Different concentrations of betaxolol switch cell fate between necroptosis, apoptosis, and senescence in human corneal stromal cells.

Betaxolol is commonly used to manage glaucoma in clinical practice. However, its long-term use may damage the cornea. Thus, the cytotoxicity and mechanisms of betaxolol in human corneal stromal cells (HCSCs) warrant further study. In this study, we used in vitro HCSCs and in vivo rabbit corneal models to investigate betaxolol cytotoxic effects and mechanism of action. At near-clinical concentrations (0.28% and 0.14%), betaxolol inhibited caspase-8 activity, activated receptor-interacting protein kinase (RIPK)1, RIPK3, and mixed-spectrum kinase-like domain (MLKL), and phosphorylated MLKL to induce necroptosis in HCSCs. Similarly, moderate concentrations of betaxolol (0.07%-0.0175%) activated caspase-8 to trigger the exogenous apoptotic pathway. Through the intrinsic apoptotic pathway, betaxolol upregulated the expression of Bcl-2 family apoptotic proteins Bax and Bad and downregulated that of anti-apoptotic proteins Bcl-2 and Bcl-xL. This subsequently disrupted the mitochondrial membrane potential and cytoplasmic transfer of cytochrome c and apoptosis-inducing factor, activated caspase-9, and induced apoptosis in HCSCs. Furthermore, continuous treatment with low betaxolol concentrations (0.00875%) for three generations of HCSCs prevented apoptosis by promoting the expression of Bcl-xL and suppressing that of Bax. However, its toxic effects initiated cellular senescence by increasing reactive oxygen species, leading to the disruption of energy metabolism and DNA damage. Finally, clinical concentrations of betaxolol had a pro-apoptotic effect on rabbit corneal stromal cells in vivo. These results suggest that betaxolol induces cytotoxicity in a concentration-dependent manner in HCSCs, and that caspase-8 and Bcl-2 family proteins may be critical switches in the conversion of different HCSC death mechanisms.