阿司匹林通过激活p38丝裂原活化蛋白激酶损伤视网膜色素上皮细胞的屏障功能

Aspirin injures barrier function of human retinal pigment epithelial cells by activating p38MAPK signaling pathway

目的探讨阿司匹林对人视网膜色素上皮细胞屏障功能的影响和潜在分子机制。方法选取第3代人视网膜色素上皮细胞:(1)细胞培养液中添加阿司匹林,浓度分别为5 mmol/L、10 mmol/L、20 mmol/L,刺激24 h后,检测细胞活力。(2)细胞培养液中添加10 mmol/L的阿司匹林,刺激时间分别是30 min、60 min、120 min,提取细胞蛋白,检测p38丝裂原活化蛋白激酶(MAPK)磷酸化水平。(3)细胞分为对照组(未刺激),阿司匹林组(10 mmol/L的阿司匹林刺激24 h),阿司匹林+SB2035080刺激组(联合组,1μmol/L的SB2035080预处理30 min,10 mmol/L的阿司匹林刺激24 h),检测细胞的跨膜电阻抗(TER)和单层细胞的通透性。结果5、10、20 mmol/L视网膜色素上皮细胞的活力与对照组比较,无统计学差异(P>0.05)。与对照组比较,阿司匹林刺激30 min、60 min、120 min的p38MAPK蛋白磷酸化程度明显增加(P<0.05)。与对照组比较,阿司匹林组上皮细胞的TER、claudin-7和ZO-1蛋白表达明显降低,细胞通透性明显增高(P<0.05)。与阿司匹林组比较,联合组TER、claudin-7和ZO-1蛋白表达明显升高,细胞通透性明显降低(P<0.05)。结论阿司匹林通过激活p38MAPK信号通路,抑制紧密连接蛋白claudin-7和ZO-1蛋白表达,从而损伤人视网膜色素上皮细胞的屏障功能。

Objective To study the effect of aspirin on barrier function of human retinal pigment epithelial cells(HRPEC)and its underlying molecular mechanism.Methods The viability of HRPEC was assayed at 24 h after the HRPEC were stimulated in cell culture medium into which aspirin was added at the concentrations of 5 mmol/L,10 mmol/L,20 mmol/L respectively.The protein was extracted from HRPEC after they were stimulated for 30 min,60 min and 120 min respectively in cell culture medium into which 10 mmol/L aspirin was added.The p38 MAPK phosphorylation level was measured.The HRPEC were divided into control group,aspirin group,aspirin+SB2035080 stimulation group.The HRPEC in control group were not stimulated with aspirin,those in aspirin group were stimulated with 10 mmol/L aspirin for 24 h,and those in aspirin+SB2035080 stimulation group were pretreated with 1μmol/L SB2035080 for 30 min and stimulated with 10 mmol/L aspirin for 24 h.The transmembrane electrical impedance(TER)of HRPEC and the permeability of monolayer cells were detected in different groups.Results No significant difference was detected in viability of HRPEC stimulated with aspirin at the concentrations of 5 mmol/L,10 mmol/L,and 20 mmol/L among the 3 groups(P>0.05).The p38 MAPK phosphorylation level was significantly higher in aspirin stimulation group than in control group(P<0.05).The TER and expression levels of claudin-7 and ZO-1 proteins were significantly lower while the permeability of monolayer cells was significantly higher in aspirin group than in control group(P<0.05).The TER and expression levels of claudin-7 and ZO-1 proteins were significantly higher while the permeability of monolayer cells was significantly lower in aspirin+SB2035080 stimulation group than in aspirin group(P<0.05).Conclusion Aspirin can inhibit the expressions of claudin-7 and ZO-1 proteins by activating the p38 MAPK signaling pathway,and injure the barrier function of HRPEC.