Effect and potential signaling pathway of hedgehog protein on the function of human retinal microvascular endothelial cell

Authors:Li Yang,  Hu Qinrui,  Wang Bin,  Xia Huika,  Zhang Xiaohu
DOI: 10.3760/cma.j.issn.2095-0160.2019.09.005
Published 2019-09-10
Cite as Chin J Exp Ophthalmol, 2019,37(9): 713-718.

Abstract

Objective

To explore the impact of Hedgehog protein on human retinal microvascular endothelial cell(HRMEC)and its signaling pathway.

Methods

The cultured HRMECs were divided into normal control group, 0.5 μmol/L agonist group and 1.0 μmol/L agonist group, and were cultured in medium with final concentration of 0, 0.5 and 1.0 μmol/L Hedgehog agonist, respectively; HRMECs cultured in high glucose medium were divided into high glucose control group, 1.5 μmol/L inhibitor group and 2.5 μmol/L inhibitor group.Erismodegib, the Smoothed inhibitor with final concentration of 0, 1.5 and 2.5 μmol/L was added into corresponding group, respectively.MTS method and Transwell cell migration method were used to detect the proliferation(A490 value)and relative mobility of HRMEC.The phosphorylation of PLCγ1, Akt and Erk proteins were detected by Western blot.

Results

The relative expression of Hedgehog protein in the high glucose control group was 6.24±0.11, which was significantly higher than 1.00±0.00 in the normal control group(t=667.573, P<0.001). The A490 value was 1.349±0.050 and 1.422±0.053, and the relative mobility rate was 2.34±0.14 and 3.59±0.32 in the0.5 μmol/L agonist group and the 1.0 μmol/L agonist group, respectively, which were significantly higher than 1.203±0.101 and 1.00±0.00 in the normal control group(all at P<0.01). The A490 value was 0.849±0.010 and 0.737±0.030, and the relative mobility rate was 0.43±0.02 and 0.27 ±0.01 in the 1.5 μmol/L inhibitor group and the 2.5 μmol/L inhibitor group, respectively, which were significantly lower than 1.000±0.040 and 1.00±0.00 in the high glucose control group(all at P<0.01). The phosphorylation ratios of PLCγ1, Akt and Erk in the 0.5 μmol/L agonist group and the 1.0 μmol/L agonist group were significantly higher than those in the normal control group(all at P<0.01). The phosphorylation ratios of PLCγ1, Akt and Erk in the 1.5 μmol/L inhibitor group and the 2.5 μmol/L inhibitor group were significantly lower than those in the high glucose control group(all at P<0.01).

Conclusions

High glucose induces the expression of Hedgehog protein in HRMEC.Hedgehog protein may regulate the function of HRMEC by regulating the phosphorylation of PLCγ1, Akt and Erk in G Protein-coupled receptors pathway.

Key words:

Diabetic retinopathy; Hedgehog protein; G Protein-coupled receptors signaling pathway; Human retinal microvascular endothelial cell

Contributor Information

Li Yang
Xiamen Eye Centre of Xiamen University, Xiamen 361001, China
Hu Qinrui
Xiamen Eye Centre of Xiamen University, Xiamen 361001, China
Wang Bin
Xiamen Eye Centre of Xiamen University, Xiamen 361001, China
Xia Huika
Department of Ophthalmology, Peking University People’s Hospital, Beijing 100044, China
Zhang Xiaohu
Xiamen Eye Centre of Xiamen University, Xiamen 361001, China
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