Antioxidant And Anti-inflammation Effects Of Astragalus Polysaccharides On EA.Hy926Cells

BackgroundsBronchopulmonary dysplasia is a kind of the most common chronic lung disease in very low birth weight infants. With the development of perinatal medicine, including prenatal glucocorticoid and postnatal exogenous surfactant application and implementation of protective ventilation strategy, the survival rate of premature increases, the incidence of BPD also showed a rising trend year by year, have very high long-term morbidity and mortality of NICU, which become one of the most difficult the problem. Survivors often have chronic pulmonary function and gas exchange abnormalities, need long-term dependence on oxygen or ventilator, bring heavy burden to the family and society, but also directly affect the future quality of life in children. Therefore, how to prevent or delay the occurrence and development of BPD, reduce the incidence of BPD is an important issue related to the social and economic development and to protect the limited medical resources in China.BPD is a multifactorial disease, its etiology and pathogenesis is very complex, involving many aspects of hyperoxia, mechanical ventilation, prenatal infection and postpartum inflammation, cause there hasn’t been an effective way for the prevention and treatment. Currently recognized as, oxidative stress and inflammatory reaction play an important role in the pathogenesis of BPD. Oxidative stress response is regulated by the imbalance between ROS generation and antioxidant enzyme degradation of ROS. Under physiological circumstances, there is a delicate balance between the human body ROS generation and antioxidant defense system. The balance will be broken when the over expression or inadequate clearance of ROS was out of the metabolic control of cells, resulting the oxidative stress injury. It was known that SOD contributes to scavenging ROS. The leading role of SOD is to translate highly reactive superoxide free radical into hydrogen peroxide and water, and then hydrogen peroxide will be transformed into water by catalase, glutathione peroxidase and glutathione reductase. Oxidative stress injury can cause lipid peroxidation, and produce MDA. SOD can scavenge oxygen free radicals to protect cells from damage; the level of its vitality indirectly reflects the ability of the body to scavenge oxygen free radicals.Inflammatory response played a very crucial role in the development of BPD. A variety of cytokines, such as IL-6, IL-8, IL-10, ICAM-1, were proposed to participate in lung inflammation, closely related to the development of BPD. NF-KB is one of the transcription factors regulating the expression of inflammatory cytokines. NF-KB can up-regulate the expression of a variety of inflammatory cytokines, which induce inflammation, and then promote the development of BPD. NF-KB is a heterodimer complex, composed by the p65/p50. NF-KB binds to the inhibitory-type1KB protein in the cytoplasm. When the endothelial cell damage,1KB was phosphorylated and degraded. Phosphorylated NF-KB translocated into cell nuclear, binds to nucleiotide sequence of KB domain, and regulates the transcription of a variety of inflammatory cytokines and adhesion molecules. The activation of NF-KB led to over-expression of inflammation-related factors and caused a significant inflammatory response. In return, the increased inflammatory mediators and cytokines, further activated NF-KB, amplifying the initial inflammatory signals. The activation of NF-KB can stimulate endothelial cells to release IL-8. IL-8is a potent neutrophil chemotactic factor, can promote and prolong the inflammatory responseAntioxidant and anti-inflammatory drugs for BPD had become a major research topic at home and abroad. One of the most studied drugs was glucocorticoids, which has anti-inflammatory and antioxidant effect, was very important to reduce the incidence of BPD. However, because of its serious adverse reaction, the clinical application of glucocorticoids was limited. Therefore, there is an urgent need to find some new efficient, safe drug to improve the present situation in the treatment of BPD.Natural traditional Chinese medicine is not only a rich source of drugs but also has the advantages of low toxicity, mild adverse reaction, cheap and efficient, has good prospects for research and development. Astragalus polysaccharide (APS) is one of the more in-depth studied of traditional Chinese medicine, which widely applied in clinic. APS was one of the most important natural effective components in Radix Astragali. In recent years more and more scholars intentioned APS because of its antioxidant, scavenging free radicals, anti-inflammatory, improve the biological activity of immune function. People pay more attention to the antioxidant and anti-inflammatory effects of Astragalus Polysaccharide. In vitro tissue culture experiments indicated that APS can induce the total antioxidant capacity of the cells as well as reduced the generation of oxygen free radicals and thus rats’ lung epithelial cells injury. APS can also protect the intestinal mucosa of rats with obstructive jaundice from oxidative stress damage which may relate to the increasing of SOD and the decreasing of MDA. Xiaoli Yue found that APS had a role in promoting the wound healing of chronic ulcers, related to reduced expression of inflammatory stimuli, wound lipid peroxidation and enhancement of the expression of SOD [2]. The vitality of SOD in dogs’ blood could be significantly improved by APS. APS can increase the activity of superoxide dismutase and reduce the content of lipid peroxide, and the cell damage caused by free radicals. Previous studies had shown that the expression of VCAM-1, ICAM-1and NF-KB mRNA of cardiac microvascular endothelial cells in reperfusion injury can be suppressed by APS. In LPS-induced inflammatory response, the generation of TNF-a and IL-8can be inhibited by APS, which may play a role in preventing the inflammation. In osteoarthritis, APS can reduce the inflammatory response of synovial cells and the generation of apoptosis. However, researches on the effect of APS on BPD were rarely reported at home and abroad. Whether APS can prevent or delay the occurrence by anti-oxidation and (or) anti-inflammatory effects of BPD, is still not completely clear.Human umbilical vein endothelial fusion of lung adenocarcinoma cells (EA.hy926cell) were permanent human umbilical vein endothelial cells. Its structure and function are similar to alveolar endothelial cells, can be used to construct the alveolar model in vitro. Research on EA.hy926cells has been widely applied in leukocyte adhesion, endothelial cell expression of oxidative stress, protein regulation and so on.Objectives1. To establish the BPD cell model in vitro.2. To investigate the effects of Astragalus Polysaccharide in prevention and treatment of premature infants with bronchopulmonary dysplasia by the research on the occurrence and development of EA.hy926cell antioxidant and anti-inflammatory effects.3. To further analysis of APS exert anti-inflammatory effect through NF-KB signal pathway, looking at the molecular level to the drug targets of APS.Methods1. EA.hy926cells were cultured in high glucose DMEM medium containing10%FBS,5%CO2,37℃incubator. EA.hy926cells were divided into three groups, the air group, the hyperoxia group and the APS group (2.5mg/ml). The air group was cultured for24,36and48hours in the5%CO2,37℃incubator. The hyperoxia group and the APS group were cultured for24,36and48hours in the37℃triple gas mixture.2. Used chemical reagents to measure the expression of SOD, MDA and ROS in each group.3. The mRNA and protein expression of ICAM-1、IL-8、NF-kB p65were determined by fluorescent quantitation real time-PCR and Western blotting.4. Data were expressed as mean±S and analyzed using SPSS13.0. Stastical comparisons between groups were performed by factorial analyses Statistical significance was determined as p<0.05.Results1. The SOD level of the hyperoxia group was less than that in the air and APS group after cultured for24and36hours, while the comparison is not statistically significant between the air group and the APS group (P>0.05). The comparison at time point of48hours is not statistically significant between the hyperoxia group and the APS group (P>0.05), but the content of SOD in the air group was significantly higher. The SOD level of the APS group at36hours was significantly higher than the one at24hours (F=28.845, P<0.01)2. The MDA content of the hyperoxia group was significantly higher than that in the air and APS group at24and36hours, while the comparison is not statistically significant between the air group and the APS group (P>0.05). The comparison is not statistically significant in three groups at48hours.3. The ROS level of the hyperoxia group was significantly higher than the air group and the APS group at24and36hours, at the mean time the ROS level of the APS group was significantly lower than the air group (P<0.01). The ROS level of the air group was significantly lower than the hyperoxia group and the APS group at48hours, while the comparison is not statistically significant between the hyperoxia group and the APS group (P>0.05). The ROS level of the APS group at48hours was significantly higher than the one at24and36hours (F=675.247, P<0.01)4. The ROS level of the hyperoxia group was significantly higher than the air group and the APS group at24and36hours, at the mean time the ROS level of the APS group was significantly lower than the air group (P<0.01). The ROS level of the air group was significantly lower than the hyperoxia group and the APS group at48hours, while the comparison is not statistically significant between the hyperoxia group and the APS group (P>0.05). The ROS level of the APS group at48hours was significantly higher than the one at24and36hours (F=675.247, P<0.01)5. Inhibition of ICAM-1transcription at different time points by APS in EA.hy926cells. The results showed that The ICAM-1gene expression levels of the hyperoxia group at24,36and48hours were significantly higher than the APS group. The ICAM-1gene expression level of the hyperoxia group at24hours was four times compared with the APS group. The ICAM-1gene expression level of the hyperoxia group at36hours was1.58times compared with the APS group. The ICAM-1gene expression level of the hyperoxia group at48hours was1.63times compared with the APS group. The comparison is not statistically significant at24,36and48hours (F=1.947,P=0.159) 6. The protein expression of NF-KBp65, ICAM-1, IL-8in the hyperoxia group at24,36,48hours were significantly higher than that in the air and APS group. While there were no significant differences of NF-KBp65and IL-8between time points.Conclusions1. APS can remove ROS producing from intracellular oxidative stress through increasing the expression of SOD in EA.hy926cells and decreasing the peroxidation of cytoplasm, which better fit the aim of antioxidant and protected cells from oxidative stress injury.2. APS can significantly reduce the content of ROS in EA.hy926cells, which improved the ability of antioxidative stress.3. The activation of NF-KB p65can be inhibited by APS, thereby reducing the expression of ICAM-1and IL-8and inflammatory response.