摘要
目的探讨同型半胱氨酸(Hcy)对人结肠腺癌(Caco-2)细胞中白三烯E_4(LTE_4)、前列素E_2(PGE_2)合成水平和核转录因子-κB(NF-κB)活性的影响。方法在Caco-2细胞中加入不同浓度(0、10、20、50μmol/L)Hcy作用不同时间(1、3、6 h),采用MTT法和检测乳酸脱氢酶(LDH)活性,观察Hcy对Caco-2细胞生长活性的影响;通过检测Caco-2细胞跨膜电阻(TEER)和样品中荧光素-5-异硫氰酸酯(FITC)浓度,观察Hcy对Caco-2细胞通透性的影响;采用酶联免疫吸附实验(ELISA)测定Caco-2细胞上清液中LTE_4和PGE_2合成水平;采用凝胶迁移实验(EMSA)测定Caco-2细胞中NF-κB活性变化。结果随Hcy作用时间延长及作用浓度增加,Caco-2细胞活性明显受到抑制;随Hcy作用浓度增加,Caco-2细胞TEER明显降低,FITC跨膜转运量明显增加,细胞上清液中LTE_4和PGE_2水平明显升高(P<0.05);Hcy(50μmol/L)处理组细胞中NF-κB活性增强,且呈时间依赖性(1、3、6 h)。结论 Hcy通过激活NF-κB信号通路,促进Caco-2细胞肠黏膜LTE_4和PGE_2合成,引起肠黏膜炎症损伤。
Objective To elucidate the effects of homocysteine (Hcy) on the synthetic levels of leukotriene E4 (LTE4) and prostaglandin E2 (PGE2) in Caco-2 cells and the activity of nuclear factor kappa B (NF-κB). Methods Caco-2 cells were cultured and treated with Hcy in different concentrations (0,10,20,50 μmol/L) and time (1,3,6 h),MTT method and LDH activity were used to observe the change of cells growth viability. The concentrations of FITC in samples and TEER of Caco-2 cells were examined to understand the effect of Hcy on Caco-2 cells permeability. The synthetic levels of LTE4 and PGE2 were measured by enzyme linked immunosorbent assay (ELISA) in cells supernatant and the activity of NF-κB was detected by Gel mobility migration (EMSA). Results After treatment with Hcy,the viability of Caco-2 cells were significantly inhibited with a time and dose dependent manner. With the increasing of Hcy concentrations,the TEER significantly decreased and the FITC transmembrane transport greatly increased,the levels of LTE4 and PGE2 in cells supernatant were markly augmented ( P <0.05);the activity of Caco-2 cells treated with Hcy (50 μmol/L) significant augmented with a time dependent (1,3,6 h) manner. Conclusion Hcy can accelerate the synthesis of LTE4 and PGE2 in intestinal mucosa by activating NF-κB signaling pathway,causing intestinal mucosal inflammation damage in Caco-2 cells.
作者
王晶
梅咏玉
丁少桢
袁静静
梅俏
许建明
Wang Jing;Mei Yongyu;Ding Shaozhen(Dept of Gastroenterology,The First Affiliated Hospital of Anhui Medical University,The Key Laboratory of Digestive Disease of Anhui Province,Hefei 230022)
出处
《安徽医科大学学报》
CAS
北大核心
2019年第5期744-748,共5页
Acta Universitatis Medicinalis Anhui
基金
国家自然科学基金(编号:81470809)
