Effects And Mechanism Of Estradiol And Testosterone Propionate On The Rabbit Lacrimal Gland Cell Apoptosis

ObjectiveThrough observing the cell apoptosis and the expression of MMP-9 of lacrimal gland epithelial cells which are cultured in vitro and treated with estradiol(E₂) and testosterone propionate(TP), and then to investigate the impact of estradiol and testosterone propionate on the mechanism of rabbit lacrimal gland epithelial cells, for further studying the effect of estrogen and androgen on dry eye. Materials and Methods1. Grouping1) There were four groups: estradiol group (E2 ), testosterone propionate group (TP), apoptosis control group(AC) and blank control group (BC) respectively. The cells of group E2 and group TP were treated with 1x10^-5,1x10^-6,1x10^-7,1x10^-8mol/L estradiol and testosterone propionate respectively , the cells of group AC were treated with H₂O₂ only but group BC with nothing.2) The cells of group AC were induced into apoptosis by H₂O₂, group E and T were treated with 1x10^-5 mol/L estradiol and testosterone propionate respectively, and group BC with nothing.2. Rabbit lacrimal gland epithelial cell culture and HE staining Rabbit lacrimal gland tissues were aseptic removal. And then the lacrimal gland cells were cultured by using tissue and mixed digestion methods, purified, observed and photographed under the inverted microscope. Cell morphology was observed by HE staining.3. Identification of lacrimal gland epithelial cellsPan-cytokeratin antibody was used to identify the cells by immunocytochemisty.4. Hoechst 33342 stainingThe lacrimal gland epithelial cells apoptosis after drug intervention were estimated using Hoechst 33342 staining.5. Assessing the activity of lacrimal gland epithelial cells with MTT assayMTT method was used to evaluate the OD values of the activity of lacrimal gland cells between different groups after treated with different concentrations of estrodiol and testosterone propionate.6. Evaluating the apoptosis of lacrimal gland epithelial cells by Annexin V and PI staining methodCells apoptosis after drug intervention were analysed by flow cytometry(FCM), and then the impacts of estrodiol and testosterone propionate on apoptosis of lacrimal gland epithelial cells were observed.7. Evaluating the expression of MMP-9 by immunocytochemistryImmunocytochemistry method was used to evaluate the expression of MMP-9 in cells induced apoptosis by H₂O₂ after estrodiol and testosterone propionate intervention.Results1. Lacrimal gland cells culture and HE stainingCultured cells were in good condition, bright body, clear membrane, strong shading. Llacrimal gland epithelial cells with HE staining were showed irregular polygon, clear cell outline, obvious nuclear staining and even visible nucleoli.2. Identification of lacrimal gland epithelial cellsImmunocytochemical analysis with DAB reaction showed positive staining for pan- cytokeratin distributing in the cytoplasm evenly.3. Hoechst 33342 stainingIn group AC, the cell staining was blue fluorescence strongly and irregular, and some cells had shed off. In group BC, the cell staining was uniform and oval, and cells had no significant shedding. Part of cell nuclei staining showed high fluorescence in group E₂ and TP.4. Assessing the cell activity with MTT assayCompared with bland control group, the activity of lacrimal gland epithelial cells decreased obviously after induced into apoptosis by 10^-4mol/L H₂O₂, the OD values were significantly different (P <0.01); Compared with group AC, the activity of lacrimal gland epithelial cells treated with different concentrations of estradiol and testosterone propionate increased, in generally the OD values of cell activity of group E₂ and TP were 0.4434±0.0981 and 0.3928±0.0518 respectively, the effects of estradiol and testosterone propionate on inhibiting cell apoptosis induced by H₂O₂ were statistically different (P<0.05); Estradiol inhibit cell apoptosis more stronger than testosterone propionate, the difference was statistically significant (P< 0.05); And the protection of estradiol was dose-dependent.5. The roles of estradial and testosterone propionate on the apoptosis of lacrimal gland epithelial cellsDifferent concentrations of estradiol and testosterone propionate could inhibit early and late cell apoptosis and necrosis of lacrimal gland epithelial cells. The apoptosis rate of group E2 and TP was lower than the apoptosis control group. In estradiol group, the apoptosis rate was in dose-dependent, the higher the concentration, the smaller the rate of apoptosis. The apoptosis rate of group E2 was lower than group TP. All the differences were statistically significant (P<0.01, P < 0.05).6. Impacts of estradiol and testosterone propionate on expression of MMP-9 of lacrimal gland cellsCompared with group BC, the integrated optical density of MMP-9 expression of lacrimal gland cells of group AC,E and T increased significantly after the cells were induced by H₂O₂; the group E and group T reduced compared with group AC; group E₂ were significantly lower than group TP; the differences all were statistically significant (P <0.01).7. the correlation analysis between MMP-9 expression and apoptosis rateIt was positive correlation between MMP-9 expression and apoptosis rate in the different intervention group(r=0.943, p<0.01).Conclusions1. Estradiol and testosterone propionate could inhibit the apoptosis of the rabbit lacrimal gland epithelial cells induced by H₂O₂.2. The effect of estrogen on inhibiting lacrimal gland cell apoptosis was in dose- dependent.3. The inhibitory impact of estrogen on lacrimal gland cells apoptosis was more obvious than androgen.4. Estradiol and testosterone propionate could inhibit the expression of MMP-9 of lacrimal gland epithelial cells induced into apoptosis by H₂O₂.5. It was positive correlation between MMP-9 expression and apoptosis rate after estradiol and testosterone propionate intervention.6. The mechanism of estradiol and testosterone propionate repression of lacrimal gland cell apoptosis may be related with MMP-9.