Inhibitory effect of rapamycin on proliferation, migration and fibrosis of human pterygium fibroblasts in vitro

Authors: Wu Di,  Sun Xiaonan,  Du Lin,  Zhang Xiaoyu,  Liu Shanshan,  Sun Jing,  Xu Lin,  Zhang Shaodan

DOI: 10.3760/cma.j.issn.2095-0160.2018.12.002
Published 2018-12-10
Cite as Chin J Exp Ophthalmol, 2018,36(12): 902-907.

Abstract

Objective

To investigate the inhibitory effect of rapamycin, an mammalian target of rapamycin (mTOR) pathway inhibitor, on the proliferation, migration and fibrosis of human pterygium fibroblasts (PFBs).

Methods

Pterygium tissues were collected from patients with primary pterygium who underwent surgical excision in Shenyang Fourth People’s Hospital from May to July 2015.The tissues were cultured in vitro and the PFBs were identified by anti-human vimentin immunofluorescence assay.The 3 to 5 generation cells were used for the experiments.The viability of cells treated with different concentrations of rapamycin was detected by methyl thiazolyl tetrazolium (MTT). The cells were divided into normal control group and rapamycin group, and the scratch wound healing test was used to evaluate migration of the PFBs.The expressions of MKI67, α-smooth muscle actin (α-SMA), fibronectin, caspase3, mammalian target of rapamycin (mTOR) and LC3B mRNA were detected by real-time quantitative PCR.

Results

The cultured cells showed morphology of long spindle and were vimentin immunopositive.The cell viability in rapamycin treated PFBs demonstrated a dose-dependent decrease.At 24 hours after culture, The cell viability in 30 μmol/L rapamycin group was (76.67±8.84)% of that in 0 μmol/L rapamycin group (P<0.001). The relative residual scratch width in 30 μmol/L rapamycin group was (35.40±11.62)% 48 hours after scratch, which was significantly greater than (2.45±0.76)% in the normal control group (P<0.05). Real-time quantitative PCR showed that the mRNA expressions of MKI67, α-SMA, fibronectin and mTOR in rapamycin group were significantly decreased when compared with those in normal control group (all at P<0.05). The expression of LC3B mRNA in rapamycin group was significantly higher than that in normal control group (P<0.05). The mRNA expression of caspase3 was not significantly different between the two groups (P=0.861).

Conclusions

Rapamycin can effectively inhibit the proliferation, migration and fibrosis of PFBs without affecting the cell survival.Detailed mechanism remains to be further studied.Rapamycin may serve as an anti-fibrosis agent to prevent the progression and recurrence of pterygium in the future.

Key words:

Pterygium; Fibroblasts; Rapamycin; Anti-fibrosis

Contributor Information

Wu Di
Department of Ophthalmology, The Fourth People’s Hospital of Shenyang, Key Lab of Ophthalmology of Shenyang, Shenyang 110031, China
Sun Xiaonan
Department of Ophthalmology, The Fourth People’s Hospital of Shenyang, Key Lab of Ophthalmology of Shenyang, Shenyang 110031, China
Du Lin
Department of Ophthalmology, The Fourth People’s Hospital of Shenyang, Key Lab of Ophthalmology of Shenyang, Shenyang 110031, China
Zhang Xiaoyu
Department of Ophthalmology, The Fourth People’s Hospital of Shenyang, Key Lab of Ophthalmology of Shenyang, Shenyang 110031, China
Liu Shanshan
Department of Ophthalmology, The Fourth People’s Hospital of Shenyang, Key Lab of Ophthalmology of Shenyang, Shenyang 110031, China
Sun Jing
Department of Ophthalmology, The Fourth People’s Hospital of Shenyang, Key Lab of Ophthalmology of Shenyang, Shenyang 110031, China
Xu Lin
Department of Ophthalmology, The Fourth People’s Hospital of Shenyang, Key Lab of Ophthalmology of Shenyang, Shenyang 110031, China
Zhang Shaodan
The Eye Hospital, School of Ophthalmology & Optometry, Wenzhou Medical University, Glaucoma Institute of Wenzhou Medical University, Wenzhou 325027, China
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