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Introduction
Berberine, also known as huangliansu, is an isoquinoline
alkaloid derived from the Chinese herb Huanglian, Huangbai
and other plants. It has multiple pharmacological actions,
such as antibacterial activity and anti-inflammatory effects,
and is used in the treatment of diarrhea and other digestive
disorders[1]. Berberine has been found to protect the gastric
mucosa and inhibit gastric ulcers[2], but the pharmacological
mechanisms for the protective effect of berberine are not
clear. Accumulating evidence from both animal and human
studies indicates that nitric oxide (NO) plays a key role in
normal wound repair. The beneficial effects of NO on wound
repair may be attributed to its functional influences on
angiogenesis and inflammation[3]. A recent study
demonstrated that NO generated from endothelial nitric oxide
synthase (eNOS) played an important role in gastric ulcer
formation and gastric healing[4]. However, NO generated from
inducible nitric oxide synthase (iNOS) participates in ulcer
formation through the production of peroxide free radicals
and their cytotoxic action[5]. At the same time, it was
reported that berberine could induce the thoracic aorta of rats
to release endothelial NO[6], and 13-methylberberine and
13-ethylberberine reduced the production of NO and the
expression of iNOS protein in a concentration-dependent
manner in lipopolysaccharide (LPS)-stimulated
macrophages[7]. We therefore inferred that it was possible for berberine to
protect the gastric mucosa and accelerate the healing of peptic
ulcers through the NO pathway. In the present study, we
attempted to investigate whether the protective effect of
berberine on gastric mucosa was related to NO and nitric
oxide synthase (NOS).
Materials and methods
Drugs and reagents Berberine was supplied by Prof
Jia-lin WANG (Department of Pharmacology, Tongji Medical
College, Huazhong University of Science and Technology,
Wuhan, China). The NO kit was supplied by Nanjing Jian
Cheng Bioengineering Company (Nanjing, China).
Animals The present study was carried out using 72
Kunming mice (Certificate No SYXK 2004-0028) weighing
18 g-22 g from the Experimental Animal Center of Tongji
Medical College. Both sexes were used. They were kept in
separate cages at room temperature and deprived of food
24 h before oral administration of ethanol but were allowed
free access to water.
Induction of gastric ulcer Gastric ulcers were produced
by oral administration of ethanol 24 h after starvation. Each
of the mice was given 0.1 mL ethanol (100%, anhydrous
alcohol).
Drugs treatments and measurement of ulcer size
Animals were divided into 3 groups of 24 mice each. Group I
received saline at a dose of 0.1 mL/kg (ip) and served as the
control group. Group II received berberine at a dose of 5
mg/kg (ip) while group III received berberine at a dose of
50 mg/kg (ip).
Drugs were given 30 min before the oral administration
of ethanol. The mice were killed by cervical dislocation 1 h,
2 h, 3 h and 6 h after oral administration of ethanol, and the
gastric juice was sucked from the stomach before the
stomach was removed. It was then opened along the greater
curvature and the mucosa of the glandular portion of the
stomach was rinsed gently with saline. Macroscopic
damage was assessed and the number of ulcers, ulcer severity
and ulcer index (UI) were recorded. Because each mouse
had many lesions or ulcers and most gastric mucosal lesions
were punctate or linear, each ulcer was graded according to
severity using a scale of 1-4, as follows: 1, punctate ulcer; 2,
linear ulcer of length £2 mm; 3, linear ulcer of length
2 mm-4 mm; and 4, linear ulcer of length
³4 mm. The UI was calculated by summing the total number of ulcers, as described
previously[8,9]. The gastric tissues were stored at -70 °C
until biochemical analysis.
Measurement of nitric oxide The content of nitric oxide
in the gastric juice and gastric tissue was measured through
the method of nitric acid reductase, and the operational
processes were carried out strictly in accordance with the NO
kit instructions.
Reverse-transcription polymerase chain reaction for the
detection of eNOS and iNOS mRNA The stomachs were
removed from the saline-treated (0.1 mL/kg) and
berberine-treated (50 mg/kg) groups both before and after ulcer
induction (1 h, 3 h and 6 h) for the determination of eNOS and
iNOS mRNA expression by reverse transcription-polymerase
chain reaction (RT-PCR) using specific primers. Total RNA
was isolated from gastric tissues using Trizol reagent (Gibco
BRL, Gathersburg, MD, USA). First-strand cDNA was
synthesized from 5 µg of total cellular RNA using
oligo-(dt)20 primers with the thermoscript RT-PCR system (Gibco BRL).
PCR cycles were carried out for amplification of eNOS, iNOS
and b-actin cDNA using a thermal cycler (Perkin-Elmer
Corporation 850 Lincoln Centre Drive, Foster City, California,
USA) and oligonucleotides (Boya, Shanghai, China). The
primers for eNOS were 5กฏ-TTC CGG CTG CCA CCT GAT CCT
AA-3กฏ (sense) and 5กฏ-AAC ATA TGT CCT TGC TCA AGG CA-3กฏ
(antisense)[10]. The b-actin primer sequences for iNOS
were 5กฏ-CGG GCA TTG CTC CCT TCC GAA AT-3กฏ(sense) and
5กฏ-CTT CAT GAT AAC GTT TCT GGC TCT-3กฏ
(antisense)[11]. The oligonucleotide primer sequences for
b-actin were 5กฏ-
TCA CCC ACA CTG TGC CCA TCT ACG A-3กฏ (sense) and
5กฏ-GGA TGC CAC AGG ATT CCA TAC CCA-3กฏ (antisense). The
number of PCR cycles was adjusted carefully to avoid
saturation of the amplification system. In the RT step, the
cellular mRNAs were reverse-transcribed into a "library" of
cDNAs. This cDNA library was then used for the analysis
of various genes in the PCR step. Briefly, total RNA (2.5 µg)
from each of the tissues was reverse transcribed into
single-stranded cDNA in a 20-µL reaction mixture containing: 4 µL
5กฏ buffer, 20 IU ribonuclease inhibitor, 10 mmol/L dNTP, 0.5
µg oligo-(dt)20 primer, 20 IU RNasin and 200 IU M-MLV
reverse transcriptase. After incubation for 1 h at 42 °C, the RT
mixture was incubated at 70 °C for 10 min to inactivate the
reverse transcriptase. PCR was then carried out using 5 µL
cDNA in a final reaction volume of 50 mL. The assay mix
contained 50 mmol/L KCl, 10 mmol/L Tris-HCl (pH 8.3),
1.5 mmol/L MgCl2, 0.2 mmol/L dNTP, 1 mmol/L of the
respective primers and 1.5 IU of Taq DNA polymerase. The PCR
cycling program was set for 1 cycle of pre-denaturation at
94 °C for 1 min, and then 35 cycles at 94 °C for 30 s, 55 °C for
30 s, 72 °C for 1 min, followed by 1 cycle at 72 °C for 5 min.
PCR products were visualized by UV illumination after
electrophoresis on a 2% agarose gel containing 0.5 µL ethidium
bromide. The location of a predicted product was confirmed
using a 100 bp DNA ladder (Gibco BRL) as a standard size
marker. The gel photographs were scanned with a
computerized densitometer (SYNGENE, London, Britain). The
signals for eNOS and iNOS mRNA were standardized against
the b-actin signal for each sample and the results are
expressed as eNOS and iNOS mRNA/b-actin mRNA ratio.
Statistical analysis All of the data were expressed as
mean±SD and the analysis was carried out using the
t-test. Values of P < 0.05 were considered statistically significant.
Results
Effects of berberine on ethanol-induced gastric
lesions The UI of ethanol-induced gastric lesions are shown in
Figures 1 and 2. In the control group, the UI at 1 h, 2 h, 3 h
and 6 h after oral administration of ethanol averaged 23.8±
1.4, 23.3±2.2, 22.3±1.2 and 20.8±1.1, respectively. The UI in
the berberine-treated groups (5 mg/kg and 50 mg/kg) were
less than the control group and this effect was
time-dependent (P< 0.05 and
P<0.01, respectively).
Change in nitric oxide content with the
time The content of NO in gastric tissue and gastric juice is shown in
Table 1. In the control group, the content of NO in gastric
juice at 1 h, 2 h, 3 h and 6 h after oral administration of
ethanol was 73.3 ±7.3 µL/L,
94.0±9.2 µL/L, 109.6±6.4 µL/L and
138.2±10.2 µL/L, respectively, while the NO content in
gastric tissue averaged
5.8±1.1 µmol/g protein,
8.3±1.1 µmol/g protein,
9.8±1.1 µmol/g protein and
11.9±1.2 µmol/g protein, respectively. However, in both gastric tissue and gastric
juice the content of NO in the berberine-treated groups (5
mg/kg and 50 mg/kg) was higher than that in the control
group at 1 h after oral administration of ethanol
(P<0.05,P<0.01), but lower at 6 h after oral administration of ethanol
(P<0.05, P<0.01).
Expression of eNOS and iNOS
mRNA Expression of iNOS and eNOS mRNA is shown in Figures 3 and 4. In the
base statement the mRNA of eNOS could be expressed while
that of iNOS could almost not be expressed. In the control
group the expression of eNOS was inhibited and iNOS was
enhanced by ethanol, while in the berberine-treated groups
the expression of eNOS could be enhanced and iNOS could
be inhibited.
Discussion
Nitric oxide plays an important role in the host defense
and inflammatory response[12,13]. It also plays an important
role in the mechanism of gastric mucosal protection and
injury induced by pressure, ethanol, stress and endo
toxins[14-17]. Endogenous NO has a dual action in the
gastrointestinal tract: protective effects by constitutive nitric
oxide synthase (cNOS)/NO and proulcerogenic effects by
iNOS/NO[8].
cNOS lies in gastric endotheliocytes in the gastric tissue,
also called eNOS[18]. NO derived from eNOS is a pivotal
mediator to accelerate gastric ulcer healing; it maintains the
integrity of the gastric epithelium, regulates gastric mucosal
blood flow, and stimulates gastric mucus secretion and
synthesis[19]. On the other hand, NO stimulates the release of
vasoactive intestinal peptide (VIP) in gastric tissue and
increases the concentration of cGMP[20]. NO/cGMP plays a
central role in producing relaxation of mouse gastric fundus
smooth muscle, and cGMP protects gastric parietal cells from
ethanol-induced cytotoxicity, implicating a basolateral
Cl- channel in the plasma membrane or that pepsinogen
secretion stimulated by a Ca2+-mediated agonist is modulated by a
NO/cGMP pathway[8]. The results of the present study have
shown that the content of NO in tissue from the
berberine-treated group was higher than control group at 1 h after oral
administration of ethanol, and further study has shown that
expression of eNOS is higher. Therefore, we might conclude
that berberine can increase NO production through
improving eNOS mRNA expression. The effect of berberine on NO
formation in the vascular system was also
demonstrated[6,21].
High expression of iNOS was found in ulcer tissue of the
control group at 3 h and 6 h after ulcer induction. A similar
phenomenon was also found by Peng
et al[22]. Large amounts of NO synthesized from the inducible isoform have been
implicated in tissue injury in the gut during inflammatory
reactions[23]. Guo
et al[24] also reported that high expression
and activity of iNOS were found to coincide with severe
inflammation in ulcer tissue. It is known that induction of
high-output iNOS usually occurs in an oxidative environment,
and thus high levels of NO have the opportunity to react
with superoxide anion
(O2-) leading to peroxynitrite
(ONOO-) formation and cell toxicity, protein tyrosine nitration,
hydroxyl radical production and tissue
damage[24,25]. The results of the present study have shown that iNOS is under
low-expression conditions at all times in the berberine-treated
groups, as the content of NO had no peak and it was kept at
a relatively steady level. This demonstrated that berberine
might decrease NO production through inhibition of iNOS
expression in the gastric ulcer tissue, prevent the abundant
release of NO that aggravates gastric mucosal injury and,
ultimately, improve the healing of ulcers. The study of Lee
et al[7] also showed that 13-methyberine and 13-ethyberberine
both inhibited iNOS expression in LPS-stimulated macrophages. In conclusion, the protective effect of
berberine results in an increase in eNOS mRNA expression, which
inhibits the iNOS mRNA expression process.
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