Background
Acute respiratory distress syndrome (ARDS), induced by many pathogenic factors, such as pneumonia, sepsis, shock etc., is one of the most common causes being treated in ICU. It is characterized by respiratory distress and progressively refractory hypoxemia [
1‐
4]. Although protective ventilation, conservative fluid management, extracorporeal membrane oxygenation (ECMO) and some other supporting therapies improved its clinical outcome, the mortality of ARDS remains as high as 30–50% [
5]. Hypercoagulation and fibrinolysis inhibition in airspace is a critical pathophysiology [
6], which are the important reasons responsible for the high mortality of ARDS. Alveolar hypercoagulation and fibrinolysis inhibition contribute to microthrombus formation in pulmonary vessels and fibrin deposits in airspace, which are associated with imbalance of V/Q ratio, decreased lung compliance, diffusion disorder, etc., resulting in refractory hypoxemia and pulmonary fibrosis [
7,
8]. Our previous studies confirmed that NF-κB signaling pathway participated in the regulation of hypercoagulation and fibrinolysis inhibition in LPS-induced alveolar epithelial cell type II (ACEII) [9.10].
Nuclear factor kappa B (NF-κB) is a ubiquitous transcriptional factor participating in regulation of immune and inflammatory responses [
11]. The mammalian NF-κB family consists of p65, c-Rel, RelB, p50 and p52, which exist in the resting state as homodimers or heterodimers primarily bound to their inhibitory protein IκBs under physiological conditions, and p65 is the main transcriptional factor. Once NF-κB signaling pathway being activatied, IκBs is degraded by the IκB kinase complex (IKKBs), unmasking the nuclear localization sequence of NF-κB and allowing NF-κB dimer to translocate into nucleus, where NF-κB binds to the promoter and enhancer regions of its target genes containing κB sites, resulting in genes transcription [
12‐
14]. In previous experiments, we found that silencing NF-κB p65 gene or regulating IKKβ modulated the LPS-stimulated expressions of TF, PAI-1 and APC in alveolar epithelial cell type II (AECII) [
9,
10].
SN50, the NF-κB cell permeable inhibitory peptide, was first synthesized by Lin et al. in 1995 [
15]. It was comprised of the hydrophilic region of the signal peptide of Kaposi fibroblast growth factor as a membrane translocating motif and a nuclear localization sequence derived from the p50 subunit of NF-κB [
15]. Chian et al. showed that SN50 protected against LPS-induced lung injury in isolated rat lung by inhibiting NF-κB nuclear translocation [
16]. Based on that finding, we speculate that SN50 would correct alveolar coagulation and fibrinolysis abnormalities via NF-κB signaling pathway in ARDS. So we tested the effects and the mechanism of SN50 on alveolar hypercoagulation and fibrinolysis inhibition in LPS-induced mouse ARDS.
Materials and methods
Animal preparation
The study was performed in accordance with animal ethics guidelines of Guizhou Medical University. Briefly, male Balb/c mice, aged 8 to 12 weeks and weighing 20 ± 2 g, were obtained from the laboratory animal center at Guizhou Medical University. The whole experiment performed in this study was conformed to the Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee.
Experimental protocols
The mice were randomly divided into 6 experimental groups: control, N-SN50, LPS, L-SN50, M-SN50 and H-SN50 group, with 12 mice in each. Mice in control and LPS group received 100 μl of PBS intraperitoneal injection. Mice in N-SN50 and M-SN50 group received 100 μl of SN50 with concentration of 30 μg/ml, while mice in L-SN50 and H-SN50 group received same amount of SN50 with concentration of 10 μg/ml and 60 μg/ml respectively. An hour later of intraperitoneal injection of SN50, mice were anaesthetized with intraperitoneal injection of chloral hydrate and then were fixed on the operating table with supine position, followed by 50 μl of LPS (4 mg/ml) being sprayed into the trachea of mice in LPS, L-SN50, M-SN50, and in H-SN50 groups by using an aerosol endotracheal drug suit (Yuyan Instruments, Shanghai, No.YAN30012), but LPS was substituted by PBS in control and N-SN50 groups. Six hours later the mice were euthanized and lung tissue and BALF were collected under anesthetization.
BALF collection
After 6 h of LPS aspiration, a tracheotomy was performed and a catheter was inserted into the trachea. One milliliter of sterile PBS buffer was slowly injected into the trachea with a sterile syringe, and the PBS was suctioned out as much as possible 30 s later. PBS injection was repeated for three times. BALF were harvested and stored at − 80 °C for testing.
Haematoxylin and eosin staining
The right lower lobe of lung was embedded in paraffin and sagittally sliced at 5 μm. The sections were stained with haematoxylin and eosin. Oedema, alveolar and interstitial inflammation and haemorrhage, atelectasis, necrosis, and hyaline membrane formation were applied for lung injury scoring under microscope (0, no injury; 1, injury in 25% of the field; 2, injury in 50%; 3, injury in 75%; and 4, injury throughout the field). The total lung injury score was calculated as the sum of these scores. Ten randomly selected high-power fields (400×) in each slide were analyzed by two investigators who were blinded to the mouse groups.
Evaluation of the lung oedema
Briefly, the whole lung was removed and cleared of all extra pulmonary tissues and the wet weight was recorded. Then the lung tissue was placed in a clean container and baked in a constant temperature oven at 65 °C for 72 h until the weight didn’t change any more, and recorded the dry weight.
Western immunoblot analysis
Total protein lysates were extracted from lung tissues using RIPA lyses buffer. The lung tissues were homogenized in PBS containing the protease inhibitor cocktail. The homogenates were centrifuged at 14, 000 rpm in 4 °C for 15 min. Supernatants of lung tissues were collected and protein concentration was measured using BCA protein assay kit (Solarbio Life Sciences, Beijing). An equal amount of protein from each sample was resolved in 10% Tris-glycine SDS polyacrylamide gel. Protein band was blotted to nitrocellulose membrane. After incubation for 1 h in blocking solution at room temperature, the membrane was incubated for 24 h with anti-TF (1:500 Santa Cruz Biotechnology, Inc.), anti-PAI-1 (1:500 Santa Cruz Biotechnology, Inc.), anti-β-actin (1:1000 Santa Cruz Biotechnology, Inc.), anti-p-IKKα/β (1:800 Santa Cruz Biotechnology, Inc.), anti- p-IκBα (1:800 Santa Cruz Biotechnology, Inc.), anti-p65 (1:800 Santa Cruz Biotechnology, Inc.), anti- p-p65(1:800 Santa Cruz Biotechnology, Inc.), at 4 °C. The secondary antibody (horseradish peroxidase-conjugated donkey anti-rabbit immunoglobulin) was added at 1:10,000 dilution and incubated at room temperature for 1 h. Peroxidase labeling was detected with the enhanced chemiluminescence Western blotting detection system (Amersham Pharmacia Biotech) and analyzed by a densitometry system. The relative protein level was normalized to β-actin (Abcam Biotechnology, Inc.).
Quantitative real time RT-PCR analysis
Total RNA was isolated using an RNeasy Mini kit including DNase I digestion enzyme. Following reverse transcription, quantitative real-time PCR analysis was performed using the ABI ViiATM 7 real-time PCR system (Thermo Fisher Scientific) with SYBR Green master mix. The primers used for analysis were synthesized by Shanghai Sangon Biotech (Table
1). We performed PCR amplification with cDNA being used as template. The reaction system was set as follows: SYBR Green Mix 10 μl, forward primer and reverse primer 0.8 μl respectively, cDNA template 0.8 μl, ddH2O 7.6 μl, which were synthesized into a system containing 20 μl reagents. Dissolution and amplification curve of the gene were recorded following the gene amplification. Expressions of target genes were calculated using the 2-ΔΔCt method.
Table 1Primer sequences of each gene
TF | AATGGGCAGATAGAGTGT | ACCGCATTCAAGTCA | 182 |
PAI-1 | ACCAACTTCGGAGTAAAA | TTGAATCCCATAGCATCT | 158 |
ELISA assay
TF, PAI-1, APC and TAT levels in BALF were determined by using ELISA kits (Jiyinmei Bio-company, Wuhan, China) according to the manufacturer’s instructions.
NF-κB p65 DNA binding activity assay
For detecting NF-κB p65 DNA binding activity, TransAMTM NF-κB p65 Chemi Transcription Factor Assay Kit was used (Active Motif, Carlsbad, CA).
Immunohistochemistry
Lung slides were deparaffinized and rehydrated in xylene and ethanol. After antigen retrieval, the lung slides were treated with 0.3% H2O2 in methanol for 30 min and subsequently incubated with blocking solution (5% goat serum) for 20 min at RT. The lung slides were incubated with anti-collagen III (1:200) for 90 min at RT. After washing three times in PBS, the lung slides were incubated with secondary antibody (1:500) labelled with polymer and horseradish peroxidase (Solarbio Life Sciences, Beijing) for 30 min at RT. The immunoreactions were visualized using a diaminobenzidine substrate kit (Dako, Carpinteria, CA). The lung slides were mounted with malinol (Muto Pure Chemicals, Tokyo, Japan). Signals were classified into 4 grades of intensity as follows: (−) negative; (+) weakly positive, (++) moderately positive, and (+++) strongly positive.
Statistical analysis
Values are represented as mean ± SEM of at six independent experiments. Statistical analysis was performed using SPSS 24.0 (IBM Corporation, Armonk, NY, USA). ANOVA and independent Student’s t-tests were used to analyze the differences between the different groups. p < 0.05 was considered statistically significant.
Discussion
Our results showed that LPS-induced lung injury experienced pulmonary edema, lung tissue deconstruction, high level pulmonary expressions of TF and PAI-1, large amount of secretions of TF, PAI-1, TAT and decreased APC production in air space, and a high content of pulmonary fiber (high collagen III), all which indicated alveolar hypercoagulaiton and fibrinolysis inhibition. Meanwhile, NF-κB pathway was activated with an increased p65 translocation from cytoplasm to nucleus and enhanced p65 DNA binding activity. Our results were consistent with published data by Chian et al. [
16]. SN50 treatment not only down-regualted TF and PAI-1 expressions in pulmonary tissue, but also inhibited secretions of TF, PAI-1 and TAT and decreased pulmonary collagen III level, and promoted APC production in BALF, demonstrating that SN50 effectively corrected hypercoagulation and fibrinolysis inhibition in LPS-induced ARDS. In exploring the mechanism of SN50, we found that SN50 decreased p65 level in nucleus and decreased p65 DNA binding activity but with no influence on p-IKKα/β, p-IκBα and p-p65 in lung tissue. Therefore, we have the reason to think that SN50 corrected hypercoagulation and fibrinolysis inhibition by preventing p65 translocation from cytoplasm into nuleus and inhibiting p65 DNA binding capacity rather than other mechanisms. Our data showed that the changes of TF, PAI-1, and of some other procoagulant factors and fibrinolytic inhibitors in BALF, as well as of lung injuries and pulmonary edema did not reach the baseline in SN50- treated mice, which implies that SN 50 partially attenuated but not abrogated LPS-induced ARDS.
SN50 is a cell-permeable peptide which can specifically inhibit the NF-κB p65 translocation [
17,
18]. Previous studies have demonstrated that it can inhibit inflammatory cells infiltration and protect rat lung against LPS induced-lung injury, attenuating traumatic brain injury in mice [
16,
19]. As far as we know, this is the first to explore the effect of SN50 on hypercoagulation and fibrinolysis inhibition induced by LPS.
NF-κB activation could be either by canonical or noncanonical pathways. Studies demonstrated that there were several methods to inhibit NF-κB pathway, including inhibition of IκB kinase complex [
9,
10,
20], decrease of IκB protein degradation [
21], etc. In our study, we noticed that SN50 treatment resulted in reduction of translocation of p65 from cytoplasm to nucleus and attenuation of p65 DNA binding activity, while p-IKKα/β, p-p65, p-Iκα kept unchanged, indicating that SN50 specifically inhibited p65 translocation from cytoplasm to nucleus and decreased p65 DNA binding activity. Experiments in vivo and in vitro have confirmed that NF-κB signal pathway was involved in many pathologic process such as anti-inflammatory response in sepsis [
22‐
24], downregulation of cytokines and MAPK activation in LPS-induced lung injury [
16]. Our previous studies confirmed NF-κB p65 also participated in regulation of LPS-induced hyperexpressions of procoagulant factor TF and fibrinolysis inhibitor PAI-1 in alveolar epithelial cell type ‖, demonstrating it was associated with alveolar hypercoagulation and fibrinolysis inhibition in ARDS. In present animal study, our data demonstrated SN50 effectively down-regulated TF and PAI-1 expressions in lung tissue, decreased concentrations of procoagulants (TF, PAI-1, TAT) and promoted activated protein C (APC) in BALF, and attenuated collagen III expression in pulmonary tissue, suggesting that SN50 is expected to be an effective target to attenuate hypercoagulation and fibrinolysis inhibition in ARDS.
Our data indicated that NF-κB was involved in regulation of hypercoagulation and fibrinolysis inhibition in ARDS. So it is pivotal to effectively block the NF-κB pathway, such that correct the abnormalities of coagulation and fibrinolysis in airspace in ARDS. As mentioned above, NF-κB pathway could be activated either by canonical or by noncanonical pathway, but whether it is canonical or noncanonical pathway, the translocation process of p65 from cytoplasm to nucleus is the common way through which NF-κB exerts its transcriptional role [
12,
25]. Our results showed that SN50 treatment induced a decrease NF-κB p65 nuclear content in lung tissue and an attenuation in p65 DNA binding activity, indicating the potential protective valuability of SN50. Our results also demonstrated that the efficacies of SN50 on alveolar hypercoagulation and fibrinolysis inhibition and the lung protection role were related with the dosage of SN50, in that in a certain range, with the increase of dose, the effect of SN50 becomes more obvious., But whether there is a more optimal dose point of SN50 or not is worth further study.
There are some limitations in our experiment. First, there was no arterial blood gas analysis making the diagnosis of ARDS insufficient. Second, we took different doses of SN50 for pretreatment in LPS-induced mice just according to some previous published article [
16,
26‐
28], but the optimal dose of SN50 to attenuate hypercoagulation and fibrinolysis inhibition remains to be elucidated, and so did for the optimal timing of SN50 administration. However, NF-κB was an important therapeutic target for the alveolar hypercoagulation and fibrinolysis inhibition and SN50 has been proven to effectively attenuate these abnormalities.
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