Fluorescence Imaging Of Liver Injury Related Reactive Molecules And Microenvironments In Cells And In Vivo

Liver is the largest metabolism and detoxification organ in the human body,which also is the vulnerable organ.Liver injury is a common clinical disease,which can gradually develop into hepatic failure,and seriously endanger life.To make accurate early diagnosis and take effective treatment of liver injury,it is necessary to have a comprehensive understanding of the occurrence and development,and related mechanism of liver injury.Lots of studies have shown that the organelle dysfunction in liver cells was closely related to the different types of liver injury.For example,peroxisomal dysfunction could cause various types of liver injury,including acute liver injury and non-alcoholic fatty liver disease(NAFLD).Meanwhile,endoplasmic reticulum stress occurs easily when liver cells are stimulated by external factors.Endoplasmic reticulum stress plays an important role in liver injury.The dysfunction of these organelles is often accompanied by variations in biomolecules and microenvironments,such as reactive nitrogen species and viscosity.Moreover,the peroxynitrite(ONOO^-)has been widely used as a biomarker of liver injury.Therefore,accurate detection of peroxisomal ONOO^-is helpful to elucidate their role with liver injury.Real-time dynamic detection of peroxisomal viscosity and polarity,and endoplasmic reticulum viscosity are conducive to study the relationship between the changes of organelle microenvironments and NAFLD,develop reliable diagnostic methods and establish a platform for pharmacodynamic evaluation.Fluorescence imaging has been widely used for in situ detection of bioactive molecules and microenvironments in living cells and in vivo due to its excellent advantages of high temporal and spatial resolution,high sensitivity and simple operation.Especially,two-photon and near-infrared fluorescence imaging techniques are more suitable for deep tissue imaging fluctuations of bioactive molecules and microenvironments in vivo,because these techniques possessed superior advantage,such as deep tissue penetration and low background interference.To date,some fluorescent probes for detecting changes of bioactive molecules,viscosity and polarity in organelles have been developed and reported.However,endoplasmic reticulum probe has been not achieved imaging viscosity in deep tissues.Extremely,fluorescence probes for detecting the changes of peroxisomal ONOO^-,viscosity and polarity has not been reported so far.Therefore,it is urgent to develop two-photon or near-infrared fluorescent probes to detect the changes of peroxisomal ONOO^-,viscosity,polarity and endoplasmic reticulum viscosity during liver injury.Based on the above reasons,we established in situ fluorescent imaging methods for targeting into peroxisomes and endoplasmic reticulum,respectively.Furthermore,the proteomic analysis further revealed oxidative modification of related proteins.These probes provide some excellent imaging tools for the early diagnosis,drug screening and relevant molecular mechanisms of liver injury.These contents were as follows:1.We synthesized a novel two-photon fluorescent probe(PX-1)for imaging peroxisomal ONOO^-in mouse liver with carbon tetrachloride(CCl₄)-induced acute liver injury.In the probe structure,the SKL sequence could bind to the corresponding receptor on the surface of the peroxisomes.The sequence could help the probe locate into peroxisomes.The 4-hydroxy-1,8-naphthalenedicarboxylic anhydride was acted as two-photon fluorophore.In vitro experiments showed that PX-1 could respond to ONOO^-with high selectivity,sensitivity and specificity.Cell imaging experiment results showed that PX-1 could accurately accumulate in peroxisomes and successfully observe dynamic changes of endogenous ONOO^-.The upregulation of peroxisomal ONOO^-in mouse liver by CCl₄-induced liver injury was clearly observed for the first time via two-photon fluorescence imaging.This work provides a new strategy for studying the peroxisomal ONOO^-and mechanisms of liver injury.2.We fabricated a near infrared fluorescent and photoacoustic dual mode probe(PV-1)for imaging peroxisomal viscosity.In the probe structure,the conjugated fluorophore was formed by merocyanine and cyano groups.The PV-1 had freely rotating single bonds,which could sense to the variations of environmental viscosity.With the increase of environmental viscosity,the single bond rotation in the probe was limited,reducing the possibility of non-radiative energy loss,enhancing the fluorescence of probe.Therefore,the PV-1 exhibited remarkable near-infrared absorption peak and fluorescence peak in high viscosity solutions.In vitro test results,the PV-1could detect environmental viscosity changes with high selectivity and sensitivity.Furthermore,cell imaging experiments indicated that the PV-1 could accurately target into peroxisomes,indicating the difference in viscosity under different conditions.Using this probe,we observed that viscosity in the liver of mice with NAFLD was significantly higher than that in normal mice by fluorescence and photoacoustic dual-mode imaging.Moreover,after treated with N-acetylcysteine(NAC),the liver viscosity in NAFLD mice decreased significantly.Therefore,NAC could alleviate NAFLD to some extent.These experimental results fully verified that this dual-mode imaging method based on the viscosity probe can be used for early warning of NAFLD and evaluation of related drugs.3.We also developed a near infrared fluorescent probe(Er-V)for in situ detection of endoplasmic reticulum viscosity.The rotation of the single bond in the probe was restricted as the environmental viscosity increases,and then enhancing near infrared fluorescence.The p-methylbenzenesulfonamide structures in the probe structure could target the sulfonamide receptors on the surface of the endoplasmic reticulum.In vitro tests manifested that the Er-V showed strong near infrared fluorescence in high viscosity media,allowing real-time detection of environmental viscosity changes.Additionally,cell imaging experiments displayed that the Er-V could aggregate in the endoplasmic reticulum.The Er-V could sense viscosity changes when endoplasmic reticulum stress occurred in liver cells.Furthermore,the Er-V successfully distinguished normal cells from oleic acid-induced cells by imaging viscosity.Moreover,these results further confirmed that endoplasmic reticulum viscosity increased when liver cell occurred lipid accumulations.Most importantly,based on this probe imaging method,the liver viscosity of NAFLD mice was successfully observed to be significantly higher than that of normal mice,verifying that endoplasmic reticulum stress is closely related in the occurrence and development of NAFLD.This work based on Er-V will promote deeper understanding of the pathogenesis of NAFLD and provide a reliable platform for early diagnosis of NAFLD.4.We created a two-photon fluorescent probe(PX-P)for detecting peroxisomal polarity changes in liver of mice with NAFLD.In the probe design,triphenylamine group,furan unit and cyanide group were served as electron donor,?bridge and electron acceptor.The SKL was acted as the targeting group of peroxisomes.The twisted intramolecular charge transfer process of excited state was easy to occur from donor to acceptor when PX-P was radiated.In vitro tests verified that PX-P showed excellent sensitivity toward environmental polarity.The fluorescence wavelength and the fluorescence intensity of PX-P gradually redshifted and decreased with the increase of environmental polarity.Cell imaging experiments showed that PX-P could accurately located into peroxisomes,indicating the change of polarity.Using this probe,we successfully distinguished between normal cells and lipid-accumulating cells.The two-photon fluorescence imaging experiment results show that the polarity in the liver with NAFLD mice was lower than that in normal mice.Additionally,proteomic analysis revealed that intracellular excess ONOO^-and hydrogen peroxide(H₂O₂)could react with different sites of peroxisome proliferator-activated receptor?(PPAR-?)protein for nitrification and oxidation,respectively.Interestingly,one of the key sites of PPAR-?protein was nitrated by excessive ONOO^-,resulting in decreasing protein activity,and thus accelerating the occurrence of NAFLD.This work not only provide an imaging tool for real-time detection of peroxisomal polarity changes,but also help to reveal the molecular mechanism of the inactivation of PPAR-?protein in the occurrence and progression of NAFLD.