Rapamycin ameliorates corneal injury after alkali burn through methylation modification in mouse TSC1 and mTOR genes

• Published in: Experimental eye research 2021-02, Vol.203, p.108399, Article 108399
• Main Authors: Li, Jiande, Du, Shaobo, Shi, Yongpeng, Han, Jiangyuan, Niu, Zhanyu, Wei, Li, Yang, Pengfei, Chen, Linchi, Tian, Huanbing, Gao, Lan
• Format: Article
• Language: English
• Subjects: Actins - genetics; Alkali burn; Animals; Blotting, Western; Burns, Chemical - genetics; Burns, Chemical - pathology; Burns, Chemical - prevention & control; Chromones - pharmacology; Cornea; Corneal Injuries - genetics; Corneal Injuries - pathology; Corneal Injuries - prevention & control; Disease Models, Animal; DNA Methylation; DNA methylation Regulation; Eye Burns - chemically induced; Fluorescent Antibody Technique, Indirect; Gene Expression Regulation - physiology; Immunosuppressive Agents - therapeutic use; Male; Mice; Morpholines - pharmacology; mTOR; Neovascularization; Phosphatidylinositol 3-Kinases - metabolism; Phosphoinositide-3 Kinase Inhibitors - pharmacology; Proto-Oncogene Proteins c-akt - antagonists & inhibitors; Proto-Oncogene Proteins c-akt - metabolism; Rapamycin; Real-Time Polymerase Chain Reaction; Sirolimus - therapeutic use; Sodium Hydroxide - toxicity; TOR Serine-Threonine Kinases - genetics; TSC1; Tuberous Sclerosis Complex 1 Protein - genetics; Vascular Endothelial Growth Factor A - genetics
• ISSN: 0014-4835; 1096-0007
• DOI: 10.1016/j.exer.2020.108399

Alkali burn to the cornea is one of the most intractable injuries to the eye due to the opacity resulting from neovascularization (NV) and fibrosis. Numerous studies have focused on studying the effect of drugs on alkali-induced corneal injury in mouse, but fewer on the involvement of alkali-induced DNA methylation and the PI3K/AKT/mTOR signaling pathway in the mechanism of alkali-induced corneal injury. Thus, the aim of this study was to determine the involvement of DNA methyltransferase 3 B-madiated DNA methylation and PI3K/AKT/mTOR signaling modulation in the mechanism of alkali-induced corneal injury in a mouse model. To this end, we used bisulfite sequencing polymerase chain reaction and Western blot analysis, to study the effects of 5-aza-2′-deoxycytidine and 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one, which inhibit methyltransferase and PI3K respectively, on DNA methylation and expression of downstream effectors of PI3K related to corneal NV, including TSC1 and mTOR genes. The results showed that, after an intraperitoneal injection of rapamycin (2 mg/kg/day) for seven days, the alkali-induced opacity and NV were remarkably decreased mainly by suppressing the infiltration of immune cells into injured corneas, angiogenesis, VEGF expression and myofibroblasts differentiation; as well as by promoting corneal cell proliferation and PI3K/AKT/mTOR signaling. More significantly, these findings showed that epigenetic regulatory mechanisms by DNA methylation played a key role in corneal NV, including in corneal alkali burn-induced methylation modification and rapamycin-induced DNA demethylation which involved the regulation of the PI3K/AKT/mTOR signaling pathway at the protein level. The precise findings of morphological improvement and regulatory mechanisms are helpful to guide the use of rapamycin in the treatment of corneal angiogenesis induced by alkaline-burn. •Rapamycin reduces the expression of VEGF and α-SMA to reduce corneal NV and opacity and Rapamycin suppresses injury-induced angiogenesis and immune cell infiltration as Rapamycin reduces the expression of VEGF and α-SMA to reduce corneal NV and injury-induced immune cell infiltration.•Highlighting a key role for methylation regulation via DNMT3b in TSC1 and mTOR.•mTOR mediates control of wound healing responses through modulating the PI3K/AKT signaling pathway axis.•Elucidation of mTOR involvement identifies novel targets for improved therapeutic management of severe corneal injury.