Autophagy dysregulation mediates the damage of high glucose to retinal pigment epithelium cells

AIM:To observe the role and mechanism of autophagy in retinal pigment epithelial cell(RPE)damaged by high glucose,so as to offer a new idea for the treatment of diabetic retinopathy(DR).METHODS:ARPE-19,a human RPE cell line cultured in vitro was divided into the normal control(NC),autophagy inhibitor 3-methyladenine(3-MA),high-glucose(HG),and HG+3-MA groups.Cell viability was detected by CCK-8 assay and the apoptosis rate was measured by flow cytometry.The protein expressions of apoptosis markers,including Bax,Bcl-2,and Caspase-3,as well as autophagy marker including microtubule-related protein 1 light chain 3(LC3),p62,and mechanistic target of rapamycin(m TOR)were detected by Western blotting.Autophagic flux was detected by transfection with Ad-m Cherry-GFP-LC3 B.RESULTS:Under high glucose conditions,the viability of ARPE-19 was decreased,and the apoptosis rate increased,the protein expressions of Bax,Caspase-3,and LC3-II/LC3-I were all increased and the expressions of Bcl-2,p62 and p-m TOR decreased,and autophagic flux was increased compared with that of the controls.Treatment with 3-MA reversed all these changes caused by high glucose.CONCLUSION:The current study demonstrates the mechanisms of cell damage of ARPE-19 through high glucose/m TOR/autophagy/apoptosis pathway,and new strategies for DR may be developed based on autophagy regulation to manage cell death of RPE cells.

吡非尼酮对内皮细胞间质转化的抑制作用及机制

目的:建立缺氧诱导内皮细胞的内皮-间质转化(EndoMT)模型,探讨吡非尼酮(PFD)在视网膜下纤维瘢痕形成过程中的抗纤维化作用及机制。方法:原代培养人脐静脉内皮细胞,鉴定后取4~7代用于实验。CoCl2诱导细胞缺氧建立纤维化模型。CCK-8法检测细胞增殖率,筛选药物浓度。将细胞分为对照组(无血清培养基培养)、CoCl2(200μmol/L)模型组、CoCl2+低浓度(0.3mg/mL)PFD组、CoCl2+高浓度(0.6mg/mL)PFD组。Western blot法检测细胞CD31、VE-cadherin、α-SMA、FSP1、p-p38和p38的蛋白表达水平。CD31/α-SMA免疫荧光双染法观察蛋白表达的变化。划痕实验观察细胞迁移能力的改变。q-PCR法检测TGF-β1、SNAI1 mRNA转录水平。结果:与CoCl2模型组相比,PFD能明显提高缺氧细胞的增殖率,抑制细胞的迁移能力(均P<0.05);PFD组细胞标志蛋白CD31、VE-cadherin表达增加,α-SMA、FSP1表达降低(均P<0.05)。免疫荧光检测显示PFD可明显抑制α-SMA和增加CD31的蛋白表达(P<0.05)。内皮细胞EndoMT过程中,p38总蛋白表达不变(P>0.05),但p-p38磷酸化蛋白表达增加、TGF-β1和SNAI mRNA转录水平增高的现象可明显被PFD抑制(均P<0.05)。高低浓度PFD组上述各现象无明显差异。结论:PFD可以抑制内皮细胞纤维化的发生,TGF-β/p38 MAPK通路可能是PFD调控EndoMT过程的机制之一,为视网膜下纤维化的治疗提供了新思路。