Bleomycin induces senescence and repression of DNA repair via downregulation of Rad51.

BACKGROUND: Bleomycin, a potent antitumor agent, is limited in clinical use due to the potential for fatal pulmonary toxicity. The accelerated DNA damage and senescence in alveolar epithelial cells (AECs) is considered a key factor in the development of lung pathology. Understanding the mechanisms for bleomycin-induced lung injury is crucial for mitigating its adverse effects. METHODS: Human lung epithelial (A549) cells were exposed to bleomycin and subsequently assessed for cellular senescence, DNA damage, and double-strand break (DSB) repair. The impact of Rad51 overexpression on DSB repair and senescence in AECs was evaluated in vitro. Additionally, bleomycin was intratracheally administered in C57BL/6 mice to establish a pulmonary fibrosis model. RESULTS: Bleomycin exposure induced dose- and time-dependent accumulation of senescence hallmarks and DNA lesions in AECs. These effects are probably due to the inhibition of Rad51 expression, consequently suppressing homologous recombination (HR) repair. Mechanistic studies revealed that bleomycin-mediated transcriptional inhibition of Rad51 might primarily result from E2F1 depletion. Furthermore, the genetic supplement of Rad51 substantially mitigated bleomycin-mediated effects on DSB repair and senescence in AECs. Notably, decreased Rad51 expression was also observed in the bleomycin-induced mouse pulmonary fibrosis model. CONCLUSIONS: Our works suggest that the inhibition of Rad51 plays a pivotal role in bleomycin-induced AECs senescence and lung injury, offering potential strategies to alleviate the pulmonary toxicity of bleomycin.

Effects of sevoflurane on lung alveolar epithelial wound healing and survival in a sterile in vitro model of acute respiratory distress syndrome.

Acute respiratory distress syndrome (ARDS) is a serious lung condition that often leads to hospitalization in intensive care units and a high mortality rate. Sevoflurane is a volatile anesthetic with growing interest for sedation in ventilated patients with ARDS. It has been shown to have potential lung-protective effects, such as reduced inflammation and lung edema, or improved arterial oxygenation. In this study, we investigated the effects of sevoflurane on lung injury in cultured human carcinoma-derived lung alveolar epithelial (A549) cells. We found that sevoflurane was associated with improved wound healing after exposure to inflammatory cytokines, with preserved cell proliferation but no effect on cell migration properties. Sevoflurane exposure was also associated with enhanced cell viability and active autophagy in A549 cells exposed to cytokines. These findings suggest that sevoflurane may have beneficial effects on lung epithelial injury by promoting alveolar epithelial wound healing and by influencing the survival and proliferation of A549 epithelial cells in vitro. Further research is needed to confirm these findings and to investigate the key cellular mechanisms explaining sevoflurane's potential effects on lung epithelial injury.

Necroptosis blockade prevents lung injury in severe influenza.

Severe influenza A virus (IAV) infections can result in hyper-inflammation, lung injury and acute respiratory distress syndrome1-5 (ARDS), for which there are no effective pharmacological therapies. Necroptosis is an attractive entry point for therapeutic intervention in ARDS and related inflammatory conditions because it drives pathogenic lung inflammation and lethality during severe IAV infection6-8 and can potentially be targeted by receptor interacting protein kinase 3 (RIPK3) inhibitors. Here we show that a newly developed RIPK3 inhibitor, UH15-38, potently and selectively blocked IAV-triggered necroptosis in alveolar epithelial cells in vivo. UH15-38 ameliorated lung inflammation and prevented mortality following infection with laboratory-adapted and pandemic strains of IAV, without compromising antiviral adaptive immune responses or impeding viral clearance. UH15-38 displayed robust therapeutic efficacy even when administered late in the course of infection, suggesting that RIPK3 blockade may provide clinical benefit in patients with IAV-driven ARDS and other hyper-inflammatory pathologies.

Bronchioalveolar organoids: A preclinical tool to screen toxicity associated with antibody-drug conjugates.

Despite extensive preclinical testing, cancer therapeutics can result in unanticipated toxicity to non-tumor tissue in patients. These toxicities may pass undetected in preclinical experiments due to modeling limitations involving poor biomimicry of 2-dimensional in vitro cell cultures and due to lack of interspecies translatability in in vivo studies. Instead, primary cells can be grown into miniature 3-dimensional structures that recapitulate morphological and functional aspects of native tissue, termed "organoids." Here, human bronchioalveolar organoids grown from primary alveolar epithelial cells were employed to model lung epithelium and investigate off-target toxicities associated with antibody-drug conjugates (ADCs). ADCs with three different linker-payload combinations (mafodotin, vedotin, and deruxtecan) were tested in bronchioalveolar organoids generated from human, rat, and nonhuman primate lung cells. Organoids demonstrated antibody uptake and changes in viability in response to ADC exposure that model in vivo drug sensitivity. RNA sequencing identified inflammatory activation in bronchioalveolar cells in response to deruxtecan. Future studies will explore specific cell populations involved in interstitial lung disease and incorporate immune cells to the culture.

FTO Deficiency Alleviates LPS-induced Acute Lung Injury by TXNIP/NLRP3-mediated Alveolar Epithelial Cell Pyroptosis.

N6-methyladenosine (m6A) plays a role in various diseases, but it has rarely been reported in acute lung injury (ALI). The FTO (fat mass and obesity-associated) protein can regulate mRNA metabolism by removing m6A residues. The aim of this study was to examine the role and mechanism of the m6A demethylase FTO in LPS-induced ALI. Lung epithelial FTO-knockout mice and FTO-knockdown/overexpression human alveolar epithelial (A549) cell lines were constructed to evaluate the effects of FTO on ALI. Bioinformatics analysis and a series of in vivo and in vitro assays were used to examine the mechanism of FTO regulation. Rescue assays were conducted to examine whether the impact of FTO on ALI depended on the TXNIP/NLRP3 pathway. In LPS-induced ALI, RNA m6A modification amounts were upregulated, and FTO expression was downregulated. In vivo, lung epithelial FTO knockout alleviated alveolar structure disorder, tissue edema, and pulmonary inflammation and improved the survival of ALI mice. In vitro, FTO knockdown reduced A549 cell damage and death induced by LPS, whereas FTO overexpression exacerbated cell damage and death. Mechanistically, bioinformatics analysis revealed that TXNIP was a downstream target of FTO. FTO deficiency mitigated pyroptosis in LPS-induced ALI via the TXNIP/NLRP3 pathway. Rescue assays confirmed that the impact of FTO on the TXNIP/NLRP3 pathway was significantly reversed by the TXNIP inhibitor SRI-37330. Deficiency of FTO alleviates LPS-induced ALI via TXNIP/NLRP3 pathway-mediated alveolar epithelial cell pyroptosis, which might be a novel therapeutic strategy for combating ALI.

Nesfatin-1 alleviates hyperoxia-induced lung injury in newborn mice by inhibiting oxidative stress through regulating SIRT1/PGC-1α pathway.

Bronchopulmonary dysplasia (BPD) is a pulmonary disease commonly observed in premature infants and it is reported that oxidative stress is a critical induction factor in BPD and is considered as a promising target for treating BPD. Nesfatin-1 is a brain-gut peptide with inhibitory effects on food intake, which is recently evidenced to show suppressive effect on oxidative stress. The present study aims to explore the therapeutic effect and mechanism of Nesfatin-1 in BPD mice. AECIIs were extracted from newborn rats and exposed to hyperoxia for 24 h, followed by treatment with 5 and 10 nM Nesfatin-1. Declined cell viability, increased apoptotic rate, upregulated Bax, downregulated Bcl-2, increased release of ROS and MDA, and suppressed SOD activity were observed in hyperoxia-treated AECIIs, which were extremely reversed by Nesfatin-1. Newborn rats were exposed to hyperoxia, followed by treated with 10 µg/kg Nesfatin-1 and 20 µg/kg Nesfatin-1. Severe pathological changes, elevated MDA level, and declined SOD activity were observed in lung tissues of BPD mice, which were rescued by Nesfatin-1. Furthermore, the protective effect of Nesfatin-1 on hyperoxia-challenged AECIIs was abolished by silencing SIRT1. Collectively, Nesfatin-1 alleviated hyperoxia-induced lung injury in newborn mice by inhibiting oxidative stress through regulating SIRT1/PGC-1α pathway.

Lung adenocarcinoma promotion by air pollutants.

A complete understanding of how exposure to environmental substances promotes cancer formation is lacking. More than 70 years ago, tumorigenesis was proposed to occur in a two-step process: an initiating step that induces mutations in healthy cells, followed by a promoter step that triggers cancer development1. Here we propose that environmental particulate matter measuring ≤2.5 µm (PM2.5), known to be associated with lung cancer risk, promotes lung cancer by acting on cells that harbour pre-existing oncogenic mutations in healthy lung tissue. Focusing on EGFR-driven lung cancer, which is more common in never-smokers or light smokers, we found a significant association between PM2.5 levels and the incidence of lung cancer for 32,957 EGFR-driven lung cancer cases in four within-country cohorts. Functional mouse models revealed that air pollutants cause an influx of macrophages into the lung and release of interleukin-1ß. This process results in a progenitor-like cell state within EGFR mutant lung alveolar type II epithelial cells that fuels tumorigenesis. Ultradeep mutational profiling of histologically normal lung tissue from 295 individuals across 3 clinical cohorts revealed oncogenic EGFR and KRAS driver mutations in 18% and 53% of healthy tissue samples, respectively. These findings collectively support a tumour-promoting role for  PM2.5 air pollutants  and provide impetus for public health policy initiatives to address air pollution to reduce disease burden.

Approaches in Hydroxytyrosol Supplementation on Epithelial-Mesenchymal Transition in TGFß1-Induced Human Respiratory Epithelial Cells.

Hydroxytyrosol (HT) is an olive polyphenol with anti-inflammatory and antioxidant properties. This study aimed to investigate the effect of HT treatment on epithelial-mesenchymal transition (EMT) in primary human respiratory epithelial cells (RECs) isolated from human nasal turbinate. HT dose-response study and growth kinetic study on RECs was performed. Several approaches on HT treatment and TGFß1 induction with varying durations and methods was studied. RECs morphology and migration ability were evaluated. Vimentin and E-cadherin immunofluorescence staining and Western blotting [E-cadherin, vimentin, SNAIL/SLUG, AKT, phosphorylated (p)AKT, SMAD2/3 and pSMAD2/3] were performed after 72-h treatment. In silico analysis (molecular docking) of HT was performed to evaluate the potential of HT to bind with the TGFß receptor. The viability of the HT-treated RECs was concentration-dependent, where the median effective concentration (EC50) was 19.04 µg/mL. Testing of the effects of 1 and 10 µg/mL HT revealed that HT suppressed expression of the protein markers vimentin and SNAIL/SLUG while preserving E-cadherin protein expression. Supplementation with HT protected against SMAD and AKT pathway activation in the TGFß1-induced RECs. Furthermore, HT demonstrated the potential to bind with ALK5 (a TGFß receptor component) in comparison to oleuropein. TGFß1-induced EMT in RECs and HT exerted a positive effect in modulating the effects of EMT.

Activation of Endothelial Large Conductance Potassium Channels Protects against TNF-α-Induced Inflammation.

Elevated TNF-α levels in serum and broncho-alveolar lavage fluid of acute lung injury patients correlate with mortality rates. We hypothesized that pharmacological plasma membrane potential (Em) hyperpolarization protects against TNF-α-induced CCL-2 and IL-6 secretion from human pulmonary endothelial cells through inhibition of inflammatory Ca2+-dependent MAPK pathways. Since the role of Ca2+ influx in TNF-α-mediated inflammation remains poorly understood, we explored the role of L-type voltage-gated Ca2+ (CaV) channels in TNF-α-induced CCL-2 and IL-6 secretion from human pulmonary endothelial cells. The CaV channel blocker, Nifedipine, decreased both CCL-2 and IL-6 secretion, suggesting that a fraction of CaV channels is open at the significantly depolarized resting Em of human microvascular pulmonary endothelial cells (-6 ± 1.9 mV), as shown by whole-cell patch-clamp measurements. To further explore the role of CaV channels in cytokine secretion, we demonstrated that the beneficial effects of Nifedipine could also be achieved by Em hyperpolarization via the pharmacological activation of large conductance K+ (BK) channels with NS1619, which elicited a similar decrease in CCL-2 but not IL-6 secretion. Using functional gene enrichment analysis tools, we predicted and validated that known Ca2+-dependent kinases, JNK-1/2 and p38, are the most likely pathways to mediate the decrease in CCL-2 secretion.

Unlike Chloroquine, Mefloquine Inhibits SARS-CoV-2 Infection in Physiologically Relevant Cells.

Despite the development of specific therapies against severe acute respiratory coronavirus 2 (SARS-CoV-2), the continuous investigation of the mechanism of action of clinically approved drugs could provide new information on the druggable steps of virus-host interaction. For example, chloroquine (CQ)/hydroxychloroquine (HCQ) lacks in vitro activity against SARS-CoV-2 in TMPRSS2-expressing cells, such as human pneumocyte cell line Calu-3, and likewise, failed to show clinical benefit in the Solidarity and Recovery clinical trials. Another antimalarial drug, mefloquine, which is not a 4-aminoquinoline like CQ/HCQ, has emerged as a potential anti-SARS-CoV-2 antiviral in vitro and has also been previously repurposed for respiratory diseases. Here, we investigated the anti-SARS-CoV-2 mechanism of action of mefloquine in cells relevant for the physiopathology of COVID-19, such as Calu-3 cells (that recapitulate type II pneumocytes) and monocytes. Molecular pathways modulated by mefloquine were assessed by differential expression analysis, and confirmed by biological assays. A PBPK model was developed to assess mefloquine's optimal doses for achieving therapeutic concentrations. Mefloquine inhibited SARS-CoV-2 replication in Calu-3, with an EC50 of 1.2 µM and EC90 of 5.3 µM. It reduced SARS-CoV-2 RNA levels in monocytes and prevented virus-induced enhancement of IL-6 and TNF-α. Mefloquine reduced SARS-CoV-2 entry and synergized with Remdesivir. Mefloquine's pharmacological parameters are consistent with its plasma exposure in humans and its tissue-to-plasma predicted coefficient points suggesting that mefloquine may accumulate in the lungs. Altogether, our data indicate that mefloquine's chemical structure could represent an orally available host-acting agent to inhibit virus entry.

Renin-angiotensin system blockade on angiotensin-converting enzyme 2 and TMPRSS2 in human type II pneumocytes.

AIMS: Angiotensin-converting enzyme (ACE) 2 is the receptor for severe acute respiratory syndrome coronavirus 2 which causes coronavirus disease 2019 (COVID-19). Viral cellular entry requires ACE2 and transmembrane protease serine 2 (TMPRSS2). ACE inhibitors (ACEIs) or angiotensin (Ang) receptor blockers (ARBs) influence ACE2 in animals, though evidence in human lungs is lacking. We investigated ACE2 and TMPRSS2 in type II pneumocytes, the key cells that maintain lung homeostasis, in lung parenchymal of ACEI/ARB-treated subjects compared to untreated control subjects. MAIN METHODS: Ang II and Ang-(1-7) levels and ACE2 and TMPRSS2 protein expression were measured by radioimmunoassay and immunohistochemistry, respectively. KEY FINDINGS: We found that the ratio Ang-(1-7)/Ang II, a surrogate marker of ACE2 activity, as well as the amount of ACE2-expressing type II pneumocytes were not different between ACEI/ARB-treated and untreated subjects. ACE2 protein content correlated positively with smoking habit and age. The percentage of TMPRSS2-expressing type II pneumocytes was higher in males than females and in subjects under 60 years of age but it was not different between ACEI/ARB-treated and untreated subjects. However, there was a positive association of TMPRSS2 protein content with age and smoking in ACEI/ARB-treated subjects, with high TMPRSS2 protein levels most evident in ACEI/ARB-treated older adults and smokers. SIGNIFICANCE: ACEI/ARB treatment influences human lung TMPRSS2 but not ACE2 protein content and this effect is dependent on age and smoking habit. This finding may help explain the increased susceptibility to COVID-19 seen in smokers and older patients with treated cardiovascular-related pathologies.

Cellular response of lung fibroblasts and epithelial cells to particulate matter10 treatment examined via comparative transcriptome analysis.

Particulate matter (PM) can be categorized by particle size (PM10, PM2.5 and PM1.0), which is an important factor affecting the biological response. Exposure to PM in the air (dust, smoke, dirt and biological contaminants) is clearly associated with lung disease (lung cancer, pneumonia and asthma). Although PM primarily affects lung epithelial cells, the specific response of related cell types to PM remains to be elucidated. The present study performed Gene Ontology (GO) analysis programs (Clustering GO and Database for Annotation, Visualization and Integrated Discovery) on differentially expressed genes in lung epithelial cells (WI­38 VA­13) and fibroblasts (WI­38) following treatment with PM10 and evaluated the cell­specific biological responses related to cell proliferation, apoptosis, adhesion and extracellular matrix production. The results suggested that short­ or long­term exposure to PM may affect cell condition and may consequently be related to several human diseases, including lung cancer and cardiopulmonary disease.

Luteolin attenuates lipopolysaccharide-induced acute lung injury/acute respiratory distress syndrome by activating alveolar epithelial sodium channels via cGMP/PI3K pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Luteolin (Lut) was recently identified as the major active ingredient of Mosla scabra, which was a typical representative traditional Chinese medicine and had been used to treat pulmonary diseases for thousands of years. AIM OF THE STUDY: This study was to explore the effects and relative mechanisms of Lut in LPS-induced acute lung injury/acute respiratory distress syndrome (ALI/ARDS). The main characteristic of ALI/ARDS is pulmonary edema, and epithelial sodium channel (ENaC) is a key factor in effective removal of excessive alveolar edematous fluid, which is essential for repairing gas exchange and minimizing damage to the peripheral tissues. However, whether the therapeutic effects of Lut on respiratory diseases are relative with ENaC is still unknown. MATERIALS AND METHODS: Alveolar fluid clearance was calculated in BALB/c mice and ENaC function was measured in H441 cells. Moreover, ENaC membrane protein and mRNA were detected by Western blot and real-time PCR, respectively. We also studied the involvement of cGMP/PI3K pathway during the regulation of Lut on ENaC during LPS-induced ALI/ARDS by ELISA method and applying cGMP/PI3K inhibitors/siRNA. RESULTS: The beneficial effects of Lut in ALI/ARDS were evidenced by the alleviation of pulmonary edema, and enhancement of both amiloride-sensitive alveolar fluid clearance and short-circuit currents. Lut could alleviate the LPS decreased expression levels of ENaC mRNA and membrane protein in H441 cells and mouse lung. In addition, cGMP concentration was increased after the administration of Lut in ALI/ARDS mice, while the inhibition of cGMP/PI3K pathway could abrogate the enhanced AFC and ENaC protein expression of Lut. CONCLUSION: These results implied that Lut could attenuate pulmonary edema via enhancing the abundance of membrane ENaC at least partially through the cGMP/PI3K pathway, which could provide a promising therapeutic strategy for treating ALI/ARDS.

Iron oxide nanoparticles exert inhibitory effects on N-Bis(2-hydroxypropyl)nitrosamine (DHPN)-induced lung tumorigenesis in rats.

Iron oxide nanoparticles (magnetite) have been widely used in industry and medicine. However, the safety assessment of magnetite has not been fully completed. The present study was conducted to assess effects of magnetite on carcinogenic activity, using a medium-term bioassay protocol. A total of 100 male Fischer 344 rats, 6 weeks old, were randomly divided into 5 groups of 20 animals each, and given a basal diet and drinking water containing 0 or 0.1% of N-bis(2-hydroxypropyl)nitrosamine (DHPN) for 2 weeks. Two weeks later, the rats were intratracheally instilled magnetite 7 times at an interval of 4 weeks, at the doses of 0, 1.0 or 5.0 mg/kg body weight, and sacrificed at the end of the experimental period of 30 weeks. The multiplicities of macroscopic lung nodules and histopathologically diagnosed bronchiolo-alveolar hyperplasia, induced by DHPN, were both significantly decreased by the high dose of magnetite. The expression of minichromosome maintenance (MCM) protein 7 in non-tumoral alveolar epithelial cells, and the number of CD163-positive macrophages in tumor nodules were both significantly reduced by magnetite. It is suggested that magnetite exerts inhibitory effects against DHPN-induced lung tumorigenesis, by the reduction of alveolar epithelial proliferation and the M2 polarization of tumor-associated macrophages.

24-Dehydrocholesterol Reductase alleviates oxidative damage-induced apoptosis in alveolar epithelial cells via regulating Phosphatidylinositol-3-Kinase/Protein Kinase B activation.

Apoptosis of alveolar epithelial cells during acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) is a critical pathological event that seriously endangers the health of patients. Suppressing apoptosis of alveolar epithelial cells was shown to alleviate functional damage of lung, and modulator of the reactive oxygen species (ROS)-induced apoptosis becomes a promising approach to the ALI therapy. Previous little studies showed that DHCR24 possessed anti-oxidative and anti-apoptotic property in ALI. Thus, H2O2 was utilized to mimic oxidative damage in vitro in alveolar epithelial cell line A549 in the present study. Our results exhibited that H2O2 treatment of A549 cells reduced the level of SOD and increased the level of ROS. Moreover, H2O2 inhibited Bcl-2 expression in A549 cells, but increased Bax and the activity of Caspase-3. In addition, the apoptosis rate in the H2O2 treatment group also increased significantly. And the expression of 24-dehydrocholesterol reductase (DHCR24) was markedly reduced in the H2O2 treatment group. Overexpression of DHCR24 can remarkably inhibit H2O2-induced oxidative stress and apoptosis. We found that overexpression of DHCR24 increased the phosphorylation level of PI3K and AKT, however, the PI3K inhibitor LY294002 (LY) eliminated the protective effect of DHCR24 in ALI. DHCR24 was down-regulated in H2O2-induced ALI, and overexpression of DHCR24 could inhibit H2O2-induced oxidative stress and apoptosis of A549 cells by activating the Phosphatidylinositol-3-Kinase/Protein Kinase B (PI3K/AKT) signaling pathway. The above exhibited a protective effect of DHCR24 on alveolar epithelial cells exposed to oxidative stress-mediated apoptosis and provided a novel therapeutic method for ALI.

Effect of cyanide ions (CN-) extracted from cassava (Manihotesculenta Crantz) on Alveolar Epithelial Cells (A549 cells).

Cassava (Manihotesculenta Crantz) is one of the most important root crops in tropical countries. It is a major source of cyanogenic glycosides viz. linamarin and lotaustralin, and these on breakdown liberate HCN and ketone. Cassava cyanide extract (CCE) from cassava leaves and tuber rinds were formulated as a biopesticide against certain borer insect pests of horticultural crops. Adenocarcinomic human alveolar basal epithelial cells (A549) were treated with three different concentrations (100, 200, 400 ppm) of CCE. The MTT and NRU assays showed dose-dependent cytotoxicity. The DCFH-DA assay does not show any free radical scavenging activity, whereas the NRR assay showed a reduction in the nitrile radicals with an increase in the concentration of the bioactive compound. A negative correlation was found between the concentration of the bioactive principles and mitochondrial and lysosomal functions. Various cellular assays demonstrated the cellular response of the CCE, and it was found that at higher concentration (400 ppm), the CCE exert a significant necrotic cell death rather than apoptosis. The results of the study indicated that the CCE have a remarkable tendency of anti-proliferative ability.

Paracrine Regulation of Alveolar Epithelial Damage and Repair Responses by Human Lung-Resident Mesenchymal Stromal Cells.

COPD is characterized by irreversible lung tissue damage. We hypothesized that lung-derived mesenchymal stromal cells (LMSCs) reduce alveolar epithelial damage via paracrine processes, and may thus be suitable for cell-based strategies in COPD. We aimed to assess whether COPD-derived LMSCs display abnormalities. LMSCs were isolated from lung tissue of severe COPD patients and non-COPD controls. Effects of LMSC conditioned-medium (CM) on H2O2-induced, electric field- and scratch-injury were studied in A549 and NCI-H441 epithelial cells. In organoid models, LMSCs were co-cultured with NCI-H441 or primary lung cells. Organoid number, size and expression of alveolar type II markers were assessed. Pre-treatment with LMSC-CM significantly attenuated oxidative stress-induced necrosis and accelerated wound repair in A549. Co-culture with LMSCs supported organoid formation in NCI-H441 and primary epithelial cells, resulting in significantly larger organoids with lower type II-marker positivity in the presence of COPD-derived versus control LMSCs. Similar abnormalities developed in organoids from COPD compared to control-derived lung cells, with significantly larger organoids. Collectively, this indicates that LMSCs' secretome attenuates alveolar epithelial injury and supports epithelial repair. Additionally, LMSCs promote generation of alveolar organoids, with abnormalities in the supportive effects of COPD-derived LMCS, reflective of impaired regenerative responses of COPD distal lung cells.

Methylprednisolone Attenuates Lipopolysaccharide-Induced Sepsis by Modulating the Small Nucleolar RNA Host Gene 5/Copine 1 Pathway.

Sepsis has become a major public health problem worldwide. Methylprednisolone sodium succinate (MP) is a commonly used drug to prevent inflammation. However, the role and underlying mechanism of MP in sepsis remain vague. MP inhibited the lipopolysaccharide (LPS)-induced production of tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-17 and suppressed cell growth in alveolar type II epithelial cells (ATII cells). Small nucleolar RNA host gene 5 (SNHG5) expression was inhibited by LPS and restored by MP. Upregulation of SNHG5 inhibited the cellular role of LPS in ATII cells, and further, downregulation of SNHG5 inhibited the cellular role of MP in ATII cells under LPS conditions. SNHG5 elevated the expression of Copine 1 (CPNE1) by enhancing the mRNA stability of CPNE1. Increasing CPNE1 expression restored the silenced SNHG5-induced inhibitor role of MP in ATII cells under LPS conditions. Finally, MP attenuated lung injury and TNF-α and IL-17 secretion in an LPS-induced sepsis mouse model. Overall, this study investigated the mechanism underlying the effect of MP treatment in sepsis and, for the first time, revealed the important role of the SNHG5/CPNE1 pathway in the development and treatment of sepsis and the potential to serve as a diagnostic and therapeutic target for sepsis.

Deficiency of CARMA3 attenuates the development of bleomycin induced pulmonary fibrosis.

BACKGROUND: Pulmonary fibrosis (PF) has attracted more and more attention due to its irreversibility and high mortality rate. Currently, there is no effective treatment option is available to reverse the disease. Caspase recruitment domain-containing membrane-associated guanylate kinase protein (CARMA3) has been recognized as a proinflammatory molecule involved in many lung diseases, such as Allergic airway inflammation and lung cancer. Bleomycin (Bleo), as an alkaline sugar peptide antibiotics, is often used as a first-line anti-tumor agent. Its toxic effect is to induce pulmonary fibrosis (PF) and its clinical symptoms, so it has been widely used in the construction of pulmonary fibrosis model. METHODS: Wild type mice (WT, n = 20) and CARMA3 knockout mice (CARMA3-KO, n = 20) were generated and injected with bleomycin or saline via trachea. The severity of fibrosis was evaluated by fibrosis markers and lung histological morphology. Furthermore, the amount of alveolar epithelial cells and inflammation in lung tissue were examined. Finally, epithelial-mesenchymal transition was further investigated. RESULTS: We found CARMA3 expression in the mice alveolar epithelial cells. And compared with WT mice, CARMA3-KO mice showed reduced deposition of collagen fibers, inflammation and destruction of alveolar epithelial cells in lung tissue. In addition, after bleomycin induction, the expressions of proinflammatory factors and collagen-related factors in CARMA3-KO mice were much lower than those in WT mice. The epithelial-mesenchymal transformation phenotype was also improved in CARMA3-KO mice compared to WT mice. CONCLUSION: Our Results shows that CARMA3 plays an important role in the pathogenesis of bleomycin-induced pulmonary fibrosis. CARMA3 could alleviate the fibrosis by improving inflammation, deposition of collagen and damage of alveolar epithelial cells, which revealed that CARMA3 may be a potential target for pulmonary fibrosis.

TSSK4 upregulation in alveolar epithelial type-II cells facilitates pulmonary fibrosis through HSP90-AKT signaling restriction and AT-II apoptosis.

Alveolar epithelial injury is one of the important pathological changes in idiopathic pulmonary interstitial fibrosis (IPF), but the regulatory mechanism remains unclear. Here, we reported that alveolar epithelial type-II cells (AT II) play important roles in pathological process of pulmonary fibrosis. Through iTRAQ (isobaric tagging for relative and absolute quantification) quantitative proteomics, TSSK4 was identified to be upregulated in bleomycin-induced fibrotic mice model, which was further confirmed in clinical IPF patients' tissue specimens. TSSK4 is a germ-related protein, but its expression in other tissues and the association with other diseases are not reported. Immunofluorescence staining showed that TSSK4 selectively expressed in AT-II cells, which are essential for inflammation-induced AT-II loss during fibrosis. Luciferase assay and other molecular biological experiments proved that TSSK4 expression is regulated by TNF-α-mediated NF-κB signaling. The TSSK4 kinase activity is found to be closely related to the function of HSP90-AKT pathway that TSSK4 can phosphorylate its substrate HSP90ß on serine 255, to inhibit the ATPase activity of HSP90ß and reduce its molecular chaperone function on AKT. Under this condition, kinase activity of AKT is diminished to interfere its survival function, subsequently facilitating AT-II cellular apoptosis through the mitochondrial death machinery. Our findings highlight the importance of TSSK4 in regulating pulmonary fibrosis by facilitating AT-II loss through HSP90-AKT signaling, all of which suggest TSSK4 and the regulating mechanism as attractive targets for the clinical intervention of pulmonary injury and fibrosis.