氟西汀对培养的人结膜上皮细胞炎性因子及丝裂原活化蛋白激酶信号通路的影响

Effect of Fluoxetine on Inflammation-Related Cytokines and MAPK Pathway in Human Conjunctival Epithelial Cells

目的:探讨氟西汀对体外培养的人结膜上皮细胞(HConEpiC)细胞炎性因子IL-1和TNF-α的表达及丝裂原活化蛋白激酶(MAPK)信号通路的影响。方法:混合消化液培养法培养人结膜上皮细胞,免疫组织化学方法进行细胞鉴定。培养至第3代或第4代,在细胞培养液中分别加入不同浓度的氟西汀(0,1,2.5,5,10,20,40μmol/L),CCK-8法检测细胞增殖率;免疫荧光检测ERK1/2、P-ERK1/2、IL-1、TNF-α在对照组及氟西汀组(5μmol/L)HConEpiC细胞中的定位;Western Blot检测HConEpiC细胞中ERK、P-ERK、IL-1、TNF-α蛋白表达水平。结果:随着氟西汀浓度的增加,HConEpiC细胞增殖率呈缓慢降低状态,以氟西汀浓度为5μmol/L进一步探讨其对蛋白量表达的影响。对细胞免疫荧光照片进行光密度分析发现,氟西汀组ERK1/2的累积光密度(IOD)值较对照组差异无统计学意义,提示氟西汀干预对HConEpiC中ERK1/2蛋白的表达无明显影响;氟西汀组PERK1/2、IL-1、TNF-α的IOD值较对照组均明显增高(P<0.05),提示氟西汀干预后HConEpiC中P-ERK1/2、IL-1、TNF-α蛋白表达增多;Western Blot实验检测各组蛋白量结果显示,氟西汀组与对照相比,ERK1/2蛋白量表达无明显差异;氟西汀组P-ERK1/2、IL-1、TNF-α蛋白表达水平较对照组升高(P<0.05)。结论:氟西汀可激活培养的正常人结膜上皮细胞内MAPK-ERK信号通路并导致炎性相关因子的产生。

Objective:To evaluate the effects of fluoxetine on inflammation-related cytokines and MAPK pathway in human conjunctival epithelial cells.Methods:Human primary conjunctival epithelial cells were isolated from donors and cultured,then identified by immunochemistry.Cultured epithelial cells were treated with different concentrations of fluoxetine(0,1,2.5,5,10,20,40μmol/L);CCK-8 method was used to detect cell proliferation rate;the expression and cellular localization of ERK1/2,P-ERK1/2,IL-1,TNF-αin the groups of fluoxetine and control were detected by immunofluorescence assay;the protein expression of ERK1/2,P-ERK1/2,IL-1,TNF-αin conjunctival epithelial cells were determined by Western blot.Results:The conjunctival epithelial cell proliferation rate were reduced slowly with increased concentrations of fluoxetine;By compari-son between the fluoxetine groups and control groups,very little difference was detected in ERK1/2,while P-ERK1/2,IL-1 and TNF-αlevels increased significantly(P<0.05);Western blot detection showed obvious difference in P-ERK1/2,IL-1,and TNF-αwhen treated with fluoxetine(P<0.05).Conclusion:Fluoxetine may activate MAPK-ERK signaling pathway and lead to the production of inflammatory-related factors in normal human conjunctival epithelial cells.