摘要
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。
AIM:To establish the hypoxia induced endothelial-mesenchymal transition(EndoMT)model of endothelial cells,and to investigate the effect and mechanism of Pirfenidone(PFD)on inhibiting the subretinal fibrosis progression.METHODS:Primary cultured human umbilical vein endothelial cells(HUVEC),4-7 passages were used for experiments after cell identification.CoCl2 induced hypoxia to establish the transformation model of endothelial cells into fibroblasts.CCK-8 was performed to detect cell proliferation rate and chose the optimal drug concentration.All cells were divided into 4 groups:control group(FBS-free),CoCl2(200μmol/L)group,CoCl2+0.3mg/mL PFD group,CoCl2+0.6mg/mL PFD group.The protein expression of CD31,VE-cadherin,α-SMA,FSP1,p-p38 and p38 were detected by Western blot.Double immunofluorescence labeling method was used to observe the CD31/α-SMA expression.Wound healing assay detected the cell migration.The q-PCR was applied to detect the mRNA levels of TGF-β1 and SNAI1.RESULTS:Compared with CoCl2 group,PFD increased cell proliferation rate and inhibited cell migration significantly under hypoxia(P<0.05).PFD decreased the protein expression of the mesenchymal markersα-SMA and FSP1,and increased the protein level of the endothelial markers CD31 and VE-cadherin(P<0.05).Double immunofluorescence results showed that PFD could reduce the expression ofα-SMA and increase the level of CD31(P<0.05).In the process of EndoMT,the p38 protein expression level was stable(P>0.05).PFD down-regulated significantly the high protein expression of p-p38,and high mRNA expression of TGF-β1 and SNAI1 compared with control group(P<0.05).There was no significant difference between the 0.3 and 0.6mg/mL PFD groups in all results above.CONCLUSION:PFD can inhibit the formation of fibrosis in endothelial cells.TGF-β/p38MAPK signaling pathway might be one of the mechanisms that PFD regulates EndoMT progression.PFD will be expected to become a potential new sight on the treatment of subretinal fibrosis.
作者
廖丁莹
付丽丽
郑玉萍
王丽君
李宏松
张文怡
王建明
赵琳
Ding-Ying Liao;Li-Li Fu;Yu-Ping Zheng;Li-Jun Wang;Hong-Song Li;Wen-Yi Zhang;Jian-Ming Wang;Lin Zhao(Department of Ophthalmology,the Second Affiliated Hospital of Xi'an Jiaotong University,Xi'an 710004,Shaanxi Province,China;Department of Gynecology and Obstetrics,the Second Affiliated Hospital of Xi'an Jiaotong University,Xi'an 710004,Shaanxi Province,China)
出处
《国际眼科杂志》
CAS
北大核心
2021年第2期204-210,共7页
International Eye Science
基金
陕西省重点研发计划项目(No.2018ZDXM-SF-045,2017SF-266)
西安市科技计划项目[No.2017116SF/YX010(10)]。
关键词
吡非尼酮
内皮细胞
内皮间质转化
视网膜下纤维化
Pirfenidone
endothelial cell
endothelial-mesenchymal transition
subretinal fibrosis
