Protection Of 78kDa Glucose Regulated Protein To Neurons In Endoplasmic Reticulum Stress

Background: Apoptosis is an important mechanism of neuronal loss in ischemic cerebrovascular disease, which is the most common life threatening neurological event in China. Recent studies suggest that endoplasmic reticulum stress (ERS) is linked to apoptosis, and caspase-12 and GFP78 are recognized as potential factors in this process. However, no direct evidence have been presented to prove GRP78 roles in apoptosis induced by ERS. In this study, we used oxygen-glucose deprivation (OGD) method to establish ischemic models of primary hippocampal neurons. In order to determine the exact effect of GRP78 on ERS, the expression of GRP78 was induced when 2-deoxy-D-glucose was exposed to the primary hippocampal neurons, and blocked by RNAi. Integrated methods were used to present evidence for GRP78 protective roles on neurons in ERS and the molecular mechanism involving caspase-12 . Methods: Firstly, primary hippocampal neurons were deprived from rats and cultured in serum-free media. Double-labelling technique of immunofluorescence was used to examine the purity and growth of primary hippocampal neurons. Then, we established OGD model of hippocampal neurons, and identified the peak apoptosis time point by TUNEL and modified CCK8 methods. RT-PCR and western blot were applied to examine the induction effect of 2-deoxy-D-glucose and blocking effects of GRP78siRNA on GRP78 expression. The role of GRP78 on the consequent apoptosis was determined by TUNEL assay. We used western blot to determine the effect of different GRP78 level on the activities of caspase-12, m-calpain and IRE1.Results: After OGD stress, the viability of hippocampal cells gradually decreased at the time points of 6h, 24h and 48h. The duration of OGD stress significantly affected the time course of apoptosis. When 3h OGD was applied, the cell viability decreased to 12.16%±3.76%. The 2hOGD distress caused peak apoptosis at the time point of 24h. After 24h incubation with 2DG, the mRNA levels of GRP78 respectively increased to 3.35±0.62 fold and 3.17±0.46 fold higher than the control group. The designed GRP78siRNA inhibited the expression of GRP78, which decreased to 37.28%±8.67% (mRNA) and 42.48%±7.66% (protein). Accordingly, the active level of caspase-12, m-calpain and IRE1 protein respectively decreased to 0.42±0.15, 0.63±0.08, and 0.31±0.06 folds of control group. The block of GRP78 by RNAi caused an increase of apoptosis to 78.36%±7.61% at 24h time point. The active level of caspase-12, m-calpain and IRE1increased to 2.13±0.28, 1.42±0.21, and 2.76±0.32 folds of the OGD control group.Conclusions: The distress of 2h OGD followed by 24h recovery caused the highest apoptosis of hippocampal neurons. ERS contributes to the loss of hippocampal neurons after OGD treatment. The chaperone GRP78 has a countering effect against ischemic neuronal loss and apoptosis, which involves the inhibition of m-calpain and IRE1 resulting in capase-12 inactivity .