The Effect And Mechanism Of Isoliquiritigenin On TGF-β1 Induced Lung Fibroblasts Activation By Activating Autophagy

Background: Idiopathic pulmonary fibrosis(IPF)is a chronic interstitial pulmonary disease with irreversible destruction of lung tissue caused by diffuse inflammation around the alveoli,epithelial cell injury,fibroblast proliferation,and extracellular matrix(ECM)overdeposition.The inflammatory profibrosis factor transforming growth factor-β1(TGF-β1)induces the activation of pulmonary fibroblasts and inhibits the autophagy of them,the insufficient autophagy of fibroblasts is one of the important causes of pulmonary fibrogenesis.TGF-β1 inhibits autophagy by activating its downstream PI3K/AKT/m TOR pathway,which promotes pulmonary fibrogenesis.Isoliquiritigenin(ISL),a natural flavonoid monomer derived from the root of liquorice,has been reported to possess anti-tumor,anti-inflammatory and antioxidant effects.Meanwhile,as a natural autophagy inducer,ISL plays a vital role through activating autophagy by inhibiting the PI3K/AKT/m TOR pathway in the treatment of tumor and other diseases.Previous studies found that ISL acted the anti-fibrosis effect of adipose tissue and renal tissue,and it alleviated mesangial matrix deposition by inhibiting TGF-β1 signaling pathway in anti-renal tissue fibrosis,however its effect and mechanism on pulmonary fibrogenesis has not been demonstrated.Objective: The purpose of this study was to investigate the effect of ISL on TGF-β1 induced activation of human embryonic lung fibroblasts(MRC-5 cells)and to investigate the specific molecular mechanism,so as to search for a new potential drug for the treatment of pulmonary fibrosis and provide a theoretical basis.Methods: MRC-5 cells were induced by TGF-β1 to establish a fibrogenesis model in vitro.The proliferation and migration ability of ISL on TGF-β1 induced MRC-5 cells was detected by MTT assay and wound healing assay,and the expression of related markers of cell differentiation and ECM production were detected by q PCR,Western blot and immunofluorescence.Western blot,immunofluorescence and transfected plasmid of GFP-LC3 into MRC-5 cells were used to detect the effect of ISL on autophagy level in MRC-5 cells treated withTGF-β1.The role of ISL-regulated autophagy in TGF-β1 induced MRC-5 cells activation was examined using autophagy inhibitor 3-Methyladenine(3-MA)and autophagy activator Rapamycin(Rap).Western blot was used to detect the expression of pathway-associated proteins and the specific molecular mechanism of ISL-regulated autophagy in TGF-β1 induced MRC-5 lung fibroblasts activation using PI3K/AKT pathway inhibitor LY294002 and activator insulin-like growth factors-1(IGF-1).Results: The MTT assay showed that ISL inhibited the proliferation of MRC-5cells induced by TGF-β1 in a dose-dependent manner.The scratch test demonstrated that ISL inhibited the migration of MRC-5 cells induced by TGF-β1.q PCR,Western blot and immunofluorescence results revealed that ISL significantly down-regulated the m RNA and protein expression of alpha-smooth muscle actin(α-SMA),collagen type I alpha 1(COLIA1)and fibronectin(FN)in TGF-β1-induced MRC-5 cells.Western blot,immunofluorescence and GFP-LC3 plasmid transfection test showed that ISL upregulated the expression of autophagy related proteins and inhibited the PI3K/AKT/m TOR pathway.Western blot results showed that Rap further enhanced the effect of ISL on reducing cell differentiation and ECM deposition,and 3-MA reversed the effect of ISL on reducing cell differentiation and ECM deposition.After the addition of LY294002 and IGF-1,Western blot analysis proved that ISL inhibited TGF-β1-induced MRC-5 cells activation through activating autophagy via PI3K/AKT/m TOR pathway.Conclusion: ISL can inhibit the proliferation and migration of MRC-5 cells induced by TGF-β1,and reduce the differentiation and ECM deposition of MRC-5cells induced by TGF-β1 through activating autophagy,suggesting that ISL can inhibit the activation of MRC-5 lung fibroblasts induced by TGF-β1.The specific molecular mechanism by which ISL inhibits TGF-β1-induced lung fibroblasts activation is mediated by activating autophagy via inhibiting PI3K/AKT/m TOR pathway.