Study On The Mechanism Of Gastrodin Delaying PC12 Cellaging By Intervening Hydrogen Peroxide-induced Ferroptosis

Neurodegenerative diseases are caused by the loss of cells and neurons in the brain and spinal cord.It mainly includes related diseases such as Alzheimer’s disease（AD）,Parkinson’s disease（PD）and Huntington’s disease（HD）.Studies have proved that aging is the key factor of most neurodegenerative diseases,and neurodegenerative diseases mainly occur in middle-aged and elderly people,which seriously threaten the health of middle-aged and elderly people.However,the relevant mechanism is still unclear.Ferroptosis,as a newly discovered way of cell death,is related to excessive accumulation of reactive oxygen species in lipids.It has been shown to be closely associated with various neurodegenerative diseases.Studies have found that the senescent tissues of nerve cells contain a large amount of iron,leading to the accumulation of excessive reactive oxygen species（ROS）,which leads to the occurrence of ferroptosis,Excessive accumulation of free iron in the nervous system will accelerate the senescence of nerve cells,leading to the death of neuron cells and accelerating the process of neurodegenerative diseases.However,it is not clear whether ferroptosis can accelerate the progression of neurotrophic diseases by promoting cellular senescence.Gastrodin is one of the main active components extracted from the dried tuber of Gastrodia elata.Previous studies have shown that GAS can play a neuroprotective role by exerting an antioxidant effect and regulating the mechanism of cell death.We still need to further explore whether GAS can delay aging by regulating ferroptosis and play a neuroprotective role.Therefore,this study induces oxidative stress through hydrogen peroxide（H₂O₂）,establishes a cell oxidative stress injury model,and further studies the relationship between cell senescence and ferroptosis as well as the regulatory effect of GAS,providing a new direction for the treatment of neurodegenerative diseases.ObjectivesTo study the protective effect of GAS on ferroptosis and senescence of PC12 cells induced by H₂O₂,and to explore the relevant mechanism of GAS on postponing PC12cell aging.MethodsThe oxidative stress injury model of PC12 cells was established by hydrogen peroxide.The viability of PC12 cells was detected by MTT assay and screened to determine the optimal treatment time and concentration of GAS and H₂O₂.The morphological changes of PC12 cells treated with GAS and H₂O₂were observed by inverted microscope.The morphologic changes of PC12 cells were detected by β-galactose aging staining.The morphological changes of PC12 cells were observed by transmission electron microscopy.The activity of superoxide dismutase（SOD）in PC12cells was detected by WST-1 method,the level of malondialdehyde（MDA）in PC12 cells was detected by thiobarbituric acid method（TBA）,the level of total glutathione in PC12 cells was detected by kit,and the level of intracellular reactive oxygen species （ROS）was detected by flow cytometry.Ageing-related proteins p53,p21,p16,FTH,GPX4,SLC7A11 and ACSL4 were detected by Western blot.Results1.H₂O₂induced PC12 cells senescenceAfter PC12 cells were damaged by H₂O₂,the proliferation of PC12 cells was inhibited,the cell morphology was changed,the activity of SA-β-gal was enhanced,and the protein expressions of p53,p16 and p21 were up-regulated.2.GAS can save PC12 cells senescence induced by H₂O₂GAS can enhance the viability of PC12 cells after H₂O₂cell injury,improve the cell morphology,reduce the activity of SA-β-gal,and down-regulate the expression of p53,p16,p21 proteins.3.Study on the senescence mechanism of PC12 cells damaged by H₂O₂by GASAfter GAS can protect ferroptosis revulsant（Erastin）injury PC12 cell vitality,ferroptosis inhibitors（Ferrostatin-1）,DFO protection with gastrodin can save PC12cell injury,H₂O₂damage mitochondrial morphological changes,mitochondrial backbone thickening,mitochondria smaller volume,double film shrinking and film density increase,GAS protection can be improved after the mitochondria,GAS can also be reduced H₂O₂damage after ROS,MDA,and improve the level of SOD and GSH.Pre-protected by GAS and Ferrostatin-1,GPX4,FTH and SLC7A11 proteins were up-regulated and ACSL4 proteins were down-regulated.Ferrostatin-1 preprotection also down-regulated the expression of p53,P21,and p16 proteins.ConclusionsH₂O₂induces oxidative stress in cells,which leads to ferroptosis and cell aging.The mechanism may be that H₂O₂can up-regulate the expression of p53,p16 and p21and down-regulate the expression of GPX4,FTH and SLC7A11 proteins.However,pretreatment with GAS and Ferrostatin-1 gradually reduced ROS levels in PC12 cells,inhibited ferroptosis,delayed cell aging and reduced cell death levels,as well as inhibited p53,p16,p21 expression and increased expression of GPX4,FTH and SLC7A11 proteins.Therefore,it can be concluded that GAS can inhibit ferroptosis through p53/SLC7A11/GPX4 pathway and delay cell senescence,reduce oxidative stress level,reduce ferroptosis and delay cell aging,thus achieving neuroprotective effect.