褐藻素对脂多糖活化小胶质细胞诱导神经元损伤的保护作用

Protective effect of fucoxanthin on neuronal injury induced by lipopolysac-charide activated microglia

目的探讨褐藻素是否对脂多糖(LPS)激活的小胶质细胞诱导神经元细胞损伤具有保护作用。方法采用LPS活化BV2小胶质细胞构建小胶质细胞激活模型。将BV2细胞分为6组:正常对照组、LPS组、低剂量褐藻素治疗组、高剂量褐藻素治疗组、正常低剂量褐藻素对照组、正常高剂量褐藻素对照组。采用酶联免疫吸附试验(ELISA)检测各组BV2细胞上清中肿瘤坏死因子(TNF)-α和白细胞介素(IL)-6水平;通过活性氧(ROS)试剂盒和线粒体膜电位试剂盒,采用流式细胞术分别检测各组BV2细胞的ROS水平和线粒体膜电位;采用CCK-8检测各组BV2细胞条件培养基与SH-SY5Y细胞共培养24 h后SH-SY5Y细胞的细胞存活率;采用膜联蛋白(Annexin)Ⅴ-异硫氰酸荧光素(FITC)试剂盒、流式细胞仪检测各组SH-SY5Y细胞的凋亡情况。结果与正常对照组相比,LPS组TNF-α、IL-6水平明显增加(P<0.05);与LPS组相比,高、低剂量褐藻素治疗组TNF-α和IL-6水平均明显降低(P<0.05)。正常对照组和正常低、高剂量褐藻素对照组TNF-α和IL-6水平均无明显差异(P>0.05)。LPS组相较于正常对照组,BV2细胞ROS水平显著升高(P<0.05);高、低剂量褐藻素治疗组较LPS组BV2细胞ROS水平则显著降低(P<0.05)。正常对照组和正常低、高剂量褐藻素对照组ROS无明显差异(P>0.05)。与正常对照组相比,LPS组BV2细胞线粒体膜电位显著降低(P<0.05);高、低剂量褐藻素治疗组与LPS组相比,BV2细胞线粒体膜电位均显著升高(P<0.05)。正常对照组和正常低、高剂量褐藻素对照组线粒体膜电位无统计学差异(P>0.05)。LPS激活的BV2细胞条件培养基与SH-SY5Y共培养后,SH-SY5Y细胞存活率明显降低,细胞凋亡率明显增加(P<0.05);高、低剂量褐藻素治疗组与LPS组相比,SH-SY5Y细胞存活率均明显升高,而凋亡率则均明显降低(P<0.05)。正常对照组和正常低、高剂量褐藻素对照组细胞存活率和凋亡率均无统计学差异(P>0.05)。结论褐藻素可能通过减少促炎因子的释放、改善JC-1、抑制氧化应激水平,抑制小胶质细胞的过度活化和减少神经元凋亡,因而具有神经保护功能。

Objective To investigate whether fucoxanthin has protective effect on neuron injury cell induced by lipopolysaccharide(LPS)-activated microglia.Methods LPS-activated BV2 microglia was used to establish microglia activation cell model.BV2 cells were divided into six groups:normal control group,LPS group,low-dose fucoxanthin treatment group,high-dose fucoxanthin treatment group,normal low-dose fucoxanthin control group and normal high-dose fucoxanthin control group.Tumor necrosis factor(TNF)-α and interleukin(IL)-6 levels in the supernatant of BV2 cells in each group were detected by enzyme-linked immunosorbent assay(ELISA);the level of reactive oxygen species(ROS)and mitochondrial membrane potentials of BV2 cells in each group were detected by flow cytometry using ROS kit and JC-1 kit;the cell viability of SH-SY5Y cells was measured by CCK-8 after co-culturing SH-SY5Y cells with BV2 cell conditioned medium in each group for 24 h;the apoptosis of SH-SY5Y cells in each group was detected by flow cytometry and AnnexinⅤ-fluorescein isothiocyanate(FITC)reagent kit.Results TNF-α and IL-6 levels in the LPS group were both significantly increased compared to those in the normal control group(P<0.05),and the high or low dose fucoxanthin treatment groups had a significant decrease in the levels of TNF-α and IL-6 when compared with the LPS group(P<0.05).There was no significant difference in TNF-α and IL-6 levels between normal control group and normal control groups with low or high dose of fucoxanthin(P>0.05).Compared with the normal control group,the LPS group showed a significant increase in the ROS level of BV2 cells(P<0.05),while the ROS levels in BV2 cells in the high-dose and low-dose fucoxanthin treatment groups were both significantly lower than those in the LPS group(P<0.05).There was no significant difference in ROS between normal control group and normal control groups with low or high dose of fucoxanthin(P>0.05).Compared to that of the normal control group,the mitochondrial membrane potential of BV2 cells in LPS group was significantly lower(P<0.05);compared with those of the LPS group,the mitochondrial membrane potentials of BV2 cells in the high-dose and low-dose fucoxanthin treatment groups were significantly increased(P<0.05).There was no significant difference in mitochondrial membrane potentials between normal control group and normal control groups with low or high dose of fucoxanthin(P>0.05).After co-culturing BV2 cells activated by LPS in conditioned medium with SH-SY5Y,the cell viability of SH-SY5Y cells was significantly decreased and the apoptosis rate was significantly increased(P<0.05),while the high-dose and low-dose fucoxanthin treatment groups both showed a significant increase in cell viability and a significant decrease in the apoptosis rate of SH-SY5Y cells compared to the LPS group(P<0.05).There was no significant difference in cell viability and apoptosis rate between normal control group and normal control groups with low or high dose of fucoxanthin(P>0.05).Conclusions Fucoxanthin might have neuroprotective function by reducing the release of proinflammatory factors,improving JC-1,inhibiting the level of oxidative stress,and then inhibiting the over activation of microglia and reducing neuronal apoptosis.