Researches On The Apoptosis-inducing Effects Of Phenylephrine And Betaxolol On The Human Corneal Stromal Cells

The number of people suffering from glaucoma and other eye diseases is increasing as the global population aging process. Meanwhile, with the development of medical technology more and more clinical drugs can be provided to patients for selection. Among them adrenergic drugs and anti-adrenergic drugs are widely used in clinical ophthalmology. In return, their side effects have become the focus of patients and medicine research. Corneal injuries often happen during administering ophthalmic drugs by means of ocular way which absorbed by the cornea. The human corneal stromal cells (HCS cells) are widely concentrated in the lamina elastica anterior of cornea, which plays a key role in maintaining the normal structure and physiological function of cornea. Therefore, to research the influence of ophthalmic drugs on HCS cells is much importance of evaluation of clinical medicine safety, establishment of low toxic drug sorting system and the development of non-toxic ophthalmic drug. Until now, the side effects of phenylephrine and betaxolol on the HCS cells have not been reported yet. In this paper, the apoptosis-inducing effects of phenylephrine and betaxolol were examined on in vitro cultured untransfected IICS cells, which were obtained by our own laboratory.In order to study the toxic effects and the molecular mechanism of phenylephrine on HCS cells, different concentrations of the drugs obtained by dilution method based on the clinical concentration of phenylephrine (100 g/L) were used to treat the HCS cells, and light microscopy observation, cell viability and cell cycle detection were used to detect the toxic effects of phenylephrine on HCS cells. The results showed that phenylephrine had significant toxic effects on the HCS cells at the concentrations above 0.78125 g/L, such as the decreased cell activity and the cell abnormal growth and proliferation. The detection of cell cycle indicated that the HCS cells were led to S phase arrest under the influence of 6.25 g/L phenylephrine. With the detections of membrane permeability, membrane location of phosphatidylserine, DNA fragmentation and cell ultrastructures, the mechanism of toxicity of phenylephrine on the HCS cells was studied. The results showed that varying degrees of apoptosis of the HCS cells were caused by phenylephrine, such as the increase of membrane permeability, the valgus of phosphatidylserine, the fragments of DNA and the structural features of apoptosis including cytoplasmic empty gun, pyknosis, chromatin condensation, apoptotic bodies. The results showed that phenylephrine does induce apoptosis of HCS cells. Apoptosis can act in several ways with many proteins participating, the expression of Bcl-2 family proteins, the mitochondrial membrane potential changes and the Caspase-3,8,9 activities were detected to determine the apoptosis pathway of the HCS cells. The results showed that the expressions of Bad and Bax were increased while the expressions of Bcl-2 and Bcl-xL were decreased with the mitochondrial membrane depolarization and the activity increase of Caspase-3,8,9. All the results revealing that phenylephrine induced the HCS cells to apoptosis by the mitochondria and death receptor pathway.Different concentrations of betaxolol (0.04375 g/L～2.8 g/L) were used to treat the HCS cells to study the toxic effects and its molecular mechanism. The results of light microscopy observation showed that betaxolol had significant effects on the HCS cells at the concentrations above 0.0875 g/L, such as the decreased cell activity and the cell abnormal growth and proliferation. The detection of cell cycle indicated that the HCS cells were led to G1 phase arrest under the influence of 0.7 g/L betaxolol. The detections of membrane permeability, membrane location of phosphatidylserine, DNA fragmentation and cell ultrastructures showed increase of membrane permeability, the valgus of phosphatidylserine, the fragments of DNA and the structural features of apoptosis including cytoplasmic empty gun, pyknosis, chromatin condensation and apoptotic bodies. The results showed that betaxolol does have apoptosis-inducing effect on HCS cells. The expression of Bcl-2 family proteins, the changes of mitochondrial membrane potential and the Caspase-3,8,9 activities were detected to determine the apoptosis pathway of the HCS cells. The results showed that the expressions of Bad and Bax were increased while the expressions of Bcl-2 and Bcl-xL were decreased with the mitochondrial membrane depolarization and the activity increase of Caspase-9,3. All the results revealing that the apoptosis of HCS cells induced by betaxolol is mitochondria-dependent.The study showed that the clinical ophthalmic drug phenylephrine and betaxolol had abvious apoptosis-inducing effect on HCS cells cultured in vitro. Phenylephrine induces apoptosis of HCS cells by the mitochondria and death receptor pathway, and the apoptosis induced by betaxolol is mitochondria-dependent. Lower concentrations and frequencies and short-term use of phenylephrine and betaxolol are in the clinical applicatioin safely.The use of phenylephrine and betaxolol should be with cautious, and avoid long-term repeated use.