Contributed equally
Inflammatory responses are vital in lung injury diseases, particularly acute respiratory distress syndrome (ARDS). Recombinant human brain natriuretic peptide (rhBNP) has been shown to exhibit anti-inflammatory effects
Acute respiratory distress syndrome (ARDS) is caused by a variety of insults, such as sepsis, major trauma, pneumonia, transfusions and aspiration of gastric contents. It is associated with a high rate of mortality according to the most recent report of the ARDS Network clinical trials (
Brain natriuretic peptide (BNP) is a member of the atria natriuretic peptide (ANP) family, and was first isolated from porcine brains (
rhBNP was obtained from Nuodikang Biological Pharmaceutical Company Ltd. (Chengdu, China). LPS and fetal calf serum (FCS) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Tissue culture supplements and medium were purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). All other materials and antibodies were purchased from Takara Biotechnology Co., Ltd. (Dalian, China). Experiments were conducted according to the National Institutes of Health (NIH) guidelines and approved by the ethics committee of the General Hospital of Shenyang Military District (Shenyang, China).
HFL-1 cells were obtained from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). The cells were cultured in 100-mm tissue culture dishes with Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FCS, 50 mg/ml penicillin, 50 mg/ml streptomycin, and 0.25 mg/ml Fungizone. The cells were passaged every 3 to 5 days. Fibroblasts used in these studies were between cell passages 10 and 20.
Cells were divided into the following groups: Control group, rhBNP (0.1
Following treatment, LDH release in the medium was measured using a previously described method (
The level of IL-1β secretion in culture media was determined by an enzyme-linked immunosorbent assay kit (R&D Systems, Inc., Minneapolis, MN, USA) according to the manufacturer's instructions. Concentrations were calculated with reference to the standard curve.
Total RNA extraction was isolated using TRIzol Reagent (Invitrogen; Thermo Fischer Scientific, Inc.) according to the manufacturer's instructions. An RNA denaturation mix consisting of isolated RNA, nuclease-free water and oligo dT primers was used. cDNA was synthesized using a reverse transcription kit (TransGen Biotech, Inc., Beijing, China) according to the manufacturer's instructions. Following reverse transcription, quantitative analysis of the IL-1β mRNA expression was analyzed by the real-time polymerase chain reaction (PCR) method. The following primers (Takara Biotechnology Co., Ltd.) were used: Forward, 5′-ATGCCTCGTGCTGTCTGACC-3′ and reverse, 5′-CCATCTTTAGGAAGACACGGGTT-3′ for IL-1β; and forward, 5′-ATGTGCCGGACCTTGGAAG-3′ and reverse, 5′-CCTCGGGTTAGCTGAGAGATCA-3′ for glyceraldehyde 3-phosphate dehydrogenase (GAPDH). PCR assays were performed in duplicate on the 7500 real-time PCR machine (Applied Biosystems; Thermo Fisher Scientific, Inc.). The cycling conditions were as follows: Incubation for 2 min at 50°C followed by another incubation step at 95°C for 10 min, 15 sec at 95°C and 1 min at 60°C for 40 cycles. Reaction specificity was evaluated by melting curve analysis, which was performed by heating the plate from 55°C to 95°C and measuring SYBR Green I (Takara Biotechnology Co., Ltd.) dissociation from the amplicons. The calculation of quantification cycles (Cq values) and further analysis of these data were performed by the Sequence Detector software (version 1.2.3; Syngene, Cambridge, UK). The relative expression of mRNA in each sample was quantified and normalized to the GAPDH mRNA levels by the 2−ΔΔCq method (
HFL-1 cells were collected in lysis buffer and centrifuged at 1,500 × g for 5 min at 5°C. Total cellular protein in the supernatant was determined using the bicinchoninic acid (Takara Biotechnology Co., Ltd.) method. Equal quantities of protein (40
All quantitative data are expressed as the mean ± standard error of the mean. Numerical data were performed using one-way analysis of variance with Tukey's post hoc test. Statistical analysis was performed using SPSS (version 15; SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference. Each experiment was performed at least three times.
To evaluate LPS-induced cell injury, the release of LDH into the medium was measured. The LDH leakage was increased in a time-dependent manner following treatment with LPS (P<0.05 vs. control,
Compared with the control group, the level of IL-1β in the culture medium was significantly increased after 24 h exposure to LPS (P<0.05). As shown in
Protein expression of key molecules in the intracellular mitogen-activated protein kinases signaling pathways, p-p38, p-JNK, and p-ERK1/2 were detected using western blot analysis. The results showed that the expression of p-p38, p-JNK and p-ERK1/2 was significantly increased in the LPS group (P<0.05 vs. control), whereas treatment with rhBNP significantly suppressed the expression of p-p38, p-JNK and p-ERK1/2 when compared with the LPS group (P<0.05,
The activity of the molecules in the NF-κB signaling pathway was determined by measuring the levels of the NF-κB p65 and p-IκB proteins in the cellular extract of HFL-1 cells. The expression of NF-κB p65 and p-IκB were increased significantly in the LPS group (P<0.05 vs. control); treatment with rhBNP significantly inhibited the LPS-induced increase of NF-κB p65 and p-IκB (P<0.05 vs. LPS); furthermore, pretreatment with the JNK inhibitor SP600125 attenuated the increase of NF-κB p65 and p-IκB (P<0.05 vs. LPS) (
Our previous studies have demonstrated that rhBNP prevents LPS-induced acute lung injury in a dog model and may be associated with adjusting the levels of endogenous antioxidant enzymes (
It is generally known that fibroblasts are important in inflammation resolution following cell injury (
BNP is a type of neuroendocrine hormone and can dilate blood vessels selectively, and aid sodium and urine excretion. RhBNP is a freeze-dried peptide produced by genetic recombination. The amino acid sequence of rhBNP is the same as endogenous BNP obtained from humans (
MAPK is vital in cell growth, differentiation, proliferation and apoptosis as an essential component of signal transduction (
NF-κB is primarily found in the cytoplasm in an inactive non-DNA-binding form that is associated with the inhibitor protein IκB in unstimulated cells. Following simulation, NF-κB translocates into the nucleus and regulates the transcription of genes, including those coding for the inflammatory molecules (
In conclusion, the results of this study demonstrated that rhBNP could inhibit HFL-1 cell injury induced by LPS by inhibiting the MAPK and NF-κB signaling pathways. These findings indicate that rhBNP protects HFL-1 cell injury in response to endotoxin insult, and that rhBNP may be used not only as a diagnostic or prognostic biomarker in critical care units, but also as a novel therapeutic agent to ameliorate lung injury.
This study was supported by the Postdoctoral Science Foundation, China (grant no. 2014M552693), and Science and Technology Project of Liaoning, China (grant no. 2013225089) awarded to Dr Zhi Song.
Effects of rhBNP on LPS-induced cell injury in HFL-1 cells. Cell injury was measured by a lactate dehydrogenase release assay. (A) Cell injury was determined at different time points following treatment with LPS. (B) rhBNP at the indicated concentrations was applied to HFL-1 cells 30 min prior to treatment with LPS for 24 h. *P<0.05 vs. control, #P<0.05 vs. the LPS group (0
Effects of LPS, rhBNP and mitogen-activated protein kinase pathway inhibitors on secretion of IL-1β in HFL-1 cells. (A) IL-1β protein levels in the culture media of HFL-1 cells with or without treatment with LPS, rhBNP, p38 inhibitor (SB203580, 20
Effects of rhBNP and LPS on phosphorylation of p38, JNK and ERK1/2 in HFL-1. (A) Expression of p-p38, p38, p-JNK, JNK, p-ERK1/2 and ERK1/2 was detected by western blot analysis. Immunostaining of p38, JNK, and ERK1/2 served as respective controls. (B) Bar graphs represents semi-quantitative densitometry from western blot analysis. *P<0.05 vs. the control group, #P<0.05 vs. the LPS group. rhBNP, recombinant human brain natriuretic peptide; LPS, lipopolysaccharide; LDH, lactate dehydrogenase; p-, phosphorylate; JNK, c-Jun NH2-terminal kinase; ERK1/2 extracellular signal-regulated kinase 1/2.
Effects of LPS, rhBNP and c-Jun NH2-terminal kinase inhibitor (SP600125) on phosphorylation of proteins in the intracellular NF-κB signaling pathway. (A) Expression of NF-κB p65 and p-IκB were analyzed by western blot analysis, and β-actin was used as a control. (B) Bar graphs represent semi-quantitative densitometry from western blot analysis. *P<0.05 vs. the control group, #P<0.05 vs. the LPS group. rhBNP, recombinant human brain natriuretic peptide; LPS, lipopolysaccharide; LDH, lactate dehydrogenase; NF-κB, nuclear factor-κB; p-, phosphorylated.