Proteome Analysis Of Hypoglycemic Brain Injury And Functional Studies Of Protein DJ-1

BACKGROUND As a common complication of glycemia control in diabetes, hypoglycemia is most likely to lead to brain damage. The term of ‘Glucose reperfusion-induced brain injury’ was put forward, making the understanding of the pathogenesis of hypoglycemic cerebral injury is much more obscure, also leading to a rethinking of the traditional clinical treatment of hypoglycemia. Research on the pathological mechanisms of brain damage in the periods of hypoglycemia and recovery may provide therapeutic targets and ideas for brain protection in the process of the clinical treatment of hypoglycemia.OBJECTIVES The present study aims to screening the differential proteins associated with hypoglycemic brain injury based on comparative proteomic analysis using isobaric tag for relative and absolute quantitation(i TRAQ) approach combined with liquid chromatography-tandem mass spectrometry(LC-MS/MS) analysis, further investigating the possible roles of these proteins to clarify the probable molecular mechanisms of hypoglycemic brain injury, providing theoretical basis for treatment of hypoglycemic cerebral injury.METHODS Hypoglycemic rat model was induced by intraperitoneal injection of insulin.Protein extractions of the brain tissues were digested and then the peptides were labeled with 4-plex i TRAQ tags reagent. Labeled samples from different groups were pooled and separated by liquid chromatography-tandem mass spectrometry(LC-MS/MS). Differentially expressed proteins between hypoglycemic and sham hypoglycemic rat’s brain were identified and further validated. At last, specific targeted si-RNA was transfected to the cultured cells to evaluate the biological roles of the protein.RESULTS By comparing the profile of protein expression between hypoglycemic and sham hypoglycemic rats’ brains, a total of 147 kinds of differentially expressed proteins in hypoglycemia were screened on a relative fold of expression more than 1.5 or less than 0.67 to that in the sham hypoglycemia. Four proteins——AQP4, NDRG1, DJ-1 and Clusterin were validated by q RT-PCR and western blot analysis in duplicate animal model, showing the consistence between the change trends of the expression and the proteomic results. AQP4 and DJ-1 were further validated on the in vitro astrocyte culture after glucose deprivation. A protective role of DJ-1 against cell death was demonstrated after using RNAi techology to knock down the DJ-1 in the astrocytes subjected to glucose deprivation. AMPK phosphorylation was elevated but autophagy flux was impaired in astrocytes after glucose deprivation. Autophagy inhibitors exacerbated the hypoglycemic astrocyte death. Knockdown of DJ-1 decreased the AMPK phosphorylation and increased the m TOR activity, and impaired the autophagy activation.CONCLUSION DJ-1 plays a protective role in hypoglycemic brain injury, reducing the astrocytic cell death after glucose deprivation which was partialy mediated by the regulation of pro-survival autophagic process.