Therapeutic Effect And Mechanism Of OPN-siRNA On Idiopathic Pulmonary Fibrosis

Idiopathic pulmonary fibrosis(IPF)is a chronic,progressive and lethal lung disease of unknown cause.At present,it is generally believed that the cause of IPF is the abnormal activation of alveolar epithelial cells caused by repeated damage to the lungs,which in turn leads to fibroblast proliferation and epithelial-mesenchymal transition(EMT),and eventually leads to the extracellular matrix(extracellular matrix(ECM)over-precipitation,damage repair abnormalities,and lung tissue remodeling.Currently,there is no specific treatment method for IPF.Therefore,clarifying the cause of IPF is of great significance for the occurrence and treatment of IPF.EMT is a biological process in which epithelial cells lose contact adhesion and apical-basal polarity,their shape changes with the drastic changes in the cytoskeleton,and acquires some mesenchymal features that invade,migrate,and produce ECM.EMT is a physiological process which is necessary for normal embryonic development.It also occurs in the process of injury,canceration and fibrosis.Studies have confirmed that EMT plays an important role in IPF,and the formation of EMT is closely related to the activation of transforming growth factor-?(TGF-?)signaling pathways.TGF-? is the most studied profibrotic cytokine that can regulate key cellular activities such as cell proliferation,differentiation,adhesion,apoptosis,migration and EMT.There are different isoforms of TGF-?(TGF-?1,TGF-?2 and TGF-?3),while TGF-?1 is the main subtype and it is most closely related to the development of IPF.TGF-? can regulate the expression of downstream target genes through the TGF-? / Smad signaling pathway and promote the transformation of EMT.In addition to Smad-mediated transcription,TGF-? can also activate other signaling cascades,including the mitogen-activated protein kinase(MAPK)pathway.However,the cause of TGF-? signal activation and EMT is not clear.OPN is a secreted protein involved in a variety of pathological and physiological processes,including bone remodeling,innate immunity,acute and chronic inflammation,and cancer.It is mainly expressed in epithelial cells and immune cells,such as macrophages,dendritic cells and T lymphocytes.Other studies confirmed that OPN has a role in promoting fibrosis and is highly expressed in IPF.However,it is unclear whether the intervention of OPN expression can affect EMT and TGF-? signaling pathways,and then inhibit IPF.In this project,we first used TGF-?1 to induce A549 cells to construct a lung IPF cell model,and observed the effects of OPN-siRNA on the expression of EMT and fibrosis-related molecules in vitro.Second,bleomycin(BLM)was used to induce IPF animal models,and then established the OPN-siRNA-based IPF model mice.Then studied the effect of OPN-siRNA on BLM-induced IPF model mice and its mechanism in vivo.1.To observe the effect of OPN-siRNA on the expression of EMT and fibrosis-related molecules in IPF cell models(1)Establishment of TGF-?1-induced IPF cell modelIn order to establish an IPF cell model,we used the protocol of TGF-?1 to stimulate A549 cells to construct an IPF cell model.QRT-PCR and Western Blot methods were used to detect the expression levels of EMT and fibrosis-related molecules,including mRNA and protein expression levels of the epithelial molecular marker E-cadherin,the interstitial molecular marker Vimentin,and the fibrosis-related molecule Fibronectin,from the mRNA and protein levels,respectively,which help us to determine the best stimulation concentrations of TGF-?1.The results showed that with the concentration of TGF-?1 at 5ng / ml,the expression level of E-cadherin was highest.And the expressions level of Vimentin and Fibronectin were lowest.The results suggest that 5ng / ml is the optimal stimulating concentration of TGF-?1,which can successfully induce the IPF cell model.Therefore,we stimulated A549 cells with 5ng / ml TGF-?1 to establish a TGF-?1-induced IPF cell model for further research.(2)Effect of OPN-siRNA on the expression of EMT and fibrosis-related molecules in the IPF cell modelIn order to investigate the effect of OPN-siRNA on the expression of EMT and fibrosis-related molecules in the IPF cell model,the OPN expression was silenced in the above-mentioned IPF cell model by OPN-siRNA,and the mRNA level of EMT and fibrosis-related molecules was detected using qRT-PCR technology.The results showed that compared with control group,the expression of E-cadherin in the TGF-?1 stimulation group was reduced,the expression of Vimentin was increased,and the expression of Fibronectin was increased.Compared with the TGF-?1 stimulation group,the expression level of cadherin increased and the expression of Vimentin and Fibronectin decreased.This suggests that silencing of OPN gene may inhibit IPF caused by TGF-?1 stimulation,but it is unclear whether it has the same effect in vivo and whether it has a therapeutic effect on IPF.2.To study the therapeutic effect and its mechanism of OPN-siRNA on IPF model miceOn the basis of in vitro studies,in order to explore whether OPN-siRNA also has the effect of inhibiting IPF in vivo and the possible mechanism,this project established a mouse model of IPF with OPN-siRNA,and studied the therapeutic effect and mechanism of OPN-siRNA on IPF in animal models.(1)Establishment of OPN-siRNA treated IPF mice modelThe experiment was divided into three groups: Control group,BLM group and BLM + OPN-siRNA group.A mouse model of pulmonary fibrosis was induced by BLM,then the mice were inhaled OPN-siRNA via the nose on the third day,and sacrificed on the fourteenth day to start the experiment.We used qRT-PCR and Western Blot to detect the efficiency of OPN silencing in BLM mouse models at the mRNA and protein levels.The results showed that the expression level of OPN in the BLM + OPN-siRNA group was significantly lower than that in the BLM group,and was statistically significant.This suggests that the IPF model of OPN-siRNA has been successfully established and can be used for the next experiments.(2)Effect of OPN-siRNA on general state of IPF mice modelWe observed the effect of OPN-siRNA on IPF mice model from the body weight and gross specimen.First,in terms of weight,the weight of the control group gradually increased,while the weight of the BLM group was basically unchanged,and the weight of the BLM + OPN-siRNA group increased significantly compared to the BLM group.Secondly,from the appearance of the mouse lungs,the lungs in the BLM group became congested and their volume became smaller,but the symptoms of lung congestion in the BLM + OPN-siRNA group were reduced and their volume recovered.(3)Effect of OPN-siRNA on lung histopathology in IPF mice modelTo evaluate whether OPN-siRNA can improve the BLM-induced idiopathic pulmonary fibrosis in mice,we observed the histopathological changes in the lungs of mice by H & E,PSR,and Masson staining.The results showed that the inflammatory cells infiltrated in the BLM group,accompanied by a large amount of fibrosis,manifested as alveolar collapse and thickening,deposition of perivascular collagen fibers,bronchoalveolar hyperplasia,and normal lung structural distortion;while in BLM + OPN-siRNA group the degree of fibrosis was significantly reduced.This suggests that OPN-siRNA may restore the physiological state of lung tissue in BLM-induced IPF mice.(4)Effect of OPN-siRNA on inflammatory cells and cytokines in BALF of IPF mice modelIn order to understand whether OPN-siRNA can affect inflammatory cells and cytokines in BALF of IPF mice model,we conducted the following research.In order to explore the effect of OPN-siRNA on inflammatory cells,counting and staining the cells in mouse BALF revealed that compared with the control group,the number of macrophages,neutrophils and lymphocytes in the BALM group were all increased,but decreased in the OPN-siRNA group.This indicates that OPN-siRNA can inhibit the inflammatory cell infiltration in the lung tissue of BLM-induced IPF mice,thereby improving the inflammatory state of the lung tissue.In order to further investigate the effect of OPN-siRNA on proinflammatory cytokines in BALF of IPF mice model,lung tissues of mice were isolated and the expression levels of IL-1? and IL-6 were detected by qRT-PCR.The results showed that compared with the control group,the expression levels of IL-1? and IL-6 in the BLM group were significantly increased.Compared with the BLM group,the expression levels of IL-1? and IL-6 decreased in BLM+OPN-siRNA group.The results indicate that OPN-siRNA can reduce the secretion of proinflammatory cytokines in mouse BALF induced by BLM,thereby reducing lung tissue inflammation and improving pulmonary fibrosis.(5)Effect of OPN-siRNA on the expression of EMT and fibrosis-related molecules in IPF mice modelIn order to further explore the effect of OPN-siRNA silencing on EMT in BLM-induced IPF mice model,we detected the epithelial molecular marker E-cadherin,mesenchymal molecular marker Vimentin and fibrosis-related molecule collagen I at the mRNA,protein level and their expression in tissues,respectively.The results showed that BLM effectively reduced the expression of E-cadherin mRNA in the lung tissue of mice,increased the expression of Vimentin and collagen I mRNA in the lung tissue of mice.OPN-siRNA could restore the expression of most E-cadherin mRNA and decrease the mRNA expression of Vimentin and collagen I.At the protein level,the results of Western Blot and immunohistochemical detection showed that the molecular expression trend was the same as the mRNA level trend.This suggests that BLM may induce the activation of epithelial cells,the generation of mesenchymal cells and promote the formation of ECM,while the silencing of the OPN gene can inhibit the above processes induced by BLM,and may play a role in suppressing IPF.(6)Exploration of the regulation mechanism of OPN-siRNA on the expression of EMT and fibrosis-related moleculesIn order to explore the mechanism of OPN-siRNA to regulate the expression of EMT and fibrosis-related molecules,we used qRT-PCR to analyze the expression levels of TGF-?1,TGF-? receptor ? and receptor ?,and further analyzed the Smad pathway mediated by TGF-?1 by Western Blot,as well as the expression of related molecules in the MAPK pathway.The results showed that BLM may induce the activation of TGF-?1 pathway,while OPN-siRNA can inhibit the activation of TGF-?1-mediated Smad2 / 3 pathway induced by BLM.But OPN-siRNA had littler effects on the MAPK signal pathway.These results suggest that OPN-siRNA may inhibit the EMT and fibrosis related molecules by suppressing the activation of TGF-?1 mediated Smad2/3,therefore,play an important role in treating IPF.The conclusions of this study are as following:(1)In vitro,OPN-siRNA can affect the expression of EMT and fibrosis-relatedmolecules in IPF cell models induced by TGF-?1.(2)OPN-siRNA can treat IPF in vivo,and its mechanism of action may beexerted by inhibiting the expression of inflammatory response,EMT andfibrosis-related molecules.(3)In vivo,OPN-siRNA can inhibit the activation of Smad2 / 3 mediated byTGF-?1 and may affect the expression of EMT and fibrosis-related molecules.In summary,this study confirms that OPN-siRNA can inhibit the activation of the Smad2 / 3 pathway mediated by TGF-?1,and then affect the expression of EMT and fibrosis-related molecules,and reduce the inflammatory response in the lung tissue,thereby significantly improving the bleomycin induced pathological changes of idiopathic pulmonary fibrosis,which provide a new way to treat IPF by targeting OPN.