PURPOSE To explore the protective effect of serine protease inhibitor A3K (SA3K) on barrier integrity of rabbit corneal endothelium disrupted by TNF-α.

METHODS New Zealand rabbits’ corneal tissues were incubated ex vivo for 24h in DMEM+10% FBS with or without TNF-α, in the absence or presence of SA3K at different concentrations. Barrier function was determined by the permeability of FITC-dextran across the corneal endothelium. The integrity of apical junctional complex (AJC, including ZO-1, VE-cadherin and F-actin) and microtubule were visualized by immunofluorescent staining. The proteins and mRNAs of ZO-1 and VE-cadherin were evaluated by western blot analysis and RT-PCR respectively. And their regulator myosin light chain (MLC) was also determined by Western blot
analysis.

RESULTS The permeability to FITC-dextran across corneal endothelium was highly increased by TNF-α (20ng/ml) compared with untreated control (P<0.01). Addition of SA3K (100-200nM) significantly decreased the TNF-α stimulated permeability in a concentration-dependent manner. Tight junctions ZO-1 and subjacent adherens junctions VE-cadherin were observed to be largely disrupted by TNF-α, but almost restored to normal integrity by SA3K. Interestingly, their linked cytoskeleton F-actin was irregularly rearranged by TNF-α while SA3K restored F-actin to normal double-band structures. SA3K attenuated the disassembly of microtubule induced by TNF-α. Furthermore, phosphorylation of MLC was found to be dramatically stimulated by TNF-α and this activation was significantly blocked by SA3K.

CONCLUSIONS  Our findings demonstrate that SA3K protects rabbit corneal endothelium from the TNF-α-induced disruption of barrier structure and function by maintaining the integrity of AJC through suppression of MLC activation. It suggests for the first time that SA3K may have a therapeutic potential in treating inflammation-disrupted barrier function.