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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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Objective:To investigate the expression of long non-coding RNA MALAT1, interleukin 6(IL-6) and apoptosis induced factor(AIF) in peripheral venous blood of premature infants with bronchopulmonary dysplasia (BPD) and its clinical significance.Methods:Preterm infants admitted to the Department of Neonatology, Shanghai Children′s Hospital from January 2015 to December 2016 were enrolled.The selection criteria included gestational age (GA) ≥28 weeks and ≤32 weeks, and birth weight (BW) < 1 500 g. According to the diagnosis, they were divided into BPD group (20 cases) and control group (20 cases). The clinical data of the two groups of premature infants were collected and analyzed, and the levels of MALAT1, IL-6 and AIF in the blood of 40 premature infants were detected by real-time polymerase chain reaction (RT-PCR). T test was used to compare gestational age, birth weight, MALAT1, IL-6 and AIF between the two groups. Results:(1)There was no significant differences in sex ( χ2=1.76), gestational age ( t= 0.17) and birth weight ( t=1.25) of premature infants in BPD group, compared with the control group (all P >0.05). (2)Compared with the control group, the expression of MALAT1 in the peripheral blood of premature infants in BPD group were significantly increased (0.273 4±0.067 3 vs. 0.375 5±0.081 9, P<0.05). (3)Compared with the control group, the expression of IL-6 in the peripheral blood of premature infants in BPD group were obviously decreased (1.448 8±0.191 8 vs.4.444 6±0.165 7, P<0.05). (4)Compared with the control group, the expression of AIF in the peripheral blood of premature infants in BPD group were remarkably decreased(0.006 8±0.002 0 vs.0.004 5±0.001 9, P<0.05). Conclusions:MALAT1 and IL-6 levels of long non-coding RNA in BPD and non-BPD preterm infants are different, which may be related to the incidence of BPD.IL-6 may be a predictor of BPD, and MALAT1 may protect premature infants with BPD.
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Objective@#To observe the expression of microRNA-125b (miR-125), tumor necrosis factor alpha (TNF-α) and interleukin-6(IL-6) in premature rats exposed to hyperoxia.@*Methods@#Eighty 1-day old Sprague Dawley (SD) rats were randomly divided into an air group and a hyperoxia group.The rats in the hyperoxia group were continuously exposed to oxygen chamber for 1-3 L/min, oxygen volume fraction was maintained at (800±50) mL/L, and the rats in air group were placed in the same room with the oxygen volume fraction at 210 mL/L.The feeding conditions were same in 2 groups.Lung tissues of premature rats were extracted at different time (1, 4, 7, 10, 14 days). The pathologic changes in the lung tissues were observed by hematoxylin-eosin (HE) staining.The levels of miR-125b and TNF-α, IL-6 in lung tissues were detected by reverse transcription polymerase chain reaction (qRT-PCT) and enzyme-linked immunosorbent assay (ELISA).@*Results@#Compared with the air group, miR-125b in the hyperoxia group increased slowly after day 1, reached the highest in day 10 (2.554±0.323), and the relative expression in day 14 decreased slightly(2.329±0.263), and there were significant differences between 2 groups at di-fferent time (all P<0.05); in particular TNF-α level increased in day 7 [(78.55±39.53) ng/L], and reached the peak at day 10 [(80.16±11.24) ng/L], and there was a significant difference(P<0.05); IL-6 levels increased at day 7 [(45.44±31.94) ng/L], and reached the peak at day 10 [(90.38±8.24) ng/L], and there was a significant difference(P<0.05). There was a no significant correlation between miR-125b and TNF-α in the hyperoxia groups (r=0.132, P>0.05), but there was significant correlation between miR-125b and IL-6 in hyperoxia groups(r=0.439, P<0.05).@*Conclusions@#The levels of miR-125b, TNF-α and IL-6 are involved in the pathological process of bronchopulmonary dysplasia induced by hyperoxia, and IL-6 may be the key factor for miR-125b.
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Objective To explore the protective effect of long non-coding RNA(lncRNA) metasta-sis associated in lung denocarcinoma transcript 1 (MALAT1) involved in hyperoxia-induced lung injury in preterm infants.Methods This study had downloaded chip data set GSE25286 (Mouse GEO Genome 430 2.0 Array) from gene expression database gene expression omnibus (GEO),according to the state of hyperoxia exposure,the MALAT1 mRNA expression in rats normal lung tissues and hyperoxic lung tissues was compared at day 14th and 29th.In chip data set GSE43830(Human Exon 1.0 ST Arrays) from GEO,the expression of multi-ple genes[cell division cycle 6(CDC6),death effector domain containing 2(DEDD2),and Cyclin B1 (CCNB1)] in WI38 cells(lung fibroblasts) was compared before and after MALAT1 was knockout.At the same time,the peripheral blood samples of premature infants were collected to verify.Totally 40 premature infants were hospitalized in the department of neonatology in our hospital from Jan 2015 to Dec 2016,the pe-ripheral blood samples of 40 premature infants were collected.RNA was extracted and Real time-PCR was performed after reverse transcription,clinical data of these 40 cases were retrospectively analyzed. Results (1) By using Affymetrix Expression console and Affymetrix Transcriptome analysis console software source files of the chip of pretreatment and difference expression gene screening,the expression of lncRNA MALAT1 gene in lung tissues of hyperoxia lung injury mice significantly upregulated[fold change(FC) =2.33,P=0.001].(2) After MALAT1 in WI38 cell was knockout,MALAT1 expression was significantly reduced(FC= -15.6,P=0.000),the expression of CDC6(FC= -2.37,P=0.001) and CCNB1(FC=-2.16,P=0.002) were down regulated,DEDD2 expression was up regulated(FC =2.46,P =0.000). (3) The results of peripheral blood samples from preterm infants showed that the expression of MALAT1 was significantly increased in preterm infants with hyperoxia-induced lung injury(0.375 5 ± 0.081 9,t =4.634, P=0.015),compared with normal preterm infants(0.273 4 ± 0.067 3).Conclusion Through inhibiting cell apoptosis,lncRNA MALAT1 can protect preterm infants with hyperoxia-induced lung injury,it may provide a new strategy for prevention and treatment of hyperoxia-induced lung injury in premature infants.