Use Of Human Corneal Epithelial Cells For Eye Irritation And Inflammation Assessment

Eye irritation is a common health complaint in the office and office-like environment.A better understanding about the cause and the mechanism of eye irritation may therefore be important to improve working environment quality.Furthermore,for the safe use of products like cosmetics,pharmaceuticals,industrial chemicals etc,it is also pivotal to get adequate data about eye irritating potential of the compounds.Till present,the information about human eye irritation in laboratory researches is largely based on Draize rabbit eye irritation test,which has been criticized for some years especially for its cruelty.Moreover,the human eye inflammatory response is not assessed by this method,and the extrapolation from rabbit eyes to human eyes may be difficult.In the present study,human corneal epithelial cells model was used as an in vitro model for the assessment of human eye irritating and inflammatory potential of lipopolysaccharide (LPS) and dust.The major methods and results are as the followings,ⅠDifferent concentrations of LPS were used to expose HCE for 6h.MTT method was used to measure cellular viability to reflect eye irritation,and ELISA method was used to measure the changes of typical cytokines IL-1β,IL-8 and TNFαto reflect inflammatory response in human corneal cells.Results showed that LPS had an influence in cellular viability,but the influence was not dose-dependent,which suggested the influence of LPS on cellular viability was not great.Stimulation of human oral epithelial primary cells by this kind of LPS showed that cellular viability was decreased insignificantly,which further confirmed that LPS had limited effect on viability of human epithelial cells.ELISA results showed that IL-1βcould be measured in the media,but the concentrations were very low and without significant changes.TNFαwas not measured in media.IL-8 was significantly elevated after exposure(p＜0.01).These results suggested that IL-1βand TNFαwere not activated by LPS exposure,but IL-8 response was activated.LPS could induce inflammatory response in human corneal epithelial cells.ⅡDifferent concentrations of dust were used to expose HCE for 24h,and MTT and ELISA were used to assess eye irritation and inflammatory response,respectively. Results showed that low concentrations of dust solutions(0.05mg/ml,0.5mg/ml) had no effect on cellular viability and cytokines release,which suggested that dust solution at low concentration did not lead to human eye irritation and inflammatory response. Higher concentrations of dust(15mg/ml and 30mg/ml) exposure resulted in significant decrease of cellular viability,as well as the significant increase of IL-1βconcentration and IL-8 concentration in dose-dependent manner.But the concentration of TNFαwas not significantly changed.These results suggested that high concentrations of dust could lead to human eye irritation,and inflammatory response like IL-1βand IL-8 response, but not TNFαresponse.ⅢHCE was exposed to dust solution at different humidity,different sediment time of dust solution and different amount.Results showed that under saturated humidity,the initial dose for dust to affect cellular viability and to induce IL-1βand IL-8 inflammatory response was much higher.Meanwhile,HCE cellular viability by dust which had been stored by 15min after sonication was significantly higher than that of freshly prepared dust(p<0.05).These results combined suggested that humidity and dust sediment have effects on the results.Furthermore,it is interesting to notice that changes in cellular viability and cytokines release by 0.2ml 15mg/ml dust exposure were similar when compared with 0.1ml 15mg/ml dust,but were obviously different than that of 0.1ml 30mg/ml dust.These results suggested that it was the dust concentration,rather than the total amount of dust,has the greater effects on cellular viability and cytokine releases when using this model as the in vitro model.ⅣHistology was used to observe morphology changes of HCE after exposure.PBS control group showed no major histological changes,and HCE showed normal thickness. SDS positive control showed histological changes in thickness and necrosis,and the 3D structure was destroyed by SDS exposure.After exposure to 500μg/ml dust for 24h,HCE showed changes in thickness.Meanwhile,5mg/ml dust exposure caused morphology changes in thickness and vacuolization,but the 3D structure remained.The results of histology were consistent with MTT results.ⅤScanning Electron Microscope(SEM) was used to scan the surface of dust used in this study.Results showed that dust contains a high content of both living and dead organic material of vegetable,animal or microbial origin.Thus the eye irritating and inflammatory effect by dust is the result of its various components.The present results suggested that this HCE model could be used in in vitro study of toxicological studies of eye irritation and inflammatory response.High concentrations of dust could result in human eye irritation,and IL-1βand IL-8 were involved in inflammatory response,which could be used as biomarkers.LPS had an effect on eye irritation.IL-8,but not IL-1β,was involved in the inflammatory response.TNFαwas not involved in neither of the two responses.