Two-Photon Imaging Of Endoplasmic Reticulum Oxidative Stress To Diagnose Pulmonary Disease

The endoplasmic reticulum（ER）is the largest organelle in eukaryotic cells and the main site for protein synthesis,folding and transportation.When the redox balance of the ER is disrupted,the synthesis and processing of proteins will have different degrees of errors,which in turn lead to a series of serious diseases.Therefore,maintaining the redox balance of the ER plays a crucial role in maintaining the normal activities of living organisms.As a typical reactive nitrogen species,peroxynitrite（ONOO^-）has strong oxidative ability and nucleophilicity,so it is an important indicator to measure the redox balance of the ER.As an important reducing thiol,cysteine（Cys）plays a important role in the scavenging of reactive oxygen species or reactive nitrogen species in vivo.However,high Cys level can also cause great damage to the biological system.Therefore,real-time detection of ONOO^-and Cys levels in the ER is necessary for the early diagnosis of diseases.Unfortunately,there are currently few probes have been reported for the detection of ONOO^-or Cys in the ER.It is urgent to develop new small-molecule fluorescent probes for real-time,in situ detection of ONOO^-and Cys in the ER.In order to explore the relationship between ER oxidative stress and diseases,we designed and synthesized two small-molecule fluorescent probes for imaging detection of ONOO^-and Cys in the ER.Both probes have good selectivity and ER targeting,and can be applied to the highly selective and sensitive detection of ONOO^-or Cys in the ER.In addition,the two probes were further applied to the detection of ONOO^-or Cys in a mouse model of lung disease.The main contents of this thesis are as follows:1 We designed and synthesized a two-photon fluorescent probe TP^ER-Cys for high-sensitivity imaging to detect level of Cys in the ER.The probe TP^ER-Cys consists of three parts:（1）Hemicyanine derivatives as probe for fluorophores;（2）Acrylate as the recognition group of Cys;（3）p-Toluene sulfonamide as the targeting group of ER.The probe can not only detect Cys in solution sensitively and rapidly,but also specifically target the ER and detect Cys in the ER with high selectivity at the in vivo level.In addition,we also successfully used the TP^ER-Cys to detect Cys in lung tissue of mice with pulmonary fibrosis by two-photon confocal microscopy.The results showed that the content of Cys in lung tissue of pulmonary fibrosis mice was significantly higher than that of normal mice.It shows that there is a close relationship between Cys and the pathogenesis of pulmonary fibrosis.Therefore,the content of Cys in lung tissue is expected to serve as an important indicator in the process of pulmonary fibrosis diagnosis and provide a potential target for the development of new drugs for the treatment of pulmonary fibrosis.2 The redox balance of the ER is crucial for the normal functioning of the cell.Earlier we introduced the detection of the reducing substance Cys in the ER.Next,we designed and synthesized a two-photon fluorescent probe TP^ER-ONOO for highly sensitive detection of oxidative species ONOO^-in the ER.When the probe TP^ER-ONOO reacts with ONOO^-,the C=C bond of the probe is selectively oxidatively cleaved to form indole aldehyde and the substance DHX-OH.DHX-OH has a smallerπ-conjugated molecular system and thus emits fluorescence in the blue band（460 nm）.In vitro experiments,the probe TP^ER-ONOO can sensitively recognize ONOO^-.In cell experiments,the probe TP^ER-ONOO has good targeting ability to the ER and has been used to detect endogenous ONOO^-in the ER.In addition,we also successfully applied the probe to the imaging of ONOO^-in lung tissue during pneumonia,and observed brighter fluorescence in the lung tissue of pneumonia mice than in normal mice.The results showed that there was a correlation between the level of ONOO^-in the lungs of mice and pneumonia in mice.Next,we will study the changes of ONOO^-in the ER when stimulated by different stimulants,and continue to search for the key factors or proteins that cause the pathogenesis of pneumonia,and further study the link between ONOO^-and the pathogenesis of pneumonia.