Figure 6: The effect of shikonin on high glucose/hypoxia damaged retinal pigment epithelial (RPE) cells.

RPE cells incubated under high or low glucose concentrations and hypoxia (1% O₂) with or without shikonin (0.01–10 μM) for 24 hr. Cell viability was compared between cells incubated under low or high glucose concentrations using the MTT assay. Data are expressed as mean ± S.D. from at least three independent experiments. *p < 0.05, ***p < 0.001 compared with low glucose control. (A) Paracellular tracer-flux assays were performed using FITC-dextran (molecular masses, 40 kDa (B) and 70 kDa (C), respectively) and dense RPE monolayers treated with or without shikonin (0.01–10 μM, 24 h). Results from at least 3 independent experiments are shown. ***p < 0.001 compared with low glucose control. ^#p < 0.05, ^##p < 0.01, and ^###p < 0.001, compared with untreated high glucose/hypoxia group. (D) RPE cells were incubated with or without shikonin (0.003–0.3 μM) for 24 h under low-glucose/hypoxic or high-glucose/hypoxic conditions. Cell lysate proteins (40 μg) from each incubation were analyzed by separation on 10% reducing SDS-PAGE gels and western blotting with specific antibodies against inflammatory proteins hypoxia inducible factor 1-α (HIF1-α), cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and myeloperoxidase (MPO). (E) Representative images of western blots showing changes in the expression of adherens junction proteins and tight junction proteins in RPE cells treated with/without shikonin under low-glucose/hypoxic or high-glucose/hypoxic conditions.