Alveolar Epithelial Cell Injury Research Articles

METTL3 aggravates cell damage induced by Streptococcus pneumoniae via the NEAT1/CTCF/MUC19 axis.

Disruption of the alveolar barrier can trigger acute lung injury. This study elucidated the association of methyltransferase-like 3 (METTL3) with Streptococcus pneumoniae (SP)-induced apoptosis and inflammatory injury of alveolar epithelial cells (AECs). AECs were cultured and then infected with SP. Furthermore, the expression of METTL3, interleukin (IL)-10, IL-6, tumor necrosis factor-alpha (TNF-α), monocyte chemoattractant protein-1 (MCP-1), long noncoding RNA nuclear paraspeckle assembly transcript 1 (NEAT1), mucin 19 (MUC19), N6-methyladenosine (m6A), and NEAT1 after m6A modification were detected by qRT-PCR, Western blot, and enzyme-linked immunosorbent, m6A quantification, and methylated RNA immunoprecipitation-qPCR analyses, respectively. Moreover, the subcellular localization of NEAT1 was analyzed by nuclear/cytosol fractionation assay, and the binding between NEAT1 and CCCTC-binding factor (CTCF) was also analyzed. The results of this investigation revealed that SP-induced apoptosis and inflammatory injury in AECs and upregulated METTL3 expression. In addition, the downregulation of METTL3 alleviated apoptosis and inflammatory injury in AECs. METTL3-mediated m6A modification increased NEAT1 and promoted its binding with CTCF to facilitate MUC19 transcription. NEAT1 or MUC19 overexpression disrupted their protective role of silencing METTL3 in AECs, thereby increasing apoptosis and inflammatory injury. In conclusion, this is the first study to suggest that METTL3 aggravates SP-induced cell damage via the NEAT1/CTCF/MUC19 axis.

MiR-21-5p modulates LPS-induced acute injury in alveolar epithelial cells by targeting SLC16A10

Sepsis is a systemic inflammatory response syndrome resulting from the invasion of the human body by bacteria and other pathogenic microorganisms. One of its most prevalent complications is acute lung injury, which places a significant medical burden on numerous countries and regions due to its high morbidity and mortality rates. MicroRNA (miRNA) plays a critical role in the body's inflammatory response and immune regulation. Recent studies have focused on miR-21-5p in the context of acute lung injury, but its role appears to vary in different models of this condition. In the LPS-induced acute injury model of A549 cells, there is differential expression, but the specific mechanism remains unclear. Therefore, our aim is to investigate the changes in the expression of miR-21-5p and SLC16A10 in a type II alveolar epithelial cell injury model induced by LPS and explore the therapeutic effects of their targeted regulation. A549 cells were directly stimulated with 10 µg/ml of LPS to construct a model of LPS-induced cell injury. Cells were collected at different time points and the expression of interleukin 1 beta (IL-1β), tumor necrosis factor-α (TNF-α) and miR-21-5p were measured by RT-qPCR and western blot. Then miR-21-5p mimic transfection was used to up-regulate the expression of miR-21-5p in A549 cells and the expression of IL-1β and TNF-α in each group of cells was measured by RT-qPCR and western blot. The miRDB, TargetScan, miRWalk, Starbase, Tarbase and miR Tarbase databases were used to predict the miR-21-5p target genes and simultaneously, the DisGeNet database was used to search the sepsis-related gene groups. The intersection of the two groups was taken as the core gene. Luciferase reporter assay further verified SLC16A10 as the core gene with miR-21-5p. The expression of miR-21-5p and SLC16A10 were regulated by transfection or inhibitors in A549 cells with or without LPS stimulation. And then the expression of IL-1β and TNF-α in A549 cells was tested by RT-qPCR and western blot in different groups, clarifying the role of miR-21-5p-SLC16A10 axis in LPS-induced inflammatory injury in A549 cells. (1) IL-1β and TNF-α mRNA and protein expression significantly increased at 6, 12, and 24 h after LPS stimulation as well as the miR-21-5p expression compared with the control group (P < 0.05). (2) After overexpression of miR-21-5p in A549 cells, the expression of IL-1β and TNF-α was significantly reduced after LPS stimulation, suggesting that miR-21-5p has a protection against LPS-induced injury. (3) The core gene set, comprising 51 target genes of miR-21-5p intersecting with the 1448 sepsis-related genes, was identified. This set includes SLC16A10, TNPO1, STAT3, PIK3R1, and FASLG. Following a literature review, SLC16A10 was selected as the ultimate target gene. Dual luciferase assay results confirmed that SLC16A10 is indeed a target gene of miR-21-5p. (4) Knocking down SLC16A10 expression by siRNA significantly reduced the expression of IL-1β and TNF-α in A549 cells after LPS treatment (P < 0.05). (5) miR-21-5p inhibitor increased the expression levels of IL-1β and TNF-α in A549 cells after LPS stimulation (P < 0.05). In comparison to cells solely transfected with miR-21-5p inhibitor, co-transfection of miR-21-5p inhibitor and si-SLC6A10 significantly reduced the expression of IL-1β and TNF-α (P < 0.05). MiR-21-5p plays a protective role in LPS-induced acute inflammatory injury of A549 cells. By targeting SLC16A10, it effectively mitigates the inflammatory response in A549 cells induced by LPS. Furthermore, SLC16A10 holds promise as a potential target for the treatment of acute lung injury.

Vinpocetine alleviated alveolar epithelial cells injury in experimental pulmonary fibrosis by targeting PPAR-γ/NLRP3/NF-κB and TGF-β1/Smad2/3 pathways

This study aimed to investigate the potential anti-fibrotic activity of vinpocetine in an experimental model of pulmonary fibrosis by bleomycin and in the MRC-5 cell line. Pulmonary fibrosis was induced in BALB/c mice by oropharyngeal aspiration of a single dose of bleomycin (5 mg/kg). The remaining induced animals received a daily dose of pirfenidone (as a standard anti-fibrotic drug) (300 mg/kg/PO) and vinpocetine (20 mg/kg/PO) on day 7 of the induction till the end of the experiment (day 21). The results of the experiment revealed that vinpocetine managed to alleviate the fibrotic endpoints by statistically improving (P ≤ 0.05) the weight index, histopathological score, reduced expression of fibrotic-related proteins in immune-stained lung sections, as well as fibrotic markers measured in serum samples. It also alleviated tissue levels of oxidative stress and inflammatory and pro-fibrotic mediators significantly elevated in bleomycin-only induced animals (P ≤ 0.05). Vinpocetine managed to express a remarkable attenuating effect in pulmonary fibrosis both in vivo and in vitro either directly by interfering with the classical TGF-β1/Smad2/3 signaling pathway or indirectly by upregulating the expression of Nrf2 enhancing the antioxidant system, activating PPAR-γ and downregulating the NLRP3/NF-κB pathway making it a candidate for further clinical investigation in cases of pulmonary fibrosis.

Serum VEGF-A levels on admission in COVID-19 patients correlate with SP-D and neutrophils, reflecting disease severity: A prospective study

Background and objectiveThe coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in significant global morbidity and mortality. This study aimed to investigate the clinical significance of serum vascular endothelial growth factor A (VEGF-A) in COVID-19 patients and its association with disease severity and pulmonary injury. MethodsWe prospectively collected data from 71 hospitalized COVID-19 patients between June 2020 and January 2021. Patients were classified as either mild or severe based on their oxygen requirements during hospitalization. Serum VEGF-A levels were measured using an ELISA kit. ResultsIn comparison to mild cases, significantly elevated serum VEGF-A levels were observed in severe COVID-19 patients. Furthermore, VEGF-A levels exhibited a positive correlation with white blood cell count, neutrophil count, and lymphocyte count. Notably, serum surfactant protein-D (SP-D), an indicator of alveolar epithelial cell damage, was significantly higher in patients with elevated VEGF-A levels. ConclusionThese results suggest that elevated serum VEGF-A levels could serve as a prognostic biomarker for COVID-19 as it is indicative of alveolar epithelial cell injury caused by SARS-CoV-2 infection. Additionally, we observed a correlation between VEGF-A and neutrophil activation, which plays a role in the immune response during endothelial cell injury, indicating a potential involvement of angiogenesis in disease progression. Further research is needed to elucidate the underlying mechanisms of VEGF-A elevation in COVID-19.

Identification of non-coding RNA signatures in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a deadly, chronic, progressive, irreversible interstitial lung disease characterized by the formation of scar tissue resulting in permanent lung damage. The average survival time following diagnosis is only 3-5years, with a 5-year survival rate shorter than that of many cancers. Alveolar epithelial cell injury followed by irregular repair is the primary pathological process observed in patients with IPF. An evident characteristic of IPF is the development of fibroblastic foci representing active fibrotic areas. Most of the cells within these foci are believed to be myofibroblasts, which are thought to be the primary source of abnormal extracellular matrix production in IPF. The lung phenotype in IPF is characterized by significantly different processes from healthy lungs, including irregular apoptosis, oxidative stress, and epithelial-mesenchymal transition (EMT) pathways. The exact cause of IPF is not fully understood and remains mysterious. It is not suppressing that non-coding RNAs are involved in the development and progression of IPF. Accordingly, here we aimed to identify non-coding RNA molecules during TGFβ-induced myofibroblast activation. Differential expression and functional enrichment analysis were employed to reveal the impact of non-coding RNAs during TGFβ-associated lung fibrosis. Remarkably, LOC101448202, CZ1P-ASNS, LINC01503, IER3-AS1, MIR503HG, CLMAT3, LINC02593, ACTA2-AS1, LOC102723692, LOC107985728, and LOC105371064 were identified to be differentially altered during TGFβ-stimulated myofibroblast activation. These findings strongly suggest that the mechanism of lung fibrosis is heavily under control of non-coding RNAs, and RNA-based therapies could be a promising approach for future therapeutic interventions to lung fibrosis.

Insights into the mechanism of action of pterostilbene against influenza A virus-induced acute lung injury

BackgroundSevere respiratory system illness caused by influenza A virus infection is associated with excessive inflammation and abnormal apoptosis in alveolar epithelial cells (AEC). However, there are limited therapeutic options for influenza-associated lung inflammation and apoptosis. Pterostilbene (PTE, trans-3,5-dimethoxy-4-hydroxystilbene) is a dimethylated analog of resveratrol that has been reported to limit influenza A virus infection by promoting antiviral innate immunity, but has not been studied for its protective effects on virus-associated inflammation and injury in AEC. PurposeOur study aimed to investigate the protective effects and underlying mechanisms of PTE in modulating inflammation and apoptosis in AEC, as well as its effects on macrophage polarization during influenza virus infection. Study design and methodsA murine model of influenza A virus-mediated acute lung injury was established by intranasal inoculation with 5LD50 of mouse-adapted H1N1 viruses. Hematoxylin and eosin staining, immunofluorescence, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, western blotting, Luminex and flow cytometry were performed. ResultsPTE effectively mitigated lung histopathological changes and injury induced by H1N1 viruses in vivo. These beneficial effects of PTE were attributed to the suppression of inflammation and apoptosis in AEC, as well as the modulation of M1 macrophage polarization. Mechanistic investigations revealed that PTE activated the phosphorylated AMP-activated protein kinase alpha (P-AMPKα)/sirtui1 (Sirt1)/PPARγ coactivator 1-alpha (PGC1α) signal axis, leading to the inhibition of nuclear factor kappa-B (NF-κB) and p38 mitogen-activated protein kinase (MAPK) signaling induced by H1N1 viruses, thereby attenuating inflammation and apoptosis in AEC. PTE also forced activation of the P-AMPKα/Sirt1/PGC1α signal axis in RAW264.7 cells, counteracting the activation of phosphorylated signal transducer and activator of transcription 1 (P-STAT1) induced by H1N1 viruses and the augment of P-STAT1 activation in RAW264.7 cells with interferon-gamma (IFN-γ) pretreatment before viral infection, thereby reducing H1N1 virus-mediated M1 macrophage polarization as well as the enhancement of macrophages into M1 phenotypes elicited by IFN-γ pretreatment. Additionally, the promotion of the transition of macrophages towards the M2 phenotype by PTE was also related to activation of the P-AMPKα/Sirt1/PGC1α signal axis. Moreover, co-culturing non-infected AEC with H1N1 virus-infected RAW264.7 cells in the presence of PTE inhibited apoptosis and tight junction disruption, which was attributed to the suppression of pro-inflammatory mediators and pro-apoptotic factors in an AMPKα-dependent manner. ConclusionIn conclusion, our findings suggest that PTE may serve as a promising novel therapeutic option for treating influenza-associated lung injury. Its ability to suppress inflammation and apoptosis in AEC, modulate macrophage polarization, and preserve alveolar epithelial cell integrity highlights its potential as a therapeutic agent in influenza diseases.

IRF4-mediated Treg phenotype switching can aggravate hyperoxia-induced alveolar epithelial cell injury

Bronchopulmonary dysplasia (BPD) is characterized by alveolar dysplasia, and evidence indicates that interferon regulatory factor 4 (IRF4) is involved in the pathogenesis of various inflammatory lung diseases. Nonetheless, the significance and mechanism of IRF4 in BPD remain unelucidated. Consequently, we established a mouse model of BPD through hyperoxia exposure, and ELISA was employed to measure interleukin-17 A (IL-17 A) and interleukin-6 (IL-6) expression levels in lung tissues. Western blotting was adopted to determine the expression of IRF4, surfactant protein C (SP-C), and podoplanin (T1α) in lung tissues. Flow cytometry was utilized for analyzing the percentages of FOXP3+ regulatory T cells (Tregs) and FOXP3+RORγt+ Tregs in CD4+ T cells in lung tissues to clarify the underlying mechanism. Our findings revealed that BPD mice exhibited disordered lung tissue structure, elevated IRF4 expression, decreased SP-C and T1α expression, increased IL-17 A and IL-6 levels, reduced proportion of FOXP3+ Tregs, and increased proportion of FOXP3+RORγt+ Tregs. For the purpose of further elucidating the effect of IRF4 on Treg phenotype switching induced by hyperoxia in lung tissues, we exposed neonatal mice with IRF4 knockout to hyperoxia. These mice exhibited regular lung tissue structure, increased proportion of FOXP3+ Tregs, reduced proportion of FOXP3+RORγt+ Tregs, elevated SP-C and T1α expression, and decreased IL-17 A and IL-6 levels. In conclusion, our findings demonstrate that IRF4-mediated Treg phenotype switching in lung tissues exacerbates alveolar epithelial cell injury under hyperoxia exposure.

Circ_0114428 knockdown inhibits ROCK2 expression to assuage lipopolysaccharide-induced human pulmonary alveolar epithelial cell injury through miR-574-5p

BackgroundSepsis-induced acute lung injury (ALI) accounts for about 40% of ALI, accompanied by alveolar epithelial injury. The study aimed to reveal the role of circular RNA_0114428 (circ_0114428) in sepsis-induced ALI.MethodsHuman pulmonary alveolar epithelial cells (HPAEpiCs) were treated with lipopolysaccharide (LPS) to mimic a sepsis-induced ALI cell model. RNA expression of circ_0114428, miR-574-5p and Rho-associated coiled-coil containing protein kinase 2 (ROCK2) was detected by qRT-PCR. Protein expression was checked by Western blotting. Cell viability, proliferation and apoptosis were investigated by cell counting kit-8, 5-Ethynyl-29-deoxyuridine (EdU) and flow cytometry analysis, respectively. The levels of pro-inflammatory factors were detected by enzyme-linked immunosorbent assay (ELISA). Oxidative stress was analyzed by lipid peroxidation Malondialdehyde (MDA) and Superoxide Dismutase (SOD) activity detection assays. The interplay among circ_0114428, miR-574-5p and ROCK2 was identified by dual-luciferase reporter, RNA pull-down and RNA immunoprecipitation assays.ResultsCirc_0114428 and ROCK2 expression were significantly increased, but miR-574-5p was decreased in blood samples from sepsis patients and LPS-stimulated HPAEpiCs. LPS treatment led to decreased cell viability and proliferation and increased cell apoptosis, inflammation and oxidative stress; however, these effects were relieved after circ_0114428 knockdown. Besides, circ_0114428 acted as a miR-574-5p sponge and regulated LPS-treated HPAEpiC disorders through miR-574-5p. Meanwhile, ROCK2 was identified as a miR-574-5p target, and its silencing protected against LPS-induced cell injury. Importantly, circ_0114428 knockdown inhibited ROCK2 production by interacting with miR-574-5p.ConclusionCirc_0114428 knockdown protected against LPS-induced HPAEpiC injury through miR-574-5p/ROCK2 axis, providing a novel therapeutic target in sepsis-induced ALI.

FGF10 protects against LPS-induced epithelial barrier injury and inflammation by inhibiting SIRT1-ferroptosis pathway in acute lung injury in mice

Pulmonary alveolar epithelial cell injury is considered the main pathological and physiological change in acute lung injury. Ferroptosis in alveolar epithelial cells is one of crucial factors contributing to acute lung injury (ALI). Therefore, reducing ferroptosis and repair epithelial barrier is very necessary. More and more evidence suggested that FGF10 plays an important role in lung development and repair after injury. However, the relationship between FGF10 and ferroptosis remains unclear. This study aims to explore the regulatory role of FGF10 on ferroptosis in ALI. Differential gene expression analysis indicated that genes associated with ferroptosis showed that FGF10 can significantly alleviate LPS induced lung injury and epithelial barrier damage by decreasing levels of malonaldehyde(MDA), and lipid ROS. SIRT1 activator (Resveratrol) and inhibitor (EX527) are used in vivo showed that FGF10 protects ferroptosis of pulmonary epithelial cells through SIRT1 signal. Furthermore, knockdown of FGFR2 gene reduced the protective effect of FGF10 on acute lung injury in mice and SIRT1 activation. After the application of NRF2 inhibitor ML385 in vitro, the results showed that SIRT1 regulated the expression of ferroptosis related proteins NRF2, GPX4 and FTH1 are related to activation of NRF2. These data indicate that SIRT-ferroptosis was one of the critical mechanisms contributing to LPS-induced ALI. FGF10 is promising as a therapeutic candidate against ALI through inhibiting ferroptosis.

Quercetin alleviates lipopolysaccharide‑induced acute lung injury by inhibiting ferroptosis via the Sirt1/Nrf2/Gpx4 pathway.

Acute lung injury (ALI) causes high morbidity and mortality rates in critically ill patients, and there are currently no effective therapeutic drugs. Ferroptosis is a newly discovered mode of regulated cell death that contributes to the progression of ALI. Quercetin possesses anti‑inflammatory and antioxidant properties. However, whether quercetin can protect against lipopolysaccharide (LPS)‑induced ALI by inhibiting ferroptosis and its underlying mechanisms remains unclear. The present study evaluated the protective effects of quercetin and underlying molecular mechanisms in LPS‑induced ALI by establishing an ALI mouse model and an alveolar epithelial cell injury model via treatment of the mice or alveolar epithelial cells with LPS. Mouse lung injury was assessed by evaluating the histological lung injury score, bronchoalveolar lavage fluid cell count and inflammatory cytokine levels; alveolar epithelial cell injury was assessed by Cell counting kit‑8, lactate dehydrogenase and EDU assays; and ferroptosis was assessed by detecting the changes in the levels of malondialdehyde, glutathione, iron, glutathione peroxidase 4 (Gpx4) and 4‑hydroxynonenal invivo and vitro. The present study indicated that quercetin effectively ameliorated LPS‑induced ALI in the mouse model by reducing histopathological changes, proinflammatory cytokine release and reactive oxygen species generation and inhibiting ferroptosis. Quercetin significantly decreased ferroptosis and improved the proliferative ability of LPS‑treated alveolar epithelial cells. Additionally, it was demonstrated that quercetin markedly enhanced the alveolar epithelial barrier, as evidenced by the upregulation of tight junction protein expression both invivo and invitro. Mechanistically, quercetin effectively activated the sirtuin 1 (Sirt1)/nuclear factor erythroid 2‑related factor 2 (Nrf2)/Gpx4 signaling pathway, and targeted invivo inhibition or invitro knockdown of Sirt1 significantly reduced the anti‑ferroptotic functions of quercetin. In conclusion, the results demonstrated that quercetin exerts its therapeutic effects against LPS‑induced ALI by inhibiting ferroptosis via the activation of the Sirt1/Nrf2/Gpx4 signaling pathway.

The novel peptide DR4penA attenuates the bleomycin- and paraquat-induced pulmonary fibrosis by suppressing the TGF-β/Smad signaling pathway.

Pulmonary fibrosis (PF), which is caused by continuous alveolar epithelial cell injury and abnormal repair, is referred to as a difficult disease of the lung system by the World Health Organization due to its rapid progression, poor prognosis, and high mortality rate. However, there is still a lack of ideal therapeutic strategies. The peptide DR8 (DHNNPQIR-NH2 ), which is derived from rapeseed, exerted antifibrotic activity in the lung, liver, and kidney in our previous studies. By studying the structure-activity relationship and rational design, we introduced an unnatural hydrophobic amino acid (α-(4-pentenyl)-Ala) into DR8 and screened the novel peptide DR4penA (DHNα-(4-pentenyl)-APQIR-NH2 ), which had higher anti-PF activity, higher antioxidant activity and a longer half-life than DR8. Notably, DR4penA attenuated bleomycin- and paraquat-induced PF, and the anti-PF activity of DR4penA was equivalent to that of pirfenidone. Additionally, DR4penA suppressed the TGF-β/Smad pathway in TGF-β1-induced A549 cells and paraquat-induced rats. This study demonstrates that the novel peptide DR4penA is a potential candidate compound for PF therapy, and its antifibrotic activity in different preclinical models of PF provides a theoretical basis for further study.

Molecular markers of type II alveolar epithelial cells in acute lung injury by bioinformatics analysis

In this study, we aimed to identify molecular markers associated with type II alveolar epithelial cell injury in acute lung injury (ALI) models using bioinformatics methods. The objective was to provide new insights for the diagnosis and treatment of ALI/ARDS. We downloaded RNA SEQ datasets (GSE109913, GSE179418, and GSE119123) from the Gene Expression Omnibus (GEO) and used R language package to screen differentially expressed genes (DEGs). DEGs were annotated using Gene Ontology (GO), and their pathways were analyzed using Kyoto Encyclopedia of Genes and Genomes (KEGG). DEGs were imported into the STRING database and analyzed using Cytoscape software to determine the protein network of DEGs and calculate the top 10 nodes for the hub genes. Finally, potential therapeutic drugs for the hub genes were predicted using the DGIdb database. We identified 78 DEGs, including 70 up-regulated genes and 8 down-regulated genes. GO analysis revealed that the DEGs were mainly involved in biological processes such as granulocyte migration, response to bacterial-derived molecules, and cytokine-mediated signaling pathways. Additionally, they had cytokine activity, chemokine activity, and receptor ligand activity, and functioned in related receptor binding, CXCR chemokine receptor binding, G protein-coupled receptor binding, and other molecular functions. KEGG analysis indicated that the DEGs were mainly involved in TNF signaling pathway, IL-17 signaling pathway, NF-κB signal pathway, chemokine signal pathway, cytokine-cytokine receptor interaction signal pathway, and others. We identified eight hub genes, including IRF7, IFIT1, IFIT3, PSMB8, PSMB9, BST2, OASL2, and ZBP1, which were all up-regulated genes. We identified several hub genes of type II alveolar epithelial cells in ALI mouse models using bioinformatics analysis. These results provide new targets for understanding and treating of ALI.

Effects of the PI3K/Akt/HO-1 pathway on autophagy in a sepsis-induced acute lung injury mouse model

Sepsis-induced lung injury is an acute hypoxic respiratory insufficiency caused by systemic infectious factors that results in alveolar epithelial cell and capillary endothelial cell injury, diffuse pulmonary interstitial edema, and alveolar edema. Heme oxygenase (HO)-1 is usually associated with inflammation and has anti-inflammatory effects. Autophagy is a degradation pathway that eliminates cellular metabolic waste and plays an important protective role during stress. The phosphatidylinositol 3-kinase/ protein kinase B (PI3K/Akt) signaling pathway plays a key role in mediating cellular responses to inflammatory reactions. Therefore, we hypothesized that HO-1 is associated with autophagy and regulated by the PI3K/Akt signaling pathway in mice with sepsis-induced lung injury. Sepsis-induced lung injury was induced in mice using cecal ligation and puncture (CLP). Hemin or Sn-protoporphyrin IX (SnPP) was administered via intraperitoneal injection before surgery. Survival rates were observed during days 1–7 after the surgery; lung histology was discerned 24 h after the surgery; pro-inflammatory and anti-inflammatory factors in plasma and lung tissue were measured using enzyme-linked immunosorbent assay (ELISA); HO-1, Beclin-1, microtubule-associated protein 1 light chain 3B (LC3B)-II, p62 and lysosome associated membrane protein (LAMP)2 protein expression levels were measured 24 h after the surgery; HO-1 and LC3B-II protein expression levels were observed using immunofluorescence 24 h after the surgery; and autophagosomes were detected using electron microscopy 24 h after the surgery. Furthermore, when PI3K inhibitors LY294002, PI3K activators Recilisib and hemin were administered before the surgery, Akt, p-Akt, HO-1, and LC3-II levels were measured 24 h post-surgery. We found that HO-1 overexpression increased the survival rate and inhibited sepsis-induced lung injury. HO-1 overexpression attenuated the levels of proinflammatory cytokines (TNF-α, IL-1β) and increased the anti-inflammatory cytokine (IL-10, HO-1) overexpression. Moreover, HO-1 overexpression was also associated with increased expression of Beclin-1, LC3B-II and LAMP2 protein expression; decreased p62 protein expression; and significantly increased autophagosome formation. The results for HO-1-downregulated mice contrasted with those mentioned above. LY294002 inhibited p-Akt/Akt, HO-1, and LC3B-II protein expression; and hemin reversed the inhibitory effect of LY294002. The protective effect of HO-1 was involved in the mediation of autophagy, which may be regulated by the PI3K/Akt signaling pathway during sepsis-induced lung injury in mice.

Monocytes and Macrophages: Emerging Mechanisms and Novel Therapeutic Targets in Pulmonary Fibrosis.

Pulmonary fibrosis (PF) results from a plethora of abnormal pathogenetic events. In Idiopathic Pulmonary Fibrosis (IPF), inhalational, environmental, or occupational exposures in genetically and epigenetically predisposed individuals, triggers recurrent cycles of alveolar epithelial cell injury, activation of coagulation pathways, chemoattraction, and differentiation of monocytes into monocyte-derived alveolar macrophages (Mo-AMs). When these events happen intermittently and repeatedly throughout the individual's life cycle, the wound repair process becomes aberrant leading to bronchiolization of distal airspaces, fibroblast accumulation, extracellular matrix deposition and loss of the alveolar-capillary architecture. The role of immune dysregulation in IPF pathogenesis and progression has been underscored in the past mainly after the disappointing results of immunosuppressant use in IPF patients, however, recent reports highlighting the prognostic and mechanistic roles of monocytes and Mo-AMs revived the interest of immune dysregulation in IPF. In this review, we will discuss the role of these cells in the onset and progression of IPF, as well as potential targeted therapies.

LncRNA THRIL, transcriptionally activated by AP-1 and stabilized by METTL14-mediated m6A modification, accelerates LPS-evoked acute injury in alveolar epithelial cells

Acute lung injury (ALI) and its extreme manifestation, acute respiratory distress syndrome (ARDS), are life-threatening diseases in intensive care units. LncRNA THRIL plays a crucial role in regulating the inflammatory response; however, the potential function of THRIL in ALI/ARDS and the associated mechanism remain unclear. In our study, we found that THRIL was upregulated in the serum of ALI/ARDS patients, and its increased expression was positively correlated with the inflammatory cytokines IL-17. In LPS-induced A549 cells, knockdown of THRIL inhibited the release of the proinflammatory cytokines TNF-α, IL-1β, IL-17, and IL-6, decreased the number of monodansylcadaverine-positive cells and LC3-II with immunofluorescence staining, decreased the expression of autophagy marker ATG7 and Beclin1, and increased expression of p62. Mechanistically, the transcription factor AP-1 bound directly to the THRIL promoter region and activated its transcription by c-Jun upon LPS exposure. Moreover, m6A modification of THRIL was increased in LPS-treated A549 cells, and METTL14 knockdown significantly abolished m6A modification and reduced stabilization of THRIL mRNA. In conclusion, our findings reveal that THRIL, transcriptionally activated by AP-1 and modified by METTL14-mediated m6A modification, induces autophagy in LPS-treated A549 cells, suggesting the potential application of THRIL for ALI/ARDS therapy.

AMPK activation improves recovery from pneumonia-induced lung injury via reduction of er-stress and apoptosis in alveolar epithelial cells

BackgroundBacterial pneumonia and related lung injury are among the most frequent causes of mortality in intensive care units, but also inflict serious and prolonged respiratory complications among survivors. Given that endoplasmic reticulum (ER) stress is a hallmark of sepsis-related alveolar epithelial cell (AEC) dysfunction, we tested if AMP-activated protein kinase (AMPK) affects recovery from ER stress and apoptosis of AECs during post-bacterial infection.MethodsIn a murine model of lung injury by P. aeruginosa non-lethal infection, therapeutic interventions included AMPK activator metformin or GSK-3β inhibitor Tideglusib for 96 h. Recovery from AEC injury was evidenced by accumulation of soluble T-1α (AEC Type 1 marker) in BAL fluids along with fluorescence analysis of ER-stress (CHOP) and apoptosis (TUNEL) in lung sections. AMPK phosphorylation status and mediators of ER stress were determined via Immunoblot analysis from lung homogenates. Macrophage-dependent clearance of apoptotic cells was determined using flow cytometry assay.ResultsP. aeruginosa-induced lung injury resulted in accumulation of neutrophils and cellular debris in the alveolar space along with persistent (96 h) ER-stress and apoptosis of AECs. While lung infection triggered AMPK inactivation (de-phosphorylation of Thr172-AMPK), metformin and Tideglusib promptly restored the AMPK activation status. In post infected mice, AMPK activation reduced indices of lung injury, ER stress and related apoptosis of AECs, as early as 24 h post administration of AMPK activators. In addition, we demonstrate that the extent of apoptotic cell accumulation is also dependent on AMPK-mediated clearance of apoptotic cells by macrophages.ConclusionsOur study provides important insights into AMPK function in the preservation of AEC viability after bacterial infection, in particular due reduction of ER-stress and apoptosis, thereby promoting effective recovery from lung injury after pneumonia.

SnRNA-Seq analysis reveals ten hub genes associated with alveolar epithelial cell injury during pulmonary acute respiratory distress syndrome

BackgroundAlveolar epithelial cell injury is a key factor in the occurrence and development of pulmonary acute respiratory distress syndrome (ARDSp). Yet the gene expression profile of alveolar epithelial cells of patients with ARDSp remains unclear. MethodsWe analyzed single nuclear RNA sequencing (snRNA-Seq) data from autopsy lung tissues of both ARDSp patients and healthy donors. Sequence data for type 2 alveolar epithelizal cells (AT2) were extracted by the Seurat package. Differentially expressed genes (DEGs) in AT2 were identified by the criteria |log2FC| ≥ 0.25 and P < 0.05 with DESeq2. A protein interaction network was constructed using Search Tool for the Retrieval of Interacting Genes (STRING) and Cytoscape software to identify hub genes. We then constructed an ARDSp rat model through induction by lipopolysaccharide (LPS) airway instillation. Left lung RNA was extracted and sequenced via Illumina Hiseq platforms. Analysis of the rat RNA sequencing data was then used to verify hub genes. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed on the identified hub genes. ResultsIn AT2, a total of 289 genes were identified as differentially expressed between those from ARDSp patients and healthy donors, and these included 190 upregulated and 99 downregulated genes. Ten hub genes were further identified (RPS27A, ACTG1, CAV1, HSP90AA1, HSPA5, CCND1, ITGA3, B2M, NEDD4L, and SEMA5A). There was a similar expression trend of HSPA5 between rat RNA and snRNA sequencing data. DiscussionARDSp altered the gene expression profile of AT2. The identified hub genes were enriched in biological processes mainly involved in cell growth and transformation. Relatedly, ferroptosis and autophagy are possibly involved in AT2 injury during ARDSp. These novel insights into ARDSp may aid the discovery of potential targets for the diagnosis and treatment of ARDSp.

Sirtuin 6 ameliorates bleomycin-induced pulmonary fibrosis via activation of lipid catabolism.

Pulmonary fibrosis is a chronic and serious interstitial lung disease with little effective therapies currently. Our incomplete understanding of its pathogenesis remains obstacles in therapeutic developments. Sirtuin 6 (SIRT6) has been shown to mitigate multiple organic fibrosis. However, the involvement of SIRT6-mediated metabolic regulation in pulmonary fibrosis remains unclear. Here, we demonstrated that SIRT6 was predominantly expressed in alveolar epithelial cells in human lung tissues by using a single-cell sequencing database. We showed that SIRT6 protected against bleomycin-induced injury of alveolar epithelial cells in vitro and pulmonary fibrosis of mice in vivo. High-throughput sequencing revealed enriched lipid catabolism in Sirt6 overexpressed lung tissues. Mechanismly, SIRT6 ameliorates bleomycin-induced ectopic lipotoxicity by enhancing lipid degradation, thereby increasing the energy supply and reducing the levels of lipid peroxides. Furthermore, we found that peroxisome proliferator-activated receptor α (PPARα) was essential for SIRT6-mediated lipid catabolism, anti-inflammatory responses, and antifibrotic signaling. Our data suggest that targeting SIRT6-PPARα-mediated lipid catabolism could be a potential therapeutic strategy for diseases complicated with pulmonary fibrosis.

Wnt3a-Loaded Extracellular Vesicles Promote Alveolar Epithelial Regeneration after Lung Injury.

Compromised regeneration resulting from the deactivation of Wnt/β-catenin signaling contributes to the progression of chronic obstructive pulmonary disease (COPD) with limited therapeutic options. Extracellular cytokine-induced Wnt-based signaling provides an alternative option for COPD treatment. However, the hydrophobic nature of Wnt proteins limits their purification and use. This study devises a strategy to deliver the membrane-bound wingless-type MMTV integration site family, member 3A (Wnt3a) over a long distance by anchoring it to the surface of extracellular vesicles (EVs). The newly engineered Wnt3aWG EVs are generated by co-expressing Wnt3a with two genes encoding the membrane protein, WLS, and an engineered glypican, GPC6ΔGPI -C1C2. The bioactivity of Wnt3aWG EVs is validated using a TOPFlash assay and a mesoderm differentiation model of human pluripotent stem cells. Wnt3aWG EVs activate Wnt signaling and promote cell growth following human alveolar epithelial cell injury. In an elastase-induced emphysema model, impaired pulmonary function and enlarged airspace are greatly restored by the intravenous delivery of Wnt3aWG EVs. Single-cell RNA sequencing-based analyses further highlight that Wnt3aWG EV-activated regenerative programs are responsible for its beneficial effects. These findings suggest that EV-based Wnt3a delivery represents a novel therapeutic strategy for lung repair and regeneration after injury.

Antifibrotic mechanism of avitinib in bleomycin-induced pulmonary fibrosis in mice

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive interstitial lung disease characterized by alveolar epithelial cell injury and lung fibroblast overactivation. At present, only two drugs are approved by the FDA for the treatment of IPF, including the synthetic pyridinone drug, pirfenidone, and the tyrosine kinase inhibitor, nintedanib. Avitinib (AVB) is a novel oral and potent third-generation tyrosine kinase inhibitor for treating non-small cell lung cancer (NSCLC). However, the role of avitinib in pulmonary fibrosis has not yet been established. In the present study, we used in vivo and in vitro models to evaluate the role of avitinib in pulmonary fibrosis. In vivo experiments first verified that avitinib significantly alleviated bleomycin-induced pulmonary fibrosis in mice. Further in vitro molecular studies indicated that avitinib inhibited myofibroblast activation, migration and extracellular matrix (ECM) production in NIH-3T3 cells, mainly by inhibiting the TGF-β1/Smad3 signalling pathways. The cellular experiments also indicated that avitinib improved alveolar epithelial cell injury in A549 cells. In conclusion, the present findings demonstrated that avitinib attenuates bleomycin-induced pulmonary fibrosis in mice by inhibiting alveolar epithelial cell injury and myofibroblast activation.