Acute lung injury (ALI) and its severe stage, acute respiratory distress syndrome (ARDS), are defined as acute pneumonia and tissue damaged disease. The clinical symptoms include acute hypoxemic respiratory failure, reduced pulmonary compliance, excessive pulmonary inflammation, pulmonary edema, and diffuse alveolar damage due to an imbalance of pulmonary gas exchange and blood flow [
The pathogenesis of ALI is very complicated as alveolar macrophages play a key role in the development of ALI. Alveolar macrophage is the most common nonparenchymal cells in lung tissue. Once activated by bacterial or viral infection, macrophages generate and release a large number of inflammatory cytokines and chemokines, at the same time, transport a large number of leukocytes to the lesion [
In this study, in vitro HPAEC model is used to study the effect of LPS on cell damage. Then a rat model of LPS-induced ALI was built and evaluated by blood gas analysis. Furthermore, we compared the inhibition efficiency between direct administration and polyaldehyde dextran-coated nanoparticles with antagonist RS102895, expecting to find a better approach to achieve high-efficiency inhibitor delivery with less extra injury. Finally, in this study, we discuss the underlying mechanism of MCP-1 in ALI rat model and provide new therapeutic ideas for the clinical treatment of ALI.
Logarithmic phase HPAEC cells in good conditions were digested and adjusted to a density of 1 × 105 cells per mL with culture media supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin and then were plated 1 mL per well into a 12-well plate. The cells were grown until confluence reached 70%–80% before the LPS treatment. The supernatant of culture media was removed, and the cells were washed once using Dulbecco’s phosphate-buffered saline (DPBS). Then cells were divided into control group and experimental groups: experimental groups were treated with different concentrations of LPS solutions (100 ng/mL, 500 ng/mL, and 1
After LPS interventions for 24 h, the supernatant was removed, and the cells were washed twice using DPBS. 1.1 mL CCK-8 working solutions (0.1 mL CCK : 1 mL culture medium) were then added to each well. Culture supernatants were collected 4 hours after incubation and added into a 96-well plate (100
The cells were harvested 24 h after LPS treatments, washed twice with binding buffer, and suspended in 500
Thirty SD rats were randomly divided into four groups: (1) normal saline control group (NC group) which rats received intraperitoneal injection of 2 mL/kg saline; (2) LPS group which rats received 10 mg/kg LPS by intraperitoneal injection; (3) antagonist RS102895 combined with LPS (R + L group) which rats received intraperitoneal injection of 2 mL/kg RS102895 and 10 mg/kg LPS injection 24 hours after the antagonist treatment; and (4) the antagonist RS102895-loaded polyaldehyde dextran nanoparticles combined with LPS (DNPR + L group) which rats received intraperitoneal injection with 2 mg/kg RS102895 formulated nanoparticle and LPS (10 mg/kg) injection 24 hours after nanoparticle injection. Rats were sacrificed 24 hours after each intervention, and samples were collected for further use.
Sodium periodate (2 g) was added to 200 mL of dextran (50 mg/mL dissolved in distilled water). After 1 hour, ethanediol was added to the mixture. Secure the mixture with the dialysis membrane bag and precipitate the distilled water for 3 days and dried for 24 hours after participation. The characteristics of polyaldehyde dextran (PAD) were tested by hydroxylamine hydrochloride method. Dried polyaldehyde dextran (1 g) was dissolved in distilled water (10 mL, 30°C) and mixed with 0.01 g/mL RS102895 solution. After stirred for 30 min, pH was adjusted to 7.4 with sodium hydroxide or hydrochloric acid. The mixture was set for dialysis for 30 min.
The drug encapsulation efficiency (EE) and loading efficiency (LE) were calculated to characterize the efficiency of NPs production. LE was defined as the ratio of the mass of RS102895 in NPs to the total mass of NPs, and EE was the percentage of the mass of RS102895 to the mass of the total used RS102895 for NPs preparation. The mass and purity of RS102895 in NPs were evaluated by HPLC (Malvern Instruments, UK). All measurements were performed in triplicate.
24 hours after LPS treatments, 0.5 mL blood was drawn from rat celiac artery by 1 mL syringe and used for blood gas analysis.
After scarifying the rats, the inferior lobe of the right lungs was excised, cleaned, and weighed to obtain wet weight (W). The lungs were then dried in 80°C oven until the weight stayed constant, and the lungs were weighed again to obtain dry weight (D). The W/D ratio was then calculated.
The left lung tissues of rats were taken, and bronchoalveolar lavage (BAL) samples were obtained by lavaging the lungs for 1 min for 3 cycles. The lavage fluid was pooled and kept at −20°C for further use. Enzyme-linked immunosorbent assay (ELISA) was performed according to the instructions of TNF-
The lung tissues of rats in each group were collected, excised, and washed three times with precooled PBS. The tissues were lysed and homogenized. The lysate of tissue was centrifuged, and the top clear supernatant was electrophoresed by SDS-PAGE. The protein strip was then transferred to the PVDF membrane and blocked with 5% skim milk. After blocking, the membrane was washed 5 times for 5 min each using 1X TBST buffer. MCP-1 antibody (1 : 1000 dilution) and
The mRNA expression of MCP-1 was measured by RT-PCR, and
SPSS 18 software was used for statistical analysis. Data were presented in mean ± standard deviation format. Between-group comparisons were carried out by one-way analysis of variance (One-way ANOVA). Multiple-group comparisons were conducted by
In this study, the LE of nanoparticle was 23.1 ± 7.1%. The EE of RS102895 nanoparticle was 40.4 ± 4.7%. The average size of RS102895 nanoparticle was found to be around 145 ± 32.9 nm.
Compared with the control group (0 ng/mL LPS), the HPAEC cell viability significantly decreased in response to the increased dose of LPS interventions. The dosage of LPS interventions was 100 ng/mL, 500 ng/ml, and 1
Effects of LPS doses on human pulmonary artery endothelial cell viability.
To further confirm the effect of LPS on cell viability, Annexin V-APC/7-AAD flow cytometry was used to detect cell apoptosis. The percentages of apoptosis were 37.10 ± 1.73 and 46.27 ± 1.55 after 100 ng/ml and 500 ng/ml LPS interventions (Figure
Effects of LPS doses on human pulmonary artery endothelial cell apoptosis. (a) Negative control (LPS 0 ng/mL); the rate of apoptosis of cells without LPS stimulating. (b) The rate of apoptosis of cells with LPS stimulating in 100 ng/mL. (c) The rate of apoptosis of cells with LPS stimulating in 500 ng/mL. (d) The rate of apoptosis of cells with LPS stimulating in 1
According to the blood gas analysis data, PaO2 and PaO2/FiO2 of LPS group were lower than those of NC group; the difference between the groups was statistically significant (
Effects of LPS and LPS/CCR2 antagonist interventions on PaO2, PaO2/FiO2, and wet/dry weight ratio in LPS-induce ALI rat models (
Group | PaO2 (mmHg) | PaO2/FiO2 (mmHg) | Wet/dry weight ratio |
---|---|---|---|
NC group | 104.30 ± 7.36 | 458.70 ± 12.05 | 4.05 ± 0.15 |
L group | 61.50 ± 7.18 |
261.30 ± 7.07 |
6.02 ± 0.44 |
R + L group | 83.30 ± 3.83 |
331.30 ± 9.87 |
5.02 ± 0.33 |
DNPR + L group | 95.78 ± 4.51# | 379.85 ± 8.19# | 4.47 ± 0.43# |
114.07 | 1027.386 | 90.662 | |
0.000 | 0.000 | 0.000 |
24 hours after LPS induced in ALI models, rats in group L were in poor physiological conditions with low activity and poor mental state, while the activities of rats in the NC group were normal. The lung wet/dry weight ratio of LPS group was significantly higher than that of the NC group (
The level of TNF-
Effects of LPS and LPS/CCR2 antagonist interventions on TNF-
Group | TNF- |
IL-1 |
---|---|---|
NC Group | 61.51 ± 6.22 | 45.38 ± 4.55 |
L Group | 440.34 ± 29.06 |
341.19 ± 27.51 |
R + L Group | 150.24 ± 25.55 |
101.08 ± 10.61 |
DNPR + L Group | 120 ± 21.43# | 86 ± 9.47# |
766.85 | 833.09 | |
0.000 | 0.000 |
The expression level of MCP-1 significantly increased in the LPS group compared to the control group (
Effects of LPS and LPS/CCR2 antagonist interventions on the expression level of MCP-1 by Western blot. The result of Western blot shows that under LPS stimulating the expression is upregulated, and direct administration of RS102895 can reduce the expression of MCP-1 to some extent but not to the condition without LPS stimulating. Nanoparticles can significantly reduce the MCP-1 expression.
The expression of MCP-1 mRNA in lung tissue was examined by real-time fluorescence quantitative PCR. Compared with the NC group, MCP-1 mRNA expression in LPS group increased by 9.13 ± 1.15 times, and the difference was statistically significant (
Effects of LPS and LPS/CCR2 antagonist interventions on MCP-1 mRNA expression by quantitative real-time PCR.
ALI is a disease characterized by acute pneumonia and tissue injury [
MCP-1, which belongs to the chemokine family, is a secretory protein that plays an important role in the development of inflammation [
However, our results also indicated that the protective effects of MCP-1/CCR2 blockage cannot counteract the effects of LPS challenge evidenced by significant differences between R + L group and NC group. The MCP-1/CCR2 pathway is only one of many pathways in systematic disease such as acute lung injury. LPS is recognized by toll-like receptors on the membrane of antigen-presenting cells, which triggers LPS/TLR/MyD88/IRAK/TRAF/NF-
In conclusion, the MCP-1/CCR2 signaling contributes to the pathogenesis of acute lung injury. In order to test whether MCP-1/CCR2 pathway could be a potential therapeutic target for ALI treatment and molecules interfering with MCP-1/CCR2 interaction could be studied and regarded as a promising ALI therapy, future clinical trials should be taken into consideration. And our method to deliver the inhibitor through self-synthesized nanoparticles may provide a more efficient way for drug delivery. Furthermore, aside from inhibition of MCP-1/CCR2 interaction, inhibition of TLR/MyD88/NF-
The data used to support the findings of this study are available from the corresponding author upon request.
Approval for the present study was obtained by the Ethics Committee of Shanghai Ninth People’s Hospital (Shanghai, China).
All subjects participating in the image acquisition signed the consent form.
We declare that we have no financial and personal relationships with other people or organizations.
Zheng Cao designed the study and performed the experiments. Qiao Wang analyzed the data. All authors read and approved the manuscript. Qiao Wang was responsible for study conception and design and revised the manuscript; Zheng Cao and Jing-Lan Liu performed the experiments and drafted the manuscript; and Zheng Cao and Shen Wu analyzed the data. All authors read and approved the final manuscript.
The article is supported by grant 51673190 from the National Natural Science Foundation of China, and the data and materials used to support the findings of this study are included in the published article.