Integration Of Fluorescence Imaging And Diagnosis And Treatment Of Hepatic Ischemia-Reperfusion Injury Related Biomarkers

As an inevitable pathological phenomenon in clinical operations such as liver resection,liver transplantation and hemorrhagic shock,hepatic ischemia-reperfusion injury（HIRI）is mainly responsible for postoperative liver dysfunction or liver failure.Traditional clinical diagnosis of HIRI is mainly through blood tests,diagnosis by imaging,and liver biopsy.However,they have the limitations of low sensitivity,poor resolution and hysteresis.Meanwhile,current treatment strategies for HIRI mainly focus on surgical treatment and drug therapy.However,due to the ambiguous contour and boundary of HIRI liver lesions,accurate intraoperative anchoring of liver injury site is a major challenge in surgical treatment.However,the anti-inflammatory and antioxidant drugs targeting HIRI have some problems,such as poor pharmacokinetics,non-specific biological distribution,poor efficacy and large side effects.Therefore,there is an urgent need to develop reliable diagnosis and treatment tools for HIRI to achieve early diagnosis,accurate intraoperative navigation and effective drug intervention for HIRI.Accumulating researches have shown that the pathological mechanism of HIRI is closely related to oxidative stress and energy metabolism.The overproduction of reactive oxygen species（ROS）and reactive nitrogen species（RNS）such as superoxide anion(O₂^·-),hypochlorous acid（HOCl）and peroxynitrite（ONOO^-）in HIRI play crucial roles in the occurrence and development of HIRI.Meanwhile,the depletion of adenosine-5’-triphosphate（ATP）is a key process in HIRI.Highly reactive ROS and RNS can cause significant changes in microenvironmental viscosity.Therefore,the detection of important oxidative stress markers(O₂^·-,HOCl,ONOO^-),energy metabolism marker（ATP）and microenvironment marker（viscosity）in living cells and in vivo during HIRI,the distinction of normal tissues from damaged tissues based on the differences in viscosity,and drug release targeting excessive ROS in HIRI,are conducive to reveal the occurrence and development mechanism of HIRI,which promote the early diagnosis of HIRI,accurate localization of lesions during surgery and effective drug treatment.Fluorescence imaging has become a powerful tool for real-time monitoring of disease-related biomarkers in living cells and in vivo.In particular,two-photon fluorescence imaging stands out in the study of targeted biomolecules and biological events in living cells and deep tissues,which can significantly reduce the photodamage and photobleaching of samples,improve the penetration depth,and produce higher contrast images.Second near-infrared window（NIR-Ⅱ）fluorescence imaging integrates centimeter tissue penetration depth,ultra-high micron resolution at millimeter depth and ultra-high signal-to-noise ratio imaging.In recent years,the potential applications of two-photon fluorescence imaging and NIR-Ⅱfluorescence imaging in diagnosis of diseases and image-guided surgery have been continuously explored.The integrated fluorescent diagnosis and treatment materials based on fluorescence imaging have both diagnostic and therapeutic functions,which can diagnose disease early,deliver therapeutic drugs to the lesion site,and visualize the progression of the disease.However,up to date,fluorescent probes that can simultaneously visualize HIRI-related biomarkers in real time and guide accurate navigation of HIRI lesion sites are rarely reported.Especially,the fluorescence probe that integrate diagnosis and treatment in HIRI has not been developed.Therefore,it is urgent to develop high-performance fluorescent diagnostic and therapeutic materials for in situ monitoring of biomarkers associated with HIRI,accurate identification and resection of liver injury sites,and releasing drug in the intervention of HIRI,which are of great significances for early diagnosis and effective treatment of HIRI,in-depth understanding of the pathogenesis and the revelation of potential targets of HIRI.Based on the above reasons,this dissertation developed a series of organic small molecule fluorescent probes with high sensitivity,high selectivity,fast response and good biocompatibility,aiming at the bottleneck problem of the precise diagnosis and treatment of HIRI in living livers.The imaging analyses of important biomarkers such as O₂^·-,HOCl,ONOO^-,ATP and viscosity in hepatocytes and mouse livers during HIRI were realized.The important signal pathways involved in HIRI-related biomarkers were discussed.The clear identification of the contour and boundary of liver lesion tissues was achieved,further promoting surgical excision.Finally,the effective alleviation and treatment of HIRI in hepatocytes were realized.The main contents of this dissertation are as follows:1.We designed and synthesized a two-color and two-photon fluorescence probe（Cy CA）for the simultaneous detection of mitochondrial O₂^·-and ONOO^-in HIRI mice livers.Cy CA has high specificity,high sensitivity,specific mitochondria-targeting capability and fast response.Take advantages of two-photon fluorescence imaging,synergistic increases in mitochondrial O₂^·-and ONOO^-levels were first observed in HIRI mouse livers.Proteomic analysis identified three nitration sites of tyrosine in arginase 1 induced by ONOO^-.ELISA assays and two-photon fluorescence imaging revealed that arginase 1 nitrated by ONOO^-could further promote the formation of ONOO^-,which ultimately aggravated intracellular redox imbalance and HIRI injury.This work illustrates the molecular mechanism of O₂^·--ONOO^--arginase 1 in HIRI process,and provided a new tool for the early diagnosis of HIRI.2.We created a dual-color and dual-reversible molecular fluorescence probe（UDP）for simultaneous and dynamic visualization of O₂^·-and ATP in HIRI process.UDP possessed excellent sensitivity,selectivity,and reversible responsiveness to O₂^·-and ATP.The dynamic visualization of synchronous O₂^·-bursts and ATP depletion in hepatocytes and mouse livers during HIRI was realized for the first time.A slight increase in ATP levels in the stage of reperfusion was confirmed.Intracellular O₂^·--succinate dehydrogenase（SDH）-mitochondrial（mito）reduced nicotinamide adenine dinucleotide（NADH）—mito ATP—intracellular ATP cascade signaling pathway in the HIRI process was first unveiled,which elucidated the synergistic effect between O₂^·-and ATP in HIRI.This work confirms the great potential of UDP in dynamically monitoring HIRI,contributing to promoting the early and accurate diagnosis of HIRI.3.We fabricated a lysosomal viscosity-activated NIR-Ⅱfluorescence probe（NP-V）for precise targeting of HIRI injury sites and resecting HIRI inflammatory lesions under intraoperative fluorescence navigation in living mice.NP-V had outstanding superiorities,including a significant fluorescence response upon viscosity,bright NIR-Ⅱfluorescence emission,specific lysosomal-targeting ability and good biocompatibility.Combined with NP-V and LW-OTf which could respond to lysosomal O₂^·-and ONOO^-,the lysosomal ROS-malondialdehyde（MDA）-cathepsin B cascade signaling pathway-mediated viscosity variation in HIRI was revealed for the first time,which demonstrated that lysosomal viscosity was an ideal biomarker for intraoperative navigation in HIRI.Precise navigation and resection of liver lesions in HIRI mice were conducted,confirming by histopathological examination.This work provides a new strategy and method for accurate intraoperative navigation of HIRI lesions.4.We developed a fluorescent diagnostic and therapeutic reagent（MB-Gly）,which could be triggered by HOCl to release methylene blue fluorophore and glycine for early detection and targeted treatment of HIRI.MB-Gly was able to visualize HOCl in hepatocytes during HIRI.MB-Gly showed antioxidative,anti-inflammatory and anti-apoptotic activities on HIRI hepatocytes due to the synergistic effect of HOCl-clearing urea bond and therapeutic glycine.This work provides a powerful tool for early diagnosis and targeted treatment of HIRI.