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BAR 501, a Novel GPBAR1 Ligand, Reverses Intestinal and Liver Inflammatory Models Demonstrating That GPBAR1 Is an Essential Modulator of Innate Immunity in Entero-Hepatic Tissues
Barbara Renga*, Sabrina Cipriani #, Adriana Carino * , Silvia Marchianò * , Angela Zampella † and Stefano Fiorucci *
*Dipartimento di Scienze Chirurgiche e Biomediche, Università degli Studi di Perugia, Nuova Facoltà di Medicina e Chirurgia , Sant’Andrea delle Fratte,Perugia, Italy
#Dipartimento di Medicina, Università degli Studi di Perugia ,Nuova Facoltà di Medicina e Chirurgia, Perugia, Italy †Dipartimento di Farmacia, Università di Napoli Federico II, Napoli, Italy
Background. GPBAR1, also known as TGR5, is a bile acid receptor expressed in various cell types including monocyte/macrophages and Kupffer cells (the resident macrophages in the liver). GPBAR1 activation leads to reduction of cytokine release by macrophages and its deletion results in enhanced intestinal inflammation. BAR501 is recently described selective GPBAR1 agonist that activates the receptor with an EC50 OF 3 Μm (J Med Chem. 2014 Oct 23;57:8477-95). Aim of the study. To investigate whether BAR501 protects against development of intestinal and liver inflammation in rodent models of immune dysfunction.Material and methods. Hepatitis was induced by intravenous injection of ConA and colitis was induced by intra-rectal injection of TNBS. At day 0, male mice GPBAR1 wild type and GPBAR-/- were treated with a single dose of intrarectal TNBS (0,5 mg/mouse in 100μl solution 50% water and 50% ethanol). BAR501 was administered daily, at doses of 15 and 30 mg/kg. Weights and colitis score were evalueted daily, at day 7 mice were sacrificed and colon collected for length measurement, evaluation of macroscopic and histological scores (H/E) and cytokine assay. For the ConA model, female wild type and GPBAR-/- mice were pre-treated for 6 days with BAR501 (15 mg/kg). At day 6 ConA was administered at dose 10 mg/kg. Animals were sacrificed after 4, 8 and 24 hours post ConA injection and blood and liver collected for plasma AST and ALT measurement, liver histopathology analysis (H/E) and for cytokines assay (rt-PCR).
Results. Hepatitis induced by ConA, was severely exacerbated in GPBAR1-/-. BAR501 rescued from death and reduced AST and ALT levels by ~90%, IL-6, and TNFα by 60-70% and ameliorated the necrotic/inflammatory score in wild type mice but not in GPBAR1-/- mice. Colitis induced by TNBS was severely exacerbated in GPBAR-/- mice. BAR501 reduced mortality, ameliorates colitis symptoms (intestinal colitis scores and histological score) and reduced TNFα mRNA in wild type but not in GPBAR-/- mice. Conclusions. BAR501 exerts potent anti-inflammatory activity and modulates innate immunity
OHHOH
OH
BAR501
GPBAR1
% survival
1 2 3 4 5 6 70
25
50
75
100Gpbar+/+CTRL
Gpbar+/+ TNBS
Gpbar+/+TNBS+ +501(15mg/kg)
Gpbar+/+ TNBS+501 (30mg/kg)
Gpbar-/- CTRLGpbar-/- TNBSGpbar -/- TNBS+501(15mg/kg)
days
% s
urvi
val
delta weights
0 1 2 3 4 5 6 7-30
-25
-20
-15
-10
-5
0
5
10
Gpbar+/+CTRLGpbar+/+ TNBS
Gpbar+/+ TNBS +501(15mg/kg)
Gpbar+/+ TNBS+501 (30mg/kg)
Gpbar-/- CTRL
Gpbar-/- TNBS
Gpbar-/- TNBS+501(15)
days
%
-1 0 1 2 3 4 5 60.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
Gpbar+/+ CTRL
Gpbar+/+ TNBS
Gpbar+/+ TNBS+501(15mg/kg)
Gpbar+/+ TNBS+501 (30mg/kg)
Gpbar-/- CTRL
Gpbar-/- TNBS
Gpbar-/- TNBS+501(15mg/kg)
TNBS
BAR501
*
* TNBS +/+ vs ctrl +/+
#
# TNBS -/- vs ctrl -/-
#
*
§
§ TNBS+ 501 -/- vs ctrl-/-
*
#
§
TNBS+ 501+/+ vs TNBS +/+
*
#
§
#
§
Time (Days)
Col
itis
scor
e
macroscopic colon score(thickness+ulceration)
CTRL
TNBS
TNBS+50
1(15)
TNBS+ 50
1 (30
)CTRL
TNBS
TNBS+50
1(15)
0.0
2.5
5.0
7.5
** *
**
Gpbar+/+
Gpbar-/-
colon lenght
CTRLTNBS
TNBS+501
(15)
TNBS+501
(30)
CTRLTNBS
TNBS+501
(15)
0.0
2.5
5.0
7.5
10.0
** *
Gpbar+/+
Gpbar-/-cm
GPBAR+/+
Gpbar1+/+ naive Gpbar1+/+ Con A Gpbar1+/+ BAR501
Gpbar1-/- naive Gpbar1-/- Con A Gpbar1-/- BAR501
0
10
20
30
40
50
% a
rea
necr
osis
*
*
- - + - - +0
100
200
300GPBAR +/+
GPBAR -/-
ConA ConA
BAR 501
IL-
6 m
RN
A r
el. e
xpre
ssio
n
0 4 8 12 16 20 240
5000
10000
15000
20000
25000Gpbar1-/- ConA+501Gpbar1-/- ConA
Gpbar1+/+ ConAGpbar1+/+ ConA+501
Time (hr)
ALT
pla
sma
leve
ls (U
I/L)
*
0 4 8 12 16 20 240
10000
20000
30000Gpbar1+/+ ConA
Gpbar1+/+ ConA+501 Gpbar1-/- ConA+501
Gpbar1-/- ConA
Time (hr)
AS
T pl
asm
a le
vels
(U/L
)
*
- - + - - + 0
50
100
GPBAR +/+
GPBAR -/-
ConA ConABAR 501
TNF
mR
NA
rel.
expr
essi
on
**
**
- - + - - +0
100
200
300GPBAR +/+
GPBAR -/-
ConA ConA
BAR 501
IL-
6 m
RN
A re
l. ex
pres
sion
*
* *
**
GPBAR-/- CTRL GPBAR-/-TNBS + 501 (15mg/kg)GPBAR-/- TNBS
10x10x 10x
GPBAR+/+ CTRL GPBAR+/+ TNBS
10x 10x
GPBAR+/+ TNBS+501(15 mg/kg) GPBAR+/+ TNBS+501(30 mg/kg)
10x 10x
A B
C D
E F
GPBAR1 receptor structure and BAR501 molecular structure
Figure 1: Effects of BAR501 on mice treated with ConA. (A-B) Plasmatic levels of transaminases (AST and ALT), (C-D) relative hepatic mRNA expression of IL6 and TNFα assayed by rt-PCR in livers from mice after 8 hours ConA treatment. (E) Hematoxylin/ eosin staining of livers after 8 hours ConA treatment . (F) % of liver necrotic areas calculated with ImageJ software, (* p<0,05).
Figure 2: Effects of BAR501 on colitis induced in mice with TNBS. (A) % delta weights, (B) colitis score, (C) % survival , (D) colon length, (E) macroscopic colon score (thickness plus ulceration), (F) hematoxylin/ eosin staining of colon. ( #, § vs Gpbar -/- CTRL; * vs Gpbar+/+ CTRL; Φ vs Gpbar+/+ TNBS; p< 0,05).
A B C
D E F
D'Amore C, et al. Design, synthesis, and biological evaluation of potent dual agonists of nuclear and membrane bile acid receptors. J Med Chem. 2014 Feb 13;57(3):937-54.