The Role Of Impaired Mitophagy In PINK1/Parkin Pathway In High Glucose-Induced Oxidative Damage Of Sensory Neurons

BackgroundDiabetic peripheral neuropathy(DPN)is one of the most common chronic complications in diabetes,and more than 50%of patients will have diabetic peripheral neuropathic pain(DPNP).DPNP seriously affects patients’ quality of life.Due to the complicated pathogenesis,it is still unable to completely relieve the pain in clinic.Mitochondrial dysfunction plays an important role in the pathogenesis of DPNP.When mitochondrial dysfunction occurs,it will trigger oxidative stress and lead to cell damage.Mitophagy is an important process to remove damaged mitochondria,which is mainly guided by PINK1/Parkin pathway.Our research group found that mitophagy was inhibited and the expression of PINK1 decreased in the Dorsal root ganglion(DRG)of DPNP mouse model.In this study,we will use mouse neuroblastoma and rat DRG hybrid cell line(ND7/23 cell line)to explore the role of mitophagy damage of PINK1/Parkdn pathway in triggering oxidative damage of sensory neurons in high glucose environment,and provide some theoretical basis for the prevention and treatment of DPNP.Objective1.To observe the effects of different concentrations of glucose on the mitophagy and oxidative damage of ND7/23 cells at different times,and to establish a high glucose model of impaired mitophagy.2.By regulating the expression of PINK1,explore the mechanism of impaired mitophagy in the PINK1/Parkin pathway that induces oxidative damage to sensory neurons in high glucose.Methods1.The changes of cell viability of ND7/23 cells after glucose concentration gradient(25 mM,50 mM,75 mM)and time gradient(24 h,48 h,72 h)were measured,and the expression changes of mitochondrial autophagy proteins and apoptosis-related proteins in PINK1/Parkin pathway after glucose concentration gradient and time gradient intervention were detected by western blot.The co-localization of COXIV-LC3B(autophagy formation)and COXIV-LAMP1(autophagosome formation),mitochondrial autophagy,mitochondrial membrane potential and mitochondrial ROS level were detected by immunofluorescence.Finally,the optimal glucose concentration and intervention time of the model were determined.2.Knock down the expression of PISK1 in ND7/23 cells by transfection of siRNA,and the grouping is set as:control group with negative control siRNA;Control group with PINK1siRNA;High glucose group with 50 mM glucose and negative control siRNA,High sugar group with 50mM glucose and PIBK1-siRNA.The changes of cell viability in each group were determined,and the expression changes of mitochondrial autophagy proteins and apoptosisrelated proteins in PINKl/Parkin pathway were detected by western blot The co-localization of COXIV-LC3B and COXIV-LAMP1,mitochondrial autophagy,mitochondrial membrane potential and mitochondrial ROS level were detected by immunofluorescence.Results1.An in vitro model of mitophagy inpaired by high glucose(50 mM glucose,48 h)was established,in which PINK1 expression,parkin phosphorylation decreased,mitophagy inhibited,apoptosis activated,mitochondrial membrane potential decreased and mitochondrial ROS increased.2.After inhibition of PINK1 expression,mitophagy of ND7/23 cells was further inhibited,apoptosis was activated,mitochondrial membrane potential was further decreased and mitochondrial ROS was further increased.Conclusion1.Under high glucose,the mitophagy of the PINK1/Parkin pathway is impaired,which will lead to aggravated mitochondrial dysfunction,lead to oxidative damage in ND7/23 cells,and activate the apoptosis pathway.2.Inhibiting the expression of PINK1 reduces the phosphorylation ratio of Parkin,further aggravates mitochondrial dysfunction,aggravates the degree of damage to mitophagy,aggravates the degree of oxidative damage,and activates the apoptosis pathway.