An NADH-selective And Sensitive Fluorescence Probe To Evaluate The Neuronal Cell Stress In Rats

Stress is an individual-level specific or non-specific adaptive response to a variety of stressors.Traumatic stress or prolonged exposure to stress is a serious risk to human health,especially in high-risk operations,where pathological stress affects worker productivity,increases errors at work and leads to frequent accidents.Severe stress disorders are characterised by complex symptoms,long duration of illness,potential group onset and lack of specific treatments,which not only damage health at the individual level,but may also have serious negative effects on social public health,battlefield decisive victories and national strategic development.According to statistics,75%～90%of the diseases prevalent in today’s society are associated with the activation of stress mechanisms.According to the 2018 WHO survey,about one in five(22%)people who have experienced war or a major catastrophic event in the last 10 years suffer from stress-related mental disorders.In the fight against the COVID-19,stress was detected in 73.4%of frontline workers.Stress disorders have become one of the major causes of harm to individual health and social development.As a result,there is a desire to further understand the nature of stress and to explore means of detecting it in real time and accurately,in order to provide a basis for early intervention in stress injury.Nicotinamide adenine dinucleotide(NADH)is a key endogenous molecule in the oxidative respiratory chain in cell mitochondria,and as the most important coenzyme and core metabolite in the organism,it is often used as a key indicator to evaluate the metabolic status of the cell.It has been found that changes in intracellular NADH levels are positively correlated with the level of stress in the organism,suggesting that the detection of changes in intracellular NADH levels may lead to an accurate assessment of the level of stress in the organism.In this paper,we have developed an ion-based fluorescent probe for the detection of intracellular NADH levels in living cells based on a review of the relevant literature at home and abroad,and initially validated and analysed its effectiveness in the assessment of cellular stress injury models.The main contents of this paper are as follows.1.Construction of a highly sensitive ionic NADH fluorescent probeThe key intermediate was synthesized using reflux treatment in ethanol,and the intermediate was modified by N-methylation using iodomethane to obtain an ionic probe C with fluorescent detection of NADH.The Probe C uses quinolinium as the NADH response site.Before interacting with NADH,both ends of its structure are positively charged and the probe has no fluorescence signal.When the probe interacts with NADH,it transforms into an electron deficient state with a positive charge at one end and an electron rich state with an amine group attached at the other end,resulting in a fluorescent luminescence at 510 nm excitation.In addition,a colour change from pale yellow to pink is visible to the naked eye upon interaction with NADH.Fluorescence imaging of intracellular NADH is achieved using the principle that changes in chemical structure cause changes in the luminescent signal.2.Evaluation of the effectiveness for the Probe CTo achieve real-time,accurate assessment of cellular stress damage,the investigators validated and further modified the specificity,sensitivity and stability of Probe C.The Probe C specifically binds NADH without interference from ions,groups and compounds commonly found in living organisms.The detection limit of the probe can reach 0.36 n M,which is far below normal physiological levels,and can detect small changes in intracellular NADH levels.The probe has a high stability after interaction with NADH.After binding to NADH,the fluorescence signal does not decay within 1hour,and no significant disturbance in fluorescence signal intensity is seen in the p H range of 4～9.In addition,the intracellular application of the probe was analysed and the results showed that a normal working dose(≤10μM)of the probe solution had a low impact on cell viability.The combination of laser confocal imaging andρ~0 cell mitochondrial function activation assays to locate the probe working sites up to the mitochondrial subcellular organelle level,which further confirms the usefulness of the Probe C.3.Construction of a stress model for PC12 cells and application of the Probe CCombining the nature of the organism’s stress response,the researchers constructed two stress injury models,namely the hormonal(corticosterone)stress injury model and the hypoxic stress injury model for PC12 cells,as well as validating the construction effect of the models by detecting the changes of the main indicators after stress.The experimental results showed that the MDA,LDH,ATP and NADH contents in both cell models showed an increasing trend,while the SOD contents showed a decreasing trend.The validation results were basically in line with the gradual change in stress intensity,indicating that the stress models were successfully constructed.The results of using Probe C to detect changes in NADH levels after stress in the cell model showed that the amount of NADH detected in the cells gradually increased with the increase of stress in the model.This indicates that the probe enables the detection and assessment of stress damage at the cellular level.In summary,a specific,sensitive and stable ionic NADH fluorescent probe was constructed by chemical synthesis.The application of the probe in stress injury models has confirmed the value of the probe in the assessment of stress injury at the cellular level.This provides a new method for the detection and assessment of molecular metabolism at the cellular stress level.