Study On The Mechanism Of Trehalose In Inhibiting Protein Aggregation Induced By Transient Ischemic Insults

Study on the Mechanism of Trehalose in Inhibiting ProteinAggregation Induced by Transient Ischemic InsultsObjective: To investigate the effect of trehalose on the protein aggregation caused by transient ischemic insults and its mechanism.Methods: Two vessels occlusion transient global ischemia rat model and SH-SY5 Y model of OGD were used.LDH release assay and trypan blue assay、Measurement of intracellular ROS、Behavioral Assessment with Morris water maze 、 Hematoxylin and Eosin staining and histological examination 、Immunohistochemical analysis、Proteasome activity assay、Gel Electrophoresis and Western Blotting and statistical analysis were used in this study.Results:(1)when compared with those in the sham group,the neurons in the ischemia group presented morphologically pink cytoplasms and polygonal pyknotic nuclei at reperfusion 72 h as revealed by microscopy combined with hematoxylin and eosin staining.48 hours pretreatment with 3.0% trehalose inhibited markedly the morphological changes caused by transient ischemia in the CA1 neurons.Statistical analysis showed as well that the percentage of living neurons at reperfusion 72 h was increased from 5.52± 2.31% to 62.56±8.17% by administration of 3.0% trehalose prior to ischemia.In Morris watermaze,the typical swimming tracks indicated trehalose made the ischemic rat search for platform in a more appropriate way,leading to shorter latency to find the platform and fewer excursions in QII.Although the rat in the ischemia group spent less time in QII than the sham operated rat did,trehalose significantly elongated their retention in the target quadrant.(2)In western blotting analysis,the increase of ubiquitin-labeled protein aggregates at reperfusion 24 h was attenuated significantly by 3.0%trehalose.Immunochemical staining revealed as well that the ubiquitin-labeled protein aggregates at reperfusion 24 h were obviously inhibited by 3.0%trehalose in the CA1 neurons.Pretreatment with 3.0% trehalose markedly reversed the decreased proteasome activity from 47.56%±4.89% to71.88%±7.12%,53.25%±4.21% to 73.35%±6.96% and 46.88%±3.79% to75.28%±7.86% at reperfusion 12 h,24 h and 48 h,respectively.(3)As revealed by LDH release assay,48 h pretreatment with trehalose at0.5 or 5.0mmol/L inhibited significantly OGD-induced cell death at reoxygenation 6h.As demonstrated by western blotting analysis,pretreatment with trehalose at 5.0mmol/L reversed OGD-induced increases in the levels of LC3 II and Beclin-1 and decrease in the selective substrate of autophagy p62 at re-oxygenation 2 h.We found that 1 hour pretreatment with autophagy inhibitor3 MA at 2mmol/L not only inhibited OGD-induced changes in the protein levels of LC3 II,Beclin-1and p62,but also attenuated the cell death at re-oxygenation6 h.(4)5.0mmol/L trehalose attenuated markedly MG-132-induced reduction of proteasome activity,increase of LDH release and formation of ubiquitin-labeled protein aggregates.(5)the images under fluorescence microscope revealed that 48 h pretreatment with trehalose at 0.5mmol/L or 5.0mmol/L significantly suppressed the increase of green fluorescence in the SH-SY5 Y cells at re-oxygenation 2h after being stressed with OGD for 24 h.Statistical analysis of the fluorescence density proved that both 0.5mmol/L and 5.0mmol/L trehalose could effectively inhibit the increase of intracellular ROS level caused by OGD.(6)As shown by western blotting analysis and blot density analysis,OGD-induced expressional upregulation of PERK,phosphorylated PERK,phosphorylated eIF2α,IRE-1 and GRP78 at re-oxygenation 2h were all suppressed in the SH-Y5 Y cells pretreated with 5.0mmol/L trehalose.This indicated that trehalose inhibited OGD-induced ER stress in SH-SY5 Y cells.Conclusion: In this study,we demonstrated that trehalose inhibited protein aggregation caused by ischemic insults via preservation of proteasomal function,which was associated with the inhibitory effect of trehalose on oxidative stress and ER stress.Therefore,proteasome played a crucial role in regulation of protein aggregation caused by ischemic insults,and trehalose is aneffective neuroprotectant against ischemic neuronal death.