Study On Mechanism Of 4-PBA Regulates Autophagy Byinhibition Endoplasmic Reticulum Stress In PC12 Cells After Mechanical Injury

Objective:To investigate the relationship between endoplasmic reticulum stress and PI3 K / AKT / m TOR signaling pathway-mediated autophagy after spinal cord injury.Background:Spinal cord injury generally brings severe dysfunction,and there is currently no particularly effective treatment.With the discovery of autophagy and the in-depth study of the mechanism of autophagy in spinal cord injury,including the study of related mechanisms in vivo and in vitro,it is found that early stage of spinal cord injury,enhanced autophagy is beneficial to spinal cord cell survival and reduces apoptosis.In the study of autophagy,from macro autophagy to the involvement of intracellular organelles in autophagy,the endoplasmic reticulum is an organelle that synthesizes and processes proteins within the cell.Under external damage factors,protein abnormalities can cause endoplasmic reticulum stress.Appeared,endoplasmic reticulum stress has been found to be associated with a variety of neurodegenerative diseases,and there is a complex link between endoplasmic reticulum stress and autophagy.In this study,we explored the relationship between endoplasmic reticulum stress and autophagy signaling pathway and the effect of using endoplasmic reticulum stress inhibitor 4-PBA on autophagy by establishing an in vitro PC12 neuron injury model.Methods:(1)PC12 cells were routinely cultured and passaged,and then NGF was added to induce,and cultured until cell axonal growth(5-7days).The PC12 neurons was identified by using neuron-specific antibody NSE and TUBB3;(2)The model of spinal cord injury was established in fifth or seventh days by Que Haiping method.Then we collected cytosolic proteins at different time points(0h,1h,4h,8h,24h)after injury,and the autophagy marker protein LC3 and autophagy-related PI3 K / AKT / m TOR signaling pathways AKT/m TOR protein,ER stress related protein(Grp78/HSPA5)were analyzed by western blot.(3)The well-differentiated PC12 neurons were pretreated with LY294002 and 4-PBA for 1 h,respectively,and a mechanical injury model was previously established and divided into 6 groups(sham group,SCI group,LY294002 group,LY294002 + SCI group,4-PBA group,4-PBA + SCI group),cytosolic proteins were collected 4h after injury.Thep-AKT / p-m TOR protein and LC3 protein were analyzed by western blot.Result:1.PC12 cells after induced by NGF were observed by an inverted microscope,and synapses were formed in the PC12 neurons.The results of immunofluorescence identification showed that most of the PC12 neurons could be stained with NSE and TUBB3.2.Induced autophagy and endoplasmic reticulum stress after injury: The results of western blot showed that the level of LC3II/I increased significantly at 4 hours(P <0.05),and endoplasmic reticulum stress-related proteins HSPA5 / GRP78 increased gradually after 4 hours(P <0.05);3.PI3 K / AKT / m TOR pathway: Western blot results showed that the level of p-AKT began to increase at 1h,peaked at 4h(p <0.05)after injury,and the level of downstream p-m TOR began to increase at 1h,reaching a peak at 4h(p <0.05).After pretreat with PI3 K inhibitor LY294002,the levels of downstream p-AKT and p-m TOR were suppressed,LC3 II / I increased significantly(p<0.05);4.Endoplasmic reticulum stress and PI3 K signaling pathway-mediated autophagy: we giving 4-PBA to inhibit endoplasmic reticulum stress,downstream p-AKT and p-m TOR levels were inhibited,LC3II/I was significantly increased,(p<0.05).The obtained effect was similar to that of PI3 K inhibitor LY294002.Conclusion:1.Autophagy and endoplasmic reticulum stress were induced after mechanical injury;and autophagy through PI3 K / AKT / m TOR signaling pathway after mechanical injury;2.After 4-PBA inhibits endoplasmic reticulum stress,it can inhibit the phosphorylation level of downstream proteins AKT / m TOR of the PI3 K signaling pathway to enhance autophagy level and help reduce apoptosis.