experiment on liver fibrosis

1. Evaluation of piperine as a potential antifibrotic agent against
CCl4 – induced rat liver fibrosis
Monu Kumar Kashyap1, Anand Kumar1, Deepak Rawat1, Mahima1 and Sapana Kushwaha1*
1​Department of Pharmaceutical Sciences, School of Biomedical and Pharmaceutical Sciences
Babasaheb Bhimrao Ambedkar University (A Central University), Vidya Vihar, Raebareli Road, Lucknow-226025, INDIA
Email id: sapna.pharm@gmail.com
ACTIVATION
TIMP
Oxidative Stress Induce LiverFibrosis
Non alcoholic fatty liver
disease (NAFLD) induce
liverfibrosis
Liver fibroproliferative disease
Chronichepatitis
Long time using NSAIDS drugs
like paracetamol
HEPATOCYTES KUPFFERCELLS
TCELLS
IL 6,TGF-β1, TNF—α,EGF, IGF
IL -6 , INF-ϒ TGF-β1,TNF--αEGF IGF
QUIESCENT HSCs ACTIVATEDHSCs
ECMSYNYHESIS
TGF-βCTGF
TNF-α, TIMP3,
TIMP1
Biomarkersof fibrogenesis
related cytokine:
TGFβ
CTGF/CCN2
PDGF
TNFα
IL-4, IL-6, IL-8, IL-18
Biomarkers of
fibrinolytic pathway:
MMPs and TIMPs
Biomarkers of
liver function:
ALT
AST
Biomarkers of ECM
synthesis:
Glycogens
Polyglycans
Collagens
Biomarkersof ECMdegradation:
Neo--epitopes
ECMDegradation
MMP
• In conclusion, the present findings
showed that piperine a natural
component of black pepper significantly
restored the liver function biomarkers
(ALT, AST & bilirubin), and oxidative
stress parameters (MDA, GSH and
SOD).
• Piperine significantly reduced the
percentage fibrotic area as shown by
Masson’s trichome staining and
improved the cellular structure as
histology of liver in CCl4 - induced
fibrosis. Further detailed molecular
studies are required to elucidate the
protective role of piperine in CCl4 -
induced liver fibrosis.
Fig. 2. Representative images of liver tissue of normal control,
CCl4, Piperine and Valsartan groups. Normal control liver has
regular smooth surface. And CCl4 liver has rough nodular
surface, appearance of micronodules and
macronodules.CCl4+Piperine liver has normal smooth surface.
CCl4 + Valsartan liver has nearly smooth surface and few
micronodules. (n = 5/group)
Fig. 4. Representative photomicrographs showing the collagen deposition in the liver,
with masson’s trichome stain. No fibrosis was observed in control group. Fibrosis area
shown by blue staining . Magnification: 40X. Right hand Bar graph showing the quantative
analysis of liver represents the percentage of fibrosis area. All the values are expressed as
mean ± SD. ***p<0.001 and *** vs , CCl4 vs. Control, CCl4 vs Piperine, CCl4 vs Valsartan
Fig. 3. Effect of piperine CCl4 induced liver
fibrosis. Representative photomicrographs
depicting histology of liver under Hematoxylin &
Eosin (H & E) staining. Magnification: 40X.
Normal control liver has normal histological
structure and architecture. CCl4 control liver has
structural damage, irregular regenerating lobules
with dense fibrotic septa, proliferation of bile duct,
and presence of centrilobular and inflammatory
cells. CCl4 + Piperine liver have mild inflammation
but no fibrotic septa. CCl4 + Valsartan liver have
partially preserved hepatocytes, small area of
necrosis, narrow fibrotic septa.
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Negreş S, Bejenaru C, Turculeanu A, Badea
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INTRODUCTION RESULTS
CONCLUSION
REFERENCES
• Liver fibrosis can progress to cirrhosis,
hepatocellular carcinoma or even death[1].
• Liver fibrosis is a dynamic and reversible process
which is characterized by an imbalance between
formation and degradation of extracellular matrix
(ECM) which is rich in fibrillar collagens (mainly
collagen I and III)[2].
• CCl4 induced the activation of kupffer cells to
activate the pro-inflammatory cytokines that
promote the activation of HSCs[3].
• Piperine is an alkaloid present in seeds of black
pepper (Piper nigrum), long pepper (P. longum),
and other pepper species belongs to family
Piperaceae. Piperine show evidence of
protection in depressive disorders,
cancer,cardiac fibrosis, obesity and diabetes [4].
Studies revealed that piperine treatment
attenuates the dyslipidemia by reducing the
serum triglyceride (TG), total cholesterol, LDL,
VLDL level in high fat diet rats [5].
Chemicals
Piperine was purchased from Sigma-Aldrich (St.
Louis, MO), USA. Valsartan was kindly gifted from
UNICHEM Pvt. Ltd, India. Carbon were purchased
from Loba Chemine, New Delhi, India. ALT, AST and
Bilirubin kit was purchased from the Robonic (India)
Pvt. Ltd., Thane, Maharastra, India. All the solvents
and chemicals were used in experiments of analytical
grades with 99% purity.
Dose schedule and preparation
The dose of CCl4 was given thrice per week for six
consecutive weeks. The dose of piperine and
valsartan has given for consecutive six weeks after the
induction of fibrosis by CCl4. Both piperine and
valsartan were prepared in phosphate buffer (pH 7.4)
and given through the oral route. At the end of the
experiment, all animals were anesthetized with mild
ether and the blood sample was collect from retro -
orbital sinus.
OBJECTIVE
MATERIAL AND METHODS
• To evaluate the possible antifibrotic effect of
Piperine against as comparison of Valsartan
carbon tetrachloride (CCl4)-induced liver fibrosis
in rat.
Normal Control
CCl4
CCl4 + Piperine CCl4 + Valsartan
SGPT (U/L)
NO
RM
AL
CO
NTRO
L
CCl4
CCl4+VALSARTAN
CCl4+PIPERINE
0
20
40
60
80
***
**
**
SGPT
(U/L)
SGOT(U/L)
0
20
40
60
80
100
NORMAL CONTROL
CCl4
CCl4+PIPERINE
CCl4+VALSARTAN
***
**
****
SGOT(U/L)
DIRECT BILIRUBIN (mg/dl)
0.0
0.1
0.2
0.3
0.4
0.5
NORMAL CONTROL
CCl4
CCl4+PIPERINE
CCl4+VALSARTAN
****
** **
DIRECT
BILIRUBIN
(mg/dl)
TOTAL BILIRUBIN (mg/dl)
N
O
R
M
A
L
CO
N
TR
O
L
C
Cl4
C
Cl4+PIPER
IN
E
C
Cl4+VA
LSA
RTA
N
0.0
0.2
0.4
0.6
****
** **
TOTAL
BILIRUBIN
(mg/dl)
GSH (µM/µg of protein)
N
O
R
M
A
L
C
O
N
T
R
O
L
C
C
L
4
C
C
l
4
+
P
I
P
E
R
I
N
E
C
C
l
4
+
V
A
L
S
A
R
T
A
N
0.000
0.005
0.010
0.015
0.020
0.025
****
****
GSH
(µM/µg
of
protein)
SOD(U/µg of protein)
N
O
R
M
A
L
C
O
N
T
R
O
L
C
C
l
4
C
C
l
4
+
P
I
P
E
R
I
N
E
C
C
l
4
+
V
A
L
S
A
R
T
A
N
0.000
0.002
0.004
0.006
0.008
0.010
****
***
**
SOD(U/µg
of
protein)
MDA/TBAR (nM/µg of protein)
N
O
R
M
A
L
C
O
N
T
R
O
L
C
C
l
4
C
C
l
4
+
P
I
P
E
R
I
N
E
C
C
l
4
+
V
A
L
S
A
R
T
A
N
0.0
0.2
0.4
0.6
0.8
1.0
****
**** **
MDA/TBAR
(nM/µg
of
protein)
Fig. 5. Effect of Piperine on serum AST and ALT levels in CCl4 -induced liver
fibrosis. All the values are expressed as mean ± SD (n = 5). ***p<0.001 ,
**p<0.01, CCl4 vs. Control, CCl4 vs Piperine, CCl4 vs Valsartan
Fig. 7. Effect of Piperine on serum Bilirubin (Total and Direct) levels in
CCl4 -induced liver fibrosis. All the values are expressed as mean ± SD (n
= 5). ***p<0.001 , **p<0.01, CCl4 vs. Control, CCl4 vs Piperine, CCl4 vs
Valsartan
Fig. 7. Effect of piperine on the GSH, MDA and SOD level in the liver of CCl4 induced
liver fibrosis. All the values are expressed as mean ± SD (n = 5). ***p<0.001, **p<0.01, , CCl4
vs. Control, CCl4 vs Piperine, CCl4 vs Valsartan
MOLECULAR MECHANISM OF LIVER FIBROSIS
Fig. 1. Schematic overview of TGFβ/Smad2/3 signaling that ultimately leads
to fibrosis. TGF β-Transforminggrowth factor-β, TRAF6- TNF receptor-
associated factor 6, TAK1- Transforming growth factor beta-activated kinase
1, MAPK-Mitogen-activated protein kinases, P13K-Phosphoinositide 3-
kinase, AKT - Protein kinase B, TNF-α- Tumor necrosis factor α, JnK- c-Jun
N-terminal kinases
STATISTICAL ANALYSIS
• Results will be shown as the mean ± standard
deviation (SD) for each group. Statistical
analysis will be performed using Graph Prism
pad (version7.05) statistical software.
• The significance of the difference between
multiples comparisons will be evaluated by
using a one-way analysis of variance
(ANOVA).
• In case, ANOVA showed significant
differences, post-hoc analysis will be
performed with Bonferroni’s test. The value of
p < 0.001 will be considered to be statistically
significant.