Objective: To investigate the role of connective tissue growth factor (CTGF) and PI3K/Akt signaling pathways in paraquat (PQ) -induced alterations in alveolar epithelial cell mesenchymalization (EMT) . Methods: In February 2023, RLE-6TN cells were divided into 2 groups, which were set as uncontaminated group and contaminated group (200 μmol/L PQ), and cellular EMT alteration, CTGF and PI3K/Akt signaling pathway related molecules expression were detected by cell scratch assay, qRT-PCR and western-blot assay. Using shRNA interference technology to specifically inhibit the expression of CTGF, RLE-6TN cells were divided into four groups: control group, PQ group (200 μmol/L PQ), interference group (transfected with a plasmid with shRNA-CTGF+200 μmol/L PQ), and null-loaded group (transfected with a plasmid with scramble- CTGF+200 μmol/L PQ), qRT-PCR and western blot were used to examine the alteration of the cellular EMT and the expression of molecules related to the activity of PI3K/Akt pathway. The PI3K/Akt signaling pathway was blocked by the PI3K inhibitor LY294002, and the expression of EMT-related molecules in cells of the control group, PQ group (200 μmol/L PQ), and inhibitor group (200 μmol/L PQ+20 μmol/L LY294002) was examined by qRT-PCR and western blot.The t-test was used to compare the differences between the two groups, while the analysis of variance (ANOVA) was applied to compare the differences among multiple groups. For further pairwise comparisons, the Bonferroni method was adopted. Results: The results of cell scratch test showed that compared with the uncontaminated group, RLE-6TN cells in the contaminated group had faster migration rate, lower mRNA and protein expression levels of E-Cadherin, and higher mRNA and protein expression levels of α-SMA, CTGF, PI3K and Akt, with statistical significance (P<0.05). After specific inhibition of CTGF expression, the mRNA and protein expression of CTGF, PI3K, Akt, and α-SMA in the cells of the interference group were significantly lower than that of the PQ group and the null-loaded group (P<0.05/6), whereas that of E-Cadherin was higher than that of the PQ group and the null-loaded group (P<0.05/6). Specifically blocking the PI3K/Akt signaling pathway, the mRNA and protein expression of PI3K, Akt and α-SMA in the cells of the inhibitor group was decreased compared with that of the PQ group (P<0.05/3), while the expression of E-Cadherin was elevated compared with that of the PQ group (P<0.05/3) . Conclusion: CTGF may promote PQ-induced alveolar epithelial cell EMT through activation of the PI3K/Akt signaling pathway. Inhibition of CTGF expression or blockade of PI3K/Akt signaling pathway activity can alleviate the extent of PQ-induced alveolar epithelial cell EMT.
目的： 探讨结缔组织生长因子（CTGF）及磷脂酰肌醇3-激酶/丝氨酸/苏氨酸激酶（PI3K/Akt）信号通路在百草枯（PQ）致肺泡上皮细胞上皮-间充质化（EMT）改变的作用。 方法： 于2023年2月，将RLE-6TN细胞分成2组，设为未染毒组和染毒组（200 μmol/L PQ），采用细胞划痕实验、qRT-PCR和Western-blot法检测细胞EMT改变、CTGF及PI3K/Akt信号通路相关分子表达情况。利用shRNA干扰技术特异性抑制CTGF的表达，将RLE-6TN细胞分成4组，分别为对照组、PQ组（200 μmol/L PQ）、干扰组（转染含shRNA-CTGF质粒+200 μmol/L PQ）和空载组（转染含CTGF-scramble质粒+200 μmol/L PQ），qRT-PCR和Western-blot法检测细胞EMT改变及PI3K/Akt通路活性相关分子的表达情况。使用PI3K抑制剂LY294002阻断PI3K/Akt信号通路，运用qRT-PCR和Western-blot法检测对照组、PQ组（200 μmol/L PQ）、抑制剂组（200 μmol/L PQ+20 μmol/L LY294002）细胞EMT相关分子表达情况。两组间差异比较采用t检验，多组间差异比较采用方差分析，进一步两两比较采用Bonferroni法。 结果： 细胞划痕实验结果显示，与未染毒组比较，PQ染毒组RLE-6TN细胞迁移速度更快，E-钙黏蛋白（E-Cadherin）的mRNA和蛋白表达量更低，α-平滑肌肌动蛋白（α-SMA）、CTGF、PI3K、Akt的mRNA和蛋白表达量更高，差异均有统计学意义（P<0.05）。特异性抑制CTGF表达后，干扰组细胞CTGF、PI3K、Akt、α-SMA的mRNA和蛋白表达量明显低于PQ组和空载组（P<0.05/6），E-Cadherin的mRNA和蛋白表达量高于PQ组和空载组（P<0.05/6）。特异性阻断PI3K/Akt信号通路，抑制剂组细胞PI3K、Akt和α-SMA的mRNA和蛋白表达量较PQ组降低（P<0.05/3），E-Cadherin表达量较PQ组升高（P<0.05/3）。 结论： CTGF可能通过激活PI3K/Akt信号通路促进PQ致肺泡上皮细胞EMT改变，抑制CTGF的表达或阻断PI3K/Akt信号通路活性，可减轻PQ所致肺泡上皮细胞EMT的程度。.

Pulmonary toxicity is a serious side effect of some specific anticancer drugs. Bleomycin is a well-known anticancer drug that triggers severe reactions in the lungs. It is an approved drug that may be prescribed for the treatment of testicular cancers, Hodgkin\'s and non-Hodgkin\'s lymphomas, ovarian cancer, head and neck cancers, and cervical cancer. A large number of experimental studies and clinical findings show that bleomycin can concentrate in lung tissue, leading to massive oxidative stress, alveolar epithelial cell death, the proliferation of fibroblasts, and finally the infiltration of immune cells. Chronic release of pro-inflammatory and pro-fibrotic molecules by immune cells and fibroblasts leads to pneumonitis and fibrosis. Both fibrosis and pneumonitis are serious concerns for patients who receive bleomycin and may lead to death. Therefore, the management of lung toxicity following cancer therapy with bleomycin is a critical issue. This review explains the cellular and molecular mechanisms of pulmonary injury following treatment with bleomycin. Furthermore, we review therapeutic targets and possible promising strategies for ameliorating bleomycin-induced lung injury.

Organoid models have become an integral part of the research methodology in the lung field. These systems allow for the study of progenitor and stem cell self-renewal, self-organization, and differentiation. Distinct models of lung organoids mimicking various anatomical regions of mature lungs have emerged in parallel to the increased gain of knowledge regarding epithelial stem and progenitor cell populations and the corresponding mesenchymal cells that populate the in vivo niche. In the distal lung, type 2 alveolar epithelial cells (AEC2s) represent a stem cell population that is engaged in regenerative mechanisms in response to various insults. These cells self-renew and give rise to AEC1s that carry out gas exchange. Multiple experimental protocols allowing the generation of alveolar organoids, or alveolospheres, from murine lungs have been described. Among the drawbacks have been the requirement of transgenic mice allowing the isolation of AEC2s with high viability and purity, and the occasional emergence of bronchiolar and bronchioalveolar organoids. Here, we provide a refined gating strategy and an optimized protocol for the generation of alveolospheres from wild-type mice. Our approach not only overcomes the need for transgenic mice to generate such organoids, but also yields a pure culture of alveolospheres that is devoid of bronchiolar and bronchioalveolar organoids. Our protocol contributes to the standardization of this important research tool.

BACKGROUND: Silicosis, characterized by interstitial lung inflammation and fibrosis, poses a significant health threat. ATII cells play a crucial role in alveolar epithelial repair and structural integrity maintenance. Inhibiting ATII cell senescence has shown promise in silicosis treatment. However, the mechanism behind silica-induced senescence remains elusive.
METHODS: The study employed male C57BL/6 N mice and A549 human alveolar epithelial cells to investigate silicosis and its potential treatment. Silicosis was induced in mice via intratracheal instillation of crystalline silica particles, with honokiol administered intraperitoneally for 14 days. Silica-induced senescence in A549 cells was confirmed, and SIRT3 knockout and overexpression cell lines were generated. Various analyses were conducted, including immunoblotting, qRT-PCR, histology, and transmission electron microscopy. Statistical significance was determined using one-way ANOVA with Tukey\'s post-hoc test.
RESULTS: This study elucidates how silica induces ATII cell senescence, emphasizing mtDNA damage. Notably, honokiol (HKL) emerges as a promising anti-senescence and anti-fibrosis agent, acting through sirt3. honokiol effectively attenuated senescence in ATII cells, dependent on sirt3 expression, while mitigating mtDNA damage. Sirt3, a class III histone deacetylase, regulates senescence and mitochondrial stress. HKL activates sirt3, protecting against pulmonary fibrosis and mitochondrial damage. Additionally, HKL downregulated cGAS expression in senescent ATII cells induced by silica, suggesting sirt3\'s role as an upstream regulator of the cGAS/STING signaling pathway. Moreover, honokiol treatment inhibited the activation of the NF-κB signaling pathway, associated with reduced oxidative stress and mtDNA damage. Notably, HKL enhanced the activity of SOD2, crucial for mitochondrial function, through sirt3-mediated deacetylation. Additionally, HKL promoted the deacetylation activity of sirt3, further safeguarding mtDNA integrity.
CONCLUSIONS: This study uncovers a natural compound, HKL, with significant anti-fibrotic properties through activating sirt3, shedding light on silicosis pathogenesis and treatment avenues.

The stemness loss-associated dysregeneration of impaired alveolar type 2 epithelial (AT2) cells abolishes the reversible therapy of idiopathic pulmonary fibrosis (IPF). We here report an inhalable mucus-penetrating lipid nanoparticle (LNP) for codelivering dual mRNAs, promoting realveolarization via restoring AT2 stemness for IPF treatment. Inhalable LNPs were first formulated with dipalmitoylphosphatidylcholine and our in-house-made ionizable lipids for high-efficiency pulmonary mucus penetration and codelivery of dual messenger RNAs (mRNAs), encoding cytochrome b5 reductase 3 and bone morphogenetic protein 4, respectively. After being inhaled in a bleomycin model, LNPs reverses the mitochondrial dysfunction through ameliorating nicotinamide adenine dinucleotide biosynthesis, which inhibits the accelerated senescence of AT2 cells. Concurrently, pathological epithelial remodeling and fibroblast activation induced by impaired AT2 cells are terminated, ultimately prompting alveolar regeneration. Our data demonstrated that the mRNA-LNP system exhibited high protein expression in lung epithelial cells, which markedly extricated the alveolar collapse and prolonged the survival of fibrosis mice, providing a clinically viable strategy against IPF.

Pulmonary hypertension (PH) refers to a group of deadly lung diseases characterized by vascular lesions in the microvasculature and a progressive increase in pulmonary vascular resistance. The prevalence of PH has increased over time. Currently, the treatment options available for PH patients have limited efficacy, and none of them can fundamentally reverse pulmonary vascular remodeling. Stem cells represent an ideal seed with proven efficacy in clinical studies focusing on liver, cardiovascular, and nerve diseases. Since the potential therapeutic effect of mesenchymal stem cells (MSCs) on PH was first reported in 2006, many studies have demonstrated the efficacy of stem cells in PH animal models and suggested that stem cells can help slow the deterioration of lung tissue. Existing PH treatment studies basically focus on the paracrine action of stem cells, including protein regulation, exosome pathway, and cell signaling; however, the specific mechanisms have not yet been clarified. Apoptotic and afunctional pulmonary microvascular endothelial cells (PMVECs) and alveolar epithelial cells (AECs) are two fundamental promoters of PH although they have not been extensively studied by researchers. This review mainly focuses on the supportive communication and interaction between PMVECs and AECs as well as the potential restorative effect of stem cells on their injury. In the future, more studies are needed to prove these effects and explore more radical cures for PH.

OBJECTIVE: Severe community-acquired pneumonia (SCAP) is a common respiratory system disease with rapid development and high mortality. Exploring effective biomarkers for early detection and development prediction of SCAP is of urgent need. The function of miR-486-5p in SCAP diagnosis and prognosis was evaluated to identify a promising biomarker for SCAP.
METHODS: The serum miR-486-5p in 83 patients with SCAP, 52 healthy individuals, and 68 patients with mild CAP (MCAP) patients were analyzed by PCR. ROC analysis estimated miR-486-5p in screening SCAP, and the Kaplan-Meier and Cox regression analyses evaluated the predictive value of miR-486-5p. The risk factors for MCAP patients developing SCAP were assessed by logistic analysis. The alveolar epithelial cell was treated with Klebsiella pneumonia to mimic the occurrence of SCAP. The targeting mechanism underlying miR-486-5p was evaluated by luciferase reporter assay.
RESULTS: Upregulated serum miR-486-5p screened SCAP from healthy individuals and MCAP patients with high sensitivity and specificity. Increasing serum miR-486-5p predicted the poor outcomes of SCAP and served as a risk factor for MCAP developing into SCAP. K. pneumonia induced suppressed proliferation, significant inflammation and oxidative stress in alveolar epithelial cells, and silencing miR-486-5p attenuated it. miR-486-5p negatively regulated FOXO1, and the knockdown of FOXO1 reversed the effect of miR-486-5p in K. pneumonia-treated alveolar epithelial cells.
CONCLUSIONS: miR-486-5p acted as a biomarker for the screening and monitoring of SCAP and predicting the malignancy of MCAP. Silencing miR-486-5p alleviated inflammation and oxidative stress induced by K. pneumonia via negatively modulating FOXO1.

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.

BACKGROUND: Acute lung injury (ALI) is characterized by acute pulmonary inflammatory infiltration. Alveolar epithelial cells (AECs) release numerous pro-inflammatory cytokines, which result in the pathological changes seen in ALI. Ophiopogonin D (OD), extracted from the roots of Ophiopogon japonicus (Thunb.) Ker Gawl. (Liliaceae), reduces inflammation; however, the efficacy of OD in ALI has not been reported and the underlying molecular mechanisms remain unclear.
OBJECTIVE: This study investigated the anti-inflammatory effects of OD, as well as the underlying mechanisms, in AECs and a mouse ALI model.
METHODS: Lipopolysaccharide (LPS) and tumor necrosis factor-α (TNF-α) were used to stimulate macrophages and A549 cells, and a mouse ALI model was established by intratracheal LPS administration. The anti-inflammatory effects and mechanisms of OD in the TNF-α-induced in vitro inflammation model was evaluated using real-time quantitative polymerase chain reaction qPCR), enzyme-linked immunosorbent assay (ELISA), western blotting, nuclear and cytoplasmic protein extraction, and immunofluorescence. The in vivo anti-inflammatory activity of OD was evaluated using hematoxylin and eosin staining, qPCR, ELISA, and western blotting.
RESULTS: The bronchoalveolar lavage fluid and lung tissue of LPS-induced ALI mice exhibited increased TNF-α expression. TNF-α induced a significantly greater pro-inflammatory effect in AECs than LPS. OD reduced inflammation and mitogen-activated protein kinase (MAPK) and transcription factor p65 phosphorylation in vivo and in vitro and promoted signal transducer and activator of transcription 3 (STAT3) phosphorylation and A20 expression, thereby inducing apoptosis signal-regulating kinase 1 (ASK1) proteasomal degradation.
CONCLUSIONS: OD exerts an anti-inflammatory effect by promoting STAT3-dependent A20 expression and ASK1 degradation. OD may therefore have therapeutic value in treating ALI and other TNF-α-related inflammatory diseases.

UNASSIGNED: Nanoplastics, an emerging form of pollution, are easily consumed by organisms and pose a significant threat to biological functions due to their size, expansive surface area, and potent ability to penetrate biological systems. Recent findings indicate an increasing presence of airborne nanoplastics in atmospheric samples, such as polystyrene (PS), raising concerns about potential risks to the human respiratory system.
UNASSIGNED: This study investigates the impact of 800 nm diameter-PS nanoparticles (PS-NPs) on A549, a human lung adenocarcinoma cell line, examining cell viability, redox balance, senescence, apoptosis, and internalization. We also analyzed the expression of hallmark genes of these processes.
UNASSIGNED: We demonstrated that PS-NPs of 800 nm in diameter significantly affected cell viability, inducing oxidative stress, cellular senescence, and apoptosis. PS-NPs also penetrated the cytoplasm of A549 cells. These nanoparticles triggered the transcription of genes comprised in the antioxidant network [SOD1 (protein name: superoxide dismutase 1, soluble), SOD2 (protein name: superoxide dismutase 2, mitochondrial), CAT (protein name: catalase), Gpx1 (protein name: glutathione peroxidase 1), and HMOX1 (protein name: heme oxygenase 1)], senescence-associated secretory phenotype [Cdkn1a (protein name: cyclin-dependent kinase inhibitor 1A), IL1A (protein name: interleukin 1 alpha), IL1B (protein name: interleukin 1 beta), IL6 (protein name: interleukin 6), and CXCL8 (protein name: C-X-C motif chemokine ligand 8)], and others involved in the apoptosis modulation [BAX (protein name: Bcl2 associated X, apoptosis regulator), CASP3 (protein name: caspase 3), and BCL2 (protein name: Bcl2, apoptosis regulator)].
UNASSIGNED: Collectively, this investigation underscores the importance of concentration (dose-dependent effect) and exposure duration as pivotal factors in assessing the toxic effects of PS-NPs on alveolar epithelial cells. Greater attention needs to be directed toward comprehending the risks of cancer development associated with air pollution and the ensuing environmental toxicological impacts on humans and other terrestrial mammals.