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中国临床药理学与治疗学 ›› 2019, Vol. 24 ›› Issue (11): 1227-1233.doi: 10.12092/j.issn.1009-2501.2019.11.003

• 基础研究 • 上一篇    下一篇

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

杨青霞,毛怡清,张燕芳,薄旭芳   

  1. 杭州师范大学附属医院 药剂科,杭州 310000,浙江
  • 收稿日期:2019-06-03 修回日期:2019-10-22 出版日期:2019-11-26 发布日期:2019-12-02
  • 通讯作者: 毛怡清,男,本科,药师,研究方向:药学。 Tel:13588333826 E-mail:maoyiqing01@163.com
  • 作者简介:杨青霞,女,本科,药师,研究方向:药学。 Tel:13567194182
  • 基金资助:

    浙江省医药卫生平台计划基金(2016HZB023)

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

YANG Qingxia,MAO Yiqing,ZHANG Yanfang,BO Xufang   

  1. Pharmacy Department, Affiliated Hospital of Hangzhou Normal University, Hangzhou 310000, Zhejiang,China
  • Received:2019-06-03 Revised:2019-10-22 Online:2019-11-26 Published:2019-12-02

摘要:

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-α mRNA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。

关键词: 干眼症, 氟西汀, 人结膜上皮细胞, Toll样受体2, 核转录因子-κB, 炎症

Abstract:

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.

Key words: xerophthalmia, fluoxetine, human conjunctival epithelial cells, Toll-like receptor 2, nuclear factor-&kappa, B, inflammation

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