Diabetes inhibits corneal epithelial cell migration and tight junction formation in mice and human via increasing ROS and impairing Akt signaling

Abstract

Corneal wounds usually heal quickly; but diabetic patients have more fragile corneas and experience delayed and painful healing. In the present study, we compared the healing capacity of corneal epithelial cells (CECs) between normal and diabetic conditions and the potential mechanisms. Primary murine CEC derived from wild-type and diabetic (db/db) mice, as well as primary human CEC were prepared. Human CEC were exposed to high glucose (30 mM) to mimic diabetic conditions. Cell migration and proliferation were assessed using Scratch test and MTT assays, respectively. Reactive oxygen species (ROS) production in the cells was measured using dichlorofluorescein reagent. Western blot was used to evaluate the expression levels of Akt. Transepithelial electrical resistance (TEER) and zonula occludens-1 (ZO-1) expression were used to determine tight junction integrity. We found that the diabetic CEC displayed significantly slower cell proliferation and migration compared with the normal CEC from both mice and humans. Furthermore, ROS production was markedly increased in CEC grown under diabetic conditions. Treatment with an antioxidant N-acetyl cysteine (NAC, 100 μM) significantly decreased ROS production and increased wound healing in diabetic CEC. Barrier function was significantly reduced in both diabetic mouse and human CEC, while NAC treatment mitigated these effects. We further showed that Akt signaling was impaired in diabetic CEC, which was partially improved by NAC treatment. These results show that diabetic conditions lead to delayed wound-healing capacity of CEC and impaired tight junction formation in both mice and human. Increased ROS production and inhibited Akt signaling may contribute to this outcome, implicating these as potential targets for treating corneal wounds in diabetic patients.

Keywords: Akt signaling; N-acetyl cysteine; ROS; cell migration; cell proliferation; corneal epithelial cells; diabetes; healing capacity.

Fig. 1

Diabetes inhibits corneal epithelial cell proliferation. Mouse primary corneal epithelial cell (mCEC) and diabetic mCEC (dmCEC) proliferation was measured using an 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) assay after growth for 1–5 days (a). Human primary CEC (hCEC) proliferation was measured using an MTT assay after growth for 1–5 days with normal glucose or high glucose (30 mM) (b). (c, d) A colony formation assay also showed higher colony-forming ability of both mCEC and hCEC over their diabetic counterparts, dmCEC and hCEC + glucose, respectively. n = 3 independent experiments. Mean ± S.D. *P < 0.05; **P < 0.01

Fig. 2

Diabetes inhibits corneal epithelial cell migration in vitro. Cell migration was observed at the indicated time points after generation of the scratch (a, b). Relative migration distances for the indicated cells were quantified (c, d). n = 4 independent experiments. Mean ± S.D. *P < 0.05; **P < 0.01

Fig. 3

Diabetic corneal epithelial cells increase the generation of intracellular reactive oxygen species (ROS). Representative images were observed and captured after 24 h of culture (a), and the ROS Brite intensity was analyzed by ImageJ software (b). n = 4 independent experiments. In cultured stratified human primary CEC (hCEC), ROS production was significantly increased (c). n = 3 or 6 independent experiments. Mean ± S.D. **P < 0.01

Fig. 4

N-acetyl cysteine (NAC) reverses the observed delay in migration under diabetic conditions. Cell migration was observed at the indicated time points after the scratch wound with or without NAC (100 µM; a, b). The relative migration distances for the indicated cells were quantified (c, d). n = 4 independent experiments. Mean ± S.D. *P < 0.05; **P < 0.01

Fig. 5

Hyperglycemia compromises corneal epithelial barrier function. Stratified human primary CEC (hCEC) have a reduced number of corneal epithelial cell (CEC) layers (a) and significantly reduced transepithelial electrical resistance (TEER) values in the presence of high glucose (b). Significantly lower TEER values were observed in cellular monolayers under diabetic conditions at each indicated time point; treatment with NAC significantly restored barrier function (c, d). Expression of zonula occludens-1 (ZO-1) was reduced following treatment with high glucose and this effect was mitigated by NAC treatment (e). n = 6 independent experiments. Mean ± S.D. *P < 0.05; **P < 0.01

Fig. 6

Downregulation of phosphorylated Akt (p-Akt) pathways in diabetic corneal epithelial cells. The indicated cells were subjected to Western blot using antibodies to p-Akt, total Akt, and β-actin (loading control; a, b). The normalized expression level was analyzed by ImageJ software. n = 3 independent experiments (c, d). Mean ± S.D. **P < 0.01