CAJ | 학술논문

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.
목적 탐토파라서정대지다당(LPS)손상적해마신경간세포(NSCs)활력급세포조망적영향,병초보연구포외신호조절격매(ERK)신호통로재기중적작용.방법 건입체외대서해마NSCs적LPS손상세포모형,분위대조조(sham조)、LPS조、LPS+파라서정조(포괄1、5、10화20μmoL/L 4개불동제량조),작용72 h후,채용WST-8시제검측세포활성,단백질인적(Western blot)검측린산화포외신호조절격매1/2(pERK1/2)적표체수평,류식세포술검측세포조망정황.위료진일보명학ERK신호통로재저일과정중적작용,재간세포배양기중첨가ERK억제제U0126,병수궤분위U0126조、U0126+ LPS조、U0126+ LPS+파라서정조(포괄1、5、10 μmol/L 3개불동제량조),작용72 h후채용류식세포술검측세포조망정황.결과 여sham조(흡광도치0.342±0.010)화LPS+파라서정5 μmol/L조(흡광도치0.338±0.016)상비,LPS조(흡광도치0.306±0.016)세포활력경저(F =23.019,P치균＜0.01),이LPS조여LPS+파라서정1μmol/L조(흡광도치0.323±0.013)、LPS+파라서정10 μmol/L조(흡광도치0.304±0.012)적세포활력접근.Western blot결과현시,LPS조(0.749 ±0.144)적pERK1/2표체수평명현저우sham조(0.982 ±0.131)급LPS+파라서정5 μmol/L조(1.092±0.113)(F=6.887,P치균＜0.05),이LPS조여LPS+파라서정1μmol/L조(0.871±0.123)급LPS+파라서정10 μmol/L조(0.766 ±0.209)적pERK1/2표체수평접근.류식세포술검측조망적결과여세포활력검측결과일치,여sham조[(13.40±2.87)％]비교,LPS조[(17.85±1.26)％]조망솔경고,5 μmol/L파라서정[(14.42±1.22)％]대LPS적촉조망작용구유완해효응(F=26.520,P＜0.05),이1μmol/L화10 μmol/L파라서정칙무차완해효응；U0126촉진NSCs적조망,이차가억제5 μmol/L파라서정대LPS손상적보호효과.결론 괄당농도적파라서정능억제LPS도치적해마NSCs손상,차저충작용가능시통과영향pERK1/2적표체실현.
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.