The Target And Mechanism Of Hydroxysafflor Yellow A To Inhibit TGF-β1-induced Activation Of Lung Fibroblasts

Objective Lung fibroblasts are the main cellular components in the connective tissue of the lung,which are involved in maintaining the normal morphology of the lung and repairing the injured tissues in time.Fibroblasts are involved in the process of pathogenesis of idiopathic pulmonary fibrosis(IPF)through abnormally proliferation,differentiation and extracellular matrix secretion.Transforming growth factor-β1(TGF-β1)is an effective factor to induce the change of fibroblast function,and it is also one of the most critical cytokines leading to pulmonary fibrosis.Therefore,it is expected to be a new method for the treatment of pulmonary fibrosis by interfering the function changes of TGF-β1 mediated lung fibroblasts.Hydroxysafflor yellow A(HSYA)is the main active water-soluble ingredient in safflor yellow(SY),the main active part of Carthamus tinctorius L.,isolated from the flowers of traditional Chinese medicine Carthamus tinctorius L..HSYA has been shown to have many pharmacological properties,such as anti-inflammation,antioxidant,antiplatelet aggregation and cardiovascular protection.Several studies have reported that HSYA is a potential antifibrotic agent in liver and kiney.In our previous study,we found that HSYA can effectively alleviate bleomycin-induced pulmonary fibrosis in rats and mice,alleviate the lung fibrosis induced by cigarette smoke and lipopolysaccharide in rats with chronic obstructive pulmonary disease,inhibit the expression of cytokines related to pulmonary fibrosis in mice with acute respiratory distress syndrome induced by lipopolysaccharide and suppress TGF-β1-induced epithelial–mesenchymal transition in A549 cells.However,it has been reported by our lab that HSYA can directly affect the biological function of lung fibroblasts induced by TGF-β1,but the target and mechanisms of its specific effects were still unclear.Therefore,the main purpose of this study was to investigate the inhibitioneffects of HSYA on TGF-β1-iuduced lung fibroblasts functions activation,which have important significance in the process of IPF,including proliferation,migration,differentiation,extracellular matrix synthesis and degradation,and explore its possible mechanisms.It will provide an experimental basis and theoretical support for the future clinical application of HSYA anti-pulmonary fibrosis therapy.Methods Part 1 HSYA inhibited TGF-β1-induced lung fibroblasts activation MRC-5 cells activated by TGF-β1 were incubated with HSYA and/or the TGF-β type Ⅰ receptor kinase inhibitor,SB431542.3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfo-phenyl)-2H-tetr azolium inner salt assay and 5-ethynyl-2’-deoxyuridine(Ed U)fluorescence staining were used to detect the effect of HSYA on TGF-β1-induced cell proliferation.Cell migration was detected by wound-healing assay.The levels of α-smooth?muscle?actin(α-SMA),a biomarker for myofibroblasts,and the main component of the extracellular matrix,collagen Ⅰ alpha 1(COL1A1)and fibronectin(FN)were measured by Real-time PCR,Western blot and immunofluorescence.Protein levels of matrix metalloproteinase-2(MMP-2),tissue inhibitor of matrix metalloproteinase-1(TIMP-1)and tissue inhibitor of matrix metalloproteinase-2(TIMP-2),which were the key enzymes regulating extracellular matrix degradation,were measured by Western blot.Part 2 Mechanisms of HSYA to inhibit the lung fibroblasts activation induced by TGF-β1 1.MRC-5 cells activated by TGF-β1 were incubated with HSYA and/or the TGF-β1 typeⅠreceptor kinase inhibitor,SB431542.The phosphorylation levels of Smad2,Smad3 were detected by Western blot.The nuclear translocation of Smad2 and Smad3 were detected by Western blot and immunofluorescence.A dual-luciferase reporter assay was used to measure typeⅠcollagen promoter binding by Smad3 after TGF-β1 stimulation in MRC-5 cells.The effect of HSYA on TGF-β1-induced mitogen-activated protein kinase(MAPK)phosphorylation level of extracellular signal-regulated kinase MAPK,c-Jun N-terminal kinase MAPK,p38 MAPK and Aktwere measured by Western blot.TGF-β1-induced mitogen-activated protein kinase(MAPK)and phosphatidylinositol-3 kinase/Akt signalling pathway activation were observed.2.To investigate the interaction of HSYA on Smad3 and extracellular signal-regulated kinase(ERK)/MAPK signaling transduction,MRC-5 cells activated by TGF-β1 were incubated with HSYA and/or ERK-specific inhibitor PD98059,Smad3 inhibitor SIS3.Part 3 To exploring the target of HSYA in inhibiting the activation of TGF-β1-induced fibroblasts MRC-5 cells activated by TGF-β1 were incubated with HSYA.Protein levels of TGF-β1 typeⅡreceptor(TβRⅡ),and TGF-β1 typeⅠreceptor(TβRⅠ)were detected by Western blot.The antagonistic effect of HSYA on the binding of fluorescein isothiocyanate-TGF-β1 to MRC-5 cell cytoplasmic receptors was measured by flow cytometry.TβRⅡ knockdown with si RNA interfered with the inhibitory effect of HSYA on TGF-β1-induced α-SMA,COL1A1,and FN expression elevation,and TGF-β1-induced Smad,and ERK/MAPK signalling pathway activation.Results Part 1 HSYA inhibited the proliferation and migration of MRC-5 cells stimulated by TGF-β1.HSYA inhibited the expression of α-SMA and attenuated TGF-β1 triggered the phenotype transformation of fibroblasts to myofibroblasts.HSYA also exert an inhibition effect on TGF-β1-stimulated COL1A1 and FN expression elevation.In addition,HSYA had no effect on the expression of MMP-2,TIMP-1 and TIMP-2 in MRC-5 cells stimulated by TGF-β1.The above results showed that HSYA affect the changes of fibroblasts biological function activation induced by TGF-β1 including cell proliferation,migration and extracellular matrix synthesis.HSYA can play an important role in inhibiting lung fibroblasts activation and remodeling of lung tissue.Part 2 1.HSYA inhibited the phosphorylation of Smad2,Smad3 and suppressed nuclear translocation of Smad2,Smad3 in TGF-β1-activated MRC-5 cells.HSYA inhibitedTGF-β1-activated the binding activity of Smad3 to SBE in the type Ⅰcollagen promoter.HSYA inhibited TGF-β1-stimulated phosphorylation of ERK MAPK.The phosphorylation level of JNK MAPK,p38 MAPK and Akt induced by TGF-β1 can not be observe,also HSYA did not interfere with this action.These results indicated that the mechanisms of the inhibitory effect of HSYA on TGF-β1-induced MRC-5 cell activation were associated with the Smad and ERK MAPK signaling pathways.2.When the ERK pathway was blocked,HSYA did not exert an effect on Smad3 phosphorylation,which suggested that the inhibition of HSYA on TGF-β1-induced Smad3 phosphorylation may dependent on ERK/MAPK signaling.When the Smad3 pathway was blocked,HSYA still inhibited the phosphorylation of ERK,it suggested that the inhibition of HSYA on ERK phosphorylation was Smad3-independent.Part 3 The results of Western blot showed that HSYA had no effect on TGF-β1-induced TβRⅡ or TβRⅠexpression elevation.Flow cytometry test revealed that HSYA could antagonize the binding of FITC-TGF-β1 to MRC-5 cell cytoplasmic receptors.After TGF-β1-induction,there was no significant difference between the data of TGF-β1-TβRⅡ si RNA group and(TGF-β1+HSYA)-TβRⅡ si RNA group and the parameters including α-SMA,COL1A1,and FN expression,and phosphorylation of Smad2,Smad3,and ERK/MAPK.So under this condition the pharmacological effect of HSYA can not be observed and it suggested that TβRⅡ might be the target of HSYA to inhibit TGF-β1-induced expression of α-SMA,COL1A1,and FN,and phosphorylation of Smad2,Smad3,and ERK/MAPK in cells.Conclusions HSYA inhibited the Smad and ERK signaling pathway might by blocking the binding of TGF-β1 to TβRⅡon the MRC-5 cell membrane,further suppressed TGF-β1-induced changes of fibroblasts signal transduction and related function changes.These effects might be beneficial to the treatment of pulmonary fibrosis.