Study On The Acting Mechanism Of Environmental Estrogens On TRALL Via Mitochondria Injury Mediated By ROS

Background and objectivesTransfusion related acute lung injury (TRALL) is a kind of rare but potentially lethal transfusion reaction, which is the primary cause of transfusion related death at present [1]. Although the harm of TRALL has been gradually recognized, the attention on the complication is still not enough [2]. Currently, the diagnosis of TRALL is dependent on clinical symptoms, which are mainly acute respiratory distress and non-cardiogenic pulmonary edema within6hours after transfusion [3]. Regarding to the pathophysiological mechanism of TRALL, the first step refers to leukocyte antibodies in the transfused blood from the plasma of donors, among which type I and type II human leukocyte antibody (HLA) are the most common, eliciting neutrophil deformation and adhesion in the recipients due to the potential disease [4]. The second step is that the pulmonary bioactive factors activated by the substrate of neutrophils leads to direct injury of pulmonary blood vessels, capillary exudates and acute lung injury [5,6]. The terminal pathway is to act on the pulmonary blood vessels and elicit transfusion related acute lung injury no matter which mechanism it is and which factors are released.Di(2-ethylhexyl)phthalate (DEHP) and its metabolite, mono(2-ethylhexyl) phthalate(MEHP) belong to phthalic acid esters (PAEs) among environmental estrogens (EEs), which are endocrine interferents widespread in the environment. They are existent in plastics of polyvinyl chloride as the plasticizer, and readily influencing the production and living of human beings since they are widely used. They could be detected in human amniotic fluid, umbilical blood, human milk, sperm and salivary fluid as the plasticizer [7]. DEHP and MEHP are absorbed directly along with intravenous administration of the content in the blood bags when the medical apparatuses containing PAEs like blood bags or transfusion bags are used, as is indicated by a report in2009in Swedish Medical Center, USA. The content of DEHP and MEHP in the supernatant of the bags in the blood donation center is tested from the first day till the valid42th day by liquid chromatography/mass spectrometry (LCMS). The results show that DEHP remarkably rises by12.6times, from34.3μM (20.0±SD) on the first day to433.2μM (131.2±SD) on the42th day, and that MEHP dramatically increases by20.2times, from3.7μM (2.8±SD) on the first day to74.0μM (19.1±SD) on the42th day, indicating that the use of stored blood products containing excessive DEHP and MEHP may be one important factor of TRALL.Apoptosis is the chief study of programmed cell death as one of the most hot field in cell biology during the past10years, which plays the key role in development of tissue homeostasis[8-14]. Bcl-2family protein is the main engager among a variety of signal pathways of apoptosis, whose main effect is to regulate permeability of outer membrane of mitochondria (OMM), releasing various apoptotic factors, cytochrome C, Smac/DIABLO, Omi/HtrA2,endonuclease g and AIF. Both of Bax as apoptosis-promoting protein and Bcl-2which is anti-apoptosis protein belong to Bcl-2family[15,16].N-acetyl-cysteine (NAC), playing an important role in the formation, which directly affects the ability of the protein extraction, is considered a powerful antioxidant that can reduce cell damage [17].In order to explore the cause and mechanism of TRALL by MEHP (DEHP could be converted to MEHP in human body), we set up a vitro contamination model to study the direct toxic effect of MEHP on human vascular endothelial cells (HUVEC) and the mechanism. Methods and results:Methods:1.Culture HUVEC in1640media which contains10%fetal bovine serum, which is contaminated by MEHP (0、6.25、62.5、25、50、100μM) of a variety of concentrations for a time after stable.2.Detect cell activity and apoptosis rate by MTT and flow cytometry to evaluate toxic effect of MEHP on cells.3.Test intracellular ROS production and the potential change of mitochondrial membrane by DCFH-DA fluorescent probes and JC-1fluorescent probes, respectively.4.Detect intracellular index of oxidative stress, including intracellular malondialdehyde (MDA) level, glutathione (GSH) and superoxide dismutase (SOD) activity by the kits.5. Detect the possible apoptotic pathways by ELISA, and detect activity of caspase-3, caspase-8and caspase-9to verify intrinsic and extrinsic apoptotic pathway.6. Detect Bax and Bcl-2in cell lysates by real-time fluorescent quantitative PCR.7. Detect the protein expression of Bax, Bcl-2and cytochrome C in cell lysates by Western blot.8. Judge whether the toxic effect of MEHP is mediated by ROS by detecting ROS production, cell activity and apoptosis rate after reducer NAC pretreatment.Results:l.Cell activity dramatically lowers down, showing dose-dependent and time-dependent decline.2.The production of ROS rises as the increase of concentration, and intracellular ROS in moderate and high dose group obviously increases. 3.MEHP induces HUVEC apoptosis, and thereby results in intracellular oxidative stress disorder.4.HUVEC apoptosis is induced by MEHP via both intrinsic and extrinsic pathway.5.RNA level of Bax and Bcl-2could be changed by MEHP.6.The ratio of Bax and Bcl-3protein in HUVEC increases as the rise of MEHP concentration, exhibiting dose-dependent rise, indicating there is apoptosis promotion present in cells, while apoptosis could be alleviated by NAC incubation.Conclusions:MEHP induces mitochondrial injury of HUVEC, oxidative stress and lipid peroxidation; it induces HUVEC apoptosis via intrinsic apoptosis pathway mediated by ROS. MEHP could induce HUVEC apoptosis and thereby leads to TRALL.