Apoptosis Of Airway Smooth Muscle Cells Research Articles

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 relevanceShegan-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 studyThis study investigated the efficacy as well as the potential mechanism of SMD in CA. Materials and methodsIn 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. ResultsThe 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. ConclusionsSMD 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.

Paclitaxel inhibits proliferation by negatively regulating Cdk1-cell cycle axis in rat airway smooth muscle cells

Objective Paclitaxel exhibits outstanding biological activities in inhibiting cell proliferation and inducing cell apoptosis. However, the effects of paclitaxel on airway smooth muscle cells (ASMCs) have not been reported yet. The purpose of this study is to determine the effects of paclitaxel on the proliferation and apoptosis of ASMCs. Methods Rat primary ASMCs were isolated and used in all the experiments. Cell Counting Kit-8 assay and Edu assay were used to analyze the cell viability and proliferation, respectively. Flow cytometry was used to detect the cell cycle and apoptosis. Quantitative real-time PCR (qRT-PCR), western blotting, and immunostaining were used to detect the expression of cyclin-dependent kinase 1 (Cdk1). Results Our study showed that paclitaxel inhibits the proliferation of ASMCs in a dose- and time-gradient-dependent manner. Further study displayed that cell cycle is arrested at G2/M phase. And Cdk1 was dramatically down-regulated by paclitaxel treatment. Cell morphological analysis showed that ASMCs are elliptical with a larger surface area after paclitaxel treatment. Nucleus morphological analysis showed that the nuclei are in a diffuse state after paclitaxel treatment. However, paclitaxel did not induce the apoptosis of ASMCs. Conclusions Our study demonstrated that paclitaxel inhibits the proliferation of ASMCs at least partly by negatively regulating Cdk1-cell cycle axis.

The miR-9-5p/KLF5/IL-1β Axis Regulates Airway Smooth Muscle Cell Proliferation and Apoptosis to Aggravate Airway Remodeling and Inflammation in Asthma.

The aim of this study was to investigate the underlying mechanism of miR-9-5p in airway smooth muscle cells (ASMCs) of asthmatic mice. An asthmatic mouse model was established through the intraperitoneal injection of ovalbumin. Histopathological changes in lung tissues of asthmatic mice were observed using HE staining. ASMCs was identified using immunofluorescencestaining and cell morphology. The mRNA expressions of miR-9-5p, KLF5, and IL-1β were measured using RT-qPCR. Additionally, CCK8 assay and flow cytometry were applied for ASMC proliferation and apoptosis, respectively. The protein levels of OPN, KLF5, and IL-1β were assessed using western blotting. The results showed that miR-9-5p was abnormally downregulated in lung tissues and ASMCs of asthmatic mice. Dual-Luciferase Reporter Assay and Chromatin immunoprecipitation confirmed that miR-9-5p targeted KLF5 that bounds to IL-1β promoter. Besides, miR-9-5p negatively regulated IL-1β mRNA and protein level via KLF5. Moreover, miR-9-5p was found to positively regulate ASMC apoptosis, negatively regulate ASMC proliferation and OPN protein expression, albeit with partial reversal by KLF5. Mechanistically, the regulation of ASMC proliferation and apoptosis by miR-9-5p is achieved by targeting KLF5/IL-1β axis.

Midkine ameliorates LPS-induced apoptosis of airway smooth muscle cells via the Notch2 pathway

Objective: To evaluate the effect of midkine on lipopolysaccharide (LPS)-induced airway smooth muscle cells (ASMCs). Methods: LPS-stimulated acute lung injury model was used to analyze the effect of midkine on ASMCs in vitro. Recombinant midkine and midkine siRNA were used to investigate the role of Notch2 signaling pathway. Cell proliferation was assessed using Cell Counting Kit-8 assay. Additionally, apoptosis was measured by flow cytometry and protein and mRNA expression of midkine and Notch2 was assessed by Western blotting and qPCR, respectively. Immunofluorescence analysis was also conducted. Results: LPS increased the mRNA and protein expression of midkine and Notch2. Midkine silencing reduced LPS-induced midkine and Notch2 expression. In addition, midkine silencing further reduced the viability and increased apoptosis of ASMCs induced by LPS, which was attenuated by recombinant midkine. Conclusions: The midkine/Notch2 signaling pathway plays a regulatory role in ASMC proliferation and apoptosis in airway inflammation.

MicroRNA-98-5p Inhibits IL-13-Induced Proliferation and Migration of Human Airway Smooth Muscle Cells by Targeting RAC1.

The dysfunction of airway smooth muscle cells (ASMCs) is one of the key factors in the pathogenesis of asthma. How miR-98-5p works in asthma has not been completely elucidated. This work focused on how miR-98-5p functions in the proliferation and migration of human ASMCs treated with interleukin-13 (IL-13). MiR-98-5p expression in plasma of asthmatic patients and IL-13-stimulated ASMCs was probed by quantitative real-time polymerase chain reaction (qRT-PCR). RAS-relevant C3 botulinum toxin substrate 1 (RAC1) protein expression in ASMCs was assessed by Western blot. The growth of ASMCs was measured by cell counting kit-8 (CCK-8) assay and 5-ethynyl-2'-deoxyuridine (EdU) assay. The migration of ASMCs was examined by Transwell assay. Besides, the apoptosis of ASMCs was analyzed by flow cytometry. The targeting relationship between miR-98-5p and RAC1 3'-UTR was verified by a dual-luciferase reporter gene assay. MiR-98-5p expression was reduced in patients' plasma and IL-13-stimulated ASMCs, and RAC1 expression was upregulated in ASMCs treated with IL-13. MiR-98-5p overexpression inhibited IL-13-induced proliferation and migration of ASMCs while promoting the apoptosis. The opposite result was observed after inhibiting miR-98-5p expression. Besides, RAC1 was identified as a direct downstream target of miR-98-5p in ASMCs. The restoration of RAC1 expression counteracted the impacts of miR-98-5p overexpression on IL-13-stimulated proliferation, migration, and apoptosis of ASMCs. MiR-98-5p inhibits IL-13-induced proliferation and migration and accelerates the apoptosis of ASMCs by downregulating RAC1 expression.

The microRNA-1278/SHP-1/STAT3 pathway is involved in airway smooth muscle cell proliferation in a model of severe asthma both intracellularly and extracellularly.

This study investigated the regulatory effects of microRNA-1278 (miR-1278) on airway inflammation, airway reconstruction, and the proliferation and apoptosis of airway smooth muscle cells (ASMCs) induced by transforming growth factor β1 (TGF-β1). The results showed that miR-1278 was upregulated in the blood and lung tissues (LTs) of patients with asthma compared with that in healthy volunteers; miR-1278 expression was also upregulated in asthmatic mice, and miR-1278 inhibition improved the LTs of asthmatic mice. Moreover, miR-1278 inhibited inflammation in asthmatic mice and counteracted the effect of TGF-β1 of induced proliferation and reduced apoptosis in ASMCs. DLRA indicated that miR-1278 targeted the 3'-UTR of Src-homology 2-containing phosphatase 1 (SHP-1). Furthermore, miR-1278 promoted ASMC proliferation, in which TGF-β1 played an important role by regulating the SHP-1/STAT3 signaling pathway. In conclusion, this study showed that miR-1278 played a critical role in the processes of airway remodeling and reduction of apoptosis by targeting SHP-1.

SREBP2 promotes the viability, proliferation, and migration and inhibits apoptosis in TGF-β1-induced airway smooth muscle cells by regulating TLR2/NF-κB/NFATc1/ABCA1 regulatory network

ABSTRACT Asthma is a respiratory disease with complex pathogenesis. Sterol-responsive element-binding proteins 2 (SREBP2) was found to bind to promoter sequences of ABCA1 to suppress ABCA1 promoter activity. This study aimed to explore the expression level of SREBP2 and ATP-binding cassette transporter A1 (ABCA1), and their effects on the development of airway smooth muscle cells (ASMCs) in asthma. ASMCs were treated with different concentrations of TGF-β1 (0, 0.5, 1, 5 and 10 ng/mL). Short hairpin SREBP2 (shSREBP2), SREBP2, shABCA1 or ABCA1 were transfected into ASMCs. Cell viability, proliferation, apoptosis, migration, and the expression of SREBP2, ABCA1 and related pathway proteins were detected by MTT assay, Brdu staining, flow cytometer, Transwell assay, qRT-PCR, and Western blotting, respectively. The results showed that TGF-β1 increased the viability, proliferation, migration and inhibited apoptosis in ASMCs. Moreover, TGF-β1 also decreased the expression of ABCA1, cleaved caspase-3, cleaved PARP, E-cadherin, and increased the expression of vimentin, TLR2, p-p65 and NFATc1. SREBP2 knockdown alleviated these TGF-β1-induced changes. SREBP2 overexpression inhibited ABCA1 expression and apoptosis, and promoted cell migration and the expression of TLR2, p-p65, NFATc1 in ASMCs. ABCA1 overexpression alleviated these SREBP2-induced promoting and inhibition effects. In conclusion, SREBP2 activates TLR2/NF-κB/NFATc1 regulatory network and promotes TGF-β1-induced cell movement through inhibiting ABCA1 expression.

Curcumol inhibits PDGF-BB-induced proliferation and migration of airway smooth muscle cells by suppressing ERK/CREB pathway.

Curcumol, possessing antiviral, antifungal, antimicrobial, anticancer, and anti-inflammatory properties, has been widely used in treating cancers and liver fibrosis. The aim of this study was to determine the effect of curcumol on the progression of asthma. Curcumol was administrated to platelet-derived growth factor (PDGF)-BB-stimulated airway smooth muscle cells (ASMCs). The proliferation of ASMCs was assessed by MTT and EdU incorporation assays. The apoptosis of ASMCs was measured by flow cytometry and Western blotting. The migration of ASMCs was evaluated by Transwell migration assay and Western blotting. The regulatory effects of curcumol on extracellular signal-regulated protein kinase (ERK)/cAMP response element-binding protein (CREB) pathway was evaluated by Western blotting. The proliferation and migration of ASMCs induced by PDGF-BB was suppressed, and the apoptosis of ASMCs was elevated by curcumol in a dose-dependent manner. The activation of ERK/CREB pathway induced by PDGF-BB was suppressed by curcumol. Curcumol could suppress ERK/CREB pathway to inhibit proliferation and migration and promote apoptosis of PDGF-BB-stimulated ASMCs. These findings suggest that curcumol may act as a potential drug for asthma treatment.

Adipose-Derived Mesenchymal Stem Cell-Derived Exosomal miR-301a-3p Regulates Airway Smooth Muscle Cells During Asthma by Targeting STAT3.

BackgroundAsthma is a chronic inflammatory disease featured by inflammation and remodeling of airway. Adipose-derived mesenchymal stem cell (ADSCs)-derived exosomal miRNAs have been suggested as promising therapeutic manners for diseases.MethodsADSCs and airway smooth muscle cells (ASMCs) were isolated from SD rats. Flow cytometry was conducted to detect the surface biomarkers of isolated cells. Exosomes were extracted by sequentially centrifuge method and identified by Western blotting and nanoparticle tracking analysis (NTA). Uptake of exosomes by ASMCs was detected by confocal assay. ASMCs were treated with platelet-derived growth factor-BB (PDGF-BB) to mimic cell remodeling and inflammation. Cell counting 8 (CCK-8), Transwell, and flow cytometry were performed to determine the viability, migration, and apoptosis of ASMCs. Release of inflammatory factors was detected by enzyme-linked immunosorbent assay (ELISA). Levels of RNAs and proteins were measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay. Interaction between miR-301a-3p and signal transducer and activator of transcription 3 (STAT3) was determined by luciferase reporter gene assay. The effect of Exosomal miR-301a-3p was analyzed in ovalbumin (OVA)-induced asthma mouse model.ResultsADSCs-derived exosomes could be effectively internalized by ASMCs. Exosomal miR-301a-3p notably suppressed the PDGF-BB-stimulated proliferation and migration of ASMCs, and enhanced apoptosis, as well as decreased the secretion of inflammatory factors. MiR-301a-3p directly targeted the 3ʹUTR region of STAT3. STAT3 overexpression reversed the suppressive effects of exosomal miR-301a-3p on ASMCs under PDGF-BB stimulation. The expression of miR-301a-3p and STAT3 was negative correlation in specimen from patients with asthma. Exosomal miR-301a-3p inhibited OVA-induced lung injury by targeting STAT3 in mice.ConclusionThis study exposed that exosomal miR-301a-3p from ADSCs could effectively alleviate PDGF-BB-stimulated remodeling and inflammation of ASMCs via targeting STAT3, presented ADSCs-derived exosomal miR-301a-3p as a promising therapeutic approach for asthma.

Azithromycin induces apoptosis in airway smooth muscle cells through mitochondrial pathway in a rat asthma model.

BackgroundThe proliferation of airway smooth muscle cells (ASMCs) is a key feature of airway remodeling in asthma. Azithromycin (AZM) has been shown to decrease bronchial hyperresponsiveness and airway inflammation in asthmatics; however, the role of AZM in ASMC proliferation remains unclear. Thus, we investigated the effect of AZM on ASMC proliferation in a rat model of experimental asthma.MethodsWe isolated ASMCs from rats sensitized and challenged by ovabulmin (OVA), and then treated with different concentrations of AZM. Cytotoxicity of ASMC was evaluated by Cell Counting Kit-8 (CCK-8) assay, morphological change was examined with laser confocal microscope after Annexin V/propidium iodide (PI) double staining, mitochondrial membrane potential was determined with JC-1 staining, and the expression of cytochrome C was examined by western blot.ResultsThe relative surface areas of airway wall and smooth muscle layers in OVA-sensitized rats were significantly increased compared to those in the control group. Furthermore, in OVA-sensitized rats, the mitochondrial membrane potential of ASMC was higher, while the expression of mitochondria cytochrome C was lower compared to that in control rats. After AZM treatment, ASMC apoptosis was increased, mitochondrial membrane potential reduced, and the protein level of cytosolic cytochrome C was increased.ConclusionsThis study demonstrated that AZM increased the apoptosis of ASMCs through a mitochondrial pathway, which might play an important role in ASMs proliferation during asthmatic remodeling.

SET knockdown attenuated phenotype modulation and calcium channel associated markers of airway smooth muscle cells in asthmatic mice.

BackgroundDysfunctional phenotype modulation and calcium channels in airway smooth muscle cells (ASMCs) are important characteristics of airway remodeling in chronic asthma. However, the mechanisms underlying these pathological processes remain unclear. SET (I2PP2A, inhibitor-2 of protein phosphatase 2A) has many significant functions and is involved in various physiological and pathological processes. This study aimed to determine the function of SET in chronic asthma.MethodsBALB/c mice were sensitized by ovalbumin injection and repeated inhalation of ovalbumin. The Penh value was measured using the Buxco whole body plethysmography system. A short hairpin RNA of the SET gene was designed and transfected into ASMCs derived from asthmatic mice. Flow cytometry of Annexin-V/propidium iodide staining was used for evaluating cell apoptosis. Western blot was adopted to measure the expression levels of ASMCs phenotype modulation markers and calcium channel-associated proteins.ResultsThe results showed that shRNA targeting SET significantly decreased the expression of SET, and enhanced the apoptosis of ASMCs. SET knockdown promoted the expression of contractile phenotype markers such as α-SMA (alpha smooth muscle Actin), SM-MHC (smooth muscle Myosin heavy chain), and calponin, and inhibited the expression of synthetic phenotype markers including vimentin and CD44. The expression of the calcium channel-related proteins STIM1 (Stromal interaction molecule 1) and Orai1 were also inhibited after SET knockdown.ConclusionsThese data demonstrated that SET participated in the development of airway dysfunction in asthma, suggesting that the silencing of SET may be a new therapeutic target for the treatment of asthma patients.

Effect of TLR7 gene expression mediating NF-κB signaling pathway on the pathogenesis of bronchial asthma in mice and the intervention role of IFN-γ.

To explore the mechanism of TLR7 mediating NF-κB signaling pathway on the pathogenesis of bronchial asthma in mice and the intervention effect of IFN-γ in the process. The experimental animals were 70 C57BL/6J female mice of clean grade, which were divided into 7 groups according to different treatment protocols, including Normal group, Asthma group, Model+1-MT group, Model+IFN-γ group, Model+TLR7 agonist group, TLR7 deficient group, and Model+TLR7 deficient group. Hematoxylin-eosin (HE) staining was used to observe the pathological changes of lung tissues. The positive expression rates of TLR7, p-IKKα and NF-κBp65 were detected by immunohistochemistry. bronchoalveolar lavage fluid (BALF) cells were classified and counted. The contents of interleukin (IL)-4, IL-10, IL-12 and interferon (IFN)-γ in BALF supernatant were detected by enzyme-linked immunosorbent assay (ELISA). Following isolation, culture and plasmid construction of airway smooth muscle cells (ASMCS) from normal mice and asthmatic mice, cells were transfected and divided into the Control group, pcDNA-TLR7 NC group, siRNA-TLR7 NC group, pcDNA-TLR7 group, siRNA-TLR7 group, Asatone group, Triptolide group, and pcDNA-TLR7 +Asatone group. The expression of TLR7, IDO, p-IKKα and NF-κBp65 was detected by real-time polymerase chain reaction (RT-PCR) and Western blot, respectively. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) was used to detect the proliferation of ASMCS. The cell cycle and apoptosis of ASMCS were detected by flow cytometry. HE staining showed successful modeling of asthma. Immunohistochemical test showed that the positive expression rate of TLR7 in the Asthma group was significantly decreased, and that of IKKα and NF-κBp65 was significantly increased, with significantly increased IL-4, IL-10, IL-12 and IFN-γ levels (all p<0.05). Model+1-MT group and Model+TLR7 deficient group had a large number of inflammatory cell infiltration, increased IL-4, IL-10, IL-12 and IFN-γ levels, decreased expression levels of TLR7 and IDO, and increased expression of p-IKKα and NF-κBp65 (all p<0.05); while the opposites results were detected in Model+IFN-γ group and Model+TLR7 agonist group (all p<0.05). Cell transfection experiments revealed that pcDNA-TLR7 group and Triptolide group had increased TLR7 expression while decreased p-IKKα and NF-κBp65, decreased proliferation level, and increased cell apoptosis (all p<0.05); while the contrary results were found in siRNA-TLR7 group and Asatone group (all p<0.05); yet without significant difference in pcDNA-TLR7+Asatone group (all p>0.05). Upregulation of TLR7 can inhibit the activation of NF-κB signaling pathway, reduces airway inflammation, inhibits ASMCS proliferation and thus promotes cell apoptosis in asthmatic mice. Besides, IFN-γ can exert a protective role in suppressing the progression of inflammation in asthma.

Exposure to ozone impacted Th1/Th2 imbalance of CD4+ T cells and apoptosis of ASMCs underlying asthmatic progression by activating lncRNA PVT1-miR-15a-5p/miR-29c-3p signaling.

This investigation attempted to elucidate whether lncRNA PVT1-led miRNA axes participated in aggravating ozone-triggered asthma progression. One hundred and sixty-eight BALB/c mice were evenly divided into saline+air group, ovalbumin+air group, saline+ozone group and ovalbumin+ozone group. Correlations were evaluated between PVT1 expression and airway smooth muscle function/inflammatory cytokine release among the mice models. Furthermore, pcDNA3.1-PVT1 and si-PVT1 were, respectively, transfected into CD4+T cells and airway smooth muscle cells (ASMCs), and activities of the cells were observed. Ultimately, a cohort of asthma patients was recruited to estimate the diagnostic performance of PVT1. It was demonstrated that mice of ovalbumin+ozone group were associated with higher PVT1 expression, thicker trachea/airway smooth muscle and smaller ratio of Th1/Th2-like cytokines than mice of ovalbumin+air group and saline+ozone group (P<0.05). Moreover, pcDNA3.1-PVT1 significantly brought down Th1/Th2 ratio in CD4+ T cells by depressing miR-15a-5p expression and activating PI3K-Akt-mTOR signaling (P<0.05). The PVT1 also facilitated ASMC proliferation by sponging miR-29c-3p and motivating PI3K-Akt-mTOR signaling (P<0.05). Additionally, PVT1 seemed promising in diagnosis of asthma, with favorable sensitivity (i.e. 0.844) and specificity (i.e. 0.978). Conclusively, lncRNA PVT1-miR-15a-5p/miR-29c-3p-PI3K-Akt-mTOR axis was implicated in ozone-induced asthma development by promoting ASMC proliferation and Th1/Th2 imbalance.

MiRNA-620 promotes TGF-β1-induced proliferation of airway smooth muscle cell through controlling PTEN/AKT signaling pathway.

Asthma is an inflammatory syndrome characterized by airway hyperresponsiveness, bronchial inflammation, and airway remodeling. The hypertrophy and hyperplasia of airway smooth muscle cells (ASMCs) are hallmarks of bronchial remodeling in asthma. In this study, the regulatory effects of microRNA-620 (miR-620) on ASMC proliferation and apoptosis in response to transforming growth factor β1 (TGF-β1) stimulation was investigated. The expression of miR-620 was significantly upregulated in TGF-β1-treated ASMCs compared with vehicle-treated cells. Downregulation of miR-620 suppressed the proliferation and increased apoptosis in TGF-β1-stimulated ASMCs. Phosphatase and tensin homolog (PTEN) was predicted and confirmed as a downstream target of miR-620. PTEN was upregulated in miR-620-inhibitor transfected ASMCs, but decreased in cells delivered with miR-620 mimics. Moreover, knocking down miR-620 alone efficiently reduced the phosphorylation of protein kinase B (AKT), decreased TGF-β1-induced proliferation and promoted apoptosis in ASMCs, whereas downregulation of PTEN in miR-620 inhibitor-transfected cells restored the activation of AKT, increased TGF-β1-triggered proliferation, and partially inhibited ASMC apoptosis. Taken together, the present study provided evidence that miR-620 increased TGF-β1-mediated proliferation and suppressed apoptosis in ASMCs via the regulation of PTEN and AKT expression. These findings suggest that miR-620/PTEN/AKT axis may be considered as a therapeutic target for asthma treatment.

Circular RNA circHIPK3 modulates the proliferation of airway smooth muscle cells by miR-326/STIM1 axis

AimsEmerging findings demonstrate the critical roles of noncoding RNA (ncRNA) in asthma development. Nevertheless, the biological roles of circular RNA (circRNA) in airway remodeling are still elusive. Here, the present research focuses on the regulation of circRNA circHIPK3 in airway smooth muscle cells (ASMCs) proliferation and migration. Materials and methodsThe sequence of circRNA was detected using Sanger sequencing. Cellular phenotypes were detected using CCK-8 assay, transwell and flow cytometer assay. The potential binding of miRNA and downstream and upstream targets was detected using dual-luciferase reporter assay. Key findingsResults showed that circHIPK3 was significantly upregulated in platelet-derived growth factor (PDGF) induced ASMCs. Functional analysis using CCK-8, transwell migration assays and flow cytometry analysis showed that circHIPK3 knockdown repressed proliferation, migration and up-regulated the apoptosis in ASMCs. Mechanistic assays showed that circHIPK3 sponged miR-326 in the cytoplasm, thereby targeting stromal interaction molecule 1 (STIM1) to regulate ASMCs' proliferation, migration and apoptosis. SignificanceCollectively, the data elucidates that circHIPK3 functions as a regulator in the airway remodeling during the asthma development through miR-326/STIM1 axis, providing a novel insight for the therapeutic target.

Exchange protein directly activated by cAMP (Epac) protects against airway inflammation and airway remodeling in asthmatic mice

Backgroundβ2 receptor agonists induce airway smooth muscle relaxation by increasing intracellular cAMP production. PKA is the traditional downstream signaling pathway of cAMP. Exchange protein directly activated by cAMP (Epac) was identified as another important signaling molecule of cAMP recently. The role of Epac in asthmatic airway inflammation and airway remodeling is unclear.MethodsWe established OVA-sensitized and -challenged acute and chronic asthma mice models to explore the expression of Epac at first. Then, airway inflammation and airway hyperresponsiveness in acute asthma mice model and airway remodeling in chronic asthma mice model were observed respectively after treatment with Epac-selective cAMP analogue 8-pCPT-2′-O-Me-cAMP (8pCPT) and Epac inhibitor ESI-09. Next, the effects of 8pCPT and ESI-09 on the proliferation and apoptosis of in vitro cultured mouse airway smooth muscle cells (ASMCs) were detected with CCK-8 assays and Annexin-V staining. Lastly, the effects of 8pCPT and ESI-09 on store-operated Ca2+ entry (SOCE) of ASMCs were examined by confocal Ca2+ fluorescence measurement.ResultsWe found that in lung tissues of acute and chronic asthma mice models, both mRNA and protein expression of Epac1 and Epac2, two isoforms of Epac, were lower than that of control mice. In acute asthma mice model, the airway inflammatory cell infiltration, Th2 cytokines secretion and airway hyperresponsiveness were significantly attenuated by 8pCPT and aggravated by ESI-09. In chronic asthma mice model, 8pCPT decreased airway inflammatory cell infiltration and airway remodeling indexes such as collagen deposition and airway smooth muscle cell proliferation, while ESI-09 increased airway inflammation and airway remodeling. In vitro cultured mice ASMCs, 8pCPT dose-dependently inhibited, whereas ESI-09 promoted ASMCs proliferation. Interestingly, 8pCPT promoted the apoptosis of ASMCs, whereas ESI-09 had no effect on ASMCs apoptosis. Lastly, confocal Ca2+ fluorescence examination found that 8pCPT could inhibit SOCE in ASMCs at 100 μM, and ESI-09 promoted SOCE of ASMCs at 10 μM and 100 μM. In addition, the promoting effect of ESI-09 on ASMCs proliferation was inhibited by store-operated Ca2+ channel blocker, SKF-96365.ConclusionsOur results suggest that Epac has a protecting effect on asthmatic airway inflammation and airway remodeling, and Epac reduces ASMCs proliferation by inhibiting SOCE in part.

Isorhynchophylline exerts anti-asthma effects in mice by inhibiting the proliferation of airway smooth muscle cells: The involvement of miR-200a-mediated FOXC1/NF-κB pathway

Hyperplasia of airway smooth muscle cells (ASMCs) is key to the progression of asthma. Isorhynchophylline (IRN) derived from Uncaria rhynchophylla can inhibit the proliferation of AMSCs. The major purpose of the current study was to assess the effect of IRN on the asthma symptoms was assessed both in vitro and in vivo, and the associated mechanism of the effect was also explored by focusing on the function of miR-200a. Asthma model was induced using ovalbumin (OVA) method and AMSC hyperplasia model was induced using TGF-β1. The effect of IRN on allergic asthma mice and the effect of IRN on the proliferation of ASMCs were investigated as well, and the changes in miR-200a level and FOXC1/NF-κB pathway were detected. The administration of IRN attenuated the eosinophils recruitment in BALF, reduced collagen deposition in lung tissues, and suppressed production of IgE and pro-inflammation cytokines. IRN also inhibited the proliferation and induced the apoptosis of ASMCs. Moreover, the administration of IRN increased the level of miR-200a while inhibited the activation of FOXC1/NF-κB pathway. However, after the inhibition of miR-200a level, the function of IRN on ASMCs was impaired. Collectively, it was demonstrated that the effect of IRN on asthma relied on the up-regulation of miR-200a, which then deactivated FOXC1/NF-κB pathway.

Bioactive molecule Icariin inhibited proliferation while enhanced apoptosis and autophagy of rat airway smooth muscle cells in vitro.

Icariin is the main active compound extracted from epimedium Flavonoids (EFs) and involved in regulation of cell behaviors (proliferation, apoptosis, and autophagy etc.) for many cell types, but the effect of Icariin on airway smooth muscle cells (ASMCs) is still unknown. The aim of the present study is to examine the role of Icariin on rat ASMCs proliferation, apoptosis and autophagy. CKK8 assay showed that Icariin inhibited rat ASMCs growth in dose-time-dependent manner, and the flow cytometry assay showed that the Icariin interfered with ASMCs cell cycle, when treated with Icariin, S phase shortened while G2 phase extended, cyclin E1 and cyclinA1 gene and protein expression decreased. Next apoptosis was detected, Flow cytometry and TdTmediated dUTP Nick-End Labeling (TUNEL) assay showed that Icariin promoted ASMCs apoptosis, and enhanced apoptosis protein cleaved-caspase-3 expression. Finally, it was found Icariin induced rat ASMCs autophagy, with enhancement expression of autophagy marker LC3 II. In conclusion, Icariin inhibited ASMCs proliferation while promoted apoptosis and autophagy, revealing its potential role in treatment of airway remodeling in asthma.

Silencing of ADAM33 restrains proliferation and induces apoptosis of airway smooth muscle cells in ovalbumin-induced asthma model.

A defibrinogen and metalloproteinase 33 (ADAM33) was reported to play an important role in asthma. Furthermore, ADAM33 may play a possible role in airway remodeling due to its high expression in myo-/fibroblasts, epithelium, as well as the airway smooth muscle cells (ASMCs). Thus, the study is supposed to investigate the effect of the downregulation of ADAM33 on the proliferation and apoptosis of ASMCs in allergic asthma. An ovalbumin-induced asthma model in rats was established for investigating the function of the silencing of ADAM33. ASMCs were cultured and divided into four groups after transfection. The messenger RNA and protein expressions of ADAM33 were measured by reverse transcription quantitative polymerase chain reaction and Western blot analysis. Cell proliferation was tested by cell counting kit-8 and cell apoptosis by TdT-mediated dUTP nick-end labeling. The allergic asthma rats showed a large number of inflammatory cell infiltration, airway smooth muscle hypertrophy and hyperplasia, and increased WA t , WA m , and numbers of bronchial smooth muscle nucleus. Additionally, increased numbers of eosinophils and neutrophils, expressions of immunoglobulin E and interleukin-4, content of airway air pressure, and NO, although decreased in expression of interferon-γ, were exhibited in rats with allergic asthma. In our study, upregulated ADAM33 was found, and after the silencing of ADAM33, decreased proliferation and increased apoptosis of ASMCs were observed. The study evidences that silencing of ADAM33 can decrease the proliferation and increase the apoptosis of ASMCs in a rat model of allergic asthma, suggesting ADAM33 represents a potential investigative focus target aiding allergic asthma.

MicroRNA-142 Inhibits Proliferation and Promotes Apoptosis in Airway Smooth Muscle Cells During Airway Remodeling in Asthmatic Rats via the Inhibition of TGF-β -Dependent EGFR Signaling Pathway

Asthma is a heterogeneous disease characterized by chronic airway inflammation resulting from airway hyper-responsiveness to diverse stimuli. In this study, we investigated whether microRNA-142 (miR-142) expression affects proliferation and apoptosis in airway smooth muscle cells (ASMCs) during airway remodeling in asthmatic rats. Thirty six Wistar rats were randomly classified into a control group and an model group. miR-142 mimics and inhibitors were constructed, and ASMCs were transfected using liposomes according to the following groups: blank, negative control (NC), miR-142 mimics, miR-142 inhibitors, si-TGF-β and miR-142 inhibitors + si-TGF-β. We verified that miR-142 targets TGF-β using a dual-luciferase reporter assay. The expression levels of miR-142, TGF-β, EGFR and apoptosis signaling pathway-related genes were determined using RT-qPCR and western blotting. Changes in cell proliferation, cell cycle progression and apoptosis were analyzed using MTT assays and flow cytometry. Rats with asthma had higher expression levels of EGFR and Akt and lower miR-142 levels. miR-142 was negatively correlated with TGF-β expression. In ASMCs, the expression of TGF-β, EGFR, Akt, phosphorylated-Akt (p-Akt), Bcl-2 and Bcl-xl and the rate of early apoptosis were decreased while expression of Bax and p21 and the proliferation rate were elevated with the upregulation of miR-142. The opposite results were observed with the downregulation of miR-142. Finally, the proliferative rate was decreased while the apoptosis rate was increased and expression levels of EGFR, Akt, p-Akt, Bcl-2 and Bcl-xl were reduced while Bax and p21 were elevated in the ASMCs transfected with miR-142 inhibitors and si-TGF-β. The results of our study suggest that miR-142 inhibits proliferation and promotes apoptosis in ASMCs during airway remodeling in asthmatic rats by inhibiting TGF-β expression via a mechanism involving the EGFR signaling pathway.