河豚毒素对BV-2细胞氧化应激损伤的诱导作用

Induction of Tetrodotoxin exposure to oxidative stress injury of BV-2 Cells

目的探究河豚毒素(TTX)暴露对小鼠小胶质细胞BV-2的氧化应激损伤的诱导作用。方法选择BV-2细胞进行TTX体外暴露。首先采用不同浓度(0、0.01、0.1、1、10、100μmol·L^(-1))TTX对BV-2细胞进行单独暴露,观察细胞形态和活力变化,利用CCK8法确定TTX联合暴露浓度。使用藜芦定(VTD)和毒毛旋花苷G(O)的混合物与TTX对BV-2细胞进行联合暴露,根据细胞形态和细胞活力验证BV-2细胞膜上离子通道类型。通过测定TTX单独暴露后乳酸脱氢酶(LDH)释放量、钙离子荧光量、活性氧(ROS)含量和细胞凋亡率水平,分析TTX暴露对BV-2细胞的影响。结果100μmol·L^(-1)TTX可改变BV-2细胞形态,抑制细胞活力。联合暴露情况下,TTX能够拮抗VTD和O混合物造成的细胞膜内外钠离子浓度差异,延缓细胞损伤状态。与对照组比较,100μmol·L^(-1)TTX可促使细胞膜通透性改变,释放4.01 U/g LDH。在100μmol·L^(-1)TTX暴露下分别升高细胞内钙离子和ROS水平,细胞相对荧光强度提升至84.78%和63.48%;100μmol·L^(-1)TTX暴露下,BV-2细胞凋亡率增加2.9倍。结论TTX暴露可以抑制BV-2细胞增殖能力,改变细胞膜的通透性,促使ROS在细胞内蓄积,诱导细胞氧化应激损伤,造成细胞凋亡。

Objective To investigate the induction of tetrodotoxin(TTX)exposure to oxidative stress damage of microglia BV-2 in mice.Methods BV-2 was selected as the cell model for TTX exposure in vitro.Firstly,BV-2 cells were exposed to TTX at different concentrations(0,0.01,0.1,1,10,100μmol·L^(-1))to observe the changes of cell morphology and vitality.CCK8 method was used to determine the combined exposure concentration of TTX.BV-2 cells were exposed using a mixture of veratridine(VTD)and ouabain(O)with TTX,and the types of ion channels on the BV-2 cell membrane were validated based on cell morphology and cell viability.The effects of TTX exposure on BV-2 cells were evaluated by determining lactate dehydrogenase(LDH)release,calcium ion fluorescence,reactive oxygen species(ROS)content and cell apoptosis rate after TTX exposure alone.Results Under experimental concentration exposure,100μmol·L^(-1)TTX could change the morphology of BV-2 cells and inhibit cell viability.Under combined exposure,TTX could counteract the difference of sodium concentration in and outside the cell membrane caused by VTD and O mixture and delay the cell injury state.Compared with the control group,100μmol·L^(-1)TTX could induce the change of cell membrane permeability and release 4.01 U/g LDH.The relative fluorescence intensity increased to 84.78%and 63.48%,respectively.The apoptosis rate increased by 2.9 times with significant difference.Conclusion Under the conditions of exposure concentration and time,TTX exposure can inhibit the proliferation of BV-2 cells,change the permeability of cell membrane,promote the accumulation of reactive oxygen species in cells,and induce oxidative stress injury of cells,resulting in cell apoptosis.