Tiotropium Can Inhibit Methacholine-induced Extracellular Matrix Production By Airway Smooth Muscle Cells

Backgrounds:Airway remodeling is one of the most important features of asthma,which contributes to airway hyperresponsiveness and airflow limitation.Studies have shown that airway smooth muscle cells contribute to airway rmodeling through cell proliferation and producing and releasing various inflammatory mediators,growth factors,and ECM proteins.Thomson thinks that ECM proteins produced by airway smooth muscle cells play a crucial role in airway remodeling.Besides,corticosteroids can only control asthma symptoms and have no beneficial effect on airway rmodeling.In generally,to investigate the mechanism of the production of ECM proteins by airway smooth muscle cells is necessary.The ECM is an intricate structure of macromolecules produced by various mesenchymal cells in the airways,including fibroblasts and airway smooth muscle cells.It acts as mechanical support to maintain the airway function.Both the quantity and the composition of the ECM are altered in the airways of chronic asthmatics.For example,deposition of collagen ?,?,and ?,fibronectin,tenascin,hyaluran,versican,and laminin are increased,whereas other components like collagen ?,elastin and decorin are decreased.ACh has been regarded as the primary parasympathetic neurotransmitter which is associated with the regulation of bronchoconstriction and mucus secretion for centuries.ACh is synthesized by different cell types including neurons,epithelial cells,airway fibroblasts and airway smooth muscle cells.Resently,increasing evidence indicates that ACh(either neuronal or nonneuronal)may also regulate airway inflammation and airway remodeling,which might contribute to the new therapeutic effectiveness of muscarinic receptor antagonists.These findings suggest that anticholinergics can not only relieve airway obstruction but also control airway inflammation and airway remodeling.?-catenin is a member of the Armadillo family of proteins which is associated with the cadherin/catenin complexes at adherens junctions and stabilizes cell-cell contacts.In addition,?-catenin regulates T-cell factor(TCF)/Lymphoid enhancer factor(LEF)-mediated gene transcription in the Wnt signaling pathway.Accumulating evidence indicates that activation of ?-catenin signaling is associated with various fibroproliferative diseases such as Idiopathic pulmonary fibrosis(IPF).Furthermore,a recent study shows that ?-catenin is required for TGF-?1-induced extracellular matrix production by airway smooth muscle cells.The present data adds to the increasing evidence indicating the importance of tiotropium in inhibiting Mch-driven airway wall remodeling.Besides,?-catenin signaling may be of importance in Mch-induced ECM production.Therefore,targeting ?-catenin-dependent gene transcription may hold promise as a new therapeutic intervention.Objective:1.To primarily culture human airway smooth muscle cells(HASMC),observe the characteristics of HASMC in vitro,and identify the cultured cells into smooth muscle cells.2.To investigate the role of mathecholine in producing extracellular matrix by airway smooth muscle cells and the effect of tiotropium on this process.3.To investigate the role of Wnt/?-catenin in producing extracellular matrix by airway smooth muscle cells.Method:Part One:Primary culture and identification of airway smooth muscle cells1.Primary culture:take the surgical specimens of lobectomy surgery patients,separate the middle-lever of smooth muscle layer of the leave/segmental bronchi under sterile conditions.Cut the tissue into about 1mm3 size and affixe them to the bottom of culture flasks with an interval of about 0.5cm.Add 20%fetal bovine serum medium into the culture flask and culture at 37?,5%CO2 in incubator.2.Subculture:When the cells climb out of the tissue block and grow to fusion,digeste the cells with 0.25%trypsin,passage them into 25cm2 flasks at a proportion of 1:1-1:3 and culture with medium containing 10%fetal bovine serum.3.Cell identification:cells were identified by immunofluorescence with specific staining of smooth muscle-specific alpha actin(a-SMA).4.Subsequent processing:3-6 generations logarithmic growth phase cells were taken to seed into Petri dishes at a density of 1×104 cell/cm2 for subsequent experiments.Part Two:Methacholine induces extracellular matrix production by airway smooth muscle cells1.Cell preparation:Cells were seeded into 35mm/60mm Petri dishes by the method above.Subsequent tests could be conducted when the 70-80%area of the dish bottom were grown with cells.2.Concentration gradient:To investigate the proper concentration of methacholine on ECM production,we stimulate cells with increasing concentration from 0.1ng/ml to 10ng/ml for 24h and then detect the expression of collagen ? ?1 protein.3.Time gradient:To investigate the proper time point of methacholine on ECM production,we stimulate cells with different time points up to 48h and then detect the expression of collagen ? ?1 protein.4.Groups:Cells were randomly divided into control group and Mch group.Detect the expression of ECM mRNA.5.Expression of collagen ? ?1 protein:The cells were seeded on 60mm culture dishes and total cellular protein was extracted.Collagen ? ?1 protein level was detected by Western Blot.Equal protein loading was verified by the analysis of GAPDH.Collagen ? ?1 deposition was quantified by densitometry and normalized to GAPDH expression.6.Expression of different ECM mRNA:The cells were seeded on 35mm culture dishes and total cellular RNA was extracted and reverse transcribed into cDNA.Different ECM mRNA levels were evaluated by quantitative RT-PCR(qRT-PCR)analysis.The CT value of each group were read and the mRNA relative expression levels were calculated as 2-??CT.7.Statistical analysis:Data were expressed as(x±s).Comparisons between groups were analyzed with one-way analysis of variance(ANOVA),and multiple comparisons were taken by q test when variances equal.Besides,welch method was used for comparisons between groups and Dunnett T3 method for multiple comparisons when variances unequal.The experimental data was analyzed by the software of SPSS 13.0.Differences were considered statistically significant at P<0.05.Part Three:Tiotropium inhibits Mch-induced extracellular matrix production by ASMC1.Cell preparation:Cells were seeded into 35mm/60mm Petri dishes by the method above.Total cellular protein was extracted and total cellular RNA was extracted when the 70-80%area of the dish bottom were grown with cells.2.Groups:Cells were randomly divided into control group,Mch droup,tiotropium group and(Mch+Tiotropium)group.Tiotropium was added 30min before the addition of Mch.3.Expression of collagen ? ?1 protein:The cells were seeded on 60mm culture dishes and total cellular protein was extracted.Collagen ? a1 protein level was detected by Western Blot.Equal protein loading was verified by the analysis of GAPDH.Collagen ? ?1 deposition was quantified by densitometryand normalized to GAPDH expression.4.Expression of different ECM mRNA:The cells were seeded on 35mm culture dishes and total cellular RNA was extracted and reverse transcribed into cDNA.Different ECM mRNA levels were evaluated by quantitative RT-PCR(qRT-PCR)analysis.The CT value of each group were read and the mRNA relative expression levels were calculated as 2-??CT.5.Statistical analysis:Data were expressed as(x±s).Comparisons between groups were analyzed with one-way analysis of variance(ANOVA),and multiple comparisons were taken by q test when variances equal.Besides,welch method was used for comparisons between groups and Dunnett T3 method for multiple comparisons when variances unequal.The experimental data was analyzed by the software of SPSS 13.0.Differences were considered statistically significant at P<0.05.Part Four:The role of Wnt/?-catenin signaling in Mch-induced extracellular matrix production by airway smooth muscle cells1.Cell preparation:Cells were seeded into 35mm/60mm Petri dishes by the method above.Total cellular protein was extracted and total cellular RNA was extracted when the 70-80%area of the dish bottom were grown with cells.2.Expression of total ?-catenin protein:We stimulate cells with increasing concentration of methacholine from 0.1ng/ml to 10ng/ml for 24h.The cells were seeded on 60mm culture dishes and total cellular protein was extracted.Total?-catenin protein level was detected by Western Blot.Equal protein loading was verified by theanalysis of ?-actin.total ?-catenin expression was quantified by densitometryand normalized to ?-actin expression.3.Expression of phosphorylated GSK3?:We stimulate cells with Mch(1ng/ml)for up to 2h.The cells were seeded on 60mm culture dishes and total cellular protein was extracted.Phosphorylated GSK3? protein level was detected by Western Blot.GSK3? activity level was calculated as the ratio of p-GSK3? and GSK3(?+?)bands gray value.4.Expression of active ?-catenin protein:We stimulate cells with Mch(1ng/ml)for up to 48h.The cells were seeded on 60mm culture dishes and total cellular protein was extracted.Active ?-catenin protein level was detected by Western Blot.Equal protein loading was verified by the analysis of ?-actin.Active ?-catenin expression was quantified by densitometryand normalized to ?-actin expression.5.Groups:Cells were randomly divided into control group,Mch group,PKF115-584 group and(Mch+PKF115-584)group.6.Expression of collagen ? ?1 protein:The cells were seeded on 60mm culture dishes and total cellular protein was extracted.Collagen ? ?1 protein level was detected by Western Blot.Equal protein loading was verified by the analysis of GAPDH.Collagen ? ?1 deposition was quantified by densitometry and normalized to GAPDH expression.7.Expression of different ECM mRNA:The cells were seeded on 35mm culture dishes and total cellular RNA was extracted and reverse transcribed into cDNA.Different ECM mRNA levels were evaluated by quantitative RT-PCR(qRT-PCR)analysis.The CT value of each group were read and the mRNA relative expression were calculated as 2-??CT.8.Statistical analysis:Data were expressed as(x±s).Comparisons between groups were analyzed with one-way analysis of variance(ANOVA),and multiple comparisons were taken by q test when variances equal.Besides,welch method was used for comparisons between groups and Dunnett T3 method for multiple comparisons when variances unequal.The experimental data was analyzed by the software of SPSS 13.0.Differences were considered statistically significant at P<0.05.Results:Part One:Primary culture and identification of Airway smooth muscle cells1.After 2 or 3 weeks,smooth muscle cells began to climb out of the tissue affixed to the bottom of the fasks.Observed under ordinary light microscope,smooth muscle cells grow by static adherence,the shape of cells was fusiform,the oval nucleus located in the center of the cells,showing one or more nucleoli.2.About one week later,the cells out of the adjacent tissues began to grow to confluens.Cells adherent 12 hours after being passaged,grow to confluens 3 or 4 days later and arranged in the law which shew a typical "peak and valley sign".3.Immunofluorescence staining shows that about 95%of cells positively expressed ?-SMA.4.Cells were seeded with a density of 1×104 cells/cm2 in Petri dishes and 70-80%area of the bottom was covered by cells after 3 days.Part Two:Methacholine induces extracellular matrix production by airwaysmooth muscle cells1.Sterimulated with increasing concentration of Mch from 0.1ng/ml to 10ng/ml for 24h.The collagen ? ?1 protein expression of control,0.1ng/ml,1ng/ml and 10ng/ml are:0.255±0.009,0.383±0.006,0.4123±0.012,0.401±0.004;The collage n ??1 protein expression of the groups with different concentrations of Mch are higher than that of control group,the difference is statistically significant(P<0.001).The collagen ? ?1 protein expression of lng/ml is the highest.2.Sterimulated with different time points up to 48h,the collagen ? ?1 protein expression of control,2h,4h,6h,16h,24h,48h are:0.372±0.090,0.512±0.070,0.537±0.010,0.529±0.011,0.550±0.015,0.561±0.002,0.443±0.066.Compared with control group,Mch can introduce collagen ? ?1 protein expression from 2h to 48h,the difference is statistically significant(P<0.001).3.Stimulated with Mch(1ng/ml)after 24h,the mRNA expression of control,collagen ? ?1,fibronectin,versican,laminin and decorin are:1.000±0.010,2.540±0.177,1.780±0.125,2.343±0.190,1.357±0.068,0.743±0.103.Comparedwith control group,the mRNA expression of collagen ? ?1(P<0.001),fibronectin(P<0.001),versican(P<0.001),laminin(P<0.01)are higher while the mRNA expression of decorin is lower.Part Three:Tiotropium inhibits Mch-induced extracellular matrix production by ASMC1.The collagen ? ?1 protein expression of control group,Mch group,tiotropium group and(Mch+tiotropium)group are:0.135±0.038,0.308±0.039,0.157±0.044,0.146±0.048.Compared with control group,the expression of collagen ? ?1 protein of Mch group is higher,the difference is statistically significant(P<0.001)and that of(Mch+tiotropium)group is lower than Mch group,the difference is statistically significant(P<0.01).2.The mRNA expression of different ECM protein(1)The mRNA expression of collagen ? ?1 of control group,Mch group,tiotropium group and(Mch+tiotropium)group are:1.000±0.305,2.520±0.375,1.220±0.195,1.333±0.215.Compared with control group,the mRNA expression of collagen ? ?1 of Mch group is higher,the difference is statistically significant(P<0.001)and that of(Mch+tiotropium)group is lower than Mch group,the difference is statistically significant(P<0.001).(2)The mRNA expression of fibronectin of control group,Mch group,tiotropium group and(Mch+tiotropium)group are:1.000±0.215,2.157±0.281,1.193±0.146,1.270±0.1913.Compared with control group,the mRNA expression of fibronectin of Mch group is higher,the difference is statistically significant(P<0.001)and that of(Mch+tiotropium)group is lower than Mch group,the difference is statistically significant(P<0.001).(3)The mRNA expression of versican of control group,Mch group,tiotropium group and(Mch+tiotropium)group are:1.000±0.213,2.240±0.305,1.230±0.178,1.277±0.260.Compared with control group,the mRNA expression of versican of Mch group is higher,the difference is statistically significant(P<0.001)and that of(Mch+tiotropium)group is lower than Mch group,the difference is statistically significant(P<0.001).Part Four:The role of Wnt/?-catenin signaling in Mch-induced extracellular matrix production by airway smooth muscle cells1.The expression of total ?-catenin protein of control,0.1ng/ml,1ng/ml and10ng/ml are:0.229±0.044,0.357±0.051,0.385±0.065,0.352±0.065.The total ?-catenin protein expression of the groups with different concentrations of Mch are higher than that of control group,the difference is statistically significant(P<0.05).The collagen ? ?1 protein expression of lng/ml is the highest.2.The expression of phosphorylated GSK3? of control,5min,15min,30min,60min and 120min are:0.729±0.112,1.138±0.111,1.157±0.117,1.188±0.109,1.233±0.140,1.380±0.075.Mch can phosphorylate GSK3? from 5-120min,the difference is statistically significant(P<0.01,P<0.01,P<0.001,P<0.001,P<0.001 respectively)compared to control.3.The expression of active ?-catenin protein of control,2h,4h,6h,16h,24h,48h are:0.279±0.061,0.307±0.044,0.318±0.024,0.365±0.053,0.442±0.053,0.457±0.476,0.418±0.030.Mch can induce the expression of active ?-catenin protein from 6h-48h,the difference is statistically significant(P<0.05,P<0.01,P<0.001,P<0.01 respectively)compared to control.4.The expression of collagen ? ?1 protein of control,Mch group,PKF115-584 group and(Mch+PKF115-584)group are:0.551 ±0.057,0.775±0.075,0.573±0.063,0.614±0.111.PKF115-584 can inhibit Mch-induced collagen ? ?1 protein expression,the difference is statistically significant(P<0.01).5.The mRNA expression of different ECM proteins(1)The mRNA expression of collagen ? ?1 of control group,Mch group,PKF 115-584 group and(Mch+PKF115-584)group are:1.000±0.105,2.453±0.228,1.153±0.146,1.367±0.166.Compared with control group,the mRNA expression of collagen ? ?1 of Mch group is higher,the difference is statistically significant(P<0.001)and that of(Mch+PKF115-584)group is lower than Mch group,the difference is statistically significant(P<0.001).(2)The mRNA expression of fibronectin of control group,Mch group,PKF115-584 group and(Mch+PKF115-584)group are:1.000±0.105,2.157±0.181,1.193±0.143,1.257±0.160.Compared with control group,the mRNA expression of collagen ? ?1 of Mch group is higher,the difference is statistically significant(P<0.001)and that of(Mch+PKF115-584)group is lower than Mch group,the difference is statistically significant(P<0.001).(3)The mRNA expression of versican of control group,Mch group,PKF115-584 group and(Mch+PKF115-584)group are:1.000±0.305,2.273±0.256,1.200±0.122,1.307±0.215.Compared with control group,the mRNA expression of collagen ? ?1 of Mch group is higher,the difference is statistically significant(P<0.001)and that of(Mch+PKF115-584)group is lower than Mch group,the difference is statistically significant(P<0.001).Conclusions:1.The period of HASMC primary culture by tissue adherence method is about one month.1×104cells/cm2 for petri dish is appropriate seeding density,which can get the required cell amount for subsequent experiments 24 hours after seeding.The cultured cells express smooth muscle-specific ?-actin.The percentage of smooth muscle cells can reach to about 95%.2.Methacholine can induce ECM proteins expression by airway smooth muscle cells.The best concentration is lng/ml and 24 hours is the appropriate time point.3.Tiotropium can inhibit methacholine-induced ECM proteins expression by airway smooth muscle cells.4.Wnt/?-catening plays a crucial role in methacholine-induced ECM proteins expression by airway smooth muscle cells,which can be inhibited by pharmacological inhibition PKF115-584.