NGHIEN CUtJ TAC DUNG PHONG NGl/A LOAN SAN TE BAO GAN VA CHONG SUY TUY CUA LINH CHI
Ngo Kieh Ehic*, Trdn Manh Hung*
TOM
TATMd ddu: Ung thtf la benh gdy ti le tie vong cao, vi the phdng ngiea ung thie, hda tri lieu va tri lieu hd trg ung thu ludn dugc chii trgng. Nhieu nghien deu dd chihig minh linh dti (Ganodenna luddum) cd tdc phdng ngiea ung thu in vitro (Yuen 2005), trong nghien cieu ndy, chiing tdi klido sdt tac dung dia lmh dti trong phong ngiea loan sdn tebdo gan vii khd ndng phuc Hdi tinh trang suy tuy in vivo.
Muc tieu: Nghien dtu ndy dat ra 2 muc tieu sau: 1) Nghien deu tdc dung cita linh chi trong phdng ngiea hqn sdn te'bdo gan, vd 2) N^ien deu tdc dung cm Unh dii tren khd ndng phuc Iwi tinh trqng suy tuy do thuoc hoa tri ung thu gay ra.
Phuang phdp: Nghien cim dugc tiim hanh tren ca sd md plidng phuang phdp gay ton thuong tien ung thu bdng N-nitrosamin (Hedtt 1997) vd phteong plidp gay suy tuy bang methotrexat (Nakamura 2003).
Ket qua: Tren mo hinh gay loqn sdn tebdo gan bdng dietylnitrosamin, lirdi chi (15% vd 30%) cd tdc dung uc dteste hitdi thdnli khdi ii; iec die Men tiegng plidn bdo bai thieang vd su hinh thdnh cdc nhdn quai dt Tren md hinh gdy suy tuy bdng methotrexat, linh chi cd tdc dung phuc hoi tong lirgng bqdi cau bi suy gidm, ddc biit linh chi cd tdc dung phuc hdi dong bach cdu trung tinh va bach cdu lympho; ngoai ra linh chi dmg cd kha nang phuc hoi tinh trqng suy gidm tieii mu.
Kii luam Ngodi tdc dung phdng ngiea va tic diesu hinlt thdnh khdi u, linh chi ding co tdc dung phuc lioi tinli trqng suy tiiy do thudc lioa tri gay ra.
Tiikhoa: linh dii, dietylnitrosamin, hda phong ngiea, suy tiiy ABSTRACT
INVESTIGATION OF PROTECTIVE EFFECTS OF LINGZHI ON UVER DYSPLASIA AND BONE- MARROW SUPPRESSION
Ngo Kien Due, Tran Manh Hung * Y Hoc XP. Ho Chi Minh * Vol. 14 - Supplement of No 1-2010:52- 58 Background: Cancer is one of the most fatal diseases, therefore cancer prevention as well as cancer chemotherapy and supportive treatment are of important. Many stiidies have proved cancer-preventive effed of lingzhi (Ganodemm luddum) in vitro (Yuen 2005) In this study, loe investigated the effect of lingzhi in prevention of hepatic dysplasia and in recovery of bone marrow suppression in vivo.
Objectives: The aims of this study were as following-1) To investigate the effed oflingzlii on prevention of liepatic dysplasia induced by dietliylnitrosamine, and 2) to investigate tlie recovery effed of lingzlii on mcthotrexate-induced bone marrow suppression.
Methods: Study was conduded in accordance with "two-stage cardnogenesis" method described by Hedit (1997) and bone marrow suppression was induced by methotrexate as suggested by Nakamura (2003).
Results: On liepatic dysplasia model induced didhylnitrosamine: lingzhi 15% or 30% showed inhibitory effeds oji tumorigenesis and abnormal cell division. On methotrexate-induced bone mairow suppression, lingzlii
' Khoa Duoc - Dai hpc Y Dirpc Tp. HCM
Dia chi lien he: D5. Ngo Kien Ehic DT: 0903 055 357 Email ngokJenduc^gmail-com
e^ed recovering action on uhite blood cell count, especially on tlu numbers of neutrophil and lympliocf, in addition, liiigdn also showed recovering effect on deaeascd platelet cell count.
Conclusion: In addiHon to cancer prevention, lingzhi abo possesses recovering effect of bone-mam, suppression induced by dtemotherapy.
Keyzvords: lingzhi, diethynitrosamme, cliemoprevention, bone_-jnarrow suppression. ^ r^:,v«>:^>T r-.»i —IT-.,. mAi IA fi't R_1 S m n C h i i o DAT VAN DE
Benh ung thu ngay cang gia tang khong chi 6 cac nuoc phat trien ma 6 ca cae nuoe dang phat trien, trong do co Viet Nam. Ti 1§ tu vong do benh ung thu chi diing sau tim mach.
Truoc day ung thu diroc coi la b^nh nan y, nhung ngay nay nho su phoi hop tot giira phau tri, xa tri, hoa tri va cae tri U?u ho tro, chat luong cugc song va ti le song cua benh nhan ung thu ngay cang dvtgc eai thien.
Trong nhihig loai duoc lieu dugc nghien euu nham phong ngira va diHu tn ung thu, lirh ehi (Ganoderma luddum) duge xem la co nhieu tac diing dang chii y. Thanh phan polysaccharid cao trong linh chi lam tang kha nang mien dich ciia CO the, lam tang chuc nang gan, co lap va diet eac te bao ung thu (Yuen 2005). Nhom hoat chat protein ma tieu bieu la Lingzhi-8 dugc chiing minh la chat chong di ung va dieu hoa mien dich hiru hieu, dong thoi duy tri sir tao khang the chong viem gan B (Kino 1989), Nhom triterpen co tac dung ha huyet ap, irc che acetj'lcholine esterase, bao ve gan, chong khoi u (Lui 1993).
Nham gop phan mo rgng kha nang ung dung cua linh chi trong phong ngira va tri Ueu ho trg ung thu, chiing toi tien hanh nghien eiru nay voi 2 muc tieu ehinh sau:
- Nghien cihi tac dung ciia linh chi trong phong ngua loan san li bao gan gay boi dietylnilrosamin - Nghien curu tac dung ciia linh chi tren kha nSng phuc hoi tinh trang suy tuy do thu6c hoa tri ung thu gay ra.
odlTL/QNG -PHl/ONG mAPNGMEN c u l l Thu vat thii nghiem
Thii vat thu nghiem la chupt nhat trang, chimg ddY, giong due, eo trgng Iupng tu 18-20 g, dugc phan bo ngau nhien thanh nhieu 16 khac
nhau, m6i 16 tii 8-15 con. Chupt dugc nuoi tronj moi truong tien hanh thuc nghiem tir 3-5 n^' de thich nghi vol moi truong.
Che p h ^ thu n g h i e m
Che ph&n thif nghiem la cao kho Linh tii toan phan do C6ng ty Co Phan Dirge Phajn OPC san xuat dat tieu chu&i kiem nghiem cap CO so. Bpt cao kho linh chi dupc pha vao nuoc cat de tao thanh cao long voi ham lugng M (15g cao kho/lOO ml nuoc) va 30%. Cac dim|
dich nay dupe sir dung eho eac 16 thii nghifM Bdng 1. Cong thiec bdo che'vd tieu chuan nguyen j » dm cao linh chi (do OPC sdn xu^) M
Cong thtPc t)ao che Linh chi (G. luadum}
10kg
Natri benzoal 5g NU'O'C u6ng difoc vira flu
Tieu chuln nguyfin l|^
Bat tieu chuln ccr sd Dat tieu chuln DuTJcflil^
Viet nam II tap 3 I Dgt tieu chuln cua Vi^n"*
smh y lk c6ng cong Phucmg, p h a p gay loan san gan bang dietylnitrosamin va k h a o sat tac dung p h o n g n g u a loan san g a n ciia cao linh chi
M6 hinh gay loan san gan dirge thtrc hien bang each tiem phiic mac (ip) dung didi dietylnitrosamin 1%, sau do tiep tuc tiem dung dich e c u 4% trong dau oliu (ip) hang hian. Danh gia mure do loan san gan bang viec phan tieh dai the va vi the te bao gan (Tran 2005).
P h u o n g p h a p gay suy tiiy bang methotrexat va k h a o sat tac d y n g phye hoi suy hiy ciia cao l i n h chi
Methotrexat duge su dung a lieu 7,5 mg/kg the trpng, tiem phue mac vao cac ngay 0, 3,6 ciia qua trinh thir nghiem. Toan bp qua trinh thii nghiem keo dai 7 ngay. Vi?e xac dinh eong thiic mau dugc tien hanh vao ngay 0 (truoc khi tiem methotrexat) va ngay 7 sau khi tiem h'eu dau tien methotrexat.
Phan tich ve mat mo hpc
Gan chugt thi nghiem dugc co dinh trong dung dich formaldehyd 10% trong dung dich dem. Quan sat dai the dupc tien hanh bSng viec so sanh hmh dang ciia gan 6 nhom gay nhiem dgc vol gan ciia nhom chugt binh thirong. Su hinh thanh cae not tang san bat thuong 6 cac nhom chugt thi nghiem ciing dupc ghi nhan va thong ke. Quan sat vi the gan bao gom quan sat cau tnic, hinh thai te bao gan trong cac tieu thuy gan de tim ra nhiing cau tnic, hinh thai bat thuong so voi gan binh thuang.
Xur ly so lieu, thong ke phan tich Cac so lieu thu duoe tu cac thi nghiem dugc xii ly theo phuong phap thong ke mo ta va trinh bay 6 dang so trung binh + sai so ehuan trung binh. Thi^c hien so sanh su khac biet ve cae chi tieu danh gia giiia hai 16 thii vat thir nghiem bang trac nghiem t-Shident (t test) hay chi binh phuang (x-).
KETQUA
Khao sat miic dp gay loan san gan a i a diethylnitrosamin va tac dung phong ngua cua Iinh chi
Chugt thi nghiem dugc chia thanh cac 16 nhu sau:
- Lo 1: ip dung dich NaCl 0,9% (1 lan/tuan x 16 tuan) + uong nuoc cat 0,2ml/l Og the trgng/ngay, n = 8
- Lo 2: ip dung dich DEN 1% va CC14 4% (theo mo ta tren) + uong nuoe eat 0,2ml/10g the trgng/ngay, n = 10
- L6 3: ip dung dich DEN 1% va CC14 4% (theo m6 ta tren) va dugc uong cao linh chi 15%
lieu 0,2 ml/lOg the trgng/ngay, n = 13 - Lo 4: ip dung dich DEN 1% va CCM 4% (theo
mo ta tren) va dugc uohg cao linh chi 30%
lieu 0,2 ml/lOg the trpng/ngay, n = 13 Sau 16 tuan thir nghi|m, viec so sanh mo hgc dugc thue hien de danh gia miic dp tang san cnia mo hinh cting nhu miic do bao ve gan cua eao linh chi.
Ve mat dai the: khong co su xuat hien cac not tang san bat thuang tren gan 6* 16 hem NaCl 0,9% (hinh 1). Tuy nhien, 6 16 DEN + e c u xuat hien nhieu not tang san bat thuang tren gan. Cac not tang san co mau trang nga, CO bo ro ret. Ty le chupt co not tang san bat thuang xuat hien o 16 DEN + CCLla 70% (7/10 chupt). Nhu vay viec ke't hap DEN va CCh co the da gay roi loan qua trinh sinh san te bao gan dan den sy tang san bat thuang ciia eae te' bao 6 CO quan nay.
6 16 u6'ng cao Linh chi 15%, chi co 1/13 chupt (7,69%) CO sir xuat hien eae not tang san va 6 16 uong cao Linh chi 30% ty le nay la 0/13 (0%). Nhu vay, so voi 16 gay loan san bang DEN + CCh voi ty Ie not tang san la 70%, eae 16 dupe phong ngira bang cao Linh chi da the hien tac d6ng lie ehe su hirdi thanh cac not tang san tren be mat gan.
ChOng (Nacr 0,9%) DEN + CCIj Linh chi 15% Linh chi 30% Hinh 1.
Hinh dnh dqi the gan chugt a Id chieng (NaCl 0,9%), Id gdy loqn sdn (DEN + CCU), Id phdng ngiea bdng Unh chi (DEN + CCL + hnh chi 15% hay 30%>)
Ve mat vi the: 6 16 tiem NaCl 0,9%, eau mao maeh hinh dang nan hoa dac thii (hinh 2).
tnie va hinh thai te bao gan the hien binh O 16 tiem DEN + CCL co su bien doi bat Jiuong vcii tinh mach trung tam va eac xoang thuong ro ret tren hinh thai va cau tnic te bao
gan. Te bao gan c6 kich thuoc nhan lon, tang sac, nhiem sac the tho va h le nhan/bao tuong tang, CO hien tirgng phan bao bat thudng (miii ten) va svr xuat hien nhan quai di, chiing to co su xuat hien cua nhiing dong te bao dot bien bat thuong.
6 ca 2 16 uong linh chi 15% va 30%, vi the gan van con bieu hien cac te bao gan co bao tuong thoai hoa, nhan te bao lan, nhiem sat chat tho, ti le nhan/bao tucmg tang. Tuy nhien quan sat vi the kh6ng phat hien cac nhan quai di chiing to khong con su hien dien ciia cae dong te'bao dpt bien bat thuong.
• ^ ' J - ^ ' • ^ •
Chting {NaCI 0,n) DEN + CC||
Hinh 2. Hmh dnh vi the gan chudt a Id dn'mg (NaCl bing linh chi (DEN + CCU + Iinh chi 35% hay 30%) Bdng 2. Tdm tdt 1^ qud gdy loqn sdn gan vd tdc dung phdng ngiea cua linh chi
n = Ndltang
san VithI
NaCI 0,9%
8 0/8 (0%) Binh t h u ^ g
DEN + ca4
10 7/10 (70%) Tang sdn kh6ng diin hlnh
Linh chi 15%
13 1/13 (7.69%)
Bao tuong thodi hoa
Linh chi 30%
13 0/13 (0%)
Bao tuwng thoai hoa Khao sat tac dpng cua linh chi tren sii phuc hbi tinh tr^ng suy tiiy gay bdi methotrexat
Trong thii nghidm nay, chupt thi nghiem dugc chia ra lam eac 16 nhu sau:
L6 gay suy giam bach cau bang methotrexat (7,5mg/kg, ip, vao cac ngay 0,3,6)
L6 gay suy giam bach cau bloig methotrexat (MTX) va uong cao Iinh ehi 15% (0.1 ml/10 g), uong sau khi tiem lieu dau tien methotrexat 1 ngay va lien tuc trong 7 ngay
L6 gay suy giam bach cau bang methotrexat (MTX) va uong cao linh ehi 30% (0.1 ml/10 g), uong sau khi tiem lieu dau tien methotrexat 1 ngay va lien tue trong 7 ngay)
Linhchi15% Linh chr 30%
0,9%>), Id gdy loqn sdn (DEN + CCU), Id phdng ngiea
Lo chiing: tiem nuac cat (0.1 ml/10 g, ip, vao eac ngay 0, 3, 6) + uong nucic eat (0.1 ml/10 g tu ngay 1 den ngay 7)
Xet nghiem huyet hpe dupc thuc hi^n vao cae thoi diem: ngay 0 (16 truoc thii nghiem), va ngay 7.
Tdc dgng cua methotrexat vd linh chi tren tong lUf?ng hach cdu
Tnroc khi tiem methotrexat (ngay 0) va ngay 7, mpt nhom chupt duge lay mau de xet ng^em cong thuc mau tren may xet nghidm huyet hgc Lo lay mau vao ngay 0 (truoc khi tiem MTX) dugc xem la 16 cung cap cac chi so huyet hgc tren cac chugt binh thuong 6 thai diem trucic khi thir nghiem. Lo lay mau o ngay 7 duge xem la 16 doi ehiing 6 cimg thoi diem cho cae 16 hem MTX va thii nghiem linh chi.
Ket qua gay suy giam tong lugng bach ciau ciia methotrexat va tic dung phuc hoi bach tfau ciia cao linh chi 15% hay 30% dugc tiinh bay trong hinh 3.
Hinh 3. Tohg hegng bach cdu dm cdc Id thie nghiem Trong thi nghiem nay, methotrexat gay suy giam ro ret tong lupng bach cau vao ngay thii 7, va 6 eac 16 cho uong linh chi, tong lugng bach eau dupc kh6i phuc tro lai gan nhir binh thuong (linh chi 15%) hay tang kha cao so voi binh thuong (linh chi 30%).
Chi so truoc thuc nghiem va 16 chiing 6 ngay 7 khong cho thay co sir dao dgng dang ke, vi the co the xem nhu ket qua thu dupc chii yeu la do tac dpng oia thuoc thii nghiem. Tir su suy giam tong lupng bach cau do methotrexat gay ra, chiing t6i tiep tuc danh gia tac dung eiia methotrexat tren timg loai bach cau rieng biet.
Tdc dong cua methotrexat vd linh chi tren so' luang cdc loai bach cdu
Doi vol mo hinh gay suy giam bach cau bang methotrexat, so lugng bac cau trung tinh cung giam manh vao ngay 7; cac loai bach cau khac ciing giam co y nghia thong ke so vcii 16 chiing, ngoai trir bach eau ua acid kh6ng co su thay doi (hinh 4).
O cac 16 cho uohg hnh chi 15% hay 30%, su suy giam bach cau do methotrexat da duge khoi phuc tro lai. Dac biet 616 eho uong cao linh chi 30%, hau nhu so lugng cac loai bach cau cJeu tang cao so vcii 16 chiing. Day la dieu ma chiing toi chua giai thich dupc vi chupt dugc nuoi trong dieu kien giong nhau nen co the loai bo nguv co nhiem trimg tren nhom duoL cho uong lmh ehi
til II
30%. Vl the CO the gia dinh rang linh chi 30% CO the kich thich su sinh san manh cac loai te'bao bach cau.
R
^ n i # : = l
Hinh 4. Solugng cac loai bach cau cita cdc 16 thie nghiem Tac dpng cua methotrexat va linh chi tren so lupng hong cau va tieu cau
Tren lidng cau: ket qua thi nghiem cho thay methotrexat trong dieu kien nay chua gay suy giam hong cau, vi the* cao linh ehi 15% hay 30% deu khong the hien tac dpng den so lugng hong cau. Ngoai ra, khi so sanh cac chi so Hb, Htc va the tich hong cau giiia cac 16 thii nghiem, ke't qua cung ehua the hien su khac biet CO y nghia thong ke.
Hinh 5. Sd'ltegng lidng cau vh tieu ciau dm cdc Id thie nghiem
Tren ticu cdu: ket qua tren cho thay ring 0,01) so voi 16 chung. O cae 16 cho uo'ng cao methotrexat giam so lugng tieu cau gan 2 lan linh chi 15% va 30% sau khi tiem methotrexat, so vcii so lugng tieu cau ciia 16 doi ehiing va so lugng tieu cau deu dugc eai thien dang ke khae biet mgt each co y nghia thong ke (p < va tang eo y nghia thong ke (p < 0,01) so voi 16
tiem methotrexat. Mac dii va>, ca 2 16 cho uong cao linh chi 15% hay 30% deu khong lam phuc hbi hoan toan sir suy giam so lugng tieu eau do methotiexat ga)' ra ve miic nhu binh thuong (so vcH 16 doi chiing).
BANLUAN
Ket qua khao sat tac dpng bao ve gan ciia cao linh chi tien mo hinh gay loan san gan bing DEN va CCh cho thay cao linh chi the hien ro tac dung bao v? tren dai the va vi the.
Viec cac not tang san khong con xuat hi|n chiing to cao linh chi co kha nang lie ehe sir hinh thanh cae not tang san nay. Mat khae quan sat vi the kh6ng phat hien nhan quai di eho thay rang viec sir dung linh chi cilng han che su hinh thanh cac dong te bao bat thuong.
Nhieu cong trinh nghien ciiu ciing da chiing nunh tac dung tang cuong he mien dieh va lie che khbi u eiia linh chi, va eo the ea che tac dgng ciia linh chi trong khia canh nay la do SM' gia tang tbng hgp interleukin-2 eo tac dung tang cuong mien dich va iic ehe khoi u (Wang 2007).
Xet nghiem cong thiie mau thuang bieu thi so lupng cac loai te bao mau voi mgt khoang dao dpng nhat dinh, tuy nhien tiong nghien Cliu nay, ciii so eac Io?i bach cau trudc thue nghidm va chi so nay d 16 doi chiing vao ngay 7 dao dpng khong dang ke, do do su khac biet ve ehi sb eac lo^i te bao mau giiia 16 gay suy giam bsieh cau bling methotiexat so vdi 16 chung va sir khac biet giira 16 cho ubng linh chi so vdi 16 gay suy giam bach cau khong the gan eho nguyen nhan ve khoang dao dgng binh thuong khi xet nghiem, ma day chinh la tac dpng eiia thubc thu nghiem. Mat khac, su giam manh bach eau trung tinh xubng den miic rat thap chung to day la do dgc tinh dac thil cua thubc hoa tri, tir do cd the eho rSng kha nang phue hbi dong bach eau ciia linh chi cung chinh la tac dyng duge ly ciia linh chi.
Dugc tien hanh trong eimg mgt dieu kien thii nghiim, tiiy nhien chi cd 16 duge cho uong i) mdl cd su gia tang mgt sb loai eao linh chi 30% r
bach cau gan gap d6i tii so binh thudng, nhu vay CO the day la tac dpng ciia linh chi kem theo mgt yeu tb anh hudng khac ehua giai thich dugc, chii khong tiie do nguyen nhan nhilm tiung. Mpt dieu kha ngac nhien la linh chi lai cd tac dung phuc hoi mgt phan sb lugng tieu cau mac dii it cd tai heu nao de cap den tac dung nay. Nhieu tai lieu chiing minh linh chi ed tac dung tang cudng mien dich, tuy nhien ket qua tir nghien ciiu nay eho thay linh chi cdn ed tac dung khoi phuc cac loai te bao mau.
KETLUAN
Nhung ket qua tren chi la bude tfau tiong viec xac dinh xem cao linh chi toan phan (mot nguyen lieu chinh trong bao che cae che pham ehua linh ehi cua OPC) cd tac dpng 6 muc d?
nao tien kha nang phong ngira loan san gan va phuc hbi tinh trang suy tiiy do thubc hda Iri gay ra. Qua viee tong hgp eac ket qua thirc nghiem chiing toi cho rang, linh ehi d cae h'eu Iugng khao sat cd tac dung tot tren cae hirong nghien cuu tien va can dugc tiep tyc nghien Cliu md rpng trong viec thir nghiem ung dung tri lieu.
T A I LIEU T H A M KHAO
1 Hecht SS (1997), A p p r o a c h e s to cancer prevention based on an u n d e r s t a n d i n g of N-ntrosaimne carcinogenesis, Proc Soc Expt Biol Med 216.181-191 2 Kmo K, YamashiU A, Yainaoka K, Watanabe J, Tanaka S,
Ko K, Shimizu K Tsunoo H (1989) Isolation and characterization of a new immuromoduiatory protem, lmg zhi-8 (L2-8), from Canodemia luadium J Bio! Chem 264' 472-4yB-
3 Lui GT, Chang ST, Busvvell TA (1993), Phannacology and clinical uses of Ganoderma, Mushroom biology and mushroom products, the Chinese Universitj- Press, Hmg Kong, p p . 267-273.
4 Nakamura K, Yamasaki M (2003), Effect of Methotrexate- induced Neutropenia cm Pulpal Inflammation m Rats.
Journal of Endodontics 28; 2 8 7 - 2 9 0 .
5 I r a n Mar\h H i m g N g o Kioi D u e (2005), Phuong phap gay tang san bat thuong te bao gan biing carixm tetradorid va djetylnitrosamm. Tap chi Duoc hgc SO: 26- 29.
6 Wang G, Zhao J, Liu ), H u a n g Y, Zhong JJ. Tang W (2007).
Enhancement of IL-2 and IFN-gamma expressitxi and NK cdls achwty involved in the anh-tumor effed <rf ganoderic a d d m vivo. Int Immunopharmacol 7 864^70
Yuen JW, Gohel MD (2005). Anticancer effects of Ganodenna luddum. a review of saentific evidence, Nutition and Cancer, 53 11-17.