NU V6l VA MU'C Dp BltU HIEN C O A MQT S6 GENE OXY HOA STRESS
Truong Tuyet Mai, Truffng Hoing Kien*, Nguyen Cdng Khin**,Yu7,uru Otsuka***, Nguyen Van Chuyen****
Vifn Dinh duffng QuSc gia, Vlft Nam
* Chi cue Vf sinh An todn Thuc phSm Qudng Ninh
** Ciic Vf sinh An toan Thucphhm, Bg Yte
*** Tru&ng Dgi hgc Ochanomizu Nhpt Bdn
**** Tru&ng Dgi hgc Plti^ nQ Nhpt Bdn
T6M TAT
Ny vdl Mb\ thflnh phin chfnh Ifl cdc polyphenol dfl 6\tqe chirng minh cd khi ning chdng oxy hda, khi nflng hgn ch6 tflng dufrng huyit trSn chuOt flfli thio iubnq. Tuy nhifin, finh hirdng cCia polyphenol chi6t tflch tCr ny vdl ddi v6l s\f bl6u hlOn cOa mft sd gene llfin quan a4n chdng oxy hda v i n chi/a di/g-c nghiSr ctni.
Trong nghifin cCru nfly, chiing tfli tifin hflnh cho chu^t nhit ICR dfli thflo dir&ng udng ni/6c ny vdi chifit tfldi (500 mg/kg tr^ng lirang ca thl, trong 10 tudn lifin tyc) vfl dflnh gifl hi$u qui cua vi$c si> dyng ny vdl d6i vdi mi>c dd biiu hifin gene oxy hda stress GPX-2, GST A1 vfl SOD-1 trfin t i bflo th^n v i gan chuOt Mi>c dO bi^u hi§n gene dich trfin Xk bflo th^n vfl gan chuOt di?p'c do bing phuong phip cDNA microanay. Kfit qui cho thfiy, mi>c dO bilu hlfin gene GPX-2, GST A l vfl SOD-1 dfl tSng ISn b nhdm chuOt dfli thflo dufimg chCrng, cao ho'n 2 lin so v6i nhdm chuOt khde mgnh. Trong khi dd, mi>c dO bifiu hifin ct!ia cflc gene nfly lgi giim xu6ng b nhdm chudt dfli thflo du'fi'ng u6ng ny vii so vdi nhdm chuOt dfli thflo dudng chung. Ny v6i cd mdt dflp irng didu hda duong tinh d6i vdi cflc gene oxy hda stress it cflc lfi bflo cua chuOt dfli thflo dudng, chung tfl khfl nflng ching oxy hda cua ny v6l trfin chu^t dfli thflo dudng.
Ti> khofl: Ny V6l, chfing oxy hofl, bifiu hlfin gene, chudt tifiu dudng
I. D A T VAN D E oxy h6a n^i sinh (cdc enzyme ch6ng oxy h6a
„ , 1 , . ., nhu superoxide dismutase (SOD), glutatiiione Polyphenols la thanh phan co nmeu
^ , , ., -. ^ J!. L-i J 1 , - S-transferase (GST), glutathione peroxidase tiong nhom thvc v^t vol rat nhieu d^ng khac / t nhau va chfc nang ho?t tfah sinh hpc da dimg, < ° ^ » ' ''^>' ^^ * ™ 8 tro ISn mat can btog, trong do khi nSng ch6ng oxy h6a dii(rc xem ""' ^ ''=" ^"^ "^o" "=*= ^'^^ *^g 't^^^^ " ' ' nhu la hojt tinh chinh ciia polyphenol [1], ^^- ^ * ' " " " S nhOng h^u qui l i t?o ra cic sin Hju qui ciia vi$c tSng dudng huyet Men ^'^^ 8™^ '^'' ""^ S V ra cic te bio ton thuong tyc trong b?nh dii thio du6ng 14 cic phin ^^ sinh la cic bien chung ciia dai thio ducmg ling reactive oxygen species (ROS), dan d^n [2-3]. Sv thay doi ciia miic dO bieu hi?n gene tang oxy h6a stress 6 rat nhieu te bio [2]. Vi?c t^ii m6i tru&ng tSng du6ng huyet l i nhan to lien giim dip iing thich h(?p tir m&ng ludi chong quan den co che benh sinh ciia b i ^ chiing dii Tic gii: Truong Tuyet Mai Phin bi$n 1: TS. Vfl Thj Thu Hi^n Dja chi: Vi^n Dinh duong quoc gia Vi$n Dinh du&ng qu6c gia.
Di$n thoiu: 094 9911 777 Phin bien 2: TS. Bili Thj Nhung Ngiy nhjn bii: 15/2/2012 Vi$n Dinh duSng quoc gia.
Ngiy dang bii: 01/6/2012
68 Tap cM Y hpc dii phdng, T^p XXH, sS 3 (130)
tiido du6ng [4]. Mtic dO bi^u hi$n gene I^ yen to c6 ^ nghia thvc t l de Iam sdng t6 sv khSc biet mtic dp bieu hi^n gene giua d6i tuvng binh thudng vk d6i tuvng d&i thdo dudng. Vi$c tAng bieu hi§n cdc gene oxy h6a stiess, nhu (GPx) Ven chuQt dai thao dudng da duvc bdo cao tiong nghifin ctiu cua Iwata et al., (2006) [4].
Ny voi c6 ten gpi khoahpc Id Cleistocalyx operculatus (COB) c6 hdm lugng polyphenol eao va dupe chung minh c6 kha nSng h ^ ehl tang duting huyet, chong oxy h6a tifin chu^t ddi thdo dudng [5-7]. Hi^u q u i ch6ng oxy h6a ctia ny voi tifin chupt ddi thdo dudng dS dupe ldm sdng td thong qua vi^c gidm chi sS peroxy hda lipid vd sv phyc hoi bo^t dpng cua ede men chong oxy hda nhu SOD and GST [6].
Tuy nhien, cho den nay, chua cd mOt nghifin cuu ndo ddnh gid sy dnh hudng ctia polyphenol ny voi doi vdi bieu hi$n gene lifin quan den oxy hda stiess d chupt ddi thdo dudng. Chinh vi v^y, mye dich ctia nghien ctiu Id ddnh gid dnh hudng cua vipc su dung chilt xuat ny vol d6i vdi bieu hifin gene dich GPX-2, GST AI vd SOD-1 tifin gan vd th|n cua chupt ddi thdo dudng de cung cap them bdng chting ve Ipi ich chong oxy hda cua ny voi khi su dyng hang ngdy tiong vi?c h5 tip phdng vd dilu trj b?nh ddi thdo dudng.
n . P H l T O N G P H A P N G H D & N ctfu 2.1. Nguyen lifu vk phuotig ph^p chiet tdch
Ny V6i va Id 6 i dupe thu mua t ^ Vi§t Nam. Sau khi thu mua, nguyen lipu dupe chpn lpc, rtia sach, lam dong khd, sau dd duoc xay nhd tiidnh bpt. Chifit tdch tiieo tJ I? 100 g ny Voi vdi 2 lit nudc, d|t trong thiing nudc 100
"C tiong 40 phtit. Sau dd, dting phuong phdp ly tdm de thu lay dung djch chilt tdch. Ldm ddng khd dung djch chilt tdch de thu dupe bpt ny V6i. BOt ny v6i khd cd hdm lupng polyphenol tiomg bhih Id 452 mg/g bpt chilt.
2.2. Chu$t ddi thdo dirdmg u6ng ny Vll 10 tu^n vd chuSn bt t l biko
Hai muoi bdy con chupt dye ICR chia thdnh 3 nhdm, nhdm chu^t khde m ^ doi chting, nhdm chuQt ddi thdo dudng chting vd nhdm chupt ddi thdo dudng uong ny vii, moi nhdm 9 con. Chupt ddi thdo dudng dupe gay ddi thdo dudng b ^ g thu6c stieptozotocin vdi lilu 100 mg/kg thl tipng. Chupt thyc nghi?m dupe nudi bdng thtic dn, nudc uing tifiu chudn d$t tiong phdng nudi ddng v§t, su dyng thftc fin chuan CE-2, cua Nippon Clea, Nh|t Bdn.
Nhdm chuOt can thipp dupe u6ng ny v6i vdi lilu lupng 500 mg bdt ny voi chiet tdch/kg cdn n&ng/ngay tiong 10 tuin lifin tyc, tiong khi dd nhdm chung ehl dupe uong nudc binh thudng.
Sau 10 tuan, tat cd chupt dupe phlu thi4t de thu ldy mdu va cdc bp phan tii^ vd gan. Mdu todn phdn dupe ly tam, tdch lay huyit thanh ldm xfit ngjiifm MDA vd cdc chi s l sinh hda bao gom dudng huyit, cholesterol vd triglyceride mdu. Cdc bp phan gan vd th?n dupe Iam ddng khd bdng nittogen lanh vd bdo qudn tai -80 °C.
2.3. Tdch I§p RNA vd qui trinh cDNA microarray
RNA tti cdc bO ph|ui gan va thgn dupe chilt tdch vd tinh Ipc bdng kit RiboPure™
(Ambion, Inc. U.S A.). Xdc djnh ning dp RNA dung djch thu dupe bdng do dp bap thu t ^ budc sdng 260/280 nm Ven mdy NanoDrop lOOOA Spectrophometer. RNA tong s l dupe chiet tdch tti bp phgn thgn h o ^ gan ctia 9 con chudt, sau dd dupe tipn ciing vdo 1 mlu d l bhih tiiudng hda cdc ed thl khdc nhau. Mau RNA chilt tdch dupe bdo qudn t^i -80 °C eho den khi sti dyng.
Dung djch mau RNA ctia ba nhdm:
nhdm chu^t khde (N), chuOt ddi thdo dudng chting (DC) vd chudt ddi thdo dudng uong ny voi (DB) dupe tiln hdnh cdc budc xti 1^
mau theo hudng din cua Applied Biosystems Chemilummescent RT Labelmg Kit (Sdn pham Tap chi Y hpc du phdng, Tap XXH. s6 3 (130) 69
s6.4340415, Applied Biosystems, Lincoln Centie Drive Foster City, CA, U.S.A.). Sau dd cdc mlu dupe tidn vdi 90 pg dimg djch DIG- labeled RT cRNA vd dupe lai t?o bdng kft vd h§ thing mdy Applied Biosystems Mouse Oligo Microarrays (Lot. MB0016 and MBOOl) (sdn phdm s6 4342142, Applied Biosystems Chemiluminesccni Detection Kit, Applied Biosystems, Lincoln Centie Drive Foster City, CA, U.S.A.). Phdt hi^n vj tri spot, djnh lupng dupe phdn tich b^ng cdch sti dyng cdc phdn mim Spotfire (Applied Biosystems, U.S.A.).
Trung binh ctia cudng dd dilm phdt hi^n dupe vti di tti trung binh ctia cudng dd nin vd ddu ra vdi UniGene vd GenBank (NgSn hdng gene) vd dupe md td tiong m^t bdng tinh dti li?u Microsoft Excel. KJ hipu gene tiong ddu ngo$c don dupe xdc djnh nhu Id homologs gen chupt cua Tmng tam Thdng tin Cdng ngh§ sinh
hpc Quic gia (NCBI) (Bdng 1). Sv tiiay dli vl mtic dd bilu hi$n gene dupe tinh todn bdng cdch chia cudng dO spot ctia nhdm chupt ddi thdo dudng uing ny vii (DB) vdi nhdm chudt khde m ^ (DB/N), ctia nhdm chupt ddi thdo dudng uing ny vii vdi nhdm chupt ddi thdo dudng chting (DB/DC) vd cua nhdm ehuOt ddi thdo dudng chting vdi nhdm chupt khde mgnh (DC/N). Vd sv tiiay dli cd f nghia khi tiiuong s l tii 2 trd lfin.
2.4. Phdn tfch s l li$u
Kit quA tifin thi nghidm dpng v^t dupe diln td theo means ± standard deviation (SD), vdi n=9. Sy khdc bipt giua 3 nhdm dupe phdn tfch bdng one way analysis of variance (ANOVA) theo Duncan's multiple-range t-test.
Vdi gid trj p < 0.05 thl dupe xem Id sy khdc bi^t cd f nghia thing kfi.
Bdng 1. Danh sdch gene dich
ID Gene Ky hl$u cua gene* T6n cfia gene Chi>c n i n g
glutathione peroxidase 2
MiSn djch, gidi d$c, bdo v$;
Chdng oxy h6a vd io^i bd gic t p d o
glutathione S-transferase, alpha 1 Midn d|ch, gidi dOc, bdo v$;
Chdng oxy hda
superoxide dismutase 1, soluble Chdng oxy hda vd logi bd glc t v do; miSn djch vd bdo v$
* Ky hifu cua gene dugc xdc dinh nhu homologs doi v&i hf gene chugt cua Trung tdm Thong tin Cong ngh? sinh hgc Quoc gia (NCBI).
m. Ktx QUA vA BAN LUAN 3.1. Cdc kit qud bda sinh
Sau 10 tuan uing ny v i i chilt tdch, dudng huyit cua nhdm chupt ddi thAo dudng da gidm m$t cdch ed y nghia thong ke so vdi nhdm ehuOt ddi tiido dudng chting (221.7 ±46.0
so vdi. 270.6 ± 40.5 mg/dL, p < 0.05) vd ndng do dudng huyet d nhdm chupt khde manh tiii duy tiinh d mtic trung bhih 127.9 mg/dL (Bang 2). Tuong tv, dli vdi ning dp cholesterol vd tiiglyceride, nhdm chupt ddi thdo dudng uong ny vii cung dS gidm xuing m$t cdch ddng kl
70
T^p chi Y hQC dv ph6ng, T^p XXH, s6 3 (130)so vdi nhdm chudt ddi tiido dudng chting. Cdc Bdng 2 cung cho tiidy nong dp TBARS kit qud ve hi$u qud gidm dudng huyit vd gidm ttong mdu, ttong gan ctia nhdm chupt ddi thdo cdc chi so cholesterol vd triglyceride cung da dudng chting tfing cd f nghTa thong kfi so vdi dupe chting minh Vong cde nghifin cftu trudc nhdm chu^t kho6 nmnh. Ngupc lai, d nhdm [5,7], ttong nghifin ctiu ndy, chung tdi cung thn chu^t ddi thdo dudng uing ny vii thl ning dupe kit qud tuong ty, dieu ndy chung td khd dd TBARS I^i gidm mdt cdch ddng kl so vdi nfing phdng chong ddi thdo dudng cua ny vii. nhdm chupt ddi thdo dudng chftng (p < 0.05).
Bdng 3. Cdc chi sd hda sinh vd chi sd chdng oxy hod trdn chudt ddi d u ^ n g sau 10 tuiln udng nif vdl
. Nhdm chudt Nhdm DTD chtitig Nhdm DTD+ Ny
^ kh6em9nh(N) (DC) VSt {DB) Blood glucose (mg/dL) 127.9 ± 12.3a 270.6 ± 40.5b 221.7 ± 46.0c Total Cholesterol (mg/dL) 93.4 ± 14.7a 117.6 ± 23.6b 103.3 ± 31.5c Triglyceride (mg/dL) 56.0 ± 13.4a 122.5 ± 31 .Ob 75.4 ± 46.7c TBARS (nmol MDA/L huySt thanh) 5,12 ± 0,67a 14,42 ± 1,91 b 9,92 ± 1,63c TBARS (nmoIMDA/gtd chtic gan) 61,0±7,3a 76,2±7,1b 68,1 ±2,1a Ket qud trinh bdy du&i dang mean ± SD (n=9). Tren ciing hdng, cdc ky tu khdc nhau biiu dien sif khdc biet CO y nghia thong kep < 0.05 (Duncan's multiple-range t-test).
3.2. Bilu hifn cua gene dich trSn t l bdo gan, chftng so vdi nhdm chupt dai thdo dudng udng thSn chu$t ddi thdo dirdng sau 10 tudn ndng ny vol vd nhdm chupt khde m^nh.
ny vol chiet tdch Bdng 5 cho thdy mdt lo9t gene oxy hda Mftc dp bilu hipn cua 3 gene tifin t l bdo stiess GPX-2, GST Al vd SOD-1 dd tdng Ifin thgn va gan chuOt dupe xae djnh bdng phuong 2 lln so vdi nhdm chupt khde manh. Ngupc phdp cDNA microarray analysis (Bdng 4). l^i, d nhdm chudt ddi thdo dudng udng ny Mftc dd bilu hifn ctia gene oxi hda stiess GPX- vii tlti mftc dp bieu hifn cua cdc gene ndy lgi 2, SOD-I ti^n ed t l bdo gan vd tiign da cd sy gidm xuing so vdi nhdm chu$t ddi tiido dudng tdng Ifin ddng k l d nhdm chupt ddi thdo dudng chftng.
T?ip chi Y hpc dv ph6ng, T^p XXH, s^ 3 (130)
Bdng 4. Bidu hl$n gene cOa t l bdo gan vd thOn sau 10 tuin udng ny vdl (don v| Il6n quan)
Tfln Gene
GPX-2
GSTA1
SOD-1
T i U o
Gan Th«n Gan Thjn Gan Than
Chu^t khde m^nh (N) 458 29 7,801 1,435 664 24
Chuft au thio dirdmg chl>ng (DC)
1,679 134 25,841
2,482 717 201
Chu$t au thio du^ng-^NvvilCDB)
296 41 20,952
2,770 416
84
So lieu dugc tinh todn tit phan mem Spotfire Inc.,con so dien td nhu gid tri trung binh cda cu&ng dg spot
Bdng 5. Sv thay ddi 2 l i n cOa biiu hl^n gene d* t l bdo thfn vd gan chu^t ddi thdo 6\rimg v6i c h u ^ khde m^nh (DB/N), vdl chu^t ddi thdo dirdng chi>ng (DB/DC), vd ir chu^t ddi thdo difdmg chOng
vd^l chudt khde m^nh (DC/N).
Ten Gene T l bdo DB/N DC/N DB/DC Gan
Th^n Gan Th5n Gan Thjn
-1.5 1.4 2.7 1.9 -1.6 2 7
3.7 4.5 3.3 1.7 1.1 8.5
-5.7 -3.3 -1 2
1.1 -1.7 -3.2
Cdcklt qud v l sy thay dli muc ddbilu ddithdodudng [4]. Cdc kit qud tiong phdng thl hifn gene Hfin quan din oxy hda stiess ti^n nghifm vd cdc kit qud sinh hda tien chudt ddi chupt ddi thdo dudng dd cho thdy vife tfing thdo dudng dS chting minh hifu qud ching oxy dudng huyet lien tyc ddng mpt vai tid trong hda cua ny voi [6, 8]. Trong nghifin cftu ndy, vifc dilu hda tfing sy bilu hifn cua cdc gene chftng tdi da thdy rdt ro hifu qud ctia ny vii GPX-2, GST AI, vd SOD-I d t l bdo tii§n vd dli vdi mftc dp bieu hifn gene oxy hda stiress, gan chuft. Cdc kit qud ndy phti hpp vdi kit thdng qua vifc gidm mftc dO bilu hifn ctia 3 qud ciia tdc gid Iwata et al., (2006) vl mftc dp gene GPX-2, GST A l , vd SOD-1 d t l bdo tiign bilu hifn gene oxy hda stiess GPx ttfin chuOt vd gan chupt ddi thdo dudng sau 10 tudn uing 72 Tap chi Y h p c d v p h d n g , B p X X n , s d 3 (130)
ny voi. Dieu ndy ed till gidi thich rdng hifu qud chong oxy hda ctia ny voi da ed feedback duong tinh ddi vdi cdc gene oxy hda stiess.
IV. KI;T LUAN
Ket qua nghien ctiu da chting td rdng vifc su dyng ny voi dli vdi ddi thdo dudng da cd ddp ting dieu hda duong tinh doi vdi cdc gene lifin quan den ching oxy hda ttfin te bdo chupt ddi thdo dudng. Chinh vi v§.y, ny voi dupe coi lfi rapt nguyfin lifu chong oxy hda tiem nfing, hd ttp tiong vifc phdng vd dilu trj bfnh ddi tiiao dudng cung nhu mdt so bfnh man tinh khdng lay khdc.
T A I L I $ U T H A M K H A O
1. Urquiaga I, Leighton F. 2000. Plant polyphenol antioxidants and oxidative stiess. Biol Res 33: 55-64.
2. Evans JL, Goldfine ID, Maddux BA, Grodsky GM. 2002. Oxidative stiess and sttess-activated signaling pathways: a unifying hypothesis of type 2 diabetes.
Endocr Rev 23(5): 599-622.
3. Fridlyand E and Philipson LH. 2005.
Oxidative reactive species in cell injury:
Mechanisms in diabetes meOitus and therapeutic approaches. Ann NY Acad Sci 1066(1): 136-151
4. Iwata K, Nishinaka T, Matsuno K, Yabe- Nishimura C. 2006. Increased gene expression of glutathione peroxidase-3
in diabetic mouse heart. Biol Pharm Bull 29(5): 1042-5.
Truong Tuyet Mai and Nguyen Van Chuyen. 2007. Anti-hyperglycemic activity of an aqueous extiact from flower buds of Cleistocalyx operculatus (Roxb.) Merr and Perry. Biosci Biotechnol Biochem 71(1):
69-76.
Truong Tuyet Mai, Nagashima Fumie and Nguyen Van Chuyen. 2009. Antioxidant activities and hypolipidemic effect of an Aqueous Exttact irom Flower Buds of Cleistocalyx operculatus (Roxb.) Merr and Perry in vitto and in Stteptozotocin- Induced Diabetic Rats. J Food Biochem 33:
790-807.
Truong Tuyet Mai, Keiko Yamaguchi, Mizuho Yamanaka, Nguyen Thi Lam, Yuzuru Otsuka and Nguyen Van Chuyen.
2010. Protective and anticataract effects of the aqueous exttact of Cleistocalyx operculatus flower buds on beta-cell of Stteptozotocin- Diabetic Rats. Joumal of Agricultiiral and Food Chemistiy 58 (7):
4162-4168.
Sone Yasuko, Moon, Joon-Kwan, Tmong Itiyet Mai, Nguyet Thu Nghiem, Asano Eri; Yamaguchi Keiko, Otsuka Yuzuru, Shibamoto Takayuki. 2011. Antioxidant' anti-inflammatory activities and total phenolic content of extracts obtained from plants grown in ^^etnam. J Sci Food Agric 91(12):2259-64.
T^p chi Y hoc dv phdng, Tap XXE, s^ 3 (130) 73
COB AND THE EXPRESSION OF OXIDATIVE STRESS GENES
Truong Tuyot Mai, Truong Hoang Kien*, Nguyen Cong Khan**, Yuzuru Otsuka***, Nguyen Van Chuyen ****
National Institute of Nutrition, Vietnam
*Quangninh Food Administration
** Food Administration, Ministry of Health
***Ochanomizu University, Tokyo, Japan
*-'<**japan Women University, Tokyo, Japan
The anti-diabetic effects of COB exttact, that its major components are polyphenols, and that it produces antioxidant, anti-hyperglycemic and anti-hyperlipidemic eSects in STZ-diabetic rats were investigated.
However, the influence of COB polyphenols on the expression of genes involved in these biological effects m diabetic rat tissues have been not investigated. Therefore, in this study, we fed STZ diabetic-male ICR mice with a long-term COB tteatment (500 mg/kd body weight, for 10 weeks) and investigated the effects of COB tieatment on Ihe target gene expression levels in liver and kidney tissues, including the oxidative stress genes of GPX-2, GST Al and SOD-1. The expression levels of target genes in mouse liver and kidney tissues were quantified by cDNA microarray analysis.
Differences in gene expression were considered to be significant at a ratio of at least 2.0. After
10 weeks of feeduig, tbe COB-tieated diabetic mice (DB) had a significant decrease in blood glucose, total cholesterol and triglyceride level compared to the contiol diabetic mice (DC).
A series of oxidative sttess genes of GPX-2, GST Al and SOD-1 was up-regulated in DC, and showed a higher than 2.0 fold change as compared to normal mice. The DB group showed its antioxidant effect by decreasing fold change values m GPX-2, GST Al and SOD- 1 to that of the DC group. These results have suggested that COB tteatment has a positive regulatory response to oxidative sttess genes in various tissues in diabetic mice. Consequentiy, COB is likely to be sufficiently safe for daily use in prevention and tieatment of diabetes and other non-communicable diseases.
Keywords: Cleistocalyx operculatus flower bud, antioxidation, gene expression,
diabetic mice
