' Nghien CIPU - Ky thuSt
r
Xay dyng mo hinh sang loc chat khang viem thong qua thu thg toll like 4 (TLR4) tren mang te bao macrophage chuot
Nguyin Thi IMai Phirffng'*, Trinh T^t Cirdng^
Tr^n Thi Nhung', Dirmig Thi Nu', DSng Ngoc Quang^
' Vien Cong nghe sinh hgc,
^Dai hgc Khoa hoc Tir nhien
^Dai hgc Suphrfm Hd Ngi
Madau
Vipt Nam la nudc cfl nguIn dupe iidu rit phong phiji. Day chinh la nguon nguydn lipu dli ddo cho cdc nghidn cdu sdng lpc nhim tim ra nhung chit cfl hogt tinh sinh dupe hpc quj, bao gim cd ede chat khdng viem mfii cfl hi#u qua eao ma khfing gay ra cdc phan Cmg phy. Vin de mlu chot cOa cdc nghidn cdu ndy Id phdi xdy dyng dupe mfit mfi hinh sdng Ipc nhanh va hipu qud.
So vdi ede mfi hinh nghidn edu in vivo, mfi hinh sdng Ipe in vitro cfi nhilu uu thi ve thdi gian, dfi phdc tgp vd hidu qua kinh t l . Do dfi, vi^e xdy dyng vd dng dyng mfl hinh ndy la rit d n thilt.
Thy the Toll-tike (TLR) cfl the nhgn dgng nhilu logi vi sinh v§t. Sau khi nhan dang, thu thi ndy nhanh chong sdn xuit ra ede chemokin, cytokin, interferon khdng lai vi sinh v$t d l bdo vp vgt chu chong Igi sy xdm nhdp cCia mim bpnh '^' \ Trong hp thy the Toll-like, TLR4 tham gia hogt dflng trong hd thing midn djeh edm dng bang lipopolysaccharid (LPS) cud vl khuln thflng qua nhdn dgng dgc hi$u nhdng phan td ngogi dflc t l (lipid A). Day Id mpt ylu t l ca ban trong ddp dng miln djch vdi vi khuln gram dm.
Bhattacharjee va Akira ^'' cung da chdng minh duoc TLR4 la thy thi nhgn dgng dae hi^u LPS.
Dya vdo nhung tfn hi§u eda thy t h i TLR4, mpt s6 hp'p chit td thye vdt eung da dupc chdng minh efl khd ndng dilu hfia ddp dng viem vd dilu trj hi$u qud chuflt bi nhilm khuln ngng vd choang nhilm trCing. Vf dy, cde chit tdch chilt td nhdn sdm Hdn Quic nhu Rbl, Rb2, Re, hp'p ehit K (C-K) ^- ^. Cdc djch chiet eCia Amaranthus sp., Eryngium campestre. Thymus pulegioldes vd Reichardia picmides cQng da
T ^ CHf DlTQC HQC - 03/2013 (S6 443 NAM 53)
duae danh gid Id efi kha nang khang viem thfing qua hp thu t h i TLR sd dyng mfl hinh nghidn cdu khdng viem TLR4 in vitro '''^.
Cl mfl hinh sang Ipc chat khdng viem su dyng thy thi TLR4, t l bde macrophage se dup'c xd ly vdi djch chilt thyc v|t vd sau dfl duac gdy cam dng viem bing LPS (phoi tu d$c hidu cOa TLR4). Vide do mde dfl anh hudng eCia ede eytoldn dupe gidi phflng ra la IL-6, TNF-a vd IL- 10 se cho phdp kit lugn djch chiet thyc vgt cfl chda chit khdng v i ^ tilm nang vd chit ndy cfl khd ndng tdc dyng khdng viem theo con dudng thu thi TLR4. Bdi bdo ndy trinh bdy kit qud sdng Ipc cdc chit khdng viem mdl td nguIn dupc lipu Vipt Nam sd dyng mfl hlnh sang lpe in vitro thflng qua thy the TLR4. Ddy Id mfi hlnh sang lpc ehit khdng viem hipn dgi, efl nhilu uu diem va cfl thi triin khai ftSt d Vipt Nam.
Nguyen lieu vd phu'ang phap nghien ci>u
Nguyen lieu
Cdc nguydn lipu thyc vgt duo'c thu thap td Ha Npi va mpt so tinh phfa Bac gim Phii Thp, Hd Tay, San La. Sau khi duac djnh danh, cdc nguyen lipu ndy duac n>a sgch, sly khfl d 50°C, nghien thdnh bpt mjn va ehilt bang dung mfli methanol theo ti Id 1:10 (1 gam nguydn li^u:
10 ml dung mfli). Djeh chilt dup'c thu hll vd
quay cit chan khflng de thu can chilt. Cdc cgn
ehilt ndy sau dfl duae hfia tan trd lal trong dung
mfii thieh ho-p vd su dyng tare tilp eho thf
nghidm sdng lpe cac chit khdng vidm. Danh
sach ede thye v§t lya chpn eho nghidn cdu
sang lpc dupc trinh bay d bang 1.
• Ngh i e n CU'U - Ky thuat
Bang 1: Danh sach cac dlch chiet thuc vat iJuvc lua chon cho nghiin cuu sang k>c cic chat khing viem sCr dung mi hlnh TLR4
1 2 3 4 5 6 7
a
g 10 11 12
Tin Viet Nam Sim Soi n>ng Ngai OJ'U Xuan Inoa (con i<hi)
Dinh Iang Mang cut San tiiuyen Bacil bp Ciie vSng Lu-gc vSng B6ng nifor:
Thanh hao
Danh sach diwc vit Ten la tinh Rhodomyrius tomentosa (Alton) Hassk
Sarcandra glabra (Thunb.) Nakai Artemisia vulgaris Linn Pseuderanthemum palatiferum (Nees) Radlk
Panax fruticosum Linn Garcinia mangostana Linn Syzygium polyanthum (Wight) Walp
Stemona tuberosa Lour.
Jasminum subtriplinerve Blume Callisia fragrans ^indl.^ Woodson
Impatiens balsamina Linn Artemisia anua Linn
Bg phin ddng Li La Ld L^
Ld Vo qud " -
li Re cii
La L^
La i " .
^ ,JA
Hoa ehat lipopolysaccharid (LPS) dup'c mua tu Hang Sigma. Moi trudng nufli ely DMEM va cdc thanh phin lien quan nhu FBS (fetal bovine serum), trypsin, khang sinh dyac mua td hdng Invitrogen. Cac kit ELISA djnh lupng cytokine IL-6, IL-10, TNF-a dup'c mua td hang Bioseienee. Kit dim t l bao (Cell Counting Klt-8 - CCK-8) dup'c mua tu hang Dojlndo Laboratories, Kumammoto, Nhgt Ban. Chupt ehung Swiss dupc mua td V\in Vp sinh Dich t l Trung uang. Cac hod chit cfln iai deu dgt mdc tinh saeh phan tieh.
Phuvng phdp nghien cdu
Tach chiit macrophage tCr tuy xwomg cua chugt
Phin xuang dCii eua chuflt sau khi da gay ehit dupe tdch ra va dupc bam mfli trudng DMEM bing xy-lanh lidn tyc vao giua xuang dui den khi thiy xuang cfl mau trang. Phan dieh thu duyc sau dfl dem Ipe va ly tam. Phin can sau khi ly tdm dupe hoa Igi bang mfli tnj'dng DMEM efl b l sung 10% moi trudng dilu kidn nufli t l bao L929 (duae xem nhu yeu t l kfch thfch M- CSF (maerophage-specific colony stimulating factor), FBS 10%, natri pyruvat 1 mM, penicillin 50 lU/ml, streptomycin 50 pg/ml va 2- mercaptoethanol 5x10"^ M trong dilu ki^n nhiet dp phfing 37''C va COa 5%. Sau khoang 5 ngdy, t l bao dupc xd ly vdi trypsin vd ly tdm d toe dfi 5000 vflng/phOt trong 10 phCit. T l bao sau dfl dwoc ehiajdeu vdo ede gilng eua dTa nhya sao eho mli gilng efi mgt dp 10^ t l bao.
Gay cam Ung viem tren ti bio macrophage v6i LPS
Te bao macrophage trong eae gilng d m|t dp 10^ / gilng dup'c u cung vdi LPS 100 r\glm(.
Sau cdc thdi gian 0 0, 3, 6, 18 va 48 gid, djch nuoi ely te bao dup'c thu lai bdng ly tdm d tie dO 5000 gf phiit trong 10 phut. Dich thu dupc sau dfl dupe xae dinh ham lup'ng cytokin sinh ra bang phuang phdp ELISA.
Dinh gia kha nang wc che qua trinh sinh cytokin cua cac djch chiet thwc v$t
Cao ehiet thye v§t dwgc hfia tan trong dung mfii huu ea thfch hp'p. Sau do, 10 pi djeh tong s l duo'c bo sung vdo cae gilng cfl chda t l bao marcophage trong 60 phut. Tidp theo, cac te bao ndy duae Ci tilp eCing vdi LPS d l gay earn dng viem. Sau thdi gian u thfeh hap vdi LPS, djeh nufii ely t l bao dup'c thu lai bang ly tdm cr tic do 5000 vfing/phiit trong 10 phiit. Hdm lup'ng cytokine dupe gidi phfing ra trong djch thu duac xae djnh bang phuang phdp ELISA.
£>inh gii dgc tinh cua cac djch r^iet thuc vat
Dpe tinh eua cdc djeh ehiet thyc vgt len t l
bao macrophage dup'c danh gid sd dyng kit
dim t l bdo (CCK-8, Dojindo Laboratories,
Kumammoto, Japan). Kha nang hip thy cua cac
mau ndy se duyc do d bude sfing 450 nm bing
mdy dpe ELISA. Gid tri hip thu d 450 nm cua
mli mau ndy duac tfnh nhu tt lp phan tram so
vdi miu khflng dupe xd ly vdi djeh ehilt hogc
dexamethason - Dex (xem nhu 100% t l bdO
song) (Yang vd cpng sy, 2008). 4
T ^ CHI DlTQC HQC - 03/2013 (S6 443 NAM SJNghien ctPu - Ky thuat
Ket qua
T d c h te bao macrophage t d t u y xwamg chu$t
Sau 24 g i d nufli e l y , te bdo macrophage tdch ra t d x u a n g dui chupt d u a c k i l m tra dudi kinh h i l n vi soi ngup'c. K i t qua thu d u p e trdn hinh 1 eho thay iupng t l bao macrophage da c h i l m chu y l u vd bdm dinh r i t tot d u d i day dTa.
T l bdo nufii cay cfi hinh thdi eua macrophage va quan the te bao la dong n h i t , ehdng tfl khflng eo htpn t u a n g nhiem Cac te bdo nay t i l p tyc d u p c nufli cay them 5 ngay nua d l thu sinh k h i l dGng eho ede thi nghipm t i l p theo.
Hinh 1 : Te bao macrophage dwgc tich ra tir tdy xwong chugt
Gay c a m d n g viem tren d o n g te bao macrophage c h u p t s d d u n g LPS
Hdm lupng cytokin cOa t l bao macrophage sau khi gay cdm dng vidm vdi LPS dupc danh gid bdng kit do ELISA dgc hieu. K i t qua thu d u p c (hinh 2A) eho t h i y vdi l i l u lupng t h l p 100 ng/ml, LPS dd kieh thfch t l bdo macrophage sinh TNF-a. Cfl t h i t h i y r i n g ham lupng cytokin nay phy thufic vdo thdi gian u te bao vdi LPS.
Lupng TNF-a sinh ra sau 18 gid dat gia tri idn ( g i n 10.000 pg/ml). Tuy nhien, khi u v u p l qua 48 g i d thi lupng TNF-o Igi giam x u l n g chf cfln khodng 6.500 pg/ml. K i t qua tuang t y cung phdt hipn t h i y vdi cdc cytokin IL-6. So lipu trdn hinh 2B chi ra r i n g lupng IL-6 smh ra hoan todn phy thufic vao thdi gian u vdi LPS. Lupng IL-6 sinh ra dgt n h i l u n h i t sau 18 gid vd gidm mgnh sau 48 gid u vdi LPS.
Bdn than t l bdo macrophage cfi chdc nang t i l t ra ed cytokin t i l n vidm va khdng vidm trong qud trinh vidm. Do v$y, de ddnh gid d u p c khd ndng gdy vidm cua LPS, hdm lupng lL-10, mfit trong nhung cytokin khdng viem cung d u o e
T^P CHf DlTQC HQC - 03/2013 (S6 443 NAIM 53)
ddnh gid. K i t qud tren hinh 2C eho thay luang IL-10 eung sinh ra nhieu n h i t sau 18 gid u vdi LPS ( >5000 pg/ml) vd giam mgnh sau 48 gid (<
4000 pg/ml). N h u vay, eac k i t qua xdc dinh ham lupng cytokin da cho t h i y t l bao macrophage hodn todn d trang thai viem khi cam dng vdi LPS vd 18 gid Id thdi gian thich hpp d l gay cam dng viem vdi LPS d eac te bao macrophage.
3 6 18 48 Thd'i gian (gi£r)
Thcrl gian (gifir)
Hinh 2: LPS kich thieh sinh TNF-a. IL-10, IL- 6 tien viem trong ti bao macrophage. A. TNFa;
B. IL-6; C. IL-10
Danh gid dpc b'nh cua cac dich c h i l t t h y c vdt Cae can ehilt thye vat dupe hfla tan vdi dung mfii huu ea d n i n g dp 10 p,g/ml vd t i l n hdnh danh gia dflc tinh cua cac djch chiet t h y c vgt len t l bao macrophage s d dung kit d i m te CCK-8 (Dojindo Laboratories, Kumammoto, Japan). K i t qua thu d u a c d bdng 1 cho t h i y khfing efi s y khde bidt ve gid tn OD tgi b u d c sfing A450 d t i t cd cac djch c h i l t nghien cdu.
Dieu nay ehung tfi cde djch c h i l t ndy d i u khfing dflc d l i vdi t l bdo. Nhu vgy, eae dich chiet ndy deu efi the s d dyng cho nghien cdu sang ipc cde ehat khdng vidm t i l m nang.
• Nghien C I J U - Ky thuat
Bang 2: Dgc tinh cua len ti bao macrophage
cae dich chiet thwc vit
TT 1 2 3 4 5 6 7 8 9 10 11 12 13
Ten dich chiet Sim Soi n>ng Ngai cuu Xuan hoa Dinh Iang Mang cut S^n thuyen
Bach bg Che vang Lu^c vang Bong nu'dc Thanh Do! chung
OD/l,ai 1.2177 ±0,0002
1,2168±0,000 1.2169±0,0003 1,216310,0001 1,2161 ±0,0002 1,2180±0,000 1,2161 ±0,0001 1,2173 ±0,0003 1,2186 ±0,0005 1,2175±0,0002 1,2160 ±0,0004 1,2169 ±0,0003 1,2178 ±0,0001 Xay d y n g mo h i n h s a n g Ipc T L R 4 Vidm tuy la dap dng mien djeh eo lpi ctia ea the song n i u thdi gian viem qua lau se gay ra hang loat roi loan trong ea t h i nhu: sung phu gay can trd qua trinh tuan hoan, can t r d qua trinh chuyen hoa, trao doi chat trong ca the. Dgc biet, n i u viem man keo dai con gay ndn suy giam ehde nang cua eae ca quan quan trpng nhu: gan, thgn... dan ddn hang loat cac phan dng eo hai eho ca the. Vi vay, v i n d l dat ra la phdi tim d u p e cac logi thuoc cfi kha nang lam giam anh hudng cua viem doi vdi ca the, hay
noi each khae la giam b f i l thdi gian vidm, han c h l s y san sinh eac san p h i m eo hgi t d qua trinh viem. Nhung loai thuoe cfl khd nang gidm vidm phai la thuoc d u p e chdng minh v d a efl kha nang Idm giam s y sinh cac cytokin tien vidm (san p h i m dau tidn eCia qua trinh viem cfi tac dyng ho t r a vidm), dong thdi, ehung khflng anh h u d n g d i n s y sinh ede cytokine khang vidm nhu IL-10, la cytokin ho trp- trong vidm giam bdt
tac hai cua vidm. • K i t qua do ham lupng cac cytokin tien viem
cua t l bao macrophage khi d u p e u vdi p h i i ti>
dae hieu LPS trong s y co mat cua eae dich chiet sdng Ipc dwgc trinh bdy d hinh 3, 4, 5. S l lipu tren hinh 3 cho thay, trong so 12 djeh chiet ti>
t h y e vat d u a e sang Ipc gom: dieh e h i l t Id sim, la soi rdng, la ngdi edu, Id xuan hoa. Id dinh Iang, vo qua mang eyt, la san t h u y i n , r l bdeh bp, la che vang, la l u a c vdng, la bong n u d c . Id thanh hao, thi mau so 1, 3, 5, 8 t u a n g dng vdi djch c h i l t t u la sim, la ngai cdu, Id eay dinh Idng va r l cay bdch bfl eo kha nang lam gidm kha mgnh l u a n g cytokin T N F a d u p c t i l t ra. Cae m l u con Igi khflng eo kha nang nay. Kha nang Idm gidm lupng cytokin T N F a eua re cay bdeh bp ( m l u so 8) thdm chi con thap han ea ehat doi chdng aspirin ( m i u so 13). Cac so li^u thu dupe eho phep k i t lugn cde djch e h i l t t d la sim, ngai cdu, dinh Iang va r l bach bp cfi khd nang de c h l qua trinh sinh cytokin T N F a eua macrophage.
10 11 12 Aspnn Hinh 3: Kha nang d c chi sinh cytokin TNF-a cda te bao macrophage dwgrc cim wng viim bing LPS trong 18 gid khi co mat eic dich chiit thwc vat (1 mg/ml). DC (-): doi chirng am (dich ti bio khdng CO LPS), DC(+): ddi chwng dwang (djch ti bao tJ vai LPS), 1. la sim, 2. li sdi rimg, 3. la ngai cwu. 4. li xuin hoa, 5. la dinh Iang, 6. vo qua mang cut, 7. la sin thuyen, 8. ri bach bfl, 9. li che ving, 10. H Iwac vang, 11. li bong nwac, 12. la thanh hao, 13. Asprin (ehit eo khi ning chong viim m^nh khdng
CO ban chit steroid 1 mg/ml). Cac thi nghiem dwgrc lip lai ba lin d0c Igp.
T^P CHI DlTQC HQC - 03/2013 (S6 443 NAM 53)
• Nghien CLFU - Ky thuat
DC(-) 0C(+) 1 3 5 8 /\sprin Hinh 4: Kha ning dc chi sinh cytokin IL-6 cua ti bao macrophage dwac cam wng viem
bing LPS trong 18 gia khi co mat cae dich chiit thwc vat (1 mg/ml).
DC (-): doi chO'ng im (djch te bio khong co LPS), DC(*): ddi chwng dwang (djch te bao u vol LPS), 1. la sim, 3. li ngai cO'u, 5. ta dinh ling, 8. ri bach bd. Asprin (ehit ed kha ning ching viim manh
khong co bin chit steroid Img/ml). Cac thi nghigm dwac tip lai ba lan die l$p.
Cac djeh e h i l t chiet t u Id sim, ngai cdu, dinh Iang va re bdeh bfi d u p c tiep tyc danh gia anh hudng ien s y sinh cytokin IL-6 cua t l bao macrophage. S l lieu thu d u p c d hinh 4 eho t h i y cde djch ehilt nay d i u efl kha nang de e h l mgnh s y sinh IL-6. Mue dp dc che mgnh IL-6 d u p e phat hipn vdi djch c h l l t la sim vd r l cay bdch bp vdi hdm lupng cytokin sinh ra chi dgt <
30% so vdi doi chdng d u a n g .
N h i m danh gia dup^: kha nang anh hudng
t d i qua trinh sinh eae cytokine khang vidm IL- 10 cua cac djch c h i l t t d la sim, ngai e d u , dinh Iang vd r l bach bfl, cdc t l bdo macrophage da d u a e gay cam dng vidm vdi LPS khi cfl mgt cua cac djch c h i l t nay. K i t qua trdn hinh 5 da cho thay ed 4 djch c h i l t ndy d i u khfing dc che cytokin IL-10. N h u vgy ede djch chiet la sim, ngai e d u , dinh Iang vd re bdeh bfi d i u cfi tiem nang khang viem thfing qua thy the TLR4.
illllll
OC(-) DC(+) 1 3 5 8 Asprin Hinh 5: Kha nang Oc ehi sinh cytokin IL-10 cua ti bio macrophage dupc cam wng viembing LPS trong 18 gia khi co mit eua cic dich chiit thgv vgt (1 mg/ml).
DC (-): doi chCmg im (dieh ti bio khdng eo LPS). DC(*): doi chwng dwang (dieh te bao u vm LPS), 1. la sim. 3. li ngii cwu, 5. li dinh Iang. 8. ri bach bg. Asprin (chit co kha ning chong viem mgnh
khdng co ban ehit steroid 1 mg/ml). Cae thi nghigm dwac tip igi ba lin die Igp Nhan xet va ket luan
- Mfl hinh sdng Ipc c h i t khang vidm tiem ndng thflng qua thy t h i TLR4 da d u a c xay dyng thdnh cfing g i m cdc budc: i) tdch t l bao macrophage t d xuang dCii ehupt; ii) gdy cdm dng viem d t l bdo macrophage vdi LPS d n i n g
T,^P CHf DlTQC HQC - 03/2013 (SO 443 NAM 53)
dp 100 ng/ml; lii) do hdm luang cytokine cua macrophage giai phflng ra khi efi mat cua ede djeh chiet thye v l t .
- Trong 12 mau djch ehilt thyc vgt dupc sdng Ipe g i m dich chiet la sim, la soi n>ng. Id ngai cdu.
Id xuan hoa. Id dinh Idng, vo qud mdng cyt, la s i n
• Nghien CIFU - Ky thuat
thuyen, r l bach bp. Id ehe vdng, id l u y c vang, la bong nude, la thanh hao thi ehi cfl djch ehiet ehilt cua Id sim, ngai cdu, dinh Iang va re bach bp cfl kha nang Idm giam luang cac cytokin t i l n viem la TNF-a va IL-6 trong t l bdo macrophage dup'c cam dng bdi LPS trong 18 gid thfing qua thu t h i TLR4. Dieh ehilt t u r l eay bdch bfi (Stemona tuberosa Lour.) cfl kha nang dc c h l mgnh han ca e h i t khang vidm doi ehdng asprin.
- Cac djch chiet t u la sim, ngai edu, dinh Iang va bdeh bfl khflng de e h l s y sinh cytokin khang viem IL-10, ehdng to ehung la nhung djch c h i l t khang viem tiem nang, can phal d u p e t i l p tuc nghien cuu.
L d i c a m e n : Cong trinh nay d u p c hoan thanh vdi s y ho trp kinh phi cua de tdi Khoa hpc Cflng nghd Vidn Khoa hpc va Cfing nghp Vidt Nam ma so VAST02.03/12-13.
Summary
As a contribution to a systematic study on screening, extraction, isolation and exploitation of novel bioactive agents from Vietnam's natural resource of medicinal plants, novel anti- inflammatory compounds were screened in vitro from therefrom by the screening model of toll like 4 receptors (TLR4) on the macrophage cells of mice. The In vitro model was successfully established with 4 of 12 potentially anti- inflammatory herbal extracts including Rhodomyrtus tomentosa (sim). Artemisia vulgaris (ngai cu-u), Panax fruticosum (dinh ling) and Stemona tuberosa Lour (bich bg). For the LPS-induced inflamation. the pro-inflammatory cytokines IL-6 and TN-a levels in the macrophage cells pretreated with the plant extracts were markedly lowered, especially with
those from Stemona tuberosa Lour. Contmrilly, no effects were recorded with IL-10. The bioactive compounds in the root extracts from Stemona tuberosa Lour is still under isolation of active compounds and characterization for inflamamtory activity.
Key words: Toll-like 4 receptor (TLR4), inflammation, lypopoiysaccharide (LPS), plant extracts, cytokines.
Tai lieu tham khao 1. Bhattacharjee R, Akira S (2005), "Toll-like receptor signaling: emerging opportunities in human diseases and medicine", Cument Immunology Reviews-1: 81-90.
2. Cuong TT, Yang CS, Yuk JM, Lee HiVl, Ko SR, Cho BG, Jo EK (2009), "Glucocorticoid receptor agonist compound K regulates dectin-1-dependent inflammatory signaling through inhibition of reactive oxygen species", Life Sciences. 85: 625-633.
3. Of arctiln by inhibiting COX-2 expression via NF-KB pathways. Journal of Inflammation. 8:16-24.
4. Ramasubramaniaraja R (2011), Inflammation and medidnal herbs, Internatbnal Joumal of Pharmaceutical Sciences Review and Research. 9:1-7.
5. Schnare M, Rollinghoff M, and Qureshl S (2006), "Toll-like receptors: sentinels of host defense against bacterial infection", International Archives of Allergy and lmmunology-139: 75-85.
6. Weiss DS, Raupach B, Takeda K, Akira S, and Zychlinsky A (2004), Toll-like receptors are temporally involved in host defense", Joumal Immunology. 172: 4463-^1469
7. Yang CS, Ko SR, Cho BG, Shin D M. Yuk JM, Li S, Kim JM, Evans RM, Jung JS, Song DK and Jo EK (2008), The ginsenoside metabolite compound K, a novel agonist of glucocorticoid receptor, induces tolerance to endotoxin-induced lethal shock, Joumal of Cellular and Molecular Medicine. ^2 1739-1753.
N g h i e n C L F U . . . (Tiip theo trang 25) (i) S. Zhu et al. (2006), '6-Substituted 6H- dlbenzo[c,/)][2.6]naphthyndin-5-ones: Reversed lactam analogues of ARC-111 with potent topoisomerase l-targeting activity and cytotoxicity', Bioorg. Med. Chem.. 14, 3131-3143; Q) Feng, W.S.
et al. (2008), '11-Substituted 2,3-dimethoxy-8,9- methylenedioxybenzoHphenanthridine derivatives as novel topoisomerase !-targeting agents', Bioorg. Med.
Chem., 16, 8598-8606; (k) Wei F. et al. (2009). '12- Substituted 2,3-dimethoxy-8,9-methylenedioxybenzo [ilphenanthridlnesn as novel topoisomerase I- targeting antitumor agents', Bhorg. Med. Chem., 17.
2877-2885.
4. Molecular Operating Environment (2008), 10.
http://vww chemcomp.com (15.02.2011).
5. Rapidminer http://rapid-l.com (15.02.2011).