Experimental Study Of Isoliquiritigenin Inhibiting Silicosis Fibrosis By Regulating EMT Via ERK1/2 Pathway

Background: Silicosis is a kind of progressive interstitial lung disease which is caused by long-term inhalation of high concentration of free silicon dioxide（SiO₂）dust during production activities,which is mainly caused by diffuse fibrosis of lung tissue.The epithelial-mesenchymal transition（EMT）induced by transforming growth factor β1（TGF-β1）in alveolar epithelial cells is an important pathogenesis of silicosis.TGF-β1can induce EMT by activating the downstream extracellular regulated protein kinase 1/2（ERK1/2）pathway and promote the development of silicosis fibrosis.Isoliquiritigenin（ISL）is a flavonoid compound extracted from the rhizome of the Chinese herbal medicine licorice,which has anti-inflammatory,anti-oxidant,anti-tumor and anti-heart,liver,kidney and pancreatic fibrosis activities.In addition,the previous studies of this research showed that ISL can inhibit the activation of human embryonic lung fibroblasts by activating autophagy of human embryonic lung fibroblasts,and has potential anti-pulmonary fibrosis effects.However,whether ISL can regulate EMT through the ERK1/2 pathway and inhibit silicosis fibrosis is rarely reported in the literature.Objective:In this study,in vitro cell experiments and in vivo animal experiments were conducted to explore whether ISL can regulate EMT to inhibit pulmonary fibrosis by interfering with the ERK1/2 signaling pathway,and to explore the effect and molecular mechanism of ISL on silicosis fibers.Methods:1.Cell experiment part: A549 cells were cultured in vitro and stimulated with 10ng/m L TGF-β1 to construct an EMT cell model.After ISL intervention,A549 cells were divided into Control group,TGF-β1 group,TGF-β1+ISL group and ISL group.By MTT assay,the suitable concentration of ISL for A549 cell experiment was screened and the effect of ISL on the proliferation ability of A549 cells induced by TGF-β1 was detected.The effect of ISL on the migration ability of TGF-β1 induced A549 cells was detected by cell scratch experiment.The effect of ISL on the morphology of A549 cells induced by TGF-β1 was observed by inverted phase contrast microscope.Real-time fluorescent quantitative PCR（Real-time PCR,RT-PCR）was used to detect the effect of ISL on TGF-β1 induced A549 cells EMT-related epithelial cadherin（E-cadherin）,Vimentin（Vimentin）,α-smooth muscle agonist protein（α-SMA）and fibronectin（FN）and its related transcription factors of Snail,Slug,Twist,ZEB1,ZEB2.Western blot（Western blot,WB）was used to detect the effect of ISL on TGF-β1induced A549 cells EMT-related protein（E-cadherin,N-cadherin,Vimentin,α-SMA,FN）and ERK1/2 pathways on the expression of phosphorylated ERK1/2 protein（p-ERK1/2）.After ERK1/2 pathway inhibitor U0126 was pretreated for 1h,the effect of U0126 on TGF-β1-induced A549 cell morphology was observed by an inverted phase contrast microscope.The effect of U0126 on the expression of E-cadherin,Vimentin,FN induced by TGF-β1 in A549 cells was detected by RT-PCR.The effect of U0126 on the expression of E-cadherin,N-cadherin,Vimentin,α-SMA,FN and p-ERK1/2proteins induced by TGF-β1 in A549 cells was detected by WB.2.Animal experiment part: C57BL/6 mice were raised,and 100 μL of 50 ㎎/m L SIO₂ suspension was instilled through the oropharyngeal trachea to construct a mouse model of silicosis fibrosis in vivo.After the modeling is completed,they are divided into Control group,SIO₂ group,and SIO₂ +ISL group and ISL group.Mice in the ISL intervention group were intraperitoneally injected with a daily dose of 5 ㎎/m L at a dose of 20 ㎎ /kg for 28 days after a comprehensive analysis of the concentration recommended in the relevant references and the preliminary animal experiment results.During the experiment,general conditions such as diet,activity,respiration and body weight of the mice were observed and recorded.Mice were dissected on the 7,14,and28 days of intraperitoneal injection and lung tissue specimens were collected.Lung tissue morphology of mice was observed,lung weight of mice was measured and lung coefficient of mice were calculated.The inflammation and collagen deposition of mouse’s lungs tissue were observed by HE and MASSON staining.WB experiment was used to detect the expression levels of collagen I（COl-1）,E-cadherin,N-cadherin,Vimentin,α-SMA,FN and p-ERK1/2 protein.Results:1.Cell experiment part: Compared with the Control group,A549 cells stimulated by TGF-β1 transformed from epithelial cells to fibroblasts,with enhanced cell proliferation and migration(（P<0.05）.The EMT-related epithelial cell markers expression level of E-cadherin decreased,and the interstitial cell markers expression levels of N-cadherin,Vimentin,α-SMA,and FN increased(（P<0.05）.The expression level of EMT-related transcription factors Snail,Slug,Twist,ZEB1 ZEB2 and p-ERK1/2 in ERK1/2 signaling pathways increased(（P<0.05）.After intervention with ISL,ISL can inhibit the proliferation and migration of A549 cells induced by TGF-β1(（P<0.05）,and prevent the transformation of A549 cells induced by TGF-β1 from epithelial phenotype cells to mesenchymal cells.ISL can increase the expression level of E-cadherin,reduce the expression levels of N-cadherin,Vimentin,α-SMA,FN(（P<0.05）.ISL can reduce the expression levels of EMT-related transcription factors and p-ERK1/2(（P<0.05）.After intervening TGF-β1-induced A549 cells with ERK1/2pathway inhibitor U0126,U0126 also prevented TGF-β1-induced A549 cell morphologic transformation from epithelial cells to fibroblasts,and up-regulated the expression of E-cadherin,down-regulated the expression of N-cadherin,Vimentin,α-SMA and FN(（P<0.05）.2.Animal experiment part: Compared with the Control group,mice stimulated by SiO₂ for 7,14 and 28 days showed loss of appetite,decreased activity,increased respiratory rate and weight loss after modeling（P<0.05）.After 1 week,the above situation gradually improved.And compared with the Control group,the surface of the lung tissue of the mice stimulated by SIO₂ was not smooth with obvious white plaque formation,and the lung weight and lung index were significantly increased（P<0.05）.The lung tissue structure was damaged and appeared obvious inflammatory cell infiltration and collagen deposition.The expression levels of EMT markers E-cadherin was reduced,COl-1,N-cadherin,Vimentin,α-SMA,FN and p-ERK1/2 in the ERK1/2pathway were increased（P<0.05）.After ISL treatment,the abnormal conditions of the mice’s appetite,activity,breathing,weight,lung tissue morphology,etc.were significantly improved and lung weight and lung index were decreased（P<0.05）.The inflammation and fibrosis of lung tissue were significantly reduced.The expression levels of E-cadherin was increased,COl-1,N-cadherin,Vimentin,α-SMA,FN and p-ERK1/2 protein were decreased（P<0.05）.Conclusion:1.ISL can inhibit the EMT of A549 cells induced by TGF-β1 through the ERK1/2 pathway;2.ISL can inhibit SiO₂-induced EMT in mice through the ERK1/2 pathway,improve lung tissue damage in mice,reduce lung tissue inflammation and collagen deposition in mice and has an anti-silicosis fibrosis effect.