Isoliquiritigenin Inhibiting Silicosis Fibrosis By Activating Ferroptosis

Background:Silicosis is characterized by an inflammatory and irreversible fibrous lung disease caused by inhalation of silica.Silicosis is the most common and serious occupational disease among mining,construction and manufacturing workers worldwide,and China has the largest number of silicosis patients in the world.Silicosis patients not only their own health and quality of life have been seriously endangered,but also brought a heavy burden to the whole society.Due to the lack of understanding of its specific mechanism,there is currently no effective treatment strategy,therefore,to explore the key mediators and molecular mechanisms of silicosis morbidity,to explore safe and effective drugs to alleviate silicosis pulmonary fibrosis is one of the current research hotspots.Objective:This study aims to explore the effect of isoliquiritin on pulmonary fibrosis and its specific mechanism through in vivo and in vitro experiments,and to provide a new plan for the treatment of pulmonary fibrosis.Methods:This study mainly includes two parts: in the first part,the pulmonary fibrosis model of silicosis mice was established by one-time tracheal dripping of SiO₂.The control group and the ISL group was given an equal volume of normal saline tracheal instillation.The ISL group and the silica dust+ISL group were given ISL intraperitoneal injection the next day,and the control group and silica dust group were given the same amount of normal saline intraperitoneally.Observe the effect of ISL on pulmonary fibrosis induced by SiO₂.The body weight of mice was recorded daily and the general condition of mice was observed.After modeling,mice were sacrificed in batch at 7,14 and 28 days,lung wet weight was weighed to calculate lung coefficient（lung weight/body weight）,and the morphological changes of lung tissue were observed by hematoxylin-eosin（HE）staining.Masson staining was used to evaluate the pulmonary fibrosis of mice.The expression levels of epithelial mesenchymal transformation（EMT）markers（E-Ca,Vimentin,COL I）and ferroptosis related proteins（GPX4）、HO-1 in lung tissues were detected by Western Blot（WB）.The second part: Epithelial-mesenchymal transition（EMT）of alveolar epithelial cells A549 was stimulated by growth transforming factor β1（TGF-β1）to construct cell fibrosis model,ISL dose was screened by MTT,and EMT,ferroptosis and related detection were detected by q PCR and Western Blot.To explore whether ISL affects the occurrence of EMT in A549 cells through ferroptosis.Results:1.In vivo experiments showed that compared with the control group and the ISL group,the mice in the silicon dust group and the silicon dust +ISL group were less mental,and their hair was untidy.The body weight of the mice in the silicon dust +ISL group decreased significantly after modeling.Compared with the control group,the lung coefficient of the mice in the silica dust group and the silica dust+ISL group increased（P<0.05）,and the lung coefficient of the silica dust+ISL group decreased compared with the silica dust group（P<0.05）.The results of HE and Masson staining showed that ISL could alleviate the damage of lung structure caused by SiO₂ and alleviate pulmonary fibrosis.Western Blot results showed that at 28 days,Vimentin and Col I fibrosis markers were increased,epithelial marker E-cadherin were reduced in the lung tissues of mice in the silica dust group compared with those in the control group.The expression of ferroptosis markers GPX4 and HO-1 were increased in the silica dust group,while ISL could reduce the increase of SiO₂-induced fibrosis indexes,increase pithelial marker E-cadherin and decrease the expression of ferroptosis markers GPX4 and HO-1.2.In vitro results showed that ISL inhibited TGF-β1-induced A549 cell proliferation in a dose-dependent manner.After TGF-β1 stimulation,A549 cells transformed from typical polygons of epithelial cells to spindle mesenchymal cells.ISL inhibited the morphological changes of A549 cells after TGF-β1 stimulation.Western Blot results showed that TGF-β1 stimulated A549 cells with decreased expression of epithelial marker E-cadherin and increased expression of stromal marker Vimentin.Western Blot results showed that after A549 cells were stimulated by TGF-β1,the expression of epithelial cell marker E-cadherin decreased,and the expression of mesenchymal cell marker Vimentin increased.ISL combined with TGF-β1 can reverse TGF-β1-induced decreased expression of E-cadherin and increased expression of Vimentin.Western Blot results showed that the expression of ferroptosis marker GPX4 and HO-1 increased after TGF-β1 stimulation of A549 cells.The combination of ISL and TGF-β1 could inhibit the expression of GPX4 and promote the expression of HO-1.The q PCR results showed that the expression of GPX4 HO-1 was increased in TGF-β1group compared with the control group,and the expression of GPX4 was decreased and HO-1 expression was increased after the combination of ISL and TGF-β1.Conclusion:1.ISL can alleviate silicosis pulmonary fibrosis caused by SiO₂;2.ISL can inhibit TGF-β1-induced EMT;3.ISL may inhibit silicosis pulmonary fibrosis by activating ferroptosis and inhibiting EMT through the HO-1/GPX4 signaling pathway.