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目的:观察扶正固本、益气类中药党参皂苷L1对缺氧缺糖再给氧所致大鼠大脑星形胶质细胞损伤是否有保护作用。方法:实验于2004-08/2005-05在北京中医药大学东直门医院中医脑病研究室完成。①选用出生1d的Wistar大鼠40只,雌雄不拘。②分离大脑星形胶质细胞进行原代培养。加入含连二亚硫酸钠10mmol/L的无糖Earle液,置37℃体积分数0.05CO2孵箱中孵育90min进行缺氧缺糖处理。然后吸弃Earle液,加入不含连二亚硫酸钠的无血清DMEM培养液,继续置37℃体积分数0.05CO2孵箱中孵育90min进行再给氧处理。③将在相同或不同细胞培养板中的不同培养孔中细胞按随机抽签法分为6组(每10孔或8孔细胞为1组):正常对照组(正常细胞培养),模型组(进行缺氧缺糖再给氧处理),尼莫地平组[在无糖Earle液和无血清DMEM培养液加入终浓度0.5mg/L尼莫地平[购自山东新华制药股份有限公司,生产批号:0211048,10mL(2mg)/支],其余处理同模型组],缺氧缺糖再给氧+党参皂苷L1高、中、低浓度组[在无糖Earle液和无血清DMEM培养液分别加入质量浓度1.3,0.13和0.013mg/L党参皂苷L1(由北京中医药大学中医内科学教育部重点实验室采用高效液相色谱法制备法分离得到,含量为96.2%),其余处理同模型组]。测定乳酸脱氢酶漏出率时缺氧缺糖处理后直接测定,而不做再给氧处理。④四唑盐比色法测定细胞存活率,Hoechst33342和碘化丙啶原位双染法荧光显微镜观察细胞形态学变化和细胞坏死率、细胞凋亡率,比色法测定乳酸脱氢酶漏出率。结果:①荧光显微镜观察细胞形态:尼莫地平组和党参皂苷L1各浓度组与模型组比较均可见红染的坏死细胞数量明显减少,尼莫地平、党参皂苷L1各浓度组与模型组比较凋亡细胞数量无明显变化。②星形胶质细胞存活率:模型组明显低于正常组、尼莫地平组和党参皂甙L1中、低浓度组(F=42.464,P<0.01)。③细胞坏死率和乳酸脱氢酶漏出率:模型组明显高于其他5组(F=69.344,24.994,P<0.01)。④细胞凋亡率:模型组明显高于正常组(F=3.311,P<0.05),与尼莫地平组和党参皂苷L1各浓度组比较,差异不明显(P>0.05)。结论:中药党参皂苷L1对缺糖缺氧再给氧造成的星形胶质细胞损伤具有保护作用,可抑制细胞坏死,但对凋亡过程无保护作用。
Objective: To observe if Fuzheng Guben and Yiqi type of Chinese herbal medicine Codonopsis saponin L1 can protect astrocytes from damage caused by oxygen-glucose deprivation and reoxygenation. METHODS: The experiment was performed at the Department of TCM Encephalopathy, Dongzhimen Hospital, Beijing University of Chinese Medicine between August 2004 and May 2005. 1 Forty Wistar rats born at 1d were selected either male or female. 2 Isolation of brain astrocytes for primary culture. Add sugar-free Earle solution containing 10 mmol/L sodium hyposulfite and incubate for 90 min in a 37°C volume fraction 0.05 CO2 incubator for hypoxia and glucose deprivation. Then, the Earle solution was discarded, serum-free DMEM containing sodium dithionite was added, and the samples were further incubated in a 0.05°C CO2 incubator at 37°C for 90 min and then reoxygenated. 3 The cells in different culture wells in the same or different cell culture plates were randomly divided into 6 groups (1 group per 10 or 8 well cells): normal control group (normal cell culture), model group ( Oxygen-glucose deprivation reoxygenation treatment), Nimodipine group [In glucose-free Earle fluid and serum-free DMEM culture medium added to a final concentration of 0.5 mg/L nimodipine [purchased from Shandong Xinhua Pharmaceutical Co., Ltd., production lot number: 0211048 , 10mL (2mg)/branch], the rest of the treatment with the model group], oxygen deficiency and glucose and then oxygen + Codonopsis saponin L1 high, medium and low concentration group [in sugar-free Earle fluid and serum-free DMEM culture fluid were added to the mass concentration 1.3, 0.13 and 0.013mg/L Codonopsis saponin L1 (obtained from the key laboratory of the Ministry of Education for Internal Medicine of the Chinese University of Traditional Chinese Medicine, Beijing University of Traditional Chinese Medicine, was separated by high performance liquid chromatography and the content was 96.2%). The rest of the treatment was the same as the model group. The determination of the leakage rate of lactate dehydrogenase was performed directly after anoxic and deprivation-glucose treatment, without reoxygenation. 4Cell viability was measured by tetrazolium salt colorimetry, Hoechst 33342 and propidium iodide were used to observe the cell morphology, cell necrosis rate and apoptosis rate by fluorescence microscope. Colorimetric method was used to determine the leakage rate of lactate dehydrogenase. . RESULTS:1The morphology of the cells was observed by fluorescence microscope: Compared with the model group, the number of necrotic cells in the red staining group was significantly reduced in the nimodipine group and the codonopsis saponin L1 group. The nimodipine and the codonopsis saponin L1 concentration groups were compared with the model group. The number of dead cells did not change significantly. 2 Astrocyte survival rate: The model group was significantly lower than the normal group, nimodipine group and the codonopsis L1 medium and low concentration group (F=42.464, P<0.01). 3 The rate of cell necrosis and leakage rate of lactate dehydrogenase: The model group was significantly higher than the other 5 groups (F=69.344, 24.994, P<0.01). 4 Apoptosis rate: The model group was significantly higher than the normal group (F=3.311, P<0.05). Compared with the nimodipine group and the Dangsaponin L1 concentration group, the difference was not significant (P>0.05). Conclusion: Codonopsis saponin L1 has a protective effect on astrocyte damage caused by anoxia-reoxygenation, and can inhibit cell necrosis, but has no protective effect on apoptosis.