帕罗西汀对脂多糖损伤的海马神经干细胞的保护作用

Protective effects of paroxetine on the lipopolysaccharide injured hippocampal-derived neural stem cell

目的 探讨帕罗西汀对脂多糖（LPS）损伤的海马神经干细胞（NSCs）活力及细胞凋亡的影响,并初步研究胞外信号调节激酶（ERK）信号通路在其中的作用.方法 建立体外大鼠海马NSCs的LPS损伤细胞模型,分为对照组（sham组）、LPS组、LPS＋帕罗西汀组（包括1、5、10和20μmoL/L 4个不同剂量组）,作用72 h后,采用WST-8试剂检测细胞活性,蛋白质印迹（Western blot）检测磷酸化胞外信号调节激酶1/2（pERK1/2）的表达水平,流式细胞术检测细胞凋亡情况.为了进一步明确ERK信号通路在这一过程中的作用,在干细胞培养基中添加ERK抑制剂U0126,并随机分为U0126组、U0126＋ LPS组、U0126＋ LPS＋帕罗西汀组（包括1、5、10 μmol/L 3个不同剂量组）,作用72 h后采用流式细胞术检测细胞凋亡情况.结果 与sham组（吸光度值0.342±0.010）和LPS＋帕罗西汀5 μmol/L组（吸光度值0.338±0.016）相比,LPS组（吸光度值0.306±0.016）细胞活力更低（F =23.019,P值均＜0.01）,而LPS组与LPS＋帕罗西汀1μmol/L组（吸光度值0.323±0.013）、LPS＋帕罗西汀10 μmol/L组（吸光度值0.304±0.012）的细胞活力接近.Western blot结果显示,LPS组（0.749 ±0.144）的pERK1/2表达水平明显低于sham组（0.982 ±0.131）及LPS＋帕罗西汀5 μmol/L组（1.092±0.113）（F=6.887,P值均＜0.05）,而LPS组与LPS＋帕罗西汀1μmol/L组（0.871±0.123）及LPS＋帕罗西汀10 μmol/L组（0.766 ±0.209）的pERK1/2表达水平接近.流式细胞术检测凋亡的结果与细胞活力检测结果一致,与sham组[（13.40±2.87）％]比较,LPS组[（17.85±1.26）％]凋亡率更高,5 μmol/L帕罗西汀[（14.42±1.22）％]对LPS的促凋亡作用具有缓解效应（F=26.520,P＜0.05）,而1μmol/L和10 μmol/L帕罗西汀则无此缓解效应；U0126促进NSCs的凋亡,而且可抑制5 μmol/L帕罗西汀对LPS损伤的保护效果.结论 适当浓度的帕罗西汀能抑制LPS导致的海马NSCs损伤,且这种作用可能是通过影响pERK1/2的表达实现.

Objective To investigate the effects of paroxetine on the cell viability and expression of the phosphorylated ERK1/2 in lipopolysaccharide LPS injured hippocampal-derived neural stem cells （NSCs）.Methods The NSCs were derived from hippocampus of fetal rats,after the primary neurospheres passaged,the cells were divided into sham,LPS and LPS ＋ paroxetine groups （include 1,5,10 and 20μmol/L concentration）,each group was treated with LPS （200 μg/L） and different concentrations of paroxetine for 72 h,then the cell viability and the protein level of pERK1/2 were measured by the kit of WST-8 and Western blot,respectively.Furthermore,in order to investigate the role of ERK pathway that involved in the effects of paroxetine,the NSCs were divided into sham,LPS,LPS ＋ paroxetine groups （include 1,5,and 10 μmol/L concentration）,U0126,U0126 ＋ LPS and U0126 ＋ LPS ＋ paroxetine groups （include 1,5,and 10 μmol/L concentration）,72 h later,the apoptosis rate was measured by Annexin-Ⅴ-FLUOS staining kit.Results The cell viability in 5 μmol/L paroxetine treated group （0.338 ±0.016） was higher than LPS group （0.306 ± 0.016） （F =23.019,P ＜ 0.01）,but there were no significant differences between other paroxetine treated groups and LPS group.When detected by Western blot,the expression of pERK1/2 in paroxetine 5 μmol/L treated groups （1.092 ±0.113） or sham group （0.982 ± 0.131） was significant higher than LPS group （0.749 ± 0.144）,respectively （F =6.887,P ＜ 0.05）.Furthermore,coincide with cell viability,the apoptosis of 5 μmol/L treated groups [（14.42 ± 1.22） ％] or sham group [（13.40 ± 2.87） ％] was significant lower than LPS group [（17.85 ± 1.26） ％] by using flow cytometry （F =26.520,P ＜ 0.05）.U0126 promoted the apoptosis of LPS treated groups,and there was no significant difference between paroxetine ＋ LPS treated groups and LPS group.Conclusions The cellular damage of NSCs injured by LPS could be restrained by paroxetine,which is possibly carried out by affecting ERK1/2 pathway.