当归挥发油通过激活ERK信号通路以减轻缺血再灌注神经细胞凋亡的作用

Function of essential oils from Angelica sinensis reduce apoptosis on the neural cells of damaged by ischemia-reperfusion like injury through up-regulation of ERK pathway

目的研究当归挥发油(EOAS)对缺血再灌注损伤神经细胞凋亡的影响。方法将高分化PC12细胞分为空白组、模型组、对照组及当大中小3个剂量实验组。除空白组外,其余各组用含10 mmol·L^(-1)连二亚硫酸钠(Na2S2O4)的无糖培养基缺氧缺糖损伤1 h,大中小3个剂量实验组加入终浓度分别为25.00,12.50,6.25μg·m L^(-1)EOAS,对照组加入10μmol·L^(-1)依达拉奉,复氧48 h。用MTT比色法检测当归挥发油对细胞增殖的影响,以试剂盒检测乳酸脱氢酶(LDH)、超氧化物歧化酶(SOD)活性及丙二醛(MDA)含量,用流式细胞仪检测细胞凋亡,以AO/PI免疫荧光染色观察凋亡细胞形态,以比色法检测细胞caspase-3的活性,以免疫印迹法检测p-ERK1/2蛋白的表达。结果与空白组相比,模型组细胞存活率降至(67.4±0.10)%,差异有统计学意义(P<0.01)。与模型组相比,对照组和大剂量实验组的细胞存活率分别升至(86.2±0.10)%,(94.5±0.05)%,差异均有统计学意义(P<0.05,P<0.01)。与空白组比较,模型组细胞LDH活性、MDA含量分别升高至(912.53±16.71)U·L^(-1)及(9.05±0.25)μmol·L^(-1);而SOD水平降至(12.53±0.29)U·m L^(-1),差异均有统计学意义(均P<0.01)。与模型组相比,大剂量实验组的LDH活性降低至(565.61±11.72)U·L^(-1),而SOD水平升高至(12.53±0.29)U·m L^(-1),差异均有统计学意义(P<0.05,P<0.01)。与模型组相比,大中2个剂量实验组MDA含量分别降至(3.32±0.68),(5.79±0.68)μmol·L^(-1),差异均有统计学意义(均P<0.01)。模型组与大中小3个剂量实验组细胞的光密度(A)分别为0.75±0.06,0.10±0.02,0.16±0.03及0.49±0.04,与模型组相比,差异均有统计学意义(P<0.05,P<0.01)。与空白组相比,模型组细胞凋亡率为(31.17±2.44)%,差异有统计学意义(P<0.01);与模型组相比,大中2个剂量组细胞凋亡率分别为(4.57±0.32)%,(5.93±0.81)%,差异均有统计学意义(均P<0.01)。与模型组相比,大剂量实验组能够明显促进细胞p-ERK1/2蛋白的表达(P<0.01)。结论 EOAS通过激活ERK信号通路抑制拟缺血再灌注神经细胞凋亡。

Objective To study the effect of essential oil from Angelica sinensis （EOAS） on neuronal apoptosis induced by ischemia - reperfusion injury. Methods The high differentiated PC12 cells were divided into blank group, model group, control group （ 10 μmol · L-1 edaravone） and large - , medium- and small- dose expreimental groups（ EOAS 25.00,12. 50, 6. 25μg · mL-1） . Except blank group, the rcmaining groups were treated with sugar- free medium contai- ning 10 mmol · L-1 sodium dithionite （Na2S204） for 1 h, reoxygenation for 48 h. The cell proliferation was measured by methyl thiazolyl tetrazolium（MTT） assay. The activity of superoxide dismutase （SOD） and lactate dehydrogenase （LDH）, contents of malondialdehyde （MDA） were measured by kit. The apotosis rate and the morphological apoptotic characteristics of cells were observed by flow cytometry and acridine orange/propidium iodide（AO/PI） double staining respectively. The relative activity of caspase - 3 were examined with colorimetric assay. The expression of p - ERK1/2 proteins was measured by Western - blot. Results Compared with the blank group, the cell viability in the model group was （67.4 ± 0. 10）% with significantly （P 〈 0. 01 ）. Compared with the model group, the cell viability in the large- dose experimental group and control group were （86. 2 ± 0. 10 ）%, （94. 5 ± 0. 05 ）% with significantly （P〈O. O5,P 〈 0.01 ）. Compared with the blank group, the LDH and MDA content in the model group were （912. 53±16.71）U · L-1and（9.05±0.25） p.mol · L -1while SOD level was （12.53 ±0.29）U ·mL -1with significantly（ all P 〈0. 01 ）. Compared with the model group,the LDH and SOD level inthe large -dose experimental group were （565.61 ± 11.72） U · L- 1 and （ 12. 53 ± 0.29 ） U · mL- 1 with significantly （ P 〈 0. 05, P 〈 0. 01 ） while MDA content in the large - , medium - dose experimental groups were （ 3.32 ± 0.68 ） , （ 5.79 ± 0. 68 ） μmol · L- 1 with significantly（ all P 〈0. 01 ）. The absorbance（A） in model group and large - , medium - and small - dose expreimental groups were O. 75 ±0. 06 and O. 10 ±0. 02,0. 16 ±0. 03,0. 49 ±0. 04. Compared with the model group,the difference had signifieandy（P 〈 0.05,P 〈 0. 01 ）. Compared with the blank group,the apoptosis rate in the model group was （31.17 ±2. 44）% with significantly（P 〈0. 01）. Compared with the model group,the apoptosis rate in the large - , medium - dose experimental groups were （4. 57 ± 0. 32 ） %, （ 5.93 ± 0. 81 ） % with significantly （ all P 〈 0.01 ）. Compared with the model group, p - ERK1/2 proteins of large - dose experimental group were up - regulated significantly （P 〈 0. 01 ）. Conclusion The EOAS could inhibit the apoptosis of neurons after ischemia - reperfusion by activating ERK signaling pathway.