The aim of this study was to explore the role of astragalus polysaccharide (APS) in the pathogenesis of bronchopulmonary dysplasia (BPD) in preterm children using an established BPD cell model. EA.hy926 cell cultures were divided into three groups: the air group as the blank control, the hyperoxia group as the experimental control and the APS group (2.5 mg/ml). The production of superoxide dismutase (SOD), malondialdehyde (MDA) and reactive oxygen species (ROS) were analyzed by biochemical assays. Real-time reverse transcription-polymerase chain reaction (RT-PCR) and western blotting were used to detect the RNA and protein expression levels of inflammatory cytokines, including interleukin (IL)-8, intercellular adhesion molecule 1 (ICAM-1) and nuclear factor (NF)-κB p65. Compared with the hyperoxia group, the ROS and MDA levels of the APS group were significantly reduced. By contrast, SOD production was significantly increased. The expression of IL-8, ICAM-1 and NF-κB p65 in the APS group was downregulated. APS acts as an antioxidant by stimulating SOD production while inhibiting lipid peroxidation in the EA.hy926 cells. Furthermore, this study demonstrated that APS retards the inflammatory response, as shown by the reduced expression of NF-κB p65, IL-8 and ICAM-1 when APS was added.
Bronchopulmonary dysplasia (BPD) is the most common complication of preterm children who accept long-term mechanical ventilation or oxygen therapy. BPD seriously affects the survival of premature children and quality of life (
Astragalus polysaccharide (APS) is a traditional Chinese medicine that has been studied in depth and is widely used in clinics. APS is one of the most important natural active materials due to its multi-targeting biological activities, including antioxidant, radical scavenging and anti-inflammatory activity and immune regulation (
The EA.hy926 cell line is an immortalized human umbilical vein endothelial cell (HUVEC) line, derived from the fusion of HUVECs and lung adenocarcinoma cells. The structural characteristics and functions are similar to lung adenocarcinoma cells and these alveolar endothelial cells are used to construct the alveoli
In the current study, we aimed to explore the role of APS in preterm children with BPD and its mechanism of action by establishing an
EA.hy926 cells were commercially available from the Shanghai Institute Cell Bank (Shanghai, China). Fetal bovine serum (FBS; Cat# 16000-044) and high glucose Dulbecco’s modified Eagle medium (DMEM; Cat# C11995) were purchased from Gibco (Carlsbad, CA, USA). APS was purchased from Tianjin Cinorch Pharmaceutical Co., Ltd. (Tianjin, China). The triple gas mixture (70% O2, 5% CO2 and 25% N2) was provided by Nanfang Hospital Oxygen Center (Guangzhou, China). The SOD and MDA test kits were provided by Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The ROS kit (C1300) was purchased from Beijing Applygen Technologies Inc. (Beijing, China). Anti-intercellular adhesion molecule 1 (ICAM-1; Cat# 3518-1), anti-interleukin (IL)-8 (Cat# 3482-1) and anti-NF-κB p65 (Cat# 2229-1) were purchased from Epitomics Inc. (Burlingame, CA, USA).
EA.hy926 cells were cultured in high glucose DMEM containing 10% FBS at 5% CO2 in a 37°C incubator. Then, EA.hy926 cells were divided into three groups: the air group, the hyperoxia group and the APS group (2.5 mg/ml). The air group were cultured for 24, 36 and 48 h in a 5% CO2, 37°C incubator. The hyperoxia group and the APS group were cultured for 24, 36 and 48 h in the 37°C triple gas mixture.
Following removal of the culture medium, the cells were washed twice with phosphate-buffered saline (PBS). Then, 1 ml H2O2 (100
Cells were sonicated by ultra-sound and mixed according to the manufacturer’s instructions. The SOD mixture was incubated in water at 37°C for 40 min and then cooled to room temperature for 10 min after adding chromogenic reagent. Absorbance at a wavelength of 550 nm was measured by the MD5 microplate reader. The MDA mixture was boiled for 80 min and then cooled by flowing water. Absorbance at a wavelength of 532 nm was measured by the MD5 microplate reader.
Total protein was extracted from the cells according to the manufacturer’s instructions (Cat# P0027; Beyotime Institute of Biotechnology, Jiangsu, China). Then, 20
The total RNA of cells was extracted using TRIzol reagent (Cat# D9108A; Takara Biotechnology Co., Ltd., Dalian, China) and cDNA was synthesized according to the instructions from the PrimeScript® RT reagent kit with gDNA Eraser (Perfect Real Time; Cat# DRR047S; Takara Biotechnology Co., Ltd.). RT-PCR primers were as follows: GAPDH, forward: 5′-agaaggctggggctcatttg-3′ and reverse: 5′-aggggccatccacagtcttc-3′; IL-8, forward: 5′-tagcaaaattga ggccaagg-3′ and reverse: 5′-aaaccaaggcacagtggaac-3′; and ICAM-1, forward: 5′-ggctggagctgtttgagaac-3′ and reverse: 5′-actgtggggttcaacctctg-3′. The reaction system was prepared according to the instructions of the SYBR® Premix Ex Taq™ kit (Perfect Real time; Cat# DRR420A; Takara Biotechnology Co., Ltd.) and then DNA was amplified as follows: 95°C for 10 min; then 95°C for 15 sec, 61°C for 15 sec and 72°C for 15 sec for 40 cycles; and finally 95°C for 1 min, 55°C for 30 sec and 95°C for 30 sec.
Data are expressed as mean ± standard deviation and analyzed using SPSS 13.0 (SPSS, Inc., Chicago, IL, USA). Statistical comparisons between groups were performed by factorial analyses. P<0.05 was considered to indicate a statistically significant difference.
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The protein expression levels of NF-κB p65, ICAM-1 and IL-8 in the APS group were significantly lower than those in the hyperoxia group at each time point. However, NF-κB p65 and IL-8 expression levels were not significantly altered over different time points.
Oxidative stress and the inflammatory response are two important events during BPD development. SOD removes the superoxide anion radical in order to protect cells from damage (
The oxidative stress response is regulated by an imbalance between ROS generation and antioxidant enzyme degradation of ROS (
APS, as a potent antioxidant, effectively inhibits the generation of oxygen free radicals, preventing membrane lipid peroxidation and reduction of biofilm injury. APS ameliorates hypoxia and prevents reperfusion lung injury (
The inflammatory response plays a crucial role in the development of BPD. A variety of cytokines, including IL-6, IL-8, IL-10 and ICAM-1, are considered to participate in lung inflammation, which is closely related to the development of BPD. NF-κB is one of the transcription factors regulating the expression of inflammatory cytokines. NF-κB upregulates the expression of a variety of inflammatory cytokines, which induce inflammation and then promote the development of BPD. NF-κB is a heterodimer complex, composed of p65/p50. NF-κB binds to the inhibitory-type IκB protein in the cytoplasm. When endothelial cell damage occurs, IκB is phosphorylated and degraded. Phosphorylated NF-κB translocates to the nucleus, binds to the nucleotide sequence of the κB domain and regulates the transcription of a variety of inflammatory cytokines and adhesion molecules (
Oxidative stress and inflammatory responses affect and promote each other. Inflammatory cells release ROS and are involved in oxidative stress. However, ROS-induced oxidative stress activates NF-κB p65 signaling and promotes the expression of pro-inflammatory cytokine genes and the chemotaxis of inflammatory cells, including neutrophils (36). Additional oxidative stress may enhance the chemotaxis of neutrophils and macrophages and stimulate cell adhesion molecule expression, inducing the inflammatory response. The mechanisms by which APS inhibits the activation of NF-κB p65 may be: i) APS has a direct anti-inflammatory role by inhibiting the expression of NF-κB p65 directly; and ii) APS eliminates oxygen free radicals, which is the main stimulant of NF-κB p65 activation. Higher concentrations of APS have an improved scavenging effect on superoxide anion free radicals and lipid free radicals, with the concentration increasing as the scavenging effect increases.
In summary, oxidative stress and inflammation are two important pathological events of BPD, independently or interactively. Lipid peroxidation of EA.hy926 cells may be reduced by APS, which removes the oxygen free radicals within the cells as an antioxidant. In addition, the activation of NF-κB p65 may be effectively blocked and the expression of cytokines, including IL-8 and ICAM-1, may be reduced by APS, which inhibits the inflammatory response. This study was limited to
This study was completed in the Clinical Trials Center of Nanfang Hospital, Guangzhou, China. Financial support was obtained from the Presidential Foundation of Nanfang Hospital (2010A001). The manuscript was reviewed by Dr Yan Hua Liang.
SOD levels in the air, hyperoxia and APS groups. The SOD level of the hyperoxia group was lower than those in the air and APS groups after culture for 24 and 36 h; however, there was no significant difference between the SOD levels of the air and APS groups (P>0.05). At 48 h, there was no statistically significant difference between the hyperoxia and APS groups (P>0.05); however, the content of SOD in the air group was significantly higher. The SOD level of the APS group at 36 h was significantly higher compared with that at 24 h (F=28.845, P<0.01). SOD, superoxide dismutase; APS, astragalus polysaccharides.
MDA levels in the air, hyperoxia and APS groups. The MDA level of the hyperoxia group was significantly higher than those in the air and APS groups at 24 and 36 h; however, there was no significant difference between the air and APS groups (P>0.05). The comparison between the three groups at 48 h was not statistically significant. MDA, malonidaldehyde; APS, astragalus polysaccharides.
ROS levels in the air, hyperoxia and APS groups. The ROS level of the hyperoxia group was significantly higher than those of the air and APS groups at 24 and 36 h while the ROS level of the APS group was significantly lower than that of the air group (P<0.01). The ROS level of the air group was significantly lower compared with those of the hyperoxia and APS groups at 48 h, while the difference between the hyperoxia and APS groups was not statistically significant (P>0.05). The ROS level of the APS group at 48 h was significantly higher compared with the ROS level at 24 and 36 h (F=675.247, P<0.01). ROS, reactive oxygen species; APS, astragalus polysaccharides.
Inhibition of ICAM-1 transcription at different time points by APS in EA.hy926 cells. The results demonstrated that the ICAM-1 gene expression levels of the hyperoxia group at 24, 36 and 48 h were significantly higher compared with those of the APS group. The ICAM-1 gene expression levels of the hyperoxia group at 24, 36 and 48 h were 4-, 1.58- and 1.63-fold higher than the ICAM-1 gene expression level of the APS group at the same time point. The ICAM-1 expression levels at 24, 36 and 48 h were not statistically significantly different (F=1.947, P=0.159). ICAM-1, intercellular adhesion molecule 1; APS, astragalus polysaccharides; RQ, relative quantity.
Inhibition of IL-8 transcription at different time points by APS in EA.hy926 cells. The results demonstrated that the IL-8 gene expression levels in the hyperoxia group at 24, 36 and 48 h were significantly higher compared with those of the APS group. The IL-8 gene expression levels of the hyperoxia group at 24 and 36 h were 3-fold higher than the IL-8 gene expression level of the APS group. The IL-8 gene expression level of the hyperoxia group at 48 h was double the level of the APS group. The IL-8 expression levels at 24, 36 and 48 h were not statistically significantly different (F=0.233, P=0.794). IL, interleukin; APS, astragalus polysaccharides; RQ, relative quantity.