新型番荔枝酰胺衍生物FLZ对去血清培养损伤星形胶质细胞的保护作用
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摘要
目的FLZ是番荔枝叶提取物番荔枝酰胺的合成衍生物,前期研究表明,该化合物在脑缺血模型上具有神经保护作用。但FLZ对星形胶质细胞的作用研究尚少。近年研究表明,星形胶质细胞在中枢神经系统疾病中具有重要的生理和病理意义。去血清培养是体外用于模拟脑缺血的常用模型之一。S100B是一种主要表达在星形胶质细胞中的Ca2+结合蛋白,在急性中风、心脏停搏、蛛网膜下腔出血中也表现出敏感、特异性强的生物标记物特点。在去血清培养损伤引起的星形胶质细胞氧化应激过程中,S100B参与其中。胞外S100B作用于细胞膜受体可引起活性氧簇(reactive oxidative speciies,ROS)生成,并由此对细胞造成氧化应激损伤。因此本论文旨在研究FLZ对去血清培养引起的星形胶质细胞S100B过量分泌的作用,以及FLZ在由此引发的星形胶质细胞氧化应激中的作用。
方法在去血清培养的原代大鼠脑皮层星形胶质细胞模型上,以MTT法检测细胞存活率,Hoechst 33342染色后观察细胞核形态变化,ELISA方法检测细胞培养液中S100B蛋白含量,DCFDA法检测细胞内ROS生成。生化法检测超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-Px)活力,及丙二醛(MDA)与谷胱甘肽(GSH)含量。
结果去血清培养致星形胶质细胞存活率显著下降(P<0.001),FLZ(1×10-7mol/L、3×10-7 mol/L、1×10-6 mol/L、3×10-6 mol/L可提高去血清培养条件下星形胶质细胞的存活率。去血清培养致原代培养的大鼠脑皮层星形胶质受损细胞核明显增多(P<0.001), FLZ各浓度均可减轻去血清培养造成的大鼠脑皮层星形胶质细胞核受损状况(P<0.001)。去血清培养4 h内,模型组星形胶质细胞培养液中S100B含量持续升高,4 h后与正常对照组相比明显增多(P<0.001).FLZ(1×10-7 mol/L,1×10-6 mol/L)组星形胶质细胞S100B分泌量缓慢升高,4 h后分泌量显著低于模型组(P<0.001)。模型组细胞内ROS含量较正常对照组升高4.11倍,FLZ (1×10-7 mol/L-3×10-6 mol/L)可显著的减少去血清培养造成的星形胶质细胞内ROS的过度生成。去血清培养致原代星形胶质细胞内SOD活力明显下降(P<0.001),脂质过氧化产物MDA明显增多(P<0.001),GSH含量明显降低(P<0.001),GSH-Px的活性升高P<0.01)。FLZ(3×10-7 mol/L-3×10-6 mol/L)可增加SOD活力,改善率分别为21%,41%,73%;减少MDA过量生成,改善率分别为39%,45%,63%；升高GSH含量,升高率分别为25%,33%,63%。(?)FLZ在1×10-7 mol/L、3×10-7 mol/L浓度下可逆转去血清损伤造成的GSH-Px活性变化,而1×10-6 mol/L和3×10-6 mol/L组对GSH-Px活性无明显影响。
结论去血清培养致原代大鼠脑皮层星形胶质细胞损伤,FLZ对去血清培养损伤星形胶质细胞有明显的保护作用。FLZ通过减少去血清培养引起的S100B过度分泌从而降低ROS过度生成,增强细胞自身抗氧化系统,减轻去血清培养星形胶质细胞引起的氧化应激损伤。
Objective Compound FLZ is a synthesized squamosamide derivative shows neuroprotective effects on rat ischemia model according to early research. However little is known about its effects on astrocytes yet. Serum deprivation is a common model mimic the ischemia situation in vitro. Recently studies shows that astrocytes play an important role in the physiology and pathology of multiple central nervous system diseases. S100B is an astrocyte-specific Ca2+ binding protein when expressed in brain. S100B showed the sensitive and specific properties as a biomarker in acute stroke, cardiac arrest and subarachnoid hemorrhage. According to early results, S100B is involved in the serum deprivation induced astrocytic oxidative stress. The extracellular S100B could induce reactive oxidative species production through binding to membrane receptors, and thereupon induce oxidative injury intracellularly. Since its significance in the clinical diagnosis of multiple nervous system diseases of S100B, this thesis is focused on the effects of FLZ on serum deprivation induced astrocytic over secretion of S100B, and effects of FLZ on the followed oxidative stress induced by S100B over secretion.
Methods Astrocytes were cultured under normal and serum-deprived condition. Firstly, the optimal concentrations of FLZ exerting protective effects against serum deprivation injury was determined by 3-(4,5-dimethylthiazol-2yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Then, the morphological changes of cell nuclei were observed under fluorescence microscope after Hoechst 33342 staining. The level of secreted S100B was determined by ELISA. Furthermore, reactive oxygen species (ROS) in cultures was measured by fluorescence probe DCFDA. Finally, activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), concentrations of malondialdehyde (MDA) and glutathione (GSH) were determined using biochemical methods.
Results Serum deprivation caused cell viability decrease significantly (P< 0.001, vs. control group), and FLZ (I×10-7 mol/L、3×10-7 mol/L、I×10-6 mol/L、3×10-6 mol/L) treatment could increase the cell viability by 80%,69%,42% and 34%, respectively. Serum deprivation increased astrocytic nuclei damage significantly (P< 0.001), and FLZ treatment attenuated the nuclei damage of primary cortical astrocytes cultures with improvement rates of 66%,82%,85%,97%, respectively. After 4 h serum deprivation, the medium S100B content of model group increased continuously and significantly (P< 0.001) vs. control group. Meanwhile, the extracellular S100B level of FLZ (1×10-7 mol/L, 1×10-6 mol/L) treated primary astrocytes increased slowly and significantly (P< 0.001) lower than model group. The intracellular ROS levels of model group rose to 4.11 fold of control group, FLZ (1×10-7 mol/L-3×10-6 mol/L) significantly attenuated the overproduction of ROS induced by serum deprivation. Cultured under serum deprivation circumstance significantly decreased the astrocytic SOD activity (P< 0.001) and GSH content (P< 0.001), meanwhile increased the MDA production (P< 0.001) and GSH-Px activity (P<0.01). FLZ (3×10-7 mol/L-3×10-6 mol/L) increased the SOD activity by 21%,41% and 73% respectively, reduced the MDA overproduction by 39%,45% and 63% respectively, and raised the GSH content by 25%,33% and 63%, respectively. FLZ (1×10-7 mol/L & 3×10-7 mol/L) could reverse the serum deprivation induced GSH-Px activity changes, while GSH-Px activities in the higher dose-treated groups were not affected by FLZ.
Conclusion Serum deprivation could decrease the cell viability of primary cortical astrocytes significantly, and FLZ showed protective effects against serum deprivation induced cell damage. FLZ decreased the overproduction of ROS through attenuating the extracellular S100B level. FLZ demonstrated anti-oxidative effects to protect the serum-deprived astrocytes by inhibiting MDA over production, GSH level and SOD activity attenuation, and GSH-Px activity elevation. In conclusion, FLZ showed (?)tive effects of serum deprivation induced primary cortical astrocytes damage through decrease S100B production and enhance cellular anti-oxidative system.
方法在去血清培养的原代大鼠脑皮层星形胶质细胞模型上,以MTT法检测细胞存活率,Hoechst 33342染色后观察细胞核形态变化,ELISA方法检测细胞培养液中S100B蛋白含量,DCFDA法检测细胞内ROS生成。生化法检测超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-Px)活力,及丙二醛(MDA)与谷胱甘肽(GSH)含量。
结果去血清培养致星形胶质细胞存活率显著下降(P<0.001),FLZ(1×10-7mol/L、3×10-7 mol/L、1×10-6 mol/L、3×10-6 mol/L可提高去血清培养条件下星形胶质细胞的存活率。去血清培养致原代培养的大鼠脑皮层星形胶质受损细胞核明显增多(P<0.001), FLZ各浓度均可减轻去血清培养造成的大鼠脑皮层星形胶质细胞核受损状况(P<0.001)。去血清培养4 h内,模型组星形胶质细胞培养液中S100B含量持续升高,4 h后与正常对照组相比明显增多(P<0.001).FLZ(1×10-7 mol/L,1×10-6 mol/L)组星形胶质细胞S100B分泌量缓慢升高,4 h后分泌量显著低于模型组(P<0.001)。模型组细胞内ROS含量较正常对照组升高4.11倍,FLZ (1×10-7 mol/L-3×10-6 mol/L)可显著的减少去血清培养造成的星形胶质细胞内ROS的过度生成。去血清培养致原代星形胶质细胞内SOD活力明显下降(P<0.001),脂质过氧化产物MDA明显增多(P<0.001),GSH含量明显降低(P<0.001),GSH-Px的活性升高P<0.01)。FLZ(3×10-7 mol/L-3×10-6 mol/L)可增加SOD活力,改善率分别为21%,41%,73%;减少MDA过量生成,改善率分别为39%,45%,63%；升高GSH含量,升高率分别为25%,33%,63%。(?)FLZ在1×10-7 mol/L、3×10-7 mol/L浓度下可逆转去血清损伤造成的GSH-Px活性变化,而1×10-6 mol/L和3×10-6 mol/L组对GSH-Px活性无明显影响。
结论去血清培养致原代大鼠脑皮层星形胶质细胞损伤,FLZ对去血清培养损伤星形胶质细胞有明显的保护作用。FLZ通过减少去血清培养引起的S100B过度分泌从而降低ROS过度生成,增强细胞自身抗氧化系统,减轻去血清培养星形胶质细胞引起的氧化应激损伤。
Objective Compound FLZ is a synthesized squamosamide derivative shows neuroprotective effects on rat ischemia model according to early research. However little is known about its effects on astrocytes yet. Serum deprivation is a common model mimic the ischemia situation in vitro. Recently studies shows that astrocytes play an important role in the physiology and pathology of multiple central nervous system diseases. S100B is an astrocyte-specific Ca2+ binding protein when expressed in brain. S100B showed the sensitive and specific properties as a biomarker in acute stroke, cardiac arrest and subarachnoid hemorrhage. According to early results, S100B is involved in the serum deprivation induced astrocytic oxidative stress. The extracellular S100B could induce reactive oxidative species production through binding to membrane receptors, and thereupon induce oxidative injury intracellularly. Since its significance in the clinical diagnosis of multiple nervous system diseases of S100B, this thesis is focused on the effects of FLZ on serum deprivation induced astrocytic over secretion of S100B, and effects of FLZ on the followed oxidative stress induced by S100B over secretion.
Methods Astrocytes were cultured under normal and serum-deprived condition. Firstly, the optimal concentrations of FLZ exerting protective effects against serum deprivation injury was determined by 3-(4,5-dimethylthiazol-2yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Then, the morphological changes of cell nuclei were observed under fluorescence microscope after Hoechst 33342 staining. The level of secreted S100B was determined by ELISA. Furthermore, reactive oxygen species (ROS) in cultures was measured by fluorescence probe DCFDA. Finally, activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), concentrations of malondialdehyde (MDA) and glutathione (GSH) were determined using biochemical methods.
Results Serum deprivation caused cell viability decrease significantly (P< 0.001, vs. control group), and FLZ (I×10-7 mol/L、3×10-7 mol/L、I×10-6 mol/L、3×10-6 mol/L) treatment could increase the cell viability by 80%,69%,42% and 34%, respectively. Serum deprivation increased astrocytic nuclei damage significantly (P< 0.001), and FLZ treatment attenuated the nuclei damage of primary cortical astrocytes cultures with improvement rates of 66%,82%,85%,97%, respectively. After 4 h serum deprivation, the medium S100B content of model group increased continuously and significantly (P< 0.001) vs. control group. Meanwhile, the extracellular S100B level of FLZ (1×10-7 mol/L, 1×10-6 mol/L) treated primary astrocytes increased slowly and significantly (P< 0.001) lower than model group. The intracellular ROS levels of model group rose to 4.11 fold of control group, FLZ (1×10-7 mol/L-3×10-6 mol/L) significantly attenuated the overproduction of ROS induced by serum deprivation. Cultured under serum deprivation circumstance significantly decreased the astrocytic SOD activity (P< 0.001) and GSH content (P< 0.001), meanwhile increased the MDA production (P< 0.001) and GSH-Px activity (P<0.01). FLZ (3×10-7 mol/L-3×10-6 mol/L) increased the SOD activity by 21%,41% and 73% respectively, reduced the MDA overproduction by 39%,45% and 63% respectively, and raised the GSH content by 25%,33% and 63%, respectively. FLZ (1×10-7 mol/L & 3×10-7 mol/L) could reverse the serum deprivation induced GSH-Px activity changes, while GSH-Px activities in the higher dose-treated groups were not affected by FLZ.
Conclusion Serum deprivation could decrease the cell viability of primary cortical astrocytes significantly, and FLZ showed protective effects against serum deprivation induced cell damage. FLZ decreased the overproduction of ROS through attenuating the extracellular S100B level. FLZ demonstrated anti-oxidative effects to protect the serum-deprived astrocytes by inhibiting MDA over production, GSH level and SOD activity attenuation, and GSH-Px activity elevation. In conclusion, FLZ showed (?)tive effects of serum deprivation induced primary cortical astrocytes damage through decrease S100B production and enhance cellular anti-oxidative system.
引文
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