Combination of curcumin and piperine prevents formation of gallstones in C57BL6 mice fed on lithogenic diet: Whether NPC1L1/SREBP2 participates in this process?

Article (PDF Available)inLipids in Health and Disease 14(1):100 · September 2015with 196 Reads
DOI: 10.1186/s12944-015-0106-2 · Source: PubMed
Abstract
A disruption of cholesterol homeostasis characterized by the physical-chemical imbalance of cholesterol solubility in bile often results in formation of cholesterol gallstones. Our earlier studies revealed that curcumin (1000 mg/kg) could prevent formation of gallstones. It has been proved that curcumin is poorly absorbed while piperine is a bioavailability-enhancer. Nevertheless, whether curcumin combined with piperine could enhance the effect of curcumin in preventing gallstones is still awaited. C57BL6 mice were fed on a lithogenic diet concomitant with curcumin at 500 or 1000 mg/kg and/or piperine at 20 mg/kg for 4 weeks. The ratio of gallbladder stone formation was recorded and samples of blood, bile, gallbladder, liver and small intestine were also collected. The volume of gallbladder and weight of liver were calculated, and blood and bile samples were analyzed through biochemical methods. Intestinal NPC1L1 and SREBP2 mRNA and protein expression were detected by real-time PCR and Western blot. Combining with piperine can significantly enhance the effect of curcumin, thus preventing the development of gallbladder stones, lowering the saturation of blood lipids and cholesterol in bile, as well as decreasing the expression of NPC1L1 and SREBP2 in both mRNA and protein levels. Curcumin can prevent the formation of cholesterol gallstones induced by high fat diet in mice and SREBP2 and NPC1L1 may participate in this process. Piperine can increase curcumin's bioavailability, thereby enhancing the effect of curcumin.
R E S E A R C H Open Access
Combination of curcumin and piperine
prevents formation of gallstones in C57BL6
mice fed on lithogenic diet: whether NPC1L1/
SREBP2 participates in this process?
Yongnan Li, Min Li, Shuodong Wu
*
and Yu Tian
Abstract
Background: A disruption of cholesterol homeostasis characterized by the physical-chemical imbalance of
cholesterol solubility in bile often results in formation of cholesterol gallstones. Our earlier studies revealed that
curcumin (1000 mg/kg) could prevent formation of gallstones. It has been proved that curcumin is poorly absorbed
while piperine is a bioavailability-enhancer. Nevertheless, whether curcumin combined with piperine could enhance
the effect of curcumin in preventing gallstones is still awaited.
Method: C57BL6 mice were fed on a lithogenic diet concomitant with curcumin at 500 or 1000 mg/kg and/or
piperine at 20 mg/kg for 4 weeks. The ratio of gallbladder stone formation was recorded and samples of blood,
bile, gallbladder, liver and small intestine were also collected. The volume of gallbladder and weight of liver were
calculated, and blood and bile samples were analyzed through biochemical methods. Intestinal NPC1L1 and SREBP2
mRNA and protein expression were detected by real-time PCR and Western blot.
Result: Combining with piperine can significantly enhance the effect of curcumin, thus preventing the development of
gallbladder stones, lowering the saturation of blood lipids and cholesterol in bile, as well as decreasing the expression of
NPC1L1 and SREBP2 in both mRNA and protein levels.
Conclusion: Curcumin can prevent the formation of cholesterol gallstones induced by high fat diet in mice and SREBP2
and NPC1L1 may participate in this process. Piperine can increase curcumins bioavailability, thereby enhancing the
effect of curcumin.
Keywords: NPC1L1, SREBP2, Cholesterol absorption, Curcumin, Piperine, Gallbladder stone
Background
A disruption of cholesterol homeostasis characterized by
the physical-chemical imbalance of cholesterol solubility
in bile often results in development of cholesterol gall-
stones. The homeostasis of cholesterol in human body
mainly depends on its synthesis, absorption from intes-
tine and secretion of the bile, of which the metabolic
process is under precise regulation [1]. Previous studies
have demonstrated that intestine is the unique organ
providing dietary and reabsorbed cholesterol for the
body, and the absorption of cholesterol often starts from
the apical membrane of its epithelial cells. In light of the
fact that there is a close relationship between cholesterol
absorption and gallstone formation [2, 3], and clinical
research has found that intracellular cholesterol trans-
port was enhanced in patients with cholesterol gallstones
[4]. So it seems that we can benefit a great deal by redu-
cing the absorption of cholesterol from intestine to
prevent gallbladder stones. Curcumin is the active
ingredient in the traditional herbal remedy and dietary
spice turmeric (Curcuma longa) [5]. And it has been
researched in many directions, such as anti-carcinoma,
anti-inflammatory, anti-oxidative as well as cardiovascu-
lar fields [610]. It has been reported that feeding litho-
genic diet supplemented with 0.5 % curcumin for
* Correspondence: wushuodong@aliyun.com
Biliary & Vascular surgery, Shengjing Hospital of China Medical University,
Shenyang City 110004, PR China
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Li et al. Lipids in Health and Disease (2015) 14:100
DOI 10.1186/s12944-015-0106-2
10 weeks could reduce the incidence of gallstone forma-
tion to 26 % as compared to 100 % incidence in group
fed with lithogenic diet alone in young male mice [11].
However, its low bioavailability, which due to poor
absorption and faster metabolic alterations, presents a
great challenge for its extensive applications [1214].
Piperine, an alkaloid amide, is one of the major active
components in black pepper. Suresh et al. [15] found
that curcumin concomitant with piperine (20 mg/kg)
not only reduced curcumins metabolic breakdown rate,
which lead to prolonged retention of curcumin in the
body, but also enhanced the intestinal absorption of
curcumin. Kumar et al. [16] found that in Caco-2cells,
curcumin could modulate Niemann-Pick C1-like 1
(NPC1L1) expression at transcriptional level with sterol
response element-binding protein 2 (SREBP2) involved
in the process. Moreover, our previous studies have
indicated that 1000 mg/kg curcumin could prevent
formation of cholesterol gallbladder stones. In this study,
we attempted to find out whether lower dose of curcu-
min combined with piperine could have the same effect
on gallstone-susceptible C57BL6 mice. Also, we explored
if NPC1L1 and SREBP2 had participated in this process.
Results
Lipid levels of bile and blood
The serum concentration of cholesterol and triglyceride
was significantly decreased in both LC500P20 and
LC1000 compared with those in LD (P< 0.001). But no
significant difference was found when mice were fed
only with curcumin (500 mg/kg) or piperine (20 mg/kg)
(Table 1).
Bile cholesterol level (calculated by molarity) in
LC500P20 and LC1000 decreased by over 60 % com-
pared with those in LD, while phospholipid and bile
acid varied slightly between each group. Moreover,
the mole fraction of cholesterol, cholesterol/phospholipid
ratio, as well as CSI values all decreased due to 1000 mg/
kg curcumin or curcumin (500 mg/kg) administration
combined with piperine (20 mg/kg) intervention (Table 2).
The relative lipid composition of pooled gallbladder bile
from mice in LD is located in the central three-phase
zone, in which the bile is composed of mixed cholesterol
monohydrate and lamellar liquid-crystalline pattern [17].
By contrast, the relative lipid compositions in LC500P20
and LC1000 are plotted in the one-phase micellar zone in
which the bile is composed of unsaturated micelles at
equilibrium (Fig. 1).
Prevention of cholesterol gallstones
Our previous research indicated that mice fed solely
on lithogenic diet for 4 weeks showed 100 % forma-
tion of cholesterol gallstones. The reduced incidence
of gallstones amounted to 30 %, 20 %, 70 % and 60 %
respectively in corresponding groups compared with
LD (Table 3). The volume of gallbladder increased
when fed on lithogenic diet compared with CD, while
showed slightly difference between each group with
or without curcumin and/or piperine. In addition, the
weight of liver decreased obviously when mice were
fed on 1000 mg/kg curcumin or 20 mg/kg piperine or
curcumin combined with piperine (Table 4).
Histopathology of gallbladder and liver
HE staining of liver revealed obvious vacuolar degener-
ation, coupled with neutrophils infiltrating into acini and
portal areas in LD. By comparison, the changes miti-
gated with curcumin-piperine group. HE staining and
sirius red saturated picric acid staining of gallbladder
indicated mucosal hyperplasia and connective tissue
expansion of lamina propria in LD, as well as a signifi-
cant increase of small vessels and collagenous fibers. In
contrast, these changes could be inhibited by curcumin
combined with piperine (Fig. 2).
Interestingly, we found that livers of mice fed on
piperine (concomitant with/without curcumin) showed a
protective effect against lithogenic diet. Livers of mice in
LD showed hepatic steatosis under light microscope,
including hepatomegaly, yellowing of liver, edge blunt-
ing, greasy touch and vacuolar degeneration. While in
Table 1 Lipid levels of bile and serum
Bile(mmol/L)* Serum(mmol/L) n = 10
Cholesterol Phospholipid Bile acid Cholesterol Triglyceride
CD 5.73 29.64 154.47 2.45 ± 0.31 0.27 ± 0.03
LD 18.64 28.93 138.64 6.86 ± 0.16** 1.59 ± 0.10**
LP20 15.92 29.79 143.24 5.80 ± 0.79*** 1.56 ± 0.12
LC500 16.29 30.13 141.59 6.56 ± 0.42 1.58 ± 0.09
LC500P20 6.39 31.95 163.29 2.89 ± 0.14*** 0.84 ± 0.10***
LC1000 6.59 32.24 161.35 2.96 ± 0.08*** 0.84 ± 0.10***
*It was calculated by molarity, and the bile was collected for each experimental group
**P< 0.001 compared with CD
***P< 0.001 compared with LD
Li et al. Lipids in Health and Disease (2015) 14:100 Page 2 of 8
piperine group, the color of liver turned red, its edge
sharpened and cell arrangement became normal under
light microscope (Fig. 3).
Curcumin combined with piperine reduces NPC1L1
expression
Both of the spice principles could inhibit NPC1L1
mRNA expression, especially when curcumin (1000 mg/
kg) or curcumin combined with piperine (1.00 ± 0.04 vs
2.64 ± 0.04, 2.11 ± 0.11, 1.69 ± 0.06, 1.22 ± 0.06, 1.33 ±
0.05 respectively, *P< 0.001 compared with CD, **P<
0.001 compared with LD) were administered (Fig. 4a).
Furthermore, results of Western blot revealed that
1000 mg/kg curcumin or curcumin combined with pip-
erine could reduce the expression of NPC1L1 protein
(Fig. 4b). Fig. 4c was grey analysis for western blot (1.00
vs 2.52 ± 0.038, 2.18 ± 0.036, 1.94 ± 0.072, 1.19 ± 0.05,
1.24 ± 0.049, *P< 0.001 compared with CD, **P< 0.001
compared with LD).
Curcumin combined with piperine reduces SREBP2
expression
Treatment with curcumin significantly inhibited SREBP-
2 mRNA expression, especially when mice were fed
on curcumin combined with piperine (1.0 ± 0.03 vs
1.34 ± 0.08, 1.22 ± 0.08, 1.19 ± 0.02, 1.08 ± 0.04, 1.16 ±
0.03 respectively, *P< 0.001 compared with CD, **P<0.01
compared with LD) (Fig. 5a). We also found that curcu-
min combined with piperine could decrease SREBP2
protein expression as 1000 mg/kg curcumin did
(Fig.5b).Fig.5cwasgreyanalysisforwesternblot
(1.00 vs 1.38 ± 0.03, 1.16 ± 0.05, 1.28 ± 0.01, 1.05 ± 0.05,
1.12 ± 0.03,*P< 0.001 compared with CD, **P<0.01
compared with LD).
Discussion
Curcumin is the active ingredient of traditional herbal
remedy and dietary spice turmeric, and has a long history
of use in traditional Chinese medicine [5]. Curcumin has
been widely investigated of its numerous biological effects,
including anti-carcinoma, anti-inflammatory and antioxida-
tive effect as well as its efficacy in cardiovascular diseases
[610]. Our previous study has evidenced that curcumin
could prevent formation of gallstones in C57BL6 mice
in a dose dependent manner, especially when taken at
1000 mg/kg. However, this curcumin intake is much
more than our normal consumption in daily life. Besides,
curcumin is poorly absorbed from intestine and can be rap-
idly metabolized in liver and intestinal wall [1214]. So it
has always been a challenge on how to enhance the oral
bioavailability of curcumin. Then, the introduction of piper-
ine cast a new light on this research.
Piperine is an alkaloid isolated from Piper nigrum
fruits, and it is the first batch of purified natural molecules
with bio-enhancer properties [18]. Piperine is pharmaco-
logically safe and has been listed in compounds generally
regarded as safeaccording to the US Food and Drug
Administration (FDA) [19]. It is also an inhibitor of
hepatic and intestinal glucuronidation, and it has been
reported that the ingestion of piperine contributed to in-
crease the serum concentration of curcumin and thereby
its bioavailability [20, 21]. Shoba et al. [22] have found that
concomitant administration of piperine 20 mg/kg could
increase curcumins (2 g/kg) bioavailability by 154 % in
rats and 2000 % in human. Later, Suresh et al. [15] found
500 mg/kg curcumin combined with 20 mg/kg piper-
ine could made an increase of curcumin absorption
from 60 %66 % to 78 % in experimental animals.
Table 2 Biliary lipid compositions of gallbladder bile
Cholesterol Phospholipid Bile salt Cholesterol Total lipid
concentration(g/dL)
CSI
(Mole%
a
) (Mole%
a
) (Mole%
a
) Phospholipid Bile salt
CD 3.02 15.61 81.37 0.20 0.04 10.1 0.546
LD 10.01 15.54 74.45 0.64 0.13 9.77 1.712
LP20 8.43 15.77 75.81 0.53 0.11 9.96 1.465
LC500 8.66 16.03 75.31 0.54 0.11 9.92 1.476
LC500P20 3.17 15.85 80.98 0.2 0.04 10.74 0.554
LC1000 3.29 16.11 80.6 0.20 0.04 10.68 0.575
a
Mole fraction
Fig. 1 The relative lipid compositions of pooled gallbladder bile.
represents relative lipid compositions of pooled gallbladder bile at
4 weeks in LD, Δfor LP20; for LC500; for LC500P20; for
LC1000; for CD
Li et al. Lipids in Health and Disease (2015) 14:100 Page 3 of 8
Moreover, Singh et al. [23] found that piperine
might enhance the bioabailability of curcumin through
P-glycoprotein. P-glycoprotein is organized in two hom-
ologous halves, each half begins with a transmembrane
domain that containing six transmembrane segments
followed by a hydrophilic nucleotide-binding domain [24].
Therefore, we adopted curcumin (500 mg/kg) in com-
bination with piperine (20 mg/kg) for our research,
and found that curcumin combined with piperine
could lower serum cholesterol and triglyceride, which
was more effective than LC500, and similar with
LC1000 (Table 1). It is similar with study of Tu et al. [25],
who found that curcumin plus piperine could decrease
levels of total cholesterol (TC), triglyceride (TG) and
low-density lipoprotein in serum, as well as increase
levels of fecal TC, TG and total bile acid compared with
administration of curcumin alone. It was found that
piperine alone also could decrease serum cholesterol,
though not as obviously as curcumin combined piper-
ine did (Table 1). Duanqjai et al. [26] found piperine
could reduce cholesterol uptake by internalizing the
cholesterol transporter protein, thus, reduced serum
cholesterol. Curcumin combined with piperine also
made cholesterol settled out (Table 2 and Fig. 1), and
finally, inhibited formation of gallstones, while piperine
taken alone did not have the effect of preventing gall-
stones (Table 3). It has been reported that curcumin
has an effect of liver protection [6], and our previous
studies indicated that the liver weight of mice fed on
lithogenic diet and curcumin was significantly de-
creased in a dose dependent manner, compared with
thosefromLD.However,evenformicefedon
1000 mg/kg curcumin, hepatic steatosis was still ob-
servedinprogress.Inthisstudy,weweresurprisedto
find that piperine had a significant effect on hepatopro-
tection. Liver of mice in LD showed a typical hepatic
steatosis, including hepatomegaly, which include yel-
lowing of liver, edge blunting, greasy touch as well as
vacuolar degeneration under light microscope. When
fed on piperine (with or without curcumin), the color
of liver turned red, its edge sharpened and cell arrange-
ment became normal (Fig. 3). It is consistent with the
perspective of Hyejeong et al. [27] who thought piper-
ine could alter liver X receptor α(LXRα)-mediated
lipogenesis, including SREBP1c. Moreover, Seoyoon et
al. [28] reported that the effect of piperine on hepatic
steatosis was probably due to reduced expression of
genes involved in lipogenesis, as well as enhanced expres-
sion levels of genes involved in fatty acid oxidation medi-
ated by adiponectin-AMPK pathway. Therefore, the
mechanism of piperine on liver-protection deserves more
intensive study and may provide a new insight into
prevention and treatment of fatty livers. Interesting, co-
administration of curcumin with piperine seemes could
not bring advantage to the curcumin effects on antidiabetic
and antioxidant activities, which Carlos et al. [29] sus-
pected might be related to changes on its biotransform-
ation. Therefore, at least part of the curcumin actions may
be related to metabolites.
Homeostasis of cholesterol in human body mainly
depends on its synthesis, absorption from intestine and
secretion of the bile, the metabolic process of which is
under precise regulation [1]. Previous studies have
revealed that intestine is the unique organ providing
dietary and reabsorbed cholesterol for the body, and
the absorption of cholesterol often starts from the
apical membrane of epithelial cells. Besides, there is a
close relationship between cholesterol absorption and
gallstone formation [2, 3]. There is also evidence that
deletion of Niemann-Pick C1-like 1(NPC1L1) gene or
use of its specific inhibitor, ezetimibe, could decrease up
to 70 % of cholesterol absorption in intestine [3032].
Our earlier studies found that curcumin could de-
crease NPC1L1 mRNA and protein expression in a
dose-dependent manner. While in this study, it was
found that 20 mg/kg piperine or curcumin combined
piperine could significantly decrease expression of
NPC1L1 mRNA and protein expression, especially in
LC500P20, which showed even lower expression than
LC1000. The expression of NPC1L1 is regulated by
SREBP2 [33] and it has been reported that the -291/+56
Table 3 Prevention of cholesterol gallstones by curcumin and
piperine
Incidence of cholesterol
gallstones(%)
Reduction in incidence of
cholesterol gallstones(%)
CD 0 -
LD 100 % -
LP20 70 % 30 %
LC500 80 % 20 %
LC500P20 30 % 70 %
LC1000 40 % 60 %
There are 10 mice per group
Table 4 Volume of gallbladder and weight of liver
Gallbladder (n= 10) Liver (n= 10)
Length(mm) Width(mm) Volume(μL) Weight(g)
CD 6.35 ± 0.91 2.75 ± 0.32 25.24 ± 5.97 0.90 ± 0.07
LD 8.19 ± 1.71 3.87 ± 0.48 65.54 ± 22.69* 1.93 ± 0.18*
LP20 7.36 ± 1.51 3.61 ± 0.70 53.90 ± 28.08 1.32 ± 0.18***
LC500 7.91 ± 0.87 3.66 ± 0.50 61.41 ± 18.44 1.90 ± 0.16
LC500P20 6.66 ± 1.36 3.09 ± 0.57 35.98 ± 15.27** 1.15 ± 0.19***
LC1000 7.18 ± 1.04 3.49 ± 0.72 49.05 ± 25.01 1.23 ± 0.13***
Values are means ± SD, with 10 samples each group
*P< 0.001 compared with CD, **P< 0.05 compared with LD, ***P< 0.001
compared with LD
Li et al. Lipids in Health and Disease (2015) 14:100 Page 4 of 8
region of the NPC1L1 gene harbors a binding site
for the SREBP2, which is essential for the basal ac-
tivity of the NPC1L1 promoter as well as cholesterol
modulation [34, 35]. Earlier studies from our labora-
tory demonstrated that curcumin could decrease
SREBP2 mRNA and protein expression in a dose-
dependent manner. In this research, it was found
that both piperine and curcumin combined piperine
could decrease SREBP2 mRNA and protein expres-
sion, especially when applied in combination, the in-
hibition rate was superior to that of 1000 mg/kg
curcumin group.
Conclusion
In summary, our present study demonstrated that
curcumin can prevent formation of gallstones, and it
Fig. 2 HE staining and sirius red saturated picric acid staining in liver and gallbladder
Fig. 3 Changes of liver and gallbladder in different groups. LD: Liver underwent hepatic steatosis and gallbladder stones formed. LP20: Liver
appeared normal, while gallbladder stones were observed. LC500P20: Liver appeared normal and no stones were seen in gallbladder. LC1000:
Liver underwent fatty degeneration and no stones were observed
Li et al. Lipids in Health and Disease (2015) 14:100 Page 5 of 8
is likely due to reduced expression of NPC1L1 that
regulated by SREBP2. Moreover, piperine, as an
enhancer of curcumin, could increase curcumin bio-
availability and make curcumin more effective in pre-
venting gallstones. Since curcumin is easy to access in
daily life and piperine is generally regarded as safe
by FDA, more research on curcumin combined with
piperine deserves to be carried out to bring new
insight into maintaining cholesterol homeostasis in
our daily diet.
Fig. 4 Curcumin combined with piperine reduces NPC1L1 expression. aNPC1L1 mRNA levels. bNPC1L1 protein expression. cGrey analysis for
western blot
Fig. 5 Curcumin combined with piperine reduces SREBP2 expression. aSREBP2 mRNA expression. bSREBP2 protein expression. cGrey analysis
for western blot
Li et al. Lipids in Health and Disease (2015) 14:100 Page 6 of 8
Materials and methods
Animals and diets
Male C57BL6 mice, 6-8 weeks old, were purchased from
Central Laboratory of Shengjing Hospital. Mice were fed
on normal rodent feedstuff (cholesterol < 0.02 %) or a
lithogenic diet [36] (2 % cholesterol plus 0.5 % cholic acid
and 15 % buffer) for 4 weeks, and divided into six groups
(10 mice/group): (1) Normal rodent feedstuff(Control diet,
CD), (2) Lithogenic diet(LD), (3) Lithogenic diet + 20 mg/kg
piperine(LP20), (4) Lithogenic diet + 500 mg/kg curcu-
min(LC500), (5) Lithogenic diet + 500 mg/kg curcu-
min + 20 mg/kg piperine(LC500P20), (6) Lithogenic
diet +1000 mg/kg curcumin(LC1000). Mice of all groups
(except CD and LD) were treated by gavage with
curcumin/piperine for 4 weeks after weighed every
dayat09:00.Thecurcumin,piperineandbothwere
administered in 1 ml 0.5 % sodium carboxymethyl
cellulose. Mice of CD and LD group received 1 ml
0.5 % sodium carboxymethyl cellulose every day. All
mice in six groups got free access to water, and were
kept under controlled condition at room temperature
(22 ± 3 °C), with a relative humidity of 60 %70 %
and a 12-h exchange of light/dark cycle. The animal
experiments were conducted according to the regulations
of the Committee on Bioethics of China Medical
University.
Collection of gallbladder, liver and small intestine
After the last gavage, all mice were fasted for 12 h and
then anesthetized using 10 % chloral hydrate. Blood was
drawn immediately through cardiac puncture and serum
was prepared by centrifugation. Each size of gallbladder
was measured by vernier caliper. Cholecystectomy was
performed and gallbladder was cautiously removed with
its adhering tissues being cleared up. Bile was collected
from each experimental group and stored at -80 °C for
subsequent extraction and analysis. Livers were excised,
washed with 0.9 % saline, blotted dry and weighted, then
they were stored at -80 °C for extraction and analysis.
Finally, a section of small intestine was resected (about
45 cm) from each mouse.
Lipid analysis
Levels of bile cholesterol, phospholipid, bile salts as well
as cholesterol and triglyceride in serum were determined
respectively following manufacturers instructions. Chol-
esterol saturation index (CSI) of the bile was calculated
according to Carey tables [37].
Histopathology
Paraffin-embedded gallbladder and liver sections (5 μm
in thickness) were used for hematoxylin and eosin stain-
ing (HE staining). They were observed at 200× magnifi-
cation under light microscope.
1 % sirius red saturated picric acid staining was used
for paraffin-embedded gallbladder sections (5 μmin
thickness). The sections were observed at 400× magnifi-
cation under light microscope.
Real-time PCR analysis
Total RNA was isolated using Trizol reagent (Takara)
following manufacturers instructions, the concentration
of RNA was calculated by spectrophotometry. cDNA
was prepared using the PrimeScript RT reagent kit with
gDNA Eraser (Takara). Real-time PCR was performed
using SYBR Green Premix Ex Taq (Takara). Sequences
for the primers used were as follows:
NPC1L1: (forward) 5-GACATCACCTTCCACCTC
TTG-3,
(reverse) 5- CTGGCATTCGACCCATGTAG-3
SREBP2: (forward) 5-TGGGGACAGATGCCAAGA
TG-3,
(reverse) 5- CACCAGACTGCCCAAGTCGA-3
β-actin: (forward) 5-CTGTGCCCATCTACGAGGGC
TAT-3 ,
(reverse) 5- TTTGATGTCACGCACGATTTCC-3
For real-time PCR, the PCR mixture was denatured at
95 °C for 10s, annealed at 60 °C for 20s and then
extended at 72 °C for 30s. This process was repeated for
a total of 40 cycles. The relation of NPC1L1 and SREBP2
mRNA expression with β-actin was calculated basing on
the threshold cycle (Ct) values.
Western blot analysis
Protein concentration was measured using the bicincho-
ninic acid (BCA) protein assay. Protein samples were
separated on 8 % SDS-PAGE gels and transferred to
polyvinylidene difluoride membranes (Millipore, USA).
Non-specific binding to the membrane was blocked for
1 h at room temperature with 5 % fat-free milk in TBST,
and then the membranes were incubated with 1:2000
NPC1L1 primary antibody (Novus Biologicals, USA) and
1:2000 SREBP2 primary antibody (Abcam) respectively
at 4 °C overnight. Then, the membrane was washed four
times with TBST and incubated with a 1:5000 dilution of
the appropriate secondary antibody at room temperature
for 45 mins. After the membrane was washed twice with
TBST, membrane-bound antibody was visualized using
an enhanced chemiluminescent kit (Millipore) according
to manufacturer instructions.
Statistical analysis
Values are given as the mean ± SD. Differences between
multiple groups were compared using one-way analysis
of variance (ANVOA). When statistical significance was
identified based on ANOVA, the Student-Newman-Keuls
Li et al. Lipids in Health and Disease (2015) 14:100 Page 7 of 8
test was used for multiple comparisons. P-values < 0.05
were regarded as statistically significant.
Abbreviations
NPC1L1: Niemann-Pick C1-like 1; SREBP2: Sterol response element-binding
protein 2; TC: Total cholesterol; TG: Triglyceride.
Competing interests
The authors declare that they have no competing interests.
Authorscontributions
YL and SW participated in the design of the study. YT, YL and ML completed
the experiment. SW and YT performed the statistical analysis. YL and ML
draft the manuscript. All authors read and approved the final manuscript.
Authorsinformation
Yongnan Li, MM. Research interests: Cholelithiasis. Min Li, MM. Research
interests: Cholelithiasis. Shuodong Wu, MD. Research interests: Cholelithiasis.
Yu Tian, MD. Research interests: Cholelithiasis.
Funding subsidize
This work was supported in part by grants from National Natural Science
Foundation of China (No. 81200318).
Received: 14 March 2015 Accepted: 25 August 2015
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Combination of curcumin and piperine prevents formation of gallstones in C57BL6 mice fed on lithogenic diet: Whether NPC1L1/SREBP2 participates in this process?.pdf
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    Black pepper (Piper nigrum L.) lowers blood lipids in vivo and inhibits cholesterol uptake in vitro, and piperine may mediate these effects. To test this, the present study aimed to compare actions of black pepper extract and piperine on (1) cholesterol uptake and efflux in Caco-2 cells, (2) the membrane/cytosol distribution of cholesterol transport proteins in these cells, and (3) the physicochemical properties of cholesterol micelles. Piperine or black pepper extract (containing the same amount of piperine) dose-dependently reduced cholesterol uptake into Caco-2 cells in a similar manner. Both preparations reduced the membrane levels of NPC1L1 and SR-BI proteins but not their overall cellular expression. Micellar cholesterol solubility of lipid micelles was unaffected except by 1 mg/mL concentration of black pepper extract. These data suggest that piperine is the active compound in black pepper and reduces cholesterol uptake by internalizing the cholesterol transporter proteins.
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