Hydrogen-rich GO-C₃N₄-Pd Nanomaterials Release Hydrogen Against Oxidative Stress Damage Of PC12 Cells Induced By Aβ_25-35

Alzheimer’s disease（Alzheimer’sdisease,AD）,a fatal neurodegenerative disease,is the most common dementia,memory loss and cognitive impairment,the later development will affect daily life.More and more studies have shown that amyloid plaques formed by abnormal deposition of Aβpeptides are the main cause of AD.Accumulation in the brain will lead to oxidative stress and inflammatory damage,mitochondrial dysfunction and then affect the function of neurons.Screening candidate drugs against Aβpeptide toxicity by cultured nerve cells in vitro is an effective strategy for the development of drugs for the treatment of AD patients.One possible treatment strategy is to prevent the formation of amyloid plaques by inhibiting the accumulation of Aβ and antagonizing the damage caused by oxidative stress.Hydrogen molecule is a safe and effective antioxidant,which can selectively scavenge high cytotoxic reactive oxygen species such as·OH,showing the potential for the treatment of AD by reducing oxidative stress.Research into hydrogen therapies has gained popularity in the last two decades.However,due to the characteristics of low solubility and easy diffusion of hydrogen,there is no effective way to realize the sustained and efficient accumulation of hydrogen in the brain of Alzheimer’s disease patients.Nanomaterials are excellent carriers for drug delivery according to their superior physical and chemical properties,can make up for the shortage of hydrogen molecules,and are promising materials for hydrogen therapy.So I synthesized hydrogen-rich GO-C₃N₄-Pd nanomaterials for the first time to antagonize the damage caused by oxidative stress to PC12 cells to inhibits the aggregation of Aβ,prevents the formation of amyloid plaques,restores mitochondrial dysfunction,blocks synapses and neuronal apoptosis,and provides a theoretical basis and scientific basis for subsequent research on hydrogen therapy for AD.Objective:to explore the protective effect of hydrogen-rich GO-C₃N₄-Pd nanomaterials on oxidative stress injury of rat adrenal pheochromocytoma cells（ratpheochromocytoma,PC12）induced by Aβ_25-35,and try to study its related mechanism.Methods:（1）the PC12 cells of passage 2-10 were inoculated in the orifice plate with Cultrex Mouse Collagen IV in advance,After 24 h,added 50ng/m L NGF to induce neuronal cells.On the 6th day,the induced neuronal cells were verified by cellular immunofluorescence and Western blotting with Microtubule-Associated Protein-2（MAP2）antibody.（2）the AD cell model was made by adding aging Aβ_25-35 solution with the final concentration of 0（control group）,10,20,40,60,80μm L/mol for 24 h to the differentiated mature neurons.The working concentration and action time of Aβ_25-35 to construct the cell oxidative stress injury model were determined by CCK8 method.（3）GO-C₃N₄-Pd composite nanomaterials were obtained by ultrasonic treatment of GO-C₃N₄-Pd powders.GO-C₃N₄-Pd composite nanomaterials were characterized by Transmission Electron Microscopy（TEM）,Scanning Electron Microscopy（SEM）and Zeta potentiometer.Hydrogen-rich GO-C₃N₄-Pd composite nanomaterials were prepared by injecting high pressure hydrogen 30min into GO-C₃N₄-Pd composite nanomaterials.（4）the AD cell models of the control group and the experimental group were set up,and the final concentrations of hydrogen-rich GO-C₃N₄-Pd composite nanomaterials were added to the experimental group at the final concentration of 20,60,200,600,2000μg/m L for 24 hours to detect the related indexes.（5）the cell survival rate of the control group and the experimental group AD cell model was detected by CCK-8.（6）the contents of oxidation index ROS,NO and antioxidant enzyme SOD in the AD cell model treated with hydrogen-rich GO-C₃N₄-Pd composite nanomaterials were detected by special kits of ROS,NO and SOD.（7）the mitochondrial membrane potential of the AD cell model treated with hydrogen-rich GO-C₃N₄-Pd composite nanomaterials was detected by JC-1 probe.The results were as follows:（1）the PC12 cells induced by NGF on the 6th day were verified by MAP2 cell immunofluorescence and Western blotting to express MAP2 protein,indicating that the cells at this time had the characteristics of mature neurons.（2）CCK8 results showed that compared with the blank control group,the survival rates of PC12 cells treated with 80μmol/m L and 100μmol/m L Aβ_25-35 were 63.05%and 46.60%respectively（P<0.0l）.In the follow-up experiment,we used 80μmol/m L Aβ_25-35 to treat PC12 cells for 24 hours to construct AD cell model.（4）CCK-8 detection results showed that 2000 ng/m L hydrogen-rich GO-C₃N₄-Pd composite nanomaterials could improve the survival rate of PC12 cells compared with the control group.（5）the results of ROS,NO and SOD special kits showed that 2000 ng/m L hydrogen-rich GO-C₃N₄-Pd composite nanomaterials could effectively reduce the contents of ROS and NO,and increase the activity of SOD（P<0.05）;（6）2000 ng/m L hydrogen-rich GO-C₃N₄-Pd composite nanomaterials could also significantly improve the decrease of mitochondrial membrane potential and restore mitochondrial function effectively.Conclusion:（1）undifferentiated PC12 cells can differentiate into mature neurons induced by NGF and assisted by Cultrex Mouse Collagen IV,which can replace primary neurons for neurobiological and neurochemical studies.（2）Aβ_25-35 can damage PC12 cells,resulting in the decrease of cell survival rate and oxidative stress injury,which can be used to construct AD cell model.（3）hydrogen-rich GO-C₃N₄-Pd composite nanomaterials can reduce the levels of ROS and NO in PC12 cells treated with Aβ_25-35,increase SOD activity,reduce cell death,improve mitochondrial membrane potential,resist the damage caused by oxidative stress,and protect PC12 cells.Hydrogen-rich GO-C₃N₄-Pd composite nanomaterial is expected to be further studied for the treatment of AD.