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.

Research progress of dopamine and dopamine receptor in pathogenesis of asthma / 中国药理学通报

Asthma is a common respiratory disease charaeterized by airway inflammation and airway hyperresponsiveness.Dopamine is an important neurotransmitter in human body.In reeent years it has been reported that dopamine reeeptor is expressed in immune eells, type I alveolar eells, airway epithelium and airway smooth muscle, and affects ovalbumin ( OVA )-indueed asthma in animals.This review introduees the role of dopamine in the occurrence and development of asthma from the aspeets of airway inflammation, pulmonary nerve regulation, airway epithelial and smooth muscle abnormalities.

Effects of budesonide on proliferation and apoptosis of airway smooth muscle cells in asthmatic rats / 解剖学报

Objective To investigate the effect of budesonide ( BUD) inhalation on the proliferation and apoptosis of airway smooth muscle cells ( ASMCs) in asthmatic rats and its molecular biological mechanism. Methods Totally 40 SD rats were randomly divided into control group, asthma model group, low (0. 25 mg/kg) and high (2 mg/kg) BUD group. The rat asthma model was induced by ovalbumin (VOA) combined with aluminium hydroxide Gel sensitization stimulation. After sensitization, the intervention group inhaled different doses of BUD before stimulation. The related parameters of lung tissue and airway were measured and calculated by medical image analysis system, immunofluorescence was used to detect the expression of type I collagen ( Col I ) and Col III in rat airway smooth muscle ( ASM ), and the protein expressions of Bcl-2, Bax, Caspase-3, phosphorylated ERK 1 and 2(p-ERK 1 / 2), p-p38 MAPK were detected by Western blotting. The proliferation activity of ASMCs was detected by MTT method, and the apoptosis rate of ASMCs was detected by flow cytometry. Results Compared with the control group, airway remodeling occurred in the asthmatic model group, and the airway wall thickness ( WAt/Pbm ), inner wall thickness ( WAi/Pbm ) and smooth muscle thickness ( WAm/Pbm ) increased, compared with the model group, the airway remodeling was inhibited in BUD intervention group, and the tracheal WAt/Pbm, WAi/Pbm and WAm/Pbm decreased in bud treatment group. BUD could decrease the proliferation activity of ASMCs, increase the apoptosis rate of ASMCs, inhibit the expression of Col I and Col III, deregulate the expression of Bcl-2, upregulate the expression of Bax and Caspase-3 ( all P<0. 05), and inhibit the activity of ERK 1/2 and p38 MAPK signal pathway. Conclusion BUD can inhibit the proliferation and the promote apoptosis of ASMCs in asthmatic rats, which may be related to the inhibition of ERK 1/2 and p38 MAPK signal pathways.

Effect of Modified Xiongxiesan on Proliferation of Airway Smooth Muscle Tissues and Expressions of MMP-9 and TIMP1 in CVA Model Rats / 中国实验方剂学杂志

Objective:To explore the effect of modified Xiongxiesan on the proliferation of airway smooth muscle tissues and the expressions of matrix metalloproteinase-9（MMP-9）， tissue inhibitor of metalloproteinase-1 （TIMP-1） in cough variant asthma （CVA） model rats. Method:A total 48 male SD rats were randomly divided into the normal control group （8 rats） and model group （40 rats）. CVA model of rats were established through the intraperitoneal administration with 2 mg ovalbumin （OVA） and 100 mg Al（OH）3， and then aerosol inhalation of 1％ OVA 15 days later. The same volume of sterile saline was given to the normal group through the intraperitoneal injection. Then 40 rats in the modeling group were randomly divided into model group， modified Xiongxiesan group （TCM group， 6 g·kg-1·d-1）， montelukast group （0.4 mg·kg-1·d-1）， chemokine receptor1/2 （CXCR1/2） inhibitor group （G31P group injected subcutaneously via the neck with a dose of 0.5 mg·kg-1 every other day）， and CXCR1/2 inhibitor and modified Xiongxiesan group （G31P+TCM group）， with 8 rats in each group. The control group and the model group were orally given distilled water 10 mL·kg-1·d-1. Then the rats were sacrificed， and lung samples were collected. Histological changes were examined by hematoxylin-eosin（HE）. Basement membrane perimeter （PBM），wall area of bronchial tube （WAt），wall area of bronchial smooth muscle （WAm） and the number of smooth muscle cells （N） were measured using image pro-plus software and standardized based on PBM. The expressions of PCNA， MMP9 and TIMP1 were detected by immunohistochemistry. Result:Compared with the control group， there were a large number of inflammatory cells infiltration and moderate hyperplasia of smooth muscle area in the model group， which however were alleviated in other groups. The expressions of PCNA and MMP-9，TIMP1 were higher in the model group，which were reduced in other groups significantly. Conclusion:Modified Xiongxiesan can reduce the thickness of airway smooth muscle tissue in the CVA model rats， which may be correlated with the inhibition of the CXCR1/2 pathway， thereby reducing the proliferative activity of smooth muscle tissue and inhibiting the expression of related matrix metalloproteinases.

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.

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 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.

Progress in the research of epigenetic regulation of airway smooth muscle cells in asthma / 基础医学与临床

Hyperplasia of smooth muscle cells is a major aspect of airway remodeling in asthma .In recent years.It is found that the epigenetic regulation plays an important role on the proliferation of airway smooth muscle cells and the secretion of inflammatory cytokines , including DNA methyltransferase inhibitors to inhibit the phenotype trans-formation;histone acetylation related with hypertrophy .In addition , the microRNA is potentially related with the regulation of a variety of physiological functions of airway smooth muscle cells of asthma model , including inhibition of proliferation and release of inflammatory factors .Hope that epigenetics can become a new target for the treatment of asthma .

Azithromycin inhibits proliferation of rat airway smooth muscle cells though TRAF6/NF-κB/VEGF signaling pathway / 重庆医学

Objective To explore the mechanism of azithromycin (AZM) for inhibiting the proliferation of rat airway smooth muscle cells (ASMCs).Methods Thirty Sprague-Dawley (SD) rats were divided into the control group,asthma model group and AZM group.The rat model of asthma was established by ovalbumin (OVA) sensitization and stimulation in vitro.The airway related parameters of rat lung tissue were determined by using the medical image analysis system.Primary passage ASMCs were isolated and cultured using the tissue-sticking method,and the vascular endothelial growth factor (VEGF) overexpression vector or tumor necrosis factor receptor-associated factor 6 (TRAF6) overexpression vector was transfected into ASMCs in the AZM group.The protein levels of VEGF,NF-κB p65 and TRAF6 were detected by Western blotting,and the proliferation of ASMCs was evaluated by CCK-8 kit.Results AZM significantly inhibited the increase of thickness of total airway wall,thickness of inner airway wall and thickness of airway smooth muscle layer in asthma rats (P<0.05),also significantly inhibited the proliferation of ASMCs in the asthma model group (P<0.05).AZM significantly inhibited the protein expression of VEGF and NF-κB p65 induced by asthma (P<0.05),and the overexpression of VEGF significantly reduced the inhibiting effects of AZM on proliferation of ASMCs (P<0.05).AZM significantly inhibited the high expression of TRAF6 induced by asthma (P<0.05),and the overexpression of TRAF6 significantly reduced the inhibiting effects of AZM on expression of VEGF and NF-κB p65 as well as proliferation of ASMCs (P<0.05).Conclusion AZM can suppress the proliferation of ASMCs,its partial mechanism may be realized through inhibiting TRAF6/NF-κB/VEGF signaling pathway.

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 .

The effect of neropeptide substance P receptor antagonist can relieve the current of reverse-mode NCX in airway smooth muscle cells / 国际儿科学杂志

Objective Investigate the effect of substance P and receptor antagonist on the current of NCX in ASMC.Methods Primary rat ASMC cultures were established.Immunofluorescence was used to identify ASMCs.Patch clamp exam was used to assess NCX currents in ASMC before and after intervented by Ach,substance P,NK-1R receptor antagonist and nimodiping,Voltage-current curves were drawn according to variation of voltage and current.PA indicates current amplitude,PF indicates cell area,and PA/PF indicates current density.Results As the voltage increased,the current density increased.When voltage up to-40mV the reverse current appeard.When the voltage is 60mV the current density in the Ach intervention group was highest,but lowest in the nimodipine intervention group;the current density in the substance P intervention group is more than in control group but lower than in Ach interevention group.The current density in the substance P receptor antagonist intervention group higher than nimodipine interevention goup,but lower than the other group (P ＜0.05).Conclusion In asthma airways,Ca2 + overload is related to the appearance of reverse-mode NCX.NK-1R antagonist plays a role in decreasing the Ca2 + concentration,which can relieve the current of reverse-mode NCX,and may benefit alleviating airway inflammation and responsiveness.As a result,NK-1R antagonist may be an attractive target for therapeutic approaches to asthma.

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.