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Study on the effect and mechanism of Pirfenidone on endothelial-mesenchymal transition in endothelial cells / 国际眼科杂志(Guoji Yanke Zazhi)
International Eye Science ; (12): 204-210, 2021.
Article in Chinese | WPRIM | ID: wpr-862412
ABSTRACT
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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.<p>

METHODS:

Primary cultured human umbilical vein endothelial cells(HUVEC), 4-7 passages were used for experiments after cell identification. CoCl<sub>2</sub> 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), CoCl<sub>2</sub>(200μmol/L)group, CoCl<sub>2</sub>+0.3mg/mL PFD group, CoCl<sub>2</sub>+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.<p>

RESULTS:

Compared with CoCl<sub>2</sub> group, PFD increased cell proliferation rate and inhibited cell migration significantly under hypoxia(<i>P</i><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(<i>P</i><0.05). Double immunofluorescence results showed that PFD could reduce the expression of α-SMA and increase the level of CD31(<i>P</i><0.05). In the process of EndoMT, the p38 protein expression level was stable(<i>P</i>>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(<i>P</i><0.05). There was no significant difference between the 0.3 and 0.6mg/mL PFD groups in all results above.<p>

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.

Full text: Available Index: WPRIM (Western Pacific) Language: Chinese Journal: International Eye Science Year: 2021 Type: Article

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Full text: Available Index: WPRIM (Western Pacific) Language: Chinese Journal: International Eye Science Year: 2021 Type: Article