Abstract
PurposeTo investigate the protective effects of thymosin β4 (Tβ4) on ethanol injured human corneal keratocytes (HCKs).MethodsHCKs and BALB/c mice were chosen as the study subject. Ethanol was used to treat the cells and corneal stroma of mice to build the ethanol injured model in vitro and vivo respectively. CCK-8 was used to evaluate the cell metabolic activity. DCFH-DA was used to detect the intracellular reactive oxygen species level. TUNEL was chose to detect the cell apoptosis rate. The cell proliferation and migration were investigated by using wound healing insert. Wound healing of corneal surface and stroma was observed by using fluorescein sodium eyedrop and HE stain. RT-qPCR, ELISA, and immunostaining were performed to detect gene and protein expression in keratocytes or corneal stroma tissue of mice.ResultsEthanol induced oxidative stress injury and cell apoptosis on HCKs, and Tβ4 can alleviate it by up-regulating the expression of Bcl-2, catalase, and CuZnSOD, and inhibiting the expression of Caspase-3. Tβ4 promotes the proliferation of HCKs and the process of corneal wound healing. It may relevant to the up-regulated expression of Ki67.ConclusionsOur study established an ethanol-injured corneal stroma model in both vitro and vivo. The present study confirmed that Tβ4 play a protective effect on the reconstruction process of ethanol-injured corneal stroma.
Highlights
Ethanol is commonly used in clinic, and in daily life
Effect of ethanol and thymosin β4 (Tβ4) on the cell metabolic activity of human corneal keratocytes (HCKs) To investigate the effect of various concentrations of ethanol and Tβ4 on the cell metabolic activity of HCKs, we performed a dose–response experiment, in which serially diluted ethanol (0, 5, 10, 15, 20 and 25%) was added to injury HCKs, and beyond that, HCKs treated with 15% ethanol was applied to culture in medium supplementing with 1 μg/ml Tβ4
The results of Cell Counting Kit-8 (CCK-8) showed that low concentration of ethanol (5%) resulted in higher cell metabolic activity (111.7 ± 6.06%) than the control group (100 ± 0.00%)
Summary
Most of ophthalmology surgeries would apply ethanol to sterilize the corneal surface and periorbital thin skin. Some types of refractive surgery, such as laser-assisted subepithelial keratectomy (LASEK), photorefractive keratectomy (PRK) and removing epithelial flap type cross linking, use 20% ethanol to loosen the connection among cornea epithelial cells and remove the epithelium. The thymosin proteins were discovered from fractionations of calf thymus tissue. Under natural conditions, they are short, highly charged, intrinsically unstructured proteins [3]. Thymosin family were found in thymus and in other tissue, such as macrophages, tumor cells and hemocytes except erythrocyte [4, 5]. Previous studies revealed that the main function of thymosin β is inhibiting
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