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Objective:To observe the expression changes of nuclear factor erythroid 2 related factor 2 (Nrf2) and glutathione peroxidase (GPX4) in human pulmonary microvascular endothelial cells (HPMEC) under different experimental conditions, and to explore the role of Nrf2 in inhibiting ferroptosis in the process of alleviating hyperoxic lung injury(HLI).Methods:Hyperoxic model was established by hyperoxia exposure.HPMEC were treated with blank control (control group), oxygen exposure at the concentration of 950 mL/L (hyperoxia group), oxygen exposure at the concentration of 950 mL/L+ 10 μmol/L Ferrostatin (ferroptosis inhibitor group) and oxygen exposure at the concentration of 950 mL/L + 10 μmol/L ML385 (Nrf2 inhibitor group). Cell viability at 24 h and 48 h was tested by the Cell Counting Kit-8 assay, and reactive oxygen species (ROS) levels were detected by a commercial ROS kit.The mRNA and protein levels of Nrf2 and GPX4 were detected by real-time quantitative polymerase chain reaction and Western blot, respectively.Differences were analyzed using the Student′s t-test for a two-group comparison or one-way ANOVA test among groups. Results:(1)Compared with the control group, significantly decreased viability and increased ROS levels were detected in hyperoxia group.Meanwhile, the mRNA (24 h: 0.750±0.010 vs.1.010±0.160, 48 h: 0.690±0.050 vs.1.000±0.070) and protein levels of GPX4 (24 h: 0.160±0.010 vs.0.290±0.010, 48 h: 0.190±0.010 vs.0.250±0.010) at 24 h and 48 h were significantly downregulated, while the mRNA (24 h: 1.740±0.050 vs.1.000±0.050, 48 h: 2.130±0.020 vs.1.000±0.030) and protein levels of Nrf2 (24 h: 0.840±0.010 vs.0.480±0.010, 48 h: 0.840±0.010 vs.0.550±0.030) at 24 h and 48 h were significantly upregulated in hyperoxia group than those of control group (all P<0.05). (2)Compared with the hyperoxia group, significantly increased viability and decreased ROS levels were detected in ferroptosis inhibitor group.Meanwhile, the mRNA (24 h: 1.520±0.110, 48 h: 1.880±0.050) and protein levels of GPX4 (24 h: 0.290±0.010, 48 h: 0.250±0.004) at 24 h and 48 h were significantly upregulated, while the mRNA (24 h: 0.780±0.040, 48 h: 0.760±0.030) and protein levels of Nrf2 (24 h: 0.480±0.010, 48 h: 0.540±0.020) at 24 h and 48 h were significantly downregulated in ferroptosis inhibitor group than those of hyperoxia group (all P<0.05). (3)Compared with the hyperoxia group, significantly decreased viability and increased ROS levels were detected in Nrf2 inhibitor group.Meanwhile, the mRNA (24 h: 0.600±0.030, 48 h: 0.590±0.003) and protein levels of GPX4 (24 h: 0.150±0.001, 48 h: 0.180±0.001) at 24 h and 48 h were significantly downregulated, while the mRNA level of Nrf2 was significantly upregulated at 24 h (3.360±0.130), but downregulated at 48 h (1.430±0.130) (all P<0.05). No significant difference was detected in the protein level of Nrf2 at 24 h and 48 h between hyperoxia group and Nrf2 inhibitor group ( P>0.05). Conclusions:Ferroptosis is involved in the development of HLI, and Nrf2 is able to alleviate hyperoxic lung injury by inhibiting ferroptosis.Therefore, inhibition of ferroptosis by Nrf2 may provide a new therapeutic target for HLI.
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ABSTRACT Objectives: This study aimed to explore the effects of miR-128b in the regulation of Lipopolysaccharide (LPS) induced apoptosis. Methods: Human Pulmonary Microvascular Endothelial Cells (HPMECs) were transfected with an miR-128b inhibitor and stimulated with LPS for 24 h. FCM was performed to detect apoptosis and Reactive Oxygen Species (ROS) production. In addition, miRNA and caspase-3 expression levels were determined using real-time quantitative polymerase chain reaction and western blotting. Results: LPS significantly induced apoptosis and ROS production and upregulated miR-128b and caspase-3 expressions in HPMECs. However, LPS-induced effects were suppressed when an miR-128b inhibitor was used. Preincu-bation with NAC decreased the LPS-induced apoptosis of HPMECs. Conclusions: These effects were mediated by miR-128b via the caspase-3 pathway.
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OBJECTIVE:To study the effects of alprostadil on the expression of vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS) in human pulmonary microvascular endothelial cells (HPMECs) with hypoxia-in-duced injury. METHODS:HPMECs were divided into group A(normal cultural environment),group B(3%O2),group C(3%O2+15 μg/L alprostadil)and group D(3%O2+45 μg/L alprostadil)for 24 h culture. Cell morphology was observed;MTT method was conducted to detect cell activity(recorded by absorbance);flow cytometry was adopted to detect apoptosis rate;Western blot was used to detect relative expressions of VEGF and eNOS in cells. RESULTS:Cell density in group A and D was relatively high, that in group B and C was low. Compared with group A,cell activities decreased and apoptosis rates increased in group B,C and D,expressions of VEGF and eNOS enhanced,eNOS/VEGF decreased (P<0.05). Compared with group B,cell activities in-creased in group C and D,VEGF expression decreased (P<0.05);apoptosis rate in group D decreased,eNOS expression de-creased and eNOS/VEGF increased. Compared with group C,cell apoptosis rate was decreased in group D,VEGF expression de-creased and eNOS/VEGF increased(P<0.05). CONCLUSIONS:Alprostadil maybe play effect on HPMECs with hypoxia-induced injury by up-regulating the expression level of eNOS/VEGF.
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β-arrestins are expressed proteins that were first described, and are well-known, as negative regulators of G protein-coupled receptor signaling. Penehyclidine hydrochloride (PHC) is a new anti-cholinergic drug that can inhibit biomembrane lipid peroxidation, and decrease cytokines and oxyradicals. However, to date, no reports on the effects of PHC on β-arrestin-1 in cells have been published. The aim of this study was to investigate the effect of PHC on β-arrestin-1 expression in lipopolysaccharide (LPS)-induced human pulmonary microvascular endothelial cells (HPMEC). Cultured HPMEC were pretreated with PHC, followed by LPS treatment. Muscarinic receptor mRNAs were assayed by real-time quantitative PCR. Cell viability was assayed by the methyl thiazolyl tetrazolium (MTT) conversion test. The dose and time effects of PHC on β-arrestin-1 expression in LPS-induced HPMEC were determined by Western blot analysis. Cell malondialdehyde (MDA) level and superoxide dismutase (SOD) activity were measured. It was found that the M3 receptor was the one most highly expressed, and was activated 5 min after LPS challenge. Furthermore, 2 μg/mL PHC significantly upregulated expression of β-arrestin-1 within 10 to 15 min. Compared with the control group, MDA levels in cells were remarkably increased and SOD activities were significantly decreased in LPS pretreated cells, while PHC markedly decreased MDA levels and increased SOD activities. We conclude that PHC attenuated ROS injury by upregulating β-arrestin-1 expression, thereby implicating a mechanism by which PHC may exert its protective effects against LPS-induced pulmonary microvascular endothelial cell injury.