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。

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 markers α-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 of α-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.