Nghiên cứu khả năng chống viêm của cây nhọ nồi và ngải cứu thông qua thụ thể TLR4

1

Antiinflammatory studies of soot and theformer
plant through TLR4 receptors

Nguyen Thi Tuoi

Hanoi University of Science, VNU; Faculty of Biology
Major: Biochemical; Code:60.42.30
Supervisors: Dr. Trinh Tat Cuong
Date of Presenting Thesis: 2012


Abstract u ngi c
n xut cytokine tiu t c hiu ca TLR4, 
 d c ch cu
ngi cnh c ch yu t t -activated protein
 c ch cu ngi c
o phn u t c hiu TLR4,  
 dng LPS.
Keywords. Sinh hc thc nghi ni cu; Th th; Ch
Thc vt 
Content

I. Lý do chọn đề tài:
ng t   chng lnh t ng
 ngoc bi
bnh. Kt qu ci b hoc triu kiy,
u thi gian tn t chuyn t

n cng hu qu . Nh
i phi k i loi
cht, n hong c n dn suy
gim cha th


2

Do vu tr m bng bt li ci v  ht
sc quan trng hic biu tr b
 bic chn g yc
tng hi thu 
  ng ph n. S dng thuc
n gc thc v gii quyt nhng lo ng
hiu qu p.
n nay, vit chn gc t thc vc
i nhng hiu qu chim Tr
i hc Khoa hc T  c
 t ch 
Wedelolactone t  nt vu khc kh ng








“Nghiên cứu khả năng chống viêm của cây nhọ nồi và ngải cứu thông qua thụ
thể TLR4”.

II. Mục tiêu đề tài
c chu
ngi cu trong t  th TLR4.
c kh i cu trong t 
th TLR4.
III.Nội dung nghiên cứu


3

 u ngi cn xut
cytokine tiu t c hiu ca TLR4,   dng
LPS.
2 c ch cu ngi cc ch yu
t t MAPK ( mitogen-activated protein kinase).
 c ch cu ngi co phn
u t c hiu TLR4,   dng LPS.
IV. Phương pháp nghiên cứu
1.Tách chiết macrophage từ tủy xương của chuột
T i th a chuc
 c b ng
u ki        u t   -CSF (macrophage-specific
           

5
M 2-mercaptoethanol.
2.Đánh giá hoạt tính độc tố của Wedelolactone tới khả năng sống của BMDM bằng kít
CCK-8
Kh  ng ca t     ng s dng Cell Counting Kit-8 (CCK-8,
     c  c  c

Dexamethasone (Dex) trong mt s thi gian nha dung dch
CCK-c  i Wedelolactone trong 1 gi. Kh p
th c  n hp th 
450nm ca mi m      l ph   i m  c x  i
Wedelolactone ho ng).
3. Đánh giá khả năng sản xuất cytokine bằng ELISA Kit
ELISA (Enzyme-Linked ImmunoSor 
mt k thu  m. Hin
c s dng r
c bi kin phm sinh hc.


4

c hi-  m

 c gt b mt
 - antibody bic "ra" qua b m c gn kt vi
enzyme
t; enzyme s bi c
4. Phương pháp Western blot
 nh mt loi protein trong mu sinh hc b   
SDS-page (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), c thc hin theo
c g
d   c min d     c g  th  n dch
(immunoblotting) 6,3.
t k thup thc hi
               
     n di s c chuy
c  vi mt dung dch ch c hiu vc

 s gng  
bng phn ng enzyme hi c v   
t s  c
dm mn s t ca mt hoc mt s
lo cn nghiu trong hn hp phc tp.
5. Phân tích ức chế của Wedelolactone trong quá trình tạo phản ứng oxy hóa[17]
c s i Laminarin hoi
c  vi Wedelolacto
t c  vi DHE (Fluorescent Dyes (dihydroethidium) - cht hu
 
2
. Nhng t  c kim tra bng laser-scanning confocal
 nh m u cc x 


5

6.Phân tích thống kế
i v lic ly t t qu c lc th hi
-u chnh Bonferroni hoc
Ai vi nhi 

c ch ng P< 0.05.
V. Kết quả nghiên cứu
1. Khả năng sống của tế bào không bị ảnh hưởng bởi Wedelolactone

Hình 12. Biểu đồ Wedelolactone không gây độc đối với tế bào BMDM
0
50
75
100
DM 10 20 30 (µg/ml )
Kh  sng
ca t 


6

BMDMi m 
5
t ng. Sau 3- c x 
i Wedelolactone   l 
bm t  liu ch ra s sai s cc lp.
2. LPS kích thích tế bào BMDM sinh cytokine

Hình 13: Biểu đồ LPS kích thích tế bào BMDM sản xuất cytokine tiền viêm và kháng
viêm.
BMDMs t chuc  vi LPS 100 (ng/ml). Dch t c  vi LPS  
nhau l gi, 4 gi, 8 gi, 18 gi, 48 gi s i vn xut
cytokine bt qu c biu hin sai s c lp.






TNF-α
0
5000
10000
15000
0 4 8 18 48 (h)
0
500
0
1000
0
1500

0
0 4 8 18 48 (h)
0
50
0
100
0
150
0
0 4 8 18 48 (h)
0
200
0
400
0
600
0
800
0
0 4 8 18 48 (h)
IL-6
INF-ү
IL-10
Quá
trình
tiết
của
cytok
ine
(pg/

ml)
Quá
trình
tiết
của
cytoki
ne
(pg/m
l)


7

3. Wedelolactone ức chế quá trình sinh cytokine thông qua thụ thể TLR4 kích thích bằng
LPS












Hình 14: Biểu đồ Wedelolactone ức chế quá trình sinh cytokine tiền viêm trong BMDM
được kích thích bằng LPS.
BMDMs t chuc  i Wedelolactone   

hoi chc khi  vi vi LPS 100 (ng/ml). Dch
sau khi t c  vi LPS 18 gi s i vn xut cytokine bng
t qu c biu hin sai s c li chng; W:
Wedelolactone).


DM 2.5 5 10 20
(µg/ml )





0
500
0
10000
15000
DM 2.5 5 10
20(µg/ml )
TNF-α
IL-6





0
5000
10000

15000
DM 2.5 5 10
20(µg/ml )





0
500
100
0
150
0
DM 2.5 5 10 20
(µg/ml )
INF-ү







0
2000
4000
6000
8000
IL-10

8


4. Wedelolactone ức chế quá trình phospho của yếu tố tự phân bào bằng LPS kích thích
thông qua thụ TLR4






Hình 16. Wedelolactone đã ức chế quá trình phospho của ERK1/2 và p38 trong BMDM
c  i Wedelolactone (20 c Dexamethasone (100nM) trong 45
 p tc  vi LPS (100ng/ml) trong 18 gi
 nh  
         i chng: SD; Wedelolactone: W;
Dexamethasone: Dex).
5. LPS kích thích hoạt động p47 phox trong quá trình tạo ra ROS


p47phox-ser345

p47phox

Hình 18. LPS kích thích quá trình phospho hóa của p47phox trong BMDM.
ERK
p38
M SD W SD Dex

P-ERK
P-p38
LPS
0 5 15 30 60 120 (min)
LPS


9

c  vi LPS (100ng/ml) ti m t 
t  nh  

6. Dầu ngải cứu không gây độc đối với BMDM







VI. KẾT LUẬN VÀ KIẾN NGHỊ
KẾT LUẬN
1. c ch n xut cytokine tic ch 
i n  th 
bng cu t c hiu LPS.
2.   c ch     a yu t t    
(extracellular-signal-regulated kinases
3.        ng c ch     a
p47phox.
4. Tinh du ngi c c ch n xut cytokine

h th u t c hiu LPS.



25
50

75
100




TỶ LỆ
TẾ
BÀO
SÔNG
(%)
ĐC 10 20 40 (µg/ml )


10

KIẾN NGHỊ
1. Tit cht thuc cu t ca th th
Glucocorticoid.
2.  u tr chut b nhim khun b
chut vi LPS ca Wedelolactone.
References
Tài liệu tiếng Việt

1. 





, , 



, , 


(2009), 















 (Premna

corymbosa Rotteneen) 



, Tp ch Dưc hc, 48(389), tr. 22-24.
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u kh  rong to bin ViTp ch Hoá
hc, 46(5A), tr. 81-90.
3.    di truyn h  
i Hc Qui.
4.  Tt Li (2000), Những cây thuốc và vị thuốc Việt Nam, NXB Y hi.
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k thui.
6.    Kỹ thuật di truyền và ứng dụng i Hc
Qui.
7.  , 

, 





 , 



 , 
(2010), 


















 (Sapium
sebiferum) 



, Tp ch Dưc hc, 50(411), tr. 36-39.
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


, 




, 
(2010), 















 (Clerodendrum
Chinese var. simplex (Mold) SL Chen) 



, Tp ch Dưc hc, 50(414), tr. 20-
23.




11

Tài liệu tiếng Anh
9.            
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human diseases Current Immunology Reviews, 1, pp. 81-90.
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ethyl acetate extract and major compounds on NF- -activation activity and LPS-
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receptor agonist compound K regulates dectin-1-dependent inflammatory signaling through
Life Sciences, 85, pp. 625-633.
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 European Musculoskeletal Review, 4(1), pp. 24-25.
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       -   

Molecular Medicine, 14, pp. 346352


12

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method for evaluation anti-  Arc International Pharmcodynamic
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lipopolysaccharide: evidence for TLR4 as the LPS Journal Immunology, 162,
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    Agricultural Food
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