Baicalein reduces the occurrence of cirrhotic endotoxemia by reducing intestinal mucosal apoptosis

Experimental animals

90 healthy and clean Sprague Dawley rats were 8 weeks old weighing 190–220 g, and
all were purchased from the Laboratory Animal Center of Xi’an Jiaotong University.
All animals were housed under controlled temperature (23?±?1) °C, humidity (55?±?5)
% and 12 h light/12 h dark cycle for 1 week before the experiment.

Experimental materials

BA (purity 99 %) was purchased from Sigma-Aldrich Chimie Company (Fallavier, France).
Glutamine (Kotobuki Pharmaceutical Co., Ltd.) was purchased from the pharmacy of the
First Affiliated Hospital of Xi’an Jiaotong University. Carbon tetrachloride analytical
reagent (AR) was purchased from Chemical Reagent Branch of Tianjin Zonghengxing Industrial
and Trading Co., Ltd. Olive oil (AR) was purchased from Sinopharm Chemical Reagent
Co., Ltd. Rat endotoxin quantitative detection kit was purchased from Beijing Jinshanchuan
Technology Development Co., Ltd. Apoptosis detection kit was purchased from Biosynthesis
Biotechnology Co., Ltd. Total RNA extraction kit was purchased from Takara Biotechnologh
(Da Lian) Co., Ltd. Caspase-3 activity detection kit was purchased from Clontech company.

Reagent preparation

CCL4 oil solution preparation: 40 ml CCL4 was dissolved in 60 ml olive oil and 40 %
CCL4 oil solution was prepared and stored at room temperature.

30 % alcoholic beverage preparation: anhydrous ethanol and animal drinking water were
prepared in proportion of 3: 7 and stored at room temperature.

Glutamine solution: 15 g glutamine was dissolved 15 ml distilled water and was prepared
as 1 g/ml solution.

The establishment of endotoxemic cirrhotic rat model

Half of male and female Sprague-Dawley rats between 190 and 220 g were obtained from
the Animal Research Center of Xi’an Jiaotong University which were maintained according
to the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal
Resources, 1996, Nat. Acad. Press) and approved by the Xi’an Jiaotong University (Xi’an,
China) Institutional Animal Care and Use Committee (IACUC). At the end of experiments,
rats were euthanized under Phenobarbital anesthesia.

Endotoxemic cirrhotic rat model was established through complex factor modeling method
using carbon tetrachloride combined with high-fat diet 27],28]. Conventional adaptive feeding of animals was done for 1 week and then an eight-week
fibrosis induction period was initiated. All rats were randomly divided into model
control group with 80 rats and normal control group with 10 rats. During the initial
two-week treatment, 40 % CCL4 olive oil solution was biweekly subcutaneously injected
for 0.5 ml/100 g body weight, combined with high-fat diet (20 % lard oil +0.5 % cholesterol
+79.5 % corn flour) and 5 % ethanol (White wine and distilled water) as drinking water,
followed by two weeks 50 % CCL4 olive oil solution of biweekly subcutaneous injection
of 0.3 ml/100 g body weight, with high-fat diet (0.5 % cholesterol +99.5 % corn flour)
and 10 % ethanol as drinking water. From the 5^th to 8^th week, 60 % CCL4 olive oil solution was biweekly subcutaneously injected for 0.3 ml/100 g
body weight, combined with high-fat diet (0.5 % cholesterol +99.5 % corn flour) and
10 % ethanol as drinking water (see in Fig. 1). At the 9^th week, 5 rats of the model control group were randomly selected, and their liver histopathology
and serum endotoxin levels were detected. After the establishment of endotoxemic cirrhotic
rat model was determined, follow-up tests were conducted. During modeling, 15 endotoxemic
cirrhotic rats died due to liver failure, and the mortality rate was 18 %. No rats
died in the normal control group.

Fig. 1. The pathological changes of liver tissue in the each group after treatment (HE staining,
10?×?40). Note: a The pathological changes of liver tissue in normal group; b The pathological changes of liver tissue in Model group; c The pathological changes of liver tissue in Glutamine treatment group; d The pathological changes of liver tissue in BA treatment group

Method of administration

After modeling, 60 rats of the model group were randomly divided into 3 groups, namely
BA treatment group, the glutamine treatment control group and the model control group
with 20 rats in each group. In all of BA treatment groups, rats were administered
for intravenous injection with BA (20 mg/kg 10]); Baicalin were mixed with Poloxamer according to the ratio of 1:5, and then after
the mortar was used for grinding for 20 min, 0.9 % NaCl was added. The solution was
adjusted to pH 7.4),and the rats were gavaged with 1 ml/100 g aqueous solution for
two weeks. 1 ml/100 g glutamine aqueous solution was drenched to the rats of the glutamine
group, and 1 ml/100 g aqueous solution was drenched to the rats of model control group.
Moreover, the rats in the glutamine and model control group also received an intravenous
injection with equivalent volume of 0.9 % saline for 2 weeks.

Specimen collection and preparation

At 14^th day after treatment, i.e. fasting for 24 h after the last administration, specimens
were collected, and the specific steps were as the following:

After rat body weight was recorded, 10 % 1 ml/100 g chloral hydrate solution was used
for anesthesia by intraperitoneal injection, and then the abdominal cavity was opened
under sterile conditions. Under pyrogen-free conditions, abdominal aorta blood was
collected into depyrogenation tube and kept for enzyme linked immunosorbent assay
(ELISA) detection.

After blood collection, the liver was exposed, and terminal ileum tissue of about
3 cm was taken from the intestinal tract. One part of this intestinal tissue was put
in 4 % paraformaldehyde solution for TUNEL assay,and the other part was put in liquid
nitrogen for later use.

Detection of serum endotoxin levels with two-step double antibody sandwich enzyme-linked
immunosorbent assay

The enzyme linked immunosorbent assay (ELISA) was used for the detection and quantification
of serum endotoxin levels of rats in all groups according to the manufacturer’s description
(range 2.5–80 pg/mL). Samples of serum (100 ?L) were dispensed into wells of 96-well
microlitre plates which had been pre-coated with rat endotoxin (LPS) antibody. After
a 30 min incubation at 37 °C, unbound proteins were washed away from the wells. A
protein-specific biotinylated antibody was then added and the plates were incubated
for 2 more hours at 37 °C. After further rinsing to remove unbound antibody, a substrate
solution A, B was added to each well and the mixture was incubated for 20 min at 37 °C.
The reaction was terminated with the addition of a stop solution. A value was read
at 450 nm in microplate reader. With a value as vertical coordinates and the standard
concentration as abscissa, standard curve was drawn. According to the value of serum
sample, its concentration was found at the standard curve, and the data were expressed
as mean value.

Serum ALT, AST and TBIL levels were detected with ELISA method.

Detection of intestinal mucosal cell apoptosis by TUNEL

Terminal deoxynucleotide transferase dUTP nick end labeling (TUNEL) was performed
to detect cellular apoptosis on terminal ileum tissue using in situ cell death detection
Kit according to the manufacturer’s instructions. Sections were post-fixed in ethanol-acetic
acid (2:1) and rinsed. Then the sections were incubated with proteinase K (100 ?g/mL),
rinsed, incubated in 3 % H₂O₂, and washed with PBS for 10 min followed by washing with permeabilization solution
(0.1 % Triton X-100, 0.1 % sodium citrate) for 5 min. After the sections were washed
twice with PBS, incubated in TUNEL reaction mixture,and rinsed again. Sections were
visualized using converter-POD with 0.02 % 3,3’-diaminobenzidine (DAB). Mayer’s hematoxylin
was used for counter-staining. The sections were finally mounted onto gelatin-coated
slides and then they were air dried overnight at room temperature. Yellow stained
nucleus was TUNEL positive cells.

TUNEL-positive cell quantitative method was used. In each group, 5 cases of well-stained
sections were selected and magnified 10 times for observation under the microscope
objective. 5 high power fields (×400) in each section were randomly selected, and
a total of 25 high power fields were photographed into computer (resolution is 800×
600). Q550CW image signal acquisition and analysis system was employed to calculate
intestinal epithelial cell apoptosis index. The number of apoptotic cells / the total
cell number?×?100 %?=?the apoptosis rate of this field, and the average rate of apoptosis
of five fields was the apoptosis rate of rat intestinal mucosal cells in the group.

Detection of apoptotic gene Bcl-2 mRNA and Bax mRNA by RT-PCR

Experimental procedures

Primer synthesis: According to Complementary DNA (cDNA) sequence of Bcl-2, Bax and
?-actin, two pairs of primers were designed respectively. Bcl-2 upstream primer was
5’-GAACTGGGGGAGGATTGTGG-3 ’, and its downstream primer was 5’-CCACCGAA CTCAAAGAAGG-3’,
and the product length was 269 bp; Bax upstream primer was 5’-GTTACAGGG TTTCATCCAGG-3
’, and its downstream primer was 5’-CAAAGTAGAAGAGGGCAACC-3, and the product length
was 272 bp; The upstream primer of housekeeping gene ?-actin was 5’-AACCCT AAG GCC
AAC CGT GAA AAG-3 ’, and its downstream primer was 5’-TCATGAGGTAGTCTGTCAG-3’, and
the product length was 241 bp. All primers were designed and synthesized by Beijing
Aoke Company.

Total RNA extraction: After the rats were sacrificed, dry roasted tweezers were used
to clip terminal ileum tissue of about 3 cm quickly from the intestinal tract. Intestinal
contents were rinsed with saline, and the tissues were cut into 2 cm?×?1 cm size and
they were cooled and preserved rapidly with liquid nitrogen. In the experiment, 100 mg
colon specimens were taken, and the tissues were ground into power in liquid nitrogen.
Total RNA was extracted by Trizol reagent extraction method with reference to the
method on the kit.

RNA was reverse transcribed into cDNA. Reverse transcription reaction was performed
according to Invitrogen’s Superscript III First-Strand Synthesis reverse transcription
kit instructions. The product obtained was stored at ?20 °C or used directly.

Amplification of the housekeeping genes: PCR reaction system (0.5 ?l Taq DNA polymerase
(5u/?l), 5 ?l 5?×?Buffer (1.5 mmol/L MgCl₂), 1 ?l P1 (100 ?mol/l), 1 ?l P2 (100 ?mol/l), 0.5 ?l dNTP (10 mmol/l), 10 ?l template
cDNA, 4 ?l 50 mmol/L MgCl₂, 28 ?l H₂O, and the total reaction system was 50 ?l). PCR reaction amplification conditions:
initial denaturation: 95 °C 30 s, denaturation: 95 °C 30 s, renaturation: 58 °C 30 s,
extension: 72 °C 30 s, 35 cycles, 72 °C for 10 min.

PCR amplification of gene fragments: reaction system (0.5 ?l Taq DNA polymerase (5u/?l),
5 ?l 5?×?Buffer (1.5 mmol/L MgCl₂), 1 ?l P1 (100 ?mol/l), 1 ?l P2 (100 ?mol/l), 0.5 ?l dNTP (10 mmol/l), 10 ?l template
cDNA, 4 ?l 50 mmol/L MgCl₂, 28 ?l ddH₂O, and the total reaction system was 50 ?l). PCR reaction amplification conditions:
initial denaturation: 95 °C 5 min, denaturation: 94 °C 45 s, renaturation: 56 °C 45 s,
extension: 72 °C 45 s, 35 cycles, 72 °C for 10 min.

Electrophoresis was performed to PCR products in a 2 % agarose gel at 80 V for 30 min.
Then PCR products were observed and photographed in the gel imager, and the Quantity
One analysis system was used for the analysis of electrophoretic bands to calculate
the values of Bcl-2 products, Bax products and ?-actin products, that is, Bcl-2 and
Bax mRNA expressions in tissues.

The above process was repeated three times.

The detection of caspase-3 activity of small intestinal tissues with colorimetric
method

100 mg fresh terminal ileum tissue was homogenized and centrifuged, and protein concentration
was determined and adjusted. DTT (1 mol/L 10 ?l) of Caspase-3 detection kit and 50 ?l
2?×?reaction buffer were added into 10ul supernatant sample protein, and then 10 ?l
1 mmol Caspase-3 substrate Ac-DEVD-pNA was added. The mixture was incubated at 37 °C
for 60 min. The fluorescence units of Caspase-3 before and after incubation were determined
with excitation wavelength 400 nm and emission wavelength 405 nm according to the
instruction of the kit. Lysis Buffer and reaction Buffer were taken as the blank control.

Statistical methods

SPSS13.0 statistical analysis software package was used. One-way analysis of variance
(one-way ANOVA) was performed, and q test (Student-Newman-Keuls test, S-N-K) was used
for pair wise comparisons. Data were all expressed with (), and when heterogeneity of variance appeared, the data were square-root transformed.
P 0.05 was considered to be statistically significant.