Effect of Amygdalin on the Proliferation of Hyperoxia-exposed Type Ⅱ Alveolar Epithelial Cells Isolated from Premature Rat

Summary: The pathogenesis of hyperoxia lung injury and the mechanism of amygdalin on type 2 alveolar epithelial cells (AEC2) isolated from premature rat lungs in vitro were investigated. AEC2 were obtained by primary culture from 20-days fetal rat lung and hyperoxia-exposed cell model was established. Cell proliferating viability was examined by MTT assay after treatment of amygdalin at various concentrations. DNA content and the proliferating cell nuclear antigen (PCNA) protein expression of AEC2 were measured by using flow cytometry and immunocytochemistry respectively after 24 h of hyperoxia exposure or amygdalin treatment. The results showed that hyperoxia inhibited the proliferation and decreased PCNA protein expression in AEC2 of premature rat in vitro. Amygdalin at the concentration range of 50-200 μmol/L stimulated the proliferation of AEC2 in a dose-dependent manner, however, 400 μmol/L amygdalin inhibited the proliferation of AEC2. Amygdalin at the concentration of 200 μmol/L played its best role in facilitating proliferation of AEC2s in vitro and could partially ameliorated the changes of proliferation in hyperoxia exposed AEC2 of premature rat. It has been suggested that hyperoxia inhibited the proliferation of AEC2s of premature rat, which may contribute to hyperoxia lung injury. Amygdalin may play partial protective role in hyperoxia-induced lung injury.

Cigarette Smoke Extract Inhibits the Proliferation of Alveolar Epithelial Cells and Augments the Expression of P21^(WAF1)

Cigarette smoking is intimately related with the development of chronic obstructive pulmonary diseases, and alveolar epithelium is a major target for the exposure of cigarette smoke extract. In order to investigate the effect of cigarette smoke extract on the proliferation of alveolar epithelial cell type Ⅱ and its relationship with P21^WAF1, the alveolar epithelial type Ⅱ cell line （A549） cells were chosen as surrogate cells to represent alveolar epithelial type Ⅱ cells. MTT assay was used to detect cell viability after interfered with different concentrations of cigarette smoke extract. It was observed cigarette smoke extract inhibited the growth of A549 cells in a dose- and time-dependent manner. The morphological changes, involving the condensation and margination of nuclear chromatin, even karyorrhexis, were observed by both Hoechst staining and electronic microscopy. Flow cytometry analysis demonstrated the increased cell percentages in G1 and subG1 phases after the cells were incubated with cigarette smoke extract. The expression of p21^WAF1 protein and mRNA was also significantly increased as detected by the methods of Western blot or reverse transcription-polymerase chain reaction respectively. In conclusion, cigarette smoke extract inhibits the proliferation of alveolar epithelial cell type Ⅱ and blocks them in G1/S phase. The intracelhilar accumulation of P21^WAF1 may be one of the mechanisms which contribute to cigarette smoke extract-induced inhibition of cell proliferation.

Electrotaxis of alveolar epithelial cells in direct-current electric fields

Purpose:This study aims to elucidate the electrotaxis response of alveolar epithelial cells(AECs)in direct-current electric fields(EFs),explore the impact of EFs on the cell fate of AECs,and lay the foundation for future exploitation of EFs for the treatment of acute lung injury.Methods:AECs were extracted from rat lung tissues using magnetic-activated cell sorting.To elucidate the electrotaxis responses of AECs,different voltages of EFs(0,50,100,and 200 mV/mm)were applied to two types of AECs,respectively.Cell migrations were recorded and trajectories were pooled to better demonstrate cellular activities through graphs.Cell directionality was calculated as the cosine value of the angle formed by the EF vector and cell migration.To further demonstrate the impact of EFs on the pulmonary tissue,the human bronchial epithelial cells transformed with Ad12-SV402B(BEAS-2B cells)were obtained and experimented under the same conditions as AECs.To determine the influence on cell fate,cells underwent electric stimulation were collected to perform Western blot analysis.Results:The successful separation and culturing of AECs were confirmed through immunofluorescence staining.Compared with the control,AECs in EFs demonstrated a significant directionality in a voltage-dependent way.In general,type I alveolar epithelial cells migrated faster than type II alveolar epithelial cells,and under EFs,these two types of cells exhibited different response threshold.For type II alveolar epithelial cells,only EFs at 200 mV/mm resulted a significant difference to the velocity,whereas for,EFs at both 100 mV/mm and 200 mV/mm gave rise to a significant difference.Western blotting suggested that EFs led to an increased expression of a AKT and myeloid leukemia 1 and a decreased expression of Bcl-2-associated X protein and Bcl-2-like protein 11.Conclusion:EFs could guide and accelerate the directional migration of AECs and exert antiapoptotic effects,which indicated that EFs are important biophysical signals in the re-epithelialization of alveolar epithelium in lung injury.

Continuous purification and culture of rat type 1 and type 2 alveolar epithelial cells by magnetic cell sorting

Purpose:The incidence of acute lung injury(ALI)in severe trauma patients is 48%and the mortality rate following acute respiratory distress syndrome evolved from ALI is up to 68.5%.Alveolar epithelial type 1 cells(AEC1 s)and type 2 cells(AEC2s)are the key cells in the repair of injured lungs as well as fetal lung development.Therefore,the purification and culture of AECls and AEC2s play an important role in the research of repair and regeneration of lung tissue.Methods:Sprague-Dawley rats(3-4 weeks,120-150 g)were purchased for experiment.Dispase and DNase I were jointly used to digest lung tissue to obtain a single-cell suspension of whole lung cells,and then magnetic bead cell sorting was performed to isolate Tla positive cells as AECls from the single-cell suspension by using polyclonal rabbit anti-Tla(a specific AECls membrane protein)antibodies combined with anti-rabbit IgG microbeads.Afterwards,alveolar epithelial cell membrane marker protein EpCAM was designed as a key label to sort AEC2s from the remaining Tla-neg cells by another positive immunomagnetic selection using monoclonal mouse anti-EpCAM antibodies and anti-mouse IgG microbeads.Cell purity was identified by immunofluorescence staining and flow cytometry.Resii沾••The purity of AECls and AEC2s was 88.3%±3.8%and 92.6%±2.7%,respectively.The cell growth was observed as follows:AECls stretched within the 12-16 h,but the cells proliferated slowly;while AEC2s began to stretch after 24 h and proliferated rapidly from the 2nd day and began to differentiate after 3 days.Conclusion:AECls and AEC2s sorted by this method have high purity and good viability.Therefore,our method provides a new approach for the isolation and culture of AECls and AEC2s as well as a new strategy for the research of lung repair and regeneration.

Role of various imbalances centered on alveolar epithelial cell/fibroblast apoptosis imbalance in the pathogenesis of idiopathic pulmonary fibrosis

There have been recent extensive studies and rapid advancement on the pathogenesis underlying idiopathic pulmonary fibrosis(IPF),and intricate pathogenesis of IPF has been suggested.The purpose of this study was to clarify the logical relationship between these mechanisms.An extensive search was undertaken of the PubMed using the following keywords:"etiology,""pathogenesis,""alveolar epithelial cell(AEC),""fibroblast,""lymphocyte,""macrophage,""epigenomics,""histone,"acetylation,""methylation,""endoplasmic reticulum stress,""mitochondrial dysfunction,""telomerase,""proteases,""plasminogen,""epithelial-mesenchymal transition,""oxidative stress,""inflammation,""apoptosis,"and"idiopathic pulmonary fibrosis."This search covered relevant research articles published up to April 30,2020.Original articles,reviews,and other articles were searched and reviewed for content;240 highly relevant studies were obtained after screening.IPF is likely the result of complex interactions between environmental,genetic,and epigenetic factors:environmental exposures affect epigenetic marks;epigenetic processes translate environmental exposures into the regulation of chromatin;epigenetic processes shape gene expression profiles;in turn,an individual’s genetic background determines epigenetic marks;finally,these genetic and epigenetic factors act in concert to dysregulate gene expression in IPF lung tissue.The pathogenesis of IPF involves various imbalances including endoplasmic reticulum,telomere length homeostasis,mitochondrial dysfunction,oxidant/antioxidant imbalance,Th1/Th2 imbalance,M1-M2 polarization of macrophages,protease/antiprotease imbalance,and plasminogen activation/inhibition imbalance.These affect each other,promote each other,and ultimately promote AEC/fibroblast apoptosis imbalance directly or indirectly.Excessive AEC apoptosis and impaired apoptosis of fibroblasts contribute to fibrosis.IPF is likely the result of complex interactions between environmental,genetic,and epigenetic factors.The pathogenesis of IPF involves various imbalances centered on AEC/fibroblast apoptosis imbalance.

Perfluorocarbon attenuates lipopolysaccharide-mediated inflammatory responses of alveolar epithelial cells in vitro

Background Toll-like receptor-4 （TLR-4） is integrally involved in lipopolysaccharide （LPS） signaling and has a requisite role in the activation of nuclear factor-κB （NF-κB）. The exact mechanisms that lend perfluorocarbon （PFC） liquids a cytoprotective effect have yet to be elucidated. Therefore we examined in an in vitro model the cytoprotective effect of PFC on LPS-stimulated alveolar epithelial cellls （AECs）. Methods AECs （A549 cells, human lung adenocarcinoma cell line） were divided into four groups： control, PFC, LPS and LPS ＋ PFC （coculture group） groups. Intercellular adhesion molecule-1 （ICAM-1） was detected by ELISA, tumor necrosis factor-g （TNF-a） and interleukin-8 （IL-8） were detected by radioimmunological methods. The expression of TLR-4 mRNA and protein was detected by real time PCR and Western blotting, respectively. The activation of NF-κB was detected by Western blotting （proteins of I-κBa and NF-κB p65）. Results ICAM-1, TNF-a and IL-8 were significantly increased in LPS-stimulated AECs groups. The expression of TLR-4 mRNA and protein in LPS-stimulated groups was markedly increased. Meanwhile, NF-κB was activated as indicated by the significant degradation of IKB-a and the significant release of NF-κB P65 and its subsequent translocation into the nucleus. There were no significant effects of PFC alone on any of the factors studied while the coculture group showed significant downregulation of the secretion of ICAM-1, TNF-a and IL-8, the expression of TLR-4 rn^NA and the activity of NF-κB. Conclusions Taken together, our results demonstrate that LPS can induce AEC-related inflammatory injury via the activation of TLR-4 and subsequent activation of NF-κB. PFC is able to protect AECs from LPS-induced inflammatory injury by blocking the initiation of the LPS signaling pathway, which is indicated by the significant decrease of TLR-4 expression and NF-κB activation.

Induction of type Ⅱ alveolar epithelial cells apoptosis in mouse by lipopolysaccharide does not require TNF-α

To examine whether lipopolysaccharide (LPS)-induced apoptosis correlates with TNF-α release by type Ⅱ alveolar epithelial cells (AEC Ⅱ), whether TNF-α knockout (TNF KO) abrogates the induction of apoptosis by LPS and whether TNF-α is sufficient to induce apoptosis in this cell type Methods AEC Ⅱ were isolated from wild type mice and TNF KO mice Cells were stimulated with LPS or recombinant murine TNF-α for 24 h TNF-α in culture supernatant was determined by ELISA following LPS stimulation Apoptosis was determined by the terminal deoxynucleotidyl transferase end-labeling (TUNEL) assay after treatment with either LPS or TNF-α Results LPS induced apoptosis in wild type AEC Ⅱ in a concentration-dependent manner LPS-induced AEC Ⅱ apoptosis was accompanied by an 11-fold increase (from 0 073±0 065 ng/ml in control to 0 94±0 14 ng/ml in 50 μg/ml of LPS, P<0 01) in TNF-α release However, increasing concentrations (5 or 25 ng/ml) of recombinant murine TNF-α failed to induce AEC Ⅱ apoptosis In addition, apoptosis did occur in AEC Ⅱ isolated from TNF KO mice following LPS stimulation Conclusions This study confirms that LPS induces TNF-α release and apoptosis in murine AEC Ⅱ in vitro Exogenous TNF-α failed to induce AEC Ⅱ apoptosis, and apoptosis occurred following LPS stimulation in cells lacking the ability to produce TNF-α Taken together, these results suggest that LPS-induced AEC Ⅱ apoptosis occurs by a TNF-α-independent mechanism

Primary Culture of Alveolar Epithelial Type Ⅱ Cells and Its Bionomic Study

To establish a better method of primary culture for alveolar epithelial type Ⅱ cells （AEC Ⅱ ） and to study its bionomics, alveolar epithelial type Ⅱ cells were isolated by digestion with trypsin and collagenase, which were then purified by plated into culture flask coated with rat immunoglobulin （i The purified AEC Ⅱ were identified by alkaline phosphatase staining, electron mi- croscopy, immunocytochemical staining of pulmonary surfactant protein A （SPA）. The SPA expression and transfection characteristics were compared with those of A549 cell line. The results showed that AEC Ⅱ could be isolated by digestion with trysin and collagenase and purified by adhesive purification by using IgG, with a yield of about 2-3 × 10^7, and a purity of about 75%-84 %. Cells could be quickly identified with AKP staining. AEC Ⅱ were different from A549 cell line in terms of SPA expression and transfection characteristics. It is concluded that adhesive purification with IgG can improve the purity of AEC Ⅱ, and AKP staining is simple in cell identification. AEC Ⅱ can not be completely replaced by A549 cells in some studies because the differences between them, such as SPA expression.

Effects of Hyperoxia on Cytoplasmic Thioredoxin System in Alveolar Type Epithelial Cells of Premature Rats

This study investigated the effects of hyperoxia on dynamic changes of thioredoxin-1 （Trx1） and thioredoxin reductase-1 （TrxR1） in alveolar type Ⅱ epithelial cells （AECⅡ） of premature rats. Pregnant Sprague-Dawley rats were sacrificed on day 19 of gestation. AECⅡ were isolated and purified from the lungs of premature rats. When cultured to 80% confluence, in vitro cells were randomly divided into air group and hyperoxia group. Cells in the hyperoxia group were continuously exposed to 95% O2/5% CO2 and those in the air group to 95% air/5% CO2. After 12, 24 and 48 h, cells in the two groups were harvested to detect their reactive oxygen species （ROS）, apoptosis, TrxR1 activity and the expressions of Trx1 and TrxR1 by corresponding protocols, respectively. The results showed that AECⅡ exposed to hyperoxia generated excessive ROS and the apoptosis percentage in the hyperoxia group was increased significantly at each time points as compared with that in the air group （P0.001）. Moreover, TrxR1 activity was found to be markedly depressed in the hyperoxia group in comparison to that in the air group （P0.001）. RT-PCR showed the expressions of both Trx1 and TrxR1 mRNA were significantly increased in AECⅡ exposed to hyperoxia for 12 and 24 h （P0.01）, respectively. At 48 h, the level of Trx1 mRNA as well as that of TrxR1 mRNA in the hyperoxia group was reduced and showed no significant difference from that in the air group （P0.05）. Western blotting showed the changes of Trx1 protein expressions in the hyperoxia group paralleled those of Trx1 mRNA expressions revealed by RT-PCR. It was concluded that hyperoxia can up-regulate the protective Trx1/TrxR1 expressed by AECⅡ in a certain period, however, also cause dysfunction of the cytoplasmic thioredoxin system by decreasing TrxR1 activity, which may contribute to the progression of oxidative stress and cell apoptosis and finally result in lung injury.

Hypoxia upregulates hypoxia inducible factor(HIF)-3α expression in lung epithelial cells: characterization and comparison with HIF-1α

The role of the hypoxia-inducible factor(HIF)subunits 1α and 2α in response to hypoxia is well established in lungepithelial cells,whereas little is known about HIF-3α with respect to transcriptional and translational regulation by hy-poxia.HIF-3α and HIF-1α are two similar but distinct basic helix-loop-helix-PAS proteins,which have been postulatedto activate hypoxia responsive genes in response to hypoxia.Here,we used quantitative real time RT-PCR and immu-noblotting to determine the activation of HIF-3α vs.HIF-1α by hypoxia.HIF-3α was strongly induced by hypoxia(1%O_2)both at the level of protein and mRNA due to an increase in protein stability and transcriptional activation,whereasHIF-1α protein and mRNA levels enhanced transiently and then decreased because of a reduction in its mRNA stabilityin A549 cells,as measured on mRNA and protein levels.Interestingly,HIF-3α and HIF-1α exhibited strikingly similarresponses to a variety of activating or inhibitory pharmacological agents.These results demonstrate that HIF-3α is ex-pressed abundantly in lung epithelial cells,and that the transcriptional induction of HIF-3α plays an important role in theresponse to hypoxia in vitro.Our findings suggest that HIF-3α,as a member of the HIF system,is complementary ratherthan redundant to HIF-1α induction in protection against hypoxic damage in alveolar epithelial cells.

Deletion of SMARCA4 impairs alveolar epithelial type II cells proliferation and aggravates pulmonary fibrosis in mice

Alveolar epithelial cells(AECs)injury and failed reconstitution of the AECs barrier are both integral to alveolar flooding and subsequent pulmonary fibrosis(PF).Nevertheless,the exact mechanisms regulating the regeneration of AECs post-injury still remain unclear.SMARCA4 is a part of the large ATP-dependent chromatin remodelling complex SWI/SNF,which is essential for kidney and heart fibrosis.We investigates SMARCA4 function in lung fibrosis by establishing PF mice model with bleomycin firstly and found that the expression of SMARCA4 was mainly enhanced in alveolar type II(ATII)cells.Moreover,we established an alveolar epithelium-specific SMARCA4-deleted SP-C-rtTA/(tetO)7-Cre/SMARCA4f/f mice(SOSM4D/D)model,as well as a new SMARCA4-deleted alveolar type II(ATII)-like mle-12 cell line.We found that the bleomycin-induced PF was more aggressive in SOSM4D/D mice.Also,the proliferation of ATII cells was decreased with the loss of SMARCA4 in vivo and in vitro.In addition,we observed increased proliferation of ATII cells accompanied by abnormally high expression of SMARCA4 in human PF lung sections.These data uncovered the indispensable role of SMARCA4 in the proliferation of ATII cells,which might affect the progression of PF.

The cell cycle inhibitor P21 promotes the development of pulmonary fibrosis by suppressing lung alveolar regeneration

The cell cycle inhibitor P21 has been implicated in cell senescence and plays an important role in the injury-repair process following lung injury.Pulmonary fibrosis(PF)is a fibrotic lung disorder characterized by cell senescence in lung alveolar epithelial cells.In this study,we report that P21 expression was increased in alveolar epithelial type 2 cells(AEC2 s)in a time-dependent manner following multiple bleomycin-induced PF.Repeated injury of AEC2 s resulted in telomere shortening and triggered P21-dependent cell senescence.AEC2 s with elevated expression of P21 lost their self-renewal and differentiation abilities.In particular,elevated P21 not only induced cell cycle arrest in AEC2 s but also bound to P300 andβ-catenin and inhibited AEC2 differentiation by disturbing the P300-β-catenin interaction.Meanwhile,senescent AEC2 s triggered myofibroblast activation by releasing profibrotic cytokines.Knockdown of P21 restored AEC2-mediated lung alveolar regeneration in mice with chronic PF.The results of our study reveal a mechanism of P21-mediated lung regeneration failure during PF development,which suggests a potential strategy for the treatment of fibrotic lung diseases.

Establishment of Surfactant-associated Protein A Suicide Gene System and Analysis of Its Activity

Summary： Alveolar epithelial type II （AT II） cells are essential for lung development and remodeling, as they are precursors for type I cells and also produce other non-repair cells （fibroblasts）. Progenitor ceils are believed to possess capability of multi-potent transdifferentiation, which is closely related to the niche, suggesting the importance of establishment of a lung progenitor cell niche model. We hypothesized that pulmonary surfactant-associated protein A （SPA） suicide gene system would cause AT II cell to kill itself through apoptosis and leave its niche. In vitro, the recombinant adeno-associated virus vectors-SPA-thymidine kinase （rAAV-SPA-TK） system was established to get targeted apoptotic AT II cells. The apoptosis of AT II cells was detected by using MTT. The results showed that cloned SPA gene promoter had specific transcriptional activity in SPA high expression cells, and SPA high expression cells （H441） transfected with TK gene had higher sensitivity to ganciclovir （GCV） than SPA low expression cells （A549）. In vivo, increased apoptosis of AT II cells induced by GCV in rAAV-SPA-TK system was observed by TUNEL. Finally, the successful packaging and application of rAAV-SPA-TK system provide experimental basis to get specific lung progenitor cell （AT II） niche in vitro and in vivo.

Potential biomarkers for diagnosis and disease evaluation of idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis(IPF)is a chronic progressive lung disease characterized by progressive lung fibrogenesis and histological features of usual interstitial pneumonia.IPF has a poor prognosis and presents a spectrum of disease courses ranging from slow evolving disease to rapid deterioration;thus,a differential diagnosis remains challenging.Several biomarkers have been identified to achieve a differential diagnosis;however,comprehensive reviews are lacking.This review summarizes over 100 biomarkers which can be divided into six categories according to their functions:differentially expressed biomarkers in the IPF compared to healthy controls;biomarkers distinguishing IPF from other types of interstitial lung disease;biomarkers differentiating acute exacerbation of IPF from stable disease;biomarkers predicting disease progression;biomarkers related to disease severity;and biomarkers related to treatment.Specimen used for the diagnosis of IPF included serum,bronchoalveolar lavage fluid,lung tissue,and sputum.IPF-specific biomarkers are of great clinical value for the differential diagnosis of IPF.Currently,the physiological measurements used to evaluate the occurrence of acute exacerbation,disease progression,and disease severity have limitations.Combining physiological measurements with biomarkers may increase the accuracy and sensitivity of diagnosis and disease evaluation of IPF.Most biomarkers described in this review are not routinely used in clinical practice.Future large-scale multicenter studies are required to design and validate suitable biomarker panels that have diagnostic utility for IPF.

Ion therapy of pulmonary fibrosis by inhalation of ionic solution derived from silicate bioceramics

Pulmonary fibrosis(PF)is a chronic and progressively fatal disease,but clinically available therapeutic drugs are limited due to efficacy and side effects.The possible mechanism of pulmonary fibrosis includes the damage of alveolar epithelial cells II(AEC2),and activation of immune cells such as macrophages.The ions released from bioceramics have shown the activity in stimulating soft tissue derived cells such as fibroblasts,endothelia cells and epithelia cells,and regulating macrophage polarization.Therefore,this study proposes an“ion therapy”approach based on the active ions of bioceramic materials,and investigates the therapeutic effect of bioactive ions derived from calcium silicate(CS)bioceramics on mouse models of pulmonary fibrosis.We demonstrate that silicate ions significantly reduce pulmonary fibrosis by simultaneously regulating the functions of AEC2 and macrophages.This result suggests potential clinical applications of ion therapy for lung fibrosis.