Authors: Dong Yahui, Chen Peng, Zhang Zhenzhen, Feng Lu, Zhou Qingjun
Interleukin-6 (IL-6) is a pleiotropic cytokine involving in inflammation and wound healing.Previous report found that IL-6 increases phosphorylated STAT3 (p-STAT3) level and promotes corneal epithelial wound healing by stimulating migration.However, the essential role of IL-6 in corneal epithelial wound healing and the expression changes in diabetic mellitus remains unknown.
This study was to explore the roles of IL-6 in corneal epithelial proliferation and wound healing in both normal and diabetic mice.
Fifty-two normal C57BL/6 mice were randomized into normal control group (32 mice) and diabetic group (20 mice). Type 1 diabetic mellitus was induced by intraperitoneal injections of 50 mg/kg streptozotocin once per day for consecutive 5 days in the mice of the diabetic group.Whole corneal epithelium was scraped in all mice, and the corneal epithelial defect area was examined by fluorescein staining in 24, 48 and 72 hours after corneal epithelium removal.Recombinant mouse IL-6 or anti-IL-6 blocking antibody of 5 μl were subconjunctivally injected according to the grouping and contrasted with PBS injection group or isotype control antibody group, respectively.TKE2 cells, a mouse corneal epithelial stem/progenitor cell line, were trypsinized and incubated in the KSFM with different concentrations of IL-6 or without IL-6, and colony formation efficency (CFE) was examined by crystal violet staining.The expressions of ΔNP63 and Ki67, specific makers of stem cells, were detected by immunofluorescine technology.The expressions of ΔNP63, Ki67 and p-STAT3 proteins were assayed in the cells by Western blot, respectively.The expression of IL-6 mRNA and protein in the regenerated corneal epithelium was detected by real time quantitative PCR and ELISA.The use and care of the mice complied with the Statement of Association for Research in Vision and Ophthalmology.
The percentage of residual corneal epithelium defect area with initial detect area was gradually shrinked over time after PBS and IL-6 injection in both normal control mice and diabetic mice, and the percentage of residual corneal epithelium defect area was significantly reduced in the IL-6 injected group compared with the PBS injected group (normal control group: Fgroup=19.982, P<0.01; Ftime =589.350, P<0.01; Diabetic group: Fgroup =25.411, P<0.01; Ftime =334.807, P<0.01). The CFE was (13.23±1.12)%, (15.87±1.30)%, (21.69±1.62)%, (25.33±1.28)% and (18.67±1.54)% in the blank control group and 10, 20, 50, 100 ng/ml IL-6-treated groups, respectively, showing a gradual increase of CFE dependent upon IL-6 concetrations (F=35.547, P<0.01). The expressions of ΔNP63, Ki67, p-STAT3 proteins in the cells were gradually increased over time after 50 ng/ml IL-6 treated for 5, 10, 15, 30 and 60 minutes, and the relative expression level of the cytokines was significnatly higher in the IL-6 cultured groups than that without IL-6 culture group (all at P<0.05). The relative expression of IL-6 mRNA in the regenerated corneal epithelilum was 0.45±0.21 and 1.00±0.16 in the diabetic group and normal control group, respectively, and compared with the normal control group, the expression of IL-6 mRNA reduced by 56% (t=3.42, P=0.03). The content of IL-6 protein in regenerated corneal epithelium of the diabetic group was (257±12) ng/μl, which was significantly lower than (323±17) ng/μl of the normal control group (t=5.60, P<0.01).
IL-6 promotes the proliferation and regeneration of corneal limbal stem cells to repair defected corneal epithelium by activating STAT3 signaling pathway in both normal and diabetic mice, while the blocking of endogenous IL-6 impairs the corneal epithelial cell activation and wound healing.