In Situ Fluorescence Imaging Reveals The Function Of Oxidative Stress Related Bioactive Molecules In Ischemic Reperfusion Injury

Ischemia reperfusion injury（IR injury）is an irreversible injury that occurs when the blood flow is restored to the tissues or organs after ischemia.This injury will aggravate the structural damage of tissues or organs,leading to the dysfunction and even death.However,at present,the pathophysiological mechanisms of IR injury are not sufficiently understood,which hinds the prevention,treatment and prognosis of IR injury.Oxidative stress is widely involved in cell signal transduction,biochemical reaction and pathophysiological processes.Existing studies have shown that oxidative stress and the following lipid peroxidation are key links in the occurrence and development of IR injury,and related bioactive molecules such as reactive oxygen species（ROS）and reactive carbonyl species（RCS）play essential regulatory roles in the pathophysiological mechanisms of IR injury.Among them,malondialdehyde（MDA）,as the highly toxic RCS,is one biomarker of oxidative stress and lipid peroxidation,as well as one of the indicators of IR injury,which is easy to induce cross-link of biomacromolecules or dysfunction of proteins.Superoxide anion radical(O₂^·-),as the first ROS produced during oxidative stress,is involved in regulating the production and levels of other ROS in IR injury,which could cause the oxidative damage to proteins.Therefore,exploring the levels,the subcellular distributions and the regulatory effects of above oxidative stress related bioactive molecules are of great significance for revealing the pathophysiological mechanisms of IR injury.Fluorescence imaging technology is widely used in the field of visualization of bioactive molecules in living cells and in vivo as its high sensitivity,extraordinary selectivity,excellent spatial-temporal resolution,simple operation,rapid response,and little biological trauma.In particular,long wavelength excitation fluorescence imaging methods such as two-photon fluorescence imaging and near-infrared fluorescence imaging,which have deep tissue penetration and low background fluorescence interference,are more suitable for the in-situ fluorescence imaging of bioactive molecules in vivo.So far,fluorescence imaging materials for detecting MDA or subcellular MDA in IR injury have not been reported.In addition,fluorescence imaging materials for the in-situ and ratio detection of O₂^·-in IR injury are rarely reported.Therefore,in order to in-situ explore the levels,the subcellular distributions and the regulatory effects of above oxidative stress related bioactive molecules in IR injury,it is urgent to develop exquisite two-photon or near-infrared fluorescence imaging materials.Aiming at the issues of in-situ imaging of oxidative stress related bioactive molecules in IR injury,this thesis developed series of fluorescence imaging materials with superior performance and excellent biocompatibility.MDA,lysosomal MDA,lipid droplet MDA in cerebral IR injury and mitochondrial O₂^·-in hepatic IR injury were in-situ visualized in living mice.The fluctuations and the distributions of these oxidative stress related bioactive molecules under cerebral or hepatic IR injury in living mice were systematically studied,and the regulatory roles of these oxidative stress related bioactive molecules were preliminarily discussed,which provided powerful imaging tools and key information for revealing the oxidative stress related molecular mechanisms in IR injury.The main contents of this thesis are as follows:1.A two-photon polymer carbon dot material,BH-PCDs,was designed for the in-situ fluorescence imaging of MDA in living mouse brains.The material BH-PCDs was made by one-pot in one step,and the level of MDA can be specifically and quickly monitored.By using two-photon fluorescence imaging technique,the increased level of MDA was observed in the brains of mice under cerebral IR injury for the first time.In addition,in-situ imaging revealed that O₂^·-regulated MDA levels in cerebral IR injury.Combined with the commercial kits,it was found that the overproduction of MDA inactivated glutamine synthetase,resulting in elevated glutamate levels and leading to glutamate neurotoxicity in the mouse brains under cerebral IR injury.This work provides a promising tool for studying MDA related molecular mechanisms in cerebral IR injury,and also reveals a regulatory role of MDA on glutamate neurotoxicity in cerebral IR injury.2.A two-photon fluorescence probe,Lyso-MCBH,was synthesized for in-situ fluorescence tracing of lysosomal MDA in living mouse brains.Lyso-MCBH used the morpholine group to specifically target the lysosomes,and Lyso-MCBH used the hydrazide group to selectively recognize MDA.In-situ fluorescence imaging results showed that the lysosomal MDA level erupted in living brains of mice under cerebral IR injury.Excessive lysosomal MDA was found to affect the efficacy of vitamin B₁₂ by blocking the transport of vitamin B₁₂ from lysosome to cytoplasm.More importantly,the expression and function of vitamin B₁₂ transporter LMBD1was proved to be associated with the excessive lysosomal MDA.This work provides a perspective strategy for exploring the lysosomal MDA related biological mechanisms in cerebral IR injury,and also proposes a regulatory effect of lysosomal MDA in vitamin B₁₂ transport under cerebral IR injury,which could provide new insight into the inefficacy of vitamin B₁₂ in cerebral IR injury.3.A near-infrared fluorescent probe,Ld-NBH,was constructed for the in-situ fluorescence capturing of the lipid droplet MDA in living mouse brains.The core of Ld-NBH was the commercial dye of lipid droplet,Nile blue,which can selectively target the lipid droplet.The recognition group of Ld-NBH was the hydrazine group.In-situ fluorescence imaging results exhibited that the MDA level in lipid droplet boosted in living brains of mice under cerebral IR injury.With the help of commercial lipid dye,excessive MDA was found to mediate the up-regulation of lipid droplet-associated protein perilipin-5（Plin 5）,eventually leading to the lipid accumulation in lipid droplet under cerebral IR injury.This work provides a promising tool for studying the lipid droplet MDA related molecular mechanisms in cerebral IR injury,and also reveals a role of MDA on regulating lipid metabolism under cerebral IR injury.4.A ratio two-photon fluorescence probe,CST,was developed for the in-situ fluorescence capturing of mitochondrial O₂^·-in living mouse livers.CST specifically targeted to the mitochondrial intima through the Safranine T group,and the caffeic acid on CST was used to selectively identify O₂^·-.Based on the change of the ratio fluorescence signal,up-regulated level of mitochondrial O₂^·-in living mouse liver under hepatic IR injury was real-time,quantitative,and in-situ detected.Combined with commercial cytoplasmic O₂^·-probe,the relationship between mitochondrial O₂^·-and cytoplasmic O₂^·-under hepatic IR injury was in-situ investigated.Further results showed that O₂^·-transported from mitochondria to cytoplasm via VDAC channel,resulting in the inactivation of aconitase in the cytoplasm.Moreover,blocking VDAC channel and increasing the activity of mitochondrial Mn-SOD could effectively inhibit the hepatic IR injury.This work provides a new method for exploring the biological mechanisms related to mitochondrial O₂^·-,and reveals a transport pathway of mitochondrial O₂^·-in hepatic IR injury,which could be a prospective therapeutic target.