党参皂苷L1对抗缺氧缺糖再给氧诱导大鼠星形胶质细胞损伤的作用

Protective effect of saponin L1 of radix codonopsis on the damage of cultured astrocytes induced by hypoxia/hypoglycemia and reoxygenation

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。

AIM： To study whether the saponin L1 of radix codonopsis, a Chinese herb of strengthening the body resistance and restoring normal functioning of the body to eonsolidate the eonstitution, has the proteetive effect on the damage of primary cultured astroeytes in rats induced by hypoxia/ hypoglycemia and reoxygenation. METHODS： The experiment was conducted in the Research Room of Eneephalopathy of Traditional Chinese Medicine, Dongzhimen Hospital of Beijing University of Traditional Chinese Medicine from August＇ 2004 to May 2005, ①Totally 40 Wistar rats born in one day with either sex were selected. ②Astroeytes in cortex were primarily cultured and put into the glucose-free Earle solution containing sodium dithionate, following by located in the incubator at 37 ℃ containing 0.05 volume fraction CO2 for 90 minutes for hypoxia and hypoglycemia. Then the Earle solution was aspirated and the serum free DMEM without sodium dithionate was add into to incubate at 37 ℃ containing 0.05 volume fraction CO2 for 90 minutes for reoxygenation. ③Astrocytes from different cultured wells were randomly divided into 6 groups （every 10 or 8 wells as one group）： Normal control group （incubated serum free DMEM without any medicine）; model group （with hypoxia/hypoglyeemia and reoxygenation）; model ＋ Nimodipine group [Nimodipine with end concentration of 0.5 mg/L was added into Earle solution and DMEM （purchased from Shandong Xinhua Pharmaceutical Co., Ltd, No. 0211048, 10 mL： 2 mg each one）]; model ＋ different end concentration saponin L1 groups [high, middle and low concentration groups were added in saponin L1 at end concentration of 1.3 mg/L, 0.13 mg/L and 0.013 mg/L, respectively, （prepared by High Performance Liquid Chromatography method by the Key Laboratory of Chinese Internal Medicine, Ministry of Edueatign, Beijing University of Traditional Chinese Medicine, with content of 96.2%）]. The lactate dehydrogenase （LAD） release was determined at hypoxia/hypoglyeemia without reoxygenation. ④ The cell survival rate was detected by MTY assay. Morphological changes of astrocytes, necrosis and apoptosis were observed with fluorescence microscope on the slide double＇stained in situ by Hoeehst 33342 and propidium iodide, and LAD release was detected by ehromometry. RESULTS： ①Appearanee of cells： Compared with the model group, the red-staining necrosis cells of the Nimodipine group and different end concentration saponin L1 groups were obviously decreased, but there was no obvious change in number of apoptotic cells. ②Cell survival rate： The model group was significantly lower than that in the normal group, Nimodipine group, middle and low end concentration saponin L1 groups （F=42.464, P 〈 0.01）. ③Cell necrosis rate and LAD release rate： in the model group, they were markedly higher than the other 5 groups （F=69.344, 24.994, P 〈 0.01）. ④ Cell apoptosis rate： The model group was higher than the normal group （F=3.311,P 〈 0.05）, but had no significant difference compared with the Nimodipine group and different end concentration saponin L1 groups （P 〉 0.05）. CONCLUSION： Saponin L1 of radix codonopsis has protective effect on the damage of astrocytes induced by hypoxia/hypoglycemia and reoxygenation, and can inhibit the necrosis of astrocytes, but cannot protect the apoptosis of astrocytes.