Z. Li, et al.
LifeSciences228(2019)11–20
factor GATA6 [10,11]. Studies have demonstrated the significant role of
peritoneal resident macrophages in maintaining the function of intes-
tine. The decreased ratio of peritoneal resident macrophages was trig-
gered in sepsis mouse model [12–14]. However, its specific mechanism
remains unclear.
Endoplasmic reticulum (ER) stress has been linked to the patho-
genesis of sepsis recently [15]. Studies have shown that ER stress can
specifically activate caspase-12-mediated apoptosis. It has been de-
monstrated in mouse model of sepsis, ER stress contributes to abnormal
apoptosis of lymphocytes, indicating that ER stress-mediated apoptosis
pathway may be a novel target in clinical prevention and therapy of
sepsis [16]. In addition, ER stress can also activate NF-κB signaling
pathway and regulate inflammatory response [17].
Rutecarpine is a significant alkaloid component of Evodia rutae-
carpa. It has extensive pharmacological effects including anti-throm-
botic, anti-cancer, anti-inflammatory, anti-allergic, muscle relaxant,
photo-aging protection, gastric mucosa protection, heart protection. It
can also improve hyperlipidemia and affect immune and allergic reac-
tions [18–21]. Previous researches have shown that rutaecarpine can
inhibit inflammatory cell infiltration, exudation and tissue hyperplasia
[21,22]. In addition, rutacarpine inhibited the protein level of proin-
flammatory cytokines TNF-α and IL-4 caused by the IgE-antigen com-
plex [23]. However, there has been no study on the therapeutic effects
of rutacarpine in sepsis.
Our study demonstrated that rutaecarpine ameliorated sepsis-in-
duced peritoneal resident macrophages apoptosis and inflammation
response through inhibiting ER stress-mediated caspase-12 and NF-κB
pathways. This is the first report about rutacarpine in ameliorating
sepsis. Our study provided new insights for drug development against
sepsis.
surgery group had abdominal incision and cecum exposure without li-
gation and puncture. 20 mg/kg rutaecarpine (Sigma-Aldrich St Louis,
MO, USA), which was dissolved in PBS, was administered in-
traperitoneally 1 h after the surgery. After 24 h, 5 mice from each of the
above 4 groups were randomly selected to extract peritoneal resident
macrophages for follow-up experiments, and others continued to ob-
serve survival rate and weight of mice. Mice were sacrificed by cervical
dislocation after 12 days. Liver samples were obtained for analysis.
2.2. Bacterial load detection
Peritoneal lavage fluid or blood was used as a stock solution, and
diluted 10 times with sterile physiological saline. 20 μL of different
concentrations of bacterial dilutions were inoculated on aerobic culture
dishes and anaerobic culture dishes (anaerobic culture dishes were
placed in anaerobic culture bags). Aerobic and anaerobic culture dishes
were cultured in a 37 °C incubator for 24 and 48 h, respectively. Count
the colonies after the end of the culture and calculate the number of
colony forming units (CFU) per mL of blood or peritoneal lavage fluid.
2.3. Inflammatory factors in peritoneal fluid
ELISA kits (R&D Systems, Minneapolis, MN, USA) were used to
detect the inflammatory factors TNF-α, IL-6 and IL-10 in peritoneal
fluid from each group of mice, and strictly followed the ELISA kit in-
structions.
2.4. HE staining
The liver tissue sections were first de-paraffinized by two xylenes for
15 min and soaked in high-to-low-concentration ethanol (100%, 90%,
80% and 70%) for 3 min each. Then, hematoxylin was stained for
1–3 min, 1% ethanol for 10 s, dilute ammonia for 20 s, and eosin
staining for 3 min. Each step above was rinsed with distilled water for
1 min. After dehydration with low to high concentration gradients
(70%, 80%, 90% and 100%) ethanol for 5 min, xylene was allowed to
clear twice for 15 min, and the solution was sealed with a neutral resin.
The staining results were observed by an optical microscope (Olympus,
Center Valley, PA, USA) and photographed. Histopathological changes
were scored to four levels based on the intensity of necrosis and dif-
fusion of the inflammatory cells according to previous research [25].
The four scored levels were interpreted as: Zero, no necrosis and in-
flammation; One, mild hepatocyte necrosis with mild inflammatory
reaction; Two, diffuse hepatocyte necrosis and intralobular necrotic
bridges along with inflammatory reaction; Three, complete destruction
of lobules, diffuse hepatic necrosis along with diffuse interlobular in-
flammatory reaction.
2. Materials and methods
2.1. Cecal ligation and puncture (CLP) surgery
Male C57BL/6 mice of 8 weeks old were used in this study. All the
animals were purchased from SJA Laboratory Animal company (Hunan,
China) and maintained in accordance with Guidelines for the Care and
Use of Laboratory Animals of the third Xiangya hospital (Hunan,
China). All procedures were performed according to the internationally
accepted ethical guidelines.
Cecal ligation and puncture (CLP) surgery was used to establish
sepsis model according to the previous reference (24). In brief, mice
were randomly divided into four groups (17 mice per group): sham
surgery group, CLP surgery group, CLP surgery with vehicle (PBS) in-
jection group and CLP surgery with rutaecarpine injection group. Mice
were anesthetized with 40 mg/kg intraperitoneal injection of 10 g/L
sodium pentobarbital. For CLP surgery groups, a 1.5 cm long long-
itudinal incision was made along the midline of the abdomen to sepa-
rate the skin and subcutaneous tissue layer. The rectus abdominis and
peritoneum were dissected at the abdominal white line, and the cecal
and its surrounding adjacent intestines were exposed. The cecum was
gently pulled out, and the cecum and mesenteric vessels were ligated
from the end of the cecum to the entire cecum length of 1/3 to 1/2
using a 4th surgical line. Before the caecum perforation, the content of
the cecum was gently pushed to the distal cecum. In the middle position
between the ligation and the cecum tip, a single penetration puncture
through the cecum was performed using a 21G needle. Then the cecal
part of the ligation was perforated, and a small amount of intestinal
contents were slightly squeezed out, so as to ensure that the puncture
hole was unobstructed. All the intestines were then returned to the
abdominal cavity, and the surgical incision was sutured layer by layer
with the 4th surgical line. Animals were resuscitated by injecting pre-
warmed normal saline (37 °C, 5 mL per 100 g body weight) sub-
cutaneously. Buprenorphine (0.05 mg per kg body weight) was injected
every 6 h for at least 2 d for postoperative analgesia. Mice in sham
2.5. TUNEL assay
The in-situ terminal labeling kit (Solarbio, Beijing, China) was used
based on the operation manual. The procedures were briefly described
as follows. The liver tissue samples were washed 3 times with PBS so-
lution. Then, 100 μL of the 10% proteinase K solution was added and
incubated at 37 °C for 30 min, followed by washing 3 times with PBS.
Then, 50 μL of TdT enzyme solution was added and incubated at 37 °C
for 60 min. After washing 3 times with PBS, the sample was mixed with
5 μL of streptavidin-fluorescein solution and 45 μL of labeling buffer
and stored at 37 °C for 30 min in the dark. After washing with PBS and
staining with DAPI, the samples were then observed and photographed
under
a fluorescence microscope (Carl Zeiss Microscopy, Jena,
Germany).
2.6. Peritoneal macrophages isolation and culture
Five mice from each group were sacrificed 24 h after the surgery by
12