The Synthesis Of A Near Infrared Fluorescent Probe For Detection Of Hydrogen Peroxide And Its Application In Pulmonary Fibrosis

Objective:Pulmonary fibrosis(PF)is a fatal chronic lung disease with unknown etiology,unclear pathogenesis and poor prognosis.Although the US Food and Drug Administration(FDA)has approved the use of pirfenidone and nidanib for the treatment of pulmonary fibrosis,but the long-term treatment effect of these two drugs is not clear,and accompanied by severe gastrointestinal side effects which are difficult to tolerate,besides there is no effective treatment other than lung transplantation.At present,there is increasing evidence that oxidative stress characterized by excessive production of reactive oxygen species(ROS)is an important molecular mechanism of pulmonary fibrosis.ROS includes two subgroups:free radical ROS represented by superoxide radicals(O2-)and non-radical ROS represented by hydrogen peroxide(H2O2).Compared with free radical ROS,H2O2 plays a more important role in physiological and pathological processes.H2O2 is less reactive,has a longer half-life,and is more susceptible to physicochemical properties in the plasma membrane and cell membrane.H2O2 plays an important role in the development of idiopathic pulmonary fibrosis.Idiopathic pulmonary fibrosis is usually studied using a bleomycin-induced fibrosis model.We designed and synthesized a novel near-infrared fluorescent probe,Mito-OH,to study changes of endogenous hydrogen peroxide in a model of pulmonary fibrosis in living cells and animals.Methods:Herein,a small molecule NIR mito-targeted fluorescent probe,the Mito-OH,is designed and synthesized for detecting hydrogen peroxide in cell models and murine models of pulmonary fibrosis.In the design of the Mito-OH,the azo-BODIPY is incorporated as a fluorophore,which exhibits the near-infrared emission spectra and has a robust photochemical stability,and low biological toxicity.The detection mechanism is based on the response of an alkyl borate or aryl borate to H2O2,releasing a fluorophore to turn on fluorescence for detection of hydrogen peroxide in a cell model and a mouse model of pulmonary fibrosis.The cytotoxicity assays of Mito-OH were performed using Cell Counting Kit-8(CCK8)prior to fluorescence imaging in biological systems.Then we selected two human non-small cell lung cancer cell lines(A549 and PC9)as cell models,and studied the changes of endogenous H2O2 in living cells by laser confocal experiment with co-implantation with probe Mito-OH.Flow cytometry analysis was used to detect changes in cellular fluorescence intensity over a period of 0 to 60 minutes.Mito-OH studies the fluctuation of H2O2 during the establishment of a pulmonary fibrosis cell model.Pulmonary fibrosis cells were established by stimulating two human embryonic fibroblast cell lines(IMR-90 and MRC-5 cells)with serum-free medium containing 5 nM of transforming growth factorβ1(TGF-β1)for 24 h,48 h and 72 h,respectively.model.During the progression of pulmonary fibrosis,not only changes in cell morphology occur,but also levels of intracellular protein markers.To further investigate the effect of endogenous hydrogen peroxide levels on fibrosis progression,IMR-90 cells and MRC-5 cells were randomly divided into three groups.Group a was a control group,and cells were cultured in serum-free medium.Group b cells were stimulated with an equal volume of serum-free medium containing TGF-β1 at a final concentration of 5 nM and cultured at 37℃ for 72 hours.Group c was based on group b,discarding the medium containing TGF-β1,and then incubating the cells with GKT137831(GKT,a small molecule-specific dual inhibitor of NOX-4/NOX-1)containing serum-free medium.Considering the good results of Mito-OH in cell experiments,we applied it to a bleomycin-induced mouse pulmonary fibrosis model to monitor changes in H2O2.After the small animals were imaged in vivo,the mice were sacrificed and organs including the heart,liver,spleen,lungs and kidneys were taken for imaging of the isolated organs.To study the morphological and pathological changes in mice,lung sections of each group of mice were subjected to hematoxylin-eosin staining(H&E staining)and Masson staining,and observed under 100-fold and 400-fold optical microscopes.Results:The high cell viability indicated that the probe was low biotoxicity to cells and abled to be used in living cells,then we performed intracellular imaging of Mito-OH with laser scanning confocal microscopy.After co-incubated with Mito-OH,A549 cells and PC9 cells displayed increasing fluorescent intensity during 0 to 60 minutes,which indicates that the probe had reacted with hydrogen peroxide in cells and released the fluorophore.The results of flow cytometry analysis were consistent with the results of the confocal microscopy images.The fluorescence intensity became stronger with the prolongation of TGF-β1 stimulation time,indicated that the level of oxidative stress in fibroblasts is getting higher and higher,and the amount of hydrogen peroxide in cells is gradually increasing.The same results also appeared in flow cytometry analysis.The expression of α-SMA,a kind of mesenchymal cell marker protein,increased gradually with the prolongation of stimulation time in the cells of experimental group.At the same time,the profibrotic factor PAI-1 shows the same trend.However,the expression of the E-cadherin,the representative of epithelial cell protein markers,showed a down-regulated trend,indicating that fibrosis was constantly evolving induced by the stimulation of TGF-β1.In murine models of bleomycin-induced pulmonary fibrosis,the intervention of GKT reduces the level of oxidative stress in lungs and makes a reduction in the production of hydrogen peroxide,which is conducive to delay the progression of the disease and alleviate the symptoms of pulmonary fibrosis.Conclusion:In this study,we have designed and synthesized a mito-targeted NIR small molecule fluorescent probe(the Mito-OH)for monitoring changes of endogenous H2O2 in living cells and in vivo.The Mito-OH is photochemical stability and highly sensitive and selective to hydrogen peroxide.The level of hydrogen peroxide was increased during the development of pulmonary fibrosis,and the NOX4 inhibitor GKT137831 was able to down-regulate hydrogen peroxide levels and delay or attenuate pulmonary fibrosis.Mito-OH has confirmed the key role of H2O2 and oxidative stress in the progression of pulmonary fibrosis in pulmonary fibrosis cells and mouse in vivo models,pointing out the direction for the treatment of pulmonary fibrosis in the future.