Shegan-Mahuang Decoction ameliorates cold-induced asthma via regulating the proliferation and apoptosis of airway smooth muscle cells through TAS2R10: An in vivo and in vitro study.

ETHNOPHARMACOLOGICAL RELEVANCE: Shegan-Mahuang Decoction (SMD) is a classical formula that has been used to effectively treat cold-induced asthma (CA) for 1800 years. Airway smooth muscle cells (ASMCs) play a crucial role in airway remodeling of CA and can be modulated through bitter taste-sensing type 2 receptors (TAS2Rs). Given that SMD contains numerous bitter herbs and TAS2R10 expression in ASMCs remains consistently high, it is pertinent to explore whether SMD regulates ASMCs via TAS2R10 to exert its CA mechanism. AIM OF THE STUDY: This study investigated the efficacy as well as the potential mechanism of SMD in CA. MATERIALS AND METHODS: In this study, experiments in vivo were conducted using the CA rat model induced by ovalbumin (OVA) along with cold stimulation. The effects of SMD and TAS2R10 expression in CA rats were evaluated using the following methods: clinical symptoms, weights, pathological staining, immunofluorescence staining (IF), enzyme-linked immunosorbent assay (ELISA), real-time quantitative polymerase chain reaction (RT-qPCR) and western blot (WB). Assays in vitro including cell counting Kit-8 (CCK-8), ELISA, flow cytometry, TUNEL staining, RT-qPCR and WB were performed to investigate potential mechanism of SMD on the proliferation and apoptosis of ASMCs through upregulation of TAS2R10. RESULTS: The administration of SMD resulted in a notable improvement in the symptoms, trends in weight, airway inflammation and airway remodeling observed in CA rats with upregulated TAS2R10. Mechanistically, we furtherly confirmed that SMD inhibits p70S6K/CyclinD1 pathway by upregulating TAS2R10. SMD furthermore blocked the G0/G1 phase, suppressed the proliferation and inducted apoptosis in ASMCs induced by platelet-derived growth factor-BB (PDGF-BB). Erythromycin (EM), a TAS2R10 agonist, can intensify these effects. CONCLUSIONS: SMD significantly ameliorates CA by upregulating TAS2R10 and inhibiting the p70S6K/CyclinD1 pathway, thereby modulating ASMCs' proliferation and apoptosis. Inspired by the Five Flavors Theory of Traditional Chinese Medicine, this study provides an updated treatment perspective for treating CA.

Long non-coding RNA MEG3 knockdown represses airway smooth muscle cells proliferation and migration via sponging miR-143-3p/FGF9 in asthma.

BACKGROUND: Asthma is a respiratory disease characterized by airway remodeling. We aimed to find out the role and mechanism of lncRNA MEG3 in asthma. METHODS: We established a cellular model of asthma by inducing human airway smooth muscle cells (HASMCs) with PDGF-BB, and detected levels of lncRNA MEG3, miR-143-3p and FGF9 in HASMCs through qRT-PCR. The functions of lncRNA MEG3 or miR-143-3p on HASMCs were explored by cell transfection. The binding sites of miR-143-3p and FGF9 were subsequently analyzed with bioinformatics software, and validated with dual-luciferase reporter assay. MTT, 5-Ethynyl-2'-deoxyuridine (EdU) assay, and Transwell were used to detect the effects of lncRNA MEG3 or miR-143-3p on proliferation and migration of HASMCs. QRT-PCR and western blot assay were used to evaluate the level of proliferation-related marker PCNA in HASMCs. RESULTS: The study found that lncRNA MEG3 negatively correlated with miR-143-3p, and miR-143-3p could directly target with FGF9. Silence of lncRNA MEG3 can suppress migration and proliferation of PDGF-BB-induced HASMCs via increasing miR-143-3p. Further mechanistic studies revealed that miR-143-3p negatively regulated FGF9 expression in HASMCs. MiR-143-3p could inhibit PDGF-BB-induced HASMCs migration and proliferation through downregulating FGF9. CONCLUSION: LncRNA MEG3 silencing could inhibit the migration and proliferation of HASMCs through regulating miR-143-3p/FGF9 signaling axis. These results imply that lncRNA MEG3 plays a protective role against asthma.

Defective mesenchymal Bmpr1a-mediated BMP signaling causes congenital pulmonary cysts.

Abnormal lung development can cause congenital pulmonary cysts, the mechanisms of which remain largely unknown. Although the cystic lesions are believed to result directly from disrupted airway epithelial cell growth, the extent to which developmental defects in lung mesenchymal cells contribute to abnormal airway epithelial cell growth and subsequent cystic lesions has not been thoroughly examined. In the present study using genetic mouse models, we dissected the roles of bone morphogenetic protein (BMP) receptor 1a (Bmpr1a)-mediated BMP signaling in lung mesenchyme during prenatal lung development and discovered that abrogation of mesenchymal Bmpr1a disrupted normal lung branching morphogenesis, leading to the formation of prenatal pulmonary cystic lesions. Severe deficiency of airway smooth muscle cells and subepithelial elastin fibers were found in the cystic airways of the mesenchymal Bmpr1a knockout lungs. In addition, ectopic mesenchymal expression of BMP ligands and airway epithelial perturbation of the Sox2-Sox9 proximal-distal axis were detected in the mesenchymal Bmpr1a knockout lungs. However, deletion of Smad1/5, two major BMP signaling downstream effectors, from the lung mesenchyme did not phenocopy the cystic abnormalities observed in the mesenchymal Bmpr1a knockout lungs, suggesting that a Smad-independent mechanism contributes to prenatal pulmonary cystic lesions. These findings reveal for the first time the role of mesenchymal BMP signaling in lung development and a potential pathogenic mechanism underlying congenital pulmonary cysts.

Congenital disorders are medical conditions that are present from birth. Although many congenital disorders are rare, they can have a severe impact on the quality of life of those affected. For example, congenital pulmonary airway malformation (CPAM) is a rare congenital disorder that occurs in around 1 out of every 25,000 pregnancies. In CPAM, abnormal, fluid-filled sac-like pockets of tissue, known as cysts, form within the lungs of unborn babies. After birth, these cysts become air-filled and do not behave like normal lung tissue and stop a baby's lungs from working properly. In severe cases, babies with CPAM need surgery immediately after birth. We still do not understand exactly what the underlying causes of CPAM might be. CPAM is not considered to be hereditary ­ that is, it does not appear to be passed down in families ­ nor is it obviously linked to any environmental factors. CPAM is also very difficult to study, because researchers cannot access tissue samples during the critical early stages of the disease. To overcome these difficulties, Luo et al. wanted to find a way to study CPAM in the laboratory. First, they developed a non-human animal 'model' that naturally forms CPAM-like lung cysts, using genetically modified mice where the gene for the signaling molecule Bmpr1a had been deleted in lung cells. Normally, Bmpr1a is part of a set of the molecular instructions, collectively termed BMP signaling, which guide healthy lung development early in life. However, mouse embryos lacking Bmpr1a developed abnormal lung cysts that were similar to those found in CPAM patients, suggesting that problems with BMP signalling might also trigger CPAM in humans. Luo et al. also identified several other genes in the Bmpr1a-deficient mouse lungs that had abnormal patterns of activity. All these genes were known to be controlled by BMP signaling, and to play a role in the development and organisation of lung tissue. This suggests that when these genes are not controlled properly, they could drive formation of CPAM cysts when BMP signaling is compromised. This work is a significant advance in the tools available to study CPAM. Luo et al.'s results also shed new light on the molecular mechanisms underpinning this rare disorder. In the future, Luo et al. hope this knowledge will help us develop better treatments for CPAM, or even help to prevent it altogether.

Effect of curcumin nanoparticles on proliferation and migration of mouse airway smooth muscle cells and airway inflammatory infiltration.

Curcumin (CUR) possesses the capability to inhibit various inflammatory factors, exert anti-inflammatory effects, and alleviate asthma attacks; however, its hydrophobicity and instability significantly impede its clinical application. In this study, we synthesized CUR-loaded nanoparticles (CUR-NPs) and evaluated their impact on the proliferation, migration, and inflammatory infiltration of mouse airway smooth muscle cells (ASMCs), while investigating their underlying mechanisms. To achieve this objective, ASMCs were isolated from BALB/c mice and subjected to TGF-ß1-induced cell proliferation and migration. Our findings demonstrate that CUR-NPs effectively regulate the release of CUR within cells with superior intracellular uptake compared to free CUR. The CCK-8 assay results indicate that the blank carrier does not exhibit any cytotoxic effects on cells, thus rendering the impact of the carrier itself negligible. The TGF-ß1 group exhibited a significant increase in cell proliferation, whereas treatment with CUR-NPs significantly suppressed TGF-ß1-induced cell proliferation. The findings from both the cell scratch assay and transwell assay demonstrated that TGF-ß1 substantially enhanced cell migration, while CUR-NPs treatment effectively attenuated TGF-ß1-induced cell migration. The Western blot analysis demonstrated a substantial increase in the expression levels of TGF-ß1, p-STAT3, and CTGF in ASMCs following treatment with TGF-ß1 when compared to the control group. Nevertheless, this effect was effectively counteracted upon administration of CUR-NPs. Furthermore, an asthma mouse model was successfully established and CUR-NPs were administered through tail vein injection. The serum levels of TGF-ß1 and the expression levels of TGF-ß1, p-STAT3, and CTGF proteins in the lung tissue of mice in the model group exhibited significant increases compared to those in the control group. However, CUR-NPs treatment effectively attenuated this change. Our research findings suggest that CUR-NPs possess inhibitory effects on ASMC proliferation, migration, and inflammatory infiltration by suppressing activation of the TGF-ß1/p-STAT3/CTGF signaling pathway, thereby facilitating inhibition of airway remodeling.

Expression of glucocorticoid receptor and HDACs in airway smooth muscle cells is associated with response to steroids in COPD.

BACKGROUND: Steroid insensitivity in Chronic Obstructive Pulmonary Disease (COPD) presents a problem for controlling the chronic inflammation of the airways. The glucocorticoid receptor (GR) mediates the intracellular signaling of inhaled corticosteroids (ICS) by interacting with transcription factors and histone deacetylases (HDACs). The aim of this study was to assess if COPD patients' response to ICS in vivo, may be associated with the expression of GR, the complex of GR with transcription factors, and the expression of various HDACs in vitro. METHODS: Primary airway smooth muscle cells (ASMC) were established from endobronchial biopsies obtained from patients with asthma (n = 10), patients with COPD (n = 10) and subjects that underwent diagnostic bronchoscopy without pathological findings and served as controls (n = 6). ASMC were also established from 18 COPD patients, 10 responders and 8 non-responders to ICS, who participated in the HISTORIC study, an investigator-initiated and driven clinical trial that proved the hypothesis that COPD patients with high ASMC in their endobronchial biopsies respond better to ICS than patients with low ASMC. Expression of GR and its isoforms GRα and GRß and HDACs was investigated in primary ASMC in the absence or in the presence of dexamethasone (10- 8M) by western blotting. The complex formation of GR with transcription factors was assessed by co-immunoprecipitation. RESULTS: Expression of GR and its isoform GRα but not GRß was significantly reduced in ASMC from COPD patients as compared to controls. There were no significant differences in the expression of GR, GRα and GRß between responders and non-responders to ICS. However, treatment with dexamethasone upregulated the expression of total GR (p = 0.004) and GRα (p = 0.005) after 30 min in responders but not in non-responders. Τhe formation of the complex GR-c-Jun was increased 60 min after treatment with dexamethasone only in responders who exhibited significantly lower expression of HDAC3 (p = 0.005) and HDAC5 (p < 0.0001) as compared to non-responders. CONCLUSIONS: These data suggest that ASMC from COPD patients who do not respond to treatment with ICS, are characterized by reduced GR-c-Jun complex formation and increased expression of HDAC3 and HDAC5. TRIAL REGISTRATION: ISRCTN11017699 (Registration date: 15/11/2016).

Heat-Induced Secretion of Heat Shock Proteins 70 and 90 Does not Affect the Expression of the Glucocorticoid Receptor in Primary Airway Cells in COPD.

PURPOSE: The response to glucocorticoids is hampered in many COPD patients by a yet unknown mechanism. Earlier we reported that short-term heat exposure of primary human bronchial epithelial cells (BEC) and airway smooth muscle cells (ASMC) of asthma patients increased the expression and secretion of extracellular heat shock proteins (eHSPs) resulting in increased expression of glucocorticoid receptor (GR) in BEC and inhibition of ASMC remodeling. The aim of the present study was to assess if the same mechanism is also present in primary airway wall cells of COPD patients. METHODS: Primary BEC and ASMC were established from endobronchial biopsies obtained from COPD patients (n = 73), who participated in the HISTORIC study, an investigator-initiated and driven clinical trial. Secretion and protein expression of HSPs was assessed by ELISA and Western blotting. Expression of total GR, its isoforms GRα and GRß and toll-like receptor 4 (TLR4) was determined by Western-blotting. RESULTS: Short heat exposure (65 °C, 10 s) of BEC resulted in a significant increase of the secretion of eHSP70 and eHSP90, while the intracellular protein was not altered. Heat treatment or exposure to eHSP70 or eHSP90 had no effect on the expression of GR and GR-isoforms. However, eHSP70 and eHSP90 significantly reduced the expression of TLR4. CONCLUSIONS: The results of this study indicate that primary airway cells from COPD patients respond differently to heat exposure and extracellular HSP70 or HSP90 than cells from asthma patients regarding the expression of GR and this may explain the reduced response to glucocorticoids in patients with COPD. TRIAL REGISTRATION: ISRCTN11017699.

Characterization of PDGF-Induced Subcellular Calcium Regulation through Calcium Channels in Airway Smooth Muscle Cells by FRET Biosensors.

The homeostasis of cellular calcium is fundamental for many physiological processes, while the calcium levels remain inhomogeneous within cells. During the onset of asthma, epithelial and inflammatory cells secrete platelet-derived growth factor (PDGF), inducing the proliferation and migration of airway smooth muscle (ASM) to the epidermal layer, narrowing the airway. The regulation of ASM cells by PDGF is closely related to the conduction of calcium signals. In this work, we generated subcellular-targeted FRET biosensors to investigate calcium regulation in the different compartments of ASM cells. A PDGF-induced cytoplasmic calcium [Ca2+]C increase was attributed to both extracellular calcium influx and endoplasmic reticulum (ER) calcium [Ca2+]ER release, which was partially regulated by the PLC-IP3R pathway. Interestingly, the removal of the extracellular calcium influx led to inhibited ER calcium release, likely through inhibitory effects on the calcium-dependent activation of the ER ryanodine receptor. The inhibition of the L-type calcium channel on the plasma membrane or the SERCA pump on the ER resulted in both reduced [Ca2+]C and [Ca2+]ER from PDGF stimulation, while IP3R channel inhibition led to reduced [Ca2+]C only. The inhibited SERCA pump caused an immediate [Ca2+]C increase and [Ca2+]ER decrease, indicating active calcium exchange between the cytosol and ER storage in resting cells. PDGF-induced calcium at the outer mitochondrial membrane sub-region showed a similar regulatory response to cytosolic calcium, not influenced by the inhibition of the mitochondrial calcium uniporter channel. Therefore, our work identifies calcium flow pathways among the extracellular medium, cell cytosol, and ER via regulatory calcium channels. Specifically, extracellular calcium flow has an essential function in fully activating ER calcium release.

High Stretch Associated with Mechanical Ventilation Promotes Piezo1-Mediated Migration of Airway Smooth Muscle Cells.

Ventilator-induced lung injury (VILI) during mechanical ventilation (MV) has been attributed to airway remodeling involving increased airway smooth muscle cells (ASMCs), but the underlying mechanism is not fully understood. Thus, we aimed to investigate whether MV-associated high stretch (>10% strain) could modulate mechanosensitive Piezo1 expression and thereby alter cell migration of ASMCs as a potential pathway to increased ASMCs in VILI. C57BL/6 mice and ASMCs were subjected to MV at high tidal volume (VT, 18 mL/kg, 3 h) and high stretch (13% strain, 0.5 Hz, 72 h), respectively. Subsequently, the mice or cells were evaluated for Piezo1 and integrin mRNA expression by immunohistochemical staining and quantitative PCR (qPCR), and cell migration and adhesion by transwell and cell adhesion assays. Cells were either treated or not with Piezo1 siRNA, Piezo1-eGFP, Piezo1 knockin, Y27632, or blebbistatin to regulate Piezo1 mRNA expression or inhibit Rho-associated kinase (ROCK) signaling prior to migration or adhesion assessment. We found that expression of Piezo1 in in situ lung tissue, mRNA expression of Piezo1 and integrin αVß1 and cell adhesion of ASMCs isolated from mice with MV were all reduced but the cell migration of primary ASMCs (pASMCs) isolated from mice with MV was greatly enhanced. Similarly, cell line mouse ASMCs (mASMCs) cultured in vitro with high stretch showed that mRNA expression of Piezo1 and integrin αVß1 and cell adhesion were all reduced but cell migration was greatly enhanced. Interestingly, such effects of MV or high stretch on ASMCs could be either induced or abolished/reversed by down/up-regulation of Piezo1 mRNA expression and inhibition of ROCK signaling. High stretch associated with MV appears to be a mechanical modulator of Piezo1 mRNA expression and can, thus, promote cell migration of ASMCs during therapeutic MV. This may be a novel mechanism of detrimental airway remodeling associated with MV, and, therefore, a potential intervention target to treat VILI.

Notoginsenoside R1 restrains the proliferation and migration of airway smooth muscle cells isolated from rats with chronic obstructive pulmonary disease.

OBJECTIVE: Chronic obstructive pulmonary disease (COPD) is a common disorder that is characterized by systemic and lung inflammation. Notoginsenoside R1 (NGR1) displays anti-inflammatory properties in numerous diseases. We aimed to explore the function and mechanism of NGR1 in COPD. MATERIALS AND METHODS: COPD rats were established through cigarette smoke exposure, lipopolysaccharide injection, and cold stimulation. Rat airway smooth muscle cells (ASMCs) were separated and identified. Then, ASMCs were treated with NGR1 (25 or 50 µM) and cigarette smoke extract (CSE). Thereafter, the vitality, proliferation, and migration of ASMCs were measured. Additionally, cell cycle, inflammation-related factors, α-SMA, and PI3K/AKT pathway-related marker expressions of the ASMCs were also detected. Molecular docking experiments were conducted to explore the interaction of NGR1 to PI3K, TGF-ß, p65, and AKT. Moreover, 740 Y-P (a PI3K/Akt pathway agonist) were used to validate the mechanism of NGR1 on COPD. RESULTS: NGR1 inhibited the proliferation and migration, but caused cell cycle arrest for CSE-triggered ASMCs. Furthermore, NGR1 not only decreased IL-1ß, IL-6, IL-8, and TNF-α contents, but also reduced α-SMA expression in CSE-stimulated ASMCs. Moreover, NGR1restrainedTGF-ß1 expression, PI3K, p65, and AKT phosphorylation in CSE-stimulated ASMCs. Molecular docking experiments showed NGR1 exhibited a strong binding ability to PI3K, TGF-ß1, p65, and AKT. Notably, the effects of NGR1 on the proliferation and migration of CSE-induced ASMCs were reversed by 740 Y-P. CONCLUSIONS: NGR1 can restrain the proliferation and migration of CSE-induced ASMCs, indicating that NGR1 may be a therapeutic candidate for treating COPD.

High Stretch Modulates cAMP/ATP Level in Association with Purine Metabolism via miRNA-mRNA Interactions in Cultured Human Airway Smooth Muscle Cells.

High stretch (>10% strain) of airway smooth muscle cells (ASMCs) due to mechanical ventilation (MV) is postulated to contribute to ventilator-induced lung injury (VILI), but the underlying mechanisms remain largely unknown. We hypothesized that ASMCs may respond to high stretch via regulatory miRNA-mRNA interactions, and thus we aimed to identify high stretch-responsive cellular events and related regulating miRNA-mRNA interactions in cultured human ASMCs with/without high stretch. RNA-Seq analysis of whole genome-wide miRNAs revealed 12 miRNAs differentially expressed (DE) in response to high stretch (7 up and 5 down, fold change >2), which target 283 DE-mRNAs as identified by a parallel mRNA sequencing and bioinformatics analysis. The KEGG and GO analysis further indicated that purine metabolism was the first enriched event in the cells during high stretch, which was linked to miR-370-5p-PDE4D/AK7. Since PDE4D/AK7 have been previously linked to cAMP/ATP metabolism in lung diseases and now to miR-370-5p in ASMCs, we thus evaluated the effect of high stretch on the cAMP/ATP level inside ASMCs. The results demonstrated that high stretch modulated the cAMP/ATP levels inside ASMCs, which could be largely abolished by miR-370-5p mimics. Together, these findings indicate that miR-370-5p-PDE4D/AK7 mediated high stretch-induced modulation of cAMP and ATP synthesis inside ASMCs. Furthermore, such interactive miRNA-mRNA pairs may provide new insights for the discovery of effective biomarkers/therapeutic targets for the diagnosis and treatment of VILI and other MV-associated respiratory diseases.

Cysteine and glycine-rich protein 2 retards platelet-derived growth factor-BB-evoked phenotypic transition of airway smooth muscle cells by decreasing YAP/TAZ activity.

Cysteine and glycine-rich protein 2 (Csrp2) has emerged as a key factor in controlling the phenotypic modulation of smooth muscle cells. The phenotypic transition of airway smooth muscle cells (ASMCs) is a pivotal step in developing airway remodeling during the onset of asthma. However, whether Csrp2 mediates the phenotypic transition of ASMCs in airway remodeling during asthma onset is undetermined. This work aimed to address the link between Csrp2 and the phenotypic transition of ASMCs evoked by platelet-derived growth factor (PDGF)-BB in vitro. The overexpression or silencing of Csrp2 in ASMCs was achieved through adenovirus-mediated gene transfer. The expression of mRNA was measured by quantitative real-time-PCR. Protein levels were determined through Western blot analysis. Cell proliferation was detected by EdU assay and Calcein AM assays. Cell cycle distribution was assessed via fluorescence-activated cell sorting assay. Cell migration was evaluated using the scratch-wound assay. The transcriptional activity of Yes-associated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ) was measured using the luciferase reporter assay. A decline in Csrp2 level occurred in PDGF-BB-stimulated ASMCs. Increasing Csrp2 expression repressed the PDGF-BB-evoked proliferation and migration of ASMCs. Moreover, increasing Csrp2 expression impeded the phenotypic change of PDGF-BB-stimulated ASMCs from a contractile phenotype into a synthetic/proliferative phenotype. On the contrary, the opposite effects were observed in Csrp2-silenced ASMCs. The activity of YAP/TAZ was elevated in PDGF-BB-stimulated ASMCs, which was weakened by Csrp2 overexpression or enhanced by Csrp2 silencing. The YAP/TAZ activator could reverse Csrp2-overexpression-mediated suppression of the PDGF-BB-evoked phenotypic switching of ASMCs, while the YAP/TAZ suppressor could dimmish Csrp2-silencing-mediated enhancement on PDGF-BB-evoked phenotypic switching of ASMCs. In summary, Csrp2 serves as a determinant for the phenotypic switching of ASMCs. Increasing Csrp2 is able to impede PDGF-BB-evoked phenotypic change of ASMCs from a synthetic phenotype into a synthetic/proliferative phenotype through the effects on YAP/TAZ. This work implies that Csrp2 may be a key player in airway remodeling during the onset of asthma.

Defective mesenchymal Bmpr1a-mediated BMP signaling causes congenital pulmonary cysts.

Abnormal lung development can cause congenital pulmonary cysts, the mechanisms of which remain largely unknown. Although the cystic lesions are believed to result directly from disrupted airway epithelial cell growth, the extent to which developmental defects in lung mesenchymal cells contribute to abnormal airway epithelial cell growth and subsequent cystic lesions has not been thoroughly examined. In the present study, we dissected the roles of BMP receptor 1a (Bmpr1a)-mediated BMP signaling in lung mesenchyme during prenatal lung development and discovered that abrogation of mesenchymal Bmpr1a disrupted normal lung branching morphogenesis, leading to the formation of prenatal pulmonary cystic lesions. Severe deficiency of airway smooth muscle cells and subepithelial elastin fibers were found in the cystic airways of the mesenchymal Bmpr1a knockout lungs. In addition, ectopic mesenchymal expression of BMP ligands and airway epithelial perturbation of the Sox2-Sox9 proximal-distal axis were detected in the mesenchymal Bmpr1a knockout lungs. However, deletion of Smad1/5, two major BMP signaling downstream effectors, from the lung mesenchyme did not phenocopy the cystic abnormalities observed in the mesenchymal Bmpr1a knockout lungs, suggesting that a Smad-independent mechanism contributes to prenatal pulmonary cystic lesions. These findings reveal for the first time the role of mesenchymal BMP signaling in lung development and a potential pathogenic mechanism underlying congenital pulmonary cysts.

PM2.5 induces lung inflammation and fibrosis via airway smooth muscle cell expression of the Wnt5a/JNK pathway.

Background: In recent years, particulate matter 2.5 (PM2.5) exposure has been considered a key dangerous factor in chronic obstructive pulmonary disease (COPD). The dysfunction of airway smooth muscle cells (ASMCs) facilitates lung inflammation and fibrosis in COPD. Therefore, we explored whether PM2.5 could promote the inflammatory response and fibrosis in ASMCs in vivo and in vitro via the wingless-related integration site 5a (Wnt5a)/c-Jun N-terminal kinase (JNK)/nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway. Methods: Wnt5a expression in the bronchoalveolar lavage fluid (BALF) of COPD patients exposed to PM2.5 was measured by enzyme-linked immunosorbent assay (ELISA). Mice were intratracheally injected with PM2.5 and a Wnt5a antagonist (BOX5). ASMCs were transfected with Wnt5a small interfering RNA (siRNA), BOX5 and the JNK inhibitor SP600125 before PM2.5 stimulation. Hematoxylin and eosin (H&E) staining was performed to measure the inflammatory response and airway fibrosis. The production of Wnt5a/JNK/NF-KB pathway factors was analyzed by Western blotting. The secretion of interleukin-6 (IL-6), IL-8 and tumor necrosis factor-α (TNF-α) was measured by ELISA. The expression levels of alpha smooth muscle actin (α-SMA), collagen I and collagen III were assessed by quantitative real time polymerase chain reaction (qRT-PCR) and Western blotting. Results: We found that the increase in Wnt5a expression in the BALF of COPD patients was positively correlated with the levels of PM2.5 exposure. The Wnt5a/JNK/NF-κB pathway was activated in the lung samples of PM2.5-induced model mice and PM2.5-exposed ASMCs, which promoted the production of α-SMA, collagen I and collagen III and increased the secretion of IL-6, IL-8 and TNF-α. Furthermore, our results showed that BOX5 could prevent these effects. Wnt5a siRNA blocked the activation of the Wnt5a/JNK/NF-κB pathway and inhibited the effects of PM2.5 on fibrosis and inflammation in ASMCs. SP600125 blocked the phosphorylation of NF-κB and inhibited inflammation and fibrosis in PM2.5-exposed ASMCs. Conclusions: These findings suggest that PM2.5 stimulation of ASMCs induces pulmonary inflammatory factor expression and collagen deposition during COPD via the Wnt5a/JNK pathway, which indicates that modulating the Wnt5a/JNK pathway could be a promising therapeutic strategy for PM2.5-induced COPD.

Vascular endothelial growth factor induces the migration of human airway smooth muscle cells by activating the RhoA/ROCK pathway.

BACKGROUND: Airway remodeling due to increased airway smooth muscle cell (ASMC) mass, likely due to enhanced proliferation, hypertrophy, and migration, has been proven to be highly correlated with decreased lung function in asthma patients. Vascular endothelial growth factor (VEGF) mediates vascular and extravascular remodeling and inflammation and has been proven to be involved in the progression of asthma. Previous studies have focused on the effects of VEGF on ASMC proliferation, but few researchers have focused on the effects of VEGF on human ASMC migration. The purpose of this study was to explore the effect of VEGF on the migration of ASMCs and its related signaling pathway mechanism to provide evidence for the treatment of airway remodeling. METHODS: We examined the effects of VEGF induction on ASMC migration and explored the mechanisms involved in ASMC migration. RESULTS: We found by wound healing and Transwell assays that VEGF promoted ASMC migration. Through the Cell Counting Kit-8 (CCK-8) experiment, we found that VEGF had no significant effect on the proliferation of ASMCs, which excluded the involvement of cell proliferation in the process of wound healing. Moreover, a cellular immunofluorescence assay showed that VEGF promoted F-actin reorganization, and Western blotting showed that VEGF improved RhoA activation and myosin phosphatase targeting subunit-1 (MYPT1) and myosin light chain (MLC) phosphorylation in ASMCs. Treatment with the ROCK inhibitor Y27632 significantly attenuated the effects of VEGF on MYPT1/MLC activation and cell migration. CONCLUSION: In conclusion, the results suggest that the promigratory function of VEGF activates the RhoA/ROCK pathway, induces F-actin reorganization, improves the migration of ASMCs, and provides a better rationale for targeting the RhoA/ROCK pathway for therapeutic approaches in airway remodeling.

Effects of Nigella sativa Oil Fractions on Reactive Oxygen Species and Chemokine Expression in Airway Smooth Muscle Cells.

BACKGROUND: many previous studies have demonstrated the therapeutic potential of N. sativa total oil fractions, neutral lipids (NLs), glycolipids (GLs), phospholipids (PLs), and unsaponifiable (IS) in asthma patients. We therefore tested its effect on airway smooth muscle (ASM) cells by observing its ability to regulate the production of glucocorticoid (GC)-insensitive chemokines in cells treated with TNF-α/IFN-γ, and its antioxidative and reactive oxygen species (ROS) scavenging properties. MATERIALS AND METHODS: the cytotoxicity of N. sativa oil fractions was assessed using an MTT assay. ASM cells were treated with TNF-α/IFN-γ for 24 h in the presence of different concentrations of N. sativa oil fractions. An ELISA assay was used to determine the effect of N. sativa oil fractions on chemokine production (CCL5, CXCL-10, and CXCL-8). The scavenging effect of N. sativa oil fractions was evaluated on three reactive oxygen species (ROS), O2•-, OH•, and H2O2. RESULTS: our results show that different N. sativa oil fractions used at 25 and 50 µg/mL did not affect cell viability. All fractions of N. sativa oil inhibited chemokines in a concentration-dependent manner. Interestingly, the total oil fraction showed the most significant effect of chemokine inhibition, and had the highest percentage of ROS scavenging effect. CONCLUSION: these results suggest that N. sativa oil modulates the proinflammatory actions of human ASM cells by inhibiting the production of GC-insensitive chemokines.

Combined Epimedii Folium and Ligustri Lucidi Fructus with dexamethasone alleviate the proliferation of airway smooth muscle cells by regulating apoptosis/autophagy.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional Chinese medicine (TCM) theory believes kidney deficiency is the root cause of chronic refractory asthma with pathological changes of airway remodeling. Our previous experiments confirmed that the combination of Epimedii Folium and Ligustri Lucidi Fructus (ELL) with the effect of nourishing Yin and Yang of the kidney could improve the pathological changes of airway remodeling in asthmatic rats, but the specific mechanism remains unclear. AIM OF THE STUDY: This research was designed to reveal the synergy of ELL and dexamethasone (Dex) in the proliferation, apoptosis, and autophagy of airway smooth muscle cells (ASMCs). MATERIALS AND METHODS: Primary cultures of ASMCs from rats were prepared and induced with histamine (Hist), Z-DEVD-FMK (ZDF), rapamycin (Rap), or 3-Methyladenine (3-MA) at generation 3-7 for 24 or 48 h. Subsequently, the cells were treated with Dex, ELL, and ELL&Dex for 24 or 48 h. The effect of various concentrations of inducers and drugs on cell viability was detected by Methyl Thiazolyl Tetrazolium (MTT) assay, cell proliferation was tested using immunocytochemistry (ICC) by detecting Ki67 protein, cell apoptosis was measured by Annexin V-FITC/PI assay and Hoechst nuclear staining, cell ultrastructure was observed by transmission electron microscopy (TEM), and immunofluorescence (IF), Western blot (WB) combined with quantitative real-time PCR (qPCR) were used for measuring autophagy and apoptosis-related genes including protein 53 (P53), cysteinyl aspartate-specific proteinase (Caspase)-3, microtubule-associated protein 1 light chain 3 (LC3), Beclin-1, mammalian target of rapamycin (mTOR) and p-mTOR. RESULTS: In ASMCs, Hist and ZDF promoted cell proliferation, significantly decreased Caspase-3 protein expression, and up-regulated Beclin-1 levels; Dex alone and in combination with ELL promoted Beclin-1, Caspase-3, and P53 expression, enhancing autophagy activity and apoptosis in Hist and ZDF-induced AMSCs. In contrast, Rap inhibited cell viability, increased Caspase-3, P53, Beclin-1, and LC3-II/I and decreased the levels of mTOR and p-mTOR with promoting apoptosis and autophagy; ELL or ELL&Dex reduced P53, Beclin-1, and LC3-II/I to down-regulate apoptosis and the excessive autophagic state of ASMCs induced by Rap. In the 3-MA model, cell viability and autophagy were reduced; ELL&Dex significantly upgraded the expression of Beclin-1, P53, and Caspase-3 and promoted apoptosis and autophagy of ASMCs. CONCLUSIONS: These results suggest that ELL combined with Dex may regulate the proliferation of ASMCs by promoting apoptosis and autophagy and be a potential medicine for the treatment of asthma.

MiR-506 targets polypyrimidine tract-binding protein 1 to inhibit airway inflammatory response and remodeling via mediating Wnt/ß-catenin signaling pathway.

BACKGROUND: Airway remodeling, which contributes to the clinical course of childhood asthma, occurs due to airway inflammation and is featured by anomalous biological behaviors of airway smooth muscle cells (ASMCs). microRNA (miRNA) plays an essential role in the etiopathogenesis of asthma. OBJECTIVE: This research was aimed to characterize miR-506 in asthma and uncover potential regulatory machinery. MATERIAL AND METHODS: The asthmatic cell model was established by treating ASMCs with transforming growth factor-beta1 (TGF-ß1) and assessed by the levels of interleukin (IL)-1ß and interferon gamma (IFN-γ). Using real-time quantitative polymerase chain reaction, mRNA expression of miR-506 and polypyrimidine tract-binding protein 1 (PTBP1) was measured. Cell counting kit-8 and Transwell migration tests were used for estimating the capacity of ASMCs to proliferate and migrate. Luciferase reporter assay was used to corroborate whether miR-506 was directly bound to PTBP1. Expression of PTBP1, collagen I and III, and essential proteins of the wingless-related integration (Wnt)/ß-catenin pathway (ß-catenin, c-MYC and cyclin D1) was accomplished by Western blot analysis. The involvement of Wnt/ß-catenin signaling in asthma was confirmed by Wnt signaling pathway inhibitor (IWR-1). RESULTS: miR-506 was poorly expressed in asthmatic tissues and cell model. Functionally, overexpression of miR-506 reduced aberrant proliferation, migration, inflammation and collagen deposition of ASMCs triggered by TGF-ß1. Mechanically, miR-506 directly targeted the 3' untranslated region (3-UTR) of PTBP1 and had a negative regulation on PTBP1 expression. Moreover, overexpression of miR-506 suppressed the induction of Wnt/ß-catenin pathway. The administration of IWR-1 further validated negative correlation between miR-506 and the Wnt/ß-catenin pathway in asthma. CONCLUSION: Our data indicated that targeting miR-506/PTBP1/Wnt/ß-catenin axis might point in a helpful direction for treating asthma in children.

MiR-506 targets polypyrimidine tract-binding protein 1 to inhibit airway inflammatory response and remodeling via mediating Wnt/airway smooth -muscle cells, childhood asthma, miR-506, PTBP1, Wnt/B-catenin-catenin signaling pathway

Background: Airway remodeling, which contributes to the clinical course of childhood asthma, occurs due to airway inflammation and is featured by anomalous biological behaviors of airway smooth muscle cells (ASMCs). microRNA (miRNA) plays an essential role in the etiopathogenesis of asthma. Objective: This research was aimed to characterize miR-506 in asthma and uncover potential regulatory machinery. Material and methods: The asthmatic cell model was established by treating ASMCs with transforming growth factor-beta1 (TGF-β1) and assessed by the levels of interleukin (IL)-1β and interferon gamma (IFN-γ). Using real-time quantitative polymerase chain reaction, mRNA expression of miR-506 and polypyrimidine tract-binding protein 1 (PTBP1) was measured. Cell counting kit-8 and Transwell migration tests were used for estimating the capacity of ASMCs to proliferate and migrate. Luciferase reporter assay was used to corroborate whether miR-506 was directly bound to PTBP1. Expression of PTBP1, collagen I and III, and essential proteins of the wingless-related integration (Wnt)/β-catenin pathway (β-catenin, c-MYC and cyclin D1) was accomplished by Western blot analysis. The involvement of Wnt/β-catenin signaling in asthma was confirmed by Wnt signaling pathway inhibitor (IWR-1). Results: miR-506 was poorly expressed in asthmatic tissues and cell model. Functionally, overexpression of miR-506 reduced aberrant proliferation, migration, inflammation and collagen deposition of ASMCs triggered by TGF-β1. Mechanically, miR-506 directly targeted the 3’ untranslated region (3-UTR) of PTBP1 and had a negative regulation on PTBP1 expression. Moreover, overexpression of miR-506 suppressed the induction of Wnt/β-catenin pathway. The administration of IWR-1 further validated negative correlation between miR-506 and the Wnt/β-catenin pathway in asthma (AU)

LXA4 inhibits TGF-ß1-induced airway smooth muscle cells proliferation and migration by suppressing the Smad/YAP pathway.

The aims of the present study were to examine the signaling mechanisms for transforming growth factor-ß1 (TGF-ß1)-induced rat airway smooth muscle cells (ASMCs) proliferation and migration and to determine the effect of lipoxin A4 (LXA4) on TGF-ß1-induced rat ASMCs proliferation and migration and its underlying mechanisms. TGF-ß1 upregulated transcriptional coactivator Yes-associated protein (YAP) expression by activating Smad2/3 and then upregulated cyclin D1, leading to rat ASMCs proliferation and migration. This effect was reversed after treatment with the TGF-ß1 receptor inhibitor SB431542. YAP is a critical mediator of TGF-ß1-induced ASMCs proliferation and migration. Knockdown of YAP disrupted the pro-airway remodeling function of TGF-ß1. Preincubation of rat ASMCs with LXA4 blocked TGF-ß1-induced activation of Smad2/3 and changed its downstream targets, YAP and cyclin D1, resulting in the inhibition of rat ASMCs proliferation and migration. Our study suggests that LXA4 suppresses Smad/YAP signaling to inhibit rat ASMCs proliferation and migration and therefore has potential value in the prevention and treatment of asthma by negatively modulating airway remodeling.

Circ_0000029 Interacts with the miR-576-5p/KCNA1 Axis to Hamper the Development of Pediatric Asthma in an Asthma-Like in vitro Assessment.

OBJECTIVE: Numerous circular RNAs (circRNAs) have been verified to execute crucial roles in "asthma-like" progression of the airway smooth muscle cells (ASMCs). The present study aimed to scrutinize the function and mechanism of circ_0000029 in pediatric asthma etiology in vitro. METHODS: A cell model of asthma was developed using ASMCs induced by platelet-derived growth factor BB (PDGF-BB). Western blotting and qRT-PCR were performed to determine the expression levels of circ_0000029, miR-576-5p, and KCNA1 in PDGF-BB-treated ASMCs. Dual-luciferase reporter, RNA-binding protein immunoprecipitation, and RNA pull-down experiments were conducted to validate targeting relationships. CCK-8 and Transwell assays were performed to evaluate the proliferative and migratory potential of ASMCs. The rate of apoptosis was analyzed using flow cytometry. RESULTS: Pronounced circ_0000029 and KCNA1 downregulation and high levels of miR-576-5p were observed in PDGF-BB-treated ASMCs. Circ_0000029 targets miR-576-5p to regulate KCNA1 expression. The loss of KCNA1 and upregulation of miR-576-5p dramatically impeded apoptosis but promoted ASMC migration and proliferation. Ectopic expression of circ_0000029 manifested the opposite outcome among ASMCs. Furthermore, KCNA1 deficiency and miR-576-5p upregulation counteracted the effects of circ_0000029 overexpression on ASMCs. CONCLUSIONS: Circ_0000029 represses the abnormal migration and growth of ASMCs by mediating miR-576-5p and KCNA1 expression levels. This suggests that the regulatory axis circ_0000029/miR-576-5p/KCNA1 is a potential target for pediatric asthma treatment.