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CoCl2-induced alterations in antioxidative and inflammatory marker expression in an siRNA-based in vitro model of
Shao-Lun Hsu1,2, Nóra Szentmáry3,4, Fabian N Fries3,5
1Dr. Rolf M. Schwiete Center for Limbal Stem Cell and Congenital Aniridia Research, Saarland University, Kirrberger Str. 100, 66424, Homburg/Saar, Germany. arinahsu@gmail.com.
Background:
Congenital aniridia is a rare disease, accompanied by aniridia associated keratopathy (AAK) in most cases. Oxidative stress and inflammation are involved in the progression of AAK. We aimed to evaluate antioxidative and inflammatory gene and protein expression in a small interfering RNA (siRNA) paired box 6 (PAX6) knockdown limbal epithelial cell (LEC) model of aniridia under cobalt chloride (CoCl2)-induced stress.
Methods:
To mimic PAX6 haploinsufficiency in congenital aniridia, PAX6 expression was knocked down by 24-hour siRNA treatment in primary human LECs. Hypoxia-mimetic conditions were induced by 50 µM or 75 µM CoCl₂ for 48 h following siRNA transfection. Messenger RNA (mRNA) expression levels were analyzed by quantitative polymerase chain reaction (qPCR), while protein expression levels were assessed by enzyme-linked immunosorbent assay (ELISA) or western blotting.
Results:
Vascular endothelial growth factor A (VEGFA) protein levels were significantly increased in PAX6 knockdown LECs compared with control siRNA-treated cells (p = 0.007). In contrast, hypoxia-inducible factor 2-alpha (HIF-2α) and interleukin-6 (IL-6) mRNA levels (p = 0.031, p = 0.001), as well as interleukin-1 beta (IL-1β), IL-6, and interleukin-8 (IL-8) protein levels (p = 0.049, p < 0.001, p < 0.001, respectively), were significantly reduced in PAX6 knockdown cells compared with control siRNA-treated LECs. CoCl₂ treatment (50 and 75 µM) reduced hypoxia-inducible factor 1-alpha (HIF-1α) mRNA expression in both groups (p = 0.019 and p = 0.007; p < 0.001 and p = 0.046, respectively). In control cells, 75 µM CoCl₂ increased IL-1β and IL-8 mRNA expression (p = 0.022, p = 0.019) as well as IL-1β protein levels (p = 0.024), whereas IL-8 protein levels decreased at both concentrations (p = 0.002 and p < 0.001). No other analyzed genes showed significant expression changes in response to CoCl₂ treatment in either the control or PAX6 knockdown groups.
Conclusions:
Our study demonstrates altered expression of hypoxia-related (HIF-1α, HIF-2α) and inflammatory (IL-1β, IL-6, IL-8) genes in response to CoCl₂ treatment or PAX6 haploinsufficiency. Further investigation is needed to elucidate the effects of PAX6 knockdown and its interaction with inflammatory pathways. This research may contribute to better understanding of congenital aniridia.
