Effect of autophagy regulator on the injury of rat hippocampal neurons induced by oxygen-glucose deprivation / 中华危重病急救医学

ABSTRACT

Objective To explore the effect of autophagy regulator on the injury of rat hippocampal neurons induced by oxygen-glucose deprivation (OGD).Methods Rat hippocampal neurons were cultivated in primary and subjected to OGD to simulate neuronal hypoxic ischemia injury for 2 hours or 6 hours followed by reperfusion for 12 hours with or without 3-methyladenine (3-MA, 20μmol/L) or rapamycin (0.2μmol/L). The morphology of neurons was observed with optical microscope. The expression of autophagy-related protein (LC3, P62) and apoptosis-related protein (cleaved caspase-3) were assessed by Western Blot analysis. The apoptosis of neurons was detected by flow cytometry, the release rate of lactate dehydrogenase (LDH) was calculated by automatic biochemical analyzer, and the cell activity was determined by methyl thiazolyl tetrazolium (MTT) colorimetric assay.Results Compared with the control group, the expression of LC3 Ⅱ/Ⅰ (gray value 3.091±0.160, 3.422±0.186 vs. 0.256±0.021), cleaved caspase-3 (gray value 0.230±0.025, 0.440±0.051 vs. 0.050±0.007), neuronal apoptotic rate, LDH release rate [(38.50±4.15)%, (59.60±5.65)% vs. (12.40±1.32)%] were increased, while the expression of P62 (gray value 0.290±0.025,0.120±0.026 vs. 0.450±0.040), neuronal activity [(71.40±7.23)%, (42.80±4.12)% vs. (100.30±2.30)%] were decreased at 2 hours or 6 hours after OGD (allP < 0.05). When the time of OGD was 2 hours and it was combined with 3-MA, the expression of LC3 Ⅱ/Ⅰ (gray value 2.281±0.121), the neuronal activity [(51.10±5.73)%] were decreased, while the expression of P62 and cleaved caspase-3 (gray scale 0.410±0.037, 0.330±0.027, respectively), neuronal apoptotic rate, the injury of neurons [LDH release rate (47.30±4.43)%] were increased (allP < 0.05). When the time of OGD was 2 hours and it was combined with rapamycin, the expression of LC3 Ⅱ/Ⅰ (gray value 3.689±0.214), the neuronal activity [(85.30±8.56)%] were increased, while the expression of P62 and cleaved caspase-3 (gray value 0.170±0.040, 0.090±0.096, respectively), neuronal apoptotic rate, the injury of neurons [LDH release rate (24.30±2.14)%] were decreased (allP < 0.05). On the contrary, when the time of OGD was 6 hours and it was combined with 3-MA, the expression of LC3 Ⅱ/Ⅰ and cleaved caspase-3 (gray value 3.021±0.178, 0.240±0.017), neuronal apoptotic rate, the injury of neurons [LDH release rate (36.60±3.45)%] were decreased, while the expression of P62 (gray value 0.350±0.060), the neuronal activity [(59.70±6.13)%] were increased (allP < 0.05). When the time of OGD was 6 hours and it was combined with rapamycin, the expression of LC3 Ⅱ/Ⅰ and cleaved caspase-3 (gray value 3.923±0.201, 0.590±0.062), neuronal apoptotic rate, the injury of neurons [LDH release rate(71.20±7.81)%] were increased, while the expression of P62 (gray value 0.070±0.008), the neuronal activity [(27.30±2.12)%] were decreased (allP < 0.05).Conclusion The enhancement of autophagy has protective effect on neurons under the condition of mild OGD, while it can aggravate the injury of neurons induced by a long-time OGD.