The Role And Mechanism Of Eupatilin In Idiopathic Pulmonary Fibrosis By Regulating Autophagy

Objective:The aim of this study is to investigate the role of Eupatilin in idiopathic pulmonary fibrosis(IPF)by constructing pulmonary fibrosis models in vitro and in vivo,and preli minarily explore its effect and possible mechanism on autophagy,providing new therapeutic targets and strategies for clinical intervention of pulmonary fibrosis.Methods:1.Human embryonic lung fibroblast MRC-5 was treated with different concentration of Eupatilin,CCK-8 method was used to detect the influence of Eupatilin on MRC-5 cell viability.2.Wound healing was used to detect the effect of Eupatilin on proliferation and migration of differentiated MRC-5 cells induced by transfor ming growth factor-β1(TGF-β1).Eupatilin treated differentiated MRC-5 cells induced by TGF-β1 and normal lung fibroblasts from IPF patients,Western blot,q PCR and immunofluorescence assay was used to detect the expression of Fibronectin,Collagen1A1(Col1A1)and alphasmooth muscle actin(α-SMA).3.Eupatilin treated differentiated MRC-5 cells induced by TGF-β1and normal lung fibroblasts from IPF patients,Western blot was used to detect the expression of microtubule-associated protein 1 light chain 3B(LC3B)and SQSTM1/ p62,and transmission electron microscope(TEM)was used to observe the autophagosomes.The autophagy inhibitor 3-MA pretreated differentiated MRC-5 cells induced by TGF-β1 and lung fibroblasts from IPF patients and then treated with Eupatilin for 24 hours.The expression of LC3 B,Fibronectin and α-SMA protein were detected by Western blot.4.6-8-week-old healthy male C57BL/6 mice were randomly divided into three groups: control group(n = 15,saline group),bleomycin treated group(n = 15,BLM+vehicle group)and bleomycin plus Eupatilin treated group(n = 15,BLM+Eupatilin group).After 28 days,protein of lung tissue was extracted and paraffin-embedded sections of lung tissue were prepared.HE staining and Masson staining were used to observe the alveolitis and the degree of pulmonary fibrosis,ELISA was used to detect the content of hydroxyproline in lung tissues,and immunohistochemistry was used to detect the staining of Fibronectin and α-SMA in lung tissues.The protein expressions of Fibronectin,α-SMA,LC3 B and SQSTM1/p62 were detected by Western blot.5.Eupatilin treated differentiated MRC-5 cells induced by TGF-β1and normal lung fibroblasts from IPF patients,the phosphorylation levels of PI3 K,Akt and m TOR were detected by Western blot;SC79 pretreated differentiated MRC-5 cells induced by TGF-β1 and lung fibroblasts from IPF patients and treated with Eupatilin for 24 h.The phosphorylation levels of Akt and m TOR,the expression of LC3 B,Fibronectin,Col1A1 and α-SMA protein were detected by Western blot.6.MRC-5 cells were cultured with TGF-β1 and incubated for 0,3,6,9,12 and 24 hours;MRC-5 cells were treated with 0(control),1,2,5 and10 ng/ml TGF-β1 and incubated for 24 hours respectively.Sestrin2 protein expression was detected by Western blot.Eupatilin treated differentiated MRC-5 cells induced by TGF-β1,the expression of Sestrin2 protein was detected by Western blot.IHC was used to detect Sestrin2 staining in bleomycin-induced pulmonary fibrosis mice.7.Differentiated MRC-5 cells induced by TGF-β1 were transfected with Sestrin2 si RNA and treated by Eupatilin for 24 hours.The phosphorylation levels of PI3 K,Akt and m TOR,and the expression of Fibronectin,α-SMA and LC3 B were detected by Western blot.Results:1.The cell viability of MRC-5 cells was not significantly affected by Eupatilin in the range of 0-100μM,but significantly decreased when the concentration was 150μM and 200μM(p < 0.05).2.Eupatilin significantly inhibited the increased proliferation and migration of MRC-5 cells induced by TGF-β1.Eupatilin significantly decreased the increased protein and m RNA expression levels of Col1A1,Fibronectin and α-SMA induced by TGF-β1(p < 0.05),and decreased the fluorescence intensity of Col1A1,Fibronectin and α-SMA.Eupatilin significantly reduced the expression levels of Col1A1,Fibronectin and α-SMA protein and m RNA in lung fibroblasts from IPF patients(p < 0.05).3.Eupatilin significantly increased the decreased ratio of LC3B-II/I and decreased the increased expression of SQSTM1/p62 in MRC-5 cells induced by TGF-β1(p<0.05).Eupatilin significantly increased the ratio of LC3B-II/I and decreased the expression of SQSTM1/p62 in IPF lung fibroblasts(p<0.05).Eupatilin increased the number of autophagosomes in MRC-5 cells and IPF lung fibroblasts.The LC3B-II/I ratio was significantly decreased after 3-MA treatment,and 3-MA significantly increased the decreased expression of Fibronectin and α-SMA(p < 0.05).4.HE and Masson staining showed that Eupatilin significantly reduced the degree of bleomycin-induced alveolitis and pulmonary fibrosis.Eupatilin significantly reduced the content of hydroxyproline in the lung tissues of bleomycin pulmonary fibrosis mice.Immunohistochemical(IHC)staining showed that Eupatilin significantly reduced the positive staining of Fibronectin and α-SMA in bleomycin pulmonary fibrosis mice.Western blot showed that Eupatilin significantly decreased the expression of Fibronectin and α-SMA protein,increased the ratio of LC3 B II/I,and decreased the expression of SQSTM1/p62 in bleomycin pulmonary fibrosis mice(p < 0.05).5.Eupatilin significantly decreased the increased phosphorylation levels of PI3 K,Akt and m TOR in MRC-5 cells induced by TGF-β1 and decreased phosphorylation levels of PI3 K,Akt and m TOR in IPF lung fibroblasts(p < 0.05).SC79 increased the decreased phosphorylation levels of Akt and m TOR reduced by Eupatilin,reduced the ratio of LC3 B II/I and increased the expression of Fibronectin,Col1A1 and a-SMA.6.When MRC-5 cells were treated with TGF-β1 for different time,Sestrin2 expression was most significantly decreased at 24 h.Different concentrations of TGF-β1 significantly reduced the expression of Sestrin2,and the Sestrin2 expression was most significantly decreased when TGF-β1 concentration was 2 and 5ng/ml.Eupatilin significantly increased the decreased expression of Sestrin2 induced by TGF-β1.IHC showed that Eupatilin significantly increased Sestrin2 staining in bleomycin pulmonary fibrosis mice.7.Silencing Sestrin2 significantly increased phosphorylation levels of PI3 K,Akt and m TOR,reduced the ratio of LC3 B II/I and increased the expression of Fibronectin and α-SMA in MRC-5 cells.Conclusion:Eupatilin induces autophagy to inhibit the differentiation of lung fibroblasts and the degree of pulmonary fibrosis in bleomycin-induced pulmonary fibrosis mice via activating Sestrin2-dependent PI3K/Akt/m TOR signaling pathway.