Cell Division Cycle 2 Protects Neonatal Rats Against Hyperoxia-Induced Bronchopulmonary Dysplasia

Zhongying Li,Yanhong Chen,Wenrong Li,Fan Yan 연세대학교의과대학 2020 Yonsei medical journal Vol.61 No.8

Purpose: Hyperoxia-induced bronchopulmonary dysplasia (BPD) is a lung disease in preterm infants. We aimed to explore therole of cell division cycle 2 (CDC2) on histopathologic changes of lung tissues, as well as the viability, apoptosis, and inflammationof lung cells in rats with hyperoxia-induced BPD. Materials and Methods: Hyperoxia-induced BPD in neonatal rats and hyperoxia-induced A549 cells were constructed. The mRNAexpression of CDC2 was detected by qRT-PCR. The fibrosis score of lung tissues was evaluated by hematoxylin-eosin staining. Theviability and apoptosis of A549 cells were detected by cell counting kit-8 assay and flow cytometry. The protein expressions of bcl-2,bax, and caspase-3 were measured by western blot. The levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and IL-1β inA549 cells were detected by enzyme-linked immunosorbent assay. The pcDNA3.1-CDC2 was injected into rats to determine therole of CDC2 in hyperoxia-induced BPD in vivo. Results: The expression of CDC2 was decreased in lung tissues of neonatal rats with hyperoxia-induced BPD and hyperoxia-inducedA549 cells. The fibrosis score was increased in the lung tissues of neonatal rats with hyperoxia-induced BPD. Overexpressionof CDC2 increased the viability and protein expression of bcl-2; and inhibited the apoptosis, inflammation, and protein expressionof bax and caspase-3 in hyperoxia-induced A549 cells. Up-regulation of CDC2 alleviated the histopathologic changes in lung tissuesof neonatal rats with hyperoxia-induced BPD. Conclusion: Overexpression of CDC2 promoted the viability and inhibited the apoptosis and inflammation of hyperoxia-inducedcells, and alleviated the histopathologic changes of lung tissues in neonatal rats with hyperoxia-induced BPD.

Effect of Short-term Exposure of Different Concentrations of Hyperoxia on Fetal Alveolar Type II Cell Death

이현수 대한신생아학회 2013 Neonatal medicine Vol.20 No.2

Purpose: Lung injury imposed by hyperoxia is the most important cause of bronchopulmonary dysplasia (BPD) in premature lungs, and hyperoxia has the chief biological effect of inducing cell death. Our objective was to investigate the response of cell death in fetal alveolar type II cells (FATIICs) exposed to different concentrations of hyperoxia for 36 h. Methods: FATIICs were isolated on embryonic day 19 and exposed to 65%- or 85%-oxygen for 36 h. Cells in room air were used as controls. FACScan was performed in hyperoxic and control samples at 0/6/12/24/36 h, and the patterns of cell death were compared at each time point using flow-cytometry. Results: Cell necrosis as measured by selective propidium iodide staining increased significantly from 12 h of 65%-hyperoxia and 6 h of 85%-hyperoxia, respectively. Cell necrosis increased 1.6-fold, 3.0-fold and 4.6-fold after 12 h, 24 h, and 36 h, respectively during 65%-hyperoxia and increased by 2.4-fold, 3.1-fold, 6.3-fold, and 8.8-fold after 6 h, 12 h, 24 h, and 36 h, respectively during 85%-hyperoxia compared to controls. Apoptotic cells as measured by selective Annexin-V staining peaked at 1.3% at 24 h of 65%-hyperoxia and peaked at 1.2% at 6 h of 85%-hyperoxia, respectively and then decreased rapidly. Conclusion: This study shows that exposure to sublethal and lethal hyperoxia increases necrosis of FATIICs remarkably from the early stage of hyperoxia. These findings support the idea that short-term exposure to oxygen from birth may contribute to the evolution of “new” BPD in preterm lungs.

OSI-027 alleviates rapamycin insensitivity by modulation of mTORC2/AKT/TGF-β1 and mTORC1/4E-BP1 signaling in hyperoxia-induced lung injury infant rats

Long Li,Liang Mulin,Yanling Liu,Wang Pan,Dang Hongxing 대한독성 유전단백체 학회 2021 Molecular & cellular toxicology Vol.17 No.3

Background The mechanism of long time and high-concentration oxygen treatment leading to acute lung injury (ALI) or developmental lung disease in infants is currently unclear. Here we found that compared with the effect of rapamycin, pan-mTOR1/2 inhibitor OSI-027, alleviates hyperoxia-induced lung injury (HILI) by modulation of mTORC2/AKT/TGF-β1 and mTORC1/4E-BP1 signaling in infant rats. Objective Infant rats were treated with continuous inhalation of 90% medical oxygen. Normal saline, rapamycin, or OSI-027 was intraperitoneally injected, and the status of lung injury was tested on days 3, 7, and 14. The activation of mTOR/AKT/TGFβ1 and mTORC1/4E-BP1 signaling was confirmed by immunohistochemistry and Western blot analysis in normal and hyperoxia-treated live precision-cut lung tissues. The inhibitory effect of OSI-027 extended to the active state of other proteins implicated in mTOR1/2 signaling was demonstrated in hyperoxia-induced injured lung tissues. Results Our data demonstrate that hyperoxia-induced serious lung inflammation and fibrosis. OSI-027 significantly attenuated the pathological process of HILI, inhibit the phosphorylation of the primary downstream targets of mTORC1/C2, and reduce the activation of TGF-β1 signaling. Conclusions The results suggest that mTORC2/AKT/TGF-β1 and the rapamycin-insensitive mTORC1/4E-BP1 (Thr37/46) signaling has an important effect during HILI with a potential meaning for the progress of novel anti-hyperoxia-injury strategies. Background The mechanism of long time and high-concentration oxygen treatment leading to acute lung injury (ALI) or developmental lung disease in infants is currently unclear. Here we found that compared with the effect of rapamycin, pan-mTOR1/2 inhibitor OSI-027, alleviates hyperoxia-induced lung injury (HILI) by modulation of mTORC2/AKT/TGF-β1 and mTORC1/4E-BP1 signaling in infant rats. Objective Infant rats were treated with continuous inhalation of 90% medical oxygen. Normal saline, rapamycin, or OSI-027 was intraperitoneally injected, and the status of lung injury was tested on days 3, 7, and 14. The activation of mTOR/AKT/TGFβ1 and mTORC1/4E-BP1 signaling was confirmed by immunohistochemistry and Western blot analysis in normal and hyperoxia-treated live precision-cut lung tissues. The inhibitory effect of OSI-027 extended to the active state of other proteins implicated in mTOR1/2 signaling was demonstrated in hyperoxia-induced injured lung tissues. Results Our data demonstrate that hyperoxia-induced serious lung inflammation and fibrosis. OSI-027 significantly attenuated the pathological process of HILI, inhibit the phosphorylation of the primary downstream targets of mTORC1/C2, and reduce the activation of TGF-β1 signaling. Conclusions The results suggest that mTORC2/AKT/TGF-β1 and the rapamycin-insensitive mTORC1/4E-BP1 (Thr37/46) signaling has an important effect during HILI with a potential meaning for the progress of novel anti-hyperoxia-injury strategies.

Fetal Alveolar Type II Cell Injury Induced by Short-term Exposure to Hyperoxia

이현수 대한신생아학회 2013 Neonatal medicine Vol.20 No.3

A High concentration of oxygen (>40%) is used as a life-saving therapy in preterm newborns since birth. By generating excess reactive oxygen species, however,hyperoxia can cause lung injury leading to bronchopulmonary dysplasia (BPD). Although hyperoxia-induced lung injury contributes to the evolution of BPD,the mechanisms by which hyperoxia contributes to the genesis of lung injury in preterm lungs are not yet fully defined, and there are no specific measures for the protection of preterm lungs against injury secondary to hyperoxia. Alveolar type II cells are key components of the alveolar structure, and they are responsible for the restoration of normal alveolar epithelium after acute lung injury. However,hyperoxia is primarily delivered to the alveolar epithelium and alveolar type II cells can be the main target for the injury secondary to hyperoxia. To date, my researches have been focused on injury of fetal alveolar type II cells exposed to hyperoxia and the role of anti-inflammatory cytokine, IL-10 minimizing fetal type II cell injury induced by hyperoxia. Based on my previous studies, this article summarizes the cellular and molecular mechanisms of fetal type II cell injury induced in the early stage of hyperoxia and the protective potency of IL-10 in fetal alveolar type II cells and neonatal lungs injured by hyperoxia.

Interleukin-10 Down-Regulates Cathepsin B Expression in Fetal Rat Alveolar Type II Cells Exposed to Hyperoxia

이현수,김천기 연세대학교의과대학 2013 Yonsei medical journal Vol.54 No.2

Purpose: Hyperoxia has the chief biological effect of cell death. We have previously reported that cathepsin B (CB) is related to fetal alveolar type II cell (FATIIC)death and pretreatment of recombinant IL-10 (rIL-10) attenuates type II cell death during 65%-hyperoixa. In this study, we investigated what kinds of changes of CB expression are induced in FATIICs at different concentrations of hyperoxia (65%- and 85%-hyperoxia) and whether pretreatment with rIL-10 reduces the expression of CB in FATIICs during hyperoxia. Materials and Methods: Isolated embryonic day 19 fetal rat alveolar type II cells were cultured and exposed to 65%- and 85%-hyperoxia for 12 h and 24 h. Cells in room air were used as controls. Cytotoxicity was assessed by lactate dehydrogenase (LDH) released into the supernatant. Expression of CB was analyzed by fluorescence-based assay upon cell lysis and western blotting, and LDH-release was re-analyzed after preincubation of cathepsin B-inhibitor (CBI). IL-10 production was analyzed by ELISA, and LDH-release was re-assessed after preincubation with rIL-10 and CB expression was re-analyzed by western blotting and real-time PCR. Results: LDH-release and CB expression in FATIICs were enhanced significantly in an oxygen-concentration-dependent manner during hyperoxia, whereas caspase-3 was not activated. Preincubation of FATIICs with CBI significantly reduced LDH-release during hyperoxia. IL-10-release decreased in an oxygen-concentration-dependent fashion, and preincubation of the cells with rIL-10 significantly reduced cellular necrosis and expression of CB in FATIICs which were exposed to 65%- and 85%-hyperoxia. Conclusion: Our study suggests that CB is enhanced in an oxygen-concentration-dependent manner, and IL-10 has an inhibitory effect on CB expression in FATIICs during hyperoxia.

Cathepsin B is activated as an executive protease in fetal rat alveolar type II cells exposed to hyperoxia

Hyeon-Soo Lee,김춘기 생화학분자생물학회 2011 Experimental and molecular medicine Vol.43 No.4

Alveolar type II cells are main target of hyperoxia-induced lung injury. The authors investigated whether lysosomal protease, cathepsin B (CB), is activated in fetal alveolar type II cells in the transitional period from the canalicular to saccular stages during 65%-hyperoxia and whether CB is related to fetal alveolar type II cell (FATIIC) death secondary to hyperoxia. FATIICs were isolated from embryonic day 19 rats and exposed to 65%-oxygen for 24 h and 36 h. The cells exposed to room air were used as controls. Cell cytotoxicity was assessed by lactate dehydrogenase-release and flow cytometry, and apoptosis was analyzed by TUNEL assay and flow cytometry. CB activity was assessed by colorimetric assay, qRT-PCR and western blots. 65%-hyperoxia induced FATIIC death via necrosis and apoptosis. Interestingly, caspase-3 activities were not enhanced in FATIICs during 65%-hyperoxia,whereas CB activities were greatly increased during 65%-hyperoxia in a time-dependent manner, and similar findings were observed with qRT-PCR and western blots. In addition, the preincubation of CB inhibitor prior to 65%-hyperoxia reduced FATIIC death significantly. Our studies suggest that CB activation secondary to hyperoxia might have a relevant role in executing the cell death program in FATIICs during the acute stage of 65%-hyperoxia.

Cathepsin B is activated as an executive protease in fetal rat alveolar type II cells exposed to hyperoxia

Lee, Hyeon-Soo,Kim, Chun-Ki Korean Society for Biochemistry and Molecular Bion 2011 Experimental and molecular medicine Vol.43 No.4

Alveolar type II cells are main target of hyperoxia-induced lung injury. The authors investigated whether lysosomal protease, cathepsin B (CB), is activated in fetal alveolar type II cells in the transitional period from the canalicular to saccular stages during 65%-hyperoxia and whether CB is related to fetal alveolar type II cell (FATIIC) death secondary to hyperoxia. FATIICs were isolated from embryonic day 19 rats and exposed to 65%-oxygen for 24 h and 36 h. The cells exposed to room air were used as controls. Cell cytotoxicity was assessed by lactate dehydrogenase-release and flow cytometry, and apoptosis was analyzed by TUNEL assay and flow cytometry. CB activity was assessed by colorimetric assay, qRT-PCR and western blots. 65%-hyperoxia induced FATIIC death via necrosis and apoptosis. Interestingly, caspase-3 activities were not enhanced in FATIICs during 65%-hyperoxia, whereas CB activities were greatly increased during 65%-hyperoxia in a time-dependent manner, and similar findings were observed with qRT-PCR and western blots. In addition, the preincubation of CB inhibitor prior to 65%-hyperoxia reduced FATIIC death significantly. Our studies suggest that CB activation secondary to hyperoxia might have a relevant role in executing the cell death program in FATIICs during the acute stage of 65%-hyperoxia.

Combination of carboplatin and intermittent normobaric hyperoxia synergistically suppresses benzo[a]pyrene-induced lung cancer

( Hea Yon Lee ),( In Kyoung Kim ),( Hye In Lee ),( Hwa Young Lee ),( Hye Seon Kang ),( Chang Dong Yeo ),( Hyun Hui Kang ),( Hwa Sik Moon ),( Sang Haak Lee ) 대한내과학회 2018 The Korean Journal of Internal Medicine Vol.33 No.3

Background/Aims: We explored the effects of intermittent normobaric hyperoxia alone or combined with chemotherapy on the growth, general morphology, oxidative stress, and apoptosis of benzo[a]pyrene (B[a]P)-induced lung tumors in mice. Methods: Female A/J mice were given a single dose of B[a]P and randomized into four groups: control, carboplatin (50 mg/kg intraperitoneally), hyperoxia (95% fraction of inspired oxygen), and carboplatin and hyperoxia. Normobaric hyperoxia (95%) was applied for 3 hours each day from weeks 21 to 28. Tumor load was determined as the average total tumor numbers and volumes. Several markers of oxidative stress and apoptosis were evaluated. Results: Intermittent normobaric hyperoxia combined with chemotherapy reduced the tumor number by 59% and the load by 72% compared with the control B[a]P group. Intermittent normobaric hyperoxia, either alone or combined with chemotherapy, decreased the levels of superoxide dismutase and glutathione and increased the levels of catalase and 8-hydroxydeoxyguanosine. The Bax/Bcl- 2 mRNA ratio, caspase 3 level, and number of transferase-mediated dUTP nick end-labeling positive cells increased following treatment with hyperoxia with or without chemotherapy. Conclusions: Intermittent normobaric hyperoxia was found to be tumoricidal and thus may serve as an adjuvant therapy for lung cancer. Oxidative stress and its effects on DNA are increased following exposure to hyperoxia and even more with chemotherapy, and this may lead to apoptosis of lung tumors.

Intratracheal Administration of Endotoxin Attenuates Hyperoxia-Induced Lung Injury in Neonatal Rats

심재원,장윤실,박원순 연세대학교의과대학 2008 Yonsei medical journal Vol.49 No.1

Purpose: This study was undertaken to determine the effects of intratracheal administration of endotoxin on hyperoxia-induced lung injury in neonatal rats. Materials and Methods: Newborn Sprague Dawley rat pups were divided into four experimental groups: normoxia control (NC), normoxia with endotoxin treatment (NE), hyperoxia control (HC), and hyperoxia with endotoxin treatment (HE) groups. In HC and HE, rat pups were subjected to 14 days of hyperoxia (>95% oxygen) within 12 hours after birth. In endotoxin treated group (NE and HE), Escherichia coli endotoxin (0.5μg in 0.03mL of saline) was given intratracheally at the 1st, 3rd and 5th postnatal day. Radial alveolar count (RAC), mean linear intercept (MLI), RAC/MLI ratios, and degree of fibrosis were measured to assess the changes in lung morphology. Results: During the research period, survival rates in both HC and HE were notably reduced 7 days after endotoxin was administered, but body weight gain was considerably reduced only in HC. On day 14, significant arrest in alveolarization, as evidenced by the decrease of RAC and RAC/MLI ratio and increase of MLI as well as increased fibrosis, were noted in HC. Although slight but significant arrest in alveolarization and increased fibrosis score were observed in NE compared to NC, the hyperoxia-induced lung damage observed in HC was significantly improved in HE. Conclusion: This study suggests that intratracheal administration of endotoxin significantly attenuated hyperoxia-induced lung injury in neonatal rats.

rIL-10 enhances IL-10 signalling proteins in foetal alveolar type II cells exposed to hyperoxia

Lee, Hyeon-Soo,Lee, Dong Gun John WileySons, Ltd 2015 JOURNAL OF CELLULAR AND MOLECULAR MEDICINE Vol.19 No.7

<P>Although the mechanisms by which hyperoxia promotes bronchopulmonary dysplasia are not fully defined, the inability to maintain optimal interleukin (IL)-10 levels in response to injury secondary to hyperoxia seems to play an important role. We previously defined that hyperoxia decreased IL-10 production and pre-treatment with recombinant IL-10 (rIL-10) protected these cells from injury. The objectives of these studies were to investigate the responses of IL-10 receptors (IL-10Rs) and IL-10 signalling proteins (IL-10SPs) in hyperoxic foetal alveolar type II cells (FATIICs) with and without rIL-10. FATIICs were isolated on embryonic day 19 and exposed to 65%-oxygen for 24 hrs. Cells in room air were used as controls. IL-10Rs protein and mRNA were analysed by ELISA and qRT-PCR, respectively. IL-10SPs were assessed by Western blot using phospho-specific antibodies. IL-10Rs protein and mRNA increased significantly in FATIICs during hyperoxia, but JAK1 and TYK2 phosphorylation showed the opposite pattern. To evaluate the impact of IL-8 (shown previously to be increased) and the role of IL-10Rs, IL-10SPs were reanalysed in IL-8-added normoxic cells and in the IL-10Rs’ siRNA-treated hyperoxic cells. The IL-10Rs’ siRNA-treated hyperoxic cells and IL-8-added normoxic cells showed the same pattern in IL10SPs with the hyproxic cells. And pre-treatment with rIL-10 prior to hyperoxia exposure increased phosphorylated IL-10SPs, compared to the rIL-10-untreated hyperoxic cells. These studies suggest that JAK1 and TYK2 were significantly suppressed during hyperoxia, where IL-8 may play a role, and rIL-10 may have an effect on reverting the suppressed JAK1 and TYK2 in FATIICs exposed to hyperoxia.</P>