Uncovering The Function Of Peroxisomal O₂?·-?in Mouse Brains With Depression Phenotypes By Fluorescence Imaging

Depression is a mental illness with high morbidity and low cure rate,which clinical manifestations are complex and changeable,and depression has a trend to recurrence.The understanding of the pathophysiology of depression is still scare due to its complex etiology,and there are many hypotheses about the molecule mechanism,oxidative stress is one of the hypotheses.The lack of clinical gold standard for the diagnosis of depression has been a difficult problem in the field of modern medicine.Peroxisome can not only produce reactive oxygen radicals?ROS?,but also remove ROS,which plays a vital role in maintaining the intracellular redox balance.Superoxide anion radical?O₂?·-??is the first ROS produced in organism,and its content represents the level of oxidative stress directly.As another important ROS in the organism,hydrogen peroxide?H₂O₂?is directly involved in the regulation of various intracellular redox signal transduction.However,the correlation between O₂?·-?and H₂O₂ is unknown in the occurrence and development process of depression.Fluorescence imaging technology has the characteristics of "seeing is believing" and can image various biomarkers in the living brains.Two-photon fluorescence imaging has the advantages of high resolution,high sensitivity,deep tissue penetration and low light bleaching,which can real-time monitor the bioactive molecules related to depression in non-invasive manner.However,due to the complex biological background in the brain and the impenetrable blood-brain barrier,the fluorescence probe used to detect peroxisomal O₂?·-?in brains has not been reported.Based on the above reasons,we designed and synthesized a two-photon fluorescence probe TCP to detect peroxisomal O₂?·-?in mouse brains with depression phenotypes.Combining with the fluorescent probe to detect H₂O₂,we visualized the concentration changes of peroxisomal O₂?·-?and intracellular H₂O₂ in the brains of mice with depression phenotypes.We further explored the relevant signaling pathways mediated by peroxisomal O₂?·-?in the occurrence and development process of depression.The following is the work carried out in this paper:1.Two-photon fluorescence imaging of peroxisomal O₂?·-?and intracellular H₂O₂ in mouse brains with depression phenotypes.We designed a two-photon fluorescent probes TCP for monitoring peroxisomal O₂?·-?in depression phenotypes mouse brains.The probe TCP uses caffeic acid as the recognition group and chromo fluorophore,small peptide?QSKL?as the targeting group to peroxisome.Using TCP,we found that under the stimulation of dimethylestradiol?2-Me?,the peroxisomal O₂?·-?content in PC12 cells was significantly higher than that in control cells.At the same time,with the probe for detecting H₂O₂,we observed that when peroxisomal O₂?·-?concentration increased,the intracellular H₂O₂ level also increased significantly.We performed fluorescence imaging on mouse brains and found that the concentrations of peroxisomal O₂?·-?and intracellular H₂O₂ in the brains of mice with depression phenotypes were both increased than that in control mice.This work provides strong evidence for the relationship between oxidative stress and depression,which is helpful to understand the pathogenesis of depression.2.Exploring the O₂?·-?mediated signaling pathways in the brains of mice with depression phenotypes.On the basis of the above work,we further study why the peroxisomal O₂?·-?could mediate the concentration of intracellular H₂O₂ and explore the effects of the increased intracellular H₂O₂ level on the downstream tryptophan hydroxylase-2?TPH2?.Taking advantage of fluorescence imaging technology and enzyme assay kit,we found that the excessive peroxisome O₂?·-?produced in the process of oxidative stress,resulting the inactivation of catalase?CAT?and leading to the intracellular H₂O₂ increased.The excess H₂O₂ further impaired the TPH2 content,ultimately inducing depression.Using protein mass spectrometry,we found oxidative damage sites of CAT and TPH2 during oxidative stress.This work is expected to improve the related signaling pathway mediated by peroxisomal O₂?·-?during the onset of depression,which is meaningful to find the key target for the treatment of depression.