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Antibiotics in Acute Pancreatitis
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In: Acute Pancreatitis ISBN: 978-1-63485-229-6
Editor: Shane Copeland © 2016 Nova Science Publishers, Inc.
Chapter 9
BENEFICIAL EFFECT AND UNDERLYING
MECHANISM OF SHENMAI INJECTION ON
ACUTE EXPERIMENTAL PANCREATITIS
Qiang Yan
, Wei Wu, Xing Yao, Guolei Zhang,
Mingjie Zhang, Feng Cen and Xu Sun
Department of general surgery, Zhejiang University Huzhou hospital,
Huzhou, Zhejiang Province, China
ABSTRACT
Aim: To explore the therapeutic effect of Shenmai injection (SMI)
on acute experimental pancreatitis and underlying mechanisms.
Methods: After induction of acute pancreatitis model with caerulein
in rat, sixty Sprague-Dawley rats were randomized into a SMI (5 ml/kg/d)
group (n = 30) and control (5 ml/kg/d saline) group (n = 30). The
expression of serum amylase, vascular endothelial growth factor (VEGR),
angiogenetic factor (AF), platelet activating factor (PAF) was measured
at d1 (day 1), d3, d5, d7 respectively after the pancreatitis. Moreover, half
of the rats in each group were executed respectively at d7 and d14 to
assess nuclear factor kappa B (NF-κB), positive expression of CD31 and
microvascular density (MVD) of pancreatic tissue.
Correspondence: Qiang YAN, M.D., Department of general surgery, Zhejiang University
Huzhou hospital, Zhejiang, China. Tel: +8613957288699, Fax: +8657220213129, E-mail:
1837803391@qq.com.
Qiang Yan, Wei Wu, Xing Yao et al.
196
Results: Expression of serum amylase in SMI group at d3, d5, d7
was significantly lower than the counterparts in control (P < 0.05) while
the PAF was downregulated at d5 and d7 in SMI group (P < 0.05).
Conversely, the levels of VEGR and AF at 5d and 7d were significantly
higher in SMI group (P < 0.05). With respect to the biopsy result, while
the mRNA expression and protein concentration of NF-κB were
significantly lower in SMI group at d7 and d14, positive expression of
CD31 and microvascular density were significantly higher in SMI group
(P < 0.05).
Conclusion: Treatment with SMI, a traditional Chinese medicine, in
experimental pancreatitis rats, would attenuate inflammatory reaction,
promote angiogenesis and consequently improve microcirculation. This
may partially explain the beneficial effect of SMI in acute pancreatitis.
Keywords: acute pancreatitis, Shenmai injection, inflammation,
microcirculation
INTRODUCTION
Acute pancreatitis is one of the most common reasons for acute abdomen.
The severe acute pancreatitis is the most severe category of pancreatitis, and is
responsible for the high mortality of pancreatitis by leading to necrosis of
pancreas, sequential activation of cytokines and consequently systemic
inflammatory reaction syndrome and eventually to multiple organ dysfunction
[1]. Despite dozens of trials have been done in the past years, the exact
pathology for pancreatitis has not been elucidated. Recently, researchers
demonstrated that microangiopathy in the early phase of acute pancreatitis was
associated with the severity of the pancreatitis and the level of elevated serum
amylase [2, 3]. The Shenmai injection (SMI), a traditional Chinese medicine
with ginsenoside as the major and active ingredient, has been shown to inhibit
the expression and release of endothelin-1 [4], to inhibit the platelet
aggregation [5] and synthesis of thromboxane [6], while facilitate the
production of the prostacyclin. Pharmacologically, therefore, the SMI may
improve microcirculation in patients with acute pancreatitis, yet few empirical
data are available about this. We sought to answer this question by examining
the cytokines level and pancreatic biopsy in experimental pancreatitis rats
treated with SMI and saline respectively.
Beneficial Effect and Underlying Mechanism …
197
MATERIALS AND METHODS
Chemicals, Animal Model and Study Design
Shenmai injection is extracted from two traditional Chinese herbs, red
ginseng and radix ophiopogonis, with ginsenoside Rg1, ginsenoside Re and
ginsenoside Rb1 as its major and active ingredients. SMI was purchased from
Chia Tai Qingchunbao Pharmaceutical Co., Ltd. (Hangzhou, China). The
appraisal of SMI was provided by the State Food and Drug Administration
(WS
3
-B-3428-98-2010Z) according to the State pharmaceutical standards.
This study was approved by the Ethical Committee on Animal
Experiments of the Zhejiang University. Male Sprague-Dawley rats (weighing
200 ± 50g) were kept at a constant room temperature (25°C) with light–dark
cycles of 12h and were allowed free access to water ad libitum. After 1 week
of acclimatization, rats were made to fast overnight before the induction of
acute pancreatitis. As previously described, seven intraperitoneal injections of
caerulein (50 μg/kg/h) were applied consecutively at 1h intervals. Rats were
randomly divided into caerulein + NS (normal saline) pancreatitis group
(Control, n = 30), and caerulein + SMI group (SMI group, n = 30). 5ml/kg/d of
SMI or isovolumic NS were infused per day for study and control groups
respectively through osmotic pumps since the last injection of caerulein.
Caudal vein blood sample taken at d1, d3, d5, d7 respectively were centrifuged
at 15,000 rpm under 4°C and then stored in a −80°C fridge for next analysis.
Half of the rats in each group were sacrificed at d7 and d14 after the last
injection of caerulein for pancreas samples collection.
Measurement of Amylase, Vascular Endothelial Growth-Factor
(VEGR), Angiogenetic Factor (AF) and Platelet Activating
Factor (PAF)
Serum amylase was measured with ELISA method. Similarly, the VEGR,
AF and PAF were detected using a commercial enzyme-linked immunosorbent
assay kits (Jingma biotech, Shanghai, China), according to the manufacturers'
protocols.
Qiang Yan, Wei Wu, Xing Yao et al.
198
RNA and Western Blot Assay for the Expression of NF-κB
Total RNA was extracted from biopsied pancreas using guanidine
isothiocyanate based Trizol reagent (Generay Corporation, Shanghai, China)
according to the manufacturer's specifications. Superscript III reverse
transcriptase (RT) (Fermentas Corporation, Canada) was used to reverse
transcribes 5 μg of total RNA. The primer sequences used for fluorescence
quantitative PCR were: Forward Primer 5'-ACGGGGAAGGTCTGAATGC-
3', Reverse Primer 5'-GGTGTCGTCCCATCGTAGGT-3'. Gene ID of the
target gene in the Genbank was 81736, and a 223-bp product was expected in
the reaction. Results were analyzed using BIO-RAD CFX Manager.
On the other hand, total 50 mg of pancreas was homogenized in RIPA
buffer (containing 1 μM phenylmethanesulfonyl fluoride) and then centrifuged
(15000 × g, 5 min). Supernatants were collected and proteins were separated
via an 8% SDS-PAGE (80 V, 30 min for concentrating gel; 120 V, 60 min for
separating gel). Proteins were then transferred to a cellulose acetate
membrane, blocked with 5% non-fat milk in buffer (20 mM Tris HCl, pH 7.4,
135 mM NaCl, 0.1% Tween) (2 h, at room temperature). Membranes were
then incubated overnight (at 4°C) with the primary antibodies: mouse anti-NF-
κB (p65) or rabbit anti-histone H1 (P13) (1:1000), followed by the secondary
antibodies: anti-mouse or anti-rabbit (1:1000), respectively (1 h, at room
temperature). Immunoreactive proteins were detected using an enhanced
chemiluminescence detection system. Autoradiographic images were analyzed
using Quantity One software.
Immunohistochemistry and Assessment of Microvascular
Density (MVD)
Immunohistochemical staining for CD31 was performed by the
streptoavidin–biotin method. The 4-μm thick sections were stained with an
automated system. Briefly, the slides were deparaffinized, and endogenous
peroxidase activity was blocked by incubation with 3% H
2
O
2
(10min).
Antigenic sites were revealed by applying citrate buffer in a pressure cooker
(120°C for 3min). To minimize nonspecific background staining, the sections
were then preincubated with milk (30min). Then, a mouse monoclonal
antibody against CD31 (JC70; 1/50 dilution; Santa, USA) was added at room
temperature for 30 minutes. The optimal working dilutions were defined on
the basis of a titration experiment. Negative controls for each tissue section
Beneficial Effect and Underlying Mechanism …
199
were prepared by omitting the primary antibody. After washing three times
with trisphosphate buffered saline (TBS), sections were incubated with
biotinylated rabbit antigoat immunoglobulin G for 15 minutes. They were then
washed three times with TBS, treated with streptavidin–peroxidase reagent for
15 minutes, and then washed with TBS three times again. Finally,
immunoreactivity was developed with diaminobenzidine for 10 minutes,
followed by haematoxylin counterstaining. Sections were then dehydrated,
mounted and examined. CD31- containing cells were identified by the
presence of a brownish yellow chromogen.
Endothelial cells of the pancreas were immunostained with the endothelial
cell marker CD31. Five equal areas of high vascularization were photographed
with a magnification of 250. The number of vessels with CD31
immunostained was counted. Immunoreactive cell groups clearly
distinguishable from the background were counted as one vessel. CD31-
positive cell groups without an evident lumen were considered as
undifferentiated vessels and included in our analysis. MVD was assessed with
the reference to the previous method by Weidner [7].
Areas with the highest
CD31-defined microvascular density (vascular hot spots) were selected with a
Leica DMD 108 digital microimaging device. Vascular hot spots were
identified at a low optical power using ×4 and ×10 objectives. Five areas of
high vascularization were photographed to assess the microvascular density
respectively. The average number of the five areas was counted as the MVD of
the sample. Measurements were performed using the Image Pro-Plus 6.0
Image Analysis System (Media-Cybernetics). Any artifacts occurring in the
samples were removed manually.
Statistical Analysis
Descriptive data were reported as either mean ± SD, or number and
percentage. With respect to the differences between the groups, categorical
variables were compared using chi-square analysis. Continuous variables were
compared using Independent Sample T test for normally distributed data.
Statistical analysis was performed, using SPSS 15.0 (Chicago, Ill, USA).
Significance was defined as a P value < 0.05.
Qiang Yan, Wei Wu, Xing Yao et al.
200
RESULTS
The average weight for the rats in the control and SMI group were
comparable, at 210.5 g and 213.8 g respectively. No mortality was observed in
both groups during the induction and treatment of acute pancreatitis.
Downregulation of Serum Amylase Expression after SMI
Administration
Serum amylase, as a routine Lab index for pancreatitis, is helpful to access
the diagnosis and prognosis. The expression levels of serum enzyme at d1, d3,
d5, and d7 were detected and the trends were shown in Table 1. The serum
enzyme expression at d1 between two groups showed no significant
differences. Although the trends were decreasing in both groups from the 1
st
day to 7
th
day, amylase of SMI group at d3, d5 and d7 were significantly lower
than counterparts in control (P < 0.01 and P < 0.05 respectively). Though
amylase is not strictly related to the severity of pancreas, SMI downregulated
the level of serum amylase and attenuated progressive pancreas damage in
caerulein-induced pancreatitis.
Table 1. Change of serum amylase, PAF, VEGF and AF in the two groups
Group
Number
Days post induction of pancreatitis
1d
3d
5d
7d
Amylase
(IU/L)
SMI
Control
30
30
7716 ± 197
7658 ± 202
5368 ± 185
**
7532 ± 235
4568 ± 158
*
5056 ± 198
3025 ± 175
*
4528 ± 210
PAF
SMI
Control
30
30
39.34 ± 6.39
39.12 ± 6.42
30.63 ± 5.04
**
37.07 ± 6.33
25.30
± 4.76
**
36.06 ± 5.57
20.64 ± 3.56
**
33.26 ± 4.87
VEGF
SMI
Control
30
30
105.19 ± 12.43
106.70 ± 15.62
119.46
± 15.63
**
110.44 ± 18.36
139.78
± 24.30
**
118.51 ± 21.70
193.37
± 22.66
*
127.07 ± 20.16
AF
SMI
Control
30
30
1356 ± 58
1340 ± 55
1522 ± 42
**
1351 ± 65
1820 ± 65
**
1402 ± 42
2018 ± 78
**
1428 ± 51
*
P < 0.05,
**
P < 0.01, vs control, PAF: platelet activating factor, VEGF: vascular
endothelial growth factor, AF: angiogenesis factor, SMI: Shenmai injection.