Background
Propofol is used for induction and maintenance of general anesthesia. It is often used with epidural or spinal anesthesia, and is often used with analgesics, muscle relaxants and inhalation anesthetics. Previous studies have used the model of ischemia-reperfusion injury to explore the inflammatory process and found that propofol exhibited antioxidant and anti-inflammatory effects [
1‐
5]. Lipopolysaccharide (LPS) is the major cause of clinical fever, it is also the key factor that cause the shock, sepsis, cholera, and other diseases like adult respiratory syndrome, multiple organ failure, or the pathological processes [
6]. Gram-negative bacteria are often used to induce inflammation in vitro, and studies have shown that LPS is a major component of the outer membrane of Gram-negative bacteria [
6]. Studies have confirmed that high-mobility group box 1 (HMGB1) is a DNA-binding protein that maintains nucleosomal structures and regulates gene transcription, which is characterized by highly conserved proteins. Recent studies have suggested that it exhibits a pronounced proinflammatory effect in the inflammatory response [
7], which is involved in the process of development of sepsis, as well as the important inflammatory mediators for the late period in the lethal effects of LPS [
8]. HMGB1 appears to activate macrophages leading to the secretion of multiple cytokines, which in turn cause extensive inflammatory responses, including intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and nitric oxide [
9].
Studies have found that propofol can block endotoxin-induced monocyte-macrophages to produce various inflammatory factors [
3,
5,
9]. LPS stimulation can induce HMGB1 release from mouse macrophages, however, propofol can inhibit this process leading to downregulation of HMGB1 mRNA and also block the activation of nuclear transcription factor-κB (NF-κB) [
10]. In addition, the propofol can inhibit the expression of toll-like receptor 4 (TLR-4) and NF-κB to block the activation of p38 mitogen-activated protein kinase and the expression of pro-inflammatory cytokines [
4]. However, the actions of propofol to regulate the expressions of interleukin-6 (IL-6), 8 (IL-8) and TNF-α and the correlation with HMGB1 in LPS-stimulated murine macrophage are unclear. Based on the importance of HMGB1 in immunoreactions and the potential relationship between HMGB1 and LPS, the aim of our study is to disclose whether LPS stimulation can lead to RAW 264.7 cells (murine macrophage) secreting more HMGB1, IL-6, IL-8 and TNF-α. However, the addition of propofol can down-regulate the expression of LPS-stimulated HMGB1, IL-6, IL-8 and TNF-α. And the levels of IL-6, IL-8 and TNF-α seems to correlate with the expression of HMGB1 in this process.
Methods
Preparation and culture of cells
Mouse monocyte/macrophage leukemia RAW264.7 cells were purchased from Shanghai Cell Biology cell bank. RPMI-1640 and fetal bovine serum (fetal bovine serum, FBS) were purchased from Gibco BRL Company. LPS was purchased from the United States (Escherichia coli O11: B4) Sigma Corporation. The isolated RAW264.7 cells were collected into the centrifuge tube. The cells were centrifuged for 10 min at 1000 r/min. The supernatants was then discarded and the cells were pooled with RPMI 1640 medium containing 20% FBS with 1% sodium pyruvate and 1% streptomycin. After the cell density was adjusted, the cells were cultured at 37 °C under 5% CO2. The culture medium was replaced by 2–3 days according to conditional of cell growth.
Grouping of cell experiments
Three days prior to the experiment, the cells were seeded into a six-well plate and replaced with serum-free medium 6 h prior to the experiment and then grouped. The experimental cells were divided into four groups, which are showed as follows: (1) Blank control group (without any intervention); (2) LPS intervention group, plus 250 ng/mL LPS; (3) Low-dose group of propofol treatment, plus 250 ng/mL LPS and 25 μmol/L propofol; (4) High-dose group of propofol treatment, plus 250 ng/mL LPS and 50 μmol/L propofol. Each group of cells was cultured at 37 °C under 5% CO2 and cells were collected after 16 h culturing (the same culture conditions).
Western blotting
The cells in the logarithmic growth phase were harvested and washed once with phosphate phosphate saline (PBS), and the total protein was extracted according to the method described in the protein extraction kit. The extracted protein was boiled for 5 min with 2× sodium dodecyl sulfate (SDS) -polyacrylamide gel electrophoresis (PAGE) sample buffer and 20% glycerol. Each protein sample was then assayed for concentration with Micro-BCA protein. The protein samples were gel electrophected with 12% SDS-PAGE and 30 μg of total cell protein was added to each lane. Proteins were transferred to the polyvinylidene difluoride membrane (PVDF membrane) by electrophoresis. The PVDF membrane was then blocked at 4 °C with TBST buffer (pH 7.4, TBS added 0.1% Tween-20) containing 5% skimmed milk powder. PVDF membranes were incubated with anti-HMGB1 antibody (1: 500 dilution) (product code: BM3965; Boster Biological Technology co.ltd, Wuhan, China) for 1 h at 37 °C. We used β-actin as the internal control. PVDF membranes were rinsed three times with TBST buffer and incubated with HRP-conjugated secondary antibody (1: 1000) at room temperature for 1 h. Positive signals were visualized using the enhanced chemiluminescence method. Quantitative expression of protein and expression images were detected and collected through the Kodak IS2000R multifunctional image workstation.
Enzyme-linked immunosorbent assay (ELISA)
According to the previous grouping method, the RAW264.7 cell suspension was added to the 6-well plate, 1 mL per well, and 6 wells in each group. The cells were incubated at 37 °C under 5% CO2. After 16 h of culturing, the supernatants were collected for further experiments. The levels of IL-6 (product code: 70-EK2062/2, Lianke Biotech Co., Ltd. Shenzhen, China), IL-8 (product code: ZK-M4810, Ziker Biotech Co., Ltd. Shenzhen, China) and TNF-α (product code: B-21672, Dice Biotech Co., Ltd. Hangzhou, China) in the supernatants of cells were measured by ELISA according to the experimental method provided by the reagent manufacturer. The concentrations of IL-6, IL-8 and TNF-α were calculated based on standard curves provided with the kits, and the results of TNF-α and IL-8 were expressed in ng/mL, and IL-6 in pg/mL.
Statistical analysis
The mean ± standard deviation (M ± SD) is calculated for the continuity variable data and the relevant statistical analysis is performed. The comparison for different expression levels of HMGB1, TNF-α, IL-6 and IL-8 in different experimental groups was calculated by the Student’s T-test (matched samples), One-WAY ANOVA (single factor analysis) and non-parametric rank correlation analysis according to different data characteristics. The statistical analysis was finished with SPSS 19.0 (SPSS, Chicago, USA) software. All tests of statistical significance were two-sided, and a p-value <0.05 was considered as statistically significant.
Discussion
Macrophages are considered to constitute an important component of immune defense. It is generally believed that when the body is inflamed, macrophages are activated and release a variety of cytokines and inflammatory mediators involved in the inflammatory response process. Lipopolysaccharide (LPS) has been recognized as one of the most potent inducers of inflammatory response [
11]. Murine macrophage RAW 264.7 cells are an example of inflammatory cells [
12]. Studies have shown that it can produce the corresponding cytokines and inflammatory mediators, such as TNF-α, IL-1β and IL-6 and so on as long as the mouse macrophage RAW264.7 cells are stimulated by LPS [
13]. Propofol is widely used in the induction and maintenance of general anesthesia, and recent studies have shown that propofol can down-regulate the expression levels of HMGB1 messenger ribonucleic acid and protein [
10]. Our study found that the expression of HMGB1 in RAW 264.7 cells in LPS intervention group was significantly increased, as compared with the blank control group after RAW 264.7 cells were stimulated by LPS for 16 h. Furthermore, compared with the blank control group, the expressions of HMGB1 in low-dose group of propofol and the high-dose group were also up-regulated. The results suggested that LPS stimulation increased the expression of HMGB1. Previous studies have found HMGB1 as an important inflammatory mediator and proinflammatory cytokine, which links in response networks of sepsis proinflammatory cytokine [
10,
14‐
17]. LPS and various cytokines can stimulate the release of HMGB1, while HMGB1 can stimulates the synthesis of pro-inflammatory cytokines [
8]. This proinflammatory functions and features of HMGB1 seem to be more useful in potential clinical application value than some of early inflammatory mediators. A series of basic studies have shown that HMGB1 is closely related to the inflammatory response, which mediates the release of multiple inflammatory mediators and proinflammatory factors and is therefore considered to be a potential therapeutic target in the sepsis study model [
7,
15‐
17].
Since propofol is a short-acting intravenous anesthetic, it has the advantage of rapid induction and rapid resuscitation and is often used in a variety of animal experiments and surgical procedures [
18]. As a new intravenous anesthetic, propofol may play a protective role in the body through the influence to cytokines produced by endotoxemia [
10]. In our study, we analyzed whether the addition of propofol affect LPS-stimulated HMGB1 expression. We found that the expression of HMGB1 in propofol low dose group and high dose group were all significantly reduced and showed a concentration-dependent pattern, compared with the LPS intervention group. The results showed that propofol down-regulated the expression of LPS-stimulated HMGB1. A large number of laboratory studies have shown that as an oxygen free radical scavenger, propofol can inhibit lipid peroxidation, regulate antioxidant enzyme system, increase the antioxidant capacity of tissues and cells [
1,
5,
15,
19]. Therapeutic concentrations of propofol also reduces nitrogen-containing compounds in mice induced monocyte-macrophage apoptosis and death [
2]. Our study illustrated that propofol can effectively inhibit LPS-stimulated HMGB1 expression in RAW 264.7 cells, which indicating that propofol has an efficacy of anti-inflammatory. Studies have shown that HMGB1 can induce the synthesis and the secretion of inflammatory mediators in monocytes/macrophages, neutrophils and dendritic cells, these mediators can also strengthen the effect of HMGB1 secretion, forming a complex regulating secretion network of cytokine [
7,
8,
16,
20,
21].
We also found that LPS stimulation increased the release of IL-6, IL-8 and TNF-α in RAW 264.7 cells. The study found that IL-6 has the effect of promoting inflammation, which is closely related to arthritis, tumor and obesity-related diseases. In addition, IL-8 is also involved in inflammatory response and plays an important role in tissue degradation [
22]. Study shows that LPS is the main component of the outer wall of Gram-negative bacteria, which activates the nuclear transcription factor NF-κB through TLR-4 on the surface of macrophages to induce the release of a large number of inflammatory mediators and cells factors, which includes IL-6, IL-8 and TNF-α [
23]. Our study showed an interesting phenomenon that the addition of propofol affects the release of LPS-stimulated IL-6, IL-8 and TNF-α. We found that either in propofol low-dose group or high-dose group, the levels of IL-6, IL-8 and TNF-α all significantly down-regulated, as compared with LPS intervention group (without propofol). The results suggested that propofol could effectively suppress LPS stimulated-RAW 264.7 cells to release IL-6, IL-8 and TNF-α, which indicated that it may play an important role of anti-inflammatory response. It has been suggested that in vitro propofol can block LPS-stimulated PBMC cells to produce IL-6 and IL-10 [
24]. In addition to anesthetic effects, propofol seems to reduce the production and release of TNF-α [
24]. In general, LPS can induce RAW 264.7 cells to IL-6, IL-8 and TNF-α, but propofol can inhibit the effect, which indicates that propofol may play a protective role in the development and progression of inflammation.
To determine whether the release of IL-6, IL-8 and TNF-α correlated with the HMGB1 expression in the course of LPS and propofol intervention, we performed a series of correlation analysis. We found that the expression of HMGB1 and the releases of IL-6, IL-8 and TNF-α in RAW 264.7 cells displayed a close linear correlation either with LPS stimulation or by addition of propofol. Studies show that adding HMGB1 in cultured human peripheral blood mononuclear cells can promote the expressions of TNF-α, IL-1, IL-6, IL-8 [
7,
15,
16]. Recent experiment has shown that HMGB1 is an important inflammatory mediator in the regulation of hepatic ischemia-reperfusion injury and plays an important role in the early stages of tissue injury [
17]. In addition, propofol can inhibit the production and release of HMGB1 in mouse macrophages stimulated by LPS via downregulating the expression of HMGB1 mRNA, and it also inhibit the activity of NF-κB [
10]. Our results suggest that LPS can lead to the release of IL-6, IL-8 and TNF-α and thus may promote the inflammatory process. However, propofol has a significant inhibitory effect on IL-6, IL-8 and TNF-α in LPS stimulated RAW 264.7 cells. And propofol also significantly inhibited the expression of HMGB1 in LPS stimulated RAW 264.7 cells. More importantly, the release of IL-6, IL-8 and TNF-α intimately correlates with the expression of HMGB1. The results suggest that in addition to anesthetic effects, propofol may prevent systemic inflammatory responses, inhibiting the infection or non-infectious factors through reducing the release of some cytokines and inflammatory mediators, which may have a protective effect on the body’s emergency status.