摘要目的 探讨自噬调节剂对氧糖剥夺(OGD)所致大鼠海马神经元损伤的影响.方法 原代培养大鼠海马神经元,采用OGD法模拟神经元缺血缺氧性损伤2 h或6 h(为模型组),干预组在OGD的同时给予自噬抑制剂6-氨基-3-甲基嘌呤(3-MA,20μmol/L)或自噬促进剂雷帕霉素(rapamycin,0.2μmol/L).复氧复糖12 h,镜下观察神经元形态学改变,蛋白质免疫印迹试验(Western Blot)检测自噬相关蛋白微管相关蛋白轻链3(LC3)、泛素结合蛋白P62和凋亡相关蛋白活化的天冬氨酸特异性半胱氨酸蛋白酶3(cleaved caspase-3)的表达,流式细胞仪检测神经元凋亡情况,全自动生化分析仪检测并计算乳酸脱氢酶(LDH)释放率,四甲基偶氮唑盐(MTT)比色法测定细胞活性.结果 与空白对照组比较,OGD 2 h或6 h神经元LC3Ⅱ/Ⅰ比值明显增加(灰度值:3.091±0.160、3.422±0.186比0.256±0.021),P62表达明显减少(灰度值:0.290±0.025、0.120±0.026比0.450±0.040),cleaved caspase-3表达明显增加(灰度值:0.230±0.025、0.440±0.051比0.050±0.007),神经元凋亡增加,LDH释放率明显增加〔(38.50±4.15)%、(59.60±5.65)%比(12.40±1.32)%〕,细胞活性明显降低〔(71.40±7.23)%、(42.80±4.12)%比(100.30±2.30)%〕,差异均有统计学意义(均P<0.05),且OGD时间越长,神经元凋亡越多,损伤越严重,活性越低.与OGD 2 h组比较,同时给予3-MA可降低LC3Ⅱ/Ⅰ比值(灰度值:2.281±0.121),增加P62、cleaved caspase-3表达(灰度值:0.410±0.037、0.330±0.027),增加神经元凋亡,加重神经元损伤〔LDH释放率:(47.30±4.43)%〕,降低神经元活性〔(51.10±5.73)%,均P<0.05〕;而给予rapamycin可增加LC3Ⅱ/Ⅰ比值(灰度值:3.689±0.214),减少P62、cleaved caspase-3表达(灰度值:0.170±0.040、0.090±0.096),减少神经元凋亡,减轻神经元损伤〔LDH释放率:(24.30±2.14)%〕,增加神经元活性〔(85.30±8.56)%,均P<0.05〕.与OGD 6 h组比较,同时给予3-MA可降低LC3Ⅱ/Ⅰ比值(灰度值:3.021±0.178),增加P62表达(灰度值:0.350±0.060),减少cleaved caspase-3表达(灰度值:0.240±0.017),减少神经元凋亡,减轻神经元损伤〔LDH释放率:(36.60±3.45)%〕,增加神经元活性〔(59.70±6.13)%,均P<0.05〕;而给予rapamycin可增加LC3Ⅱ/Ⅰ比值(灰度值:3.923±0.201),减少P62表达(灰度值:0.070±0.008),增加cleaved caspase-3表达(灰度值:0.590±0.062),增加神经元凋亡,加重神经元损伤〔LDH释放率:(71.20±7.81)%〕,降低神经元活性〔(27.30±2.12)%,均P<0.05〕.结论 轻度缺血缺氧情况下,自噬增强能对体外培养的大鼠海马神经元起到一定的保护作用,而在长时间或致死性缺血缺氧情况下,自噬增强会加重神经元损伤.

abstractsObjective 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.