Introduction
In clinical settings, sepsis always occurs secondary
to severe infection, trauma, burn, and major surgery. Severe sepsis
and septic shock can lead to multiple organ dysfunction syndromes
and death. The annual death frequency caused by sepsis can reach
100,000 worldwide, and the cost of treatment can reach tens of
billions of dollars (1). The
abnormal immune mechanism in sepsis has become a hot research topic
and macrophages that play an important role in antigen presentation
in the process of organism immunity, constitute a communication
bridge. According to the condition of induced differentiation,
macrophages can be classified to M1 type or M2 macrophage, both of
which have different activation pathways, producing a series of
cytokines and inducing organism immune response to change toward
different directions after activation (2). Previous findings have shown that Th2
immune response is enhanced in patients with trichinellosis, even
manifesting as immunosuppression. This type of change can remit the
occurrence of immune disease (3).
Due to high risk and potentially lethal trichinization, the
application of the recombinant Trichinella protein may bring
new research directions for use in immune diseases (4).
Materials and methods
Experimental animals
Eighteen clean Sprague-Dawley (SD) rats (Shanghai
Shenggong Technology Co., Ltd., Shanghai, China) from the same race
and brood, aged 6–8 weeks with a weight of 190±10 g, were selected
and randomly divided into the sham operation (n=5), control (n=5)
and experimental (n=8) groups. Differences pertaining to comparison
among age, weight and health state were not statistically
significant (P>0.05). The rats in the sham operation group
underwent cecum division and suture with routine therapy for cure.
The rats in the control and experimental groups underwent cecal
ligation and puncture (CLP). The experimental group was given
recombinant Trichinella 53-kDa protein in advance, and the
control group was given the same dose of placebo.
Preparation of the CLP rat model
Preoperative abrosia was carried out for 12 h, with
isoflurane inhalation anesthesia to induce and maintain the
anesthesia state of the rats. The rats were placed in supine
position, the skin of hypogastrium was disinfected with iodine, and
the rats underwent direct shear operation to cut the skin, muscular
layer and peritoneum gradually below the middle of linea alba
approximately 0.3 cm. Using a tweezer the muscle layer was lifted
to explore the abdominal cavity, and the end of appendix was
removed and placed outside of the abdominal wall, then ligatured
1.6 cm from the cecum end with the suture line. Two holes were
punctured vertically to the cecum, which was then drained. The
cecum was returned to the abdominal cavity and the abdomen was
sutured. An immediate neck and back subcutaneous injection of 50
ml/kg compound sodium chloride was carried out and the rats were
placed in the cage. The rats were given water and food after 4 h.
The animals were kept indoors at a temperature of 20±1°C, humidity
60±10% and light from 6:00 a.m. to 6:00 p.m. (5).
Approval for the animal studies was received from
the ethics committee of the Second Hospital of Dalian Medical
University.
Preparation and depuration of
decombinant Trichinella 53-kDa protein
The prepared pET-28a(+)-TsP53-E. coli BL21
glycerine bacteria were defrosted, inoculated and merged in 3 ml LB
liquid medium for culture. Kanamycin solution (3 µl) was then
added, single distributed bacteria were selected for cloning, and
the culture was agitated for 12 h. When the culture medium was
turbid, the liquid was obtained and the culture continued.
Subsequently, 3 ml 1 M IPTG inducer was added, followed by lysating
it on ice using ultrasound pyrolysis apparatus (Nanjing Xianou
Instrument Manufacturing Co., Ltd., Nanjing, China), the
supernatant was discarded and the sediment was kept after three
cycles of centrifugation at 3,000 × g for 20 min. Then, 30 ml 8 M
urea solution was added to the sediment and was resuspended, which
was placed on a shaker to clear the liquid. The supernatant was
obtained by filtering the 0.45 µm microporous membrane (Xi'an
DingChang Co., Ltd., Xi'an, China), as the chromatographic sample
of Ni-NTA-HIS metal chelate. The dialyzed protein sample was added
to the ToxinEraser™ endotoxin to remove the resin chelating for 1
h, prior to being eluted. The process was repeated to condense the
rTsP53 protein sample (6).
Preparation of rat anti-rTsP53 protein
polyclonal antibody
A quantity of 200 µg purified rTsP53 protein sample
was emulsified in Freund's complete adjuvant, which was then
injected into the SD rats subcutaneously, at 3 and 5 weeks. The
peripheral blood of the animals was kept and the rTsP53 protein
antibody was detected using ELISA (7).
Detection of bone marrow-derived
macrophages (BMDMs) M1 and M2
The rats were sacrificed by cervical dislocation,
and an annular incision was performed under the abdominal skin of
rats, with the lower limb skin by blunt separation. The knee joint
was cut, and the connection of the hip and spine was examined by
careful separation to expose the femur and hip. The partial,
residual muscle and tendon tissue were removed. The two ends of the
bone were cut, one of which was injected with 2.5 ml Dulbecco's
modified Eagle's medium (DMEM) without any sodium pyruvate using a
2.5 ml syringe needle. The marrow tissue was placed in a 15 ml
centrifuge tube with medium liquid, until the marrow cavity became
white. The supernatant of 30% L929 cell culture medium and the
cells were added. The cells were cultivated at 7°C, in a 5%
CO2 environment for 96 h, changing the liquid and
removing the components that did not adhere. Macrophage BMDMs of
the bone marrow were obtained (8).
Then, induced differentiation of the macrophages were carried out
as per the instructions.
The survival rate difference of the rats was
analyzed at 6 and 12 h, and proportion of the macrophages expressed
among the groups. The expression level of Th1-type cytokines IFN-γ
and tumor necrosis factor (TNF)-α, as well as Th2-type cytokines
interleukin-4 (IL-4) and IL-13 were compared.
Statistical analyis
Data were analyzed using SPSS 19.0 statistical
software (SPSS Inc. Chicago, IL, USA). Data were presented as mean
± standard deviation. Comparison between groups were carried out
using the t-test. Countable data were expresses as cases or
percentage, and comparison of groups were carried out using the
Chi-square (χ2) test. P<0.05 was statistically
significant.
Results
Comparison of the survival rates in
three groups of rats
The survival rate of rats in the experimental group
was higher than that of the control group at 6 and 12 h, and the
difference was statistically significant (P<0.05) (Table I).
 | Table I.Comparison of the survival rates in
three groups of rats [case (%)]. |
Table I.
Comparison of the survival rates in
three groups of rats [case (%)].
| Groups | Cases | 6 h (%) | 12 h (%) |
|---|
| Sham operation | 5 | 5 | 5 |
| Control | 5 | 2 (40.0) | 1 (20.0) |
| Experimental | 8 | 6 (75.0) | 5 (62.5) |
|
χ2 |
| 3.925 | 4.152 |
|
P-value |
| <0.001 | <0.001 |
Comparison of the expression ratio of
macrophages
After 72 h of the blank medium, we obtained
PE-CCR7(−), FITC-CD206(−) macrophages (A group). Through the
activation of 20 ng/ml IFN-γ for 72 h, we obtained PE-CCR7(+)
macrophages (B group). Through the activation of 10 ng/ml
IL-3/IL-14 for 72 h, we obtained FITC-CD206(+) macrophages (C
group); following culture with rTsP53 protein for 72 h, we obtained
FITC-CD206(+) macrophages (D group); and following culture of 20
µg/ml IFN-γ and 5 µg/ml rTsP53 protein for 72 h, we obtained
PE-CCR7(+) macrophages (E group). The expression ratio of the
macrophage in the experimental group was higher than that of the
control group, and the difference was statistically significant
(P<0.05) (Table II).
| Table II.Comparison of the expression ratio of
different macrophages (%). |
Table II.
Comparison of the expression ratio of
different macrophages (%).
| Groups | A | B | C | D | E |
|---|
| Control | 81.7±16.4 | 19.8±5.2 | 69.4±15.5 | 2.3±0.2 | 10.9±2.4 |
| Experimental | 92.6±10.3 | 32.5±6.4 | 82.7±14.2 | 5.2±0.7 | 16.8±3.3 |
|
t-test | 3.526 | 3.784 | 4.152 | 4.829 | 4.628 |
|
P-value | <0.001 | <0.001 | <0.001 | <0.001 | <0.001 |
Comparison of the expression level of
Th1- and Th2-type cytokines
The expression level of the Th1-type cytokines IFN-γ
and TNF-α of the experimental group was higher than that of the
control group, while the expression level of the Th2-type cytokines
IL-4 and IL-13 was higher in the control group. The difference was
statistically significant (P<0.05) (Table III).
| Table III.Comparison of expression level of Th1-
and Th2-type cytokines. |
Table III.
Comparison of expression level of Th1-
and Th2-type cytokines.
| Groups | IFN-γ (ng/l) | TNF-α (ng/l) | IL-4 (pg/ml) | IL-13 (ng/l) |
|---|
| Control | 725.6±51.2 | 352.8±25.9 | 125.3±15.8 | 24.6±5.9 |
| Experimental | 429.3±46.3 | 125.3±36.4 | 365.9±24.3 | 64.2±3.4 |
|
t-test | 4.526 | 5.268 | 5.024 | 5.928 |
|
P-value | <0.001 | <0.001 | <0.001 | <0.001 |
Discussion
Clinical studies (9,10)
suggesting that an abnormal immune mechanism leading to excessive
activation of the organism inflammation/anti-inflammatory reaction
process can cause tissue damage or immune paralysis has become an
important field of investigation in sepsis research. A new
treatment concept of immunomodulation has emerged as a result.
Macrophages serve as antigens presenting in the body's immune
process, and a series of cytokines that macrophages produce further
regulate the body's immune reaction process. Therefore, the study
of macrophages and immune response may identify the deeper
mechanisms of the occurrence and development of sepsis, leading to
new treatments and concepts (11,12).
Function of abnormal immunologic
mechanism on sepsis
Anti-inflammatory media have been used to study
animals with sepsis, for example, classic TNF-α, with the result
showing that the anti-inflammatory medium treatment by TNF-α is
able to improve the prognosis of partial sepsis in animals
(13). At the same time, previous
findings showed (14) that besides
the classic inflammatory medium, IL-4, IL-10 and IL-13 improved
significantly patients with SIRS. Due to the findings of early
inflammation/anti-inflammatory medium research, it was found that
intervention by single regulation of inflammation/anti-inflammatory
cytokine did not correct perfectly the prognosis of sepsis. Since a
complicated intervention network of each other existed in
inflammation/anti-inflammatory cytokines, similar to the waterfall
release of inflammatory cytokines in SIRS, intervention at any
point of the inflammatory cytokine pathway may lead to
unpredictable and unmanageable alteration of the entire pathway.
This type of complex neuroendocrine network change in body
determines the treatment of sepsis and should not simply utilize
the regulation of inflammation/anti-inflammatory cytokine (15,16).
Function of macrophage active state on
sepsis
In sepsis, macrophage can phagocytose pathogenic
microorganisms directly, which breaks pathogenic microorganisms
through enzyme and bio-activator that by itself produced changes
and promotes the process of immune response in body through the
activator and its function of antigen presenting (17). Thus, macrophages are important immune
cells that constitute ‘a connecting link between the preceding and
the following’ (18).
A significant feature of macrophages is its obvious
heterogeneity, which can form different directions of
differentiation according to the micro-environmental state of its
tissue. In terms of the conditions of induced differentiation, the
original macrophage, also known as M0 macrophage, is formed by the
induced differentiation of the early macrophage colony-stimulating
factor, which can be divided into classically activated M1
macrophage, and alternatively activated M2 macrophage (19). Each has a different pathway of
activation, and a series of cytokines from activation induce an
immune response to change towards different directions (20). M1 macrophage, its production of Th1
immune response and other substances after its activation can
damage tissue. However, if the M1 macrophage and related Th1 immune
response are not regulated, it may lead to the amplification of
body inflammatory reaction with SIRS, severe sepsis, and MODS
(19). M2 macrophage can be
classified as a, b and c according to its function and mechanism,
of which, M2a macrophage is activated by IL-4 and IL-13 mainly with
the function of promoting wound healing through the generation of
extracellular matrix. Thus, IL-4 and IL-13 are the important
characteristic cytokines of Th2 immune response (21). As mentioned earlier, in various
inflammatory diseases of the body, M1/M2 macrophage can reflect a
balanced state of inflammation/anti-inflammatory reaction in the
body. M1 macrophage and inflammation reaction can remove the
pathogenic microorganism by producing various bioactive substances.
The M2 macrophage and anti-inflammatory reaction can promote tissue
repair and control the inflammatory response, avoiding to some
extent the effect of excessive damage (22). The M2 macrophages and
anti-inflammatory response promote tissue repair and control the
inflammatory response to a certain extent to avoid the effect of
excessive damage (23).
Effect of recombinant Trichinella
53-kDa protein on macrophages
Previous findings have shown (23) that, the enhancement of Th2 immune
response or even immunosuppression in patients and animals with
sepsis can alleviate the occurrence of certain autoimmune diseases.
In a study using chronic trichinization Dark Agouti (DA) rats, it
was found that experimental cerebrospinal meningitis was
alleviated, and Th2 immune response was activated in the model
(19). The secretory protein of
Trichinella can influence immunosuppression in vitro,
and rTsP53 protein is a type of secretory protein in
Trichinella that improves the prognosis of sepsis by
activating M2 macrophages in the body (21).
The results of the present study show that the
survival rate of rats in the experimental group was higher than
that of the control group at 6 and 12 h, and the difference was
statistically significant. The expression ratio of the macrophages
received from the different handling methods of the rats in the
experimental group was higher than that of the control group, and
the difference was statistically significant. The expression level
of the Th1-type cytokines IFN-γ and TNF-α of rats in the
experimental group was higher than that of the control group. By
contrast, the expression level of the Th2-type cytokines IL-4 and
IL-13 was higher than that of the control group, and the difference
was statistically significant. Therefore, recombinant
Trichinella 53-kDa protein can increase the survival rate
after the infection by CLP sepsis in rats, which may be associated
with the improvement of the macrophages and the adjustment of the
expression of the Th2 cytokines.
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