Mechanism of rhynchophylline solid lipid nanoparticles inhibiting the proliferation of airway smooth muscle cells in asthmatic model mice / 中国药房

OBJECTIVE To study the inhibitory effect mechanism of rhynchophylline solid lipid nanoparticles （Rhy-SLN） on the proliferation of airway smooth muscle cells （ASMCs） in asthmatic model mice. METHODS Asthma model was prepared by ovalbumin+calmogastrin sensitization. The primary isolation and culture of ASMCs were performed， and morphological observation and identification were also conducted [when α -smooth muscle actin （α -SMA） appeared red and Desmin appeared green in ASMCs， indicating successful cultivation of ASMCs]. The cells were divided into blank group （ASMCs of normal mice）， model group （ASMCs of asthma model mice）， Rhy-SLN group （ASMCs of asthma model mice）， recombinant suppressors of cytokine signaling 1 （SOCS1） overexpression group （ASMCs of asthma model mice transfected with SOCS1 vector）， SOCS1-RNAi group （ASMCs of asthma model mice transfected with SOCS1-RNAi vector） and SB203580 group [p38 mitogen-activated protein kinase （p38 MAPK） inhibitor， ASMCs of asthma model mice]. The cells of each group were added into the corresponding culture medium containing drug （10 μmol/L） or not containing drug for 24 hours. MTT method was used to detect the proliferation of ASMCs in asthmatic mice； Western blot assay was used to detect the protein expressions of α-SMA， interleukin-1β （IL-1β）， SOCS1， p38 MAPK and phosphorylated p38 MAPK （p-p38 MAPK） in ASMCs. RESULTS The primary ASMCs of mice varied in shape and size， presenting irregular， spindle and triangular shapes；α-SMA appeared red and Desmin appeared green， indicating successful cultivation of ASMCs. Compared with model group， ASMCs absorbance values and protein expressions of α -SMA， p38 MAPK， and p-p38 MAPK were reduced significantly in Rhy- SLN group， SOCS1 overexpression group and SB203580 E-mail：wangmeng106@163.com group， while protein expression of SOCS1 （except for group） was increased significantly （P＜0.05）； protein expressions of IL-1β was reduced significantly in ASMCs （P＜ 0.05）. ASMCs absorbance values and protein expressions of α-SMA， SOCS1， p38 MAPK and p-p38 MAPK were increased significantly in SOCS1-RNAi group （P＜0.05）. CONCLUSIONS Rhy-SLN can inhibit the proliferation of ASMCs， the mechanism of which may be associated with overexpression of SOCS1 and inhibiting the protein expressions of IL-1β and p38 MAPK.

Biomechanical Issues of Mechanical Ventilation in Covid-19 Therapy / 医用生物力学

The critically ill coronavirus disease 2019 (Covid-19) patients usually present acute respiratory distress syndrome (ARDS), or even acute respiratory failure, and require mechanical ventilation (MV) to provide support for breathing. However, clinical studies have found an extraordinarily high mortality rate (>50%) for those Covid-19 patients who underwent MV. Considering the mechanical nature of MV, the high mortality rate is highly possible to be associated with mechanical stretch-induced lung injury during MV. Thus, it is imperative to understand the MV-induced pathological alterations in the respiratory system and corresponding mitigation measures in order to improve the therapy of critically ill Covid-19 patients. Ventilator-induced lung injury in therapy of critically ill Covid-19 patients involves several biomechanical factors and mechanisms, including changes in respiratory parameters, inflammatory cytokines storm, ciliary-mucus system, airway smooth muscle cells, lung fibrosis, and stretch-activated cell signaling. It is hoped that these biomechanical issues can be diligently investigated, so as to provide insights for optimizing the therapy for Covid-19 as well as other respiratory diseases.

Effect of Saposhnikoviae Radix-Mume Fructus Drug-containing Serum in Regulating Phenotypic Transformation of Airway Smooth Muscle Cell Proliferation Model / 中国实验方剂学杂志

Objective:To observe the effect of Saposhnikoviae Radix-Mume Fructus containing-serum in regulating the phenotypic transformation of airway smooth muscle cells (ASMCs) proliferation model, in order to explore the mechanism of combined administration of "Saposhnikoviae Radix, Mume Fructus" in inhibiting airway remodeling, and reveal the compatibility mechanism of traditional Chinese medicine. Method:The proliferation model of ASMCs was established by platelet derived growth factor (PDGF) induction. The rats were given normal saline, Saposhnikoviae Radix-Mume Fructus, Saposhnikoviae Radix-Mume Fructus(15.425, 15.425, 30.85 g·kg-1·d-1) to prepare drug serum respectively. Four generations of logarithmic phase human bronchial smooth muscle cells (HBSMC) were collected and divided into blank control group, cell model group, normal rat serum group and normal rat serum cell model group, hormone intervention group, Saposhnikoviae Radix serum group, Mume Fructus serum group, Saposhnikoviae Radix-Mume Fructus serum group. The cells were given corresponding treatment. Immunofluorescence staining and Western blot were adopted to detect ASMCs deflating marks protein α-actin and osteopontin (OPN) expressions, and phenotypic transformation was observed; the levels of vascular endothelial growth factor(VEGF), transforming growth factor-β(TGF-β) and interleukin-6(IL-6) secreted by ASMCs were detected by enzyme linked immunosorbent assay (ELISA). Result:Compared with blank group and normal rat serum group, the fluorescence intensity and protein expression of α-actin in model group and normal rat serum cell model group were low, whereas the fluorescence intensity and protein expression of OPN were high, and the concentrations of VEGF, TGF-β and IL-6 increased significantly (Pβ and IL-6 (PConclusion:The combined administration of "Saposhnikoviae Radix-Mume Fructus" has an inhibitory effect on airway remodeling, which may be related to the inhibition of the transformation of ASMCs from contractile phenotype to synthetic phenotype, so as to reduce the release of active substances, such as VEGF, TGF-β and IL-6.

Grape seed proanthocyanidin extract inhibits platelet-derived growth factor induced proliferation of airway smooth muscle cells / 中国小儿急救医学

Objective To explore the effect of grape seed proanthocyanidin extract(GSPE) on the proliferation of airway smooth muscle cells( ASMCs) induced by platelet-derived growth factor( PDGF) and the underlying molecular mechanism. Methods ASMCs of primary rat were cultured. MTT and flow cytom-etry were used to detect the cell proliferation activity and cell cycle distribution of ASMCs which were treated with PDGF and GSPE respectively. The expression levels of cyclin D1,extracellular regulated protein kinases ( ERK)1/2,p-ERK1/2 and β-actin protein in each group ASMCs were analyzed using western blotting assay after ERK1/2 inhibitor PD98059 intervention. Results Compared with control group,cell proliferative activ-ity,S phase fraction and the expression of cyclin D1 and p-ERK1/2 protein increased in PDGF induced group (P<0. 05). These effects induced by PDGF could be reversed by GSPE. PD98059 also could block PDGF induced higher expression of p-ERK1/2 and cyclin D1 proteins in rat ASMCs. Conclusion GSPE can inhib-it PDGF induced cell proliferation and via ERK1/2 signaling pathway in rat ASMCs,which provide a new way for treatment of bronchial asthma.

Chitosan and carboxymethylated derivative nanoparticles as delivery systems for biological products: preparation, characterization, stability and in vitro/in vivo evaluation / Nanopartículas de quitosana e derivado carboximetilado como sistemas de fornecimento (delivery) de produtos biológicos: preparo, caracterização, estabilidade e avaliação in vitro/in vivo

Chitosan is a biocompatible and biodegradable mucoadhesive polymer with unique advantages, such as the distinct trait of opening the junctions to allow paracellular transport of antigen and good tolerability. However, the poor solubility of chitosan in neutral or alkalinized media has restricted its applications in the pharmaceutical field. Chitosan can be easily carboxymethylated to improve its solubility in aqueous media, while its biodegradability and biocompatibility are preserved. Apart from this, carboxymethyl chitosan (CMCS) can be easily processed into nanoparticles which highlight its suitability and extensive usage for preparing different drug delivery formulations. The present study deals with the development and characterization of a delivery system based on CMCS nanoparticles using ovalbumin as model protein. We demonstrated that ovalbumin loaded nanoparticles were successfully synthetized using calcium chloride as a cross-linker by ionic gelation. The nanoparticles exhibited an average size of approximately 169 nm and presented a pseudo-spherical shape. The nanoparticles size increased according to the addition of CaCl2 due to the strong electrostatic attraction. During storage the nanoparticles size increased was attributed to swelling and aggregation. The loading efficiency of ovalbumin was found to be 17%. Confocal microscopy clearly showed the association between ovalbumin and CMCS chains into nanoparticles. Therefore, we suggest these nanoparticles can be considered as an attractive and promising carrier candidate for proteins and antigens. The major challenge that limits the use of such carriers is their instability in an aqueous medium. Thus, the next step of this work was to determine the robustness of several formulations using distinct freeze-drying protocols. This study demonstrated that mannitol in concentration of 10% (w/v) is well suited to preserve ovalbumin loaded CMCS nanocapsules from aggregation during lyophilization and subsequent reconstitution. Importantly, the results showed that an annealing step has a huge impact on porosity of freeze-dried cake by nearly complete crystallization of mannitol, once the crystalline matrix prevents the partial collapse and the formation of larger pores observed without annealing. Therefore, the usual observation that annealing increases the pore size due to growth of ice crystal size does not always apply, at least when crystallization of solute is involved. Since all characterizations and stability studies had been performed, the main purpose of this study was to develop a stable antigen delivery system for oral immunization using CMCS and inactivated rabies virus (RV) as the antigen. RV loaded nanoparticles was found to enhance both systemic (IgG) and local (IgA) immune responses against RV after oral delivery in mice. The effective doses 50% were 50-times higher than the negative controls, indicating that the immune response started only after the third boosting dose. Furthermore, enough neutralizing antibodies was produced to be protected against the harmful effects of the rabies virus. It is therefore concluded, that the CMCS nanoparticles formulated in this study, are suitable for oral vaccine delivery, and can be suggested as a promising delivery system for a diverse range of antigens as well as a gene/protein delivery system, especially for those positively charged. Since several approaches show that effective intervention in airway allergic inflammation can be achieved with allergen-activated interleukin-10-secreting cells, the final part of this work was dedicated to assessing whether IL-10 loaded chitosan nanoparticles (IL10-CSNPs) could be used as a possible inhalable therapeutic tool for preventing exacerbations in asthmatic patients. As positive controls, we also assess whether interleukin 17A and interleukin 9 have the ability to stimulate human airway smooth muscle (HASM) cell contractility using magnetic twisting cytometry (MTC). Significant decreased baseline cell stiffness was observed in HASM cells pre-treated with IL-10, but not with IL10-CSNPs, whereas treatment with IL-17A significantly enhanced baseline cell stiffening. Our findings reveal a previously unknown mechanism underlying immunotherapy for prevention and treatment of asthma

A quitosana é um polímero mucoadesivo biocompatível e biodegradável, com vantagens únicas, tais como a característica distinta de abrir as junções que permitim o transporte paracelular de antígenos e boa tolerabilidade. No entanto, sua baixa solubilidade em meios neutros ou alcalinizados tem restringido suas aplicações no campo farmacêutico. A quitosana pode ser facilmente carboximetilada para melhorar de sua solubilidade em meios aquosos, enquanto sua biodegradabilidade e biocompatibilidade são preservadas. Além disso, a carboximetilquitosana (CMCS) pode ser facilmente processada na forma de nanopartículas, o que destaca sua adequabilidade para uso extensivo no preparo de sistemas de delivery de medicamentos. O presente estudo trata do desenvolvimento e caracterização de um sistema de delivery baseado em nanopartículas de CMCS utilizando ovalbumina como proteína modelo. Nós demonstramos que as nanopartículas carregadas com ovalbumina foram sintetizadas com sucesso utilizando cloreto de cálcio como agente de reticulação por gelificação iônica. As nanopartículas exibiram um tamanho médio de aproximadamente 169 nm e apresentaram uma forma pseudo-esférica. O tamanho das nanopartículas aumentou de acordo com a adição de CaCl2 devido à forte atração eletrostática. Durante o armazenamento, o tamanho aumentado das nanopartículas foi atribuído a incorporação de água e agregação. A eficiência de encapsulamento da ovalbumina foi de aproximadamente 17%. A microscopia confocal mostrou claramente a associação entre ovalbumina e a cadeias de CMCS nas nanopartículas. Sugerimos, portanto, que tal sistema pode ser considerado como candidato atraente e promissor para o carreamento de proteínas e antígenos. O principal desafio que limita o uso desses carreadores consiste na instabilidade em meio aquoso. Assim, o próximo passo deste trabalho foi determinar a robustez de várias formulações utilizandose diferentes protocolos de liofilização. Este estudo demonstrou que o manitol em uma concentração de 10% (p/v) é adequado para preservar da agregação as nanocápsulas de CMCS carregadas com ovalbumina durante a liofilização e subsequente reconstituição. Mais importante, os resultados mostraram que uma etapa de annealing tem um enorme impacto sobre a porosidade da amostra liofilizada devido a quase completa cristalização do manitol, uma vez que a matriz cristalina evita o colapso parcial e a formação de poros maiores observados na ausência do annealing. Portanto, a observação comum de que o annealing aumenta o tamanho doporos devido ao crescimento dos cristais de gelo nem sempre se aplica, pelo menos quando a cristalização de um soluto está envolvida. Uma vez que todas as caracterizações e estudos de estabilidade foram realizados, o principal objetivo deste estudo foi desenvolver um sistema estável de delivery de antígeno para imunização oral utilizando CMCS e vírus rábico inativado (RV) como antígeno. Verificou-se que as nanopartículas carregadas com RV aumentam as respostas imune sistêmica (IgG) e local (IgA) contra o RV após administração oral em camundongos. As doses efetivas 50% foram 50 vezes maiores que os controles negativos, indicando que a resposta imune foi iniciada apenas após a terceira dose da vacina. Além disso, foram produzidos anticorpos neutralizantes suficientes para proteção contra os efeitos nocivos do vírus rábico. Conclui-se, portanto, que as nanopartículas de CMCS formuladas neste estudo, são adequadas para o delivery oral de vacinas, e podem ser sugeridas como um sistema promissor de delivery para uma gama diversa de antígenos, bem como para o delivery de genes/proteínas, especialmente para aqueles carregados positivamente. Uma vez que diversas abordagens mostram que uma intervenção efetiva em casos de inflamação alérgica de vias aéreas pode ser conseguida por meio de células secretoras de interleucina 10 (IL-10) mediante ativação por alergenos, a parte final deste trabalho esteve dedicada a avaliação de nanopartículas de quitosana carregadas com IL-10 (IL10-CSNPs) como possível ferramenta terapêutica inalável para prevenção de exacerbações em pacientes asmáticos. Como controles positivos, avaliou-se adicionalmente se as interleucinas 17A (IL-17A) e 9 (IL-9) possuem a capacidade de estimular a contratilidade de células humanas de músculo liso de vias aéreas humanas (HASM) por meio de citometria de torção magnética (MTC). Uma diminuição significativa da rigidez celular basal foi observada em células HASM pré-tratadas com IL-10, mas não com IL10-CSNPs, enquanto que o tratamento com IL-17A aumentou significativamente a magnitude rigidez celular basal. Nossos resultados revelam um mecanismo previamente desconhecido subjacente à imunoterapia para prevenção e tratamento da asma

Effect of sanguinarine on biomechanics of rat airway smooth muscle cells / 生物医学工程学杂志

This study aimed to evaluate the effect of sanguinarine on biomechanical properties of rat airway smooth muscle cells (rASMCs) including stiffness, traction force and cytoskeletal stress fiber organization. To do so, rASMCs cultured were treated with sanguinarine solution at different concentrations (0.005~5 μmol/L) for 12 h, 24 h, 36 h, and 48 h, respectively. Subsequently, the cells were tested for their viability, stiffness, traction force, migration and microfilament distribution by using methylthiazolyldiphenyl-tetrazolium bromide assay, optical magnetic twisting cytometry, Fourier transform traction microscopy, scratch wound healing method, and immunofluorescence microscopy, respectively. The results showed that at concentration below 0.5 μmol/L sanguinarine had no effect on cell viability, but caused dose and time dependent effect on cell biomechanics. Specifically, rASMCs treated with sanguinarine at 0.05 μmol/L and 0.5 μmol/L for 12 and 24 h exhibited significant reduction in stiffness, traction force and migration speed, together with disorganization of the cytoskeletal stress fibers. Considering the essential role of airway smooth muscle cells (ASMCs) biomechanics in the airway hyperresponsiveness (AHR) of asthma, these findings suggest that sanguinarine may ameliorate AHR via alteration of ASMCs biomechanical properties, thus providing a novel approach for asthma drug development.

S100A8 inhibits PDGF-induced proliferation of airway smooth muscle cells dependent on the receptor for advanced glycation end-products

BACKGROUND: Airway remodeling is a key feature of asthma, characterized by increased proliferation of airway smooth muscle cells (ASMCs). S100A8 is a calcium-binding protein with a potential to regulate cell proliferation. Here, the effect of exogenous S100A8 protein on the proliferation of ASMCs induced by platelet-derived growth factor (PDGF) and the underlying molecular mechanism was investigated. METHODS: Rat ASMCs were cultured with or without a neutralizing antibody to the receptor for advanced glycation end-products (RAGE), a potential receptor for S100A8 protein. Purified recombinant rat S100A8 protein was then added into the cultured cells, and the proliferation of ASMCs induced by PDGF was detected by colorimetric-based WST-8 assay and ampedance-based xCELLigence proliferation assay. The expression levels of RAGE in ASMCs were analyzed using western blotting assay. RESULTS: Results showed that exogenous S100A8 inhibited the PDGF-induced proliferation of rat ASMCs in a dose- dependent manner with the maximal effect at 1 µg/ml in vitro. Furthermore, when ASMCs was pre-treated with anti-RAGE neutralizing antibody, the inhibitory effect of S100A8 on PDGF-induced proliferation was significantly suppressed. In addition, neither the treatment with S100A8 or PDGF alone nor the pre-treatment with rS100A8 followed by PDGF stimulation affected the expression levels of RAGE. CONCLUSIONS: Our study demonstrated that S100A8 inhibits PDGF-induced ASMCs proliferation in a manner dependent on membrane receptor RAGE.

Effects of Wnt/β-catenin signal pathway on asthma airway remodeling / 中国病理生理杂志

AIM:To explore the effect of Wnt/β-catenin signaling pathway in airway smooth muscle cells (ASMC) on asthmatic airway remodeling.METHODS:The asthmatic airway remodeling model in rats was established and the ASMC was isolated and cultured.The protein expression of β-catenin,glycogen synthase kinase-3β (GSK-3β),cMyc and cyclin D1 in the ASMC was determined by Western blot.After depressing the interaction between β-catenin and p300/CBP,the cell activity was measured by CCK-8 assay and the change of cell cycle distribution was analyzed by flow cytometry.Meanwhile,the protein expression of c-Myc and cyclin D1 in the ASMC was determined by Western blot after inhibiting P38 mitogen-activated protein kinase (MAPK) activity.RESULTS:The protein levels of β-catenin,c-Myc and cyclin D1 were significantly increased in asthma group while the protein level of GSK-3β was decreased in the same group (P ＜ 0.05).After depressing the interaction between β-catenin and p300/CBP,the cell activity of ASMC was decreased in asthma group compared with control group (P ＜ 0.05),and the change of the cell cycle distribution in asthma group was also more obvious (P ＜ 0.05).After inhibiting P38 MAPK activity,the protein levels of c-Myc and cyclin D1 were all decreased compared with control group in ASMC asthma and control rats (P ＜ 0.05).CONCLUSION:Wnt/β-catenin signaling pathway may participates in airway remodeling in asthma by increasing the protein expression of c-Myc and cyclin D1,reacting with the P38 MAPK signaling pathway and regulating the growth of ASMC.

Piperine inhibits Ang Ⅱ-induced cellproliferation and migration of rat airway smooth muscle cells / 基础医学与临床

Objective To explore the effects of piperine on cell proliferation and migration in angiotensin Ⅱ (Ang Ⅱ)-treated rat airway smooth muscle cells (ASMCs).Methods The primary ASMCs of rats were cultured by improved tissue-piece digestion inoculation and trypsin digestion.MTT assay was used to detect the effects of Ang Ⅱ and Ang Ⅱ receptor antagonist losartan on cell proliferation activity.After treatment with Ang Ⅱ and piperine, the cell proliferation activity, the cell cycle distribution and the cell migration were detected by MTT, flow cytometry and Transwell assay respectively.ERK1/2 inhibitor PD98059 and losartan were then applied to determine the expression of cyclin D1, MMP-9, p-ERK1/2, ERK1/2, and β-actin proteins by Western blot assay.Results After 24 h culture, Ang Ⅱ treatment promoted the cell proliferative activity in rat ASMCs (P<0.05), and the promotive effect of 10-7 mol/L Ang Ⅱ was the most significant.Additionally, losartan blocked the Ang Ⅱ-induced cell proliferative activity in rat ASMCs (P<0.05).10-7 mol/L Ang Ⅱ treatment resulted in the elevated cell proliferative activity, higher S phase fraction, increased migrated cell number, and enhanced expression of cyclin D1, MMP-9and p-ERK1/2 proteins (P<0.05);these effects were dose-dependently reversed by piperine.Both PD98059 and losartan blocked Ang Ⅱ-induced expression of p-ERK1/2, cyclin D1 and MMP-9 proteins in rat ASMCs.Conclusions Piperine may inhibit Ang Ⅱ-induced cell proliferation and cell migration via ERK1/2 signaling pathway in rat ASMCs.

Role of Wnt/β-catenin signaling pathway in migration of asthmatic rat airway smooth muscle cells / 局解手术学杂志

Objective To investigate the role of Wnt/β-catenin signaling pathway in migration ability of asthmatic rat airway smooth muscle cells (ASMCs).Methods The asthmatic rat airway smooth muscle cells were cultured in vitro.The expression of Wnt/β-catenin in ASMCs was detected by RT-PCR and Western blot.Migration ability of ASMCs was detected by wound healing assay and transwell migration assay.Results Compared with normal rats,the expression level of Wnt/β-catenin in chronic asthmatic rats ASMCs was obvious higher.The β-catenin signaling pathway by specific siRNA could reduce beta-catenin mRNA and protein expression level of ASMCs.The knockdown of Wnt/β-catenin was able to decreases the migration ability of ASMCs.Conclusion Abnormal activation of Wnt/β-catenin signaling pathway may participants in the migration ability of ASMCs of chronic asthma rats.

Effects of Wnt/β-catenin signal pathway on asthma airway remodeling / 中国病理生理杂志

AIM:To explore the effect of Wnt/β-catenin signaling pathway in airway smooth muscle cells (ASMC) on asthmatic airway remodeling.METHODS:The asthmatic airway remodeling model in rats was established and the ASMC was isolated and cultured.The protein expression of β-catenin,glycogen synthase kinase-3β (GSK-3β),cMyc and cyclin D1 in the ASMC was determined by Western blot.After depressing the interaction between β-catenin and p300/CBP,the cell activity was measured by CCK-8 assay and the change of cell cycle distribution was analyzed by flow cytometry.Meanwhile,the protein expression of c-Myc and cyclin D1 in the ASMC was determined by Western blot after inhibiting P38 mitogen-activated protein kinase (MAPK) activity.RESULTS:The protein levels of β-catenin,c-Myc and cyclin D1 were significantly increased in asthma group while the protein level of GSK-3β was decreased in the same group (P ＜ 0.05).After depressing the interaction between β-catenin and p300/CBP,the cell activity of ASMC was decreased in asthma group compared with control group (P ＜ 0.05),and the change of the cell cycle distribution in asthma group was also more obvious (P ＜ 0.05).After inhibiting P38 MAPK activity,the protein levels of c-Myc and cyclin D1 were all decreased compared with control group in ASMC asthma and control rats (P ＜ 0.05).CONCLUSION:Wnt/β-catenin signaling pathway may participates in airway remodeling in asthma by increasing the protein expression of c-Myc and cyclin D1,reacting with the P38 MAPK signaling pathway and regulating the growth of ASMC.

Effects of siRNA silencing of inhibitor of apoptosis protein gene on the proliferation of airway smooth muscle in bronchial asthma mice / 重庆医学

Objective To investigate the effects of siRNA silencing of inhibitor of apoptosis protein gene on the proliferation of airway smooth muscle in bronchial asthma mice .Methods Tweenty female BALB/c mice were randomly divided into experimen‐tal group and control group .Mice in the experimental group were constructed asthma models .The trachea and bronchi were collect‐ed for cell culture .Respectively ,the cultured airway smooth muscle cells in the experimental group and the control group were grouped:PBS control group (added to PBS solution) ,missense chain control group (transfected with missense chain) and siRNA transfection group (transfected with siRNA Survivin) .The expressions of Survivin mRNA ,Survivin protein and caspase‐9 protein in airway smooth muscle cells were detected .The levels of IL‐6 and CCL5 in cell supernatant were detected .Results RT‐PCR re‐sults showed that ,the relative expression levels of Survivin RNA of airway smooth muscle cells in the siRNA transfection group of the experimental group were lower than the PBS control group and missense chain group ,the differences were statistically signifi‐cant (P<0 .05) .Western blot results showed that ,the expressions of Survivin proteins in the siRNA transfection group of the ex‐perimental group were lower than the PBS control group and missense chain control group ,while the caspase‐9 protein were higher , In the experimental group ,the synthesis and secretion levels of IL‐6 and CCL5 of airway smooth muscle cells in the siRNA transfec‐tion group were lower than the PBS control group and missense chain control group ,the cell proliferations at 2-4 d in the siRNA transfection group were lower than the PBS control group and missense chain control group ,the proportion of G0/G1 phase in the siRNA transfection group were higher than the PBS control group and missense chain control group ,but the proportion of S/G2 phase were lower than the PBS control group and missense chain control group ,cells apoptosis rates of the siRNA transfection group were lower than the PBS control group and missense chain control group ,the differences were statistically significant (P<0 .05) .Conclusion siRNA specific silencing Survivin gene could accelerate the airway smooth muscle cell apoptosis ,and inhibit cell abnormal proliferation and secretion .

Platelet derived growth factor down-regulated sarcoplasmic reticulum Ca2+atpase to promote the phenotype modulation of human airway smooth muscle cells / 实用医学杂志

Objective To investigate of the effect and mechanisman of SERCA2 on the phenotype modulation of HASMCs. Methods HASMCs were starved for 5 days and divided into different groups ,then we observed morphology change of the cells from the microscope and detected a-actin、SERCA2 and p-ERK by Western Blot,cells proliferation was observed by CCK-8 method. Results Compared with the control group,PDGF could reduce a-actin of HASMCs and increased the cells proliferation ability ,TSG could significantly inhibit the effect (P<0.01), PDGF could also significantly inhibit SERCA2 protein and increased the expression p-ERK (P<0.01), while U0126 significantly inhibited the effect (P < 0.01). Conclusion PDGF may induce HASMCs phenotype modulation through the regulation of SERCA2 and p-ERK.

Research on Detecting Doubling Time of Human and Rat Airway Smooth Muscle Cells with A Real Time Cellular Analyzer / 现代检验医学杂志

Objective To detect the doubling time of airway smooth muscle cells of human and rat by the xCELLigence in-strument,a real time cellular analyzer.Methods The airway smooth muscle cells were separated by collagenase-pancreatin digestion from the rat airway.Then,added to the different holes of E-plate of xCELLigence instrument with the planting destiny of 3 000 cells/well.and so were the human airway smooth muscle cells.The E-plate was then placed on the xCELLi-gence instrument to monitor cell proliferation for 100 hours to calculate the doubling time by using the RTCA Software Package 2.0 software.Results The doubling time of human airway smooth muscle cell calculated by the real time cellular analyzer was 23.96±0.47 h,which was consistent with the data provided by the reference.The doubling time of rat airway smooth muscle cell was 18.62±0.15 h,and the computational process was simple,time-saving and also effective.Conclusion The xCELLigence instrument can be used to calculate doubling time of airway smooth muscle cells of human and rat, which provides experimental methods and reference data for the basic respiratory disease research.

Simultaneous application of BrdU and WST-1 measurements for detection of the proliferation and viability of airway smooth muscle cells

BACKGROUND: BrdU is a commonly used reagent in cell proliferation assays, and WST-1 measurement is widely used to detect cell viability. However, no previous study has formally reported the combination of the two assays, which may be used to detect the proliferation and viability simultaneously. In this study, we examined the effect of adding BrdU 2 h prior to the WST-1 assay and tried to test the possibility of the combined detection using rat airway smooth muscle cells. RESULTS: The WST-1 measurements obtained from the combined detection were consistent with those obtained from the separate detection, which suggested that the addition of BrdU 2 h prior to the WST-1 analysis did not affect the WST-1 results. The BrdU measurements obtained from the combined detection also demonstrated the same trend as that obtained from the separate detection, and dosages of 200, 400 and 800 ng/ml testing reagent significantly inhibited the proliferation of rat airway smooth muscle cells. CONCLUSIONS: Our study suggests that the BrdU and WST-1 measurements can be applied simultaneously without mutual interference, which may increase the efficacy and consistency of these measurements to a certain extent.

Influence of extracellular matrix proteins and PDGF-BB on airway smooth muscle cells / 医用生物力学

Objective To investigate the influence of three extracellular matrix (ECM) proteins, namely, laminin (LN), collagen type I (Col I), fibronectin (FN) on the morphology and contractility changes in airway smooth muscle cells (ASMCs) induced by platelet-derived growth factor-BB (PDGF-BB). Methods ASMCs were seeded on the culture dish coated with LN, Col I, or FN, respectively, and divided into two groups to be cultured either in the absence or presence of PDGF-BB (10 mg/L) for 0~5 d. Subsequently, cell morphology was examined by the optical microscopy and quantified as the ratio of cell width to length, and the KCl/histamine-induced contractile responses of the cell were measured by optical magnetic twisting cytometry (OMTC). Results ASMCs cultured in the presence of PDGF-BB generally appeared in longer and thinner cell shapes, namely, a smaller ratio of cell width to length, but the cell width/length ratio for ASMCs adhered on LN was relatively bigger than that on Col I or FN. In the absence of PDGF-BB, contractility of ASMCs to KCl increased with the duration of culture, which was independent of the ECM proteins. In contrast, in the presence of PDGF-BB, contractility of ASMCs to KCl or histamine decreased in all situations, but degree of the decrease was smaller for ASMCs adhered on LN than those on Col I or FN. Conclusions The morphology and contractility changes in ASMCs induced by PDGF-BB are influenced by ECM proteins on which cells are grown. For ASMCs adhered on LN, the morphology and contractility changes are relatively smaller than those on Col I and FN. The differential effect of ECM proteins on PDGF-BB induced changes in morphology and contractility of ASMCs is important to fully understand the interactions between ECM proteins, inflammatory factors, ASMCs, and their relation to the pathophysiological mechanism of asthma.

Transforming growth factor-1 induces angiogenesis in vitro via VEGF production in human airway smooth muscle cells.

Increase in size and number of bronchial blood vessels as well as hyperaemia are factors that contribute to airway wall remodelling in patients with chronic airway diseases, such as asthma and chronic obstructive pulmonary diseases (COPD). Expression of transforming growth factor 1 (TGF-1), a multifunctional cytokine as well as vascular endothelial growth factor (VEGF), a key angiogenic molecule, has been shown in the inflammed airways in patients with chronic airway diseases. TGF-1 has been implicated in the regulation of extracellular matrix, leading to airway remodelling in patients with chronic airway diseases. However, the role of TGF-1 in regulating VEGF expression in patients with chronic airway diseases, as well as the underlying mechanisms are not yet well established. We investigated whether TGF-1 stimulates VEGF expression in vitro and hence could influence vascular remodelling. Cultured human airway smooth muscle cells (HASMC) were serum deprived for 60 h before incubation with 5ng/ml of TGF-1 for different time points. Control cells received serum-free culture medium. TGF‑1, treatment resulted in time dependent HASMC cell proliferation with maximal values for DNA biosynthesis at 24 h and cell number at 48 h. Northern blot analysis of VEGF mRNA expression showed increased levels in cells treated with TGF-1 for 4 to 8 h. TGF-1 also induced a time-dependent release of VEGF proteins in the conditioned medium after 48 h of treatment. Furthermore, the ability of HASMC-released VEGF proteins to induce human umbilical vein endothelial cells proliferation was inhibited by VEGF receptor antagonist, confirming that TGF-1 induced VEGF was biologically active. We conclude that TGF-1 in addition to an extracellular matrix regulator also could play a key role in bronchial angiogenesis and vascular remodelling via VEGF pathway in asthma.

Effects of platelet-derived growth factor, basic fibroblast growth factor and epidermal growth factor on the proliferation, migration and phenotypic modulation of airway smooth muscle cells / 中华微生物学和免疫学杂志

Objective To investigate the effects of platelet-derived growth factor(POGF),basic fibroblast growth factor(bFGF)and epidermal growth factor(EGF)on the proliferation,migration and phenotypie modulation of airway smooth muscle cells(ASMCs)in vitro.Methods Cultured human ASMCs were stimulated with PDGF.bFGF,EGF or DMEM(control).Cell pmliferation was detected by BrdU incorporation and cell count.Cell migration was measured using Boyden's chamber.Reverse transcription-poly-merase chain reaction(RT-PCR)and Western blot analysis were used for the detection of sm-α-actin and sm-MHC mRNA and protein expression in ASMCs.Resuits The mitotic index(%BrdU-positive per total number of cells)of ASMCs stimulated with PDGF.bFGF and EGF were significantly higher than those in control(P<0.001 or P<0.05).And the cell number of ASMCs stimulated with PDGF,bFGF and EGFwere also significantly higher than those in control(P<0.05).The number of migrating cells treated with PDGF,bFGF and EGF were higher than that in eontrol(P<0.05).The expressions of sm-α-actin mRNA treated with bFGF and EGF were significantly lower than those in control.but it was higher than control when treated with PDGF.The expressions of sm-MHC mRNA treated with bFGF and EGF were significantly lower than those in control,but it was higher than control when treated with PDGF(P<0.01 orP<0.05).Theexpressions of sm-α-actin protein treated with bFGF and EGF were significantly lower than those in control,but it was higher than control when treated with PDGF(P<0.05).The expressions of sm-MHC protein treated with bFGF and EGF were significantly lower than those in control.but it was higher than control when treated with PDGF(P<0.05).Conclusion PDGF,bFGF and EGF can directly induce ASMCs,proliferation and migration in vitro;and meanwhile,the expressions of cells,contractive phenotype increased treated with PDGF and decreased treated with bFGF and EGF.

The role of PDK in regulating cytokine secretion by extracellular matrixes / 中华微生物学和免疫学杂志

Objective To investigate the regulatory effects of extracellular matrixes, including fi-bronectin(FN), and collagen Ⅰ (ColⅠ ) on the immunologic function of human airway smooth muscle cells (HASMCs) passively sensitized with asthmatic serum, and the role of phosphoinosifide 3-kinase (PI3K). Methods Primarily cultured HASMCs were inoculated on the blank plates or on the plates coated with difference matrix proteins, added 10% asthmatic serum to passively sensitized non-asthmatic HASMCs and 10% non-asthmatic serum treated HASMCs as control, cell pretreated with PI3K inhibitor LY294002 for 30 min. The expressions of RANTES, Eotaxin, TGF-β1 mRNA were observed by RT-PCR and RANTES, Eotaxin, TGF-β1 protein in the ceil culture supernatunts was detected by enzyme-linked immtmosorbent assay (ELISA). Results Compared with the control serum group, the expressions of RANTES, Eotaxin, TGF-β1 mRNA of HASMCs and those protein in HASMCs culture supematants were significantly increased in the asthmatic serum group and the control serum + FN group and the control serum + Col Ⅰ group ( P < 0.05 ). The expressions of RANTES, Eotaxin, TGF-β1 mRNA of HASMCs and protein in HASMCs culture superna-tants were significantly increased in the asthmatic serum + FN group and the asthmatic serum + Col Ⅰ group. 50 μmoL/L LY294002 could significantly inhibit the expressions of RANTES, Eotaxin, TGF-β1 mRNA of HASMCs and protein in HASMCs culture supematants(P < 0.05). Conclusion These results suggest ex-tracellular matrixe may regulate immunomodulatory function of HASMCs passively sensitized with asthmatic serum and PI3K signaling pathway may play an important role in the process.

Effect of Cigarette Smoke Extract on the Role of Protein Kinase C in the Proliferation of Passively Sensitized Human Airway Smooth Muscle Cells / 华中科技大学学报（医学）（英德文版）

Summary: To investigate the effect of cigarette smoke extract (CSE) on the role of protein kinase C (PKC) in the proliferation of passively sensitized human airway smooth muscle cells (HASMCs). After synchronization of cultured HASMCs, they were divided into a group A and Group B. The group A was treated with normal human serum and served as controls and the group B was treated with the serum of asthma patients. The group A was further divided into group of A1, A2 and A3 and the group B was sub-divided into the group of B1, B2, B3, B4 and B5. No other agents were added to the group A1 and B1. The cells of group A2 and B2 were stimulated with 5 % CSE for 24 h. HASMCs from group A3 and B3 were treated with PKC agonist PMA (10 nmol/L) and CSE (5 %) for 24 h. PKC inhibitor Ro-31-8220 (5 μmol/L) was added to the HASMCs of group B4 for 24 h. The cells from group B5 were stimulated with Ro-31-8220 (5 μmol/L) and CSE (5 %) for 24 h. The proliferation of HASMCs isolated from group A and B was examined by cell cycle analysis, MTT colorimetric assay and 3H-TdR incorporation test. The expression of PKC-α in each group was observed by Western blotting and RT-PCR, respectively. The results showed that the percentage of S phase, absorbance (A) value, the rate of 3H-TdR incorporation, the ratios of A value of PKC-α mRNA and the A value of PKC-α protein in HASMCs from group B1, B2 and B3 were significantly increased compared to those of group A1, A2 and A3 correspondingly and respectively (P<0.01). The proliferation of HASMCs of group A2 and B2 stimulated with CSE and group A3 and B3 stimulated with CSE and PMA were also significantly enhanced when group A1, A2 and A3 and group B1, B2 and B3 compared to each other (P<0.05, P<0.01, respectively). The percentage of S phase, absorbency (A) value, 3H-TdR incorporation rate, the ratios of A value of PKC-α mRNA and the A value of PKC-α protein in HASMCs from group B4 treated with Ro-31-8220 and group B5 treated with CSE and Ro-31-8220 were significantly decreased as compared to those of group B1 and B2 correspondingly and respectively (P<0.05, P<0.01). It was concluded that CSE can enhance the passively sensitized HASMC proliferation and the expression of PKC alpha. PKC and its alpha subtype may contribute to this process. Our results suggest cigarette may play an important role in ASMCs proliferation of asthma through PKC signal pathway.