氟西汀对人结膜上皮细胞TLR2/NF-κB信号通路和炎性因子的影响

Effects of fluoxetine on TLR2/NF-κB signaling pathway and inflammatory factors in human conjunctival epithelial cells

目的:观察氟西汀对人结膜上皮细胞Toll样受体2(toll-like receptor 2,TLR2)/核转录因子-κB(NF-κB)信号通路和炎性因子的影响,探讨五羟色胺再摄取抑制剂(selective serotonin reuptake inhibitor,SSRI)引起干眼症的潜在机制。方法:将结膜上皮细胞分为空白对照组和药物组,药物组采用氟西汀进行干预。采用CCK-8法明确氟西汀对人结膜上皮细胞半数抑制率作为后续实验浓度。采用RT-PCR法检测TLR2、NF-κB、白细胞介素(interleukin,IL)-1β、IL-2、IL-6和肿瘤坏死因子(tumor necrosis factor,TNF)-α的mRNA水平;免疫荧光法和Western blot法检测TLR2和NF-κB的蛋白表达水平;ELISA法检测IL-1β、IL-2、IL-6和TNF-α的蛋白表达水平。结果:10^-9~10^-5mol/L范围内氟西汀对结膜上皮细胞具有增殖抑制作用,且具有浓度依赖性。RT-PCR法检测结果显示,氟西汀组TLR2、NF-κB、IL-1β、IL-2、IL-6和TNF-αmRNA水平均高于空白对照组(P<0.05)。药物组TLR2和NF-κB的累积光密度(IOD)值明显高于空白对照组(P<0.05),提示药物组TLR2和NF-κB蛋白表达量高于空白对照组。Western blot法检测结果显示药物组TLR2和NF-κB的蛋白表达水平均高于空白对照组(P<0.05)。ELISA检测结果显示药物组IL-1β、IL-2、IL-6和TNF-α蛋白表达水平均高于空白对照组(P<0.05)。结论:本研究发现氟西汀可能通过激活结膜上皮细胞TLR2/NF-κB信号通路,促进炎症因子水平,这可能是氟西汀所致干眼症潜在的生物学机制。

AIM:To observe the effects of fluoxetine on Toll-like receptor 2(TLR2)/nuclear transcription factor-kappa B(NF-κB)signaling pathway and inflammatory factors in human conjunctival epithelial cells,and to explore the potential mechanism of SSRI-induced xerophthalmia.METHODS:Conjunctival epithelial cells were divided into blank control group and drug group.Fluoxetine was used to intervene in drug group.CCK-8 method was used to determine the half inhibition rate of fluoxetine on human conjunctival epithelial cells as a follow-up concentration.The levels of TLR2,NF-kappa B,interleukin(IL)-1beta,IL-2,IL-6 and tumor necrosis factor(TNF)-alpha were detected by RT-PCR,the levels of TLR2 and NF-kappa B protein were detected by immunofluorescence and Western-blot,and the levels of IL-1beta,IL-2,IL-6 and TNF-alpha protein were detected by ELISA.RESULTS:Fluoxetine can inhibit the proliferation of conjunctival epithelial cells in a concentration-dependent manner in the range of 10^-9 to 10^-5 mol/L.RT-PCR results showed that the levels of TLR2,NF-kappa B,IL-1beta,IL-2,IL-6 and TNF-alpha in fluoxetine group were higher than those in blank control group(P<0.05).The IOD values of TLR2 and NF-kappa B in the drug group were significantly higher than those in the blank control group(P<0.05),suggesting that the expressions of TLR2 and NF-kappa B in the drug group were higher than those in the blank control group.Western blot analysis showed that the expression levels of TLR2 and NF-kappa B in the drug group were higher than those in the blank control group(P<0.05).ELISA results showed that the expression levels of IL-1beta,IL-2,IL-6 and TNF-alpha in the drug group were higher than those in the blank control group(P<0.05).CONCLUSION:Fluoxetine may promote the level of inflammatory factors by activating TLR2/NF-κB signaling pathway in conjunctival epithelial cells.This may be the potential biological mechanism of fluoxetine-induced xerophthalmia.