Mouse Alveolar Epithelial Cells Research Articles

Mitochondrial dysfunction-associated alveolar epithelial senescence is involved in CdCl2-induced COPD-like lung injury

An earlier study found that respiratory cadmium chloride (CdCl2) exposure caused COPD-like lung injury. This study aimed to explore whether mitochondrial dysfunction-mediated alveolar epithelial senescence is involved in CdCl2-induced COPD-like lung injury. Adult C57BL/6 mice were exposed to CdCl2 (10 mg/L) aerosol for six months. Beta-galactosidase-positive cells, p21 and p16 were increased in CdCl2-exposed mouse lungs. The in vitro experiments showed that γ-H2AX was elevated in CdCl2-exposed alveolar epithelial cells. The cGAS-STING pathway was activated in CdCl2-exposed alveolar epithelial cells and mouse lungs. Cxcl1, Cxcl9, Il-10, Il-1β and Mmp2, several senescence-associated secretory phenotypes (SASP), were upregulated in CdCl2-exposed alveolar epithelial cells. Mechanistically, CdCl2 exposure caused SIRT3 reduction and mitochondrial dysfunction in mouse lungs and alveolar epithelial cells. The in vitro experiment found that Sirt3 overexpression attenuated CdCl2-induced alveolar epithelial senescence and SASP. The in vivo experiments showed that Sirt3 gene knockout exacerbated CdCl2-induced alveolar epithelial senescence, alveolar structure damage, airway inflammation and pulmonary function decline. NMN, an NAD+ precursor, attenuated CdCl2-induced alveolar epithelial senescence and SASP in mouse lungs. Moreover, NMN supplementation prevented CdCl2-induced COPD-like alveolar structure damage, epithelial-mesenchymal transition and pulmonary function decline. These results suggest that mitochondrial dysfunction-associated alveolar epithelial senescence is involved in CdCl2-induced COPD-like lung injury.

LPS-induced monocarboxylate transporter-1 inhibition facilitates lactate accumulation triggering epithelial-mesenchymal transformation and pulmonary fibrosis

The epithelial-mesenchymal transformation (EMT) process of alveolar epithelial cells is recognized as involved in the development of pulmonary fibrosis. Recent evidence has shown that lipopolysaccharide (LPS)-induced aerobic glycolysis of lung tissue and elevated lactate concentration are associated with the pathogenesis of sepsis-associated pulmonary fibrosis. However, it is uncertain whether LPS promotes the development of sepsis-associated pulmonary fibrosis by promoting lactate accumulation in lung tissue, thereby initiating EMT process. We hypothesized that monocarboxylate transporter-1 (MCT1), as the main protein for lactate transport, may be crucial in the pathogenic process of sepsis-associated pulmonary fibrosis. We found that high concentrations of lactate induced EMT while moderate concentrations did not. Besides, we demonstrated that MCT1 inhibition enhanced EMT process in MLE-12 cells, while MCT1 upregulation could reverse lactate-induced EMT. LPS could promote EMT in MLE-12 cells through MCT1 inhibition and lactate accumulation, while this could be alleviated by upregulating the expression of MCT1. In addition, the overexpression of MCT1 prevented LPS-induced EMT and pulmonary fibrosis in vivo. Altogether, this study revealed that LPS could inhibit the expression of MCT1 in mouse alveolar epithelial cells and cause lactate transport disorder, which leads to lactate accumulation, and ultimately promotes the process of EMT and lung fibrosis.

The suppression of HSPA8 attenuates NLRP3 ubiquitination through SKP2 to promote pyroptosis in sepsis-induced lung injury

BackgroundAcute lung injury (ALI) is strongly associated with hospitalization and mortality in patients with sepsis. Recent evidence suggests that pyroptosis mediated by NLRP3(NOD-, LRR- and pyrin domain-containing 3) inflammasome activation plays a key role in sepsis. However, the mechanism of NLRP3 inflammasome activation in sepsis-induced lung injury remains unclear.Resultsin this study, we demonstrated that NLRP3 inflammasome was activated by the down-regulation of heat shock protein family A member 8 (HSPA8) in Lipopolysaccharide (LPS) and adenosine triphosphate (ATP)-treated mouse alveolar epithelial cells (AECs). Geranylgeranylacetone (GGA)-induced HSPA8 overexpression in cecum ligation and puncture (CLP) mice could significantly reduce systemic inflammatory response and mortality, effectively protect lung function, whilst HSPA8 inhibitor VER155008 aggravated this effect. The inhibition of HSPA8 was involved in sepsis induced acute lung injury by promoting pyroptosis of AECs. The down-regulation of HSPA8 activated NLRP3 inflammasome to mediate pyroptosis by promoting the degradation of E3 ubiquitin ligase S-phase kinase-associated protein 2 (SKP2). In addition, when stimulated by LPS and ATP, down-regulated SKP2 promoted pyroptosis of AECs by further attenuating ubiquitination of NLRP3. Adeno-associated virus 9-SKP2(AAV9-SKP2) could promote NLRP3 ubiquitination and degradation, alleviate lung injury and inhibit systemic inflammatory response in vivo.Conclusionin summary, our study shows there is strong statistical evidence that the suppression of HSPA8 mediates alveolar epithelial pyroptosis by promoting the degradation of E3 ubiquitin ligase SKP2 and subsequently attenuating the ubiquitination of NLRP3 to activate the NLRP3 inflammasome, which provides a new perspective and therapeutic target for the treatment of sepsis-induced lung injury.

N-acetylcysteine Attenuates Cigarette Smoke-induced Alveolar Epithelial Cell Apoptosis through Reactive Oxygen Species Depletion and Glutathione Replenish In vivo and In vitro.

Chronic obstructive pulmonary disease (COPD) is the third leading cause of death worldwide. N-acetylcysteine (NAC) is well known for its antioxidant properties, along with potential protective effects on COPD. However, the molecular mechanism of NAC against the apoptosis of alveolar epithelial cells (AECs) in COPD remains unclear. This study aimed to explore the anti-apoptosis effect of NAC in COPD mice and alveolar epithelial cells. In the present study, the mouse model of COPD was established by cigarette smoke (CS), and mouse alveolar epithelial (MLE-12) cells were treated with cigarette smoke extract (CSE). TdT-mediated dUTP nick-end labeling (TUNEL) assay, reverse transcription polymerase chain reaction (RT-PCR), and western blot were performed to evaluate the effects of NAC on apoptosis, endoplasmic reticulum (ER) stress, and mitochondrial dysfunction. Meanwhile, LButhionine- sulfoximine (BSO), a glutathione (GSH) inhibitor, was used to uncover the mechanism of COPD treatment by NAC. We found that NAC pretreatment could attenuate the protein levels of apoptosis, ER stress, and mitochondrial dysfunction-related genes caused by CS in vivo. Meanwhile, CSE could decrease MLE-12 cell viability, which was prevented by apoptosis inhibitor ZVAD-FMK but not necroptosis inhibitor necrostatin-1. Pretreatment of MLE-12 cells with NAC increased cellular GSH levels, inhibited cellular and mitochondrial reactive oxygen species (ROS) accumulation, and decreased protein level of apoptosis, ER stress, and mitochondrial dysfunctionrelated genes. Moreover, experiment results showed that BSO could completely reverse the beneficial effects of NAC. Our study confirmed that NAC can attenuate CS-induced AEC apoptosis via alleviating ROS-mediated ER stress and mitochondrial dysfunction pathway, and the mechanism was found to be related to replenishing the cellular GSH content.

BPA promotes lung fibrosis in mice by regulating autophagy-dependent ferroptosis in alveolar epithelial cells

BackgroundBisphenol A (BPA) is an industrial chemical that is commonly found in daily consumer products. BPA is reportedly associated with lung diseases. However, the impact of BPA on pulmonary fibrosis (PF) and its possible mechanisms of action both remain unclear. MethodsA PF mouse model was induced by bleomycin (BLM). Mouse lung fibroblasts (MLG 2908) and mouse alveolar epithelial cells (MLE-12) were treated with BPA to establish a PF cell model. Tissue staining, CCK-8 assays, western blot experiments and relevant indicator kits were used to detect and evaluate the effect of BPA on PF. ResultsBPA dose-dependently promoted oxidative stress and induced ferroptosis, leading to PF. The ferroptosis inhibitor Fer-1 partly attenuated the effect of BPA. In addition, among the two main cell types associated with the progression of PF, MLE-12 cells are more sensitive to BPA than are MLG 2908 cells, and BPA induces ferroptosis in MLE-12 cells. Furthermore, BPA promoted autophagy-mediated ferroptosis by activating the AMPK/mTOR signaling pathway, thereby exacerbating the progression of PF. The autophagy inhibitor CQ1 partly attenuated the effect of BPA. ConclusionBPA promotes the progression of PF by promoting autophagy-dependent ferroptosis in alveolar epithelial cells, which provides a new theoretical basis for understanding BPA-induced PF.

GCN5L1 regulates pulmonary surfactant production by modulating lamellar body biogenesis and trafficking in mouse alveolar epithelial cells

BackgroundThe pulmonary surfactant that lines the air–liquid surface within alveoli is a protein–lipid mixture essential for gas exchange. Surfactant lipids and proteins are synthesized and stored in the lamellar body (LB) before being secreted from alveolar type II (AT2) cells. The molecular and cellular mechanisms that regulate these processes are incompletely understood. We previously identified an essential role of general control of amino acid synthesis 5 like 1 (GCN5L1) and the biogenesis of lysosome-related organelle complex 1 subunit 1 (BLOS1) in surfactant system development in zebrafish. Here, we explored the role of GCN5L1 in pulmonary surfactant regulation.MethodGCN5L1 knockout cell lines were generated with the CRISPR/Cas9 system. Cell viability was analyzed by MTT assay. Released surfactant proteins were measured by ELISA. Released surfactant lipids were measured based on coupled enzymatic reactions. Gene overexpression was mediated through lentivirus. The RNA levels were detected through RNA-sequencing (RNA-seq) and quantitative reverse transcription (qRT)- polymerase chain reaction (PCR). The protein levels were detected through western blotting. The cellular localization was analyzed by immunofluorescence. Morphology of the lamellar body was analyzed through transmission electron microscopy (TEM), Lysotracker staining, and BODIPY phosphatidylcholine labeling.ResultsKnocking out GCN5L1 in MLE-12 significantly decreased the release of surfactant proteins and lipids. We detected the downregulation of some surfactant-related genes and misregulation of the ROS–Erk–Foxo1–Cebpα axis in mutant cells. Modulating the activity of the axis or reconstructing the mitochondrial expression of GCN5L1 could partially restore the expression of these surfactant-related genes. We further showed that MLE-12 cells contained many LB-like organelles that were lipid enriched and positive for multiple LB markers. These organelles were smaller in size and accumulated in the absence of GCN5L1, indicating both biogenesis and trafficking defects. Accumulated endogenous surfactant protein (SP)-B or exogenously expressed SP-B/SP-C in adenosine triphosphate-binding cassette transporterA3 (ABCA3)-positive organelles was detected in mutant cells. GCN5L1 localized to the mitochondria and LBs. Reconstruction of mitochondrial GCN5L1 expression rescued the organelle morphology but failed to restore the trafficking defect and surfactant release, indicating specific roles associated with different subcellular localizations.ConclusionsIn summary, our study identified GCN5L1 as a new regulator of pulmonary surfactant that plays a role in the biogenesis and positioning/trafficking of surfactant-containing LBs.

CircUBR1 knockdown relieves ventilator-induced lung injury through regulating miR-20a-5p/GGPPS1 pathway

ObjectiveTo assess the influences and underlying mechanism of circular RNA UBR1 (circUBR1) in ventilator-induced lung injury (VILI). MethodsIn mice and mouse alveolar epithelial cells, VILI model was established. CircUBR1 and miR-20a-5p expression was assessed via quantitative real time polymerase chain reaction. Western blot and immunohistochemistry were applied to assess geranylgeranyl diphosphate synthase 1 (GGPPS1) protein expression. In lung tissues, the histopathological changes were utilized using hematoxylin and eosin staining. Cell counting kit-8 assay and flow cytometer were applied to detect cell proliferation and apoptosis. The levels of inflammatory cytokines [interleukin (IL)-1β, IL-18, IL-6, and tumor necrosis factor (TNF)-α] were measured by western blot and enzyme-linked immunosorbent assay. ResultsIn lung tissues of VILI mice, circUBR1 and GGPPS1 expression were upregulated, while miR-20a-5p expression was downregulated. In vivo, circUBR1 knockdown alleviated lung injury, inhibited cell apoptosis, and decreased the levels of inflammatory cytokines. In cells treated with cyclic stretch (CS), circUBR1 knockdown promoted cell viability, inhibited cell apoptosis, and reduced inflammatory cytokines. CircUBR1 could sponge miR-20a-5p, and GGPPS1 was the target gene of miR-20a-5p. In addition, in cells treated with CS, downregulation of miR-20a-5p or the overexpression of GGPPS1 reversed the promotive effect of circUBR1 knockdown on cell viability and the inhibitive effect of circUBR1 knockdown on cell apoptosis and inflammation production. ConclusionsIn VILI, knockdown of circUBR1 attenuated lung injury and inflammation via regulating the miR-20a-5p/GGPPS1 pathway. Our study may provide a potential therapeutic target for treatment of VILI.

Engineering single-domain antibodies targeting Gasdermin E activation by the chemotherapeutic agent cis-diaminodichloroplatinum.

As mediators of pyroptosis, gasdermins (GSDMs) are closely associated with systemic cytotoxicity or so-called side effects and are also involved in the inflammatory response during chemotherapy. Using in situ proximity ligation assay followed by sequencing (isPLA-seq), which we recently developed, we screened a single-domain antibody (sdAb) library and identified several sdAbs against Gasdermin E (GSDME) that specifically recognize the N-terminal domain (1-270 aa) of GSDME (GSDME-NT). One of them mitigated the release of inflammatory damage-associated molecular patterns (DAMPs) and cytokines, including high mobility group protein b1 (Hmgb1) and interleukin-1β (Il-1β), in isolated mouse alveolar epithelial cells (AECs) cells upon chemotherapeutic agent cis-diaminodichloroplatinum (CDDP) treatment. Further investigation showed that this anti-GSDME sdAb also alleviated CDDP-induced pyroptotic cell death and lung tissue injury and decreased systemic Hmgb1 release in C57/BL6 mice, due to GSDME inactivation. Collectively, our data define an inhibitory role of the specific sdAb against GSDME, providing a potential strategy for systemically alleviating chemotherapeutic toxicities in vivo. This article is protected by copyright. All rights reserved.

IFN-γ mediated epithelial injury during influenza pathogenesis

Abstract Influenza pneumonia is developed due to an exuberant host response accompanied by an impaired repair, causing profound airway lung and vascular injuries that impact gas exchange and require hospitalization. However, a significant knowledge gap remains in delineating the host response mechanisms regulating the intricate inflammatory balance in the lung that either favor viral clearance or develops into immune-mediated lung pathology. Substantial emerging data reveal that dysregulated interferon (IFN) signaling, including excessive IFN-II (IFN-g) production, promotes lung injury and impairs lung repair during influenza pathogenesis. In our ongoing work, we have discovered that IFN-g exerts a direct pathologic effect by promoting epithelial injury resulting in the exacerbation of pulmonary inflammation and loss of barrier integrity during influenza. The notable findings from our study are as follows. a.IFN-g causes the cell death of infected (IAV) and un-infected (bystander) alveolar epithelial cells at a comparable rate, contributing to the disruption of pulmonary barrier. b.Molecularly , we identified that IFN-g suppressed IL6-STAT3 axis, which exerts a protective effect against influenza epithelial injury. c.Notably, the protective effect of IFN-g deficiency was abrogated by neutralizing IL-6. d.Finally, using in vitrohuman and mouse alveolar epithelial cells we show that IL-6 treatment can ameliorate IFN-g mediated injury of IAV-infected or un-infected epithelial cells (bystander injury). Our data is the first to demonstrate suppression of the IL-6-STAT3 axis as a potential molecular basis of pathologic IFN-g response in lung inflammation and repair to the best of our understanding. National Institute of Health

Effect of FTY-720 on Pulmonary Fibrosis in Mice via the TGF-β1 Signaling Pathway and Autophagy.

We investigated whether FTY-720 might have an effect on bleomycin-induced pulmonary fibrosis through inhibiting TGF-β1 pathway, and up-regulating autophagy. The pulmonary fibrosis was induced by bleomycin. FTY-720 (1 mg/kg) drug was intraperitoneally injected into mice. Histological changes and inflammatory factors were observed, and EMT and autophagy protein markers were studied by immunohistochemistry and immunofluorescence. The effects of bleomycin on MLE-12 cells were detected by MTT assay and flow cytometry, and the related molecular mechanisms were studied by Western Blot. FTY-720 considerably attenuated bleomycin-induced disorganization of alveolar tissue, extracellular collagen deposition, and α-SMA and E-cadherin levels in mice. The levels of IL-1β, TNF-α, and IL-6 cytokines were attenuated in bronchoalveolar lavage fluid, as well as protein content and leukocyte count. COL1A1 and MMP9 protein expressions in lung tissue were significantly reduced. Additionally, FTY-720 treatment effectively inhibited the expressions of key proteins in TGF-β1/TAK1/P38MAPK pathway and regulated autophagy proteins. Similar results were additionally found in cellular assays with mouse alveolar epithelial cells. Our study provides proof for a new mechanism for FTY-720 to suppress pulmonary fibrosis. FTY-720 is also a target for treating pulmonary fibrosis.

Ginseng saponin metabolite 20(S)-protopanaxadiol relieves pulmonary fibrosis by multiple-targets signaling pathways

BackgroundPanax ginseng Meyer is a representative Chinese herbal medicine with antioxidant and anti-inflammatory activity. 20(S)-Protopanaxadiol (PPD) has been isolated from ginseng and shown to have promising pharmacological activities. However, effects of PDD on pulmonary fibrosis (PF) have not been reported. We hypothesize that PDD may reverse inflammation-induced PF and be a novel therapeutic strategy. MethodsAdult male C57BL/6 mice were used to establish a model of PF induced by bleomycin (BLM). The pulmonary index was measured, and histological and immunohistochemical examinations were made. Cell cultures of mouse alveolar epithelial cells were analyzed with Western blotting, co-immunoprecipitation, immunofluorescence, immunohistochemistry, siRNA transfection, cellular thermal shift assay and qRT-PCR. ResultsThe survival rate of PPD-treated mice was higher than that of untreated BLM-challenged mice. Expression of fibrotic hallmarks, including α-SMA, TGF-β1 and collagen I, was reduced by PPD treatment, indicating attenuation of PF. Mice exposed to BLM had higher STING levels in lung tissue, and this was reduced by phosphorylated AMPK after activation by PPD. The role of phosphorylated AMPK in suppressing STING was confirmed in TGF-β1-incubated cells. Both in vivo and in vitro analyses indicated that PPD treatment attenuated BLM-induced PF by modulating the AMPK/STING signaling pathway. ConclusionPPD ameliorated BLM-induced PF by multi-target regulation. The current study may help develop new therapeutic strategies for preventing PF.

CircHECTD1 attenuates apoptosis of alveolar epithelial cells in acute lung injury

AbstractCircular RNAs (circRNAs) play important roles in many lung diseases. This study aimed to investigate the role of circHECTD1 in acute lung injury (ALI). The mouse and cell models of ALI were induced by lipopolysaccharide (LPS). The apoptosis of alveolar epithelial cells (AECs) was detected by flow cytometry. The relationships between circHECTD1, miRNAs, and target genes were assessed by RNA pull-down, luciferase reporter gene, and RNA-FISH assays. circHECTD1 was downregulated in LPS-induced human and mouse AECs (HBE and MLE-12). The knockdown of circHECTD1 increased the apoptotic rates and the expressions of miR-136 and miR-320a, while its overexpression caused opposite effects in LPS-induced HBE and MLE-12 cells. Mechanistically, circHECTD1 bound to miR-320a and miR-136. miR-320a targeted PIK3CA and mediated the effect of circHECTD1 on PIK3CA expression. miR-136 targeted Sirt1 and mediated the effect of circHECTD1 on Sirt1 expression. Silencing PIK3CA and/or Sirt1 reversed the effect of circHECTD1 overexpression on the apoptosis of LPS-induced HBE and MLE-12 cells. In vivo, overexpression of circHECTD1 alleviated the LPS-induced ALI of mice. Our findings suggested that circHECTD1 inhibits the apoptosis of AECs through miR-320a/PIK3CA and miR-136/Sirt1 pathways in LPS-induced ALI.

Bryodulcosigenin attenuates bleomycin-induced pulmonary fibrosis via inhibiting AMPK-mediated mesenchymal epithelial transition and oxidative stress.

Fibrosis is a pathological result of a dysfunctional repair response to tissue injury and occurs in several organs, including the lungs. Bryodulcosigenin (BDG) is a cucurbitane-type triterpene isolated from Siratia grosvenori and has clear-cut anti-inflammatory effects, yet its benefit of pulmonary fibrosis (PF) remains unclear. In this study, we investigated the protective effects of BDG (10mg/kg/day, for 14 days) against TGF-β1-stimulated mouse alveolar epithelial MLE-12 cells and bleomycin (BLM)-induced PF mice. In vitro experiments showed that BDG could inhibit epithelial-mesenchymal transition (EMT) and oxidative stress. In vivo experiments indicated that BDG could ameliorate BLM-induced PF in mice as evidenced by characteristic structural changes in histopathology, increased collagen deposition and reduced survival and weight of mice. The abnormal increased expressions of TGF-β1, p-Smad2/3, α-SMA, COL-I, and NOX4 and decreased expressions for Sirt1 and p-AMPK were improved in BDG treatment. But these beneficial effects could be eliminated by co-treatment with Compound C (CC, a selective AMPK inhibitor). Molecular docking technology also revealed the potential of BDG to activate AMPK. In summary, AMPK activation modulated by BDG not only ameliorated TGF-β1/Smad2/3 signaling pathways but also partially mediated the suppression effects on EMT and oxidative stress, thus mediating the anti-fibrotic effects.

BML-111 alleviates inflammatory response of alveolar epithelial cells via miR-494/Slit2/Robo4 signalling axis to improve acute lung injury

Acute lung injury (ALI) is a common, variously induced lung cell injury with high mortality. It is also an early stage of acute respiratory distress syndrome. BML-111 is a lipoxin A4 receptor agonist that plays an important role in inflammation. However, its function on ALI remains unclear. To explore whether BML-111 is involved in ALI and its regulatory molecular mechanism, we constructed an in vitro ALI model by stimulating primary mouse alveolar epithelial cells (AECs) with lipopolysaccharide (LPS). The downstream target of microRNA (miR)-494 was predicted by Targetscan. The apoptosis and expression of inflammatory cytokines were analysed by RT-qPCR, Western blot, and ELISA. BML-111 treatment alleviated LPS-induced apoptosis and the production of inflammatory cytokines, such as tumour necrosis factor α, interleukin (IL)-6, IL-1β, in primary mouse AECs via downregulating miR-494. MiR-494 targeted and downregulated slit guidance ligand 2 (Slit2) in primary mouse AECs. BML-111 activated the Slit2/roundabout guidance receptor 4 (Robo4) axis via downregulating miR-494 to reduce LPS-induced damage in AECs. This study elucidated that miR-494 on BML-111 alleviated LPS-induced ALI in primary mouse AECs via downregulating miR-494 and subsequently activating the Slit2/Robo4 axis. These findings provided a new idea for the prevention and treatment of ALI and respiratory distress syndrome.

Punicalagin suppresses inflammation in ventilator-induced lung injury through protease-activated receptor-2 inhibition-induced inhibition of NLR family pyrin domain containing-3 inflammasome activation.

Punicalagin is recorded to be a potent anti-inflammatory drug, while its effect on inflammation existing in ventilator-induced lung injury (VILI) requires further verification. Rats were pretreated with punicalagin, followed by VILI modeling. Lung histopathological examination was performed with hematoxylin-eosin staining accompanied by the lung injury score. The lung wet/dry (W/D) weight ratio and total bronchoalveolar lavage fluid (BALF) protein level were measured. After transfection with protease-activated receptor-2 (PAR2) overexpression plasmids, mouse alveolar epithelial MLE-12 cells were treated with punicalagin and then subjected to cyclic stretching. Punicalagin's cytotoxicity to MLE-12 cells were measured by MTT assay. The levels of inflammatory cytokines (tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6), PAR2, NLR family pyrin domain containing-3 (NLRP3), and apoptosis-associated speck-like protein containing a CARD (ASC) in the BALF, lung tissues or cells were analyzed by enzyme-linked immune-sorbent assay (ELISA), qRT-PCR or/and western blot. Punicalagin treatment attenuated VILI-induced lung histopathological changes and counteracted VILI-induced increases in the lung injury score, W/D weight ratio and total protein level in BALF. Also, punicalagin treatment counteracted in vivo VILI/cyclic stretching-induced increases in the levels of PAR2, inflammatory cytokines, NLRP3, and ASC. PAR2 overexpression potentiated the cyclic stretching-induced effects, while punicalagin treatment revoked this PAR2 overexpression-induced potentiation effect. In turn, PAR2 overexpression partly resisted the punicalagin treatment-induced counteractive effects on the cyclic stretching-induced effects. Punicalagin suppresses inflammation in VILI through PAR2 inhibition-induced inhibition of NLRP3 inflammasome activation.

Beneficial effects of aloperine on inflammation and oxidative stress by suppressing necroptosis in lipopolysaccharide-induced acute lung injury mouse model

RationaleAlveolar epithelial cell death, inflammation, and oxidative stress are typical features of acute lung injury (ALI). Aloperine (Alo), an alkaloid isolated from Sophora alopecuroides, has been reported to display various biological effects, such as anti-inflammatory, immunoregulatory, and anti-oxidant properties. In this study, we investigated the effects and mechanisms of Alo in treating a lipopolysaccharide (LPS)-induced ALI in a murine model. MethodsThe effects of Alo in LPS-induced ALI were investigated in C57BL/6 mice. The RIPK1 inhibitor (Nec-1) and the RIPK3 inhibitor (GSK’872) were used to evaluate the relationship of necroptosis, NF-κB activation, and PDC subunits in LPS-treated mouse alveolar epithelial cells (MLE-12). Then the effects of Alo on necroptosis, inflammation, and oxidative stress of LPS-stimulated MLE-12 cells were evaluated. ResultsAlo significantly attenuated histopathological lung injuries and reduced lung wet/dry ratio in LPS-induced ALI mice. Alo also remarkedly reduced total protein and neutrophils recruitment in bronchoalveolar lavage fluid of ALI mice. Meanwhile, Alo ameliorated the LPS-induced necroptosis in the lungs of ALI mice. The RIPK3 inhibitor GSK’872, but not the RIPK1 inhibitor Nec-1, reversed LPS-induced p65 phosphorylation and translocation to the nucleus in MLE-12 cells. GSK’872 also reversed the LPS-induced increase in ROS and binding of RIPK3 and PDC subunits in MLE-12 cells. Moreover, Alo down-regulated the levels of p-RIPK1, p-RIPK3, p-MLKL, p-p65, the translocation of p65 to the nucleus, and reduced the expression of IL-6 and IL-8 in LPS-stimulated MLE-12 cells. Alo also inhibited the binding of RIPK3 and PDC-E1α, PDC-E1β, PDC-E2, and PDC-E3 and the ROS production in LPS-treated MLE-12 cells. ConclusionThe present study validated the beneficial effects of Alo on LPS-induced ALI , suggesting Alo may be a new drug candidate against ALI.

A model of the aged lung epithelium in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is an age-related disorder that carries a universally poor prognosis and is thought to arise from repetitive micro injuries to the alveolar epithelium. To date, a major factor limiting our understanding of IPF is a deficiency of disease models, particularly in vitro models that can recapitulate the full complement of molecular attributes in the human condition. In this study, we aimed to develop a model that more closely resembles the aberrant IPF lung epithelium. By exposing mouse alveolar epithelial cells to repeated, low doses of bleomycin, instead of usual one-time exposures, we uncovered changes strikingly similar to those in the IPF lung epithelium. This included the acquisition of multiple phenotypic and functional characteristics of senescent cells and the adoption of previously described changes in mitochondrial homeostasis, including alterations in redox balance, energy production and activity of the mitochondrial unfolded protein response. We also uncovered dramatic changes in cellular metabolism and detected a profound loss of proteostasis, as characterized by the accumulation of cytoplasmic protein aggregates, dysregulated expression of chaperone proteins and decreased activity of the ubiquitin proteasome system. In summary, we describe an in vitro model that closely resembles the aberrant lung epithelium in IPF. We propose that this simple yet powerful tool could help uncover new biological mechanisms and assist in developing new pharmacological tools to treat the disease.

The Spike Glycoprotein of SARS-CoV-2 Binds to β1 Integrins Expressed on the Surface of Lung Epithelial Cells.

The spike glycoprotein attached to the envelope of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) binds to and exploits angiotensin-converting enzyme 2 (ACE2) as an entry receptor to infect pulmonary epithelial cells. A subset of integrins that recognize the arginyl–glycyl–aspartic acid (RGD) sequence in the cognate ligands has been predicted in silico to bind the spike glycoprotein and, thereby, to be exploited for viral infection. Here, we show experimental evidence that the β1 integrins predominantly expressed on human pulmonary epithelial cell lines and primary mouse alveolar epithelial cells bind to this spike protein. The cellular β1 integrins support adhesive interactions with the spike protein independently of ACE2, suggesting the possibility that the β1 integrins may function as an alternative receptor for SARS-CoV-2, which could be targeted for the prevention of viral infections.

Studies on the effects of bone marrow stem cells on mitochondrial function and the alleviation of ARDS.

Mesenchymal stem cells (MSCs) can alleviate acute respiratory distress syndrome (ARDS), but the mechanisms involved are unclear, especially about their specific effects on cellular mitochondrial respiratory function. Thirty mice were allocated into the Control, LPS, and LPS + Bone marrow mesenchymal stem cell (BMSC) group (n = 10/group). Mouse alveolar epithelial cells (MLE-12) and macrophage cells (RAW264.7) were divided into the same groups. Pathological variation, inflammation-related factors, reactive oxygen species (ROS), ATP levels, and oxygen consumption rate (OCR) were analyzed. Pathologic features of ARDS were observed in the LPS group and were significantly alleviated by BMSCs. The trend in inflammation-related factors among the three groups was the LPS group > LPS + BMSC group > Control group. In the MLE-12 co-culture system, IL-6 was increased in the LPS group but not significantly reduced in the LPS + BMSC group. In the RAW264.7 co-culture system, IL-1β, TNF-α, and IL-10 levels were all increased in the LPS group, IL-1β and TNF-α levels were reduced by BMSCs, while IL-10 level kept increasing. ROS and ATP levels were increased and decreased respectively in both MLE-12 and RAW264.7 cells in the LPS groups but reversed by BMSCs. Basal OCR, ATP-linked OCR, and maximal OCR were lower in the LPS groups. Impaired basal OCR and ATP-linked OCR in MLE-12 cells were partially restored by BMSCs, while impaired basal OCR and maximal OCR in RAW264.7 cells were restored by BMSCs. BMSCs improved the mitochondrial respiration dysfunction of macrophages and alveolar epithelial cells induced by LPS, alleviated lung tissue injury, and inflammatory response in a mouse model of ARDS.

ATF4 Mediates Mitochondrial Unfolded Protein Response in Alveolar Epithelial Cells.

Although endoplasmic reticulum (ER) unfolded protein response (UPRER) is well known, mitochondrial unfolded protein response (UPRmt) has not been recognized in alveolar epithelial cells. Furthermore, ER stress and mitochondrial dysfunction are frequently encountered in alveolar epithelial cells from an array of lung disorders. However, these two scenarios have been often regarded as separate mechanisms contributing to the pathogeneses. It is unclear whether there is interplay between these two phenomena or an integrator that couples these two signaling cascades in the stressed alveolar epithelial cells from those pathologies. In this study, we defined UPRmt in alveolar epithelial cells and identified ATF4 (activating transcription factor 4), but not ATF5, as the key regulator of UPRmt. We found that UPRER led to UPRmt and mitochondrial dysfunction in an ATF4-dependent manner. In contrast, mitochondrial stresses did not activate UPRER. We found that alveolar epithelial ATF4 and UPRmt were induced in aged mice with experimental pulmonary fibrosis as well as in patients with idiopathic pulmonary fibrosis. Finally, we found that the inducible expression of ATF4 in mouse alveolar epithelial cells aggravated pulmonary UPRmt, lung inflammation, body weight loss, and death upon bleomycin-induced lung injury. In conclusion, ER stress induces ATF4-dependent UPRmt and mitochondrial dysfunction, indicating a novel mechanism by which ER stress contributes to the pathogeneses of a variety of pulmonary disorders.