Role Of RAGE-Mediated Autophagy In Hydrogen Peroxide-Induced Oxidative Stress In HT22 Murine Hippocampal Neuronal Cells

BackgroundOxidative stress injury is involved in the occurrence and development of a variety of nervous system diseases and is an important target for research and treatment of central nervous system diseases.The receptor for advanced glycation end products(RAGE)is a transmembrane protein and belongs to the immunoglobulin superfamily of receptors.RAGE can be combined with a variety of ligands and is widely involved in the inflammatory cascade And cell migration process.RAGE is constitutively expressed in the brain during embryonic development;it is downregulated in adulthood,and its level is increased in elderly individuals.In fact,the expression of RAGE is low under physiological conditions,and under external stimulation,RAGE participates in the inflammatory cascade through upregulation.In recent years,studies have shown that the expression of RAGE increases after cerebral ischemia,and the survival rate of neurons can be improved by regulating RAGE.RAGE plays an important role in the process of neuronal oxidative stress injury,however,its specific mechanism is not yet clear.Cellular autophagy can remove excess and damaged organelles,degenerated proteins,help cells adapt to changing nutritional conditions,and maintain the homeostasis of cells.However,autophagy is a double-edged sword.Studies have shown that in certain Under conditions,such as oxidative stress and excessive inflammatory response will cause excessive activation of autophagy,thereby promoting apoptosis.Recent studies have shown that RAGE-induced autophagy plays an important role in the early metastasis of pancreatic cancer.Increased autophagy leads to the induction of IL-6,activation of STAT3 and its translocation to mitochondria,leading to increased production of adenosine triphosphate(ATP)and Cancer cell proliferation;RAGE deficiency can promote cell autophagy through the AMPK/mTOR signaling pathway.AimIn this study,by constructing a H2O2-induced oxidative damage model of HT22 hippocampal neuronal cells,we observed the expression level and autophagy level of HT22 hippocampal neuronal cells,and targeted the knockdown of RAGE gene by lentivirus to deeply explore the RAGE-regulated cellular autophagy The role and related mechanisms of meta-oxidative stress injury.MethodsThis project intends to study the following contents through in vitro experiments:(1)To construct an oxidative damage model of H2O2-induced HT22 hippocampal neuron cells,to simulate neuronal oxidative damage,to apply CCK8 cell activity to detect the degree of cell damage,and to apply Western Blot to detect RAGE expression levels;(2)Construction of the lentiviral vector RAGE knockdown HT22 hippocampal neuron cell line,the efficiency of HT22RAGE knockdown was verified by quantitative RTPCR(qRT-PCR)and Western Blot;(3)RAGE inhibition Agent,NF-κB inhibitor and RAGE(-/-)model,respectively observe the expression levels of HMGB1,NF-κB,and autophagy-related protein Beclin-after H2O2-induced oxidative damage to HT22 hippocampal neuron cells 1.The expression level of LC3B and p62,the expression level of LC3B-Ⅱ was detected by immunofluorescence;the number of autophagosomes was observed by transmission electron microscopy to study the role of RAGE/NF-κB-regulated autophagy in the model of oxidative damage of hydrogen peroxide-induced HT22 cells effect.Results(1)After H2O2 induces oxidative damage to HT22 hippocampal neurons,the expression level of RAGE rises and the level of autophagy increases.The degree of damage is dependent on the concentration of H2O2.(2)In the model of H2O2-induced HT22 cell damage,the level of autophagy is increased,and by knocking down the RAGE gene or using RAGE inhibitors,the effect of H2O2-induced oxidative damage-mediated autophagy is significantly reduced,and the H2O2-induced cell oxidative damage is reduced,Increased cell survival rate of HT22 hippocampal neurons.(3)In the model of oxidative damage of HT22 hippocampal neurons induced by H2O2,inhibition of RAGE reduces the expression of HMGB1 and the activity of NF-κB,and inhibition of NF-κB activity reduces the effect of H2O2 on the expression of HMGB1;meanwhile,H2O2 mediates autophagy Lead activation also decreased significantly.ConclusionH2O2-induced oxidative stress promotes cell RAGE protein expression and regulates autophagy by activating the NF-κB/HMGB 1 signaling pathway;inhibiting RAGE protein expression helps reduce H2O2-induced excessive autophagy levels and promotes HT22 hippocampal neurons Survive.