刺五加皂甙对自由基作用和无血清培养条件下两种神经元衰老模型细胞的保护作用

Protective effects of acanthopannx senticousus saponins on two kinds of aged neuron models under free radicals function under the condition of serum free culture

目的:对体外小鼠胎鼠皮质神经元采用自由基作用和无血清培养条件建立衰老模型,观察刺五加皂甙对衰老神经元的保护作用。方法:实验于2000-05/11在南通大学航海医学研究所生化教研室完成。选择孕15～17d的小鼠胎鼠,在无菌条件下,分离大脑皮质,进行原代细胞培养。自由基作用建立神经元衰老模型:①模型组:H2O2和FeSO4加入到被培养7d的神经细胞内。②刺五加皂甙组:在H2O2和FeSO4处理前后24h加入12.5,25,50mg/L的刺五加皂甙。③正常对照组:不加FeSO4和H2O2及刺五加皂甙。无血清培养建立神经元衰老模型:①模型组:加入无血清的L15培养基。②刺五加皂甙组:从无血清培养前24h和无血清培养过程中分别加入12.5,25,50mg/L的刺五加皂甙。③正常对照组:在无血清处理前测各项指标。观察刺五加皂甙对两种衰老条件下的神经元存活率,乳酸脱氢酶活性,超氧化物歧化酶活性和丙二醛含量的影响。并在光镜和电镜下观察神经细胞形态。结果:自由基作用条件下:①神经细胞的存活率:12.5,25,50mg/L刺五加皂甙组高于模型组。②乳酸脱氢酶活性和丙二醛含量:12.5,25,50mg/L刺五加皂甙组均低于模型组犤(0.542±0.020),(0.505±0.022),(0.502±0.076),(0.613±0.063)μkat;(10.20±0.51),(9.17±0.9),(8.95±1.72),(11.46±1.23)μmol/g,P<0.05～0.01犦。③超氧化物歧化酶活性:12.5,25,50mg/L刺五加皂甙组明显高于模型组犤(32.91±1.71),(32.91±1.71),(36.10±5.37),(30.37±1.83)NU/mg,(P<0.05～0.01)犦。无血清培养条件下:①神经细胞的存活率:12.5,25,50mg/L刺五加皂甙组高于模型组。②乳酸脱氢酶活性和丙二醛含量:12.5,25,50mg/L刺五加皂甙组均低于模型组犤(0.333±0.018),(0.302±0.027),(0.309±0.064),(0.385±0.044)μkat;(7.07±0.18),(6.33±0.48),(6.64±1.58),(8.38±1.02)μmol/g,P<0.05～0.01犦。③超氧化物歧化酶活性:12.5,25,50mg/L刺五加皂甙组明显高于模型组犤(33.98±1.52),(37.85±2.71),(38.40±6.29),(31.23±2.07)NU/mg,P<0.05～0.01犦。显微镜及扫描电镜观察,加用刺五加皂甙保护的神经细胞损伤明显减轻,部分细胞形态基本趋于正常。结论:刺五加皂甙通过降低脂质过氧化物含量,增加自由基清除力;增强细胞膜稳定性,提高皮质神经元的存活率来发挥对神经元的保护作用,改善其功能,从而延缓了神经细胞的衰老。形态学变化也表明刺五加皂甙能明显减轻衰老神经元细胞的损伤,减缓其衰老。

AIM： The cortex neuron of ex vivo foetus mice were made into aged models with free radicals function and serum free culture condition. To observe the protective effects of acanthopannx senticousus saponins （ASS） on the aged neuronal cells. METHODS： The experiment had been completed in the Biochemistry Department of Sailing Medical Science Institute of Nantong University from May to November 2000. The cerebral cortex from 15-17 days old foetus mice which were selected and dissociated under sterile condition was used to primary cell culture. The aged models of neuron with free radicals effect were found： ① Model group： Nerve cells cultured for 7 days were treated with H2O2 and FeSO4. ② ASS group： The neurons were treated with 12.5, 25, 50 mg/L ASS 24 hours before and after disposal of FeSO4 and H2O2. ③ Normal control group： The neurons were not treated with FeSO4, H2O2 and ASS. The aged model of serum free damage to cultured cortical neuronal cells was made： ① Model group： Neurons were treated with L15 medium with serum free. ② ASS group： The neurons were treated with 12.5, 25, 50 mg/L ASS respectively 24 hours before culture with serum free and during the culture with serum free. ③ Normal control group： The every index of neurons were examined before culture with serum free. We observed the effects of ASS on the neuronal viability, lactate dehydrogenase （LDH） activity, superoxide dismutase （SOD） activity and malondiadehyde （MDA） under the condition of two kinds of aged models. We observed the neurons form by using light microscope and electron microscope. RESULTS： Under the condition of free radicals effect： ① The neuronal viability： The neuronal viability were significantly higher in the 12.5, 25, 50 mg/L ASS group as compared with model group. ② The activity of lactate dehydrogenase （LDH） and content of malondialdehyde （MDA）： It was lower significantly in the 12.5, 25, 50 mg/L ASS group as compared with model group. [（0.542±0.020）, （0.505±0.022）, （0.502±0.076）, （0.613±0.063） μkat; （10.20±0.51）, （9.17±0.9）, （8.95±1.72）, （11.46±1.23） μmoL/g, （P 〈 0.05-0.01）]. ③ Superoxide dismutase （SOD） activity： It was significantly higher in the ASS 12.5, 25, 50 mg/L group as compared with model group. [（32.91±1.71）, （32.91±1.71）, （36.10±5.37）, （30.37±1.83） NU/mg, （P〈 0.05-0.01 ）]. Under the culture condition of serum free： ① The neuronal cells viability： The neuronal viability was significantly higher in the 12.5, 25, 50 mg/L ASS group as compared with model group. ② The lacate dehydrogenase （LDH） activity and malondialdehyde （MDA） content： The Lacate dehydrogenase （LDH） activity and MDA content were significantly lower in the 12.5, 25, 50 mg/L ASS group as compared with model group. [（0.333±0.018）, （0.302±0.027）, （0.309±0.064）, （0.385±0.044） μkat; （7.07±0.18）, （6.33±0.48）, （6.64±1.58）, （8.38±1.02） μmol/g, （P 〈 0.05-0.01）]. ③ Superoxide dismutase （SOD） activity： The superoxide dismutase （SOD） activity was significantly higher in the ASS group 12.5, 25, 50 mg/L as compared with model group. [（33.98±1.52）, （37.85±2.71）, （38.40±6.29）,（31.23±2.07） NU/mg,（P 〈 0.05-0.01）]. By using microscope and scanning electron microscope to observe, injury in nerve cells which treated with ASS protection mild markedly reduced and some neurons form was similar to normal basically. CONCLUSION： The protective effects of ASS on the cortical neurons by decreasing lipid peroxidation content, increasing free radicals clearance, reinforcing cell membrane stability and rising neuronal viability were led to improve its function and postpone the aged of neurons cells. The changes of morphology also indicates that the ASS has the effects of relieving injury of aged neurons markedly and deferring the aged of neurons cells.