Recent evidence has shown that C-X-C chemokine receptor type 4 (CXCR4) plays a crucial role in acute lung injury (ALI). Macrophages are key factors in the pathogenesis of ALI. The aim of this study was to investigate the role of CXCR4 in macrophages after lipopolysaccharide (LPS) stimulation and confirm that CXCR4 knockdown can inhibit inflammatory cytokines by suppressing mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) signaling pathway activation. In this study, we found that CXCR4 expression in lung tissue of ALI was significantly increased using immunofluorescence. We also found that the expression of CXCR4 in macrophages sorted from bronchoalveolar lavage fluid (BALF) of ALI was obviously upregulated through RT-qPCR. After CXCR4 knockdown using siRNA, we found that the expression of interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) was obviously down regulated in macrophages. Additionally, the phosphorylation of p38, Erk, and p65 was significantly decreased after CXCR4 knockdown through western blotting. Taken together, the present study suggests that CXCR4 knockdown may inhibit inflammatory cytokine expression in macrophages by suppressing MAPK and NF-κB signaling pathway activation. Therefore, CXCR4 knockdown may have potential clinical value in treating ALI.

Aim of the Study: Matrix metalloproteinases (MMPs) play a critical role in chronic obstructive pulmonary disease (COPD). This study investigated the role of mitogen-activated protein kinases (MAPKs) in MMP-9 secretion of BEAS-2B cells, a human bronchial epithelial cell line and U937 cells, a human myeloid leukaemia cell line, which could differentiate into macrophage, after LPS stimulation, and some details of involved signaling. Materials and Methods: MTT assay was used to measure cell viability. U937 cells were incubated for 48h with 100ng/ml PMA, and had a resting period of 24h with culture medium without PMA for differentiation of U937 cells into macrophages. For the experiments, U937 cells or BEAS-2B cells were pretreated with several inhibitors and then stimulated by LPS. Western blotting, quantitative real-time PCR, enzyme-linked immunosorbent assay (ELISA) and DNA binding activity assay were used for measuring the protein expression, RNA expression, cytokine production and DNA binding activity, respectively. Results: We found LPS induced MMP-9 expression and secretion were completely blocked by stress-activated protein kinase/jun kinase (SAPK/JNK) and extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitors but not by p38 inhibitor. LPS-induced transactivation of AP-1 was also inhibited by JNK inhibitor SP600125 and ERK1/2 inhibitor PD98059. Conclusions: The present study suggests that in BEAS-2B cells and U937 cells, LPS probably activates ERK1/2 pathway and JNK pathway, which in turn initiate AP-1 activity, and leading to MMP-9 expression. Thus the ERK1/2 inhibitor and JNK inhibitor may have potential clinical value in treating COPD.

To explore the differentiation of airway inflammation, bronchial hyperresponsiveness and anti-asthma therapy responses between the cough variant asthma (CVA) and classic asthma (CA) patients with FEV1% ≥80% predicted. In the first monocentre retrospective cross-sectional study, 402 patients with suspicion of CA and 544 patients with chronic cough were enrolled. Further prospective monocentre study was conducted and 66 patients of suspected asthma with negative bronchial dilation test (BDT) but positive bronchial challenge (BCT) test were enrolled and followed up for 4 weeks. CA patients had higher fractional exhaled nitric oxide (FENO) values than CVA patients (36.0 ppb vs. 24.0 ppb, p < 0.0001). The predictive value of FENO for positive BCT was significantly lower in chronic cough patients compared to those with suspicion of CA (AUC = 0.603 vs. 0.728). Following four weeks anti-asthma therapy, both the CVA and CA groups showed significant improvement in both the large and small airway function and symptom relief. There was no significant difference between the respective groups. The two most valuable spirometric variables for predicting a positive response to anti-asthma treatment were the improvements of FEV1 (ΔFEV1, cut-off values = 90 ml for CA and 110 ml for CVA) and FEV1% (ΔFEV1%, cut-off values = 3.49% for CA and 2.59% for CVA) after BDT in baseline of CA and CVA patients, respectively. Patients with CVA exhibited lower levels of airway eosinophilic inflammation compared to those with mild CA. Most patients with mild CA and CVA could benefit promptly from anti-asthma treatment. Additionally, an improvement in FEV1 and FEV1% during BDT can potentially predict positive responses to anti-asthma therapy in both groups.

Background: Ozone exposure is a major risk factor for chronic obstructive pulmonary disease (COPD). In this study, we investigated the potential role of targeting mTOR signaling in the treatment of COPD induced by ozone exposure. Methods: The public database was chosen to explore the expression of mTOR mRNA, S6K1 mRNA, and LC3B mRNA in COPD patients, and potential correlations with FEV 1 (%pred). In an ozone-exposed mouse model, large airway and small airway function were evaluated by spirometry. After intraperitoneal injection of a mTOR inhibitor known as rapamycin, the emphysema index, and inflammation scores in lung tissue were measured. Inflammatory cell infiltration in bronchoalveolar lavage fluid (BALF) and levels of cytokines in the lung tissue were also observed. Airway remodeling in the lung tissue was detected using Masson's trichrome stains and immunohistochemical staining. Mucus hypersecretion was evaluated by PAS staining. The protein expression of the mTOR pathway and autophagy marker LC3B in the lung tissue was determined through Western blot. Results: mTOR mRNA and S6K1 mRNA were upregulated in patients with COPD compared to the control subjects, whereas LC3B mRNA showed a downward shift in patients with COPD. Mice that received mTOR inhibitor treatment displayed higher FEV 50 /FVC, FEF 25 , FEF 50 , FEF 75 , and MMEF. The mTOR inhibitor rapamycin improved the emphysema index and inflammation scores in mice lung tissue. Moreover, it significantly inhibited inflammatory cell infiltration in BALF, IL-1β, TNF-α, and NF-κb in the lung tissue of ozone-exposed mice. The mTOR inhibitor significantly suppressed mucus hypersecretion in large and small airways and decreased the protein expression of collagen I and α-SMA in the lung tissue of ozone-exposed mice. Notably, mTOR repression also decreased the protein expression of S6K1 and increased LC3B expression in the lung tissue. Conclusion: The mTOR inhibitor rapamycin ameliorates ozone-induced airway inflammation and emphysema in a LC3B-dependent manner. mTOR inhibition may offer a promising therapeutic approach for preventing ozone-induced COPD by mitigating airway inflammation, reducing airway remodeling, and alleviating mucus hypersecretion.

Background: Small open reading frames (smORFs) in long noncoding RNA (lncRNA) have been demonstrated to encode small peptides. However, it has yet to be investigated whether the engendering peptides correlate directly with inflammatory-fibrosis in COPD. Methods: A combined CS exposure and LPS intratracheal instillation mice model was established and typeⅡ alveolar epithelial (ATⅡ) cells was isolated. Gene expression was analyzed via transcriptome sequencing, and the smORF embedded the lncRNA was identified through preliminary bioinformatics analysis. We named the smORF as peptide MSTRG.1695.1-aa. The morphologic changes, fibrosis injury, cytokines, lung function, EMT proteins in COPD mice and peptide MSTRG.1695.1-treated mice were measured. MSTRG.1695.1-aa in the serum of COPD patients were detected. Results: We identify a key lncRNA-MSTRG.1695.1, with a reduction in ATⅡ cells, and discover a negative correlation between the p38-MAPK pathway and MSTRG.1695.1 in ATII cells. We report that the smORF embedded in MSTRG.1695.1 encodes a micropeptide MSTRG.1695.1-aa. MSTRG.1695.1-aa suppresses the progression of EMT via the p38-MAPK pathway in ATⅡ cells. Exogenous MSTRG.1695.1-aa dramatically mitigates inflammatory cytokine production, fibrosis injury, and EMT proteins expression on the lung tissue in COPD mice. High homologously peptides of MSTRG.1695.1-aa in the serum of COPD patients are detected, which are positive correlated with lung function. Conclusions: MSTRG.1695.1-aa represents a compelling therapeutic target for lung injury in COPD and a putative serum marker for evaluation of early pulmonary fibrosis in COPD patients.