Y HOC VIET NAM THANG 5 - SO 2/2015
TOI DEN CO TAG DUNG DIT PHONG UNG THU* TREN MO HINH UNG THir GAN NGUai THU'C NGHIEM
T O M TAT
Muc dich: Nghien cu'u nay cd muc dich lam sang to kha nang du" phdng ung thU gan cua toi den.
Phu'dng phap: Chuot thieu hut miln djch (athymic mouse) du'dc udng djch tdi den hang ngay vdi lieu 1 g/kg va 10 g/kg the trong, trong 1 thang. Sau do, dpng vat du'dc ghep 10* te bao ung thu" gan ngu'di.
Ket qua: vdi lieu tdi den 10 g/kg the trpng/hang ngay, keo dai 1 thang, ti le chupt hinh thanh khdi u giam ro ret so vdi nhdm chiTng (p<0,05). Hdn niJa d lieu nay, tdi den co kha nang dc che phat trien ciia khdi u, keo dai thdi gian sdng va tl le chupt song so vdi nhdm chimg (p<0,05J. Ket luan: Tdi den du'dng udng cd tac dyng dd phong ung thu' gan tren thu'c nghiem.
TWkhda: tdi den, Hep 3B, du" phdng, ung thu" te bao gan
S U M M A R Y
CANCER PREVENTIVE EFFECTS OF BLACK GARLIC ON H U M A N HEPATOCARCINOMA
A N I M A L MODEL
Aims: This study investigated the cancer preventive effect of black garlic on hepatocellular carcinoma. Methods: Athymicnude mice drank daily the of black garlic solution by the doses of l g or 10 g/kg body weight in 1 month. Then, the nude mice were inoculated 10* human hepatocellular carcinoma cells. Results: The results showed that with dose of 10 g/kg daily of black garlic solution were significantly reduced in the rate of nude mice beanng tumor development and volume of tumors in nude mice.
Moreover, by this dose, the black garlic also inhibited significantly the tumor grow and prolong alive time of nude mice beanng tumor in companson to control group (p<0.05). Conclusions: Drinking the black garlic shown its effect to prevent liver cancer development in vivo.
Keywords: black garlic, Hep 3B, prevention, hepatocellular carcinoma
I. OAT V A N OE
Sir dung tdi thu'dng xuyen cd kha nang dy"
phdng mpt so benh trong y hpc hien dai, cung nhu" Y hpc co truyen 6 nu'de ta va y hpc Trung
* Vien YhpcCotruyen Quan dpi Chiu trach nhiem chi'nh: Pham Xuan Phong Email: [email protected] Ot: 0912083747 Ngay nhan bai: 05/3/2015
Ngay phan bien khoa hpc; 20/3/2015 Ngay duyet bai: 28/3/2015
Pham Xuan Phong*
Hoa. Tdi du'pc len men goi la tdi den cd ham lu'dng mdt sd nhdm chiTc da thay doi manh so vdi tdi tiidng thu'dng chu'a len men, trong d d : ham lu'dng flavonid, thiosufat, va polyphenol toan phan tang tir 1,5- 2,5 lan so vdi tdi tu'di tru'dc khi len men [ 1 ] . Dinh lu'dng S- Allyl-L- Cystein mdt hdp chat cd tac dung sinh hpc chi'nh cd trong tdi thu'dng cho thay ham lu'dng nay tang 6 i l n so vdi tdi tu'di tru'dc khi len men [ 2 ] , [3]. Ngoai ra trong thanh phan hda hpc ciia tdi den sau khi len men ham lu'dng mpt so acid amin tu" do tang ro ret so vdi tru'dc klii len men nhu': Acid aspartic, threonin, Methiolin, Arginin...
Cac ket qua nghien ciTu danh gia tac dung sinh hpc cua tdi den gan day cho thay tdi den cd tac dung chdng oxy hda manh tren ddng vat thy'c nghiem thdng qua cac chi so nhu^ SOD, GSH, TAS, MDA. Ngoai ra, djch chiet tdi den cd nguon gdc vLing dao Ly Sdn cdn cd tac dung kfeh thfch mien djch tren md hinh chupt gay suy giam mien djch bang chieu tia xa. Trong nghien cu'u nay, Chung tdi tiep tuc danh gia hieu qua ciia tdi den trong dd phdng ung thu' gan tren chupt thieu hut mien djch.
II. OOI T U p N G VA PHU'OTMG PHAP NGHIEN COU 2 . 1 . 0 6 1 t u ' d n g
- Te bao unq thu" bleu md gan ngu'di ddng Hep3B (HB-8064, ATCC, Hoa Ky). Day la mpt ddng ung thu' bieu md gan du'dc phan lap tir benji nhan nam 8 tudi bi ung thu'gan.
- Mau bpt cao khd chu3 50% tdi den dat TCCS (so Id 010313) do Trung tam nghien cuU iiVig dung san xuat thudc - Hpc vien Quan y cung cap.
- Mdi tirdng nuoi cay te bao: mdi tru'dng nudi cay te bao DMEM, bo sung 10% huyet thanh bao thai be (FBS), 1 % dung djch penicillin/ streptomycin.
- Chupt thieu hut mien djch (Athymic BALB/c mice, nu/nu) la ctiupt dot blen gen dan den thieu hut quan the te bao lympho T (nude mice, Foxnl™). Chupt du'dc nhap khau tir Cdng ty Sigma, Hoa Ky, nudi trong dieu kien phdng sach, khong khf du'dc Ipc va cd ap lu'c du'dnq tinh.
Nhiet dp phdng dirdc duy tri d 25 ± 0 , 5 ^ , do am 55 ± 5%, anh sang du'dc tu" ddng dieu khieri bat liic 7h00, tat luc 19hd0. thiTc an {Zeigler, Hoa Ky) va/iu'dc udng du'dc t i e t j r i i n g tru'dc khi sir dung. Mdi long chupt du'dc de tren he thong gia cd thdng khf dpc lap va Ipc qua mang bao dam kha nang each ly tdt vdi mam benh.
Y HOC VIET NAM THANG 5 - SO 2/2015
SS lifdng chuot thieu hut mien dich: 30 con 2.2. Phu'dng phap nghien ciJu 2.2.1. Tao khoi ung thif gan ngWdi tren dill chuot
- NUOI cay te bao ung thu' gan ngu'di ngu'di dong Hep 3B, dydc nuoi cay trong moi trycfng DMEI>1 bo sung 10% FBS, 1% penicillin va streptomycin.
Te bao du'dc nuoi cay, tang sinh trong chai co dien tich d6y chai 75 cm^ dat trong tii vo trUng, duy tri nhiet do 370C va C02 5%. Thay moi trtfdng nuoi cay 3 ngay/lan. Khi te bao d?t mat do 80% dien tich be mat chai, tach khoi chai nuoi bang Trypsin EDTA 0,05% va cay cliuyen sang chai nuoi cay mdi. Khi so lifdng te bao du idn, thu hoach te bao va dem so lu'dng te bao thu difdc, dieu chinh mat do te bao dat mat do 10' te bao/ml.
- Sau khi thu hoach te bao ung thy, chupt nude du'dc co dinh va tiem djch chiJa te bao vao du'di da dill phai. Qua trinh thao tac du'dc thifc hien trong dieu kien vo triJng tuyet doi.
2.2.2, Theo doi vi xic dinh sif hinh
thinh khoi ung thu' tren chuot,
Khoi ung thy tao thanh tren chuot dydc danh gia theo doi su" phat trien tai vj tri tieni (diJi phai) 2 lan moi tuan, bang quan sat, sd nan va do kich thydc khoi u bang thydc chinh xac NSK.
+ Kich thydc khoi u; khoi u dydc do theo hai kich thydc dai x rong bang thydc chinh xac NSK (Nhat Ban), 2 @n/tuan.
kich thydc u dydc tinh theo cong thifc: V=D X R^ X 0,5
Trong do: V: the tich khoi u;
D: chieu dai khoi u;
R: chieu rong khoi u
2,2.3. Phan nhom vi each sir dung tdi den Chuot nude dyflc ngau nhien chia thanh cac nhom va uong vien nang toi den nhy sau:
- Nhom chiing: 10 chuot nude, hang ngay dydc cho uong nUdc cat tydng ifng vdi _the tich 0,lml/10 g the trong, lien tuc trong 4 tuan.
- Nhom dy phong 1: 10 chuot nude, hang ngay dydc cho uong nydc hoa tan bot cua vien nang toi den vdi lieu tydng irng Ig/kg the trpng/ngay, dydc pha vdi the tich 0,lml/10 g the trong.
Chuot dydc udng lien tuc trong 4 tuan.
- Nhdm dif phong 2: 10 chuot nude, hang ngay dydc cho uong nydc hoa tan bot cua vien nang tdi den vdi lieu tydng irng lOg/kg the trpng/ngay, dydc pha vdi the tich 0,lml/10 g the trong. Chupt dydc uong Hen tuc trong 4 tuan.
Sau khi uong lien tuc tdi den 4 tuan, chupt dydc ghep te bao ung thy gan Hep 3B va theo doi ti le mpc khoi u, toe dp phat trien khoi u, thS gian song, ti le song chet ciia cac nhdm. Thi nghiem difng d thdi diem sau khi bat dau dieu trj bang tdi den 3 thang.
III. K£T QUA
3,1. Ti le moc u va kich thu'dc khoi u gan tren cac nhom chuot
Nghien cu'u dydc tien hanh tren 30 chuot nude, sau khi chupt nhdm dif phdng 1 va 2 d y * uong dung djch pha bpt vien nang toi den vS lieu 1 va lOg/kg the trpng/ngay x 1 thang lien tuc, nhdm chifng dydc udng nydc cat tydng ifng viSi thdi gian tren. Sau dd, so chupt nay du'dc ghep te bao ung thy gan ngydi Hep 3B.
Bang J. Ti le chupt cd khoi u gan Hep 3B ciia cac nhdm chupt Chu'ng (1)
Du' phong 1 (2) Du' phong 2 (3)
P
So lu'Ong (con) 10/10 10/10 6/10
Ti le (%) 100 100 60
P31,7<0,05Nhan xit: Trong so chupt dydc ghep te bao ung thy, nhdm di/ phpng 2 cd 40% so chupt khong phat trien khoi u. Cac nhdm khac, so chupt cd u diu dat 100%. Trong suot qua trinh thi nghiem, so chupt khdng mpc u nay cd dien blen binli thydng, khdi cd diii tai vj tri ghep dydc kiem tra khi ket thiic thi nghiem cho thay khong cd hinh anh ciia khoi u.
Bang 2. Kich thydc khoi u qan Hep3E Ngay(D)
Chu'ng (1) Du' phong 1 (2) Dy phong 2 (3)
P
DO 78,3 76,3 92,7
>0,05
D14215,4 339,9 204,2
>0,05
D21312,8 378,1 217,3
>0,05 cua cac
D35
344,0 406,3 311,0
>0,05 nhdm chuot
D42
507,0 507,2 354,6
>0,05
D49609,3 626,3 375,3
Pl-3<0,05 D56
907,8 882,5 433,0
Pl-3<0,05 D63
1203,5
949,9 458,9
Pl-2,3<o,os
Y HOC VIET NAM THANG 5 - SO 2/2015 Nhin xet: Ket qua cho thay khdi u ciia cac
nhdm chupt van tang dan theo thdi gian, den ngay 63 thi cd 1 chupt nhdm chii'ng chet. Qua trinh do kfeh thu'dc khdi u du'dc dirng lai. Trong giai doan dau den ngay thir 42 (D42), kfeh thu'dc
trung binh khdi ung thu' ciia cac nhdm chupt khdng cd s i / khac biet thdng ke.
O ngay thir 63 (D63) Nhdm du' phdng 1, lieu Ig/kg can nang, cho thay sd khac biet cd y nghTa thdng ke ve kich thu'dc trung binh l<hdi u so vdi nhdm chirng vdi p<0,05).
Bleu do 1. Dien blen ve ki'ch thu'dc khdi u gan Hep3B
Nhin xet: Nhdm chupt dd phdng 2, cd ki'ch thu'dc trung binh khdi u nhd hdn nhom chirng va nhdm dir phdng 1 mdt each cd y nghia thong ke tir lan do ngay 42. The tfch trung binh khdi u cua nhdm dir phong 2 chi xap xi 1/3 so vdi nhdm chu'ng.
3 . 2 , Ti Ie sdng chet va thdi gian song trung binh ciia cac nhom chuot Bleu do 2. Ti le sdng chet ciia cac nhdm chupt
~\ lesongi%';
— • - Chung
~ ^ Du phong 2
••E) DU'' phong 1
~hb'\ gian mgay;
Nhinxet'Tai thdi diem ket thiic thi nghiem (sau ghep te bao u 3 thang), nhdm chirng, dd phdng 1 va du" phdng 2 cdn lai so chupt lan lu'dt la 3 0 % ; 5 0 % va 70%. Tuy nhien, khdng cd sd khac biet cd y nghTa thong ke. (p>0,05).
Bing 3. Thdi gian song trung binh ciia chupt den thdi diem ket thuc thf nghiem Thdi gian song
t r u n g bJnh ( X ± SD)
Chu'ng
76,5 ± 11,0 79,4 ± 12,1
Du* phdng 1 Du* phdng 2
m I (3)
85,6 ± 7,3 <0,05 Nhin xit: Ket qua cho thay thdi gian song trung binh ciia chupt d y phdng 2 dai hdn cd y nghTa thong ke so vdi nhdm chtfng (p<0,05).
Y HOC VIET NAM THANG 5 - SO 2/2015
IV. BAN LUAN
Ket qua nghien ciru ciia chiing tdi cho thay, sau khi du'dc udng du" phdng tdi den keo dai 1 thang vdi lieu 10 g/kg/ngay, cd tdi 40% chudt nude dupe ghep te bao ung thu" gan khdng xuat hien khdi ung thU. Oong thdi, vdi lieu tdi den nay ed tac dung kim ham si/ phat trien kfeh thu'dc khdi ung thu". Chupt du'dc dd phdng bang tdi den, ed ti le sdng cao hdn va thdi gian sdng dai hdn so vdi nhdm chirng. Cac nghien ciTu cho thay, trong tdi cd nhieu thanh phan khac nhau nhd cac hdp chat lu'u huynh hu'u cd va selen cd tac dung u'c che su" tang sinh ciia te bao ung thd, dieu chinh chu ky te bao va gay apoptosis 2,3. Kim va cdng sd (2012) cho thay hoat chat chinh ciia tdi cd the gay chet te bao theo chu'dng trinh (apoptosis) tren ddng te bao ung thu' gan Hep 3B bdi sd dieu hda cac gdc ty* do thdng qua cac gay tdn thu'dng mang ty the, giam bdc Id cac gen chdng apoptosis nhu" Bcl-2 va Bcl-xl 4. Dong thdi, dich chiet tdi gay tang bleu Id cae gen gay chet theo chu'dng trinh (apoptosis) nhu' capase 3, capase 8 va capase 9, tir do thuc day te bao ung thu'gan chet 4.
Nghien ciru tu'dng td tren ddng ung thU te bao gan Hep G2, cac tac gia cho thay kha nang kim ham sir phat trien ddng te bao nay, cac bang chuYig cho thay nong dp protein p53 ndi bao tang dang ke trong cac te bao du'dc dieu trj bang Alllcin tir tdi va gay giam nong dp Bcl-2, Ngu'dc lai, Alllcin lam tang bde Id AMPK/TSC2 va con du'dng tfn hieu Beclin-1 trong ddng te bao ung thu' bieu md gan Hep G2 thiic day qua trinh chet te bao 4.
Tren thyc nghiem, S-allylcysteine (SAC) cd tac dung ire che si/ phat trien eiia ddng ung thd phoi khdng te bao nhd NSCLC A-549. SAC cho thay kha nang ire che ddng te bao nay thdng qua dc ehe si/ hoat hda eac phan tir mTOR, NF- KB, va cyclin D l [7], Dong thdi, SAC cdn dc che sy di can ciia ddng ung t h u phdi khdng te bao nhd tren chupt mang khdi ung t h u nay. Cac tac gia eung phat hien cd ehe chdng ung t h u ciia tdi tren ung thd phdi cung tUdng t y nhu cac ddng ung t h u khac. Li va CS (2011) cung phat hien kha nang ciia DATS tir tdi cd kha nang gay chet te bao ung thU theo con dUdng apoptosis thdng qua cd che gay Ore che giam bleu hien ciia Bcl-2
va lam tang bleu hien eiia ti le Bax/Bel-2 va tang hoat ddng ciia eac gen ma hda capase 3, capase 8 va capase 9 5.
V. KET LUAN
Chudt thieu hut mien dich, sau khi dUdc udng d y phdng tdi den keo dai 1 thang vdi lieu 10 g/kg/ngay, 4 0 % chudt nude dddc ghep te bao ung thU gan ngUdI khdng xuat hien khdi ung thU. Vdi lieu tdi den dU phdng 10 g/kg/ngay, ed tac dung kim ham sU phat trien kfeh thUdc khdi ung t h u . Chupt dUdc dU phdng bang tdi den, cd t i le sdng eao hdn va thdi gian sdng dai hdn so vdi nhdm ehirng.
TAI LIEU T H A M K H A O
1. Ho Anh Sdn, Vu Binh Du'dng (2014), "Nghien ciru tac dung ire che te bao ung thu" dai trang ciia dich chiet toi den va tac S-allyl-L-cystein phan lap ti/tdi den". Tap chi du'dc hpc, 460,27-31.
2. Nian H, Delage B, Ho E, Dashwood RH (2009), "Modulation of histone deacetylase activity by dietary isothiocyanates and allyl sulfides: studies with sulforaphane and garlic organosulfur compounds". Environ Moi Mutagen., 50(3), pp. 213-21.
3. Stan SD, Singh SV ( 2 0 0 9 ) , 'Transcriptional repression and inhibition of nuclear translocation of androgen receptor by diadyl trisulfide In human prostate cancer cells", Clin Cancer Res., 15(15), pp. 4895-903.
H. Kim H3, Han MH, Kim GY, Choi YW, Choi YH (2012), "Hexane extracts of garlic cloves induce apoptosis through the generation of reactive oxygen species in Hep3B human hepatocarcinoma cells", Oncol Rep, 28(5),1757- 1763.
5. Lee BC, Park BH, Kim SY, Lee YJ (2011),
"Role of Bim in diallyl trisulfide-induced cytotoxicity in human cancer cells", J Cell Biochem., 112 (1),118-127.
6. Mousa AS, Mousa SA (2005), "Anti- angiogenesis efficacy of the garlic ingredient alliin and antioxidants: role of nitric oxide and p53", Nutr Cancer; 53:104-10.
7. Tang FY, Chiang EP, Pai MH (2010).
Consumption of S-Allylcysteine Inhibits the Growth of Human Non-Small-Cell Lung Carcinoma in a Mouse Xenograft Model", J Agric food Chem, 9(24), 1156-1164.
