Effect Of Nature Borneol Combined With Benzalkonium Chloride On The Apoptosis Of Rabbit Corneal Epithelial Cells And Its' Mechanism

Backgroud:To ensure asepsis,preservative is often added into ophthalmic preparation.Benzalkonium chloride,a cationic surfactants of quaternary ammonium salt,is commonly used in ophthalmic preparations as a preservative.Borneol is widely used in Chinese medicine eye drops as a algefacient,and is contained in all commercially Chinese medicine eye drops[2],which usualy contained benzalkonium chloride also.Modern pharmacological studies have shown that benzalkonium chloride can directly lead to corneal and conjunctival epithelial cell death or apoptosis[3].Borneol alone or combination with other medicines,can increase the apoptosis of tumor cells[4-7],and borneol can also enhance cell toxicity of benzalkonium chloride on rabbit corneal epithelial cell[8].Azone,an excellent penetrating agent of skin,is now rarely used in ophthalmic preparations for enhancing ocular surface injury combined with benzalkonium chloride[9,10].The effect of borneol,who also has an effect of enhancing penetration,is still not clear combined with benzalkonium chloride[11-16].Based on the widely use of borneol in Chinese medicine ophthalmic preparation and the widely use of preservative such as benzalkonium chloride,the further toxicological mechanism research of the combination effec of borneol and benzalkonium chloride has the urgent clinical significance and important theoretical value for preventing drug-induced dry eyes and evaluating the safety of Chinese medicine ophthalmic preparation.Cell apoptosis is an independent order cell death controlled by genes.Mitochondrial pathway and death receptor pathway is the two main ways of cell apoptosis,the body also has other apoptotic pathways,such as P53 pathway and so on[17].Mitochondrial apoptosis pathway is mainly mediated by Bcl-2 family proteins,through caspase cascade reaction eading to apoptosis eventually[18].Death receptor pathway induces apoptosis by the formation of death signal complex of extracellular ligand and membrane receptor.Such as Fas and Fas ligand(FasL)combination,through the death receptor pathway,caused caspase cascade reaction,eventually activated caspase-3,caused apoptosis.Studies have shown that increase of P53 can cause apoptosis by inducing Fas receptor expression and starting the death receptor pathwayC[19,20],also by activating the Bcl-2 family and starting the mitochondrial apoptotic pathways[21].Based on the mechanism and pathway of apoptosis,the effect of combination of natural borneol and benzalkonium chloride on the cell activity,cell apoptosis,and the expression of apoptosis related gene and protein in rabbit corneal epithelial cell was investigated,which has urgent clinical significance and important theoretical value for the safety evaluation of the Chinese medicine ophthalmic preparation.Objective:To investigate the effect of the combination of natural borneol and benzalkonium chloride on cell activity,cell apoptosis,and the expression of apoptosis related gene and protein in rabbit corneal epithelial cell,which providing preliminary research for the safety evaluation of the Chinese medicine ophthalmic preparation.Methods:The cell viability of rabbit corneal epithelial cell was determined by MTT method,cell apoptosis was determined by flow cytometry with Annexin V/PI staining,and apoptosis related gene and protein expression of Fas,P53,Bcl-2,Caspase3,Caspase9 was detected by Real time RT-PCR method and Western Blot method.Results:Our studies showed,nature borneol(100,200,400 ?g/mL)combined with benzalkonium chloride(2,4,8 u g/mL)can dose-dependent enhance the inhibition effec of benzalkonium chloride(2,4,8 ?g/mL)on cell activity(P<0.01),also can dose-dependent enhance cell apoptosis rate(P<0.01)and mRNA expression of Fas,P53,caspase-9 and caspase-3 on rabbit corneal epithelial cells(P<0.01).Nature borneol(100,200,400 u g/mL)also can dose-dependent enhance Fas and caspase-3 protein expression(except 100 ?g/mL of borneol combined with 4 ? g/mL benzalkonium chloride group,P<0.05 or P<0.01)of benzalkonium chloride(2,4,8 ? g/mL).The combination of nature borneol(100,200,400 ? g/mL)and benzalkonium chloride(2,4,8 ?g/mL)also present the similar dose dependence trend on caspase-9 protein expression,but there was no statistically significant difference(P>0.05).Conclusion:Natural borneol can dose-dependent enhance the effec of benzalkonium chloride on cell toxicity(P<0.01),cell apoptosis(P<0.01)and cell apoptosis-related gene(P<0.01)and protein(P<0.05 or P<0.01)expression in rabbit corneal epithelial cells,which maybe related to death receptor pathway and mitochondrial apoptotic pathway mediated by P53 and Fas.