Pulmonary Apoptosis Research Articles

Abstract We120: NHE1 Regulates Apoptosis Susceptibility in Sugen-Hypoxia Pulmonary Arterial Smooth Muscle Cells via Modulation of CHOP

Pulmonary hypertension (PH) is caused by lung vascular remodeling involving increased pulmonary arterial smooth muscle cell (PASMC) proliferation and apoptosis resistance. Na +/ H + exchanger (NHE) isoform 1 (NHE1) is a membrane protein regulating intracellular pH (pH i ) via movement of H + . Our lab found increased NHE activity is associated with hyperproliferation in PASMCs from the Sugen Hypoxia (SuHx) animal model of PH. In this study, we tested the hypothesis that increased NHE1 activity also causes apoptosis resistance in SuHx PASMCs via dysregulation of pH i . PASMCs isolated from distal pulmonary arteries of control and SuHx rats were treated with NHE inhibitor, [5-(N-ethyl-N-isopropyl)-amiloride; EIPA;1 uM], NHE1 inhibitor [cariporide;10 uM] or vehicle for 24 h before analyzing cell apoptosis via Hoechst staining under baseline and stimulated (250 uM H 2 O 2 ) conditions. Control PASMCs were infected with adenovirus containing green fluorescent protein (GFP), wild-type NHE1 (WT) or NHE1 lacking ion translocation activity (E262I). Protein expression and localization were assessed via immunoblotting. NHE activity was assessed from Na + -dependent recovery following an ammonium pulse. At baseline, apoptosis in SuHx PASMCs was similar to control cells but following H 2 O 2 stimulation , SuHx PASMCs were resistant to apoptosis (p<0.001). EIPA increased basal and stimulated apoptosis in SuHx cells. Surprisingly, treatment with cariporide did not alter SuHx apoptosis, suggesting that EIPA but not cariporide was able to sensitize SuHx cells to apoptosis, despite both drugs significantly inhibiting NHE activity, suggesting EIPA affects PASMC apoptosis through a mechanism other than pH i regulation. To test this possibility, control PASMCs were infected with GFP, WT or E262I. NHE activity was increased in cells with WT, but not E262I, when compared to GFP (p<0.001). Overexpression of either WT or E262I inhibited apoptosis following treatment with H2O2 (p<0.001). We next explored CAAT/enhancer binding protein homologous transcription factor (CHOP), known to be involved in induction of apoptosis. CHOP expression was increased by H2O2 in control cells but was decreased in SuHx PASMCs. EIPA increased CHOP levels in SuHx cells, whereas, expression of both WT and E262I constructs inhibited the H 2 O 2 -induced CHOP increase in control PASMCs. Our results indicate that NHE1 regulates apoptosis susceptibility in PASMCs during PH via modulation of CHOP rather than through pH i regulation.

Urolithin A alleviates respiratory syncytial virus-induced lung infection in neonatal mice by activating miR-136-mediated Sirt1 signaling

To observe the therapeutic effects of urolithin A (UA) on respiratory syncytial virus (RSV)-induced lung infection in neonatal mice and explore the underlying mechanisms. Babl/c mice (5-7 days old) were subjected to nasal instillation of RSV and received intraperitoneal injection of saline or 2.5, 5 and 10 mg/kg UA 2 h after the infection and then once daily for 2 weeks. Bronchoalveolar lavage fluid (BALF) was then collected for detection of inflammatory cells and mediators, and lung pathology was evaluated with HE staining. RSV-infected BEAS-2B cells were treated with 2.5, 5 or 10 µmol/ L UA. Inflammatory factors, cell viability, apoptosis and autophagy were analyzed using ELISA, CCK-8 assay, TUNEL staining, flow cytometry, Western blotting and immunofluorescence staining. The cellular expressions of miR-136 and Sirt1 mRNAs were detected using qRT-PCR. A dual-luciferase reporter system was used to verify the binding between miR-136 and Sirt1. In neonatal Babl/c mice, RSV infection caused obvious lung pathologies, promoted pulmonary cell apoptosis and LC3-Ⅱ/Ⅰ, Beclin-1 and miR-136 expressions, and increased the total cell number, inflammatory cells and factors in the BALF and decreased p62 and Sirt1 expressions. All these changes were alleviated dose-dependently by UA. In BEAS-2B cells, RSV infection significantly increased cell apoptosis, LC3B-positive cells and miR-136 expression and reduced Sirt1 expression (P<0.01), which were dose-dependently attenuated by UA. Dual-luciferase reporter assay confirmed the binding between miR-136 and Sirt1. In RSV-infected BEAS-2B cells with UA treatment, overexpression of miR-136 and Ex527 treatment both significantly increased the inflammatory factors and cell apoptosis but decreased LC3B expression, and these changes were further enhanced by their combined treatment. UA ameliorates RSV-induced lung infection in neonatal mice by activating miR-136-mediated Sirt1 signaling pathway.

Nasal instillation of polystyrene nanoplastics induce lung injury via mitochondrial DNA release and activation of the cyclic GMP-AMP synthase-stimulator of interferon genes-signaling cascade

Nanoplastics (NPs) are a common type of degraded plastic material associated with adverse health effects such as pulmonary injury. However, the molecular mechanism(s) underlying lung injury as caused by NPs remains uncertain. Thus, we herein investigated the pulmonary toxicity of NPs on RAW264.7 cells and C57BL/6 mice. Our in vitro study indicated that NPs induced oxidative stress, cell death, inflammation, and the activation of the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING)-signaling pathway. Mice in our in vivo study displayed significant pulmonary fibrosis, inflammation, apoptosis, necrosis, and excessive double-stranded DNA release into serum and bronchoalveolar lavage fluid. Our mechanistic exploration uncovered cGAS-STING-signaling activation as the leading cause of NPs-induced pulmonary fibrosis. The current study opens an avenue toward elucidating the role of the cGAS-STING-signaling pathway in NPs-induced pulmonary injury.

Genetic variants of the BMPR2 gene and their contribution to the development of pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a devastating disease characterized by a progressive increase in pressure in the pulmonary arteries. This leads to vascular remodeling and, eventually, right heart failure. Variants in the BMPR2 gene, which encodes the bone morphogenetic protein receptor type 2, are the most common genetic cause of hereditary and idiopathic PAH. These variants disrupt the transforming growth factor-beta (TGF-β) signaling pathway, triggering abnormal proliferation of pulmonary artery smooth muscle cells and apoptosis of endothelial cells. This results in complex vascular remodeling, characterized by thickening of the vascular walls, formation of plexiform lesions, and in situ thrombosis. Endothelial dysfunction also contributes to an imbalance between vasoconstrictors and vasodilators, exacerbating pulmonary hypertension. While current therapies aim to restore this balance, they have a limited impact on the underlying vascular remodeling. Emerging strategies seek to restore the functionality of BMPR2 variants or enhance their signaling through agonists and read-through molecules. Additionally, a higher penetrance of the disease has been observed in women carrying BMPR2 variants, suggesting an interaction with sex hormone metabolism. The research aims to understand the role of BMPR2 gene variants in the development of PAH and to explore emerging strategies to restore the functionality of these variants or enhance their signaling.

Long non-coding RNA Small Nucleolar RNA Host Gene 4 ameliorates cigarette smoke-induced proliferation, apoptosis, inflammation, and airway remodeling in alveolar epithelial cells through the modulation of the mitogen-activated protein kinase signaling pathway via the microRNA-409-3p/Four and a Half LIM Domains 1 axis

The long non-coding RNA (lncRNA) Small Nucleolar RNA Host Gene 4 (SNHG4) has been demonstrated to be significantly downregulated in various inflammatory conditions, yet its role in chronic obstructive pulmonary disease (COPD) remains elusive. This study aims to elucidate the biological function of SNHG4 in COPD and to unveil its potential molecular targets. Our findings reveal that both SNHG4 and Four and a Half LIM Domains 1 (FHL1) were markedly downregulated in COPD, whereas microRNA-409-3p (miR-409-3p) was upregulated. Importantly, SNHG4 exhibited a negative correlation with inflammatory markers in patients with COPD, but a positive correlation with forced expiratory volume in 1s percentage (FEV1%). SNHG4 distinguished COPD patients from non-smokers with high sensitivity, specificity, and accuracy. Overexpression of SNHG4 ameliorated cigarette smoke extract (CSE)-mediated inflammation, apoptosis, oxidative stress, and airway remodeling in 16HBE bronchial epithelial cells. These beneficial effects of SNHG4 overexpression were reversed by the overexpression of miR-409-3p or the silencing of FHL1. Mechanistically, SNHG4 competitively bound to miR-409-3p, mediating the expression of FHL1, and consequently improving inflammation, apoptosis, oxidative stress, and airway remodeling in 16HBE cells. Additionally, SNHG4 regulated the miR-409-3p/FHL1 axis to inhibit the activation of the mitogen-activated protein kinase (MAPK) pathway induced by CSE. In a murine model of COPD, knockdown of SNHG4 exacerbated CSE-induced pulmonary inflammation, apoptosis, and oxidative stress. In summary, our data affirm that SNHG4 mitigates pulmonary inflammation, apoptosis, and oxidative damage mediated by COPD through the regulation of the miR-409-3p/FHL1 axis.Graphical

Sinomenine attenuates bleomycin-induced pulmonary fibrosis, inflammation, and oxidative stress by inhibiting TLR4/NLRP3/TGFβ signaling

Objective The present work concentrated on validating whether sinomenine alleviates bleomycin (BLM)-induced pulmonary fibrosis, inflammation, and oxidative stress. Methods A rat model of pulmonary fibrosis was constructed through intratracheal injection with 5 mg/kg BLM, and the effects of 30 mg/kg sinomenine on pulmonary inflammation, fibrosis, apoptosis, and 4-hydroxynonenal density were evaluated by hematoxylin and eosin staining, Masson’s trichrome staining, TUNEL staining, and immunohistochemistry. Hydroxyproline content and concentrations of inflammatory cytokines and oxidative stress markers were detected using corresponding kits. MRC-5 cells were treated with 10 ng/ml PDGF, and the effects of 1 mM sinomenine on cell proliferation were assessed by EdU assays. The mRNA expression of inflammatory cytokines and the protein levels of collagens, fibrosis markers, and key markers involved in the TLR4/NLRP3/TGFβ signaling were tested with RT-qPCR and immunoblotting analysis. Results Sinomenine attenuated pulmonary fibrosis and inflammation while reducing hydroxyproline content and the protein expression of collagens and fibrosis markers in BLM-induced pulmonary fibrosis rats. Sinomenine reduced apoptosis in lung samples of BLM-challenged rats by increasing Bcl-2 and reducing Bax and cleaved caspase-3 protein expression. In addition, sinomenine alleviated inflammatory response and oxidative stress in rats with pulmonary fibrosis induced by BLM. Moreover, sinomenine inhibited the TLR4/NLRP3/TGFβ signaling pathway in lung tissues of BLM-stimulated rats. Furthermore, TLR4 inhibitor, TAK-242, attenuated PDGF-induced fibroblast proliferation and collagen synthesis in MRC-5 cells. Conclusion Sinomenine attenuates BLM-caused pulmonary fibrosis, inflammation, and oxidative stress by inhibiting the TLR4/NLRP3/TGFβ signaling, indicating that sinomenine might become a therapeutic candidate to treat pulmonary fibrosis.

TRPC4 aggravates hypoxic pulmonary hypertension by promoting pulmonary endothelial cell apoptosis

Pulmonary hypertension (PH) is a devastating disease that lacks effective treatment options and is characterized by severe pulmonary vascular remodeling. Pulmonary arterial endothelial cell (PAEC) dysfunction drives the initiation and pathogenesis of pulmonary arterial hypertension. Canonical transient receptor potential (TRPC) channels, a family of Ca2+-permeable channels, play an important role in various diseases. However, the effect and mechanism of TRPCs on PH development have not been fully elucidated. Among the TRPC family members, TRPC4 expression was markedly upregulated in PAECs from hypoxia combined with SU5416 (HySu)-induced PH mice and monocrotaline (MCT)-treated PH rats, as well as in hypoxia-exposed PAECs, suggesting that TRPC4 in PAECs may participate in the occurrence and development of PH. In this study, we aimed to investigate whether TRPC4 in PAECs has an aggravating effect on PH and elucidate the molecular mechanisms. We observed that hypoxia treatment promoted PAEC apoptosis through a caspase-12/endoplasmic reticulum stress (ERS)-dependent pathway. Knockdown of TRPC4 attenuated hypoxia-induced apoptosis and caspase-3/caspase-12 activity in PAECs. Accordingly, adeno-associated virus (AAV) serotype 6-mediated pulmonary endothelial TRPC4 silencing (AAV6-Tie-shRNA-TRPC4) or TRPC4 antagonist suppressed PH progression as evidenced by reduced right ventricular systolic pressure (RVSP), pulmonary vascular remodeling, PAEC apoptosis and reactive oxygen species (ROS) production. Mechanistically, unbiased RNA sequencing (RNA-seq) suggested that TRPC4 deficiency suppressed the expression of the proapoptotic protein sushi domain containing 2 (Susd2) in hypoxia-exposed mouse PAECs. Moreover, TRPC4 activated hypoxia-induced PAEC apoptosis by promoting Susd2 expression. Therefore, inhibiting TRPC4 ameliorated PAEC apoptosis and hypoxic PH in animals by repressing Susd2 signaling, which may serve as a therapeutic target for the management of PH.

Peficitinib alleviated acute lung injury by blocking glycolysis through JAK3/STAT3 pathway

Peficitinib is a selective Janus kinase (JAK3) inhibitor recently developed and approved for the treatment of rheumatoid arthritis in Japan. Glycolysis in macrophages could induce NOD-like receptor (NLR) family and pyrin domain-containing protein 3 (NLRP3) inflammasome activation, thus resulting in pyroptosis and acute lung injury (ALI). The aim of our study was to investigate whether Peficitinib could alleviate lipopolysaccharide (LPS)-induced ALI by inhibiting NLRP3 inflammasome activation. Wild type C57BL/6J mice were intraperitoneally injected with Peficitinib (5 or 10 mg·kg−1·day−1) for 7 consecutive days before LPS injection. The results showed that Peficitinib pretreatment significantly relieved LPS-induced pulmonary edema, inflammation, and apoptosis. NLRP3 inflammasome and glycolysis in murine lung tissues challenged with LPS were also blocked by Peficitinib. Furthermore, we found that the activation of JAK3/signal transducer and activator of transcription 3 (STAT3) was also suppressed by Peficitinib in mice with ALI. However, in Jak3 knockout mice, Peficitinib did not show obvious protective effects after LPS injection. In vitro experiments further showed that Jak3 overexpression completely abolished Peficitinib-elicited inhibitory effects on pyroptosis and glycolysis in LPS-induced RAW264.7 macrophages. Finally, we unveiled that LPS-induced activation of JAK3/STAT3 was mediated by toll-like receptor 4 (TLR4) in RAW264.7 macrophages. Collectively, our study proved that Peficitinib could protect against ALI by blocking JAK3-mediated glycolysis and pyroptosis in macrophages, which may serve as a promising candidate against ALI in the future.

Pasteurella multocida activates Rassf1-Hippo-Yap pathway to induce pulmonary epithelial apoptosis

Pasteurella multocida is an opportunistic zoonotic pathogen that primarily causes fatal respiratory diseases, such as pneumonia and respiratory syndromes. However, the precise mechanistic understanding of how P. multocida disrupts the epithelial barrier in mammalian lung remains largely unknown. In this study, using unbiased RNA-seq analysis, we found that the evolutionarily conserved Hippo-Yap pathway was dysregulated after P. multocida infection. Given the complexity of P. multocida infection associated with lung injury and systemic inflammatory processes, we employed a combination of cell culture models, mouse models, and rabbit models to investigate the dynamics of the Hippo-Yap pathway during P. multocida infection. Our findings reveal that P. multocida infection activates the Hippo-Yap pathway both in vitro and in vivo, by upregulating the upstream factors p-Mst1/2, p-Lats1, and p-Yap, and downregulating the downstream effectors Birc5, Cyr61, and Slug. Conversely, pharmacological inhibition of the Hippo pathway by XMU-MP-1 significantly rescued pulmonary epithelial cell apoptosis in vitro and reduced lung injury, systemic inflammation, and mouse mortality in vivo. Mechanistic studies revealed that P. multocida induced up-regulation of Rassf1 expression, and Rassf1 enhanced Hippo-Yap pathway through phosphorylation. Accordingly, in vitro knockdown of Rassf1 significantly enhanced Yap activity and expression of Yap downstream factors and reduced apoptosis during P. multocida infection. P. multocida-infected rabbit samples also showed overexpression of Rassf1, p-Lats1, and p-Yap, suggesting that P. multocida activates the Rassf1-Hippo-Yap pathway. These results elucidate the pathogenic role of the Rassf1-Hippo-Yap pathway in P. multocida infection and suggest that this pathway has the potential to be a drug target for the treatment of pasteurellosis.

Down-regulating lncRNA KCNQ1OT1 relieves type II alveolar epithelial cell apoptosis during one-lung ventilation via modulating miR-129-5p/HMGB1 axis induced pulmonary endothelial glycocalyx.

Endothelial glycocalyx (EG) maintains vascular homeostasis and is destroyed after one-lung ventilation (OLV)-induced lung injury. Long noncoding RNAs (lncRNAs) are critically involved in various lung injuries. This study aimed to investigate the role and regulatory mechanism of KCNQ1 overlapping transcript 1 (KCNQ1OT1) in OLV-induced lung injury and LPS-induced type II alveolar epithelial cell (AECII) apoptosis. The rat OLV model was established, and the effects of KCNQ1OT1 on OLV-induced ALI in vivo were explored. Bax and Caspase-3 expression in rat lung tissues was measured by immunochemistry (IHC). AECIIs were isolated from rat lungs and treated with LPS or normal saline (control) for in vitro analysis. The expression of KCNQ1OT1, miR-129-5p, and HMGB1 was measured by quantitative real-time PCR (qRT-PCR) or Western blot (WB). Cell proliferation and apoptosis were examined by 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di- phenytetrazoliumromide (MTT) and flow cytometry. The downstream targets of KCNQ1OT1 were predicted by bioinformatics, and the binding relationship between KCNQ1OT1 and miR-129-3p was verified by dual-luciferase reporter assays. The potential target of miR-129-5p was further explored on the Targetscan website and revealed to target HMGB1. Enzyme-linked immunosorbent assay (ELISA) or WB was adopted to determine the levels of IL-1β, TNF-α, MDA, SOD, heparanase (HPA), matrix metalloproteinase 9 (MMP9), heparan sulfate (HS) and syndecan-1 (SDC-1). KCNQ1OT1 and HMGB1 were up-regulated during OLV-induced lung injury, and their expression was positively correlated. KCNQ1OT1 knockdown reduced OLV-induced pulmonary edema and lung epithelial cell apoptosis, increased vascular permeability, reduced IL-1β, TNF-α, MDA, and SOD levels and glycocalyx markers by targeting miR-129-5p or upregulating HMGB1. Overexpressing KCNQ1OT1 promoted cell apoptosis, reduced cell proliferation, aggravated inflammation and oxidative stress, and up-regulated HMGB1, HPA and MMP9 in LPS-treated AECIIs, while the HMGB1 silencing showed the opposite effects. MiR-129-5p mimics partially eliminated the KCNQ1OT1-induced effects, while recombinant HMGB1 restored the effects of miR-129-5p overexpression on AECIIs. Additionally, KCNQ1OT1 was demonstrated to promote the activation of the p38 MAPK/Akt/ERK signaling pathways in AECIIs via HMGB1. KCNQ1OT1 knockdown alleviated AECII apoptosis and EG damage during OLV by targeting miR-129-5p/HMGB1 to inactivate the p38 MAPK/Akt/ERK signaling. The findings of our study might deepen our understanding of the molecular basis in OLV-induced lung injury and provide clues for the targeted disease management.

Arsenic exposure and pulmonary function decline: Potential mediating role of TRAIL in chronic obstructive pulmonary disease patients

BackgroundEnvironmental arsenic (As) exposure is strongly related to the progression of chronic obstructive pulmonary disease (COPD). Pulmonary epithelial cells apoptosis is implicated in the pathophysiological mechanisms of COPD. However, the role of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), one biomarker of apoptosis, remains unclear in As-mediated pulmonary function alternations in COPD patients. MethodsThis study included 239 COPD patients. The serum level of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) was measured by enzyme-linked immunosorbent assay (ELISA). The blood As level was determined through inductively coupled plasma mass spectrometry (ICP-MS). ResultsBlood As levels exhibited a negative and dose-dependent correlation with pulmonary function. Per unit elevation of blood arsenic concentrations was related to reductions of 0.339 L in FEV1, 0.311 L in FVC, 1.171% in FEV1/FVC%, and 7.999% in FEV1% in COPD subjects. Additionally, a positive dose-response correlation of blood As with serum TRAIL was found in COPD subjects. Additionally, the level of serum TRAIL was negatively linked to lung function. Elevated TRAIL significantly mediated As-induced decreases of 11.05%, 13.35%, and 31.78% in FVC, FEV1, and FEV1%, respectively among the COPD patients. ConclusionBlood As level is positively correlated with pulmonary function decline and serum TRAIL increase in individuals with COPD. Our findings suggest that elevated TRAIL levels may serve as a mediating mechanism through which As contributes to declining lung function in COPD patients.

High-intensity intermittent training ameliorates methotrexate-induced acute lung injury

Inflammation and oxidative stress are recognized as two primary causes of lung damage induced by methotrexate, a drug used in the treatment of cancer and immunological diseases. This drug triggers the generation of oxidants, leading to lung injury. Given the antioxidant and anti-inflammatory effects of high-intensity intermittent training (HIIT), our aim was to evaluate the therapeutic potential of HIIT in mitigating methotrexate-induced lung damage in rats. Seventy male Wistar rats were randomly divided into five groups: CTL (Control), HIIT (High-intensity intermittent training), ALI (Acute Lung Injury), HIIT+ALI (pretreated with HIIT), and ALI + HIIT (treated with HIIT).HIIT sessions were conducted for 8 weeks. At the end of the study, assessments were made on malondialdehyde, total antioxidant capacity (TAC), superoxide dismutase (SOD), glutathione peroxidase (Gpx), myeloperoxidase (MPO), interleukin 10 (IL-10), tumor necrosis factor-alpha (TNF-α), gene expression of T-bet, GATA3, FOXP3, lung wet/dry weight ratio, pulmonary capillary permeability, apoptosis (Caspase-3), and histopathological indices.Methotrexate administration resulted in increased levels of TNF-α, MPO, GATA3, caspase-3, and pulmonary edema indices, while reducing the levels of TAC, SOD, Gpx, IL-10, T-bet, and FOXP3. Pretreatment and treatment with HIIT reduced the levels of oxidant and inflammatory factors, pulmonary edema, and other histopathological indicators. Concurrently, HIIT increased the levels of antioxidant and anti-inflammatory factors.

Abstract 17443: Wwox is a Critical Regulator of Pulmonary Artery Smooth Muscle Cell Homeostasis

Introduction: Pulmonary artery smooth muscle (PASMC) proliferation and apoptosis resistance are characteristic of pulmonary arterial hypertension (PAH). Expression loss of the tumor suppressor WW-domain containing oxidoreductase (WWOX) promotes cell survival and metabolic reprograming in cancer. We hypothesized that loss of WWOX expression in PASMC promotes pulmonary vascular remodeling by similar mechanisms. Methods and Results: mRNA and protein levels of WWOX are decreased in PASMC from patients with PAH, and in rat models of pulmonary hypertension (Sugen/ hypoxia and monocrotaline). PASMC-specific conditional Wwox knockout mice (WWOX-SMC KO) were treated with tamoxifen or vehicle for 5 days and exposed to 10% hypoxia for 4 weeks. WWOX-SMC KO animals developed worse hypoxia induced PH with increased right ventricular systolic pressure, right ventricular hypertrophy and pulmonary vascular remodeling, compared to control animals. WWOX loss via siRNA decreased mRNA and protein levels of BMPRII, upregulated HIF 1alpha expression, increased hPASMC proliferation and migration. RNAseq revealed dysregulation in multiple pathways in WWOX-silenced hPASMCs, including cell-cycle regulation, TGF-β, mTOR and STAT3 signaling. Loss of WWOX showed decreased basal and maximal respiration, ATP production, reduced glycolytic capacity (40% in relation to control) and decreased mRNA expression of glycolytic enzymes (HK2, PFK1, PKM), generating 58% less lactate, and reduced mRNA expression of lactate dehydrogenase A. These cells exhibited increased mRNA and protein expression of glutaminase-1 and citrate lyase and had higher dependency on fatty acid oxidation and glutaminolysis compared to glucose, suggesting that these pathways are used when WWOX is suppressed. We also found mitochondrial hyperpolarization (JC-1 assay), decreased Sirtuin 3 expression, increased expression of acetylated manganese-superoxide dismutase, high production of reactive oxygen species and mitochondrial fission, indicating that loss of WWOX contributes to mitochondrial dysfunction. Conclusion: Loss of WWOX promotes cell cycle progression, proliferation, and metabolic reprogramming in PASMCs. Further studies are ongoing to better understand these effects.

MiR-23a-3p alleviates cigarette smoke extract-induced pulmonary vascular endothelial cell apoptosis by targeting DNAJB1 in emphysema.

Cigarette smoke (CS) is an important risk factor for chronic obstructive pulmonary disease, including emphysema. MicroRNAs (miRNAs) are important regulators of emphysema progression. However, miR-23a-3p role in emphysema is unclear. CS exposure was used to construct emphysema mice models, and cigarette smoke extract (CSE)-induced pulmonary vascular endothelial cells (PMVECs) were used to mimic emphysema cell models. Mouse lung tissue was stained by immunohistochemical staining, hematoxylin and eosin staining, and TUNEL staining. MiR-23a-3p and DnaJ homolog subfamily B member 1 (DNAJB1) levels were tested using quantitative real-time PCR. DNAJB1 and apoptosis-related markers' protein levels were examined via western blot analysis. Cell viability and apoptosis were analyzed by MTT assay and flow cytometry. The interaction between miR-23a-3p and DNAJB1 was evaluated by dual-luciferase reporter assay and RIP assay. MiR-23a-3p was downregulated, and DNAJB1 was upregulated in CS-induced emphysema mice models and CSE-induced PMVECs. MiR-23a-3p overexpression promoted viability and repressed apoptosis in CSE-induced PMVECs. MiR-23a-3p targeted DNAJB1 and negatively regulated DNAJB1 expression. Moreover, DNAJB1 knockdown repressed CSE-induced PMVECs apoptosis, and miR-23a-3p inhibitor reversed this effect. Additionally, miR-23a-3p alleviated lung tissue injury and improved emphysema in mice by reducing DNAJB1 expression. MiR-23a-3p alleviated emphysema progression, which could inhibit CSE-induced PMVECs apoptosis by targeting DNAJB1.

Pentastatin, a matrikine of the collagen IVα5, is a novel endogenous mediator of pulmonary endothelial dysfunction.

Deposition of basement membrane components, such as collagen IVα5, is associated with altered endothelial cell function in pulmonary hypertension. Collagen IVα5 harbors a functionally active fragment within its C-terminal noncollageneous (NC1) domain, called pentastatin, whose role in pulmonary endothelial cell behavior remains unknown. Here, we demonstrate that pentastatin serves as a mediator of pulmonary endothelial cell dysfunction, contributing to pulmonary hypertension. In vitro, treatment with pentastatin induced transcription of immediate early genes and proinflammatory cytokines and led to a functional loss of endothelial barrier integrity in pulmonary arterial endothelial cells. Mechanistically, pentastatin leads to β1-integrin subunit clustering and Rho/ROCK activation. Blockage of the β1-integrin subunit or the Rho/ROCK pathway partially attenuated the pentastatin-induced endothelial barrier disruption. Although pentastatin reduced the viability of endothelial cells, smooth muscle cell proliferation was induced. These effects on the pulmonary vascular cells were recapitulated ex vivo in the isolated-perfused lung model, where treatment with pentastatin-induced swelling of the endothelium accompanied by occasional endothelial cell apoptosis. This was reflected by increased vascular permeability and elevated pulmonary arterial pressure induced by pentastatin. This study identifies pentastatin as a mediator of endothelial cell dysfunction, which thus might contribute to the pathogenesis of pulmonary vascular disorders such as pulmonary hypertension.NEW & NOTEWORTHY This study is the first to show that pentastatin, the matrikine of the basement membrane (BM) collagen IVα5 polypeptide, triggers rapid pulmonary arterial endothelial cell barrier disruption, activation, and apoptosis in vitro and ex vivo. Mechanistically, pentastatin partially acts through binding to the β1-integrin subunit and the Rho/ROCK pathway. These findings are the first to link pentastatin to pulmonary endothelial dysfunction and, thus, suggest a major role for BM-matrikines in pulmonary vascular diseases such as pulmonary hypertension.

Activation of Nrf2/ARE pathway by Anisodamine (654-2) for Inhibition of cellular aging and alleviation of Radiation-Induced lung injury

BackgroundRadiation-induced lung injury (RILI) is a common side effect of thoracic tumor radiotherapy, including early-stage radiation-induced lung injury (RP) and late-stage radiation-induced pulmonary fibrosis (RIPF). Currently, it is urgently needed to clarify the pathogenesis of RILI and find safe and effective RILI treatment methods. Irradiation causes DNA damage and oxidative stress in tissues and cells, induces cellular senescence, and promotes the occurrence and development of RILI. In recent years, Anisodamine (654-2) has shown potential therapeutic value in acute lung injury, acute kidney injury, chlamydial pneumonia, and COVID-19. However, there is currently no research on the mechanism of 654-2-mediated cellular senescence and its preventive and therapeutic effects on RILI. PurposeThis study aimed to investigate the protective effect and mechanism of 654-2 on X-ray-induced RILI. MethodsIn vivo experiments involved a mouse RILI model with 18 Gy X-ray irradiation. Mice were divided into control, model, medication (control + 654-2), and treatment (model + 654-2) groups. And mice in medication and treatment groups were intraperitoneal injection of 5 mg/kg 654-2 every other day until being sacrificed at week 6. In vitro experiments used MLE-12 cells irradiated with 16 Gy and divided into control, model, and model + 654-2（2 μM and 10 μM） groups. Various assays were performed to evaluate lung tissue morphology, fibrosis, apoptosis, cytokine expression, cellular senescence, protein expression, and antioxidant capacity. Results654-2 mitigated pulmonary pathological damage, inflammation, DNA damage, cellular senescence, and apoptosis in RILI mice and MLE-12 cells. It restored epithelial cell proliferation ability and enhanced antioxidant capacity. Additionally, 654-2 activated the Nrf2/ARE pathway, increased Nrf2 phosphorylation, and upregulated antioxidant gene expression. Inhibition of Nrf2 reversed the effects of 654-2 on ROS production, antioxidant capacity, and cell senescence. Conclusion654-2 can activate the Nrf2/ARE pathway, enhance cellular antioxidant capacity, and inhibit cellular senescence, thereby exerting a protective effect against RILI.

Human pulmonary microvascular endothelial cell DDAH1-mediated nitric oxide production promotes pulmonary smooth muscle cell apoptosis in co-culture.

Bronchopulmonary dysplasia (BPD) is the most common chronic lung disease in preterm infants, and pulmonary hypertension (PH) develops in 25%-40% of patients with BPD, increasing morbidity and mortality. BPD-PH is characterized by vasoconstriction and vascular remodeling. Nitric oxide (NO) is a pulmonary vasodilator and apoptotic mediator made in the pulmonary endothelium by NO synthase (eNOS). Asymmetric dimethylarginine (ADMA) is an endogenous eNOS inhibitor, primarily metabolized by dimethylarginine dimethylaminohydrolase-1 (DDAH1). Our hypothesis is that DDAH1 knockdown in human pulmonary microvascular endothelial cells (hPMVEC) will result in lower NO production, decreased apoptosis, and greater proliferation of human pulmonary arterial smooth muscle cells (hPASMC), whereas DDAH1 overexpression will have the opposite effect. hPMVECs were transfected with small interfering RNA targeting DDAH1 (siDDAH1)/scramble or adenoviral vector containing DDAH1 (AdDDAH1)/AdGFP for 24 h and co-cultured for 24 h with hPASMC. Analyses included Western blot for cleaved and total caspase-3, caspase-8, caspase-9, β-actin; trypan blue exclusion for viable cell numbers; terminal deoxynucleotide transferase dUTP nick end labeling (TUNEL); and BrdU incorporation. Small interfering RNA targeting DDAH1 (siDDAH1) transfected into hPMVEC resulted in lower media nitrites, cleaved caspase-3 and caspase-8 protein expression, and TUNEL staining; and greater viable cell numbers and BrdU incorporation in co-cultured hPASMC. Adenoviral-mediated transfection of the DDAH1 gene (AdDDAH1) into hPMVEC resulted in greater cleaved caspase-3 and caspase-8 protein expression and lower viable cell numbers in co-cultured hPASMC. Partial recovery of hPASMC viable cell numbers after AdDDAH1-hPMVEC transfection was observed when media were treated with hemoglobin to sequester NO. In conclusion, hPMVEC-DDAH1-mediated NO production positively regulates hPASMC apoptosis, which may prevent/attenuate aberrant pulmonary vascular proliferation/remodeling in BPD-PH.NEW & NOTEWORTHY BPD-PH is characterized by vascular remodeling. NO is an apoptotic mediator made in the pulmonary endothelium by eNOS. ADMA is an endogenous eNOS inhibitor metabolized by DDAH1. EC-DDAH1 overexpression resulted in greater cleaved caspase-3 and caspase-8 protein expression and lower viable cell numbers in co-cultured SMC. After NO sequestration, SMC viable cell numbers partially recovered despite EC-DDAH1 overexpression. EC-DDAH1-mediated NO production positively regulates SMC apoptosis, which may prevent/attenuate aberrant pulmonary vascular proliferation/remodeling in BPD-PH.

Rapamycin prevents lung injury related to acute spinal cord injury in rats

Severe injury occurs in the lung after acute spinal cord injury (ASCI) and autophagy is inhibited. However, rapamycin-activated autophagy's role and mechanism in lung injury development after ASCI is unknown. Preventing lung injury after ASCI by regulating autophagy is currently a valuable and unknown area. Herein, we aimed to investigate the effect and possible mechanism of rapamycin-activated autophagy on lung damage post-ASCI. An experimental animal study of rapamycin's effect and mechanism on lung damage after ASCI. We randomly divided 144 female wild-type Sprague–Dawley rats into a vehicle sham group (n = 36), a vehicle injury group (n = 36), a rapamycin sham group (n = 36), and a rapamycin injury group (n = 36). The spine was injured at the tenth thoracic vertebra using Allen's method. At 12, 24, 48, and 72 h after surgery, the rats were killed humanely. Lung damage was evaluated via pulmonary gross anatomy, lung pathology, and apoptosis assessment. Autophagy induction was assessed according to LC3, RAB7, and Beclin 1 levels. ULK-1, ULK-1 Ser555, ULK-1 Ser757, AMPK α and AMPK β1/2 were used to investigate the potential mechanism. After rapamycin pretreatment, the lung showed no obvious damage (e.g., cell death, inflammatory exudation, hemorrhage, and pulmonary congestion) at 12 h and 48 h after injury and Beclin1, LC3 and RAB7 levels increased. After rapamycin pretreatment, ULK-1, ULK-1 Ser555, and ULK-1 Ser757 levels increased at 12 h and 48 h after injury compared with the vehicle group, but they decreased at 12 h after injury compared with the rapamycin sham group. After rapamycin pretreatment, AMPKα levels did not change significantly before and after injury; however, at 48 h after injury, its level was elevated significantly compared with that in the vehicle group. Rapamycin can prevent lung injury after ASCI, possibly via upregulation of autophagy through the AMPK–mTORC1–ULK1 regulatory axis.

Characteristics of Mycobacterium tuberculosis serine protease Rv1043c in enzymology and pathogenicity in mice

The serine proteases of Mycobacteria tuberculosis (Mtb) are important contributors to the process of bacterial invasion and its pathogenesis. In the present study, we systematically characterized the role of the Rv1043c protein in Mycobacterium infection by purifying the Rv1043c protein in Escherichia coli and constructing a Mycobacterium smegmatis (Msg) strain overexpressing Rv1043c (Msg_Rv1043c). We found that Rv1043c had serine protease activity and localized to the surface of Mtb. We determined that the optimal pH and temperature for the Rv1043c serine protease were 9.0 and 45°C, respectively. Moreover, the serine protease activity of Rv1043c was enhanced by divalent metal ions of Ca2+ and Mg2+. Site-directed mutagenesis studies demonstrated that the serine 279 residue in Rv1043c plays a catalytic role. Additionally, mouse model studies confirmed that Rv1043c significantly enhanced the survival of Msg in vivo, induced pulmonary injury and lung cell apoptosis, and promoted the release of pro-inflammatory cytokines interleukin-1β and interleukin-6 in mice. This study presents novel insights into the relationship between mycobacterial serine protease and the pathogenesis of the disease.

Nintedanib Ameliorates Bleomycin-Induced Pulmonary Fibrosis, Inflammation, Apoptosis, and Oxidative Stress by Modulating PI3K/Akt/mTOR Pathway in Mice

Idiopathic pulmonary fibrosis (IPF) seriously threatens human life and health, and no curative therapy is available at present. Nintedanib is the first agent approved by the US Food and Drug Administration (FDA) in order to treat IPF; however, its mechanism of inhibition of IPF is still elusive. According to recent studies, nintedanib is a potent inhibitor. It can antagonize platelet-derived growth factor (PDGF), basic fibroblast growth factor (b-FGF), vascular endothelial growth factor (VEGF), etc., to inhibit pulmonary fibrosis. Whether there are other signaling pathways involved in IPF remains unknown. This study focused on investigating the therapeutic efficacy of nintedanib in bleomycin-mediated pulmonary fibrosis (PF) mice through PI3K/Akt/mTOR pathway. Following the induction of pulmonary fibrosis in C57 mice through bleomycin (BLM) administration, the mice were randomized into five groups: (1) the normal control group, (2) the BLM model control group, (3) the low-dose Nintedanib administration model group, (4) the medium-dose nintedanib administration model group, and (5) the high-dose nintedanib administration model group. For lung tissues, morphological changes were found by HE staining and Masson staining, ELISA method was used to detect inflammatory factors, alkaline water method to estimate collagen content, and western blotting for protein levels. TUNEL staining and immunofluorescence methods were used to analyze the effect of nintedanib on lung tissue and the impacts and underlying mechanisms of bleomycin-induced pulmonary fibrosis. After 28 days, bleomycin-treated mice developed significant pulmonary fibrosis. Relative to bleomycin-treated mice, nintedanib-treated mice had markedly reduced degrees of PF. In addition, nintedanib showed lung-protective effects by up-regulating antioxidant levels, down-regulating inflammatory protein expression, and reducing collagen accumulation. We demonstrated that nintedanib ameliorated bleomycin-induced lung injury by inhibiting the P13K/Akt/mTOR pathway as well as apoptosis. In addition, significant improvement in pulmonary fibrosis was seen after nintedanib (30/60/120 mg/kg body weight/day) treatment through a dose-dependent way. Histopathological results further corroborated the effect of nintedanib treatment on remarkably attenuating bleomycin-mediated mouse lung injury. According to our findings, nintedanib restores the antioxidant system, suppresses pro-inflammatory factors, and inhibits apoptosis. Nintedanib can reduce bleomycin-induced inflammation by downregulating PI3K/Akt/mTOR pathway, PF, and oxidative stress (OS).