…Š›’‡”–”‘’Š›ǤȋƒȌƒ†ȋ„Ȍƒ

‹†—…‡†…Šƒ‰‡•‹›‘…ƒ”†‹ƒŽˆ—…–‹‘‹…ƒ”†‹ƒ…Š›’‡”–”‘’Š›ǡƒ••Š‘™„›ƒƒŽ›•‹•‘ˆ–Š‡Ž‡ˆ–˜‡–”‹…—Žƒ”

‡Œ‡…–‹‘ˆ”ƒ…–‹‘ȋ Ȍƒ†–Š‡‹–‡”˜‡–”‹…—Žƒ”•‡’–ƒŽ†‹ƒ•–‘Ž‹…–Š‹…‡••ȋ†ȌǤȋ…Ȍ‡’”‡•‡–ƒ–‹˜‡ƒ•Ǧ

•‘–”‹…Š”‘‡•–ƒ‹‹‰‘ˆŠ‡ƒ”–•‡…–‹‘•ˆ”‘Ǧ‹†—…‡†…ƒ”†‹ƒ…Š›’‡”–”‘’Š›ǤŠ‡„ƒ”‰”ƒ’Š•Š‘™•–Š‡

“—ƒ–‹ϐ‹…ƒ–‹‘‘ˆ–Š‡ϐ‹„”‘–‹…ƒ”‡ƒ•‹Š‹•–‘Ž‘‰‹…ƒŽ•‡…–‹‘•ȋ”‹‰Š–’ƒ‡ŽǢαͺȌǤƒ‰‹ϐ‹…ƒ–‹‘ǣͶͲέʹͲͲǤ

…‘–”‘Ž‰”‘—’ǡ–Š‡‹–‡”˜‡–”‹…—Žƒ”•‡’–ƒŽ†‹ƒ•–‘Ž‹…–Š‹…‡••ȋ†Ȍ™ƒ•‹…”‡ƒ•‡†‹–Š‡Ǧ

Ͷ™ǡǦͶ™Ǧ…ƒŽ‹†‘Žƒ†ǦͶ™Ǧ…ƒŽŠ‡šʹ͵ͳ‰”‘—’•ǤŠ‡Ž‡ˆ–˜‡–”‹…—Žƒ”‡Œ‡…–‹‘ˆ”ƒ…–‹‘™ƒ•

†‡…”‡ƒ•‡†‹–Š‡ǦͶ™ƒ†ǦͶ™Ǧ…ƒŽ‹†‘Ž‰”‘—’•ƒˆ–‡”ƒ†‹‹•–”ƒ–‹‘ˆ‘”Ͷ™‡‡•ǡ

…‘’ƒ”‡†™‹–Š–Š‡…‘–”‘Ž‰”‘—’ȋ ‹‰Ǥͷƒǡ„ȌǤ

‡ƒŽ•‘ˆ‘—†–Šƒ––Š‡‡š’”‡••‹‘‘ˆƒ‹Š‡ƒ”––‹••—‡•™ƒ••‹‰‹ϐ‹…ƒ–Ž›‹…”‡ƒ•‡†

‹–Š‡ǦͶ™ƒ†ǦͶ™Ǧ…ƒŽ‹†‘Ž‰”‘—’•ȋ ‹‰Ǥ͸ƒȌǤ‘‡š’Ž‘”‡–Š‡in vivo‡ˆˆ‡…–‘ˆƒ‘

Fig 6.Š‡‡ˆˆ‡…–‘ˆƒ‘‡š–”ƒ…‡ŽŽ—Žƒ”ƒ–”‹š’”‘†—…–‹‘‹…ƒ”†‹ƒ…Š›’‡”–”‘’Š›ǤȋƒȌ‡•–‡”„Ž‘––‹‰

ƒƒŽ›•‹••Š‘™‹‰ƒ‡š’”‡••‹‘‹…ƒ”†‹ƒ…Š›’‡”–”‘’Š›‘†‡ŽǤš’”‡••‹‘‘ˆȋ„ȌǦʹǡȋ…ȌǦ͵ƒ†

ȗ’δͲǤͲͷ˜•Ǥ…‘–”‘Ž‰”‘—’ǡ͓’δͲǤͲͷ˜•ǤͶ™Ϊ…ƒŽ‹†‘Ž‰”‘—’Ǥ

…ƒ”†‹ƒ… ϐ‹„”‘•‹• ‹ …ƒ”†‹ƒ… Š›’‡”–”‘’Š›ǡ ™‡ †‡–‡…–‡† –Š‡ ‡š’”‡••‹‘ ‘ˆ •‡˜‡”ƒŽ ‘Ž‡…—Žƒ”

‹†‹…ƒ–‘”•‘ˆ…ƒ”†‹ƒ…ϐ‹„”‘•‹•ǤŠ‡‡š’”‡••‹‘‘ˆ„‘–ŠǦʹǡ͵ƒ†Ǧͻ‹–Š‡ǦͶ™ƒ†Ǧ

Ͷ™Ǧ…ƒŽ‹†‘Ž‰”‘—’•™ƒ•‹…”‡ƒ•‡†…‘’ƒ”‡†–‘–Š‡ǦͶ™Ǧ…ƒŽŠ‡šʹ͵ͳƒ†…‘–”‘Ž‰”‘—’•Ǥ

Š‡•‡ ”‡•—Ž–• ˆ—”–Š‡” †‡‘•–”ƒ–‡ –Šƒ– ƒ…–‹˜ƒ–‹‘ ‘ˆ ƒ ‹• ‹˜‘Ž˜‡† ‹ –Š‡ ˆ‘”ƒ–‹‘ ‘ˆ

…ƒ”†‹ƒ…ϐ‹„”‘•‹•‹Ǧ‹†—…‡†…ƒ”†‹ƒ…Š›’‡”–”‘’Š›ȋ ‹‰Ǥ͸„ǡ…ǡ†ȌǤ

Discussion

Š‡ ‡š’”‡••‹‘ ‘ˆ ƒ ‹ ˜ƒ”‹‘—• …‡ŽŽ• ‹• ”‡Žƒ–‡† –‘ —Ž–‹’Ž‡ …‡ŽŽ—Žƒ” ˆ—…–‹‘•ǡ •—…Š

ƒ• …‡ŽŽ ’”‘Ž‹ˆ‡”ƒ–‹‘ǡ ƒ’‘’–‘•‹• ƒ† –Š‡ ”‡‰—Žƒ–‹‘ ‘ˆ •›•–‡‹… …ƒŽ…‹— Š‘‡‘•–ƒ•‹• ȏͳͷȐǤ

‘”‹•–ƒ…‡ǡ”‘™‡–ƒŽǤŠƒ˜‡…‘ϐ‹”‡†–Šƒ–ƒ‹•ƒ…–‹˜ƒ–‡†„›‡š–”ƒ…‡ŽŽ—Žƒ”…ƒŽ…‹—‹

’ƒ”ƒ–Š›”‘‹† …‡ŽŽ• ƒ† ‹• ”‡•’‘•‹„Ž‡ ˆ‘” –Š‡ ”‡Ž‡ƒ•‡ ‘ˆ  ȏͳ͸ǦͳͺȐǤ —” ’”‡˜‹‘—• •–—†‹‡•

†‡‘•–”ƒ–‡†–Šƒ––Š‡ƒ…–‹˜ƒ–‹‘‘ˆƒ‹†—…‡†…ƒ”†‹‘›‘…›–‡ƒ’‘’–‘•‹•†—”‹‰‹•…Š‡‹ƒȀ

”‡’‡”ˆ—•‹‘‹Œ—”›ƒ†‹ˆƒ‹Ž‹‰Š‡ƒ”–•ȏ͹ǡͺȐǤ—”’”‡•‡–•–—†›†‡‘•–”ƒ–‡•ǡˆ‘”–Š‡ϐ‹”•–

–‹‡ǡ–Š‡‡š‹•–‡…‡‘ˆƒ‹”ƒ–…ƒ”†‹ƒ…ϐ‹„”‘„Žƒ•–•ǤŠ‹•…‘…Ž—•‹‘‹•„ƒ•‡†‘•‡˜‡”ƒŽŽ‹‡•

‘ˆ ‡˜‹†‡…‡ǣ ȋƒȌ ƒ ’”‘–‡‹• ™‡”‡ ‹†‡–‹ϐ‹‡† ‹ ‡‘ƒ–ƒŽ …ƒ”†‹ƒ… ϐ‹„”‘„Žƒ•–• „› ‡•–‡”

„Ž‘– ƒƒŽ›•‹•Ǣ ȋ„Ȍ ƒ ’”‘–‡‹• ™‡”‡ •Š‘™ –‘ „‡ Ž‘…ƒ–‡† ‹ ‡‘ƒ–ƒŽ …ƒ”†‹ƒ… ϐ‹„”‘„Žƒ•–•

„› ‹—‘Š‹•–‘Ž‘‰‹…ƒŽ ƒ† ‹—‘ϐŽ—‘”‡•…‡…‡ •–ƒ‹‹‰Ǣ ȋ…Ȍ ‡š–”ƒ…‡ŽŽ—Žƒ” …ƒŽ…‹— ƒ†

…ƒŽ‹†‘Žǡƒ•’‡…‹ϐ‹…ƒ…–‹˜ƒ–‘”‘ˆƒǡ‹…”‡ƒ•‡†‹–”ƒ…‡ŽŽ—Žƒ”…ƒŽ…‹—Ž‡˜‡Ž•ǡ–Š‡”‡„›ƒ…–‹˜ƒ–‹‰

–Š‡Ǧ͵’ƒ–Š™ƒ›ƒ†‹†—…‹‰–Š‡”‡Ž‡ƒ•‡‘ˆ…ƒŽ…‹—ˆ”‘Ǣȋ†Ȍ‡š–”ƒ…‡ŽŽ—Žƒ”…ƒŽ…‹—

ƒ† …ƒŽ‹†‘Ž ’”‘‘–‡† ‡‘ƒ–ƒŽ …ƒ”†‹ƒ… ϐ‹„”‘„Žƒ•– ’”‘Ž‹ˆ‡”ƒ–‹‘ǡ  ‹‰”ƒ–‹‘ ƒ† 

•‡…”‡–‹‘–Š”‘—‰Š–Š‡ƒ…–‹˜ƒ–‹‘‘ˆƒǢƒ†ȋ‡Ȍƒ’Žƒ›‡†ƒ”‘Ž‡‹Ǧ‹†—…‡†…ƒ”†‹ƒ…

ϐ‹„”‘•‹•‹”ƒ–•Ǥ

CaʹΪ •‹‰ƒŽ• ƒ”‡ ™‡ŽŽ •–—†‹‡† ‹ …ƒ”†‹ƒ… ›‘…›–‡• ȏͳͻǡ ʹͲȐǤ ‘™‡˜‡”ǡ ƒʹΪ •‹‰ƒŽ‹‰

’ƒ–Š™ƒ›•ƒ”‡‘–™‡ŽŽ—†‡”•–‘‘†‹‡‘ƒ–ƒŽ…ƒ”†‹ƒ…ϐ‹„”‘„Žƒ•–•Ǥ‹‡–ƒŽǤŠƒ˜‡”‡˜‡ƒŽ‡†–Šƒ–

CaʹΪƒ…–‹˜‹–›‹Š—ƒ…ƒ”†‹ƒ…ϐ‹„”‘„Žƒ•–•‹•‡†‹ƒ–‡†„›—Ž–‹’Ž‡ƒʹΪ•‹‰ƒŽ‹‰’ƒ–Š™ƒ›•ǡ

‹…Ž—†‹‰͵

•‡•‡•…Šƒ‰‡•‹‡š–”ƒ…‡ŽŽ—Žƒ”…ƒŽ…‹—…‘…‡–”ƒ–‹‘ǡ–”‹‰‰‡”‹‰ƒ‹…”‡ƒ•‡‹‹–”ƒ…‡ŽŽ—Žƒ”

…ƒŽ…‹—ȏͳͺȐǤƒǡ‹„‘–Š”ƒ–…ƒ”†‹ƒ…›‘…›–‡•ƒ†Š—ƒƒ–”ƒŽ‰ƒ•–”‹…‡ŽŽ•ǡ‹••‡•‹–‹˜‡

–‘…Šƒ‰‡•‹‡š–”ƒ…‡ŽŽ—Žƒ”…ƒŽ…‹—…‘…‡–”ƒ–‹‘ȏͳͷǡʹʹȐǤ—””‡•—Ž–•‹‡‘ƒ–ƒŽ…ƒ”†‹ƒ…

ϐ‹„”‘„Žƒ•–•ƒ”‡•‹‹Žƒ”–‘–Š‡”‡•—Ž–•†‡•…”‹„‡†ƒ„‘˜‡Ǥ

Š‡ •ƒ”…‘’Žƒ•‹…Ȁ‡†‘’Žƒ•‹… ”‡–‹…—Ž—ǡ ƒ •’‡…‹ƒŽ‹œ‡† …ƒŽ…‹—Ǧ•–‘”‹‰ ‘”‰ƒ‡ŽŽ‡ǡ ‹•

™‡ŽŽ‘™–‘„‡‹–‹ƒ–‡Ž›‹˜‘Ž˜‡†‹”‡‰—Žƒ–‹‰ƒʹڏ‘˜‡‡–™‹–Š‹…‡ŽŽ•Ǥ͵•ƒ†

›•’ƒ”–‹…‹’ƒ–‡‹–Š‡”‡Ž‡ƒ•‡‘ˆƒʹΪˆ”‘–Š‡•ƒ”…‘’Žƒ•‹…Ȁ‡†‘’Žƒ•‹…”‡–‹…—Ž—ȏʹ͵ǡ

ʹͶȐǤ–‡”‡•–‹‰Ž›ǡ‹‡–ƒŽǤˆ‘—†–Šƒ–›•ƒ›‘–„‡‹˜‘Ž˜‡†‹ƒʹΪƒ…–‹˜‹–›‹Š—ƒ

…ƒ”†‹ƒ… ϐ‹„”‘„Žƒ•–•Ǥ Š‡•‡ ”‡•—Ž–• †‡‘•–”ƒ–‡† –Šƒ– ‹–”ƒ…‡ŽŽ—Žƒ” …ƒŽ…‹— ‹ …ƒ”†‹ƒ…

ϐ‹„”‘„Žƒ•–• ƒ”‡ ‡†‹ƒ–‡† „› ͵• ȏʹͳȐǤ —” ”‡•—Ž–• ”‡˜‡ƒŽ –Šƒ– ‹…”‡ƒ•‡† ‹–”ƒ…‡ŽŽ—Žƒ”

…ƒŽ…‹—…‘…‡–”ƒ–‹‘•™‡”‡‹Š‹„‹–‡†„›͹͵ͳʹʹƒ†ʹǦǤƒ††‹–‹‘ǡ–Š‡ƒƒ…–‹˜ƒ–‘”

…ƒŽ‹†‘Ž‹†—…‡†•‹‰‹ϐ‹…ƒ–Ž›‹…”‡ƒ•‡†‹–”ƒ…‡ŽŽ—Žƒ”…ƒŽ…‹—…‘…‡–”ƒ–‹‘•ǤŠ‡•‡”‡•—Ž–•

†‡‘•–”ƒ–‡–Šƒ–ƒ‹•‹˜‘Ž˜‡†‹‹…”‡ƒ•‹‰–Š‡‹–”ƒ…‡ŽŽ—Žƒ”ƒʹΪ…‘…‡–”ƒ–‹‘–Š”‘—‰Š

–Š‡Ǧ͵’ƒ–Š™ƒ›Ǥ

ƒ”†‹ƒ… ϐ‹„”‘•‹• ‹• ˜‹–ƒŽ ˆ‘” ˜‡–”‹…—Žƒ” ”‡‘†‡Ž‹‰ ȏʹͷȐǤ ƒ”†‹ƒ… ϐ‹„”‘„Žƒ•–•ǡ Š‹‰ŠŽ›

ƒ…–‹˜‡…‡ŽŽ•–Šƒ–‡šŠ‹„‹–‹…”‡ƒ•‡†’”‘Ž‹ˆ‡”ƒ–‹˜‡ǡ‹‰”ƒ–‘”›ƒ†•‡…”‡–‘”›’”‘’‡”–‹‡•ȏʹ͸ǡʹ͹Ȑǡ

”‡’”‡•‡––Š‡Žƒ”‰‡•–…Žƒ••‘ˆ…‡ŽŽ•”‡•‹†‹‰‹–Š‡Š‡ƒ”–Ǥ”‘Ž‹ˆ‡”ƒ–‹‘‘ˆ…ƒ”†‹ƒ…ϐ‹„”‘„Žƒ•–•‹•

–Š‡ƒ‹…Šƒ”ƒ…–‡”‹•–‹…‘ˆ›‘…ƒ”†‹ƒŽϐ‹„”‘•‹•ǤŽ–Š‘—‰Š–Š‡‹’‘”–ƒ…‡‘ˆϐ‹„”‘•‹•‹˜ƒ”‹‘—•

…ƒ”†‹ƒ…†‹•‡ƒ•‡•Šƒ•„‡‡™‡ŽŽ‡•–ƒ„Ž‹•Š‡†ǡ–Š‡ƒʹΪ•‹‰ƒŽ‹‰‡…Šƒ‹••‹˜‘Ž˜‡†‹…ƒ”†‹ƒ…

ϐ‹„”‘•‹•ƒ”‡‘–‘™Ǥ‘‡•–—†‹‡•”‡˜‡ƒŽ‡†–Šƒ–͹‹•Ž‘…ƒ–‡†‹–Š‡‡„”ƒ‡‘ˆ

…ƒ”†‹ƒ…ϐ‹„”‘„Žƒ•–•ƒ†‡†‹ƒ–‡•Š‹‰Š‡”ȏƒʹΪȐi‘•…‹ŽŽƒ–‹‘ˆ”‡“—‡…‹‡•‹†‹…ƒ–‹˜‡‘ˆŠ‹‰Š‡”

•–‹ˆˆ‡••ǡ™Š‹…Š™‘—Ž†”‡•—Ž–‹‘”‡‡ˆϐ‹…‹‡–”‡‘†‡Ž‹‰ȏʹͺǦ͵ͲȐǤƒʹڏƒ›’Žƒ›

ƒ ‡••‡–‹ƒŽ ”‘Ž‡ ‹ …ƒ”†‹ƒ… ϐ‹„”‘„Žƒ•–• ’”‘Ž‹ˆ‡”ƒ–‹‘ ƒ† †‹ˆˆ‡”‡–‹ƒ–‹‘ ȏͳͻȐǤ —” ”‡•—Ž–•

…‘ϐ‹”–Šƒ–ƒ…–‹˜ƒ–‹‘‘ˆƒ…‘—Ž†’”‘‘–‡‹‰”ƒ–‹‘ƒ†‡Ž‡˜ƒ–‡–Š‡‡š’”‡••‹‘Ž‡˜‡Ž•

‘ˆǦʹƒ†ͻ‹‡‘ƒ–ƒŽ…ƒ”†‹ƒ…ϐ‹„”‘„Žƒ•–•Ǥ‘ˆ—”–Š‡”†‡‘•–”ƒ–‡–Š‡‹˜‘Ž˜‡‡–‘ˆ

FDUGLDFÀEUREODVWVZLWKFDOKH[DVSHFLÀFLQKLELWRURI&D58DQG$3%DWWHQXDWHG

the calindol- and extracellular calcium- induced increase in intracellular calcium ([Ca2+]i)

&DUGLDFÀEUREODVWSUROLIHUDWLRQDQGPLJUDWLRQZHUHDVVHVVHGE\077''LPHWK\OWKLD]RO...

2-yl)-2,5-diphenyltetrazolium bromide), cell count and the cell scratch assay ECM production

was detected by expression of matrix metalloproteinase-3 and -9 (MMP-3 and -9) Activation

RI &D5... LQ D PRGHO RI FDUGLDF K\SHUWURSK\ LQGXFHG E\ LVRSURWHUHQRO ,62

Methods and Results:,PPXQRÁXRUHVFHQFHLPPXQRKLVWRFKHPLVWU\DQG:HVWHUQEORWDQDO\VLV

UHYHDOHGWKHSUHVHQFHRI&D5LQFDUGLDFÀEUREODVWV&DOFLXPDQGFDOLQGRODVSHFLÀFDFWLYDWRU