五味子乙素和五味子醇甲对PC12细胞氧化损伤的保护作用

Protective effects of schisandrin B and schisandrin on oxidative damage in PC12 Cells

目的:观察五味子乙素和五味子醇甲对过氧化氢(H2O2)PC12细胞氧化损伤的保护作用。方法:实验于2005-08/2006-07在首都医科大学附属北京中医医院中心实验室完成。①PC12细胞培养在RPMI1640培养液中,加入2.5,5,10,20μmol/L等不同浓度的五味子乙素和五味子醇甲,与细胞培养24h,然后加入0.1,0.2,0.3,0.5mmol/L不同浓度过氧化氢(H2O2)溶液,37℃作用30min。模型组细胞只加入H2O2;正常对照组只加入等量的培养基。②采用倒置显微镜观察PC12细胞形态学变化,四甲基偶氮唑盐比色法测定细胞生存率,用自动生化分析仪测定乳酸脱氢酶的活性,按试剂盒说明书用慧星法测定细胞内DNA损伤。结果:①五味子乙素及五味子醇甲对PC12细胞形态学的影响:倒置相差显微镜观察到PC12细胞被H2O2损伤后,细胞形态学发生变化,表现为胞体肿胀、突起断裂、细胞膜溶解等。经五味子乙素及五味子醇甲处理后细胞,状态明显改善,细胞数目较多,而且细胞形态较完整。②五味子乙素及五味子醇甲对细胞生存率的影响:PC12细胞经0.1,0.2,0.3,0.5mmol/L不同浓度H2O2处理30min后,与正常对照组相比,四甲基偶氮唑盐法测定其生存率,分别降低了4%,13%,40%,57%,(P<0.05或P<0.01)。不同浓度的五味子乙素及五味子醇甲(2.5～20μmol/L)均能提高细胞的生存率,并呈浓度依赖关系,浓度为5,10,20μmol/L时,与模型组比较,具有明显的差异(P<0.05或P<0.01),特别是10μmol/L五味子乙素及五味子醇甲的作用较强。③五味子乙素及五味子醇甲对细胞内乳酸脱氢酶和DNA损伤的影响:用0.3mmol/LH2O2处理细胞后细胞内乳酸脱氢酶漏出率高于正常对照组(P<0.01)。大量的DNA被损伤,与正常对照组比较,差异有显著性(P<0.01)。细胞与10.0μmol/L五味子乙素及五味子醇甲作用后发现细胞内乳酸脱氢酶漏出率低于模型组(P均<0.01)。DNA损伤明显抑制,与模型组比较,差异有显著性(P均<0.01)。结论:五味子乙素及五味子醇甲均能有效地减轻H2O2对神经细胞的氧化损伤作用。推测保护作用机制是通过清除氧自由基,减轻其对DNA的损伤等方面实现的。

AIM： To observe the protective effects of schisandrin B （Sch B） and schisandrin （SCH） on oxidative damage induced by hydrogen peroxide （H2O2） in PC12 cells. METHODS： The experiment was conducted in the Central Laboratory, Beijing Hospital of Traditional Chinese Medicine Affiliated to Capital Medical University from August 2005 to July 2006. ①PC12 cells were incubated in RPMI 1640 medium, which were incubated for 24 hours after adding Sch B and SCH in different concentrations （2.5, 5, 10, 20μmol/L） respectively, then treated with 0.1, 0.2, 0.3, 0.5 mmol/L H2O2 at 37 ℃ for 30 minutes. The cells in the model group were only added H2O2; the cells in the control group were cultivated with only medium of the same volume. ②The morphology of PC12 cells were observed with inverted microscope and the cell viabihty was measured by methylthiazolyl tetrazolium （MTT） colorimetry, the activity of lactic dehydrogenase （LDH） was detected by automatic biochemistry analyzer, DNA si,ngle breaks by comet assay according to the instruction of the test kit. RESULTS： ①Effect of Sch B and SCH on morphology of PC12 cells： Results under the inverted microscope showed that the cell morphology changed after injured with H2O2; the cells swelled with disrppted enation and dissolved cell membrane. The call morphology was improved after treated with Sch B and SCH; the number of cells was increased with complete appearance. ②Effect of Sch B and SCH on the viability of cells： After the cells were intervened with 0.1, 0.2, 0.3, 0.5 mmol/L H2O2 for 30 minutes, the viability measured by MTT decreased 4%, 13%, 40%, 57% respectively compared with the control group （P 〈 0.05 or P 〈 0.01）. Sch B and SCH at different concentrations （2.5-20 μmol/L） could increase the viability of cells in a dose-dependent manner. There were significant differences compared with the model group when in the concentrations of 5, 10, 20μmol/L （P 〈 0.05 or 0.01）, especially the Sch B and SCH of 10μmol/L. ③Effect of Sch B and SCH on LDH and DNA injury in cells： The releasing rate of LDH was higher than the control group after treated with 0.3 mmol/L H2O2 （P 〈 0.01）. DNA damage was great, which had significant differences compared with the control group （P 〈 0.01）. After treated with l0 μmol/L Sch B and SCH, the releasing rate of LDH decreased and lower than the model group （all P 〈 0.01）, and DNA damage were inhibited; there was obvious difference compared with the model group （all P 〈 0.01）. CONCLUSION： Both Sch B and SCH can relieve the oxidative injury caused by H2O2, which is achieved by eliminating the oxygen free radical and lessening the damage of cellular DNA in PC12 cells.