Zhou Tianen, Zeng Chaotao, Fang Jiajun, Jiang Longyuan, Yu Tao
Department of Emergency Medicine, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou 510120, Guangdong, China. Corresponding author: Yu Tao, Email:
Zhonghua Wei Zhong Bing Ji Jiu Yi Xue. 2017 Aug;29(8):738-743. doi: 10.3760/cma.j.issn.2095-4352.2017.08.013.
To explore the effect of autophagy regulator on the injury of rat hippocampal neurons induced by oxygen-glucose deprivation (OGD).
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, 20 μmol/L) or rapamycin (0.2 μmol/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.
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 (all P < 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 (all P < 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 (all P < 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 (all P < 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 (all P < 0.05).
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.
探讨自噬调节剂对氧糖剥夺（OGD）诱导的大鼠海马神经元损伤的影响。
原代培养大鼠海马神经元，进行OGD处理以模拟神经元缺氧缺血损伤2小时或6小时，随后进行12小时再灌注，分别加入或不加入3-甲基腺嘌呤（3-MA，20 μmol/L）或雷帕霉素（0.2 μmol/L）。用光学显微镜观察神经元形态。通过蛋白质免疫印迹分析评估自噬相关蛋白（LC3、P62）和凋亡相关蛋白（裂解的半胱天冬酶-3）的表达。通过流式细胞术检测神经元凋亡，用自动生化分析仪计算乳酸脱氢酶（LDH）释放率，用甲基噻唑基四氮唑（MTT）比色法测定细胞活性。
与对照组相比，OGD 2小时或6小时后，LC3 II/I的表达（灰度值：3.091±0.160、3.422±0.186比0.256±0.021）、裂解的半胱天冬酶-3的表达（灰度值：0.230±0.025、0.440±0.051比0.050±0.007）、神经元凋亡率、LDH释放率[（38.50±4.15）%、（59.60±5.65）%比（12.40±1.32）%]升高，而P62的表达（灰度值：0.290±0.025、0.120±0.026比0.450±0.040）、神经元活性[（71.40±7.23）%、（42.80±4.12）%比（100.30±2.30）%]降低（均P<0.05）。当OGD时间为2小时并联合3-MA时，LC3 II/I的表达（灰度值：2.281±0.121）、神经元活性[（51.10±5.73）%]降低，而P62和裂解的半胱天冬酶-3的表达（灰度值分别为0.410±0.037、0.330±0.027）、神经元凋亡率、神经元损伤[LDH释放率：（47.30±4.43）%]升高（均P<0.05）。当OGD时间为2小时并联合雷帕霉素时，LC3 II/I的表达（灰度值：3.689±0.214）、神经元活性[（85.30±8.56）%]升高，而P62和裂解的半胱天冬酶-3的表达（灰度值分别为0.170±0.040、0.090±0.096）、神经元凋亡率、神经元损伤[LDH释放率：（24.30±2.14）%]降低（均P<0.05）。相反，当OGD时间为6小时并联合3-MA时，LC3 II/I和裂解的半胱天冬酶-3的表达（灰度值：3.021±0.178、0.240±0.017）、神经元凋亡率、神经元损伤[LDH释放率：（36.60±3.45）%]降低，而P62的表达（灰度值：0.350±0.060）、神经元活性[（59.70±6.13）%]升高（均P<0.05）。当OGD时间为6小时并联合雷帕霉素时，LC3 II/I和裂解的半胱天冬酶-3的表达（灰度值：3.923±0.201、0.590±0.062）、神经元凋亡率、神经元损伤[LDH释放率：（71.20±7.81）%]升高，而P62的表达（灰度值：0.070±0.008）、神经元活性[（27.30±2.12）%]降低（均P<0.05）。
在轻度OGD条件下，自噬增强对神经元有保护作用，而长时间OGD诱导的神经元损伤时自噬增强则会加重神经元损伤。
