Regulation Of TGF-β1-induced Transformation Of Lung Fibroblasts To Myofibroblasts By Irisin And Its Mechanism

ObjectiveAsthma is an allergic disease characterized by airway inflammation,airway obstruction and excessive mucus production involving multiple inflammatory cells and cellular components.Repeated chronic inflammation predisposes asthmatics to airway remodeling.Airway remodeling is a key pathological change in the development of bronchial asthma and is one of the important factors in its recurrent attacks and persistence.During the formation of airway remodeling,fibroblast-to-myofibroblast transition(FMT)is a key step in the process of airway remodeling under the effect of various inducing factors.The differentiated myofibroblasts produce large amounts of α-smooth muscle actin(α-SMA)and extracellular matrix(ECM),such as fibronectin and collagen Ⅰ,III and IV.Irisin is a small-molecule peptide hormone that is secreted by skeletal muscle and other tissues after exercise and runs with the blood circulation to all parts of the body.Studies have shown that irisin can exert powerful anti-inflammatory,anti-oxidative stress,and anti-apoptotic effects in a variety of diseases.Also,there are many studies showing that irisin can play a potentially protective role in fibrosis of the heart,kidney,liver and pancreas.Similar to airway remodeling in asthma,a key step in fibrotic diseases is the differentiation of fibroblasts to myofibroblasts.However,no reports on the inhibition of fibroblast to myofibroblast differentiation by irisin in lung tissue have been seen yet.In this experiment,we cultured human embryonic lung fibroblasts(HFL-1)with transforming growth factor-β1(TGF-β1)in vitro to investigate whether irisin could play a role in the phenotypic transformation of HFL-1 cells,and to explore the possible molecular mechanisms involved for the treatment of asthma airway remodeling.To find new possible drugs and provide a theoretical basis for the treatment of asthma airway remodeling.MethodsHuman embryonic lung fibroblasts HFL-1 were cultured in vitro,and HFL-1cells were induced with 10ng/ml TGF-β1,while the cells were intervened with different concentrations of irisin(25,50,100,200)ng/ml for 24,48 and 72 hours.The cell proliferation rate was detected using CCK8 kit.Subsequently,the cells were divided into blank control group(NC group),TGF-β1 group(10ng/ml),and TGF-β1+irisin group(10ng/ml TGF-β1 and 100ng/ml irisin).The migration ability of cells in different groups was detected by cell scratching assay.The expression ofα-SMA in cells of different treatment groups was detected by immunofluorescence assay.Protein expression levels of α-SMA,collagen Ⅰ,Smad2,p-Smad2,Smad3,p-Smad3 in cells of different treatment groups were detected by Western blot assay.Results(1)The results of CCK8 experiment showed that the proliferation rate of cells in the TGF-β1 group increased significantly(P<0.05)compared with the NC group for the same duration of action;the proliferation rate of cells in each group of irisin +TGF-β1 decreased gradually(P<0.05)with the increase of irisin drug concentration compared with the TGF-β1 group.The inhibitory effect of irisin on the proliferation of HFL-1 cells was most significant when the concentration of irisin was 100ng/ml.Under the same drug concentration,the proliferation inhibition effect of irisin gradually increased with time after co-cultivation of HFL-1 cells with 100ng/ml of irisin and HFL-1 cells for 24,48 and 72 h under the induction of TGF-β1.48 h was chosen as the follow-up experiment time in combination with the cell growth status.(2)The results of cell scratching assay showed that TGF-β1 could effectively promote the migration of HFL-1 cells compared with NC group(P<0.01);compared with TGF-β1 group,the migration of HFL-1 cells was significantly inhibited by the intervention of irisin(P<0.05).(3)Immunofluorescence results showed that the fluorescence intensity ofα-SMA was significantly higher in the TGF-β1 group compared with the NC group(P<0.001);the fluorescence intensity of α-SMA in HFL-1 cells decreased under the intervention of irisin compared with the TGF-β1 group(P<0.01).(4)Western blot results showed that compared with the NC group,the expression levels of a-SMA and collagen Ⅰ proteins were increased in the TGF-β1group(P<0.001),and the expression levels of p-Smad2 and p-Smad3 were also increased(P<0.001);compared with the TGF-β1 group,under the intervention of irisin,the expression levels of HFL-1 cells,the protein expression levels of a-SMA and collagen Ⅰ were decreased(P<0.05),and the expression levels of p-Smad2 and p-Smad3 were also decreased(P<0.01).ConclusionIrisin inhibits TGF-β1-induced proliferation,migration and conversion of HFL-1cells to myofibroblasts,and the mechanism of its action may be related to the TGF-β/Smad2/3 signaling pathway.