A Research On Mechanisms For β-elemene To Inhibit The Proliferation Of Primary Human Airway Granulation Fibroblasts

Background and objectives: Benign central airway stenosis is a disease which the major treatments are endoscopic interventions,however many patients are easily to recur.Hyperplastic granulation tissues are the main culprits of this phenomenon.Our previous study proved that a Chinese traditional anti-cancer drug—β-elemene,could effectively inhibit the growth of human airway granulation tissues and granulation fibroblasts,however the mechanism was still unclear.The purpose of this study was to explore the mechanism for β-elemene to slow down the growth of primary human airway granulation fibroblasts,which could provide theoretical basis for seeking the target to treat benign central airway stenosis and developing new drugs for benign central airway stenosis.Methods: After primary human airway fibroblasts and primary human airway granulation fibroblasts were treated with different doses of β-elemene for the same time or primary human airway granulation fibroblasts were treated with the same dose of β-elemene for different times,the morphology of cells was observed and the proliferation rate of cells was measured.After primary human airway granulation fibroblasts were treated with control or β-elemene for the same time,differentially expressed m RNAs and involved pathways were screened out.For primary human airway fibroblasts and primary human airway granulation fibroblasts,the expressions of screened out TGF-β signaling,canonical Wnt signaling,and fibrosis-related genes,were measured.After primary human airway granulation fibroblasts were treated with different doses of β-elemene for the same time,the expressions of TGF-β signaling and fibrosis-related genes,were measured.After primary human airway granulation fibroblasts were treated with the same dose of TGF-β(the activator of TGF-β signaling) for different times or different doses of TGF-β for the same time,the expressions of TGF-β signaling-related genes were measured.After primary human airway granulation fibroblasts were treated with control,β-elemene,SB431542(the inhibitor of TGF-β signaling),β-elemene plus SB431542,and β-elemene plus TGF-β(2.5 ng/ml,24 h)for the same time,the morphology of cells was observed.The proliferation rate of cells and expressions of TGF-β signaling and fibrosis-related genes,were measured.After primary human airway granulation fibroblasts were treated with different doses of β-elemene or DKK-1(the inhibitor of canonical Wnt signaling)for the same time,the expressions of canonical Wnt signaling-related genes were measured.After primary human airway granulation fibroblasts were treated with different doses of Li Cl(the activator of canonical Wnt signaling)for the same time,the expressions of canonical Wnt signaling-related genes were measured.After primary human airway granulation fibroblasts were treated with β-elemene or β-elemene plus 10 m M Li Cl for the same time,the morphology of cells was observed.The proliferation rate of cells and expressions of canonical Wnt signaling and fibrosis-related genes,were measured.Results: β-Elemene had a dose-dependent but no time-dependent inhibitive effect on the growth of primary human airway granulation fibroblasts and did not affect primary human airway fibroblasts.For primary human airway granulation fibroblasts,the differentially expressed genes between control and β-elemene treated cells were closely related to fibrosis,including TGF-β,Smad2,Smad3,Wnt3 a,GSK-3β,β-catenin,α-SMA,and Col-I.These genes were mainly involved in TGF-β signaling and canonical Wnt signaling.Compared to primary human airway fibroblasts,primary human airway granulation fibroblasts had more activated TGF-β signaling,Wnt signaling,and fibrosis-related genes.For primary human airway granulation fibroblasts,β-elemene inhibited the activation of TGF-β signaling and fibrosis-related genes in a dose-dependent manner.2.5 ng/ml TGF-β for 24 hours had the largest promotive effect on TGF-β signaling.The inhibitive effect of SB431542 on cell growth,TGF-β signaling,and fibrosis-related genes was similar to β-elemene.β-elemene plus SB431542 strengthened the inhibitive effect.2.5 ng/ml TGF-β for 24 hours attenuated the inhibitive effect of β-elemene on cell growth,TGF-β signaling,and fibrosis-related genes.For primary human airway granulation fibroblasts,β-elemene inhibited the activation of canonical Wnt signaling in a dose-dependent manner.These effects were similar to DKK-1.10 m M Li Cl had the largest promotive effect on canonical Wnt signaling.10 m M Li Cl attenuated the inhibitive effect of β-elemene on cell growth,canonical Wnt signaling,and fibrosis-related genes.Conclusions: β-Elemene is a safe and effective drug.It only inhibits the growth of primary human airway granulation fibroblasts and does not affect primary human airway fibroblasts.Compare to primary human airway fibroblasts,primary human airway granulation fibroblasts have more activated TGF-β signaling and canonical Wnt signaling.β-Elemene inhibits the proliferation of primary human airway granulation fibroblasts via attenuation of TGF-β signaling and canonical Wnt signaling.These two pathways can be promising targets to treat benign central airway stenosis.