Effect of Maotai liquor in inducing metallothioneins and on hepatic stellate cells.

Abstract

To explore the possible mechanism why drinking Maotai liquor dose not cause hepatic fibrosis.
After being fed with Maotai for 56 days consecutively, the male SD rats were decollated for detecting the biological indexes, and the livers were harvested to examine the liver indexes and the level of hepatic metallothioneins (MT). Hepatic stellate cells (HSC) proliferation and collagen generation were also observed.
Hepatic MT contents were 216.0 ng.g(-1)+/-10.8 ng.g(-1) in the rats of Maotai group and 10.0 ng.g(-1)+/-2.8 ng.g(-1) in the normal control group, which was increased obviously in Maotain group (P<0.05). In the rats with grade CCL(2) poisoning induced by Maotai, hepatic MT content was 304.8 ng.g(-1)+/-12.1 ng.g(-1) whereas in the controls with grade CCL(4) poisoning, it was 126.4 ng.g(-1)+/-4.8 ng.g(-1) (P<0.05). MDA was 102.0 nmol.g(-1)+/-3.4 nmol.g(-1) in Maotai group and 150.8 nmol.g(-1)+/-6.7 nmol.g(-1) in the control group (P<0.05). When both of the groups were suffering from grade CCL(4) poisoning, hepatic MT contents was negatively correlated with MDA (r=-0.8023, n=20, P<0.01). The 570 nmA values of each tube with HSC regeneration at concentrations of 0, 10, 50, 100, and 200 g.L(-1) of Maotai were 0.818, 0.742, 0.736, 0.72, 0.682, and 0.604, respectively. From the concentration of 10 g.L(-1), Maotai began to show obvious inhibitory effects against HSC, and the inhibition was concentration-dependent (P<0.05, P<0.01). Type I collagen contents in HSC were 61.4, 59.9, 50.1, 49.2, 48.7, 34.4 microg.g(-1) at concentrations of 0, 10, 50, 100, and 200 g.L(-1) of Maotai. At the concentration of 100-200 g.L(-1), Maotai had obvious inhibitory effect against the secretion of type I collagen (P<0.05). Gene expression analysis was conducted on cells with Maotai concentrations of 0, 50, 100g.L(-1) respectively and the ash values of beta-actin gene expression were 0.88, 0.74, and 0.59, respectively,suggesting that at the concentration of 100g.L(-1), Maotai could obviously inhibit gene expression of type I procollagen (P<0.05), but the effect was not obvious at the concentration of 50 g.L(-1) (P>0.05). At the concentration of 10 g.L(-1), HSC growth in vitro inhibition rates were 16.4+/-2.3 in Maotai group and -8.4+/-2.3 in the control group (P<0.05).
Maotai liquor can increase metallothioneins in the liver and inhibit the activation of HSC and the synthesis of collagen in many aspects, which might be the mechanism that Maotai liquor interferes in the hepatic fibrosis.

Full text

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PO Box 2345, Beijing 100023, China World J Gastroenterol 2002;8(3):520-523
Fax: +86-10-85381893 World Journal of Gastroenterology
E-mail: wjg@wjgnet.com www.wjgnet.com Copyright © 2002 by The WJG Press ISSN 1007-9327
• BASIC RESEARCH •
Effect of Maotai liquor in inducing metallothioneins
and on hepatic stellate cells
Ming-Liang Cheng, Jun Wu, Hai-Qin Wang, Lie-Ming Xue, Ying-Zhi Tan, Liu Ping, Cheng-Xiu Li,
Neng-Hui Huang, Yu-Mei Yao, Lan-Zheng Ren, Lan Ye, Ling Li, Mei-Lin Jia
www.wjgnet.com
Ming-Liang Cheng, Jun Wu, Hai-Qin Wang, Yu-Mei Yao, Department of
Infectious Diseases, Affiliated Hospital, Guiyang Medical College, Guiyang
550004, Guizhou Province, China
Lie-Ming Xue, Ying-Zhi Tan, Liu Ping, Shanghai University of Traditional
Chinese Medicine. Shanghai 200020, China
Cheng-Xiu Li, Neng-Hui Huang, Lan-Zheng Ren, Lan Ye, Ling Li, Mei-
Lin Jia, Department of pharmacology, Guiyang Medical College, Guiyang
550004, Guizhou Province, China
Supported by The primary sciences and technology project of Guizhou
province, No. 19992015
Correspondence to: Ming-Liang Cheng, Professor Department of
Infectious Diseases, Affiliated Hospital, Guiyang Medical College, Guizyang
550004, Guizhou Province, China. chengml@21cn.com
Telephone: +86-851-6782199(H), +86-851-6855119 Ext. 3263 (O)
Received 2002-05-02 Accepted 2002-05-25
Abstract
AIM: To explore the possible mechanism why drinking
Maotai liquor dose not cause hepatic fibrosis.
METHODS: After being fed with Maotai for 56 days
consecutively, the male SD rats were decollated for
detecting the biological indexes, and the livers were
harvested to examine the liver indexes and the level
of hepatic metallothioneins (MT). Hepatic stellate cells
(HSC) proliferation and collagen generation were also
observed.
RESULTS: Hepatic MT contents were 216.0ng·g-1±10.8
ng·g-1 in the rats of Maotai group and 10.0ng·g-1±2.8
ng·g-1 in the normal control group, which was increased
obviously in Maotain group (P<0.05). In the rats with
grade CCL2 poisoning induced by Maotai, hepatic MT
content was 304.8ng·g-1±12.1ng·g-1 whereas in the
controls with grade CCL4 poisoning, it was 126.4ng·g-1
±4.8ng·g-1 (P<0.05). MDA was 102.0nmol·g-1±3.4
nmol·g-1 in Maotai group and 150.8nmol·g-1±6.7
nmol·g-1 in the control group (P<0.05). When both of
the groups were suffering from grade CCL4 poisoning,
hepatic MT contents was negatively correlated with
MDA (r=-0.8023, n=20, P<0.01). The 570nmA values
of each tube with HSC regeneration at concentrations
of 0, 10, 50, 100, and 200g·L-1 of Maotai were 0.818,
0.742, 0.736, 0.72, 0.682, and 0.604, respectively. From
the concentration of 10g·L-1, Maotai began to show
obvious inhibitory effects against HSC, and the inhibition
was concentration-dependent (P<0.05, P<0.01). Type
I collagen contents in HSC were 61.4, 59.9, 50.1, 49.
2, 48.7, 34.4µ µ µ µ µg·g-1 at concentrations of 0, 10, 50,
100, and 200g·L-1 of Maotai. At the concentration of
100-200g·L-1, Maotai had obvious inhibitory effect
against the secretion of type I collagen (P<0.05).
Gene expression analysis was conducted on cells with
Maotai concentrations of 0, 50, 100g·L-1 respectively
and the ash values of β β β β β-actin gene expression were
0.88, 0.74, and 0.59, respectively,suggesting that
at the concentration of 100g·L-1, Maotai could
obviously inhibit gene expression of type I procollagen
(P<0.05), but the effect was not obvious at the
concentration of 50g·L-1 (P>0.05). At the concentration of
10g·L-1, HSC growth in vitro inhibition rates were 16.4±2.3
in Maotai group and -8.4±2.3 in the control group (P<0.05).
CONCLUSION: Maotai liquor can increase metallothioneins in
the liver and inhibit the activation of HSC and the synthesis
of collagen in many aspects, which might be the mechanism
that Maotai liquor interferes in the hepatic fibrosis.
Cheng ML, Wu J, Wang HQ, Xue LM, Tan YZ, Ping L, Li CX, Huang NH,
Yao YM, Ren LZ, Ye L, Li L, Jia ML.Effect of Maotai liquor in inducing
metallothioneins and on hepatic stellate cells. World J Gastroenterol 2002;
8(3):520-523
INTRODUCTION
Long term alcohol abusing may result in alcoholic liver diseases, and
the volume and duration of drinking has a close relationship with
alcoholic liver diseases[1-5]. According to recent studies, the main cause
of alcoholic hepatic injury[6-12] is due to acetaldehyde and hydroxy
free radicals oxidized from alcohol which can injure the hepatocytes
and activate the lipid peroxidation. The necrosis, inflammation, alcohol,
its metabolites and lipid peroxidation are all able to activate Kupffer
cells to secrete many cytokines which in turn activate hepatic stellate
cells to produce various components of extracelluar matrix (ECM).
When a large amount of ECM is deposited in the liver, it will lead to
hepatic fibrosis[13-18]. Now many studies have demonstrated that
metallothionein (MT) has the cytoprotective effect of clearing away
the oxygen-derived free radicals[19-24]. It is found[25,26] that alcohol can
induce the increase of metallothionein in rats liver, but the mechanism
has not been elucidated. MT is endogenous anti-injury substance and
plays a role in the defence of stress reaction[27]. Maotai liquor has its
unique brewing technique, at the same time, there are multiple
microorganisms in the special geographical situations which are able to
absorb abundant amino acids, vitamins and many essential microelements
[28]. It was reported by Li Xinyan et al that drinking Maotai liquor 150g
for ten years daily did not result in significant damage to the liver,
moreover, it could help protect one’s health. Epidemiological study
showed that no one died of liver disease in those workers who had
drunk Maotai liquor for about 30 years. No obvious hepatic fibrosis or
cirrhosis of liver was found in the epidemiological study of 99 workers
who had a long history of drinking, or in the pathological examination of
their liver needle biopsies nor were they seen in rats fed with Maotai
liquor for a successive 56 days[28]. In order to explore the effect of
Maotai liquor on the liver, we observed its effect on hepatic stellate cell
proliferation in vitro, collagen generation,gene expression and growth of
human hepatic stellate cells, and the effect of Maotai liquor in inducing
metallothioneins in the rats liver and the relationship between it and
CCL4 hepatic injury were also studied in order to demonstrate the
possible mechanism in the inhibition on the hepatic fibrosis.
MATERIALS AND METHODS
M aterials
Male SD rats, weighing (300±20)g, were purchased from the

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Experimental Animal Center of the Third Military Medical University,
Chongqing, China. Maotai liquor (530±2)g·L-1, was produced by
Guizhou Maotai Distillery with bar code 6902952880026 provided
by Section 4 of Guizhou Oil & Foodstuff Export and Import Company.
MT standard was provided by Dr. Jie Liu in National Institute of
Environmental Health Sciences, U. S. A. 3-[4,5-dimethylthiazol]-2,5-
diphenyltetrazolium bromide. MTT were bought from Sigma Co., U.
S.A; trypsin was purchased from Difco. U. S. A and; 199 culture
medium and MEM culture medium without calcium are the products
of Gibco Co., U. S. A; newborn bovine serum (NBS) was produced
by Shanghai Huamei Co.; type I rat tail collagen standard (diluted
slowly with Naª-2CO3/NaHCO3 to 10 -200ng.L-1) and rabbit anti-
rat type I collagen antibody (diluted 1:500 with 0.01mol.L-1 PBS)
were products of Cambiolem Co.; horseradish peroxidase marker
labeled goat anti-rabbit antibody (IgG-HRP, diluted 1:1000 with
PBS containing 100ml.L-1 NBS) was purchased from Hua Mei
Company; Total protein determination agent (dcproteinassag) was
the product of Biorad Co., U. S. A.; RT-PCR reaction agent and PCR
marker were purchased from Promega Co.; diethylpyrocarbonate,
guanidine sulfocyanate, saturated mixture of phenol and chloroform,
and agarose were bought from Shanghai Sangon Co. Freezing High-
speed Centrifuge (1.0R, 22R), CO2 incubator and ultra low
temperature freezer were purchased from German Heraeus Co.;
inverted microscope was produced by Japanese Olympus Co.;
thermostat water bath, thermostat water bath vibrator were produced
by Shanghai Medical Equipment Factory; Labsystems Multiskan
MS Enzyme Marker Device, was produced in Finland; Danbury CT
ultrasonic membrane breaker was the product of Sonicsmaterials
Co.; 90mm culture plate, 60mm culture plate, 6-well, 24-well, and
96-well culture plate were the products of Danish Nunc Co.; Beck
Wallac 1410 Liquid Scintillation Counter was the product of Beckman
Co. U. S. A; Watson-Marlow 101U Constant current pump was
produced in U. S. A.
Effect of maotai liquor on liver MT of rats
Forty SD male rats were divided into two groups averagely. 20 rats
were fed with Maotai liquor at 2mL·kg-1 diluted 1:1 by distilled water
once everyday for 56 days. The other 20 rats in the control group
were fed with saline at the same volume. After all rats had been fed for
the last time, 10 rats of each group were given mixture of CCl4 and
olive oil in a volume ratio of 1:1 at 2.5mL·kg-1. All rats were sacrificed
after the last feed to get blood for testing biological indexes, to calculate
liver indexes by liver quantity, and to determine the content of MT in
the liver by saturation method of Cd- hemoglobin, and the lipid
peroxidation product of aldehyde measured by the method of
thiobarbiturate.
Isolation and culture of hepatic stellate cells (HSC)
HSC were isolated by in situ perfusion. The test of cell proliferation
was done by MTT. When monolayer HSC appeared in the 96-well
culture plate, they were cultured in the medium containing Maotai
liquor with different concentration of 1-400mg·L-1 and 50g·L-1 NBS
for 18 hours, then 20µL MTT (5g·L-1) was added in each well and
continued the culture for 4 hours. After that, suspension was removed
and the plate was aired, then 100µL acid isopropanol with 0.05mol·L-
1 HCl was added to each well, little black crystals were dissovled by
agitating which formed the steady purple solution. The absorbance
(A) in each well was determined by ELSIA at the wave-length of
570nm. There were 4 well in each sample. When the subculturing
HSC grew into full monolayer in the 24-well culture plate, then they
were cultured in the medium containing Maotai liquor with different
concentration from 1mg·L-1 to 400mg·L-1 and 50g·L-1 NBS for 24
hours.
Determination of type I collagen and total protein
After the culture was ended, it was centrifugated at 450×g at 4
for 20 minutes. Both the suspension and cell layer were collected
separately. The collected cells were dissolved by 0.2mol.L-1 NaOH
0.5ml in each well and washed with 0.5ml double distilled water.
Ultrasonic membrane-breaking was done for 10s at 40
collagen in the suspension was determined by ELISA (the enzyme
labelling plate was coated with type I collagen standard and samples
at different concentration were kept overnight at 4
was used for incubation after they bound to type I collagen antibody;
pyrocatechol oxidation was used for staining, and value A was obtained
at 492nm wavelength by Labsystem ELISA equipment, which
automatically calculated the standard curve and contents of each
specimen.). Total protein in the cell layer was determined by DC
protein assay kit.
. Type I
; then IgG-HRP
Semiquantitative RT-PCR
Total RNA was isolated from HSC by phenol-chloroform extraction
and isopropanol precipitation. The primer of procollagenβ1(I) was
synthesized, purificated and evaluated by Shanghai Sangon Company
(See Table 1). One µg of total RNA was reversely transcribed according
to the instructions of the RT-PCR Kit at 48
mixture contained 50pmo1·L-1 primer of procollagen β1[I] or
GAPDH, 1µL 10mmol·L-1 dNTPs, 2µL 25mmol·L-1 MgSO4, 5 units
AMV reverse transcriptase, 5 units Tfl DNA polymerase and 10mL
AMV/Tfl buffer. The PCR conditions included an initial
denaturation-2min at 94
, 30 cycles consisting of (a) 30s
denaturation at 94 ;(b)1 min primer annealing at 60
elongation at 38 ; and one final step of 7min at 68
product or DNA marker mixed with 5µL loading buffer were
elcectrophoresed on a 1.5% agarose gel, visualized by UV and
quantified densitometrically. Procollagenβ1[I], pre-albumin, or
hydroxyproline expression was calculated by determining the ratio
of Procollagen α1[I], relative to β-actin mRNA.
for 45min. The PCR
; (c) 2min
. The PCR
Table 1 Primer sequence and expected PCR product length
Primer designationSequence Product length
α1(I)upstream CA CCCTCA A GA GCCTGA GTC 253bp
α1(I)downstream GTT CGGGCTGA TGTA CCA GT
β-actin upstream A CA TCTGCTGGA A GGTGGA C 163bp
β-actin downstreamGGTA CCA CCA TGTA CCCA GG
The effect of Maotai liquor on human HSC
80000 human HSC cells were inoculated in each well in the 96-well
plate, and they were cultured in the DMEM medium with 100mL·L-1
FBS at 37 in a humidified atmosphere containing 5% CO2 for 24
hours. Then they were continuously cultured in the DMEM medium
with 20mL·L-1 FBS for 24hours. At last they were cultured in the
medium containing different concentration of Maotai liquor (Maotai
liquor was diluted to 0.5g·L-1, 1g·L-1, 5g·L-1, 10g·L-1, 50g·L-1 by DMEM
medium with 20mL·L-1 FBS or alcohol). Cells cultured in the DMEM
medium with 20mL·L-1 FBS was used as the control. After they were
cultured for 20 hours, 20µL MTT (5g·L-1) was added in each well and
the culture continued for 4 hours more. Then the medium was removed,
100µL acid isopropanol with 0.05mol·L-1 HCl was added in each well,
after little black crystals were dissovled, value A was obtained at
570nm wavelength by Labsystem ELISA equipment and the inhibiting
rate of cell proliferation was calculated.
Statistics analysis
Data were analyzed by t test and q test.
Cheng ML, et al. Maotai and metallothioneins 521

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RESULTS
Effect of Maotai liquor on rats liver MT content
Maotai liquor could induce the MT in rats liver to increase to 22 times
of its original level (See Table 2). It is thought that lipid peroxidation
of cell memb rane and the intracellular accumulation of Ca2+ are the
important links of hepatocellular damage. In the control group, MDA
in the liver was obviously increased after they were intoxicated by
CCl4, merely Maotai liquor did not influence MDA in rats’ liver.
MT in rats fed with Maotai liquor when intoxicated by CCl4 was
increased obviously more than those rats they were not fed with
Maotai liquor, but MDA was decreased. There was a negative
relationship between MT and MDA in rats liver intoxicated by CCl4
in the control and trial group (r=-0.8023, n=20, P<0.01).
Table 2 The effect of Maotai liquor on MT and MDA in rats liver
(n=10,x±s)
Group MT(ng·g-1) MDA(ng·g-1)
Control group 10.0±2.8 60.2±3.1
Maotai liquor group216.0±10.8b
60.1±2.4
CCL4 Group 126.4±4.8b
150.8±6.7b
CCL4 + Maotai liquor group 304.8±12.1bd
102.0±3.44bd
bP<0.01,vs control group;dP<0.01,vs CCL4 group(analysis of variance and q test)
Effect of Maotai liquor on rats’ HSC
We normally obtained (3-5)×107 HSC from one rat. Lipid droplets
in the primary HSC were obvious, but during the course of culture,
lipid droplets decreased. And as they were passaged, HSC became
extended, and looked like myofibroblasts. Each concentration of
Maotai liquor had no effect on the shape of HSC. The absorbance
(A) at 570nm of HSC in the medium with 0mg·L-1, 10mg·L-1,
50mg·L-1, 100mg·L-1 and 200mg·L-1 Maotia liquor were 0.818,
0.742, 0.736, 0.72, 0.682 and 0.604, respectively. From the
concentration of 10g·L-1, Maotai liquor had obvious inhibiting effect
on the proliferation of HSC, and the effect was enhanced with the
increase of the concentration of Maotai liquor (P<0.05, P<0.01).
The content of type I collagen of HSC in the medium with 0mg·L-1,
10mg·L-1,50mg·L-1,100mg·L-1 and 200mg·L-1 Maotia liquor were 61.
4µg·g-1,59.9µg·g-1,49.2µg·g-1,50.1µg·g-1,48.7µg·g-1 and 34.4µg·g-1,
respectively. The 100-200g·L-1 Maotai liquor had obvious inhibiting
effect on the secretion of type I collagen (P<0.05). The analysis of
gene expression of HSC in the 0mg·L-1, 50mg·L-1 and 100mg·L-1
Maotia liquor group showed that the relative density of β-actin
(n=3) analyzed by computer were 0.88,0.74 and 0.59, respectively.
The result showed that 100g·L-1 Maotia liquor could significantly
inhibit the gene expression of type I procollagen (P<0.05), but 50g·L-1
Maotia liquor did not have such effect (P>0.05).
Table 3 The effect of Maotai liquor on HSC of human (n=3, x±s)
Group Inhibiting Rate of HSC
Alcohol group (10g·L-1) -8.4±2.3
Maotai liquor group (0.5g·L-1) -4.52±0.3
Maotai liquor group (1g·L-1) 12.4±10.4b
Maotai liquor group (5g·L-1) 17.4±1.6b
Maotai liquor group (10g·L-1) 16.4±2.3b
bP<0.01,vs alcohol group
Effect of Maotai liquor on the growth of HSC (See Table 3)
There were three experiment groups in our study: blank control
group, control group and trial group. First alcohol was diluted to 530
g·L-1 (the same concentration as Maotai liquor) and then it was
dispensed to different concentrations which was the same as Maotai
liquor. We used 0.5g·L-1,1g·L-1, 5g·L-1 and 10g·L-1 Maotai liquor to
study the different inhibiting effect on HSC. Our result showed that
there was a significant difference between the 10g·L-1 alcohol group
and 10g·L-1 Maotai group (P<0.01). That was Maotai liquor had a
significant inhibiting effect on the proliferation of HSC, but alcohol
did not have such effect.
DISCUSSION
MT is a low molecular weight metal-binding protein with rich cysteine
existing widely in the biosphere and can be induced in vivo by many
factors[29-32]. MT is also a non-enzyme protein which has bioactive
functions of binding heavy metals, clearing free radicals, anti-oxidation
and cytoprotection. It is now the most powerful bioactive substance
which can remove free radicals. In recent years, a lot of research studies
show that MT can protect hepatic cells from injury and be helpful in
repairing hepatocytes without inducing hepatic fibrosis[33-38]. Induced
by Maotai liquor, the increased MT in rats’ liver was able to decrease
the lipid peroxidation product of MDA in the liver intoxicated by
CCl4. Our result showed that there was a negative correlationship
between MT and MDA in the liver and it proved that Maotai liquor
was able to induce MT to antagonize the effect poisoning by CCl4. It
may be one of the possible mechanisms in explaining why long term drinking
proper volume of Maotai would not cause hepatic fibrosis or cirrhosis.
Hepatic fibrosis is an important pathologic process resulted from
many chronic liver diseases and may process to liver cirrhosis. It is
also the key point that many chronic liver diseases are hard to cure
completely. Many investigations showed that the activation of HSC
is the key point in the pathological process of hepatic fibrosis. HSC
was activated by many pathological factors so that alterations in the
phenotype and function of HSC happened. HSC was highly
proliferated and secreted a large amount of extracellular matrix deposited
in the liver which would result in hepatic fibrosis. Collagen was the
most important component in the extracellular matrix[46-48]. The degree
of increase of collagen was type I>type III>type IV. The cell
proliferation and the enhanced generation of collagen were the main
characteristics of activation of HSC. [3H]thymidine incorperation was
able to reflect the cell division and proliferation. In the inhibiting
experiments of different concentration of Maotai liquor in both HSC
from rats and HSC from human beings showed that Maotai liquor had
a concentration-dependent inhibiting effect on the proliferation of HSC.
The generation of collagen was that first the collagen gene was transcribed
and then translated into procollagen and the product secreted in the
extracellular matrix. Our experiment showed that Maotai liquor had
inhibiting effect on the expression of collagen gene and the secretion of
collagen protein, but 10g·L-1 alcohol had no such inhibiting effect. All
those showed that Maotai liquor could inhibit the activation of HSC in
many links. The inhibiting effect of Maotai liquor in the activation of
HSC and the generation of collagen may be the possible reasons why
Maotai liquor can interfere with the process of hepatic fibrosis.
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