自噬对视网膜色素上皮细胞表达bFGF及TGF-β2的调控作用

Autophagy regulates the expression of bFGF and TGF-β2 in retinal pigment epithelium

目的观察缺氧条件下自噬对视网膜色素上皮细胞(retinal pigment epithelium,RPE)表达碱性成纤维细胞生长因子(basic fibroblast growth factor,b FGF)及转化生长因子β2(transforming growth factor,TGF-β2)的调控作用。方法将体外培养生长良好的ARPE-19细胞随机分为4组:对照组(常氧条件下培养),缺氧组(在1%O_2/5%CO_2/94%N_2缺氧箱中培养24h),缺氧+3-MA组(加入10 mmol/L自噬抑制剂3-甲基腺嘌呤,3-MA)及缺氧+CQ组(加入50μmol/L自噬抑制剂氯喹,CQ),后三组均在缺氧箱中培养24 h。采用Western blot法检测细胞自噬相关蛋白微管相关蛋白1轻链3B(microtubule associated protein 1 light chain 3 B,LC3B)、Beclin-1及p62的表达,ELISA法检测细胞上清液中b FGF及TGF-β2的浓度。结果正常对照组、缺氧组、缺氧+3-MA组和缺氧+CQ组细胞中LC3B-Ⅱ/LC3B-Ⅰ比值分别为0.24±0.02,0.50±0.05,0.34±0.01和0.67±0.05;Beclin-1相对表达量分别为0.32±0.040,0.97±0.05,0.78±0.06和0.54±0.01;p62相对表达量分别为1.03±0.02,0.32±0.10,0.78±0.08和0.96±0.02;b FGF的浓度(pg/ml)分别为1 771.01±110.38,3 709.79±262.68,3 026.24±230.39和2 610.01±183.52;TGF-β2的浓度(pg/ml)分别为256.11±27.63,516.85±57.94,439.45±17.53和353.58±33.12。与正常对照组相比,缺氧组细胞的LC3B-Ⅱ/LC3B-Ⅰ比值、Beclin-1的蛋白表达明显增加,p62明显下降(P<0.05),b FGF及TGF-β2水平升高(P<0.05)。与缺氧组相比,缺氧+3-MA组或缺氧+CQ组抑制自噬能明显减少或增加LC3B-Ⅱ/LC3B-Ⅰ的表达,减少Beclin-1的表达,增加p62的表达,同时减少b FGF及TGF-β2的表达。结论缺氧导致RPE细胞自噬激活,进一步调控RPE细胞b FGF及TGF-β2的表达。

Objective To investigate the regulation effect of autophagy on basic fibroblast growth factor（b FGF） and transforming growth factor β2（TGF-β2） expression in retinal pigment epithelium（RPE） exposure to hypoxia.Methods ARPE-19,a RPE cell line,was randomly divided into four groups：control group,hypoxia group,hypoxia ＋ 3-methyladenine（3-MA） group,and hypoxia ＋chloroquine（CQ） group.The ARPE-19 cells were incubated in a normoxic incubator in control group,and a hypoxic incubator with1% O_2/5% CO_2/94% N_2 for 24 h in hypoxia group.The ARPE-19 cells were treated with 10 mmol/L 3-MA in the hypoxic incubator for 24 h in hypoxia ＋ 3-MA group,and 50 μmol/L CQ in the hypoxic incubator for 24 h in hypoxia ＋ CQ group.Western blot was performed to detect the expression of autophagy-associated factors,including microtubule associated protein 1 light chain 3 B（LC3 B）,Beclin-1 and p62.The concentrations of b FGF and TGF-β2 in the culture supernatant were determined by ELISA after 24 h culture.Results In control group,hypoxia group,hypoxia ＋ 3-MA group and hypoxia ＋ CQ group,the LC3 B-II/LC3 B-I ratio was 0.24 ±0.02,0.50 ± 0.05,0.34 ± 0.01 and 0.67 ± 0.05,respectively.The relative expression of Beclin-1 was 0.32 ± 0.040,0.97 ± 0.05,0.78 ± 0.06 and 0.54 ± 0.01 in control group,hypoxia group,hypoxia ＋ 3-MA group and hypoxia ＋ CQ group,respectively.The relative expression of p62 was 1.03 ± 0.02,0.32 ± 0.10,0.78 ± 0.08 and 0.96 ± 0.02 in control group,hypoxia group,hypoxia ＋ 3-MA group and hypoxia ＋ CQ group,respectively.The concentration of b FGF（pg/ml） was 1 771.01 ± 110.38,3 709.79 ± 262.68,3026.24 ± 230.39 and 2 610.01 ± 183.52 in control group,hypoxia group,hypoxia ＋ 3-MA group and hypoxia ＋ CQ group,respectively.The concentration of TGF-β2（pg/ml） was 256.11 ± 27.63,516.85 ± 57.94,439.45 ± 17.53 and 353.58 ± 33.12 in control group,hypoxia group,hypoxia ＋ 3-MA group and hypoxia ＋ CQ group,respectively.Compared to control group,the LC3 B-Ⅱ/LC3 B-Ⅰ ratio and Beclin-1 expression were significantly increased,while p62 level was significantly decreased in hypoxia group（all P〈 0.05）.The concentrations of b FGF and TGF-β2 were increased in hypoxia group compared to control group（P 〈0.05）.Compared to hypoxia group,the LC3 B-Ⅱ/LC3 B-Ⅰ ratio was significantly reduced in hypoxia ＋ 3-MA group while increased in hypoxia ＋ CQ group.Compared to hypoxia group,the expression of Beclin-1 was significantly reduced in hypoxia ＋ 3-MA or hypoxia ＋ CQ groups,while expression of p62 was increased.The concentrations of b FGF and TGF-β2 were significantly decreased in hypoxia ＋ 3-MA group and hypoxia ＋ CQ group compared with hypoxia group.Conclusion Hypoxia may elevate autophagy in RPE cells,and the autophagy may regulate the expression of b FGF and TGF-β2 in RPE cells.