Fine particulate matter (PM2.5) exposure contributes to over 4 million premature deaths annually, yet the mechanistic role of lung microbiota in PM2.5-induced pulmonary inflammation remains poorly understood. In collaboration of 16S rRNA and single-cell RNA multi-omics analysis and in vivo/in vitro experimental validation with antibiotic intervention strategies, the study here examined PM2.5-microbiota interactions in murine PM2.5 exposure models and cellular systems. It was found that PM2.5 exposure induced lung microbiota dysbiosis characterized by Gram-negative bacterial expansion, particularly Proteobacteria dominance, accompanied by reduced microbial diversity. scRNA analysis revealed coordinated activation of TLR4/MyD88/NLRP3 inflammatory signaling pathways and p53/p21/p16-mediated cell cycle arrest. Moreover, PM2.5 exposure activated NLRP3 inflammosome-dependent macrophage pyroptosis as evidenced by increased interleukin (IL)-1β, IL-18, caspase-1, and GSDMD expression. In vitro studies demonstrated that the inflammatory changes induced by PM2.5 exposure were statistically indistinguishable from those of LPS-positive controls, confirming endotoxin-like mechanisms. Critically, antibiotic pretreatment effectively attenuated PM2.5-induced inflammatory responses, cell cycle arrest, and tissue pathology, which established causality between microbiota disruption and pulmonary dysfunction. In conclusion, this study revealed lung microbiota dysbiosis as a critical mediator of PM2.5-induced pulmonary inflammation through Gram-negative bacterial expansion and subsequent endotoxin-like activation of inflammatory cascades, thereby providing novel mechanistic insights and potential microbiome-targeted therapeutic strategies for air pollution-associated respiratory diseases.

Background: Inflammation and the tumor immune microenvironment contribute to lung adenocarcinoma (LUAD) progression, but the relationship among inflammation-linked transcriptional heterogeneity, patient survival, and immune-state variation remains incompletely defined. Objective: We aimed to identify inflammation-associated LUAD subtypes, derive a parsimonious survival-stratification signature, and characterize its immune and pathway context across public transcriptomic cohorts. Methods: Expression profiles and clinical data were obtained from TCGA-LUAD, GTEx normal lung, and GEO datasets GSE11969, GSE30219, GSE31210, and GSE40791. A curated set of 596 inflammation-related genes was used for consensus clustering. Differential-expression analysis, functional enrichment, univariate Cox regression, and LASSO-Cox modeling were integrated to construct a gene-expression risk score. The prognostic dataset comprised 730 cases and was randomly divided into training (n=502) and internal-validation (n=228) sets; 85 GSE30219 cases formed an external-validation cohort. Immune-cell enrichment, gene set enrichment analysis (GSEA), gene set variation analysis (GSVA), and pan-cancer analyses were used for biological contextualization. Results: The LUAD-versus-control comparison identified 1,305 differentially expressed genes, including 498 upregulated and 807 downregulated genes. Consensus clustering resolved two inflammation-associated subtypes and 67 subtype-associated genes, of which 64 were higher and 3 were lower in Cluster 1 relative to Cluster 2. Thirty-three genes overlapped between the tumor-control and subtype contrasts. LASSO-Cox regression selected CHRDL1, FDCSP, CXCL13, CYP4B1, and S100P. The 1-, 3-, and 5-year areas under the time-dependent receiver operating characteristic curve were 0.6625, 0.6581, and 0.6658 in the training set; 0.7422, 0.6537, and 0.6761 in internal validation; and 0.6560, 0.6387, and 0.6753 in external validation. Risk groups differed across multiple T-cell, B-cell, natural-killer-cell, myeloid, dendritic-cell, macrophage, and granulocyte signatures. Positive GSEA signals included cell cycle (normalized enrichment score [NES]=2.67; adjusted P=1.42 x 10⁻⁸), DNA replication (NES=2.52; adjusted P=2.52 x 10⁻⁷), and mismatch repair (NES=2.20; adjusted P=1.77 x 10⁻⁴). Conclusions: The five-gene expression score separated LUAD survival groups and captured coordinated proliferative and immune transcriptional states. Its moderate discrimination supports further biological and clinical validation rather than immediate clinical application.

Background Stroke stands as the second leading cause of death worldwide. Currently, extensive research has been conducted on stroke risk factors. However, when stroke patients contend with multiple risk factors, the impact on clinical indicators remains uncertain. Objectives This study seeks to investigate potential significant variations among distinct ranges of clinical indicators in instances where stroke patients experience multiple risk factors and various ischemic stroke subtypes. Material and methods The research encompassed 440 stroke patients admitted to the First People's Hospital of Wenling City, Zhejiang Province, China. These patients were classified based on the type and quantity of risk factors and subtypes of ischemic stroke they presented. The χ 2 test was employed to assess the relationship between the risk of comorbid diseases and clinical indicators in stroke patients. Results The results of our study have underscored a significant correlation between various comorbid risk factors in stroke patients and the patients' age ( P < 0.010). Furthermore, we observed noteworthy disparities in the plasma levels of IL-2, IL-4, IL-6, IL-10, TNF-α, and INF-γ between patients devoid of risk factors and those presenting with comorbid risk factors associated with stroke. Significant differences in INF-γ were observed between the two subtypes of ischemic stroke, namely lacunar infarction and cardioembolic stroke. Conclusion Age is correlated with an elevated risk of stroke. Individuals exhibiting multiple stroke risk factors and diverse ischemic stroke subtypes commonly present with abnormal lipid levels and imbalances in Th1/Th2 cytokines. These factors significantly contribute to the onset and progression of stroke. Furthermore, inflammatory responses, particularly those induced by atherosclerosis, play a pivotal role in the genesis of stroke and exert a substantial influence on its prognosis.

Aims: To develop a model based on breast MRI to stratify axillary lymph node metastasis (ALNM) in breast cancer. Patients & methods: A total of 134 eligible patients were used to build a predicting model, which was validated with an independent group of 57 patients and evaluated for accuracy and sensitivity. Results: A model based on breast MRI was developed and yielded total accuracy of 82.5% and sensitivities of 94.3, 64.3 and 62.5% to predict patients with no, low and heavy ALNM burden, respectively, in the validation group. Conclusion: A noninvasive model based on breast MRI was developed to preoperatively stratify ALNM in breast cancer; its performance needs to be validated and improved in future research.

IntroductionThis study is aimed to explore the effects of Adenosine A2a receptors (A2aR) on hypoxia-induced pulmonary hypertension (HPH) via mitochondrial ATP-sensitive potassium channels (MitoKATP) in vivo and in vitro.Material and methodsUsing wild-type (WT) and A2aR-deficient (A2aR-/-) mice; hypoxic pulmonary artery smooth muscle cells (PASMCs) were induced by a 24-hours hypoxia exposure. Mice and PASMCs were treated with the A2aR agonist CGS21680, MitoKATP blocker 5-hydroxydecanoic acid sodium salt (5HD), or MitoKATP agonist diazoxide. Mitochondrial morphology was observed by electron microscopy. The mitochondrial membrane potential (Δψm); invasive hemodynamic parameters; right ventricular (RV) hypertrophy index; pulmonary arterial remodeling index; proliferative and apoptotic indexes; protein expression levels of A2aR, Bax, Bcl-2, and Caspase-9; and release of cytochrome C from the mitochondria to the cytoplasm were measured.ResultsIn vitro, hypoxia induced the opening of MitoKATP. The up-regulation of A2aR reduced the opening of MitoKATP, and the blocking of MitoKATP or activating A2aR promoted mitochondria-dependent apoptosis of PASMCs. In vivo, compared with WT mice, A2aR-/- mice displayed increased RV systolic pressure, RV hypertrophy index, and pulmonary arterial remodeling index. The expression levels of Bax, cytochrome C, and Caspase-9 were higher and Bcl-2 expression was lower in A2aR-/- mice than in WT mice. CGS21680 could reverse hypoxia-induced hemodynamic changes, RV hypertrophy, and pulmonary arterial remodeling as well as abnormal proliferation and apoptosis resistance in WT mice with pulmonary hypertension (PH).ConclusionsA2aR induced the mitochondrial-dependent apoptosis pathway and inhibited PASMC proliferation by blocking MitoKATP, thereby inhibiting pulmonary vascular structural remodeling and reducing PH.

Background Baicalin is a flavonoid compound that exerts specific pharmacological effect in attenuating the proliferation, migration, and apoptotic resistance of hypoxia-induced pulmonary artery smooth muscle cells (PASMCs). However, the underlying mechanism has not been fully elucidated yet. Although our previous studies had indicated that activation of A2aR attenuates CXCR expression, little is known about the relationship between A2aR and SDF-1/CXCR4 axis in hypoxic PASMCs. In this study, we aimed to investigate the effect of A2aR on the SDF-1/CXCR4 axis in hypoxic PASMCs, the mechanism underlying this effect, and whether baicalin exerts its protective functions though A2aR. Methods Rat PASMCs were cultured under normoxia/hypoxia and divided into nine groups: normoxia, hypoxia, hypoxia + AMD3100 (a CXCR4 antagonist), hypoxia + baicalin, hypoxia + negative virus, normoxia + A2aR knockdown, hypoxia + A2aR knockdown, hypoxia + CGS21680 (an A2aR agonist), and hypoxia + A2aR knockdown + baicalin. Lentiviral transfection methods were used to establish the A2aR knockdown model in PASMCs. Cells were incubated under hypoxic conditions for 24 h. Expression levels of A2aR, SDF-1, and CXCR4 were detected using RT-qPCR and western blot. The proliferation and migration rate were observed via CCK-8 and Transwell methods. Cell cycle distribution and cell apoptosis were measured by flow cytometry (FCM) and the In-Situ Cell Death Detection kit (Fluorescein). Results Under hypoxic conditions, levels of A2aR, SDF-1, and CXCR4 were significantly increased compared to those under normoxia. The trend of SDF-1 and CXCR4 being inhibited when A2aR is up-regulated was more obvious in the baicalin intervention group. Baicalin directly enhanced A2aR expression, and A2aR knockdown weakened the function of baicalin. SDF-1 and CXCR4 expression levels were increased in the hypoxia + A2aR knockdown group, as were the proliferation and migration rates of PASMCs, while the apoptotic rate was decreased. Baicalin and CGS21680 showed opposite effects. Conclusions Our data indicate that baicalin efficiently attenuates hypoxia-induced PASMC proliferation, migration, and apoptotic resistance, as well as SDF-1 secretion, by up-regulating A2aR and down-regulating the SDF-1/CXCR4 axis.

Recent studies have shown that both adenosine monophosphate activated protein kinase (AMPK) and the mammalian target of rapamycin (mTOR) are energy sensors and are related to autophagy. Our recent reports have shown that salidroside can exert protective effects against hypoxia-induced pulmonary arterial smooth muscle cell (PASMC) proliferation and apoptosis resistance through the AMPK pathway. This study aims to explore the relationship among AMPK, mTOR and ULK1 in PASMCs under hypoxic conditions and to investigate whether the protective effects of salidroside are related to the autophagic cell death pathway.

Previous studies in our lab have demonstrated that Adenosine A2a receptor (A2aR) gene-knockout mice were vulnerable to pulmonary fibrosis induced by bleomycin (BLM). Inhibition of the SDF-1/CXCR4 axis has been reported to protect the lungs from fibrogenesis in BLM-exposed mice. Little is yet known about the relationships between A2aR and the SDF-1/CXCR4 axis in idiopathic pulmonary fibrosis (IPF). This study probes the role of A2aR in the fibrotic process and explores the relationship between A2aR and the SDF-1/CXCR4 axis in BLM-induced pulmonary fibrosis in mice. In the study, A2aR-/- and A2aR+/+ BALB/c mice were exposed to BLM by intratracheal instillation, and CGS-21680 (CGS), an A2aR agonist, was administered daily for 28 days to the A2aR+/+ mice in the BLM-induced fibrosis group. Activation of A2aR produced an anti-fibrotic effect as indicated by the evaluations of the lung architecture, microstructure and ultrastructure. The quantitative analysis indicated that treatment with CGS significantly reduced the collagen content in lungs. To explore the potential mechanisms, the expression levels of A2aR, SDF-1, and CXCR4 were subsequently determined using ELISA, in situ hybridization (ISH), immunohistochemical staining and western blotting techniques. Administration of CGS markedly suppressed the elevated expression levels of SDF-1 and CXCR4. Moreover, the A2aR-/- mice developed more severe pulmonary fibrosis than the normal mice when exposed to BLM. Furthermore, the SDF-1/CXCR4 axis was aberrantly uninhibited in the knockout mice. Together, these findings indicated that A2aR alleviated BLM-induced lung fibrosis, at least partially via the SDF-1/CXCR4 pathway, which could be a potential therapeutic target for the treatment of IPF.

Objectives: To provide detailed histological characteristics of newly formed periodontal tissues and determine the expression level of Cbfa1 following Emdogain (EMD) application in alveolar bone defects in beagle dogs. Methods: Eight one-year-old male beagles were used. Two untreated beagles served as blank control. Alveolar bone defects were created in the other six dogs in mesial and distal aspects of the mandibular second premolars bilaterally. The left side of the jaw was treated with EMD and the right side with open flap debridement (OFD) (control group). Then, the periodontal regeneration and Cbfa1 expression were investigated using hematoxylin-eosin staining and immunohistology methods. Results: There was significantly more periodontal regeneration in EMD-treated group than in OFD group. The intensity and number of Cbfa1-positive fibroblasts in OFD group were significantly less than those in EMD-treated group and blank control (P<0.05), while the difference between EMD group and blank control was not significant (P>0.05). Conclusion: EMD can induce significant periodontal regeneration. The osteogenic function of EMD may involve upregulation of Cbfa1 expression.

Abstract Purpose: The carcinogenic capacity of B[a]P/B[a]PDE is supported by epidemiologic studies. However, the molecular mechanisms responsible for B[a]P/B[a]PDE-caused lung cancer have not been well investigated. We evaluated here the role of novel target PHLPP2 in lung inflammation and carcinogenesis upon B[a]P/B[a]PDE exposure. Experimental Design: We used the Western blotting, RT-PCR, [35S]methionine pulse and immunohistochemistry staining to determine PHLPP2 downregulation following B[a]P/B[a]PDE exposure. Both B[a]PDE-induced Beas-2B cell transformation model and B[a]P-caused mouse lung cancer model were used to elucidate the mechanisms leading to PHLPP2 downregulation and lung carcinogenesis. The important findings were also extended to in vivo human studies. Results: We found that B[a]P/B[a]PDE exposure downregulated PHLPP2 expression in human lung epithelial cells in vitro and in mouse lung tissues in vivo. The ectopic expression of PHLPP2 dramatically inhibited cell transformation upon B[a]PDE exposure. Mechanistic studies showed that miR-205 induction was crucial for inhibition of PHLPP2 protein translation by targeting PHLPP2-3′-UTR. Interestingly, PHLPP2 expression was inversely associated with tumor necrosis factor alpha (TNFα) expression, with low PHLPP2 and high TNFα expression in lung cancer tissues compared with the paired adjacent normal lung tissues. Additional studies revealed that PHLPP2 exhibited its antitumorigenic effect of B[a]P/B[a]PDE through the repression of inflammatory TNFα transcription. Conclusions: Our studies not only first time identify PHLPP2 downregulation by lung carcinogen B[a]P/B[a]PDE, but also elucidate a novel molecular mechanisms underlying lung inflammation and carcinogenesis upon B[a]P/B[a]PDE exposure. Clin Cancer Res; 21(16); 3783–93. ©2015 AACR.

Myosin light chain phosphatase with its regulatory subunit, myosin phosphatase target subunit 1 (MYPT1) modulates Ca(2+)-dependent phosphorylation of myosin light chain by myosin light chain kinase, which is essential for smooth muscle contraction. The role of MYPT1 in vascular smooth muscle was investigated in adult MYPT1 smooth muscle specific knock-out mice. MYPT1 deletion enhanced phosphorylation of myosin regulatory light chain and contractile force in isolated mesenteric arteries treated with KCl and various vascular agonists. The contractile responses of arteries from knock-out mice to norepinephrine were inhibited by Rho-associated kinase (ROCK) and protein kinase C inhibitors and were associated with inhibition of phosphorylation of the myosin light chain phosphatase inhibitor CPI-17. Additionally, stimulation of the NO/cGMP/protein kinase G (PKG) signaling pathway still resulted in relaxation of MYPT1-deficient mesenteric arteries, indicating phosphorylation of MYPT1 by PKG is not a major contributor to the relaxation response. Thus, MYPT1 enhances myosin light chain phosphatase activity sufficient for blood pressure maintenance. Rho-associated kinase phosphorylation of CPI-17 plays a significant role in enhancing vascular contractile responses, whereas phosphorylation of MYPT1 in the NO/cGMP/PKG signaling module is not necessary for relaxation.

Lymphatic absorption is a highly regulated process driven by both an extrinsic mechanism (external force) and an intrinsic mechanism (lymphatic vessel contractility). The lymphatic muscle is a specialized smooth muscle with unique mechanical properties. To understand the molecular mechanism and relative contribution of smooth muscle contraction in lymphatic absorption, we analyzed mice with a smooth muscle-specific deletion of Mylk, a critical gene for smooth muscle contraction. Interestingly, the knockout mice were significantly resistant to anesthesia reagents. Upon injection in the feet with FITC-dextran, the mutant mice displayed a 2-fold delay of the absorption peak in the peripheral circulation. Examining the ear lymphatic vessels of the mutant mice revealed a reduction in the amount of fluid in the lumens of the lymphangions, suggesting an impairment of lymph formation. The Mylk-deficient lymphatic muscle exhibited a significant reduction of peristalsis and of myosin light chain phosphorylation in response to depolarization. We thus concluded that MLCK and myosin light chain phosphorylation are required for lymphatic vessel contraction. Lymphatic contractility is not an exclusive requirement for lymphatic absorption, and external force appears to be necessary for absorption.