Mechanism Of Scutellarin Ameliorates Pulmonary Fibrosis Induced By Bleomycin Through Inhibiting NF-κB/NLRP3

Background:Idiopathic pulmonary fibrosis(IPF)is a chronic,progressive and irreversible lung disease,characterized by progressive pulmonary fibrous scarring and usual interstitial pneumonia,which not only seriously affects the quality of life of patients,but also becomes the "fatal killer" of patients.According to statistics,IPF affects about 3 million people in the world,and the incidence rate increases sharply with age.However,there is a lack of effective treatment.Only pirfenidone and nintedanib are approved to treat IPF in China,and their effect on prolonging survival time is not ideal.The pathogenesis of IPF is not clear.Studies have found that epithelial mesenchymal transformation(EMT)and inflammatory reaction are the key steps in the pathogenesis of IPF.It is reported that scutellarin,a flavonoid,plays an important role in inhibiting EMT and inflammatory response,has anticancer activity,can effectively inhibit the migration,invasion,adhesion and angiogenesis of cancer cells,and reverse EMT.In addition,scutellarin also prevents isoproterenol induced myocardial fibrosis by inhibiting cardiomyocyte EMT.It is suggested that scutellarin can inhibit inflammatory response and EMT process,and has anti fibrosis effect;However,its application in idiopathic pulmonary fibrosis has not been reported.In conclusion,scutellarin plays an important role in regulating EMT and inflammatory response,which may delay the development of IPF by regulating EMT and inflammatory response in IPF.Therefore,we intend to explore whether scutellarin can ameliorate the progress of IPF by inhibiting EMT and inflammatory response.Part 1 Scutellarin ameliorated the damages undergone from pulmonary fibrosis induced with BLMObjective: To explore the effect of scutellarin on the damages undergone from pulmonary fibrosis induced with BLM.Methods: BLM was firstly used to induce BALB/c mouse to establish the pulmonary fibrosis model followed by treating them respectively with 30,60 and 90 mg/kg of scutellarin,and the lung tissue structure and the collagen deposition in the model mice were then analyzed by HE and Mason’s staining methods.Furthermore,the expressions of α-SMA and type I collagen in the mice lung tissues were studied by real-time fluorescent quantitative PCR(QPCR)and Western blotting(WB).Meanwhile,after establishing the pulmonary fibrosis model of human lung epithelial A549 cells and rat lung epithelial RLE-6TN cells induced with BLM,scutellarin of 0.1,0.2 or 0.4 m M dose was respectively treated these cells,and the cells viability,the expressions of α-SMA and collagen I were further assayed by MTT,QPCR and WB techniques.Results: After treated with 30,60 and 90 mg/kg of scutellarin,the results of HE and Masson staining indicated that scutellarin not only destroyed alveolar structure and inflammatory infiltration in the model mice,but also the fibrous collagen deposition in their lung tissues were improved as dose-dependent.Meanwhile,scutellarin could significantly decrease the m RNA and the protein expressions of α-SMA and type I collagen in the both mice lung tissues of pulmonary fibrosis model and RLE-6TN and A549 cells pretreated with BLM.Conclusion: Scutellarin can alleviate the fibrosis and collagen deposition induced by BLM in a dose-dependent manner,reduce the infiltration of inflammatory cells and inhibit the expression of α-SMA and collagen I.In other words,scutellarin can improve BLM induced pulmonary fibrosis both in vivo and in vitro.Part 2 Scutellarin suppress inflammation of pulmonary fibrosis induced with BLM through NF-κB/NLRP3 pathwayObjective: To investigate the inhibition role of scutellarin on the inflammation of pulmonary fibrosis induced with BLM whether through the NF-κB/NLRP3 pathway.Methods: The model of pulmonary fibrosis were established as above.After treating the model mice with scutellarin of 30,60 and 90mg/kg dose,the expressions of phosphorylated protein 65(p-p65),p65,IκBα,NLRP3,caspase-1,caspase-11,GSDMDNterm,ASC,IL-1β and IL-18 in the lung tissue of the model mice were respectively determined by the techniques of WB,immunohistochemistry(IHC),QPCR.As Same as these,the expressions of p-p65,p65,IκBα,NLRP3,caspase-1,caspase-11,GSDMDNterm,ASC,IL-1β and IL-18 in both RLE-6TN and A549 cells following pretreatment with BLM were also assayed.Additionally,ELISA was carried out to quantify the amount of IL-1β and IL-18 either in the serum of the model mice or in the supernatants of RLE-6TN and A549 cells pretreated with BLM.Results: Scutellarin could down-regulate the expression of p-p65 in lung tissues of the pulmonary fibrosis model mice,and induce the expression of IκBα in a dose-dependent manner.Meanwhile,scutellarin could not only decrease the expressions of NLRP3,caspase-1,ASC,GSDMDNterm,IL-1β and IL-18 induced with BLM,but also could dose-dependently inhibit the production of IL-1β and IL-18 in the serum of the model mice.As similarity with this,except decreasing the expressions of p-p65,NLRP3,caspase-1,GSDMDNterm,ASC,IL-1β and IL-18,scutellarin also could reduce the secretion of IL-1β and IL-18 resulted from RLE-6TN and A549 cells pretreated with BLM in a dose-dependent manner.Conclusion: In the model of pulmonary fibrosis in vivo and in vitro,scutellarin could decrease the expressions of a variety of factors involved in the NF-κB/NLRP3 pathway,such as p-p65、NLRP3、IL-1β and IL-18 induced with BLM,this might be suggested that the role of scutellarin on the inflammation of pulmonary fibrosis induced with BLM would be closely associated with NF-κB/NLRP3 pathway.Part 3 Scutellarin inhibits the epithelial-mesenchymal transformation induced with BLM in vivo and in vitroObjective: To study whether scutellarin alleviates the epithelialmesenchymal transformation(EMT)induced with BLM.Methods: The mice,RLE-6TN and A549 cells of the pulmonary fibrosis model established as above were treated with scutellarin.Subsequently,the expressions of fibronectin,vimentin,E-cadherin,N-cadherin,MMP-2,MMP-9 and Snail in the lung tissues of the model mice,as well as in the cells of RLE-6TN and A549,were checked by the means of QPCR,WB and IHC.Furthermore,the activity of MMP in the cells of RLE-6TN and A549 was tested.Results:Scutellarin could decrease the expressions of fibronectin,vimentin,N-cadherin,MMP-2,MMP-9 and Snail in the lung tissues of the model mice,as well as in RLE-6TN and A549 cells pretreated with BLM.In contrast,after treatment with scutellarin,the expression of E-cadherin was increased in the lung tissues of the model mice,as well as in RLE-6TN and A549 cells pretreated with BLM.However,scutellarin could reduce the activity of MMP in BLM-induced RLE-6TN and A549 cells.Conclusion: Scutellarin is able to regulate the expressions of a series of molecules associated with EMT induced with BLM in vivo and in vitro,and maintain the expression of epithelial cell markers,which might be thought that scutellarin could alleviate the EMT of pulmonary fibrosis induced with BLM.Part4 Overexpression of NLRP3 reverses the inhibitory effect of scutellarin on inflammation and EMTObjective: To clarify the molecular mechanism of scutellarin in alleviating inflammatory response and EMT process in BLM induced pulmonary fibrosis by inhibiting NLRP3 pathway.Methods: The RLE-6TN and A549 cells overexpressed NLRP3 was treated with scutellarin following by assaying the expressions of p-p65,p65,IκBα,NLRP3,caspase-1,ASC,GSDMDNterm,IL-1β and IL-18 in addition to fibronectin,vimentin,E-cadherin,N-cadherin,MMP-2,MMP-9 and Snail with the help of QPCR and WB techniques.Meanwhile,the activity of MMP in the cells was detected.Results: Overexpression of NLRP3 could not only decreased the induction of p-p65,caspase-1,ASC,GSDMDNterm,IL-1β,IL-18,fibronectin,vimentin,N-cadherin,MMP-2,MMP-9 and snail,but also reduce the effect on the activity of MMP in the RLE-6TN and A549 cells induced with scutellarin.Conclusion: Overexpression of NLRP3 can reverse the protective effect of scutellarin on pulmonary epithelial cell fibrosis,eliminate the anti-inflammatory,promote EMT and exacerbate accumulation of ECM.These results fully suggest that the inhibiting of NF-κB/ NLRP3 pathway may be responsible for the effect of scutellarin alleviate BLM induced fibrosis.