Nicotine exposure exacerbates silica-induced pulmonary fibrosis via STAT3-BDNF-TrkB-mediated epithelial-mesenchymal transition in alveolar type II cells.

The addictive substance nicotine, found in cigarettes and some e-cigarettes, plays a vital role in pro-inflammatory and fibrotic processes. However, the part played by nicotine in the progression of silica-induced pulmonary fibrosis is poorly understood. We used mice exposed to both silica and nicotine to investigate whether nicotine synergizes with silica particles to worsen lung fibrosis. The results revealed that nicotine accelerated the development of pulmonary fibrosis in silica-injured mice by activating STAT3-BDNF-TrkB signalling. Mice with a history of exposure to nicotine showed an increase in Fgf7 expression and alveolar type II cell proliferation if they were also exposed to silica. However, newborn AT2 cells could not regenerate the alveolar structure and release pro-fibrotic factor IL-33. Moreover, activated TrkB induced the expression of p-AKT, which promotes the expression of epithelial-mesenchymal transcription factor Twist, but no Snail. In vitro assessment confirmed activation of the STAT3-BDNF-TrkB pathway in AT2 cells, exposed to nicotine plus silica. In addition, TrkB inhibitor K252a downregulated p-TrkB and the downstream p-AKT and restricted the epithelial-mesenchymal transition caused by nicotine plus silica. In conclusion, nicotine activates the STAT3-BDNF-TrkB pathway, which promotes epithelial-mesenchymal transition and exacerbates pulmonary fibrosis in mice with combined exposure to silica particles and nicotine.

C-X-C-Chemokine-Receptor-Type-4 Inhibitor AMD3100 Attenuates Pulmonary Inflammation and Fibrosis in Silicotic Mice.

Background: Silicosis is a severe pulmonary disease caused by inhaling dust containing crystalline silica. The progression of silicosis to pulmonary fibrosis is usually unavoidable. Recent studies have revealed positivity for the overexpression of C-X-C chemokine receptor type 4 (CXCR4) in pulmonary fibrosis and shown that the CXCR4 inhibitor AMD3100 attenuated pulmonary fibrosis after bleomycin challenge and paraquat exposure. However, it is unclear whether AMD3100 reduces crystalline silica-induced pulmonary fibrosis. Methods: C57BL/6 male mice were instilled intranasally with a single dose of crystalline silica (12 mg/60 µL) to establish an acute silicosis mouse model. Twelve hours later, the mice were injected intraperitoneally with 5 mg/kg AMD3100 or control solution. Then, the mice were weighed daily and sacrificed on day 7, 14, or 28 to collect lung tissue and peripheral blood. Western blotting was also applied to determine the level of CXCR4, while different histological techniques were used to assess pulmonary inflammation and fibrosis. In addition, the level of B cells in peripheral blood was measured by flow cytometry. Results: CXCR4 and its ligand CXCL12 were upregulated in the lung tissues of crystalline silica-exposed mice. Blocking CXCR4 with AMD3100 suppressed the upregulation of CXCR4/CXCL12, reduced the severity of lung injury, and prevented weight loss. It also inhibited neutrophil infiltration at inflammatory sites and neutrophil extracellular trap formation, as well as reduced B-lymphocyte aggregates in the lung. Additionally, it decreased the recruitment of circulating fibrocytes (CD45+collagen I+CXCR4+) to the lung and the deposition of collagen I and α-smooth muscle actin in lung tissue. AMD3100 also increased the level of B cells in peripheral blood, preventing circulating B cells from migrating to the injured lungs. Conclusion: Blocking CXCR4 with AMD3100 delays pulmonary inflammation and fibrosis in a silicosis mouse model, suggesting the potential of AMD3100 as a drug for treating silicosis.

Vitamin D3 suppresses astrocyte activation and ameliorates coal dust-induced mood disorders in mice.

BACKGROUND: Pneumoconiosis patients exhibit significantly more anxiety and depression than healthy individuals. However, the mechanism of coal dust-induced anxiety and depression remains unclear. METHODS: A pneumoconiosis mouse model with anxiety- and depression-like behaviors were established after 28 days of exposure to coal dust. Vitamin D3 treatment (1200 IU/kg/week) was administered intraperitoneally for 3 months starting from the first coal exposure. Tail suspension test (TST), open field test (OFT), and elevated plus-maze (EPM) test were used to assess anxiety- and depression-like behaviors. Theserum concentration of 25(OH)D3 and fibrillary acid protein (GFAP) expression were determined. In addition, the morphology and distribution of GFAP and neurogenic differentiation factor1 expression (NeuroD1) in different cerebral hippocampus were observed. RESULTS: In coal dust-exposed mice, immobility time decreased in OFT and increased in TST,and the frequency of entering the open arm decreased in the EPM compared with the control mice. Coal dust increased hippocampal GFAP expression and astrocyte activation and reduced neurogenic differentiation factor1 expression (NeuroD1). In addition, Vitamin D3 significantly alleviated anxiety- and depressive-like behaviors in TST and EPM test, decreased GFAP expression level, modified hippocampal astrocyte activation pattern, and advanced brain-derived neurotrophic factor (BDNF) distribution and expression in CA1 and CA3 of the hippocampus. CONCLUSIONS: Taken together, our results suggest that, by inhibiting the over-activation of astrocytes and increasing BDNF and neuron protection, vitamin D treatment ameliorates coal-dust-induced depressive and anxiety-like behavior, which is the first evidence that vitamin D may be a new approach for treating mood disorders caused by particulate matter.

Coal dust exposure triggers heterogeneity of transcriptional profiles in mouse pneumoconiosis and Vitamin D remedies.

BACKGROUND: Coal dust particles (CDP), an inevitable by-product of coal mining for the environment, mainly causes coal workers' pneumoconiosis (CWP). Long-term exposure to coal dust leads to a complex alternation of biological processes during regeneration and repair in the healing lung. However, the cellular and complete molecular changes associated with pulmonary homeostasis caused by respiratory coal dust particles remain unclear. METHODS: This study mainly investigated the pulmonary toxicity of respirable-sized CDP in mice using unbiased single-cell RNA sequencing. CDP (< 5 µm) collected from the coal mine was analyzed by Scanning Electron Microscope (SEM) and Mass Spectrometer. In addition, western blotting, Elisa, QPCR was used to detect gene expression at mRNA or protein levels. Pathological analysis including HE staining, Masson staining, immunohistochemistry, and immunofluorescence staining were performed to characterize the structure and functional alternation in the pneumoconiosis mouse and verify the reliability of single-cell sequencing results. RESULTS: SEM image and Mass Spectrometer analysis showed that coal dust particles generated during coal mine production have been crushed and screened with a diameter of less than 5 µm and contained less than 10% silica. Alveolar structure and pulmonary microenvironment were destroyed, inflammatory and death (apoptosis, autophagy, and necrosis) pathways were activated, leading to pneumoconiosis in post 9 months coal dust stimulation. A distinct abnormally increased alveolar type 2 epithelial cell (AT2) were classified with a highly active state but reduced the antimicrobial-related protein expression of LYZ and Chia1 after CDP exposure. Beclin1, LC3B, LAMP2, TGF-ß, and MLPH were up-regulated induced by CDP, promoting autophagy and pulmonary fibrosis. A new subset of macrophages with M2-type polarization double expressed MLPH + /CD206 + was found in mice having pneumoconiosis but markedly decreased after the Vitamin D treatment. Activated MLPH + /CD206 + M2 macrophages secreted TGF-ß1 and are sensitive to Vitamin D treatment. CONCLUSIONS: This is the first study to reconstruct the pathologic progression and transcriptome pattern of coal pneumoconiosis in mice. Coal dust had obvious toxic effects on lung epithelial cells and macrophages and eventually induced pulmonary fibrosis. CDP-induced M2-type macrophages could be inhibited by VD, which may be related to the alleviation of the pulmonary fibrosis process.

A suitable silicosis mouse model was constructed by repeated inhalation of silica dust via nose.

Silicosis as the serious occupational disease is highly necessary to construct a suitable mouse model for disclosing mechanism of occurrence and development in this disease. Here, the volume-effect relationship and volume-based survival curves in mice who inhaled silica suspension intranasally were analyzed. Notable, the optimal volume 80 µl repeated-inhalation by nose to silica suspension in the inbred mouse C57BL/6 J with the highest susceptibility to silicosis led to a great entrance into the lung and a high survival rate after instillation. After repeated-exposure to 20 mg/mL, 80 µl silica for 16 days and then fed without silica exposure until 31 days, weight of mice showed a trend of first decrease and then recover. Moreover, the degree of pulmonary inflammation and fibrosis in mice were analyzed by pathological and immunohistochemistry staining. Transforming growth factor-beta (TGF-ß), smooth muscle alpha-actin (α-SMA) and collagen type-I (collagen I, Col-I) were significantly increased in the silica-exposed mouse lung at post-exposure day 16 compared with the controls. Sirius red stain and Micro-CT analysis showed that lung fibrosis formed at post-exposure day 31. This study highlights the critical importance of perfusion volume and repeated nasal drops in inducing inflammatory response and pulmonary fibrosis in silicosis.

Maternal Nicotine Exposure Alters Hippocampal Microglia Polarization and Promotes Anti-inflammatory Signaling in Juvenile Offspring in Mice.

Accumulating evidence reveal that maternal smoking or perinatal nicotine replacement therapy impairs hippocampal neurogenesis, neural development, and cognitive behaviors in the offspring. Microglia is a source of non-neural regulation of neuronal development and postnatal neurogenesis. In this study, we explored the impact of nicotine on the microglia during the development of hippocampus. Developmental nicotine exposure in a mouse model was conducted by supplementing nicotine in the drinking water to mother mice during gestation and lactation period. We found that juvenile offspring with maternal nicotine exposure presented physical and neurobehavioral development delay and an increase in anxiety-like behavior in the open field test on postnatal day (PND) 20. To further detect possible developmental neurotoxic effects of nicotine in offspring and underlying mechanism, whole genome microarray analysis of the expression profile of the hippocampus was performed on postnatal day 20. Significant alterations in the expression of genes related to inflammatory, neurotransmitter, and synapsis were observed in the hippocampus after maternal nicotine exposure, as compared to the vehicle control. Concurrently, an increase in microglial markers and the presence of M2 polarity state in the hippocampus of the nicotine offspring were observed by histological analysis and confocal z-stacking scanning. The M2 microglial polarization state was further confirmed with in vitro primary microglia culture by cytokine array, and double-positive expression of BDNF/Iba1 in microglia by immunohistochemical staining in the juvenile offspring hippocampus was visualized. We also found that nicotine offspring showed an increase of neurite length in the molecular layer and CA1 by Tuj1 staining, as well as an increase in the expression of synapse associated protein, PSD95, but the expression of NeuroD1 in CA1 and CA3 reduced. In summary, maternal nicotine exposure dysregulates immune-related genes expression by skewing the polarity of M2 microglia in the hippocampus, which may cause abnormal cognitive and behavioral performance in the offspring.

Blockade of 5-Hydroxytryptamine 2A Receptor Attenuates Precipitation of Naloxone-Induced Withdrawal Symptoms in Opioid-Exposed Mice.

Heroin dependency has become a global problem and has caused significant clinical and socioeconomic burdens along with devastating medical consequences. Chronic drug exposure alters the expression and functional activity of 5-hydroxytryptamine (serotonin) 2A receptors (5-HT2ARs) in the brain. Furthermore, pharmacological blockade of 5-HT2ARs reduces cue-induced cocaine craving behaviors. In this study, we explored the influence of 5-HT2ARs on heroin-withdrawal behaviors in mice. Black C57BL/6J mice were given gradually increasing (10-50 mg/kg over 4.5 days) doses of heroin to induce heroin dependency, after which naloxone was given to precipitate withdrawal symptoms. MDL100907, a selective and potent 5-HT2AR antagonist, attenuated naloxone-precipitated withdrawal symptoms in these mice. In addition, 5-HT2AR protein levels increased significantly in the medial prefrontal cortex (mPFC), while phosphorylation of extracellular signal-regulated kinase (p-ERK) decreased in the mPFC after heroin exposure. In conclusion, these results suggest that 5-HT2ARs might be involved in the development of opioid dependency and that pharmacological blocking of 5-HT2ARs might be a new therapeutic strategy for heroin dependency.