Protective effect and molecular mechanism of liquiritin on oxybuprocaine-induced apoptosis of human corneal endothelial cells

This study was designed to investigate the protective effect and possible molecular mechanism of liquiritin on oxybuprocaine-induced apoptosis of human corneal endothelial cells (HCECs). In this study, the effect of oxybuprocaine on the proliferation of HCEC-12 was detected using cell counting kit-8 (CCK-8). The inductive effect of oxybuprocaine on HCEC-12 apoptosis and protective effect of liquiritin against oxybuprocaine-induced HCEC-12 apoptosis were tested by Annexin V/propidium iodide (PI) staining and flow cytometry. The production of reactive oxygen species (ROS) was analyzed by 2,7-dichlorodi-hydrofluorescein diacetate (DCFH-DA) staining and fluorescent-activated cell sorting (FACS), and the expression of nuclear factor-κB (NF-κB) p65 and apoptosis-related proteins, caspase-3 and Bax, was determined by western blot analysis. Our results show that liquiritin resisted the inhibitory effect of oxybuprocaine on the proliferation of HCEC-12, and cell activity had the most significant increase in pretreatment with liquiritin group in the concentration of 8 mg/ml; compared with that in oxybuprocaine group. Apoptosis in pretreatment with liquiritin was distinctly decreased and liquiritin resisted the production of ROS in HCEC-12 induced by oxybuprocaine. Investigation of molecular mechanism revealed that the pretreatment with liquiritin and pyrrolidinedithiocarbamic acid (PDTC) obviously blocked the expression of NF-κB p65 in nuclear protein increased by oxybuprocaine and the expression levels of total proteins, caspase-3 and Bax.Moreover, tumor necrosis factor-α (TNF-α) blocked the inhibitory effect of liquiritin on the expression of NF-κB p65 in nuclear protein and total proteins, caspase-3 and Bax, thus obstructing the protective effect of liquiritin on corneal epithelial cells. The results of this study indicated that liquiritin reduces the expression of apoptosis protein and increases the expression of anti-apoptotic protein through inhibiting NF-κB signal pathway, thus resisting HCEC-12 apoptosis induced by oxybuprocaine.