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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 EOAS on neuronal apoptosis induced by ischemia-reperfusion injury. Methods Differentiated PC12 cells were divided into three groups: blank group, model group, control group and experimental group. Except for the blank group, the other groups were treated with glucose-free medium containing 10 mmol·L -1 sodium dithionite (Na 2 S 2 O 4) for 1 h. The experimental groups were divided into three groups: 25.00,12.50,6.25μg · m L -1 EOAS, while the control group was treated with 10μmol·L -1 edaravone for 48 hours. The effects of Angelica sinensis volatile oil on the cell proliferation were detected by MTT colorimetric assay. The activity of lactate dehydrogenase (LDH), superoxide dismutase (SOD) and the content of malondialdehyde (MDA) were detected by kit, and detected by flow cytometry The apoptotic cells were observed by AO / PI immunofluorescence staining. The activity of caspase-3 was detected by colorimetric assay. The expression of p-ERK1 / 2 protein was detected by Western blotting. Results Compared with the blank group, the survival rate of the model group decreased to (67.4 ± 0.10)%, the difference was statistically significant (P <0.01). Compared with the model group, the cell viability of the control group and the high-dose experimental group increased to (86.2 ± 0.10)% and (94.5 ± 0.05)%, respectively, with statistical significance (P <0.05 and P <0.01). Compared with the blank group, the activity of LDH and MDA in model group increased to (912.53 ± 16.71) U · L -1 and (9.05 ± 0.25) μmol·L -1, respectively, while the level of SOD decreased (12.53 ± 0.29) U · m L -1, the differences were statistically significant (all P <0.01). Compared with the model group, the LDH activity of the high-dose experimental group decreased to (565.61 ± 11.72) U · L -1 and the SOD level increased to (12.53 ± 0.29) U · m L -1, The differences were statistically significant (P <0.05, P <0.01). Compared with the model group, the MDA content in large and medium dose groups decreased to (3.32 ± 0.68) and (5.79 ± 0.68) μmol·L -1, respectively (all P <0.01) . The optical density (A) of the three experimental groups were 0.75 ± 0.06, 0.10 ± 0.02, 0.16 ± 0.03 and 0.49 ± 0.04, respectively, which were significantly different from the model group (P < 0.05, P <0.01). Compared with the blank group, the apoptotic rate of the model group was (31.17 ± 2.44)%, the difference was statistically significant (P <0.01); compared with the model group, the apoptosis rates of the two dose groups were ( 4.57 ± 0.32)% and (5.93 ± 0.81)%, respectively (all P <0.01). Compared with the model group, high-dose experimental group can significantly promote the expression of p-ERK1 / 2 protein (P <0.01). Conclusion EOAS inhibits the neuronal apoptosis induced by ischemia / reperfusion through activation of ERK signaling pathway.