Study on the effect and mechanism of Pirfenidone on endothelial-mesenchymal transition in endothelial cells

Abstract

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 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.

Keywords

• pirfenidone
• endothelial cell
• endothelial-mesenchymal transition
• subretinal fibrosis